Phenolic Content and Phenolic Acid Composition of Einkorn and Emmer Ancient Wheat Cultivars—Investigation of the Effects of Various Factors

Abstract

Interest in ancient wheat species is growing because of their unique agronomic and nutritional qualities, and they could be potential sources of antioxidants. The aim of this research was to determine the total, bound, and free phenolic content (TP, FP, BP), the bound and free phenolic acid (BPA, FPA) content, and the phenolic acid (PA) composition of einkorn and emmer cultivars sourced from a two-year pesticide-free organic variety trial. TPs, FPs, and BPs were analyzed using spectrophotometry, and PAs were determined using HPLC/MS/MS. The results showed that highest mean TP, FP, and BP contents were found in an emmer cultivar, while generally, einkorn varieties had lower phytonutrient values than emmer and bread wheat control. Emmer had the highest TPA, FPA, and BPA contents, followed by control wheat and einkorn landraces. Our gap-filling research was the analysis of the individual PA values in all free and bound fractions. Ferulic acid was the predominant phenolic acid, followed by p-coumaric acid, syringic acid, sinapic acid, and p-hydroxybenzoic acid, whereas salicylic acid and caffeic acid had the lowest concentrations. In the future, we propose to continue this research to gain deeper insights into the changes in phytonutrient properties related to the growing conditions of these cultivars.

1. Introduction

Wheat has served as a staple crop and nutrient source since prehistoric times. Growing demand for sustainably produced foods has boosted interest in organic systems and ancient grains with traditional traits and unique flavors [1]. In the past decade, ancient wheat species such as spelt (Triticum aestivum subsp. spelta L.), einkorn (Triticum monococcum L. subsp. monococcum), and emmer (Triticum turgidum subsp. dicoccum Schrank) have been also identified as promising candidates for increasing the biodiversity of cultivated cereals [2,3].

These wheat species have retained their ancient features more than common wheat because they have been less affected by modern breeding [2,4]. Their high adaptability to low-input systems is a key agroecological trait [5]. Although their grain yield is lower than that of modern cultivars [6], interest persists due to their resilience to biotic and abiotic stresses [4]. They exhibit favorable agronomic traits, such as low water and nutrient requirements [7,8] and strong disease resistance [4,9].

Einkorn and emmer have a beneficial nutritional profile [10,11,12,13]. They are potential sources of antioxidants and other bioactive phytochemicals that promote human health [14,15]. Phenolic compounds (PCs)—classified as phenolic acids (PAs), coumarins, flavonoids, stilbenes, tannins, and lignans—are well-studied for their health benefits [16]. In cereals, these components occur mostly in insoluble bound forms attached to cell wall polymers, with limited free forms, reducing their bioavailability [17,18]. Therefore, they are not readily available to demonstrate their positive health-related impacts [19]. Although ancient wheats may contain higher levels of certain phytochemicals, current evidence does not confirm superior health benefits compared to modern wheat [20,21,22]. The health-promoting characteristics of both modern and ancient wheats are determined by a complex interplay of agronomic and environmental factors, such as genetic background, cultivation practices, processing techniques, and individual dietary contexts. Therefore, further research is needed to elucidate these multifactorial interactions and determine the most effective ways to incorporate ancient and modern wheats into a healthy diet.

The most typical form of PCs present in cereals is PAs [23,24]. PAs are one of the most abundant phytochemicals in wheat [25]. The most commonly found PAs in wholegrain wheat flour are ferulic acid (FA), vanillic acid, 4-hydroxybenzoic acid (p-HBA), syringic acid, para-coumaric acid, caffeic acid, and sinapic acid [25]. FA is found in the leaves and seeds of many crops, particularly cereals, and is the dominant PA in wheat, representing more than 90% of all PAs [26]. PAs can occur in soluble-free, soluble-conjugated, and insoluble-bound forms [27]. Most PAs in wheat (90–95%) are insoluble [28,29].

Similar to PCs, PAs participate in plant defense responses to biotic and abiotic stresses [30]. The composition and concentration of PCs and PAs in wheat have been proved to be influenced by many factors, including genotype, environmental conditions, meteorological conditions, sprouting process, cultivation method (organic, integrated, or conventional), and harvest time [31,32,33,34].

A number of papers have studied the relationship between meteorological conditions and the content of PCs and PAs in wheat [32,35]. However, there are relatively limited data on ancient wheats in this respect. A study on the metabolomics of wheat has shown that PCs are mostly dependent on temperature stress, especially high temperature, which was found to affect the concentration of PCs [33]. Antioxidant characteristics of wheat grown at different sites showed a negative correlation between solar radiation and antioxidant capacity [36]. These results suggest that changes in solar radiation caused by weather patterns, such as passing cold fronts, may affect wheat’s biosynthesis and accumulation of PCs.

Despite the work done so far and cited above, the relationship between genotype, growing site, meteorological conditions, and PC, especially PA content in einkorn and emmer, has not yet been extensively examined. To gain a deeper understanding of the synergistic effects of these parameters, the TP, BP, and FP content, BPA and FPA content, and PA composition in einkorn and emmer cultivars were studied in our two-year trial system, combining on-station and on-farm experiments. The first part of the research statistically compares the effects of the studied genotypes (cultivars), crop year, two growing sites, and meteorological conditions on the PC content and PA composition. The second part of this study is a statistical comparison of the effects of ancient wheat genotypes, crop year, and ten cropping sites on the same phytonutrients. Knowledge of these relationships can provide insight into the factors influencing the antioxidant characteristics and the nutritional value of these two ancient wheat species. Currently, there is a lack of comprehensive literature review or meta-analysis focusing on the PC and PA composition of ancient wheat cultivars. Therefore, the present study may provide a foundational reference to be used for in-depth meta-analyses in the future.

2. Materials and Methods

2.1. Plant Materials and Experimental Methodology

Our research investigated ancient cereal species, einkorn and emmer, and modern bread wheat as a control, based on their quality characteristics (PC and PA profiles). As some differences between materials with different genetic backgrounds may be expected even within species, we included a wider range of accessions, not only registered varieties but also variety candidates and genetically more variable (not homogeneous) landraces in the analyses. We will refer to these diverse categories as ‘cultivars’, as this term may include all of these. The variety set included 6 einkorn landraces and varieties (Triticum monococcum L. subsp. monococcum) and 8 winter and 3 spring emmer accessions (Triticum turgidum subsp. dicoccum Schrank), while as controls, 2 modern wheat varieties (Triticum aestivum) were used (

Table 1. The analyzed emmer and einkorn cultivars according to their cultivar type, country of origin, and their cultivation sites between 2019 and 2020.

Species	Name of Cultivar/Code of Accession	Common Name/Cultivar Type—Country of Origin	Growing Site
Winter einkorn	Mv ALKOR	registered variety—HU	M, Ze
	Mv MENKET	registered variety—HU	M
	Mv ESZTENA	registered variety—HU	M
	GT-2139	unknown/landrace—CH	Ps, B, Z, C
	NÖDIK einkorn (RCAT 074129)	landrace from Morocco (COLL. SCHIEMANN)	P, B
	Tifi	registered variety—NL	F
Winter emmer	Mv HEGYES	registered variety—HU	M, Ze, S
	Roter (RCAT 004664)	Emmer roter/German landrace	Ps
Winter emmer	GT-143	Schwarzwerdender/landrace—CH	B, M
	GT-381	Schwarzer Samtemmer/landrace—CH	B, M
	GT-831	Blauemmer/landrace—CH	M, Ps, Ze
	GT-1399	Grauer/landrace—CH	M, B, N
	GT-1400	Schwarzbehaarter/landrace—CH	M, B, Ze
	GT-1402	Weisser behaarter/landrace—CH	B
Spring emmer	GT-1669	Schwarzer Eschikon/landrace—CH	M
	HOLLAND spring emmer	variety candidate—NL	M
	GT-1971	Weisser/landrace—CH	M
Winter wheat	Mv KÁPLÁR	registered variety—HU	M
	Mv UNCIA	registered variety—HU	M

Codes of the growing sites: B: Bugac, C: Csonkahegyhát, F: Füzesgyarmat, M: Martonvásár, N: Nagykáta, P: Páprád, Ps: Pásztó, S: Szolnok, Z: Zalaszentlászló, Ze: Želiezovce (Slovakia).

).

Landraces with GT codes originate from the ProSpecieRara Community Genebank collection (Basel, Switzerland), while those with RCAT were obtained from the National Centre for Biodiversity and Gene Conservation (formerly NöDiK, Hungary). Registered varieties were generously offered by the Agricultural Institute of the HUN-REN Centre for Agricultural Research (HUN-REN ATK Martonvásár, Hungary) and the Louis Bolk Institute (Driebergen-Rijsenburg, The Netherlands). Figure A1 in the Annexes illustrates the growing years of each cultivar. According to this, it can be seen that the cultivars Mv Menket, Mv Esztena, Mv Alkor, and GT-2139 of the einkorns, and the cultivars Mv HEGYES, HOLLAND sping emmer, GT-831, GT-381, GT-1971, GT-1669, GT-143, GT-1400, and GT-1399 of the emmer were grown in 2019 and 2020 in two annual experiments at the selected locations. The other cultivars were tested for only a one-year period.

2.1.1. On-Station and On-Farm Trials

The on-station experiment was conducted at the HUN-REN Centre for Agricultural Research in Martonvásár (Figure 1), where a total of 9 emmer and 3 einkorn landraces were grown in meso-plot trials in 2019 and 2020.

The plots were dedicated to the propagation of the seeds, with a plot size of 1 m × 50 m in 4 replications for winter emmers and einkorns and 1 plot for each spring emmer. The cultivation method was conventional, although there was no chemical plant protection treatment and a relatively low mineral NPK (N—nitrogen, P—phosphorus, K—potassium, for each 70 kg/ha) supply level was applied. The soil type is chernozem at this site. The cumulative amount of precipitation was low in Martonvásár in 2019 and 2020, especially in the last 100 days of the vegetation period. The average temperature was higher in 2019 than in 2020 (Appendix A, Figure A2). Regarding the other trial sites, they are part of the on-farm trial network (On-farm Living Laboratory) of the Hungarian Research Institute of Organic Agriculture (ÖMKi), accredited by the European Network of Living Labs (ENoLL). With its participatory approach, the On-farm Living Laboratory is oriented toward the objectives and preferences of farmers and their respective value chains. The specific features of this experiment are that not all varieties were sown at all on-farm locations based on the farmers’ possibilities and choices, and even the test locations could vary between years. Seeds (typically a bag of 20–30 kg of each variety) were made available to participating farmers, who were instructed to sow them in parallel strips (each corresponding to the width of their sowing machinery) in a homogenous section of their fields, one seed lot in one stripe, without replication. The agronomic management practices adopted on each farm were site-specific and adhered to operating protocols routinely followed by individual farmers, using the farming facilities and equipment locally available, with varying plot sizes, usually between 0.1 and 0.6 ha (1000–6000 m²).

Our research included nine on-farm sites. One of them was in the Slovakian Uplands, Želiezovce, where the same four landraces were grown as in the Martonvásár on-station trial in 2019 and 2020. Želiezovce represents an optimal agroecological site, where the clay loam soil profile has undergone significant improvement over the past 30 years as a result of sustained organic management practices, including soil and water conservation measures and the consistent application of farm-sourced organic amendments (manure).

Additional research results from other sites of the On-farm Living Laboratory are also presented in this article. This includes the on-farm trial in Pásztó, where a narrow layer of low-fertility, clay loam soil is present. This organic farm is managed extensively, implying strong weed presence due to the no-plough–minimum tillage strategy applied. Füzesgyarmat is located within the Great Hungarian Plain in East Hungary, and it has a loam soil type. This organic farm applies reduced tillage, while nutrient availability is limited. Although the water supply is slightly better than at Pásztó, the site is more exposed to inland water. Bugac has meadow loam soil and is a characteristic, structureless sandy soil type with low levels of organic matter and nutrients. It is located in an area with a strong tendency to drought, categorized by the UN as semi-desert [37]. Farming without ploughing takes place here as well. Nagykáta is characterized by sandy loam soils; the nutrient level here is also low. Although this is not a certified organic farm, they comply with organic principles. In the year 2019/2020, two producers from Western Hungary joined the experiment. Both are conventional farms that grow einkorn under chemical-free conditions and do not use nutrient supplementation via artificial fertilizers. Csonkahegyhát is an area with an acidic pH (pH = 4.6) and loamy brown forest soil with medium nutrient levels. Zalaszentlászló has a neutral pH and loamy brown forest soil. The Páprád site has a meadow soil type, is slightly acidic, medium nutrient-rich, and has sandy loam soil. The soil type of the Szolnok site is meadow soil and clay loam in physical composition, with medium water absorption and poor water conductivity, high water storage capacity, high water holding capacity, low acidity, and rich in organic matter.

In the first year, 2018/2019, there were four on-farm sites (1–6 landraces and varieties per site). In the second crop year, 2019/2020, nine on-farm sites (1–4 cultivars per site) participated. We only included locations in the analysis where both ancient wheat species were tested.

2.1.2. Meteorological Characteristics of the Examined Period

The FieldClimate program was used to collect meteorological data for the on-farm and on-station experimental sites. Meteorological data were collected from 1 March to 31 July in 2019 and 2020. The cumulative precipitation was higher in 2019, with more rainy days (52.5%) than in 2020 (47.6%). However, there was no significant difference in the amount of precipitation on rainy days between years (ANOVA p = 0.740) and between sites (p = 0.414) (Appendix A, Figure A2a). On average, the temperature was higher in 2019, especially in June and July. The average temperature differed significantly between years (ANOVA p < 0.001) and between sites (p < 0.001) (Appendix A, Figure A2b). The minimum temperature was higher in 2019 and significantly differed from 2020 (p < 0.001), and different sites showed significantly different minimum temperatures as well (p < 0.001) (Appendix A, Figure A2c). The daily maximum temperature tended to be more varied in 2019, with a higher value than in 2020 in the months of March, June, and July. The temperature between years differed significantly (p = 0.002) and also between sites (p < 0.001) (Appendix A, Figure A2d).

2.2. Total, Free, Bound Phenolic and Phenolic Acid Determination

Representative composite grain samples were collected from individual plot harvests at different trial locations. Where plot-specific harvesting was not feasible due to technical limitations (such as in Želiezovce), samples were instead obtained at full maturity from three 1 m² randomly selected subplots within each cultivar’s area. In the case of wheat controls, einkorns and emmers in Martonvásár, the samples from four plot replicates were also mixed into one composite sample for each cultivar. This way, we reduced the plot variability since we were more interested in the year, site, and cultivar effect. Next, each sample was divided into four technical replicates from each cultivar for further analysis. These were then analyzed for quality at the Slovak University of Agriculture in Nitra, Institute of Agronomic Sciences, following the outlined procedures.

All samples, whether harvested manually or using a combine harvester, were dehulled with a KMPP300 laboratory dehuller (JK Machinery, Prague, Czech Republic), then stored at 4 °C until further evaluation. The grains were ground into wholemeal flour using an FQC-109 lab mill (Kapacitív Kft., Budapest, Hungary) equipped with a 250 μm sieve. Four replicate 10 g samples were transferred into 50 mL ST 50 tubes and defatted twice with hexane (2:1 v/w) at 1000 rpm using the ULTRA-TURRAX Tube Drive Control disintegrator (IKA, Staufen, Germany). Filtration was performed with Whatman No. 1 paper, and the remaining flour was left to dry at ambient temperature.

2.2.1. Method for the Extraction of Free and Bound Phenolic Compounds

The extraction of FP and BP was carried out in four replicates following the modified approach of Van Hung et al. [38]. For FPs, 2 g of the defatted flour was treated with 15 mL of 80% methanol, then sonicated for 15 min (Bandelin DT 100, Berlin, Germany). The mixture was centrifuged at 8965× g (9000 rpm) using a Hettich Universal 320 centrifuge (Tuttlingen Germany), and the supernatant was collected. This extraction was repeated twice, and all supernatants were pooled, concentrated under vacuum with a RVO 400 evaporator (INGOS, Praha, Czech Republic), and brought to 10 mL with 60% methanol. The extracts were frozen at −20 °C for 4 to 5 days.

To extract BPs, the residual matter was subjected to alkaline hydrolysis using 15 mL of 4 N NaOH, then sonicated for 15 min. Acidification followed with 6 M HCl to adjust the pH to 2. The BPs were extracted four times using diethyl ether, and the combined ether fractions were evaporated to dryness. The residue was reconstituted in 10 mL of 60% methanol and stored at −20 °C for up to 7 days.

2.2.2. Phenolic Acid Analysis Using HPLC

PA content was quantified and identified using an AGILENT 1260 HPLC/MS/MS system (Santa Clara, CA, USA) equipped with a DAD detector, 6410 MS/MS detector, autosampler, and multi-column thermostat, as per Brandolini et al. [27]. The separation was achieved using a Symmetry© C18 column (5 µm, 4.6 × 250 mm, WATERS, Milford, MA, USA). The mobile phases used were 0.2% formic acid in water (A) and 0.2% formic acid in methanol (B).

The gradient elution was as follows: 10–26% B (0–5 min), 26–65% B (5–10 min), 65% B (10–25 min), and 65–10% B (25–30 min). A 1 mL/min flow rate and a 40 µL injection volume were used. DAD detection was set at 280 and 320 nm. MS was run in ESI mode at 325 °C (gas), 200 °C (evaporator), 5 L/min gas flow, 60 psi nebulizer pressure, and 2500 V capillary voltage. Identification of PAs was based on retention time comparison with standards and confirmation based on MS/MS ion mass. Mass Hunter software B.07.01. (AGILENT, Santa Clara, CA, USA) controlled the system.

Calibration was performed using standards of ferulic, sinapic, caffeic, syringic, salicylic, p-coumaric acids, and p-hydroxybenzoic at concentrations of 10, 50, 100, and 200 µg/mL in 60% methanol. Detection limits were established at 0.09, 0.08, 0.94, 0.03, 0.05, and 0.06 µg/mL, respectively. The results were expressed as µg/g of dry matter (DM).

2.2.3. Determination of Total Phenolics

TP, FP, and BP contents were determined using the Folin–Ciocalteu method described by Van Hung et al. [38] with slight adaptations. Each reaction contained 150 µL of extract, 750 µL of deionized water, and 300 µL of Folin–Ciocalteu’s reagent. After 8 min of incubation at room temperature, 300 µL of 20% sodium carbonate solution was added. The samples were left in the dark for 2 h, centrifuged at 16,060× g (13,000 rpm) for 10 min, then absorbance was measured at 765 nm using a UV-1800 spectrophotometer (SHIMADZU, Kyoto, Japan). Ferulic acid served as the calibration standard, and the results were reported as ferulic acid equivalents (FAE) per gram of dry matter. Analyses were performed in quadruplicate.

2.2.4. Statistical Evaluation

All analysis was performed with R statistical software version 4.2.3 [39]. Graphs were made with the package ggplot2 [40], data processing was performed with the package dplyr [41], and unbalanced design ANOVA Type III analysis was performed with the library car [42].

The analyses were divided into two main parts, i.e., (1) the effects of genotypes and years of selected cultivars, grown in both years in two selected sites (Martonvásár and Želiezovce), and (2) the effects of cultivar, site, and year at all sites. The impact of genotypes and years on the content of PCs and acids in the grains of selected wheat cultivars that were grown in two years at the Martonvásár and Želiezovce sites were subjected to ANOVA Type I. Residuals were inspected to ensure the assumptions of both ANOVAs were met, and the significance was set at a 95% confidence interval. Residuals were also inspected for violations of independence, heteroscedasticity, and normality. The significance was also set at a 95% CI.

To understand the effects of cultivar, site, and year, two types of analysis of variance (ANOVA Type I and III) were used. Since not all cultivars were sown at all sites and in both years, cultivars that were grown at least in more than one site or more than one year of observation were subjected to ANOVA Type III, which can deal with unbalanced data [42,43,44]. This type of ANOVA works by adjusting every effect for all other effects, and the least squares estimates of the value replace the missing data. This way, the sum of squares from Type III will be similar to the ones of Type I. The free and bound forms of the measured PC and acid of all cultivars at different sites and years also showed a wide range of variability; therefore, a free-to-bound PC and acid ratio was used to normalize the data. Then, the ratio was used as a response variable in an ANOVA Type III to check the effects of cultivars, sites, and year.

3. Results and Discussion

The data in this research were taken from multiple sites, on-farm and on-station, involving several emmer and einkorn cultivars. Since the basis of the on-farm trial system was a participatory approach and relied on farmers’ preferences, some varieties at some sites were not cultivated in two consecutive years. Each year was treated as a separate trial system; the data were analyzed as a whole dataset and as a subset with complete year replication. The results discussed in the following section start with the effects of genotype, crop year, growing site, and meteorological conditions on the PC and PA content in the sites Martonvásár and Želiezovce. Then, the second part of this section will focus on the effects of genotype and crop year on the ancient wheats’ PC and PA content, including all sites.

3.1. Total, Free, and Bound Phenolic Compound Concentrations (TPs, FPs, and BPs)

In the Martonvásár test location (which was managed conventionally, but without chemical pesticide treatment), the average values of the two years (2019 and 2020) showed that the highest mean total PC was detected in an emmer cultivar with 826 µg FAE g⁻¹ DM (GT-1669) (

Table 2. Influence of wheat cultivars and cropping years on the concentration of phenolic compounds (µg FAE g⁻¹ DM) of ancient wheat grown under conventional conditions in Martonvásár in 2019–2020.

Species, Cultivars	Phenolic Compounds (µg FAE g−1 DM)
	Free	Bound	Total
T. aestivum
Mv Káplár	121.1 ± 3.8 a	748.2 ± 58.7 a	869.3 ± 59.2 a
Mv Uncia	100.1 ± 7.8 bc	638.4 ± 60 bc	738.5 ± 67.7 b
T. monococcum
Mv Alkor St	56.3 ± 35.2 f	352.1 ± 224.7 fg	408.3 ± 259.8 gh
Mv Esztena	47.3 ± 31.1 g	321.9 ± 213 g	369.2 ± 243.9 h
Mv Menket	70.2 ± 20 e	482.6 ± 162.7 e	552.8 ± 182.5 f
T. dicoccum
GT 1399	83.7 ± 11.5 d	576.9 ± 85.1 cd	660.6 ± 93.6 bcd
GT 1400	52.8 ± 3.7 fg	368.7 ± 33.2 fg	421.6 ± 35.5 gh
GT 143	61.4 ± 7.4 f	396.1 ± 19.3 f	457.5 ± 18.3 g
GT 1669	107.8 ± 15.3 b	718.5 ± 56.2 ab	826.3 ± 50.8 a
GT 1971	94.6 ± 50.2 c	534.5 ± 243.4 de	629.2 ± 292.8 cde
GT 381	80.3 ± 15.7 d	533.7 ± 53.3 de	614 ± 65 def
GT 831	59 ± 15.3 f	354 ± 50.7 fg	412.9 ± 64.7 gh
Holland emmer	82.5 ± 4.2 d	483.6 ± 63.6 e	566.1 ± 65.5 ef
Mv Hegyes St	95.4 ± 42.8 c	598.6 ± 264.1 cd	694 ± 305.5 bc
p cultivars (C)	***	***	***

Within each column, mean values sharing the same letter do not differ significantly at the 5% probability level (p < 0.05), as determined by Tukey’s Honestly Significant Difference (HSD) post hoc test following one-way ANOVA. Differences among treatments were statistically significant at the 0.1% level (*** p < 0.001).

). However, a similarly high value was observed in the control common wheat samples with 869 µg FAE g⁻¹ DM for Mv Káplár and 739 µg FAE g⁻¹ DM for Mv Uncia in 2019 and 2020 (Figure 2).

The TP values of emmer landraces varied between 413 µg FAE g⁻¹ DM (GT-831) and 826 µg FAE g⁻¹ DM (GT−1 669). At the same time, all einkorn landraces achieved lower values, ranging from 369 µg FAE g⁻¹ DM (Mv Esztena) to 553 µg FAE g⁻¹ DM (Mv Menket). The TP content was lower in 2020 (Figure 2) for all einkorn cultivars and most emmer cultivars (GT-1400, GT-1971, GT-831, Holland, Mv Hegyes) than in 2019. On the contrary, some emmer cultivars (GT-143, GT-1669, GT-381) showed higher TP values in 2020.

The results of the on-farm experiments conducted under organic conditions with four varieties in the same years in Želiezovce (

Table 3. Influence of ancient wheat cultivars and cropping years on the concentration of phenolic compounds (µg FAE g⁻¹ DM) of ancient wheat grown under organic conditions in Želiezovce in 2019–2020.

Species, Cultivars	Phenolic Compounds (µg FAE g−1 DM)
	Free	Bound	Total
T. monococcum
Mv Alkor St	30.9 ± 5.1 c	178.5 ± 15 c	209.4 ± 10.8 c
T. dicoccum
GT 1400	52 ± 4.6 a	355.9 ± 17.1 a	408 ± 15.6 a
GT 831	45.9 ± 12.5 b	275.6 ± 40.5 b	321.5 ± 51.3 b
Mv Hegyes	26 ± 2.1 d	192.2 ± 17.2 c	218.3 ± 17.3 c
p cultivars (C)	***	***	***

Within each column, mean values sharing the same letter do not differ significantly at the 5% probability level (p < 0.05), as determined by Tukey’s Honestly Significant Difference (HSD) post hoc test following one-way ANOVA. Differences among treatments were statistically significant at the 0.1% level (*** p < 0.001).

, Figure 3) showed lower TP contents in einkorn (209 µg FAE g⁻¹ DM (Mv Alkor) and emmer varieties, ranging from 218 µg FAE g⁻¹ DM (Mv Hegyes) to 408 µg FAE g⁻¹ DM (GT-1400), averaged over the two years. Emmer GT-831 at this site showed significantly lower TP in 2020 (Figure 3). This cultivar was also present in Martonvásár and showed a similar trend. It is noteworthy that apart from these two sites, one of the emmer landraces in Füzesgyarmat under organic on-farm conditions in 2019 reached the highest TP content value with 1254 µg FAE g⁻¹ DM (GT-831).

In our research, the differences in FPs between emmer and einkorn cultivars were significant in Martonvásár. The results revealed that control common bread wheat had high FP concentrations (121 µg FAE g⁻¹ DM, Mv Káplár and 100 µg FAE g⁻¹ DM, Mv Uncia in 2019 and 2020, respectively). The concentration of FPs in emmer GT-1669 was also higher (108 µg FAE g⁻¹ DM) than in other ancient wheat species’ varieties. Meanwhile, einkorn had the lowest values (47 µg FAE g⁻¹ DM, Mv Esztena) under conventional conditions. The proportion of free phenolic fractions relative to the total phenolic content varied between 12.5% in the emmer cultivar GT-1400 and 15.0% in GT-1669, indicating genotype-dependent variation in phenolic compound distribution.

BPs accounted for 86.6% of the TPs, with the highest concentration detected in the control wheat (748 µg FAE g⁻¹ DM, Mv Káplár in 2019 and 638 µg FAE g⁻¹ DM, Mv Uncia in 2020) grown at Martonvásár. The GT-1669 emmer variety was noteworthy, with a mean 719 µg FAE g⁻¹ DM result (in 2019 and 2020), whereas half of the emmer varieties had higher BP concentrations than einkorns. The einkorn cultivars’ values ranged from 322 µg FAE g⁻¹ DM in Mv Esztena to 483 µg FAE g⁻¹ DM in Mv Menket. BPs contributed to the TPs in the range of 84.9% (emmer GT-1971) to 87.5% (emmer GT-1400). The control bread wheat had the highest TP concentration, followed by emmer, while einkorn (Mv Esztena) had the lowest value measured in this conventional on-station trial. Among the same cultivars of ancient wheat (Mv Alkor einkorn, GT 1400, GT 831, Mv Hegyes emmer) under organic conditions at Želiezovce, emmer varieties also produced higher TP values than einkorn cultivars.

The effect of year on TP concentrations was more pronounced at Martonvásár than at Želiezovce. The meteorological data from each growing site showed significant temperature differences between the years (Appendix A, Figure A2). Total rainfall was higher in 2019 than in 2020, although the difference in rainfall on rainy days was not as great. The average temperature and precipitation were higher in 2019 than in 2020 at both growing sites. The meteorological data are in line with our findings from samples originating from Martonvásár, as most varieties produced more TPs and acids in 2019.

A previous study of ÖMKi in 2018 revealed significant differences in TP content between einkorn and emmer, which are in line with our current results [4]. The average TP content was higher in emmer (2519 GAE g⁻¹ DM) than in einkorn (2490 GAE g⁻¹ DM) [4]. In partial agreement with the results of our research, Lacko-Bartošová et al. [45] found the highest TP content in bread wheat (1903 µg FAE g⁻¹ DM) rather than in emmer cultivars (e.g., 1669 µg FAE g⁻¹ DM, Farvento). Serpen et al. [46] reported that emmer had higher TP content than einkorn and bread wheat controls. In contrast, Lachman reported the opposite trend, with spring wheat (502–601 µg GAE g⁻¹ DM) having lower concentrations of total TPs than emmer (584–692 µg GAE g⁻¹ DM) and einkorn (507–612 µg GAE g⁻¹ DM) [14]. Contrary to our results, Zrcková et al. [47] reported that the TP ranged from 618 mg kg⁻¹ DM (common wheat cv. Annie) to 793 mg kg⁻¹ DM (T. monococcum GEO) and TPAs from 701 mg kg⁻¹ DM (cv. Annie) to 875 mg kg⁻¹ DM (Schwedisches einkorn), averaged over a two-year period, with the results in the following order: einkorn > emmer > common wheat > spelt. Pehlivan Karakas et al. [48] demonstrated that einkorn exhibits a higher TP and total flavonoid content, as well as enhanced antioxidant characteristics, in comparison to modern wheat species, including common bread wheat and durum wheat.

3.2. Total, Free, and Bound Phenolic Acids (TPAs, FPAs, and BPAs)

PAs represent the predominant class of PCs in cereal grains. The sum of the FPA and BPA forms of these compounds represents the TPAs. Analysis of variance showed that there were significant differences between all tested cultivars of einkorn, emmer, and common wheat in the concentrations of TPAs, FPAs, and BPAs. Contrary to the results for TP, emmer GT-1669 had the highest content, followed by wheat control Mv Káplár (in 2019) and Mv Uncia (in 2020) in the Martonvásár samples’ two-year mean data (Figure 4).

The concentrations of TPAs for einkorn were slightly below the range of most emmer cultivars, based on the results of the two-year research period. The results from the Želiezovce on-farm site showed that the TPA concentrations of einkorn Mv Alkor and emmer varieties ranged between 117 and 228 µg g⁻¹ DM (Figure 5).

The lowest FPA and BPA contents under conventional conditions were observed in the Mv Esztena einkorn cultivar. The highest FPA concentration was observed in the GT-1669 emmer cultivar, followed by the control wheat Mv Káplár. Similarly, emmer landrace samples under organic conditions had higher TPA, FPA, and BPA values than einkorn cultivars.

In Martonvásár, the average share of FPAs in TPAs was 7.2% in einkorn, 7.5% in emmer, and 7.5% in control bread wheat. The proportion of BPAs in the total concentration of PAs was high and represented 92.5% in control wheat and emmer and 92.7% in einkorn on average. In Želiezovce, the average FPA content was 7.9% for einkorn and 8.6% for emmer (the highest level was measured in GT-1400). The share of BPA in the TPA concentration was high, averaging 92.6% in einkorn and emmer.

In general, the year and cultivar variables showed similar effects on PA content as on PCs (Figure 4 and Figure 5). However, the effect of years slightly differed in the case of emmer cultivars in Martonvásár. Three emmer cultivars (i.e., GT-143, GT-1669, Holland) showed different trends of PA development between the years than in the case of PCs.

Our research found a significant effect of cropping year on free, bound, and total PAs, with a higher level in 2019 than in 2020 in Martonvásár. In 2019, significantly higher TPA, FPA, and BPA values were observed than in 2020 in the conventionally grown einkorn landrace, Mv Alkor and two other emmer varieties, GT-1971, Mv Hegyes. Also, for the samples from the Želiezovce site, the year 2019 resulted in significantly higher free, bound, and total PAs than 2020.

Research examining the PA contents of ancient wheats in recent years has led to ambiguous results. According to Li et al. [35], the mean concentration of TPAs was highest in emmer, followed by einkorn, with the lowest levels observed in spelt. In contrast, a study by Brandolini [27] reported the highest TPA concentrations in spelt, moderately lower levels in einkorn, and the lowest concentrations in emmer. Einkorn and emmer exhibited approximately two-fold higher concentrations of BPAs compared to those of soft wheat, durum wheat, and spelt; among the BPA fractions, p-coumaric acid constituted roughly 50% of the total [49]. According to Baranski et al. [3] almost all free and bound PAs were significantly higher in einkorn than in emmer and spelt wheat. The significant interaction between varieties and growing years showed different reactions of the varieties to changing meteorological conditions. Other researchers reported similar results [45,50], where bound forms of PAs represented the major share of all PAs, while free PAs were detected as only up to 10% of the total PAs. According to Shewry and Hey [15], ancient wheat species have phenolic components similar to bread wheat, including total ferulic acid. GT-1669 showed the highest free, bound, and total PA contents among the examined emmer landraces. Consistent with our findings, emmer exhibited the highest concentration of TPAs among the wheat genotypes [35,45,47]. In our study, the emmer cultivar GT-1669 showed total phenolic acid (TPA) concentrations in the highest range among the lines tested. This emmer cultivar, commonly known by its original name, ‘Schwarzer Eschikon’, is a blackish-flaked spelt landscape variety. Its characteristically higher TP content and TPA, BPA, and FPA contents can be explained by its genetically determined high content of TP compounds containing coloring agents that give the straw a dark purple-blackish color. It is established that darker cereal phenotypes are frequently associated with higher TP contents, likely due to the greater synthesis and accumulation of these bioactive compounds [14,45,51]. On the basis of contemporary scientific knowledge and dietary context, the observed elevation in TPA in GT-1669 is nutritionally meaningful: regular consumption of products made from this emmer can substantially boost PA and thus dietary antioxidant intake compared to typical cereal-based foods.

The effect of growing years on PA concentration has scarcely been investigated in the literature. According to Fernandez-Orozco et al. [52], the growth year in Hungary had a dominant impact, especially on the free and conjugated PA contents in wheat varieties. Research by Lacko-Bartošová et al. [45] demonstrated that the effect of growing year was more evident in the concentration of FPAs compared to bound ones. Warmer weather without precipitation deficiency during the ripening period was related to the increase in PAs concentrations. The extremely dry and hot weather during the maturity stages of wheat species had a negative impact on free and bound PAs [45]. Furthermore, in a three-year study, Stracke et al. [53] found that the effect of crop year was the most important factor influencing PA content. Another study [27] showed that minimal precipitation during wheat’s heading and ripening phases correlates with higher TP. Shewry and Hey [15] mentioned a significant relationship between the concentration of bioactive compounds and environmental parameters (e.g., precipitation and temperature) because climate, farming techniques, and underlying genes all influence plant metabolic pathways.

3.3. Individual Phenolic Acids

An interesting aspect of our research is that the individual PA concentrations of the total, free, and bound fractions were analyzed. Even though the total individual PA concentrations differed between the cultivars (

Table 4. Effects of different wheat and ancient wheat cultivars and growing years on the total individual phenolic acid concentration (µg g⁻¹ DM) of samples originating from Martonvásár (2019, 2020).

Species, Cultivars	Ferulic Acid	p-HBA 1	p-Coumaric Acid	Syringic Acid	Sinapic Acid	Salicylic Acid	Caffeic Acid
T. aestivum
Mv Káplár	358.8 ± 20 a	11.8 ± 0.2 a	28.5 ± 0.7 a	10.9 ± 0.5 a	21.7 ± 0.5 a	14 ± 0.6 a	10.1 ± 0.4 b
Mv Uncia	322 ± 31.6 b	10.5 ± 0.2 b	29 ± 0.5 a	9.4 ± 0.2 b	22.1 ± 0.8 a	14.6 ± 0.3 a	9.3 ± 0.4 cd
T. monococcum
Mv Alkor St	208.6 ± 149.7 d	6.9 ± 4.7 g	17.3 ± 11.8 e	5.9 ± 4.2 g	13.6 ± 9.7 de	8.6 ± 5.9 f	6.3 ± 4.3 gh
Mv Esztena	176.3 ± 123.7 e	6 ± 4 h	13.8 ± 9.4 g	5.2 ± 3.5 h	11.1 ± 7.3 g	7.4 ± 4.9 g	5.6 ± 3.8 i
Mv Menket	235.6 ± 64.8 c	7.4 ± 2 f	20.3 ± 5.4 d	6.7 ± 1.6 f	14.4 ± 2.8 d	9.6 ± 2.8 e	6.7 ± 1.8 g
T. dicoccum
GT-1399	303.1 ± 50.4 b	9.7 ± 1.7 d	24.8 ± 3.8 bc	8.7 ± 1.3 cd	18.9 ± 3.4 b	12.2 ± 1.8 c	9.4 ± 1.6 c
GT-1400	199.7 ± 23.1 d	6.8 ± 0.8 g	15.8 ± 1.2 f	5.6 ± 0.5 g	12.4 ± 1.1 f	8.1 ± 0.7 f	6.1 ± 0.4 hi
GT-143	200.1 ± 8.7 d	6.8 ± 0.3 g	16.9 ± 0.7 ef	5.7 ± 0.2 g	12.8 ± 0.5 bf	8.3 ± 0.6 f	5.7 ± 0.3 i
GT-1669	365 ± 13.3 a	11.8 ± 0.5 a	29.1 ± 1.2 a	10.5 ± 0.3 a	22.1 ± 0.8 a	14.1 ± 1 a	10.9 ± 0.7 a
GT-1971	295.9 ± 141.5 b	9.9 ± 4.5 bcd	25.1 ± 11.8 cd	8.8 ± 4.1 cd	18.4 ± 7.4 b	13 ± 5.9 b	9 ± 3.8 cd
GT-381	298.2 ± 32.8 b	9.7 ± 0.8 cd	23.6 ± 1.3 d	8.5 ± 1.1 d	18 ± 2.8 b	11.9 ± 0.8 c	8.4 ± 0.6 e
GT-831	200.5 ± 22.1 d	6.5 ± 0.9 g	16.5 ± 2.7 g	5.7 ± 1 g	12.3 ± 1.9 f	8.3 ± 1.4 f	6 ± 0.6 hi
Holland emmer	252.9 ± 15.7 c	8.2 ± 0.2 e	20.4 ± 0.8 e	7.6 ± 0.2 e	15.9 ± 0.8 c	10.4 ± 0.7 d	7.5 ± 0.2 f
Mv Hegyes St	300.9 ± 125 b	10.2 ± 4.4 bc	24.7 ± 10.1 bc	9 ± 3.6 bc	18.8 ± 8.7 b	13.1 ± 6.3 b	8.8 ± 3.4 de
p cultivars (C)	***	***	***	***	***	***	***
2019	308.9 ± 73.6 a	10.2 ± 2.4 a	25.3 ± 6.1 a	8.9 ± 2.2 a	19 ± 4.6 a	12.9 ± 3.2 a	9.2 ± 2.2 a
2020	210.6 ± 90.4 b	6.9 ± 2.8 b	17.3 ± 7.1 b	6.1 ± 2.6 b	13.4 ± 5.6 b	8.5 ± 3.5 b	6.2 ± 2.4 b
p year (Y)	***	***	***	***	***	***	***
p C × Y	***	***	***	***	***	***	***

¹ p-hydroxybenzoicacid. All values are expressed as means ± standard deviations (SDs), based on four replicates and data collected over two consecutive years (n = 14 for varieties, n = 28 for years). Mean values within columns that share the same letter are not significantly different at the 5% significance level (p < 0.05), as determined by Tukey’s Honestly Significant Difference (HSD) test following one-way ANOVA. Statistical significance is denoted as *** p < 0.001.

and

Table 5. Effects of ancient wheat cultivars and cropping years on total individual phenolic acid concentration (µg g⁻¹ DM) of samples originating from Želiezovce (2019, 2020).

Species, Cultivars	Ferulic Acid	p-HBA 1	p-Coumaric Acid	Syringic Acid	Sinapic Acid	Salicylic Acid	Caffeic Acid
T. monococcum
Mv Alkor St	105.3 ± 5.8 c	3.3 ± 0.1 c	8.5 ± 0.3 c	3.1 ± 0.1 c	6.6 ± 0.2 c	4.1 ± 0.1 c	3.2 ± 0.1 b
T. dicoccum
GT-1400	177.1 ± 10.8 a	5.9 ± 0.5 a	15.1 ± 1.3 a	5.4 ± 0.2 a	11.9 ± 0.8 a	7.3 ± 0.5 a	5.1 ± 0.3 a
GT-831	164.8 ± 33.6 b	5.7 ± 1.1 b	13.8 ± 2.4 b	4.9 ± 0.9 b	10.1 ± 2.0 b	6.9 ± 1.3 b	5.1 ± 1 a
Mv Hegyes	92.0 ± 5.0 d	3.0 ± 0.1 d	7.6 ± 0.3 d	2.7 ± 0.1 d	5.9 ± 0.3 d	3.6 ± 0.1 d	2.7 ± 0.1 c
p cultivars (C)	***	***	***	***	***	***	***
2019	139.2 ± 46.2 a	4.6 ± 1.6 a	11.5 ± 3.8 a	4.2 ± 1.4 a	8.8 ± 2.8 a	5.6 ± 2.0 a	4.2 ± 1.4 a
2020	130.4 ± 36.3 b	4.3 ± 1.4 b	11 ± 3.4 b	3.8 ± 1.2 b	8.4 ± 2.6 b	5.3 ± 1.6 b	3.9 ± 1.0 b
p year (Y)	***		***	***	***	***	***
p C × Y	***		***	***	***	***	***

¹ p-hydroxybenzoicacid. All values are expressed as means ± standard deviations (SDs), based on four replicates and data collected over two consecutive years (n = 4 for varieties, n = 8 for years). Mean values within columns that share the same letter are not significantly different at the 5% significance level (p < 0.05), as determined by Tukey’s Honestly Significant Difference (HSD) test following one-way ANOVA. Statistical significance is denoted as *** p < 0.001.

), their percent share showed a similar trend. Ferulic acid accounted for 77.2–78.9% of the total individual PAs measured, followed by p-coumaric (6.1–7.0%), sinapic acid (4.8–5.3%), salicylic acid (3.0–3.5%), p-HBA (2.5–2.7%), caffeic acid (2.2–2.5%), and syringic acid (2.1–2.4%). The predominance of ferulic acid has also been reported in other scientific publications [26,33,54].

The bound form of the seven PAs was always higher than their free form. The share of seven PAs in their bound form was similar to the total share; however, their free form showed a different trend. The free form of ferulic acid accounted for 57.3–59.2%, followed by p-coumaric (14.3–16%), syringic acid (9.9–11.1%), sinapic acid (6.2–7.1%), p-HBA (4.4–5.1%), salicylic acid (3.0–3.7%), and caffeic acid (1.5–2.1%).

Baranski et al. [3] reported that ferulic acid was dominant among all PCs in wheat, representing over 72% of free PAs and over 95% of bound ones. Lacko-Bartošová et al. [45] reported higher free PA concentrations in emmer than in wheat. Ferulic acid was the predominant free phenolic acid (66.3%) detected across the wheat genotypes, followed by syringic acid (11.7%), sinapic acid (7.4%), p-hydroxybenzoic acid (5.3%), salicylic acid (3.8%), p-coumaric acid (3.6%), and caffeic acid (2.1%). In contrast, Li et al. [35] found a lower rate of FPAs in emmer than in bread wheat. It has been pointed out that there are a lack of data on the presence of FPAs in ancient wheat, but it is also recommended that further studies are carried out with various ancient and modern wheat species and varieties [15]. At the Želiezovce organic site, a similar trend was observed, with ferulic acid being the most dominant aggregate in the measured samples (Table 5).

The growing year significantly influenced the concentrations of all individual FPAs in Martonvásár and in Želiezovce, as well. Significantly higher concentrations of all PAs were reported in 2019 than in 2020. The spring period of the 2019 growing year (especially May) was colder, but June and July were warmer than usual, with a seasonal mean temperature of 22.3 °C, which was 2.1 °C higher than the 1981–2010 average, making it the second warmest summer since 1901. The rainfall was about 11% less than the long-term average. Following an unusually cool May, June 2020 experienced a positive temperature anomaly of approximately +0.6 °C relative to the long-term average, while July temperatures returned to near-normal seasonal values. Figure A2 in Appendix A represents the precipitation data for each year. The maturation period of the 2019 growing season was characterized by the most favorable agroclimatic conditions for the biosynthesis and accumulation of PAs. A significant interaction between cultivars and growing years in both sites was observed in all PAs at p < 0.001. The 2019 growing season resulted in the highest concentrations of all individual PAs across all wheat cultivars, indicating a strong environmental influence on phenolic acid biosynthesis.

According to research by Lacko-Bartošová et al. [45], the caffeic acid content of bread wheat was one of the lowest (2.14 µg g⁻¹ DM), and significant differences were found among other cultivars and breeding lines. Meanwhile, Barański et al. [3] reported that the amount of caffeic acid did not differ among ancient wheat species such as spelt, einkorn, and emmer.

Several studies investigated the influence of environmental factors (air temperature, rainfall) on the levels of TPs and PAs in ancient wheat, but the results were inconsistent. High temperature was negatively correlated with TPs [45], while Alexieva et al. [55] re-ported an increased level of soluble PCs in wheat exposed to UV-B stress and drought. Baranski et al. [3] found a more pronounced influence of year of cultivation on the amount of FPAs (except syringic acid) in ancient wheat species compared to BPAs, for which the year effect was found to be important for p-HBA, salicylic, p-coumaric, and syringic acids. No statistically significant differences were observed among wheat species in the concentration of bound caffeic acid. Climate, meteorological conditions, farming systems, agronomic practices such as crop rotation and tillage, and also genotype may affect plant metabolic pathways; moreover, secondary metabolite concentration depends on crop species [46]. Due to the absence of a comprehensive literature review or meta-analysis on this topic, definitive conclusions cannot yet be drawn.

3.4. Effects of Cultivars, Site, and Year on Phenolic Compounds and Phenolic Acid Contents

For a more in-depth understanding of the effects of cultivar and year on PCs and PAs, data from two growing years and cultivars from Martonvásár and Želiezovce were analyzed separately. Analysis of variance Type I showed that the %F (free % in total), %B (bound % in total), and TPs were significantly affected by all tested cultivars and accessions of einkorn, emmer, and common wheat (

Table 6. Results of analysis of variance Type I on the phenolic compound, total phenolic acids, and seven types of phenolic acid against wheat cultivar and year at the sites Martonvásár and Želiezovce (2019, 2020).

Response Variables	Significance of Different Explanatory Variables at Martonvásár			Significance of Different Explanatory Variables at Želiezovce
	Cultivar (C)	Year (Y)	C × Y	C	Y	C × Y
Phenolic Compound
Percent free (%F)	***		***	***		***
Percent bound (%B)	***		***	***		***
(log) total	***	***	***	***	***	***
Phenolic Acids
All acids %F	***		***	***	**	***
All acids %B	***		***	***	**	***
(log) Total acids	***	***	***	***	***	***
Individual Acids
Ferulic acid %F	***		**	**	*	***
Ferulic acid %B	***		**	**	*	***
(log) Total ferulic acid	***	***	***	***	**	***
p-HBA 1 %F	***		***	***	**	***
p-HBA 1 %B	***		***	***	**	***
(sqrt) Total p-HBA 1	***	***	***	***	***	***
p-coumaric acid %F	***	*	***	***	***	***
p-coumaric acid %B	***	*	***	***	***	***
Total p-coumaric acid	***	***	***	***	*	***
Syringic acid %F	***		***	***
Syringic acid %B	***		***	***
(sqrt) Total syringic acid	***	***	***	***	***	***
Sinapic acid %F	***	*	***	***	*	***
Sinapic acid %B	***	*	***	***	*	***
Total sinapic acid	***	***	***	***	***	***
Salicylic acid %F	***	***	**	***		**
Salicylic acid %B	***	***	**	***		**
(sqrt) Total salicylic acid	***	***	***	***	***	***
Caffeic acid %F	***	*	***	***		**
Caffeic acid %B	***	*	***	***		**
(log) Total caffeic acid	***	***	***	***	***	***

¹ p-hydroxybenzoicacid. The significance codes were based on different confidence intervals: (*) p < 0.1, (**) p < 0.01, and (***) p < 0.001.

).

Statistical analyses also revealed a significant effect of the growing year (Y) on most parameters tested at the site Želiezovce. Meanwhile, there were fewer parameters affected by the year at the site Martonvásár. In all parameters tested at both sites, the interaction between cultivars and year (C × Y, i.e., G × E) always showed medium to high significance.

ANOVA results showed a consistent effect of cultivar and interaction between year and cultivars in all parameters measured (TP, PA, individual PA) at both sites. Overall, the year effect was consistent for the total PCs, PAs, and individual acids. However, the year effect was not consistent for the free and bound forms of phenolics. Here, the consistent significant effect of interaction between year and cultivars implies that some cultivars showed different results (relative to the other trial entries) in the two years.

3.5. Trends in the Phenolic Compound and Phenolic Acid Contents of All Species at All Sites

Nineteen wheat cultivars grown in 2019 and 2020 at different sites showed a wide range of PC and PA contents (Figure 6 and Figure 7). The results of samples from other on-farm locations (where only one year of data were available) were not relevant for drawing conclusions but were suitable for observing trends. The PC and PA values followed the same trend, where their bound form (PC: 82.5–89.0%, PA: 91.9–93.6%) was consistently higher than their free form (PC: 11.0–17.5%, PA: 6.4–8.1%).

In both PC and PA, the highest value was shown by emmer GT-831 grown in Füzesgyarmat (PC: 1254.0 ± 108.9 µg FAE g⁻¹ DM, PA: 748.1 ± 0.02 µg g⁻¹ DM), and the lowest value was shown by einkorn Mv Esztena (PC: 117 ± 2.0 µg FAE g⁻¹ DM, PA: 66.9 ± 3.5 µg g⁻¹ DM). The PC and PA values of emmer GT-831 (as well as others) differed in each site and year, showing the site and year effect. The three sites, i.e., Bugac (2019), Füzesgyarmat (2019), and Martonvásár (2019, 2020), showed higher PC and PA contents than the others (Figure 6 and Figure 7). Meanwhile, wheat cultivars grown in 2020 showed lower PC and PA contents than in 2019, as demonstrated by the sites Martonvásár, Pásztó, and Želiezovce.

Similar to the PC and PA results, for all of the individual phenolic acids measured, the proportion of bound forms was higher than that of the free ones (62.4–94.9% vs. 5.1–37.6%). The bound-to-free-PA ratio was the highest in ferulic and caffeic acid (multiple wheat varieties). On the contrary, the lowest bound-to-free-PA ratio was shown by syringic acid (einkorn variety GT-2139 and emmer variety Roter). The overall mean bound-to-free ratio of each PA, starting from the highest to the lowest, was ferulic acid < caffeic acid < salicylic acid < sinapic acid < p-hydroxybenzoic acid < p-coumaric acid < syringic acid. It seems that the TPA content is positively correlated with the bound-to-free ratio of PAs, meaning that high PA content is connected to the production of more bound than free PAs.

3.6. Effects of Cultivar, Site, and Year on Phenolic Compound and Phenolic Acid Content

The analysis of variance Type III showed that the concentrations of PCs, TPAs, and individual PAs were affected by the cultivar and moderately to highly affected by the site (

Table 7. The result of analysis of variance Type III on the phenolic compound, total phenolic acid, and individual phenolic acid contents against wheat cultivar, site, and year (2019, 2020).

Response Variables	Significance of Different Explanatory Variables
	Cultivar	Site	Year
Phenolic Compound
Percent free (%F)	***	**
Percent bound (%B)	***	**
(log) total	***	***	***
Phenolic Acids
All acids %F	***	**
All acids %B	***	**
(log) total acids	***	***	***
Individual Acids
Ferulic acid %F	***
Ferulic acid %B	***
(log) Total ferulic acid	***	***	***
p-HBA 1 %F	***	**
p-HBA 1 %B	***	**
Total p-HBA 1	***	***	***
p-coumaric acid %F	***	**
p-coumaric acid %B	***	**
(log) Total p-coumaric acid	***	***	***
Syringic acid %F	***	**
Syringic acid %B	***	**
(sqrt) Total syringic acid	***	***	***
Sinapic acid %F	***	***	*
Sinapic acid %B	***	***	*
(log) Total sinapic acid	***	***	***
Salicylic acid %F	***	**
Salicylic acid %B	***	**
(sqrt) Total salicylic acid	***	***	***
Caffeic acid %F	***	***
Caffeic acid %B	***	***
(log) Total caffeic acid	***	***	***

¹ p-hydroxybenzoicacid. Note that the model structure did not allow for interactions between explanatory variables. The significance levels are indicated as follows: (*) p < 0.1, (**) p < 0.01, and (***) p < 0.001.

).

The only response variable not affected by the site was the percent bound (%B) and percent free (%F) ferulic acid content. Meanwhile, the year only slightly affected the sinapic acid %B and %F and had a high impact on all forms of the TPs. The consistently strong effect of cultivar, site, and year on all forms of TPs may be caused by the high variance between each cultivar grown at different sites and years. On the other hand, even though the TPs may differ across cultivars/site/year, the proportion of their free and bound forms showed less variance but was still affected by cultivar and site.

Note that not all wheat cultivars were present at all sites during both years of observation. Therefore, the mean PCs are visualized in Figure 8 to showcase the site effect.

A more pronounced effect of sites was observed when the mean PC content and individual PA contents of certain cultivars were plotted at different sites (Figure 8); for PAs, samples from the same sites also showed similar patterns. In general, sites showing higher contents of PCs and PAs were Bugac, Martonvásár, Füzesgyarmat, and Želiezovce. Uniquely, these sites have differing soil characteristics and farming methods.

4. Conclusions

In our research, the control common wheat (T. aestivum) had high TP, BP, and FP contents, although one emmer landrace (GT 1699) also had similarly high values. Einkorn had lower values. In general, we saw that among the ancient wheats, emmer had higher PC and PA contents than einkorn. This was in line with the results of our on-farm research, as well. Contrary to the results for TPs, emmer had the highest TPA, FPA, and BPA contents, followed by control wheat, then einkorn landraces. These on-station measurements were in line with on-farm results.

Our gap-filling research was the analysis of the individual PA values in all free and bound fractions. Ferulic acid was the predominant PA (common wheat < einkor < emmer), followed by p-coumaric acid, syringic acid, sinapic acid, and p-HBA, whereas salicylic acid and caffeic acid had the lowest concentrations. Outstandingly high free and bound individual PAs were measured in emmer landrace GT-1669.

The average temperature and precipitation were higher in 2019 than in 2020 at all growing sites. The meteorological data support our finding from the Martonvásár samples, that most varieties produced more PCs and acids in 2019.

The present research did not focus on the effects of different cultivation methods on the phytonutrient contents of ancient wheat; however, a systematic comparison of organic and conventional ancient wheat from on-farm partners and small plot variety trials in the Living Laboratory of ÖMKi would be justified in the future to analyze and better understand the variations in phytonutrient characteristics according to the farming systems in a larger sample size and over several years.

Overall, re-integrating ancient wheat species’ varieties into future cropping systems, e.g., through organic breeding programs focused on their beneficial traits (such as phytonutrient content and favorable protein composition) could pave the way for a more sustainable food system. Moreover, re-introducing einkorn and emmer in sustainable food production offers an opportunity to increase food diversity and support ecological balance. As consumer preferences continue to shift towards more environmentally friendly farming practices, these ancient grains can serve as exemplary models in the quest for sustainable agriculture.