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

Received: 27 March 2025 Revised: 25 April 2025 Accepted: 29 April 2025 Published: 1 May 2025 Citation: Györéné Kis, G.; Bencze, S.; Mikó, P.; Lacko-Bartošová, M.; Setiawan, N.N.; Lugasi, A.; Drexler, D. Phenolic Content and Phenolic Acid Composition of Einkorn and Emmer Ancient Wheat Cultivars—Investigation of the Effects of Various Factors. Agriculture 2025,15, 985. https://doi.org/10.3390/ agriculture15090985 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Phenolic Content and Phenolic Acid Composition of Einkorn and Emmer Ancient Wheat Cultivars—Investigation of the Effects of Various Factors Gyöngyi Györéné Kis 1,* , Szilvia Bencze 1, Péter Mikó 2, Magdaléna Lacko-Bartošová 3, Nuri Nurlaila Setiawan 1, Andrea Lugasi 4and Dóra Drexler 1 1ÖMKi, Hungarian Research Institute of Organic Agriculture, 1038 Budapest, Hungary; [email protected] (S.B.); [email protected] (N.N.S.); [email protected] (D.D.) 2HUN-REN Centre for Agricultural Research, 2462 Martonvásár, Hungary; [email protected]en.hu 3Institute of Agronomic Sciences, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture, 949 76 Nitra, Slovakia; [email protected] 4Faculty of Commerce, Hospitality and Tourism, Budapest University of Economics and Business, 1054 Budapest, Hungary; [email protected] *Correspondence: [email protected] 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 phydroxybenzoic 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. Keywords: ancient wheat; einkorn; emmer; organic farming; antioxidants; phenolic compounds; phenolic acids 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 Agriculture 2025,15, 985 https://doi.org/10.3390/agriculture15090985 Agriculture 2025,15, 985 2 of 26 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 – 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 – 22 ]. The healthpromoting 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 [31–34]. 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 Agriculture 2025,15, 985 3 of 26 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). 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 Agriculture 2025,15, 985 4 of 26 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.  Figure 1. Map of the study sites (green points) located in Hungary and Slovakia in 2019 and/or 2020. The two sites indicated in bold (Martonvásár and Želiezovce) were selected for more detailed analysis (see Table 1for cultivars planted on each site and Section 2.2.4 for the selected sites analysis). 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. Agriculture 2025,15, 985 5 of 26 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 ) Agriculture 2025,15, 985 6 of 26 (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 m 2 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 µ m 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 µ m, 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 µ L 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 Agriculture 2025,15, 985 7 of 26 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 µ g/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 µ L of deionized water, and 300 µ L of Folin–Ciocalteu’s reagent. After 8 min of incubation at room temperature, 300 µ L 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 – 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 Agriculture 2025,15, 985 8 of 26 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 µ g FAE g −1 DM (GT-1669) (Table 2). However, a similarly high value was observed in the control common wheat samples with 869 µ g FAE g −1 DM for Mv Káplár and 739 µ g FAE g −1 DM for Mv Uncia in 2019 and 2020 (Figure 2). Table 2. Influence of wheat cultivars and cropping years on the concentration of phenolic compounds ( µ g FAE g −1 DM) of ancient wheat grown under conventional conditions in Martonvásár in 2019–2020. Species, Cultivars Phenolic Compounds (µg FAE g−1DM) 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 pcultivars (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). The TP values of emmer landraces varied between 413 µ g FAE g −1 DM (GT-831) and 826 µ g FAE g −1 DM (GT − 1 669). At the same time, all einkorn landraces achieved lower values, ranging from 369 µ g FAE g −1 DM (Mv Esztena) to 553 µ g 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, Figure 3) showed lower TP contents in einkorn (209 µ g FAE g −1 DM (Mv Alkor) and emmer varieties, ranging from 218 µ g FAE g −1 DM (Mv Hegyes) to 408 µ g 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 Agriculture 2025,15, 985 9 of 26 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 µ g FAE g −1 DM (GT-831). Agriculture2025,15,xFORPEERREVIEW8of27   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).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).  Figure2.Effectsofgrowingyearonmean±SDtotalphenoliccompoundofselectedcerealcultivars atthesiteMartonvásár(2019,2020).Notethatthetwocommonwheatvarietieswereonlypresent inoneyear(MvKáplárin2019andMvUnciain2020);therefore,theywerenottestedforyeareffect butareshownasacomparison.Significant(ANOVAp-value)differencesbetweenthetwoyearsof measurementsareannotatedineachcultivar,withsignificantvaluesappearinginbold. 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 Figure 2. Effects of growing year on mean ± SD total phenolic compound of selected cereal cultivars at the site Martonvásár (2019, 2020). Note that the two common wheat varieties were only present in one year (Mv Káplár in 2019 and Mv Uncia in 2020); therefore, they were not tested for year effect but are shown as a comparison. Significant (ANOVA p-value) differences between the two years of measurements are annotated in each cultivar, with significant values appearing in bold. Table 3. Influence of ancient wheat cultivars and cropping years on the concentration of phenolic compounds ( µ g FAE g −1 DM) of ancient wheat grown under organic conditions in Želiezovce in 2019–2020. Species, Cultivars Phenolic Compounds (µg FAE g−1DM) 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 pcultivars (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). 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 µ g FAE g −1 DM, Mv Káplár and 100 µ g FAE g −1 DM, Mv Uncia in 2019 and 2020, respectively). The concentration of FPs in emmer GT-1669 was also higher (108 µ g FAE g −1 DM) than in other ancient wheat species’ varieties. Meanwhile, einkorn had the lowest values (47 µ g FAE g −1 DM, Mv Esztena) under conventional conditions. The Agriculture 2025,15, 985 16 of 26 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. 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 (Figures 6and 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%). Agriculture 2025,15, 985 17 of 26 Agriculture2025,15,xFORPEERREVIEW17of27   Nineteenwheatcultivarsgrownin2019and2020atdifferentsitesshowedawide rangeofPCandPAcontents(Figures6and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(Figures6and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.  Figure6.Heatmapsofthemeantotalphenoliccompoundof19wheatvarietiesmeasuredat10sites in2years(2019,2020)ofmeasurements.Thedarkershaderepresentsahighermeanphenoliccompoundvalue.ThesiteswereB:Bugac,C:Csonkahegyhát,F:Füzesgyarmat,M:Martonvásár,N: Nagykáta,P:Páprád,Ps:Pásztó,S:Szolnok,Z:Zalaszentlászló,andZe:Želiezovce.SeeFigure1and Table1formoredetailsonthesitesandthecultivars. Figure 6. Heatmaps of the mean total phenolic compound of 19 wheat varieties measured at 10 sites in 2 years (2019, 2020) of measurements. The darker shade represents a higher mean phenolic compound value. The sites were B: Bugac, C: Csonkahegyhát, F: Füzesgyarmat, M: Martonvásár, N: Nagykáta, P: Páprád, Ps: Pásztó, S: Szolnok, Z: Zalaszentlászló, and Ze: Želiezovce. See Figure 1and Table 1for more details on the sites and the cultivars. In both PC and PA, the highest value was shown by emmer GT-831 grown in Füzesgyarmat (PC: 1254.0 ± 108.9 µ g FAE g −1 DM, PA: 748.1 ± 0.02 µ g g −1 DM), and the lowest value was shown by einkorn Mv Esztena (PC: 117 ± 2.0 µ g FAE g −1 DM, PA: 66.9 ±3.5 µg g−1DM ). 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 (Figures 6and 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. Agriculture 2025,15, 985 18 of 26 Agriculture2025,15,xFORPEERREVIEW18of27    Figure7.Heatmapsofthemeantotalphenolicacidof19wheatvarietiesmeasuredat10sitesin2 years(2019,2020)ofmeasurements.Thedarkershaderepresentsahighermeanphenolicacidvalue. ThesiteswereB:Bugac,C:Csonkahegyhát,F:Füzesgyarmat,M:Martonvásár,N:Nagykáta,P: Páprád,Ps:Pásztó,S:Szolnok,Z:Zalaszentlászló,andZe:Želiezovce.SeeFigure1andTable1for moredetailsonthesitesandthecultivars. 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-tofree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).  Figure 7. Heatmaps of the mean total phenolic acid of 19 wheat varieties measured at 10 sites in 2 years (2019, 2020) of measurements. The darker shade represents a higher mean phenolic acid value. The sites were B: Bugac, C: Csonkahegyhát, F: Füzesgyarmat, M: Martonvásár, N: Nagykáta, P: Páprád, Ps: Pásztó, S: Szolnok, Z: Zalaszentlászló, and Ze: Želiezovce. See Figure 1and Table 1for more details on the sites and the cultivars. 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). 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 *** *** *** Agriculture 2025,15, 985 19 of 26 Table 7. Cont. Response Variables Significance of Different Explanatory Variables Cultivar Site Year 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 8to 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. Agriculture 2025,15, 985 20 of 26 Agriculture2025,15,xFORPEERREVIEW20of27    Figure8.Meanphenoliccompoundcontentsofselectedwheatvarietiesacrossdifferentsites(B: Bugac,C:Csonkahegyhát,F:Füzesgyarmat,M:Martonvásár,N:Nagykáta,P:Páprád,Ps:Pásztó,S: Szolnok,Z:Zalaszentlászló,Ze:Želiezovce). 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 Figure 8. Mean phenolic compound contents of selected wheat varieties across different sites (B: Bugac, C: Csonkahegyhát, F: Füzesgyarmat, M: Martonvásár, N: Nagykáta, P: Páprád, Ps: Pásztó, S: Szolnok, Z: Zalaszentlászló, Ze: Želiezovce). 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 Agriculture 2025,15, 985 21 of 26 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. Author Contributions: Conceptualization, S.B., P.M., and D.D.; methodology, M.L.-B. and S.B.; software, N.N.S.; validation, G.G.K., S.B., and D.D.; formal analysis, N.N.S.; investigation, S.B.; resources, M.L.-B.; data curation, G.G.K. and N.N.S.; writing—original draft preparation, G.G.K.; writing—review and editing, S.B., A.L., D.D., P.M., and M.L.-B.; visualization, N.N.S. and G.G.K.; supervision, D.D.; project administration, S.B. and P.M.; funding acquisition, D.D. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the EU’s Horizon 2020 projects LIVESEEDING N ◦ 101059872 and DIVINFOOD N ◦ 101000383, and the MNVH (Hungarian National Rural Network) project, N◦VP-20.2.-16-2016-00001. Data Availability Statement: Upon request, the corresponding author will provide the datasets used and analyzed in the current study. Acknowledgments: The authors would like to express their gratitude to the seed suppliers for providing the sowing material used in this research: community gene bank ProSpecieRara (Basel, Switzerland), National Centre for Biodiversity and Gene Conservation (formerly NöDiK, Tápiószele, Hungary), Agricultural Institute of the HUN-REN Centre for Agricultural Research (HUN-REN ATK Martonvásár, Hungary), and the Louis Bolk Institute (Driebergen-Rijsenburg, the Netherlands). Special thanks to the HUN-REN Centre for Agricultural Research in Martonvásár and all on-farm partners of the ÖMKi for participating in this research, and to the Slovak University of Agriculture laboratory for carrying out the analytical assays. Conflicts of Interest: The authors affirm that there are no conflicts of interest associated with this publication. Abbreviations The abbreviations used in this manuscript are listed below: ANOVA Analysis of Variance B Bugac BP Bound phenolic compound BPA Bound phenolic acid C Csonkahegyhát CI Confidence interval DAD Diode array detector DM Dry matter F Füzesgyarmat FA Ferulic acid FAE Ferulic acid equivalents FP Free phenolic compound FPA Free phenolic acid GAE Gallic acid equivalents HPLC High-performance liquid chromatography M Martonvásár N Nagykáta P Páprád PA Phenolic acid PC Phenolic compound p-HBA 4-hydroxybenzoic acid Ps Pásztó S Szolnok Agriculture 2025,15, 985 22 of 26 TP Total phenolic compound TPA Total phenolic acid Z Zalaszentlászló Ze Želiezovce Appendix A Agriculture2025,15,xFORPEERREVIEW22of27   HPLCHigh-performanceliquidchromatography MMartonvásár NNagykáta PPáprád PAPhenolicacid PCPhenoliccompound p-HBA4-hydroxybenzoicacid PsPásztó SSzolnok TPTotalphenoliccompound TPATotalphenolicacid ZZalaszentlászló ZeŽeliezovce AppendixA  FigureA1.Listof19wheatandancientwheatcultivarsusedintheresearch.Thegreyshadeindicates thesitesandyearsofthegrowncultivars. Figure A1. List of 19 wheat and ancient wheat cultivars used in the research. The grey shade indicates the sites and years of the grown cultivars. Agriculture2025,15,xFORPEERREVIEW23of27    (a)   (b) Figure A2. Cont. Agriculture 2025,15, 985 23 of 26 Agriculture2025,15,xFORPEERREVIEW23of27    (a)   (b) Agriculture2025,15,xFORPEERREVIEW24of27     (c)   (d) FigureA2.Annualweatherparameters:(a)precipitation,(b)maximum,(c)minimum,and(d)averagetemperature,measuredfrom10sites.(a)Precipitationattheresearchsites(2019and1March– 31July2020).(b)Averagetemperatureattheresearchsites(2019and1March–31July2020).(c) Minimumtemperatureattheresearchsites(2019and1March–31July2020).(d)Maximumtemperatureattheresearchsites(2019and1March–31July2020).   Figure A2. Cont. Agriculture 2025,15, 985 24 of 26 Agriculture 2025, 15, x FOR PEER REVIEW 24 of 27 (c) (d) Figure A2. Annual weather parameters: (a) precipitation, (b) maximum, (c) minimum, and (d) average temperature, measured from 10 sites. (a) Precipitation at the research sites (2019 and 1 March– 31 July 2020). (b) Average temperature at the research sites (2019 and 1 March–31 July 2020). (c) Minimum temperature at the research sites (2019 and 1 March–31 July 2020). (d) Maximum temperature at the research sites (2019 and 1 March–31 July 2020). Figure A2. Annual weather parameters: (a) precipitation, (b) maximum, (c) minimum, and (d) average temperature, measured from 10 sites. (a) Precipitation at the research sites (2019 and 1 March–31 July 2020). (b) Average temperature at the research sites (2019 and 1 March–31 July 2020). (c) Minimum temperature at the research sites (2019 and 1 March–31 July 2020). (d) Maximum temperature at the research sites (2019 and 1 March–31 July 2020). References 1. Cheng, A. Review: Shaping a sustainable food future by rediscovering long-forgotten ancient grains. 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