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Article

Chemical Composition and Genome Pattern as a Means of Identifying the Origin of Preserved Wild Garlic (Allium ursinum L.) in Poland

by
Emilia Bernaś
1,
Jacek Słupski
1,*,
Piotr Gębczyński
1,
Katarína Ražná
2 and
Jana Žiarovská
2
1
Faculty of Food Technology, University of Agriculture in Krakow, Balicka Street 122, 30-149 Kraków, Poland
2
Institute of Plant and Environmental Sciences, Faculty of Agrobiology and Food Resources, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(1), 20; https://doi.org/10.3390/agriculture14010020
Submission received: 8 November 2023 / Revised: 18 December 2023 / Accepted: 19 December 2023 / Published: 22 December 2023
(This article belongs to the Section Agricultural Product Quality and Safety)
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Abstract

:
In Poland, the mountainous and hilly regions until recently were territories with unbalanced and poor diet quality, especially in early spring. Wild edible plants were or remain influential in supplementing a human diet. One of the earliest wild edible plants to appear in the growing season, characteristic of a broad swath of Europe from Ukraine to Germany, remains wild (ramsons) garlic (Allium ursinum L.). This paper assesses the impact of the vegetation site and preservation methods on the content of chlorophylls, β-carotene, vitamin C, total polyphenols, and antioxidant activity (ABTS, DPPH, and FRAP) of wild garlic leaves. In addition, the connection between its genome pattern and chemical composition was assessed. The plants were collected in three locations in the Carpathian foothills, and the reference sample was plants collected in the lowlands. The site from which the raw material was harvested has a statistically significant effect on the chemical composition of the preserved leaves of wild garlic. The profiles of the analysed components differed in the material from a different site. Wild garlic leaves from mountainous areas (400–600 m above sea level) were characterized by a higher content of the analysed ingredients than leaves obtained from lowland sites (200 m above sea level). Moreover, the material from the lowlands had a different genetic pattern from the leaves from the mountainous areas.

Graphical Abstract

1. Introduction

Plant-based foods play an increasingly important role in modern consumer lifestyles due to changing behaviour and eating habits. The reasons for these changes vary; for example, changes in worldview that preclude the consumption of any or some animal products or beliefs related to the perception that raising animals for food causes more significant environmental damage than growing plants. Many consumers are also open to current nutritional recommendations that call for increasing the proportion of plant products in everyday food, mainly fruits and vegetables [1]. It may be easier to change eating habits by expanding the range of available plant products with unknown or forgotten plant species [2]. These may include species that are difficult to cultivate or are unprofitable in large-scale farming [3,4].
The food group in question may include plants growing in the natural environment, i.e., in forest areas, wastelands, or abandoned/neglected human settlements. According to the Food and Agriculture Organization (FAO) [5], this group of plants is referred to as wild edible plants. Such plants are usually very well known to local communities, especially in low-income areas, as they often supplement people’s daily food. Such a situation was common in the past and now can be observed in poorly developed agricultural mountainous regions [6,7]. In Europe, an example is the Carpathian Mountains and their foothills. Therefore, the experiences of the people living in these areas can be used to promote plant foods by popularising forgotten edible plant species.
One of the most characteristic plants for the areas mentioned above is wild (ramsons) garlic (Allium ursinum L.). Wild garlic is a plant of the family Amaryllidaceae (subfamily Allioideae) that belongs to the economically important genus and subgenus Allium, which includes over 850 species [8]. Almost all these species are present in temperate Europe, the Mediterranean region, the Near and Middle East, the Caucasus, and northern Asia, with only a few species being found outside this area [9,10]. In the wild, it grows on the floor of wet, usually mountainous forests [11,12].
The characteristic and dominant flavour of Allium and its possible nutritional effects and medical applications have attracted food and medical researchers [13]. Wild garlic’s properties are similar to those of common cultivated garlic. Primarily, the plant’s large, green leaves are gathered. There has been a growing interest in using wild garlic as a functional food and dietary supplement, rich it in health-promoting substances. Plants of the genus Allium, including wild garlic, contain active biological substances, phenolic and sulphur compounds, dietary fibre, and essential oils that are potentially useful for humans and animals [14,15,16]. The leaves contain free and bound forms of phenolic acids (protokatechuic, 4-OH-benzoic, vanillic, caffeic, syringic, coumaric, ferulic, and sinapic) and flavonoids, predominantly in kaempferol derivatives [17]. Allicin is synthesised by wild garlic and garlic following damage to tissues and is derived from the non-proteinogenic amino acid alliin (S-allylcysteine sulfoxide), in a reaction that is catalysed by the enzyme alliinase. Allicin is a compound related to the biological activity of garlic. Allicin and other thiosulfinates immediately decompose into oil-soluble diallyl sulfide, diallyl disulfide, diallyl trisulfide and dithiins, ajoene, and others [18,19]. Allicin (diallyl thiosulfinate) as an organosulfur compound found in processed garlic is highly active, unstable, volatile, and quickly transformed into other compounds under the influence of gastric juices and in the large intestine, which is then converted by glutathione and/or S-adenosylmethionine into volatile allyl sulfide methyl excreted through the lungs and skin [20]. Allicin is the main and characteristic odorous substance of fresh garlic. It is believed that the decrease in allicin content in thermally treated garlic is due to the fact that allicin and diallyl disulfide are compounds unstable at high temperatures [21].
Wild garlic is an interesting food product due to its early vegetation (March/April). It is one of the earliest plants in forests and surrounding areas. It is also increasingly grown in home gardens and is used in the local cuisine. European people consume wild garlic as a fresh or processed vegetable or condiment. The leaves are added mainly for salads, soups, and as a component of pie fillings [22]. In addition to its culinary uses, wild garlic is also increasingly used as an additive to processed food products. In Poland, Turkey, and Czechia, the leaves are added to local rennet cheeses [23,24,25].
This study aimed to compare the genome and chemical composition of the wild garlic (Allium ursinum L.) leaves collected in four locations in southern Poland.

2. Materials and Methods

2.1. Material

The research material consisted of whole leaves (the above-ground part) of wild (ramsons) garlic. Fresh material was collected from undisturbed wild habitats, from land belonging to private owners located in four sites in Poland, ranging from 200 to 600 m above sea level (asl): Krakow (K) (50.08382753062322, 19.96169005992687), an old, wild orchard at 200 m; Limanowa (L) (49.74124708140615, 20.34037997486435), a privately-owned forest at 400 m; Sucha Beskidzka (S) (49.70863251066192, 19.572269556064363), a private woodland at 500 m, and Ropa (R) (49.578459093112706, 21.048966974785234), a private forest at 600 m. The last three places are situated in the West Beskid Mountains, a part of the West Outer Carpathians.
The leaves were harvested before the plant flowered in late March and early April 2020. The leaves were collected once, early in the morning, and fully developed. The size of the leaves was approximately 10 to 20 cm in length. After picking, the fresh material was packed into a cooled box and transferred to the laboratory within 60–120 min. The raw material was processed on the same day. As part of preparing the raw material for freezing and drying, the leaves were washed in cold tap water. Surface water was removed by gentle centrifugation in a leafy-vegetable centrifuge and ambient air blowing from a fan. Any leaves that were mechanically damaged or diseased during these actions were removed. They were frozen by a convective shock freezer, a jet of cold air (−40 °C). Before freezing, the leaves were packed in polyethylene bags in batches of about 300 g and pressed to form a layer 1–1.5 cm thick. The bags were frozen for about 1.5 h until the temperature reached −25 °C inside the package.
Convection drying was carried out in a ProfiLine-type chamber dryer with airflow parallel to the sieves (Hendi, De Klomp, The Netherlands). The charge of the material was 2 kg per 1 m2 of the screen. The drying temperature was 40 ± 1 °C, and the time was about 40 h until the humidity reached 10%. Freeze-drying was performed in a Gamma 1–16 LSC freeze dryer (Christ, Osterode am Harz, Germany). Before placing the garlic leaves into the freeze dryer, they were air-cooled in the freeze dryer trays in a shock freezer (as described above) to speed up the drying. They were lyophilised with the following parameters: an initial product temperature of −30 °C, a condenser temperature of −52 °C, a shelf temperature during main drying of 20 °C, a shelf temperature during final drying of 30 °C, and a drying time of 6 h. Drying was carried out for 24 h until the moisture content was about 5%.

2.2. Methods

The gravimetric method determined the dry matter (DM) content according to the AOAC standard [26]. The analysed material was ground with double-distilled water (1:1). The samples were dried in an air dryer to a constant weight at a temperature of 98 °C.
Vitamin C was measured using high-performance liquid chromatography. The analysed material was prepared using the PN-EN 14130 [27] standard. Vitamin C was determined as the sum of L-ascorbic acid and dehydroascorbic acid, using L-cysteine to reduce dehydroascorbic acid. Analysis was performed on a La-Chrome Merck-Hitachi liquid chromatograph (Tokyo, Japan). The set was equipped with an L-7612 degasser, programmable L-7250 autosampler, L-7100 pump, L-7360 column oven thermostat, L-7420 UV-VIS detector, D-7000 interface with D-7000 HPLC System Manager software and a Bionacom Velocity C18 PLMX (4.6 × 250 mm, 5 µm) Bionacom Ltd. (Coventry, UK) column with a pre-column. Isocratic elution was run with a flow of 0.6 mL/min of 0.1% metaphosphoric acid solution as a mobile phase. The measurement was done at a wavelength of 254 nm. An external standard was used for the identification and quantitative analysis of vitamin C: L-ascorbic acid dissolved in 20% metaphosphoric acid at concentrations of 1.0–10.0 µg/mL.
The colourimetric method was used to determine the content of chlorophyll a and b, and β-carotene [28]. A sample of the material was homogenised with a mixture of acetone and hexane (4:6) for 2 min. The entire homogenate was transferred to a 25-mL volumetric flask with the same solvents up the mark and centrifuged at 4 °C, 5000 rpm, for 10 min. After centrifugation, the absorbance (A) was measured at four wavelengths: 453, 505, 645, and 663 nm. The content of individual compounds was calculated from the following equations:
Chlorophyll a = 0.999A663 − 0.0989A645 [mg/100 mL],
Chlorophyll b = −0.328A663 + 1.77A645 [mg/100 mL],
β-carotene = 0.216A663 − 0.304A505 + 0.452A453 [mg/100 mL].
To determine total polyphenols (TP) and antioxidant activity, sample extracts were prepared using 80% ethanol. Two grams of material homogenized with double-distilled water (1:1, w/w) was mixed with 25 mL of 80% ethanol, extracted for 30 min under reflux, and centrifuged for 10 min at 4200× g (MPW-352RH, Rotor 12177, MPW Med. Instruments, Warsaw, Poland), and stored at −30 °C until use.
Polyphenols were determined by the Folin–Ciocalteu method [29]. Wild garlic extracts (0.5 mL) were mixed with 4.5 mL of distilled water, 0.125 mL of Folin–Ciocalteu reagent (Sigma-Aldrich, Darmstadt, Germany), and 0.25 mL 25% sodium carbonate (Na2CO3) (Warchem, Warszawa, Poland). After 60 min, the absorbance was measured at 675 nm (Shimadzu UV-160 UV-VIS, Tokyo, Japan). The content of polyphenols was calculated from the standard curve prepared for gallic acid expressed as gallic acid equivalent (GAE) g/100 g DM.
The antioxidant activity was determined using ABTS, DPPH, and FRAP assays. The evaluation of this parameter against the ABTS radical (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid) was carried out according to Re et al. [30]. The solution of ABTS cation chromophore was prepared by facilitating a reaction between 100 mL of the 7.0 mM ABTS solution and 100 mL of 2.45 mM K2S2O8. The solution was kept in darkness at 25 °C for 16 h. The ABTS solution was adjusted to an absorbance value of 0.700 ± 0.020 at 734 nm by dilution with the phosphate buffer (50 mM/L, pH 7.4) before being used. Next, 2 mL of the ABTS solution was mixed with 1 mL of different concentrations of each extract sample. A mixture composed of the ABTS solution and ethanol was used as the control. The mixtures were incubated at room temperature for 10 min in the dark. The absorbance value of the mixture was measured at 734 nm. The results are expressed in μmol of Trolox (2-carboxyl-6-hydroxy-2,5,7,8-tetramethylchroman, a water-soluble analogue of α-tocopherol) in 1 g of the sample. Trolox was prepared in phosphate-buffered saline (PBS) (50 mM/L; pH 7.4). Antioxidant activity was then calculated according to the following formula:
ABTS = ((A0 − Asample) × CTrolox)/A0 − ATrolox
where A0 represents the absorbance of the control and Asample is the absorbance of the mixture containing the extract sample, CTrolox is the concentration of Trolox solution, ATrolox is the absorbance of the mixture containing Trolox solution.
In turn, in the second determination, the ability to reduce the persistent free radical DPPH’ (1,1-diphenyl-2-picrylhydrasyl) of an 80% ethanol solution was analysed (Pekkarinen et al. 1999) [31]. The absorbance was measured at a wavelength of 516 nm. The activity level was determined in µmol of Trolox (2-carboxy-6-hydroxy-2,5,7,8-tetramethylchroman) in 1 g. Trolox was prepared in phosphate-buffered saline (PBS) (50 mM/L; pH 7.4). Antioxidant activity was then calculated ABTS radical was calculated according to the formula previously described in the ABTS assay.
The antioxidant activity against the FRAP (ferric reducing antioxidant power) was defined as the ability to reduce iron, and it was measured in µmol of Fe2+ in 100 g of DM [32]. The FRAP reagent was prepared using a mixture containing 10 mM 2,4,6-tripyridyl-s-triazine solution in 20 mL of 40 mM HCl, 20 mL of 20 mM ferric (III) chloride, and 5 mL of 300 mM acetate buffer (pH 3.6). Each 0.4 mL of extract was mixed with 3.6 mL of FRAP reagent. The mixture was then incubated for 10 min in a water bath at 37 °C. After this time, the absorbance was measured at 595 nm. Antioxidant activity was then calculated according to the following formula:
FRAP = [(A − A0) − 0.0017]/2.123 µmol Fe2+/1 mL
where A0 represents the absorbance of the control and Asample is the absorbance of the mixture containing the extract sample.

2.3. DNA Analysis

2.3.1. DNA Extraction

Total genomic DNA was isolated from the lyophilised samples of wild garlic leaves (Allium ursinum) with the GeneAll Exgene™ isolation kit (Cambio, Cambridge, UK) following the manufacturer’s instructions (Plant SV, Seoul, Republic of Korea). One sample originated from an urban area of Krakow (sample K), and the others were grown in different mountainous regions of Poland (samples R, L, and S). The quantity and quality of the extracted DNA were measured with a P-Class NanoPhotometer® (Implen GmbH, Munchen, Germany).

2.3.2. CDDP and PBA Marker Assay

DNA markers based on conserved regions of DNA or gene families were used in the study. PBA primer combinations (CYPA1F + CYPA1R; CYP2BF + CYP2BR; CYP2CF + CYP2CR) were used as designed by Inui et al. [33]. CDDP primer combinations (WRKY F/R1; WRKY F/R2; WRKY F/R2b; WRKY F/R3; WRKY F/R3b) were used as designed by Xie et al. [34]. Polymerase chain reactions (PCRs) were performed with MyTaq 2x master mix (Bioline, Meridian Bioscience, Memphis, TN, USA) with 100 ng of DNA and 320 nmol of each primer. Amplification reactions were performed in a C1000 thermocycler (BioRad, Hercules, CA, USA) with the following temperature and time profile: 95 °C for 5 min (95 °C for 60 s; 50 °C for 60 s; 72 °C for 120 s) 45 times, plus 72 °C for 10 min for the final elongation.

2.3.3. Microsatellite Markers Assay

The extracted DNA was diluted to a concentration of 10 ng/µL. The microsatellite loci were amplified by two combinations of primers, amplifying the (AG)9 and (AG)16 repeat motifs [35]. The PCRs were performed in 25 µL, containing 2.5 µL of 10× Dream Taq reaction buffer (plus 50 mM of MgCl2) (ThermoFischer Scientific, Waltham, MA, USA), 0.5 µL of a mix of all four dNTPs (10 mM each), 0.5 µL forward and reverse primer (10 µM), 0.1 µL Dream Taq DNA polymerase (5 U/µL), 1.0 µL of bovine serum albumin (10 mg/mL), 1.0 µL of 10 ng/µL genomic DNA, and sterilised water up to a final volume of 25 µL.
PCR reactions were performed using a C1000 Thermal Cycler (BioRad, Hercules, CA, USA) under the following conditions: initial denaturation at 94 °C for 3 min; 40 cycles of 94 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min; 8 cycles of 94 °C for 30 s, 53 °C for 45 s, and 72 °C for 1 min; a final extension step of 72 °C for 5 min. The resulting PCR products were run on 1.5% agarose gel together with the size standard 100 bp Ready-to-use DNA Ladder (Bioron, GmbH, Römerberg, Germany). The fragments were scored by GeneTool software (version 4.3.10.0, Syngene, Synoptics Ltd., Cambridge, UK).

2.3.4. Direct Sequencing

Standard barcoding primers were used for the amplification [36]. The PCR amplification of the ITS regions was carried out in a SureCycler 8800 thermal cycler (Agilent, Santa Clara, CA, USA). The PCRs were performed with Phusion High-Fidelity PCR Master Mix (ThermoFisher Scientific) under the following conditions: 98 °C for 30 s (98 °C for 10 s, 55 °C for 40 s, and 72 °C for 40 s) 35 times, then 72 °C for 10 min final elongation. After the control display of the specificity of amplicons on 1.5% agarose gel, the product was purified with an ExpinTM Combo Kit (GeneAll, Seoul, Republic of Korea) according to the manufacturer’s instructions. GenSeq sequenced the ITS amplicons.

2.3.5. Data Analysis

The amplified fragments were separated in 6% PAGE stained by GelRed (Biotium, Fremont, CA, USA); they were analysed online using GelAnalyser software (version 19.1). The amplified loci were scored by 0–1 matrices and compared for amplification patterns. ITS raw sequences were trimmed and analysed using BioEdit software (version 7.0.9.0). Individual sequences were BLASTed against Allium sp. taxa in the NCBI database.
All samples were analysed in three independent replications (n = 3). Results were analysed statistically using a double-factor analysis of variance (ANOVA) on the basis of Duncan’s range test (p < 0.05 The independent variables were the place of origin (altitude above sea level) and the method of preservation. Calculations were performed using Statistica 13.0 Pl (TIBCO Software Inc., Palo Alto, CA, USA).

3. Results and Discussion

The frozen leaves of wild garlic after the harvest had a dry matter (DM) content ranging from 9.8–12.6 g/100 g of fresh matter (FM) (Table 1). The leaves obtained in Krakow and Sucha Beskidzka had significantly higher DM content than those from Limanowa and Ropa. The last two had amounts similar to the value reported by Jędrszczyk et al. [37] and Leahu [38], 9.79–10.77 g/100 g and 9.75 g/100 g, respectively; these levels were higher than that found by Gîtin et al. (2012). However, according to Błażewicz-Woźniak [39], the DM content in wild garlic leaves (6.69–15.7 g/100 g FM) largely depended on the ecotype and the year of cultivation. After drying to a constant weight, the air-dried leaves contained 85.5–92.7 g of DM, and the freeze-dried leaves had 94.3–96.9 g of DM per 100 g of FM. Tomšik et al. [40] were able to dry wild garlic leaves up to 91.19 g and 92.03 g of DM per 100 g of FM using air at 40 °C and 60 °C, respectively.
In the frozen and dried leaves of wild garlic, the sum of chlorophyll a and b was 638–1532 mg/100 g DM, depending on the location and processing methods. The fraction of chlorophyll a ranged from 68% to 74% (Table 1), and the chlorophyll a:b ratio was 2–3:1, which is consistent with a study by Srichaikul et al. [41]. For comparison, Luta et al. [42] reported 174 mg of chlorophyll/100 g FM in fresh wild garlic leaves, whereas Lachowicz et al. [43] 364.3–408.7 mg chlorophyll a per 100 g DM with the chlorophyll a:b ratio 1.5–1.9:1. Regardless of the processing method, the highest level of total chlorophyll was in the frozen material collected in the Limanowa region (a mountainous area), an average of 19%–89% more; the lowest content was in the leaves collected near Sucha Beskidzka (also mountainous). Among the preservation methods, freezing turned out to be the best and air-drying the worst way to protect the chlorophylls in garlic leaves. On average, the frozen product contained 15% more chlorophylls than the air-dried product. The decomposition of these pigments likely occurred due to the thin wild garlic leaves’ tissue aeration and exposure to high temperatures (40 °C) for tens of hours. According to Rocha et al. [44], a short steam blanching (15 s) can reduce the degradation of chlorophyll in basil leaves dried at high temperatures (60 °C). Still, if the samples were not blanched before drying, drying at low temperatures was the best method to prevent the degradation of chlorophylls.
In wild garlic leaves, 155–221 mg of β-carotene per 100 g of DM was measured (Table 1). The most considerable amount of this discriminant was found in the leaves collected near Limanowa (a mountainous area). At the same time, in the case of other frozen products, the location did not affect the amount of β-carotene. Lachowicz et al. [43] found the content of 57.7–82.9 b-carotene in 100 g DM of leaves, depending on the harvest date. The preservation method usually had little effect on the level, with the best process being freeze-drying. For comparison, Luta et al. [42] found only 5.3 mg of total carotenoids in 100 g of fresh wild garlic leaves, using 80% acetone for the extraction. In turn, Stajner et al. [45] showed that cultivated leaves of Allium vienale contain over two times more chlorophyll a and chlorophyll b in 100 g of FM than wild growing. Carotenoids are essential not only for the colouration of food but also for their acknowledged roles in health. They are vitamin A sources and a critical factor for human vision. In addition, they have an essential role in preventing various diseases associated with oxidative stress, such as cancer, cardiovascular diseases, and other chronic diseases [46,47].
Vitamin C is one of the most critical water-soluble vitamins with different essential biological functions. It is one of the most active antioxidants, which helps the body neutralise free radicals. It takes part in the biosynthesis of collagen, carnitine, hormones, immune response, and iron absorption. It is vulnerable to light, air, and high temperature and decomposes completely at approximately 190 °C [48]. The content of vitamin C in fruits, vegetables, and herbs is highly variable and dependent on many factors: species, variety, climate, a season of the year, method of storage after harvest, and country of origin [49]. Stajner et al. [45] noted that cultivated leaves of Allium vienale contain over three times as much vitamin C (18 mg/100 g FM) as wild-growing varieties. In turn, Jędrszczyk et al. [37] showed that the amount of vitamin C in wild garlic depends on the part of the plant because the leaves and stems contain 7–15 and 4–6 mg of vitamin C/100 g FM, respectively. In the wild garlic leaves analysed in this study, 20–1673 mg of vitamin C/100 g DM were measured, depending on the harvest site and the method of preservation (Table 2). For comparison, in fresh wild garlic leaves, Luta et al. [42] found only 80 mg of vitamin C per 100 g of FM. They determined the amount of vitamin C using titration, not chromatography.
The most vitamin C was found in the frozen material collected in the vicinity of Sucha Beskidzka (a mountainous area), whilst the lowest was in the material from Krakow (an urban area). Among the drying methods used, air-drying was the least favourable, usually resulting in almost complete decomposition of vitamin C. Vitamin C’s low resistance to high temperatures might be related to its chemical structure [49]. Wolska et al. [50] noted that the brewing temperature significantly impacted the vitamin C content in infusions of nettle leaves. Brewing temperatures of 25–50 °C were more favourable than 70–80 °C, and the optimal dewatering time was 10 min. According to Lee and Kader [51], depending on the species, L-ascorbic acid (AA) accounted for 52–93% and 73–100% of the total vitamin C in fruits and vegetables, respectively. In the product prepared from wild garlic leaves, the vitamin C contained up to 45–50%, 25–75%, and 41–54% L-ascorbic acid in frozen, air-dried, and freeze-dried leaves, respectively; the rest was L-dehydroascorbic acid (DHAA). As a rule, a minor proportion of AA was recorded in leaves collected in Krakow (an urban area).
The total polyphenol (TP) content in frozen wild garlic leaves obtained from the four sites was in the range of 1.64–1.95 g gallic acid equivalent (GAE)/100 g DM (Table 3). However, no difference was found between the frozen product gathered in Krakow and Sucha Beskidzka, whilst the product from Limanowa and Ropa contained significantly more of these compounds. The observed TP content was higher than that previously found in fresh leaves harvested in Poland—1.48 g/100 g DM [52]—but they were in the range of 1.31–2.11 g/100 g reported in garlic leaves from Montenegro and Bosnia and Herzegovina [53]. These values, on the other hand, were lower than those of the leaves of wild garlic harvested in Serbia [54,55,56]—0.92–6.05 g/100 g DM, depending on the solvent used for extraction—and of garlic cultivated in Poland [37]—2.78–3.83 g/100 g DM. According to Gîtin et al. [57] and Pop et al. [58], there was even 24.6–27.0 g TP/100 g DM in garlic leaves.
The TP content in dried wild garlic leaves was 1.41–2.14 g/100 g DM. Compared to frozen leaves, air drying resulted in a slight increase in TP content in the leaves from Ropa and a decrease in leaves from Sucha Beskidzka. In contrast, the freeze-dried leaves of plants showed a reduction in TP content regardless of the place of harvest. The decline was not statistically significant in the case of dried products from raw materials obtained in Krakow. According to Tomšik et al. [54], the TP content in frozen and dried leaves was much higher (9.18–10.10/100 g DM) than in this study.
The antioxidant activity of wild garlic depends on the harvest date. Jędrszczyk et al. [37] found that the antioxidant activity against DPPH differed almost twice between the harvest years. Similarly, Lachowicz et al. [52] found higher antioxidant activity against ABTS and DPPH but lower using the FRAP method in leaves obtained in June than in March. Kovarovič et al. [59] found that the antioxidant activity of wild garlic leaves against the DPPH radical is higher than that of white and yellow onion, leeks, and common garlic but half that of red onion. Krivokapič et al. [56] demonstrated that the antioxidant activity (IC50) of wild garlic leaves against DPPH was in the range of 111–391 μg/mL of the extract, depending on the solvent used for extraction. Similar conclusions were reached by Pop et al. [58]. According to Tomšik et al. [40], wild garlic leaves dried at 40 °C, and 60 °C did not differ in their antioxidant activity against DPPH.
The antioxidant activity measured against the cation radical ABTS in the frozen leaves was at 347–511 µmol Trolox/g DM; the lowest level was observed in the leaves harvested in Krakow, whilst the highest was in those from Limanowa. The drying contributed to a significant increase in the antioxidant activity—2.8 to 5.4 times more—compared to frozen leaves. These results were significantly higher in air-dried leaves than in freeze-dried leaves.
The antioxidant activity measured against the free radical DPPH in frozen leaves was 11–27 µmol Trolox/g DM. The dried leaves had a higher level of this index (36–131 µmol Trolox/g DM), and, as in the case of the measurements against ABTS, the freeze-dried leaves had higher antioxidant activity than air-dried ones.
The ferric-reducing antioxidant power (FRAP) of frozen wild garlic leaves was 707–798 µmol Fe2+/g DM. In both air-dried and freeze-dried leaves, a significant increase in this indicator was found; in the case of air-dried leaves from Limanowa it was even twofold, compared to frozen leaves. The advantage of air-drying was confirmed only for leaves from Limanowa and Ropa, while in the case of freeze-drying in leaves from Sucha Beskidzka. The drying method did not affect the antioxidant activity only in the leaves harvested in Krakow.

3.1. CDDP and PBA Marker Profiling

Different marker systems were developed within the trend toward gene-targeted functional markers rather than markers of non-coding regions. The conserved DNA-derived polymorphism technique (CDDP) and P450-based analogue markers (PBA) target candidate plant genes [33,60]. Similar to RAPD and ISSR techniques, the method is based on the single primer amplified region principle, as a single primer is used as a forward and reverse primer.
Different CDDP and PBA fingerprint profiles were obtained in accessions from the Krakow control sample and samples from the mountainous areas (Table 4). Monomorphic profiles with no fingerprint changes were only for two primer combinations of CDDP analyse.

3.2. Barcoding Analysis of ITS Region

Primers for barcoding analyse were selected for their putative amplification of the DNA sequences that codify for small- (18S, SSU) and large-subunit (28S, LSU) ribosomal RNA (rRNA). The primers were selected for their previous use; they are considered standard primers for amplifying these regions in various species of fungi and higher plants [36,61]. According to many authors, thanks to their variability, ITS regions yielded sufficient molecular information, which can be directly used as identifiers or molecular markers in the phylogenetic analysis [61,62,63]. In our case, the ITS product was variable in some nucleotide positions when compared to the ITS sequence of Allium ursinum stored in the NCBI database (Figure 1). For comparison purposes, registered NCBI database sequences were used, and their sequences were compared with the BLAST algorithm.

3.3. Microsatellite Markers Assay

Based on Kumar et al. [64], it was expected that clonal propagation would have little effect on the level of variation in the population structure. In our study, there were differences in the DNA fingerprinting of the garlic samples originating from different environmental conditions (Figure 2). Our results are consistent with the variability in Indian garlic germplasm consisting of accessions obtained from public research organisations and farmers’ markets (natural landraces). Similarly, cluster analysis based on microsatellite markers grouped garlic accessions according to their flowering behaviour, botanical variety, and ecophysiological characteristics [65]. The potential of SSR markers in plant germplasm evaluation is also supported by the study of da Cunha et al. [66], where hierarchical clustering (UPGMA dendrogram) analysis reflected the classification of garlic accessions according to maturity time (growth cycle).

4. Conclusions

  • Regardless of the processing method, the highest levels of chlorophylls, β-carotene, and vitamin C were found in the frozen product from raw material collected in Limanowa (400 m asl) and Sucha Beskidzka (500 m asl) mountainous areas. The preservation method significantly impacted chlorophyll and vitamin C content and less on β-carotene content. The best way for preventing chlorophyll degradation was freezing; freeze-drying was best for β-carotene. In the case of vitamin C, air drying resulted in almost complete decomposition. In contrast, freezing and freeze-drying leaves only resulted in significant differences in the raw material collected in the Sucha Beskidzka (mountainous area), where freezing became the better choice.
  • There was no difference in the total polyphenol content in the frozen leaves between those obtained in Krakow (200 m asl) and Sucha Beskidzka (500 m asl). In contrast, the frozen leaves harvested from Limanowa (400 m asl) and Ropa (600 m asl) contained significantly more of these compounds. Convection drying of the leaves resulted in a slight increase in TP content in the leaves from Ropa and a decrease in the leaves from Sucha Beskidzka, compared to the frozen leaves. In contrast, lower TP content was found in the freeze-dried leaves regardless of the collection site. Drying contributed to a significant increase in antioxidant activity in comparison to freezing. The results were significantly higher for the air-dried leaves than the freeze-dried ones.
  • Marker analysis of coding region-based CDDP and PBA techniques showed sequence variability in the samples from the mountainous areas—Limanowa, Sucha Beskidzka, and Ropa—compared to the sample from Krakow. One of the reasons for this variability might be environmental conditions.
  • Considering the level of total polyphenols and antioxidant activity, the best preservation method was usually air drying; in the case of vitamin C and chlorophylls, it was freezing; in the case of β-carotene, freeze-drying was best. Regardless of the method of preservation, the preserved leaves collected in the Limanowa area (400 m asl) were determined by the highest level of total polyphenols, chlorophylls, β-carotene, and antioxidant activity (FRAP), and those picked in the Sucha Beskidzka area (500 m asl) had the highest levels of vitamin C.
  • Considering the content of the analysed ingredients in the products, each of the preservation methods can be successfully used to process wild garlic. Only in the case of air-dried leaves can a significant reduction in the vitamin C content be expected.

Author Contributions

Conceptualization and methodology, P.G., E.B. and J.S.; validation, E.B., J.S., K.R. and J.Ž.; material preparation, P.G., E.B. and J.S.; formal analysis and investigation, P.G., E.B., J.S., K.R. and J.Ž.; data curation, E.B., J.S., K.R. and J.Ž.; writing—original draft preparation, visualization, review and editing, P.G., E.B., J.S., K.R. and J.Ž. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Polish National Agency for Academic Exchange in the project PPI/APM/2018/1/00010/U/001.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available within the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Agriculture 14 00020 g001
Figure 1. Polymorphism analysis of Allium ursinum samples by SSR marker. Highlighted are differences in DNA profile between control and mountainous samples of wild garlic. K, Krakow; control sample; R, Ropa; L, Limanowa; S, Sucha Beskidzka; samples from mountainous areas.
Figure 1. Polymorphism analysis of Allium ursinum samples by SSR marker. Highlighted are differences in DNA profile between control and mountainous samples of wild garlic. K, Krakow; control sample; R, Ropa; L, Limanowa; S, Sucha Beskidzka; samples from mountainous areas.
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Agriculture 14 00020 g002
Figure 2. Polymorphism analysis of Allium ursinum samples by SSR marker. Differences in the DNA profile between control and mountainous samples of wild garlic are marked with frames. K, Krakow (200 m asl); control sample, L, S, R—samples from the mountainous areas—Limanowa (400 m asl), Sucha Beskidzka (500 m asl), and Ropa (600 m asl) respectively.
Figure 2. Polymorphism analysis of Allium ursinum samples by SSR marker. Differences in the DNA profile between control and mountainous samples of wild garlic are marked with frames. K, Krakow (200 m asl); control sample, L, S, R—samples from the mountainous areas—Limanowa (400 m asl), Sucha Beskidzka (500 m asl), and Ropa (600 m asl) respectively.
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Table 1. Content of dry matter, chlorophylls, and β-carotene in wild garlic leaves depending on the place of collection and method of preservation *.
Table 1. Content of dry matter, chlorophylls, and β-carotene in wild garlic leaves depending on the place of collection and method of preservation *.
Component ProductLocation
Krakow
(200 m asl)		Limanowa (400 m asl)Sucha Beskidzka (500 m asl)Ropa (600 m asl)Mean
Dry matter
g/100 g FM		frozen12.0 b10.7 a12.6 b9.8 a11.3 A
air-dried91.3 e87.5 d85.5 c92.7 f89.2 B
freeze-dried94.5 g94.3 g95.4 g96.9 h95.3 C
mean65.9 B64.2 A64.5 A66.4 B
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
Chlorophyll a
mg/100 g DM		frozen914 e1081 f641 b936 e893 C
air-dried711 c1078 f450 a830 d767 A
freeze-dried817 d1047 f617 b920 e850 B
mean814 B1069 D569 A895 C
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
Chlorophyll b
mg/100 g DM		frozen379f g430 hi255 c414 h370 B
air-dried342 de454 i188 a324 d327 A
freeze-dried329 de401 gh220 b358 ef327 A
mean350 B428 C221 A365 B
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
Total chlorophyll (a + b)
mg/100 g DM		frozen1293 e1511 f896 b1350 e1263 C
air-dried1053 c1532 f638 a1154 d1094 A
freeze-dried1146 d1448 f837 b1278 e1177 B
mean1164 B1497 D790 A1261 C
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
β-carotene
mg/100 g DM		frozen180 bc213 e187 bcd155 a184 A
air-dried188 bcd221 e171 ab185 bcd191 AB
freeze-dried178 bc220 e193 cd204 de199 B
mean182 A218 B184 A181 A
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
* FM, fresh matter; DM, dry matter; asl, above sea level; (n = 3); different small letter for each object within individual component represents significant differences between means at p < 0.05; different capital letters in column or row represent significant differences between means for the type of product from a different location and different product from a specific location, respectively, at p < 0.05. In assessing the significance of the impact of factors, “+” means significance; ns, no significance.
Table 2. Content of vitamin C in wild garlic leaves depending on the place of collection and method of preservation [mg/100 g DM] *.
Table 2. Content of vitamin C in wild garlic leaves depending on the place of collection and method of preservation [mg/100 g DM] *.
Component ProductLocation
Krakow
(200 m asl)		Limanowa (400 m asl)Sucha
Beskidzka
(500 m asl)		Ropa (600 m asl)Mean
L-ascorbic acid (AA)frozen379 cd473 d840 f441 cd533 C
air-dried15 a50 ab14 a130 b52 A
freeze-dried365 c455 cd619 e445 cd471 B
mean253 A326 B491 C339 B
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
L-dehydroascorbic acid (DHAA)frozen462 e497 g833 i542 h584 C
air-dried5 a41 b40 b170 c64 A
freeze-dried516 g481 f537 h521 g514 B
mean328 A339 B470 D411 C
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
Vitamin C
(sum AA and DHAA)		frozen841 c969 de1673 g983 e584 C
air-dried20 a91 a54 a300 b64 A
freeze-dried881 cd936 de1156 f966 de514 B
mean328 A339 B470 D411 C
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
* DM, dry matter; asl, above sea level; (n = 3); different small letter for each object within individual component represents significant differences between means at p < 0.05; different capital letters in column or row represent significant differences between means for the type of product from a different location and a different product from a specific location, respectively, at p < 0.05. In assessing the significance of the impact of factors, “+” means significance; ns, no significance.
Table 3. Total polyphenol content and antioxidant activity in wild garlic leaves depending on the place of collection and method of preservation *.
Table 3. Total polyphenol content and antioxidant activity in wild garlic leaves depending on the place of collection and method of preservation *.
Component ProductLocation
Krakow
(200 m asl)		Limanowa (400 m asl)Sucha
Beskidzka
(500 m asl)		Ropa (600 m asl)Mean
Total
polyphenol
g/100 g DM		frozen1.64 cd1.95 e1.68 d1.91 e1.80 B
air-dried1.73 d2.01 ef1.47 ab2.14 f1.84 C
freeze-dried1.59 bcd1.51 abc1.45 ab1.41 a1.49 A
mean1.65 B1.83 C1.53 A1.82 C
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method preservation+
I × II+
ABTS
ABTS µmol Trolox/g DM		frozen347 a511 b397 ab418 ab418 A
air-dried1860 e1955 e1919 e1889 e1905 C
freeze-dried1636 d1434 c1725 d1523 c1580 B
mean1281 AB1300 AB1347 B1276 A
Assessment of the importance of factorsfactor I (place of origin)ns
factor II (method of preservation)+
I × II+
DPPH
ABTS µmol Trolox/g DM		frozen20 ab12 a27 bc11 a18 A
air-dried124 f99 e131 f110 e116 C
freeze-dried36 cd48 d42 d48 d43 B
mean60 AB53 A67 B56 A
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
FRAP
µmol Fe2+/g DM		frozen707 a798 b716 a717 a734 A
air-dried1295 e1609 g1050 c1379 f1333 C
freeze-dried1283 e1205 d1248 de1046 c1195 B
mean1095 C1204 D1004 A1047 B
Assessment of the importance of factorsfactor I (place of origin)+
factor II (method of preservation)+
I × II+
* DM, dry matter; asl, above sea level; (n = 3); different small letter for each object within individual component represents significant differences between means at p < 0.05; different capital letters in column or row represent significant differences between means for the type of product from a different location and a different product from a specific location, respectively, at p < 0.05. In assessing the significance of the impact of factors, “+” means significance; ns, no significance.
Table 4. Marker analysis of coding regions-based techniques CDDP and PBA.
Table 4. Marker analysis of coding regions-based techniques CDDP and PBA.
CDDP Analyse
Primer combinationResults of differentiationType of profile change
F/R1−Monomorphic profile
F/R2+Locus insertion in sample R
F/R2b−Monomorphic profile
F/R3+Locus deletion in sample R
F/R3b+Locus deletion in sample R
Locus insertion in sample L
Locus insertion in sample S
PBA analyse
Primer combinationResults of differentiationType of profile change
CYPA1F+R+Locus deletion in sample R
Locus deletion in sample L
Locus deletion in sample S
CYP2BF+R+Locus deletion in sample R
Locus deletion in sample L
Locus deletion in sample S
Locus insertion in sample R
Locus insertion in sample L
Locus insertion in sample S
CYP2CF+R+Locus deletion in sample R
Locus deletion in sample L
Locus deletion in sample S
Locus insertion in sample R
Locus insertion in sample L
Locus insertion in sample S
“−” no fingerprint changes, “+” fingerprint changes.
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MDPI and ACS Style

Bernaś, E.; Słupski, J.; Gębczyński, P.; Ražná, K.; Žiarovská, J. Chemical Composition and Genome Pattern as a Means of Identifying the Origin of Preserved Wild Garlic (Allium ursinum L.) in Poland. Agriculture 2024, 14, 20. https://doi.org/10.3390/agriculture14010020

AMA Style

Bernaś E, Słupski J, Gębczyński P, Ražná K, Žiarovská J. Chemical Composition and Genome Pattern as a Means of Identifying the Origin of Preserved Wild Garlic (Allium ursinum L.) in Poland. Agriculture. 2024; 14(1):20. https://doi.org/10.3390/agriculture14010020

Chicago/Turabian Style

Bernaś, Emilia, Jacek Słupski, Piotr Gębczyński, Katarína Ražná, and Jana Žiarovská. 2024. "Chemical Composition and Genome Pattern as a Means of Identifying the Origin of Preserved Wild Garlic (Allium ursinum L.) in Poland" Agriculture 14, no. 1: 20. https://doi.org/10.3390/agriculture14010020

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