Changes in Amino acids, Minerals and Volatile Composition of Wild Garlicaccording to Forest Cultivation Environment

1. Overview of wild garlic samples and test strips

The wild garlic used in this experiment was from broad-leaf forests (Acer palmatum, Aesculus turbinata) and coniferous forests (Chamaecyparis obtusa, Pinus koraiensis) from Gyeongsang National University Jirisan Experimental Forest (located in Sancheong-gun, Gyeongsangnam-do).

Bare ground, not a forest, was used as a negative control for forestry cultivation (Fig. 1). The test sites were set with five different sites at 10 m × 10 m each: C. obtusa forest (CO), A. palmatum forest (AP), A. turbinata forest (AT), and the bare ground located in one place and P. koraiensis forest (PK), located 2.3 ㎞ away from the main test site.

Fig 1.

Five test strips of this study. A and E; experimental site locations in Korea, B; A. turbinata forest, C; P. koraiensis forest, and D; Bare ground. 1; A. turbinata, 2; C. obtusa, 3; Bare ground, 4; A. palmatum, 5; P. koraiensis.

The average elevation of the wild garlic plantation was 168 m (140 - 223 m), 77% (30% - 95%) of the tree layer, 16% (5% - 30%) of the shrub layer, and 35% (10% - 90%) of the herbaceous layer (Table 1). These sites were selected to investigate the effect of dominance species and canopy cover on the composition and amount of chemical compounds produced by wild garlic.

Table 1.

Location and vegetation table of five sites.

The soil chemical properties of experimental sites were analyzed according to National Institute of Agricultural Science (NIAS, 2010). Soil pH was highest in CO and AP soil, and lowest in PK soil (Table 2).

Table 2.

Soil chemical properties of experimental sites.

The amount of organic matters was highest in PK soil, and lowest in CO soil. In CO soil, total N content and cation exchange capacity were lower than those of other forests. AP soil exhibited high total N and available P. BG soil had high level of available P, whereas its cation exchange capacity was lower than that of other forests.

Wild garlic seeds were collected from the Jirisan Experimental Forest of Gyeongsang National University in July 2017. They were sown in forest soil under a greenhouse and grown for two years. In May 2020, each of the 200 seedlings were planted in five test sites. Before planting, all above-ground parts of the wild garlic in each site were collected, washed, and used for component analysis.

The analysis was also conducted in May of the following year to investigate the change in the chemical compound content of wild garlic. All parts above ground including leaves were freeze-dried at -72℃, ground, prepared into a uniform powder of 80 mesh, and then stored frozen at -20℃ for use in analysis.

2. Analysis of minerals

The analysis of mineral components was performed by pretreatment using the dry decomposition method of the Korea Food and Drug Administration (MFDS, 2022).

1 g of each of the above-ground freeze-dried wild garlic was placed in a 250 ㎖ flask, and 25 ㎖ of wet decomposition solution (HNO₃ : H₂SO₄ : HClO₄ = 10 : 1 : 4) was added and boiled in a heating mantle for over 3 hours. The residue and liquid were separated using Whatman filter paper, and the liquid was finally adjusted to 100 ㎖ using distilled water.

The treated samples were analyzed using an inductively coupled plasma spectrometer (ICP spectrometer, OPTIMA 4300DV, Perkin Elmer, Massachusetts, USA).

3. Free amino acid determination

Free amino acid analysis was performed by slightly modifying the method of Jeong et al. (2006).

To 3 g of wild garlic sample, 100 ㎖ of 75% Et-OH was added to homogenize, extracted by shaking at 50℃ for 2 hours, cooled to 4℃, and centrifuged at 6,000 rpm for 10 minutes. Then, The supernatant was concentrated using a rotary vacuum concentrator and adjusted to 25 ㎖ with distilled water. Then, sulfosalicylic acid (50 ㎎ per 1 ㎖ of sample) was added, allowed to stand for 1 hour, and centrifuged at 8,000 rpm for 10 minutes.

Then, the supernatant was filtered through a 0.22 ㎛ membrane filter and analyzed with an amino acid analyzer (LKB 4150 Alpha, LKB Ltd., Cambridge, England).

4. Volatile compound analysis

0.5 g of wild garlic sample was mixed with 1.5 ㎖ of tertiary distilled water and analyzed using a GC/MS (Clarus 600/Claus 600T, Perkin Elmer, Massachusetts, USA) equipped with headspace.

The initial oven temperature was 40℃ and maintained for 3 minutes, raised at 5℃/min to 40℃, raised at 10℃/min to 280℃, and maintained at 300℃ for 3 minutes. Helium was used as the carrier gas, the ion source temperature was 230℃, and the injector temperature was 250℃. Headspace conditions were oven temperature 120℃, transfer line temperature 125℃, and compound identification of wild garlic samples was confirmed by comparing NIST05S and Wiley7 Library.

5. Statistical analysis

Analysis of mineral, amino acid, and volatile compound was repeated at least three times and expressed as mean and standard deviation, and the results were analyzed by one-way ANOVA using the SPSS 20.0 software package (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA).

Means of regional ingredient compounds were compared at a 5% significance level using Duncan's Multiple Range Test (DMRT) comparisons. Means of the ingredient compounds before and after planting were compared using a t-test, also at a 5% significance level.

In addition, Pearson’s correlation analysis was conducted to investigate the effect of soil chemical properties on the content of minerals, amino acids, and volatile compounds of wild garlic.

1. Changes in mineral contents in forestry cultivation sites

The mineral composition of wild garlic leaves planted in five sites showed slight differences (Table 3). The most abundant mineral was potassium, followed by calcium and phosphorus. Among the five sites, the one with the most mineral content was CO forest, followed by PK and AT forest. The mineral composition changed after planting wild garlic (Table 3).

Table 3.

The mineral content of wild garlic cultivated in different experimental sites (㎎/g, dry weight).

The mineral content was compared between before planting and after planting to investigate the change in the content influenced by forestry cultivation sites (Fig. 2). After planting wild garlic, the content of minerals in the above-ground increased at five experimental sites. In the wild garlic cultivated in CO forest, K, Mn, and P content increased.

Fig. 2.

Mineral content before and after planting wild garlic at five planting sites. Bars indicate means of 3 replications and error bars indicate standard deviations. BP; before planting, AP: after planting, ns: not significantly different according to t-test.Signifcantly different according to t-test (*p < 0.05).

K, which showed the highest content among the mineral components, decreased in BG forest, while its content increased in CO, AP, AT, and PK forests (Fig. 2). Fe content increased in all of the cultivation areas except CO forest and BG, and Al content decreased only in CO forest cultivation areas. In particular, the P component increased in all cultivation environments.

2. Changes in free amino acid content in forest cultivation sites

The free amino acid content of wild garlic differed depending on the cultivation environment (Table 4).

Table 4.

The free amino acid content of wild garlic cultivated in different experimental sites (㎎/g, dry weight).

The most abundant free amino acid was urea, followed by L-asparagine, L-alanine, and L-leucine. Hydroxy-L-proline was detected in CO forest with high content, but it was not detected in the other sites. It was previously detected in AT and PK forest before planting, but not detected after planting. In AP forest, urea (5.03 ㎎/g), L-aspartic acid (2.10 ㎎/g), L-asparagine (3.84 ㎎/g), L-alanine (3.81 ㎎/g) and L-valine (3.09 ㎎/g) content increased, and amino acid content increased in wild garlic planted in AT forest included urea (13.14 ㎎/g), L-aspartic acid (6.03 ㎎/g), L-threonine (3.15 ㎎/g), and L-asparagine (2.38 ㎎/g). In BG, the content of urea (12.62 ㎎/g), L-threonine (3.65 ㎎/g), L-asparagine (3.12 ㎎/g), and L-glutamic acid (3.06 ㎎/g) increased. The urea, L-threonine, L-asparagine, and L-glutamic acid contents of wild garlic grown in PK forest were commonly increased.

The content of each free amino acid changed after wild garlic was planted in five different types of forest. In AP, BG, and PK forest, the content of over 24 kinds of free amino acid increased after planting: the content of 28, 24, 24 kinds of free amino acid increased in each forest. However, the content of only 14 kinds of free amino acid increased in CO forest. In AT forest, the number of free amino acid of which the content increased was less than that of CO forest. The content of urea, L-aspartic acid, L-threonine, L-asparagine, L-glutamic acid, γ- aminobutyric acid, and ethanolamine increased in AT forest and that of the other free amino acid decreased or was not detected.

3. Changes in volatile compound content in forestry cultivation sites.

20 volatile compounds were detected in wild garlic planted in five types of forest (Table 5). The most abundant volatile compound was dimethyl trisulfide, followed by disulfide, dimethyl disulfide, methyl 2-propenyl, and 2-hexenal.

Table 5.

Volatile compound components of wild garlic collected from planting sites.

The number of volatile compounds detected in wild garlic before planting remained unchanged after it was cultivated in the forests for a year (Table 6). However, in the case of single compounds, there was significant increase or decrease in the content between before and after planting of wild garlic. There was an increase in the content of some volatile compounds after cultivation in the forests. The content of 11, 8, 10, 8, and 4 volatile compounds increased in wild garlic planted in CO, AP, AT, BG, and PK, respectively.

Table 6.

The number of volatile compounds, the content of total volatile compounds, and the content of dimethyl trisulfide, the main compound, before and after planting wild garlic.

4. Relationship between soil properties and minerals, amino acids, and volatile composition

Soil properties of each experimental site affected minerals, amino acids, and volatile composition (Table 7). pH negatively affected mineral content of wild garlic except manganese. Calcium had positive relationships with total N and cation exchange capacity, whereas sodium, aluminium, iron, and zinc had negative relationships with exchangeable cations.

Table 7.

Pearson’s correlation analysis between soil properties and change in amino acids, minerals and volatile composition.

Amino acid content was strongly affected by pH, exchangeable Ca²⁺, and Mg²⁺. In contrast, total N slightly affected amino acid content, which had significant correlations with only 6 of 41 components. Among soil properties, available P and exchangeable Na⁺ had positive correlation with the content of almost all type of amino acid. However, some amino acid such as L-citrulline, L-α-amino iso-n-butyric acid, cystathionine, L-arginine had no significant correlations with any soil properties.

The volatile compound content was strongly affected by pH, exchangeable Mg²⁺, whereas available P and cation exchange capacity had little impact on volatile compound compositions: less than only 3 types of volatile compounds had significant correlations with these soil properties. Among volatile compound, disulfide, methyl 2-propenyl, disulfide, methyl 1-propenyl, and tetrasulfide had no significant correlations with any soil properties.

1. Effects of forest cultivation on the mineral content of wild garlic

Differences in mineral content were seen between before planting and after planting wild garlic. The most abundant minerals in wild garlic leaves are potassium, calcium, and phosphorus. Among the five regions, the region with the most mineral content was CO forest, followed by PK and AT forest.

It was confirmed that the results of this study were similar to the study by Kim et al. (2012), who reported that among the inorganic components of wild garlic, the potassium and calcium content was very high compared to other inorganic elements. However, the content of inorganic components in wild garlic slightly differed in each research report. Hong and Kim (2018) reported that among the inorganic components of wild garlic, the contents of potassium, calcium, magnesium, phosphorus, sulfur, iron, zinc, and sodium are 24,562.43 ㎎/㎏, 10,538.41 ㎎/㎏, 2,001.38 ㎎/㎏, and 1,656.38 ㎎/㎏, 1,485.71 ㎎/㎏, 203.69 ㎎/㎏, 41.10 ㎎/㎏, and 28.95 ㎎/㎏ respectively, which was said to have a higher content than daylily, slightly different from the content in this study.

Fig. 3.

Amino acid content before and after planting wild garlic at five planting sites. Bars indicate means of 3 replications and error bars indicate standard deviations. *Significantly different according to t-test (*p < 0.05).

A significant increase in mineral content was observed in the AT forest, despite its lower levels of available phosphorus and exchangeable cations (K⁺, Ca²⁺, Mg²⁺) compared to other sites. These results suggest that factors beyond soil mineral content, such as total nitrogen content, cation exchange capacity (CEC), and canopy coverage, may have collectively influenced nutrient uptake.

The AT forest exhibited higher CEC and total nitrogen content than other sites, which may have facilitated increased mineral retention and absorption. In nitrogen-rich soils, root growth is enhanced, leading to an expansion of surface area for nutrient uptake, thereby improving mineral absorption efficiency. Additionally, the organic matter content in the AT forest could have played a role in increasing mineral solubility and mobility (Robinson et al., 2005; Oney-Montalvo et al., 2020). Organic acids released during decomposition can enhance the availability of P and other cations.

Canopy structure and shading effects may have also contributed to the increase in mineral content. Wild garlic is sensitive to high temperatures, and shading helps regulate surface temperature, reducing transpiration-induced water stress. Trees with a higher leaf area index (LAI) reduce surface temperature more effectively than those with lower values (Armson et al., 2013). It seems that the trees in AT forest had the largest leaves and LAI among all sites, providing better shading and creating a more favorable microclimate for wild garlic growth.

These findings indicate that soil nitrogen content, CEC, and shading effects may have collectively led to the observed increase in mineral content. However, additional research is necessary to elucidate the precise mechanisms involved. Future studies should examine microbial activity, root structure, and long-term mineral dynamics to better understand these interactions. Additionally, experiments incorporating a broader pH range and controlled fertilizer applications would be beneficial in further investigating soil-plant interactions within forest cultivation systems.

2. Effects of forest cultivation on the free amino acid composition of wild garlic

The amino acid content of wild garlic varied depending on the cultivation environment. The most abundant amino acid in wild garlic was urea, followed by L-asparagine, L-alanine, and L-leucine. This trend was different from the previous studies. Cho et al. (2011) reported that wild garlic's total free amino acid content was 535.39 ㎎/100 g and among the 38 free amino acids isolated, asparagine, glutamic acid, and lysine were contained in high amounts. In addition, Park et al. (1998) reported that the highest free amino acid content in wild garlic were arginine, followed by glutamic acid, and asparagine.

In the CO forest, hydroxy-L-proline content significantly increased, which is a major component of hydroxyproline-rich glycoproteins (HRGPs) involved in cell wall strengthening and stress responses (MacLean et al., 2009; Deepak et al., 2010). This increase could be linked to the allelopathic effects of C. obtusa (Kato-Noguchi et al., 2024) and nitrogen-limited conditions. C. obtusa is known to release secondary metabolites that alter soil chemistry, and the low nitrogen content in the CO forest may have induced stress responses, leading to an increase in hydroxy-L-proline synthesis.

Amino acid synthesis in plants is influenced by soil mineral content. Deficiencies in iron (Fe) and zinc (Zn) have been shown to increase L-asparagine and glutamic acid synthesis, while copper (Cu) and manganese (Mn) deficiencies lead to an overall rise in amino acid levels (Possingham, 1956). Additionally, nitrogen (N) fertilization has been reported to increase arginine, serine, alanine, and γ-aminobutyric acid (Hoff et al., 1974).

In this study, changes in the content of various free amino acids including urea, L-asparagine, L-aspartic acid, γ-aminobutyric acid, and L-glutamic acid were observed, and nitrogen and phosphorus enrichment in AP forest and BG likely promoted amino acid synthesis. In contrast, the D,L-β-aminoisobutyric acid in the CO and PK forest suggests that nitrogen assimilation mechanisms may have been activated under nitrogen-limited conditions.

3. Effects of environmental factors on the volatile compound composition

Change in volatile compound composition in plants occurs in response to various environmental factors, such as soil nutrients (Chrysargyris et al., 2017), foliar diseases (Ponmurugan et al., 2016), and drought stress (Chandrasekaran et al., 2022).

The most abundant volatile compound in wild garlic planted at five sites was dimethyl trisulfide, disulfide, dimethyl, disulfide, methyl 2-propenyl, and 2-hexenal. This result also has some differences from previous studies. The volatile compounds of wild garlic shoot grown in Ulleung Island in Korea are known as dimethyl trisulfide (5.2% - 10.3%), diallyl disulfide (0.1% - 12.1%) and dimethyl pentasulfide (0% - 0.3%), which is somewhat different from this study (Yang et al., 2014). Park et al. (1998) reported that the main volatile compounds in wild garlic from Ulleung Island and Mt. Odae are 3-(methylthio)-I-propene and 2-vinyl-4H-1,3-dithin.

Additionally, in this study, there was no difference in the type and total content of volatile compounds between planting areas, but differences were seen in single compounds. This also is somewhat different from previous studies. Park et al. (1998) found that wild garlic from Mt. Odae or Mt. Jiri contained significantly higher amounts of sulfur compounds than that from Ulleung Island, which has oceanic geographical conditions. In addition, Yang et al. (2014) also reported differences in the types and total contents of volatile compounds between Ulleung Island regions. PCA analysis showed that the habitat of wild garlic plants significantly affected the proportion of alk(en)yl groups (methyl, m-propyl and allyl radicals) in bulb and shoot tissues.

In this study, the contents of amino acids and volatile compounds differed depending on the planting area. Since the study sites were not far apart, the metabolite differences were likely due to differences in upper vegetation. As for upper vegetation, fallen leaves, and compounds released during leaf decomposition enter the soil, affecting wild garlic's growth and metabolite biosynthesis.

Oney-Montalvo et al. (2020) also reported that the physical and chemical composition of the soil affects the profile and concentration of polyphenols, antioxidant activity, and maturity of habanero pepper (Capsicum chinense Jacq.). Additionally, three types of soil significantly affected the concentration of volatile compounds (1-hexanol, hexyl-3-methyl butanoate, 3,3-dimethyl-1-hexanol, and cis-3-hexenyl hexanoate) (Oney-Montalvo et al., 2022).

This study used forestry cultivation method for the efficient production of wild garlic, a useful wild vegetable and medicinal plant. The results indicated that the content of minerals, amino acids, and volatile compounds are affected by the cultivation environment for wild garlic. The mineral content was negatively correlated with pH, which indicates low pH could increase mineral content of wild garlic. High content of available P and exchangeable Na⁺ in soil led to increase in the content of most of amino acids. The contents of exchangeable cations were related to a decrease in mineral content, whereas amino acids with long retention time were positively correlated with them.

Soil pH influences nutrient availability in soil and nutrient uptake by plants (Barrow and Hartemink, 2023). Normally, low soil pH induces adsorption of anions and cations, reducing nutrient availability. However, uptake of some nutrients, such as phosphorus and sulfur, can increase with decreasing soil pH (Vange et al., 1974; Barrow and Hartemink, 2023). This could have increased the content of most minerals in wild garlic even in low soil pH in this study. However, since the difference in pH among test sites was small (5.0 - 5.4), sites with a wider pH range should be included in forestry cultivation to better determine the effect of soil pH on the mineral content of wild garlic.

Generally, plant can absorb more minerals from soil containing higher content of exchangeable cations. However, the test sites used in this study contained fewer exchangeable cations compared to its native habitat in Ulleung Island, which contained 2.1 ± 1.4 cmol_c․㎏^-1 Ca²⁺ and 1.0 ± 0.8 cmol_c․㎏^-1 Mg²⁺ (Hur et al., 2012). This led to a decrease in the content of Mg, Ca, Na in leaves after transplanted in almost all test sites, which could distort the results of correlation analysis in this study.

Therefore, soil improvement, such as fertilization and amendment, should be employed to enhance mineral uptake of wild garlic to obtain more accurate and reliable correlation analysis data, which will better reflect the relationship between soil mineral content and plant mineral absorption.

This study found that the content of minerals, amino acids, and volatile compounds differed depending on the environment of forest cultivation. However, this study showed very short-term results, so long-term monitoring is likely necessary. Additionally, analysis of other secondary metabolites (e.g. flavonoid) using liquid chromatography will further support these findings. The results of this study will contribute to a better understanding of the effects of environmental factors on compounds in wild garlic. This will enable the distribution and production of quality wild garlic seeds, as well as the standardization and distribution of other valuable plants in the future.