Changes in Phytochemical Compositions and Biological Activities After Fermentation According to the Harvest Periods of Mountain-Cultivated Ginseng Sprouts

Abstract

This study investigated how harvest timing within the growing season and lactic acid bacterial fermentation influence the phytochemical composition and biological activities of mountain-cultivated ginseng sprouts (MCGS). Various nutritional and bioactive constituents were examined, and in vitro assays were conducted before and after lactic acid bacterial fermentation. Although all samples were derived from 5-year-old plants grown under the same cultivation conditions, differences in harvest timing within the same season may be associated with progressive environmental variation rather than plant age. Nevertheless, harvest timing exerted a relatively limited effect on overall metabolite variation, whereas fermentation significantly enhanced functional properties across all harvest stages. Fermentation increased total phenolic content (4.27 → 7.21 mg/g), total flavonoid content (0.47 → 1.38 mg/g), and Maillard reaction products (2.02 → 2.84 OD_420nm), contributing to enhanced antioxidant capacity and increased inhibitory activities against pancreatic lipase and α-glucosidase. Notably, the levels of bioactive ginsenosides Rg3 and compound K increased markedly after fermentation (0.67 → 1.62 mg/g and 0.68 → 3.37 mg/g, respectively), despite a decrease in total ginsenoside content, indicating selective bioconversion during fermentation. Overall, these findings suggest that fermentation serves as the primary driver of functional enhancement in MCGS, while harvest timing within the growing season may play a secondary modulatory role.

1. Introduction

Ginseng (genus Panax) is a highly valued medicinal plant that has been cultivated and consumed for centuries, particularly in East Asia and North America. Owing to its recognized health-promoting properties and high commercial value, ginseng represents one of the most economically important herbal commodities in the global nutraceutical and functional food markets [1]. In addition to its traditional medicinal use, ginseng is widely incorporated into various industrial sectors, including functional foods, dietary supplements, beverages, cosmetics, and pharmaceutical preparations, underscoring its broad industrial applicability [1,2]. Among the various Panax species, Panax ginseng C.A. Meyer and P. quinquefolius (American ginseng) are the most widely used ginseng species in East Asia and North America, respectively, and both are valued for their extensive medicinal and nutritional benefits [2]. In East Asian countries, including Korea, China, and Japan, P. ginseng has long been utilized as a traditional herbal resource and functional ingredient, with diverse pharmacological activities reported, including antioxidant capacity, anti-inflammatory and anticancer properties, regulation of glucose metabolism, and immune-related functions [2,3].

Ginsenosides (triterpenoid saponins) are the major bioactive constituents of ginseng, along with phenolic acids, flavonoids, and polysaccharides, and serve as key quality markers for ginseng-based products [4]. Ginsenosides are generally classified into three groups based on their aglycone structures: protopanaxatriol (PPT) types, including Re, Rg1, Rg2, Rf, F1, and Rh1; protopanaxadiol (PPD) types, including Rd, Rb1, Rb2, Rc, Rg3, F2, and compound K (CK); and oleanane-type saponins, represented by ginsenoside Ro [5]. Among these ginsenosides, F2, Rg3, and CK have received considerable attention. F2 and Rg3 have been associated with hepatoprotective, antidiabetic, anticancer, anti-inflammatory, and cardiovascular protective effects, whereas CK has been highlighted for its high bioavailability and multifunctional properties, including immunomodulatory, neuroprotective, and antitumor activities [1,3,6,7]. Notably, CK is a microbial bioconversion product that is absent in fresh ginseng, and its high bioavailability has further increased interest in its pharmacological relevance [3,8].

Fermentation of mountain-cultivated ginseng sprouts (MCGS) using lactic acid bacteria (LAB) can enhance their functional properties by promoting the bioconversion of PPT-type into PPD-type ginsenosides [9]. In particular, previous research using various strains of LAB, including Lactobacillus, Leuconostoc, and Bifidobacterium species, has also demonstrated improved ginsenoside bioavailability through similar fermentation processes [10]. Beyond ginsenoside bioconversion, fermentation using LAB provides multiple biochemical advantages that contribute to the overall improvement of ginseng functionality [5]. Fermentation using LAB not only produces β-glucosidase but also promotes the formation of other hydrolytic enzymes such as esterases and proteases, thus facilitating the breakdown of complex macromolecules into bioavailable forms [11,12]. Moreover, the accumulation of organic acids during fermentation reduces the pH, thereby generating an acidic environment that promotes phenolic stability and may further accelerate Maillard-type reactions, resulting in the generation of antioxidant Maillard reaction products (MRPs) [13,14]. These synergistic effects collectively improve the digestibility, absorption, and physiological efficacy of fermented ginseng [13,15]. Additionally, previous studies have demonstrated that fermentation with LAB enhances antioxidant capacity in ginseng [14]. Notably, strain-specific differences have been reported in fermentation efficiency and metabolite conversion. Among these strains, Lactiplantibacillus plantarum P1201 and Levilactobacillus brevis BMK484 have previously demonstrated effective bioconversion of PPT-type to PPD-type ginsenosides and improvement of antioxidant properties in ginseng materials [2]. Therefore, these strains were selected in the present study to evaluate fermentation-driven functional enhancement in MCGS. Hence, although fermentation-driven bioconversion of ginsenosides and enrichment of bioactive compounds have been extensively recognized [5,16], the effect of seasonal and environmental variations during harvest on the metabolic profiles of MCGS remains poorly understood [2,17].

Based on previous reports indicating that ginsenoside levels in wild-simulated ginseng tend to fluctuate during the active growing season (May–July) [17], we hypothesized that harvest timing within this period, reflecting progressive environmental changes, may influence the baseline metabolite composition and functional potential of MCGS. Although all samples were obtained from 5-year-old plants, differences in collection timing within the same growing season may correspond to environmental variation and associated physiological and metabolic adjustments, which could in turn affect their responsiveness to lactic acid bacterial fermentation [17]. Accordingly, evaluating multiple harvest periods was considered essential to clarify whether fermentation-driven functional enhancement is modulated by harvest timing and environmental conditions rather than plant age. Therefore, the aim of this study was to investigate how harvest period and fermentation influence the phytochemical profile, antioxidant capacity, and enzyme inhibitory characteristics of MCGS. Specifically, MCGS samples harvested from May to July were compared to determine the impact of developmental stage on their functional attributes. Furthermore, the potential of fermentation to facilitate bioconversion and enhance the accumulation of bioactive constituents was examined to support the application of fermented and aged MCGS (FAMCGS) as a functional food ingredient.

2. Materials and Methods

2.1. Plant Materials, Strains, Reagents, and Instruments

2.1.1. Plant Materials and Strains

MCGS were harvested from PhilBio Co., Ltd. (Baekjeonmyeon, Hamyang, Republic of Korea) in 2020. Based on the general harvest period of mountain-cultivated ginseng aerial parts, the samples were collected at the following four harvest period points, 17 May, 31 May, 21 June, and 13 July 2020, to explore the effects of harvest timing. All samples were obtained from 5-year-old mountain-cultivated ginseng grown under the same cultivation conditions. The term “harvest period” in this study refers to different collection time points within the same growing season and represents harvest timing associated with seasonal environmental variation, rather than differences in plant age or developmental stage. For each harvest date, approximately 100 individual sprouts were collected and randomly divided into two equal groups (n = 50 each). One group was used as MCGS, whereas the other group was subjected to fermentation to produce FAMCGS. Each group per harvest date constituted one independent biological batch. After harvest, MCGS samples were thoroughly washed under running water, followed by drying at 50 °C for 48 h prior to subsequent analyses (Supplementary Figure S1). Two LAB strains, L. plantarum P1201 and L. brevis BMK484, originally isolated as described by Lee et al. [2], were employed as fermentation starters in this study. Each strain was maintained and propagated using Man, Rogosa, and Sharp (MRS) broth or agar medium (Difco, Becton Dickinson Co., Sparks, MD, USA).

2.1.2. Reagents and Instruments

Ginsenoside standards, including Rg1, Re, Ro, Rf, F3, Rg2, Rh1, Rb1, Rc, F1, Rb2, Rb3, Rd, Fd2, F2, Rg3, CK, and Rh2, were purchased from KOC Biotech (Daejeon, Korea). Folin–Ciocalteu phenol, diethylene glycol, DPPH, ABTS, 2,4,6-tri (2-pyridyl)-1,3,5-triazine (TPTZ), p-nitrophenyl-α-D-glucopyranoside (p-NPG), and p-nitrophenyl butyrate (p-NPB) were purchased from Sigma-Aldrich (St. Louis, MO, USA). High-performance liquid chromatography (HPLC)-grade H₂O, methanol, and acetonitrile were acquired from J.T. Baker (Phillipsburg, NJ, USA). pH and total acidity measurements were conducted using an Orion Star™ A211 pH Benchtop Meter (Thermo Fisher Scientific, Waltham, MA, USA). Fatty acid composition was analyzed by gas chromatography (GC, Agilent 7890A system, Agilent Technologies Inc., Wilmington, DE, USA). Amino acid composition was determined using an automatic amino acid analyzer (L-8900, Hitachi High-Technologies Corp., Tokyo, Japan). Mineral content was evaluated using a liquid chromatography–inductively coupled plasma mass spectrometer (NexION 350 ICP-MS, PerkinElmer Inc., Waltham, MA, USA). Ginsenoside content was determined by HPLC (Agilent 1260 system, Agilent Technologies Inc., Santa Clara, CA, USA). In addition, total phenolic content (TPC), total flavonoid content (TFC), browning index, antioxidant activities, and digestive enzyme inhibitory activities were measured using a UV–visible spectrophotometer (UV-1800 240 V, Shimadzu Corp., Kyoto, Japan). A rotary evaporator (Labconco, Kansas City, MO, USA) was used to concentrate the extracts, and the resulting solutions were filtered through a 0.45-μm membrane filter (Dismic-25CS, Toyoroshikaisha Ltd., Tokyo, Japan).

2.2. Bioprocessing of MCGS Using LAB

FAMCGS were produced through sequential steaming, aging, and lactic acid bacterial fermentation. Initially, washed MCGS were steamed with an appropriate amount of water at 100 °C for 1 h to obtain steamed samples. The steamed MCGS were subsequently transferred to an aging chamber and maintained at 75 °C for 72 h, and this steaming–aging cycle was repeated three times. Following aging, the samples were placed in a fermentation vessel, and distilled water corresponding to twice the sample weight was added to achieve hydration for 1 h, adjusting the moisture content to approximately 50%. After sterilization by autoclaving at 120 °C for 30 min, L. plantarum P1201 and L. brevis BMK484, previously precultured in 10% (w/v) malt medium at 30 °C for 48 h, were co-inoculated at a 1:1 ratio, with each strain added at 2.5% (v/v), resulting in a total inoculum of 5% (v/v). Fermentation was conducted at 30 °C for 120 h (Supplementary Figure S1). Upon completion of fermentation, the samples were dried at 55 °C for 48 h, pulverized into powder, and stored at −40 °C until further analysis. This bioprocessing protocol was adapted from the method described by Lee et al. [2] with minor modifications.

2.3. Determination of Physicochemical Properties and Viable Cell Numbers

pH and total acidity were determined according to a previously reported method by Jeong et al. [18], with minor modifications. For pH analysis, 0.5 g of MCGS or FAMCGS was mixed with 24.5 mL of distilled water, and the pH value was subsequently recorded. Total acidity was assessed by titrating the sample–water mixture with 0.1 N NaOH until a pH of 8.2 ± 0.1 was achieved. The volume of NaOH consumed was converted into lactic acid equivalent and expressed as a percentage (%) using the following equation:

lactic acid (%) = 0.9 × volume of 0.1 N NaOH (mL)/1 mL of sample.

(1)

Next, viable cell numbers were determined immediately after fermentation, before drying. Briefly, 5 g of FAMCGS was diluted in 45 mL of sterile distilled water, followed by serial dilutions. Appropriate dilutions were plated onto MRS agar and incubated at 30 °C for 48 h for colony development. The number of colonies formed was expressed as log CFU/g. All experiments were conducted in triplicate, and results were expressed as mean values.

2.4. Analysis of Fatty Acids (FAs)

FA analysis was performed following a previously reported protocol by Lee et al. [2], with minor modifications. All reagent volumes were proportionally scaled according to the reduced sample amount. For hydrolysis, 0.5 g of each sample was treated with 1.5 mL of 0.5 N methanolic NaOH and heated at 100 °C for 10 min using a heating block (Thermo Fisher Scientific, Rockford, IL, USA). Following cooling, 1 mL of 14% BF₃ in methanol was added, and the mixture was vortexed and reheated for 30 min to achieve fatty acid methylation. After the addition of isooctane (1 mL) and centrifugation, the supernatant was dehydrated, filtered, and subjected to GC analysis. Nitrogen served as the carrier gas at a flow rate of 1 mL/min, with an injection volume of 20 μL. The initial oven temperature was set at 140 °C and then increased at 4 °C/min to 230 °C and held for 35 min.

2.5. Analysis of Free Amino Acids (FAAs)

FAA analysis was performed according to a previously described protocol by Jeong et al. [18] with minor modifications. For analysis, 0.5 g of the centrifuged supernatant was combined with 2.5 mL of HPLC-grade H₂O and subjected to hydrolysis at 60 °C for 1 h. The hydrolysate was filtered using a glass filter, and the filtrate was concentrated under reduced pressure at 60 °C with a rotary evaporator. Then, the concentrate was reconstituted in 1 mL of sodium citrate buffer (pH 2.2), treated with 1 mL of 10% 5-sulfosalicylic acid dihydrate, and incubated for 2 h at 4 °C. The final solution was passed through a 0.45-μm membrane filter prior to analysis with an automatic amino acid analyzer.

2.6. Analysis of Minerals

Mineral content was determined following a previously reported protocol by Lee et al. [2], with minor modifications. For mineral determination, 0.5 g of MCGS or FAMCGS obtained at different harvest periods was treated with 10 mL of 70% nitric acid. The samples were digested by microwave-assisted decomposition, diluted to a final volume of 50 mL with triple-distilled water, filtered, and subsequently analyzed using inductively coupled plasma mass spectrometry.

2.7. Analysis of Ginsenosides

Ginsenoside profiling was carried out using HPLC according to a previously reported method by Lee et al. [2], with minor modifications. Briefly, 0.5 g of the sample was extracted twice with 10 mL of 50% methanol at 30 °C for 1 h each, followed by centrifugation. The resulting supernatants were combined and then filtered through a 0.45-μm membrane filter. The filtrate was concentrated under reduced pressure at 60 °C with a rotary evaporator prior to further analysis. The dried extract was reconstituted in 2 mL of HPLC-grade H₂O and filtered again through the 0.45-μm membrane filter. Chromatographic separation was achieved using a TSKgel ODS-100Z column (4.6 × 250 mm, 5 μm, Tosoh Corp., Tokyo, Japan). The mobile phase consisted of HPLC-grade H₂O (solvent A) and acetonitrile (solvent B), and the gradient program based on the percentage of solvent A was as follows: 0 min, 81%; 0–10 min, 81%; 15 min, 80%; 40 min, 77%; 42 min, 70%; 75 min, 65%; 80 min, 30%; and 90–100 min, 10%. The injection volume was 10 μL, and the column temperature was maintained at 30 °C. Detection was performed at 203 nm using a diode array detector (DAD, Agilent Technologies Inc., Santa Clara, CA, USA), with a flow rate of 1 mL/min. The wavelength of 203 nm was selected because ginsenosides exhibit strong absorbance in the low-UV region (approximately 198–205 nm), enabling sensitive and simultaneous detection of multiple ginsenosides, as widely reported in previous chromatographic analyses of ginsenosides [5]. Under these conditions, comprehensive profiling of 21 ginsenosides was achieved in the present study.

2.8. Analysis of TPC, TFC, and MRPs

TPC, TFC, and MRPs were analyzed according to a previously reported method by Lee et al. [2], with minor modifications. For extraction, each sample was treated with 20 volumes of 50% methanol and maintained at 25 °C ± 1 °C for 24 h. The extract was then filtered through a 0.45-μm membrane filter to obtain the supernatant. TPC was quantified by mixing 0.5 mL of the diluted extract with 0.25 mL of Folin–Ciocalteu reagent and 0.5 mL of 25% Na₂CO₃ solution. This mixture was incubated for 1 h at 30 °C ± 1 °C, after which the absorbance was measured at 750 nm using a UV–Vis spectrophotometer. TFC was determined by mixing 0.5 mL of the diluted extract with 1.0 mL of diethylene glycol and 0.1 mL of 1 N NaOH. This mixture was incubated for 1 h at 37 °C ± 1 °C, after which the absorbance was measured at 420 nm using a UV–Vis spectrophotometer. MRP levels, reflecting nonenzymatic browning, were assessed by recording the absorbance of the extract at 420 nm using a UV–Vis spectrophotometer.

2.9. Analysis of Antioxidant and Enzyme Inhibitory Activities

2.9.1. DPPH Radical Scavenging Activity

DPPH radical scavenging activity was assessed following a previously reported method by Jeong et al. [18], with slight modifications. For the assay, 1.2 mL of DPPH solution (0.4 mM in methanol) was mixed with 0.3 mL of the centrifuged extract and allowed to react in the dark for 30 min prior to absorbance measurement at 525 nm.

2.9.2. ABTS Radical Scavenging Activity

ABTS radical scavenging activities were determined using a modified version of the method described by Jeong et al. [18]. The ABTS solution was generated by combining 7.4 mM ABTS with 2.6 mM potassium persulfate (1:3, total volume 10 mL) and incubating the mixture in the dark for 12 h. The resulting ABTS solution was diluted with methanol to an absorbance of 0.8 ± 0.1 at 732 nm. Subsequently, the ABTS solution was mixed with the extract at a ratio of 9:1 (total volume 1 mL), and absorbance was recorded at 732 nm after 3 min.

2.9.3. Hydroxyl Radical (∙OH) Scavenging Activity

·OH scavenging activity was evaluated following a previously reported method by Jeong et al. [18] with slight modifications. For the assay, the sample (1400 μL) was reacted with 10 mM FeSO₄–EDTA, 10 mM 2-deoxyribose, and 10 mM H₂O₂ (200 μL each) and incubated at 37 °C for 4 h. Following the addition of thiobarbituric acid (1%) and trichloroacetic acid (2.5%), the mixture was heated at 100 °C for 10 min, and absorbance was recorded at 520 nm.

2.9.4. FRAP Assay

FRAP was assessed according to a previously described procedure by Jeong et al. [18] with minor modifications. This assay was conducted using a reagent mixture composed of acetate buffer (30 mM, pH 3.6), TPTZ solution (10 mM in 40 mM HCl), and 20 mM FeCl₃ at a ratio of 10:1:1 (v/v/v). The sample (50 μL) was combined with FRAP reagent (950 μL), incubated at 37 °C for 15 min, and absorbance was subsequently measured at 590 nm.

2.9.5. α-Glucosidase Inhibitory Activity

Inhibitory activity against α-glucosidase and pancreatic lipase was evaluated following a previously reported protocol by Lee et al. [19] with slight modifications. For the assay, 50 μL of 200 mM sodium phosphate buffer (pH 6.8) was combined with 70 μL of α-glucosidase solution (1 U/mL) and 30 μL of the sample. Following preincubation at 37 °C for 10 min, 100 μL of 10 mM p-NPG prepared in the same buffer was added, and the reaction mixture was further incubated at 37 °C for 10 min. The reaction was terminated by the addition of 750 μL of 100 mM Na₂CO₃, and absorbance was recorded at 420 nm.

2.9.6. Pancreatic Lipase Inhibitory Activity

Pancreatic lipase inhibitory activity was evaluated following the same experimental framework applied to the α-glucosidase inhibition assay, with pancreatic lipase used as the enzyme and 10 mM p-NPB employed as the substrate. For both enzymatic assays, the negative control consisted of the corresponding extraction solvent in place of the sample. Enzyme inhibition was calculated according to the following equation:

enzyme inhibition (%) = [1 − (Abs_sample/Abs_control)] × 100.

(2)

2.10. Statistical Analysis

All experiments were performed independently in triplicate (n = 3), and results are presented as mean ± standard deviation (SD). Significant differences among groups were assessed by one-way ANOVA followed by Tukey’s HSD test (p < 0.05) using SAS software (version 9.4; SAS Institute, Cary, NC, USA). Multivariate analyses, including principal component analysis (PCA) and heatmap visualization, were conducted using R software (version 4.3.3; R Core Team, 2023).

3. Results and Discussion

3.1. Comparison of Physicochemical Properties and Viable Cell Numbers of MCGS Before and After Fermentation According to Harvest Periods

Table 1. Comparisons of physicochemical properties of raw material and fermentation according to the harvest periods of mountain-cultivated ginseng sprouts.

Analysis Item 1	Harvest Periods
	17-May		31-May		21-June		13-July
	MCGS 2	FAMCGS	MCGS	FAMCGS	MCGS	FAMCGS	MCGS	FAMCGS
pH	5.81 ± 0.05 a	4.22 ± 0.01 b	5.78 ± 0.04 a	4.20 ± 0.05 b	5.80 ± 0.01 a	4.23 ± 0.02 b	5.83 ± 0.01 a	4.11 ± 0.03 b
Acidity (%, as lactic acid)	1.52 ± 0.01 c	3.15 ± 0.02 b	1.58 ± 0.02 c	3.79 ± 0.01 a	1.53 ± 0.00 c	3.78 ± 0.01 a	1.52 ± 0.01 c	3.85 ± 0.01 a
Viable cell number (log CFU/g)	nm 3	10.46 ± 0.15 a	nm	10.55 ± 0.21 a	nm	10.12 ± 0.11 b	nm	10.07 ± 0.15 b

¹ Data are expressed as the mean ± SD, and differences are analyzed using Tukey’s test, n = 3. The results without common superscript letters (a to c) within the same row are statistically different (p < 0.05). ² Abbreviation: MCGS, mountain-cultivated ginseng sprouts and FAMCGS, fermented aging mountain-cultivated ginseng sprouts. ³ nm: not measured because viable cell counts are determined only for fermented samples.

shows the physicochemical properties of MCGS and FAMCGS according to harvest periods. The pH of MCGS exhibited no significant difference across the harvest periods (pH 5.78–5.83). However, the pH of FAMCGS was significantly lower than that of MCGS due to fermentation, although there was no significant difference according to the harvest periods (pH 4.11–4.23). The acidity of FAMCGS ranged from 3.15% to 3.85%, showing a sharp increase compared to that of MCGS. The viable cell number revealed no significant variation according to the harvest periods.

Fermentation of MCGS resulted in a significant reduction in pH and a marked increase in acidity, indicating active LAB metabolism [20]. Such changes are generally detected during fermentation by LAB and contribute to improved microbial safety and extended shelf-life [21]. The consistently high viable cell number further suggests that beneficial microorganisms remain abundant after fermentation, thereby improving the functional value of the product [22]. Notably, the physicochemical characteristics showed no significant differences according to harvest period, consistent with previous findings reported for various medicinal plant materials [23]. Comparable patterns have also been reported in other fermented products, suggesting that fermentation plays a key role in determining final physicochemical characteristics and product quality [5,24].

3.2. Comparison of Fatty Acid Profiles of MCGS Before and After Fermentation According to Harvest Periods

The fatty acid (FA) profiles of MCGS and FAMCGS according to harvest periods are demonstrated in

Table 2. Comparisons of fatty acid compositions of raw material and fermentation according to the harvest periods of mountain-cultivated ginseng sprouts.

Contents 1 (mg/100 g)	Harvest Periods
	17-May		31-May		21-June		13-July
	MCGS 2	FAMCGS	MCGS	FAMCGS	MCGS	FAMCGS	MCGS	FAMCGS
Saturated Fatty Acids
Lauric acid (C12:0)	5.82 ± 0.05 b	8.03 ± 0.01 a	3.15 ± 0.02 c	7.24 ± 0.09 a	3.16 ± 0.03 c	2.12 ± 0.01 d	nd	3.12 ± 0.01 c
Myristic acid (C14:0)	5.51 ± 0.05 b	6.82 ± 0.02 a	4.22 ± 0.02 c	6.62 ± 0.02 a	2.85 ± 0.02 d	4.71 ± 0.00 c	4.41 ± 0.04 c	5.71 ± 0.01 b
Palmitic acid (C16:0)	120.03 ± 2.52 c	146.24 ± 2.52 b	147.44 ± 1.65 b	126.13 ± 2.51 c	110.26 ± 2.41 d	162.71 ± 3.51 a	141.17 ± 1.21 b	124.80 ± 2.41 c
Stearic acid (C18:0)	26.64 ± 0.21 d	41.93 ± 0.41 b	49.17 ± 0.51 b	37.54 ± 0.32 c	37.51 ± 0.31 c	64.44 ± 1.25 a	64.05 ± 2.11 a	44.32 ± 0.22 b
Arachidic acid (C20:0)	3.71 ± 0.03 c	3.43 ± 0.03 d	3.32 ± 0.03 d	3.21 ± 0.01 d	3.04 ± 0.03 d	4.71 ± 0.01 b	5.12 ± 0.01 a	4.61 ± 0.01 b
Behenic acid (C22:0)	7.45 ± 0.08 a	6.63 ± 0.06 b	5.91 ± 0.06 c	5.82 ± 0.01 c	4.95 ± 0.02 d	6.02 ± 0.00 c	6.70 ± 0.01 b	6.31 ± 0.02 b
Tricosanoic acid (C23:0)	2.63 ± 0.01 a	2.12 ± 0.01 b	nd	nd	nd	nd	nd	nd
Lignoceric acid (C24:0)	5.32 ± 0.01 a	4.52 ± 0.04 b	4.36 ± 0.02 c	3.71 ± 0.01 d	3.02 ± 0.01 d	4.56 ± 0.01 b	3.61 ± 0.01 d	4.14 ± 0.00 c
Total	177.11	219.72	217.57	190.27	164.79	249.27	225.06	193.01
Unsaturated fatty acids
Palmitoleic acid (C16:1n7)	nd 3	nd	nd	nd	nd	3.80 ± 0.01 a	nd	2.32 ± 0.01 b
Oleic acid (C18:1n9c)	27.41 ± 0.02 d	32.41 ± 0.01 c	32.91 ± 0.31 c	39.32 ± 0.25 b	34.02 ± 0.01 c	42.22 ± 0.04 b	61.81 ± 1.21 a	59.53 ± 1.21 a
Linoleic acid (C18:2n6c)	221.34 ± 5.01 a	226.91 ± 2.25 a	200.02 ± 4.21 c	227.43 ± 5.26 a	192.90 ± 3.51 d	210.11 ± 4.52 b	192.72 ± 2.25 d	223.70 ± 4.21 a
γ-Linolenic acid (C18:3n6)	nd	nd	nd	nd	2.37 ± 0.01 a	nd	nd	nd
α-Linolenic acid (C18:3n3)	95.54 ± 1.02 b	130.41 ± 1.25 a	103.04 ± 1.21 a	93.60 ± 11.06 b	47.93 ± 0.61 d	75.61 ± 1.42 c	22.41 ± 0.41 d	37.32 ± 0.65 d
Eicosadienoic acid (C20:2)	2.41 ± 0.02 a	2.22 ± 0.02 b	2.33 ± 0.02 a	2.20 ± 0.01 b	nd	1.76 ± 0.00 c	nd	1.71 ± 0.00 c
Arachidonic acid (C20:4n6)	2.67 ± 0.01 a	2.01 ± 0.01 c	2.14 ± 0.01 b	2.01 ± 0.01 c	1.62 ± 0.01 d	2.75 ± 0.00 a	1.84 ± 0.01 d	2.33 ± 0.01 b
Total	349.37	393.96	340.44	364.56	278.84	336.25	278.78	326.91
Total fatty acids	526.48	613.68	558.01	554.83	443.63	585.52	503.84	519.92

¹ Data are expressed as the mean ± SD, and differences are analyzed using Tukey’s test, n = 3. The results without common superscript letters (a to d) within the same row are statistically different (p < 0.05). ² Abbreviation: MCGS, mountain-cultivated ginseng sprouts and FAMCGS, fermented aging mountain-cultivated ginseng sprouts. ³ nd: not detected.

and Figure 1. Eight saturated fatty acids (SFAs) and seven unsaturated fatty acids (UFAs) were detected. In the samples harvested on 17 May and 21 June, the SFA content increased approximately 1.24- and 1.51-fold, respectively, after the fermentation of MCGS to FAMCGS. However, the SFA content exhibited a decreasing trend when MCGS harvested on 31 May and 13 July were subjected to fermentation to produce FAMCGS. The UFA contents increased at all harvest periods when MCGS was fermented to FAMCGS (17 May: from 349.37 to 393.96 mg/100 g; 31 May: from 340.44 to 364.56 mg/100 g; 21 June: from 278.84 to 336.25 mg/100 g; 13 July: from 278.78 to 326.91 mg/100 g). As presented in the PCA plots, PC1 primarily reflected fermentation effects, clearly separating MCGS and FAMCGS, and PC2 captured harvest–time variations, with partial clustering detected across different harvest periods. The variance explained by PC1 and PC2 was 47.63% and 29.52% in the UFA dataset and 45.80% and 27.10% in the SFA dataset, respectively (Figure 1A,B). These trends were further confirmed by the heatmap, which showed UFA-enriched clusters in FAMCGS and SFA-enriched clusters in MCGS, with specific FAs such as tricosanoic acid (unique to MCGS and FAMCGS harvested on 17 May), palmitoleic acid (FAMCGS harvested on 21 June and 13 July), and γ-linolenic acid (MCGS harvested on 21 June) driving the differentiation and reinforcing the PCA findings (Figure 1C,D).

Environmental variation during the active growing period (May–July), including changes in temperature, light availability, and other seasonal factors, is known to influence photosynthetic activity, carbohydrate allocation, and secondary metabolite accumulation in ginseng aerial tissues [17], potentially contributing to temporal variation in phytochemical composition. Such harvest-period-associated environmental and physiological variation may therefore partly explain the observed differences in fatty acid profiles, ginsenoside content, and functional activities among the different harvest periods in the present study. Nevertheless, the magnitude of harvest-period-associated variation appeared limited when compared with the effects of fermentation. Lee et al. [2] also reported no significant differences in FA composition across harvest periods, a finding that is consistent with our study, showing that various factors, particularly fermentation, rather than harvest period, were the primary drivers of variation [13]. Fermentation exerted a significant effect on the FA composition of MCGS, resulting in an overall increase in UFA content across all harvest periods, which is consistent with the trends reported by Glenn-Davi et al. [25]. LAB produce lipolytic enzymes such as lipases and esterases, which hydrolyze complex lipids and release FAs, and desaturase-mediated modifications may contribute to the observed enrichment of UFAs [14,26]. Furthermore, β-oxidation and chain-elongation processes could explain the selective appearance or disappearance of certain long-chain fatty acids, such as tricosanoic acid and γ-linolenic acid, across harvest periods and fermentation stages [27]. Interestingly, these FAs were detected only under specific harvest or processing conditions, indicating that both harvest period and fermentation conditions contribute to shaping FA profile diversity [28,29]. Additionally, the observed increase in unsaturated fatty acids (UFAs) in FAMCGS may be nutritionally relevant, as UFAs have been associated with cardiovascular protection and anti-inflammatory properties [25]. Altogether, the enrichment of UFAs suggests that FAMCGS may offer enhanced nutritional quality and potential health benefits.

3.3. Comparison of Free Amino Acid Profiles of MCGS Before and After Fermentation According to Harvest Periods

The free amino acid (FAA) profiles of MCGS and FAMCGS according to harvest periods are presented in Supplementary Table S1 and Figure 2. A total of 23 nonessential amino acids (NEAAs) and 8 essential amino acids (EAAs) were detected. Among the MCGS samples, the total NEAA content was the highest in the harvest of 17 May (720.44 mg/100 g), followed by that of 13 July (563.61 mg/100 g), 31 May (500.68 mg/100 g), and 21 June (455.67 mg/100 g). The most abundant NEAA was arginine, whose content was 149.32–200.59 mg/100 g. Similarly, the EAA content followed the same harvest-dependent trend as the NEAAs, with the highest level observed in MCGS harvested on 17 May (307.26 mg/100 g). Among the EAAs, valine exhibited the highest levels of 23.05–61.38 mg/100 g. In contrast, the FAMCGS samples showed generally decreased NEAA and EAA contents across all harvest periods. As presented in the PCA plots, PC1 (75.71% for NEAAs and 68.42% for EAAs) primarily reflected fermentation effects, clearly separating MCGS and FAMCGS, and PC2 (12.37% for NEAAs and 14.89% for EAAs) captured harvest–time variations, with the MCGS sample harvested on 17 May clustering separately due to its generally higher amino acid levels (Figure 2A,B). Consistently, these patterns were confirmed by the heatmaps, highlighting elevated signals in early-harvested MCGS and reduced levels in FAMCGS, thus emphasizing the PCA findings (Figure 2C,D).

Distinct variations in FAA profiles were observed depending on harvest period and fermentation, with early-harvested MCGS (17 May) showing the highest NEAA and EAA levels. Importantly, all samples were obtained from 5-year-old plants harvested within the same growing season; therefore, differences among harvest periods reflect collection timing within the season, representing progressive environmental variation rather than differences in plant maturity. The higher FAA levels observed in samples harvested earlier in the season may be associated with active nitrogen metabolism and protein biosynthesis during the early growing phase [30,31]. The overall reduction in FAA levels in FAMCGS across all harvest periods suggests microbial utilization during fermentation [32]. Similarly, decreased FAA levels after fermentation have been reported previously [33], showing a comparable trend to that observed in the present study. The reduction in the content of FAAs may also be partially attributed to their participation in Maillard reactions during fermentation, where amino acids react with reducing sugars to form MRPs [34]. LABs that are used commonly in fermentation metabolize amino acids such as arginine and valine for growth and acid resistance, potentially resulting in decreased levels in the final product [35]. These results emphasize the potential of early-harvested MCGS as a nutritionally advantageous raw material for fermentation-based products. Nevertheless, the observed reduction in major amino acid levels after fermentation suggests that amino acids may not represent the primary determinants of the commercial value of ginseng [13].

3.4. Comparison of Mineral Compositions of MCGS Before and After Fermentation According to Harvest Periods

The mineral compositions of MCGS and FAMCGS according to harvest periods are illustrated in Supplementary Table S2 and Figure 3. A total of 11 minerals were detected. Potassium (K) and calcium (Ca) accounted for approximately 41.89% and 26.73% of the total mineral content of MCGS and FAMCGS, respectively, indicating the highest proportions. Conversely, zinc (Zn) (0.01–0.02 mg/100 g), manganese (Mn) (0.03–0.06 mg/100 g), and titanium (Ti) (0.03–0.06 mg/100 g) exhibited the lowest levels among all minerals detected. The total mineral content of MCGS harvested on 17 May and 13 July increased after fermentation (from 30.92 to 31.03 and from 23.13 to 24.48 mg/100 g, respectively), indicating a harvest-dependent response to fermentation. PC1 (53.67%) primarily reflected fermentation effects, clearly separating MCGS and FAMCGS, and PC2 (18.81%) accounted for minor harvest–time differences (Figure 3A). The heatmap further confirmed these patterns, demonstrating that mineral distributions clustered primarily by fermentation treatment, with FAMCGS enriched in K, Ca, S, and Al compared with MCGS (Figure 3B).

Phosphorus (P) and Ca were found to be the most abundant minerals in both MCGS and FAMCGS, which is consistent with their vital roles in plant physiology and human health [36]. In the present study, the relatively high levels of Ca, P, and magnesium (Mg) detected in FAMCGS are remarkable because these minerals contribute to bone health, fluid and electrolyte balance, muscle and nerve function, and metabolic regulation [37]. In contrast to our findings, Kim et al. [38] reported P and sulfur (S) as the predominant mineral components in ginseng roots, whereas Jiang et al. [39] and Cho et al. [40] identified K and Ca as the most abundant minerals, which is consistent with our findings. These discrepancies may be attributed to differences in the examined plant parts, as Kim et al. [38] focused on root tissues, whereas Cho et al. [40] examined whole plant materials, similar to the present study, which included both aerial and root tissues, along with potential influences of cultivation conditions and processing methods. Overall, these results suggest that FAMCGS provide not only phytochemical advantages but also valuable dietary mineral supplementation, thereby improving their utility as a functional food ingredient [2].

3.5. Comparison of Ginsenoside Contents of MCGS Before and After Fermentation According to Harvest Periods

The ginsenoside profiles of MCGS and FAMCGS according to harvest periods are shown in Figure 4 and Figure 5 and

Table 3. Comparisons of ginsenoside compositions of raw material and fermentation according to the harvest periods of mountain-cultivated ginseng sprouts.

Contents 1 (mg/g)	Harvest Periods
	17-May		31-May		21-June		13-July
	MCGS 2	FAMCGS	MCGS	FAMCGS	MCGS	FAMCGS	MCGS	FAMCGS
Protopanaxtriol types
Ginsenoside Rg1	3.32 ± 0.03 a	0.71 ± 0.00 c	3.58 ± 0.03 a	0.48 ± 0.01 d	2.48 ± 0.02 b	0.46 ± 0.00 d	2.87 ± 0.02 b	0.50 ± 0.00 d
Ginsenoside Re	9.89 ± 0.09 a	1.66 ± 0.01 d	7.95 ± 0.05 b	0.92 ± 0.02 e	7.45 ± 0.07 b	0.86 ± 0.00 e	3.72 ± 0.03 c	0.82 ± 0.00 e
Ginsenoside Rf	0.64 ± 0.00 a	0.58 ± 0.00 b	0.57 ± 0.00 b	0.46 ± 0.01 c	0.52 ± 0.00 b	0.40 ± 0.00 d	0.49 ± 0.00 c	0.45 ± 0.00 c
Ginsenoside F5	0.87 ± 0.00 a	0.32 ± 0.00 c	0.74 ± 0.00 a	0.20 ± 0.01 d	0.59 ± 0.00 b	0.17 ± 0.00 d	0.35 ± 0.03 c	0.13 ± 0.00 d
Ginsenoside F3	2.95 ± 0.02 a	0.80 ± 0.00 c	2.15 ± 0.02 b	0.36 ± 0.00 d	1.82 ± 0.01 b	0.44 ± 0.00 d	1.06 ± 0.01 c	0.26 ± 0.00 d
Ginsenoside Rg2	0.98 ± 0.01 c	2.46 ± 0.01 a	0.91 ± 0.00 c	1.59 ± 0.01 b	0.64 ± 0.01 d	1.52 ± 0.01 b	0.48 ± 0.00 d	1.23 ± 0.01 c
Ginsenoside Rh1	0.44 ± 0.00 c	1.26 ± 0.01 a	0.53 ± 0.00 c	0.88 ± 0.00 b	0.44 ± 0.01 c	0.86 ± 0.00 b	0.34 ± 0.00 d	0.72 ± 0.00 b
Ginsenoside F1	1.19 ± 0.01 a	0.61 ± 0.00 c	0.77 ± 0.00 b	0.35 ± 0.00 d	0.72 ± 0.04 b	0.35 ± 0.01 d	0.53 ± 0.02 c	0.26 ± 0.00 d
Protopanaxtriol	1.07 ± 0.01 c	2.70 ± 0.02 a	2.25 ± 0.01 b	2.24 ± 0.01 b	0.36 ± 0.01 d	1.63 ± 0.01 c	0.25 ± 0.01 d	1.21 ± 0.01 c
Total	21.35	11.10	19.45	7.48	15.02	6.69	10.09	5.58
Protopanaxdiol types
Ginsenoside Rb1	6.49 ± 0.06 a	5.03 ± 0.05 b	6.56 ± 0.05 a	4.09 ± 0.04 c	5.52 ± 0.05 b	3.24 ± 0.03 d	5.39 ± 0.05 b	3.45 ± 0.03 d
Ginsenoside Rc	2.93 ± 0.02 a	1.66 ± 0.01 c	2.97 ± 0.02 a	1.26 ± 0.01 c	2.61 ± 0.02 b	1.06 ± 0.01 d	2.29 ± 0.02 b	1.03 ± 0.01 d
Ginsenoside Rb2	3.08 ± 0.03 a	1.83 ± 0.01 c	2.85 ± 0.03 b	1.34 ± 0.01 c	2.41 ± 0.01 b	0.97 ± 0.00 d	2.07 ± 0.02 c	0.89 ± 0.00 d
Ginsenoside Rb3	0.42 ± 0.00 b	0.34 ± 0.00 c	0.36 ± 0.00 c	0.28 ± 0.00 d	0.53 ± 0.02 a	nd 3	0.37 ± 0.00 c	nd
Ginsenoside Rd	6.39 ± 0.02 a	5.48 ± 0.04 b	5.89 ± 0.02 b	4.10 ± 0.01 c	4.97 ± 0.01 c	3.84 ± 0.03 d	3.27 ± 0.01 d	2.76 ± 0.01 e
Ginsenoside Rd2	4.08 ± 0.04 a	2.75 ± 0.02 d	4.72 ± 0.04 a	2.22 ± 0.01 d	3.78 ± 0.03 b	2.19 ± 0.02 d	1.73 ± 0.01 e	1.48 ± 0.01 e
Ginsenoside F2	4.31 ± 0.04 b	6.89 ± 0.06 a	2.25 ± 0.02 c	4.72 ± 0.04 b	2.39 ± 0.01 c	4.31 ± 0.04 b	1.65 ± 0.01 d	4.27 ± 0.04 b
Ginsenoside Rg3	0.38 ± 0.00 d	1.84 ± 0.01 a	1.52 ± 0.01 b	1.73 ± 0.01 a	0.45 ± 0.00 c	1.54 ± 0.01 b	0.39 ± 0.00 d	1.36 ± 0.01 b
Compound K	0.74 ± 0.00 d	4.00 ± 0.04 a	0.69 ± 0.01 d	3.52 ± 0.03 b	0.85 ± 0.00 d	3.11 ± 0.03 b	0.45 ± 0.00 e	2.84 ± 0.01 c
Ginsenoside Rh2	0.45 ± 0.00 b	0.37 ± 0.00 c	0.35 ± 0.00 c	0.32 ± 0.00 c	0.53 ± 0.00 a	0.59 ± 0.00 a	0.15 ± 0.00 d	0.25 ± 0.00 d
Protopanaxdiol	0.83 ± 0.00 b	1.09 ± 0.01 a	1.04 ± 0.01 a	0.89 ± 0.00 b	1.07 ± 0.00 a	0.84 ± 0.00 b	0.44 ± 0.00 d	0.47 ± 0.00 d
Total	30.10	31.28	29.20	24.47	25.11	21.69	18.20	18.80
Oleanane types
Ginsenoside Ro (3)	1.91 ± 0.01 b	3.01 ± 0.01 a	1.00 ± 0.01 c	2.23 ± 0.02 b	1.27 ± 0.01 c	2.02 ± 0.02 b	0.70 ± 0.00 d	2.04 ± 0.02 b
Total ginsenosides	53.36	45.39	49.65	34.18	41.40	30.40	28.99	26.42

¹ Data are expressed as the mean ± SD, and differences are analyzed using Tukey’s test, n = 3. The results without common superscript letters (a to e) within the same row are statistically different (p < 0.05). ² Abbreviation: MCGS, mountain-cultivated ginseng sprouts and FAMCGS, fermented aging mountain-cultivated ginseng sprouts. ³ nd: not detected.

. A total of 21 ginsenosides were detected, comprising 8 PPT-type, 11 PPD-type, and 1 oleanane-type compounds. Overall, most ginsenoside compounds exhibited a decreasing trend in their levels after fermentation from MCGS to FAMCGS. Among the PPT-type ginsenosides, Rg2 (peak 7) and Rh1 (peak 8) showed a slight increase in their levels after fermentation (Figure 4). In the harvest of 17 May, the levels of Rg2 and Rh1 were 2.46 and 1.26 mg/g, respectively, which increased approximately 2.51- and 2.86-fold after fermentation, respectively. Among the PPD-type ginsenosides, F2 (peak 16), Rg3 (peak 17), and CK (peak 19) showed increased levels after fermentation. Specifically, Rg3 levels increased approximately 1.60- to 2.59-fold in FAMCGS compared with those in MCGS across all harvest periods. F2 exhibited the highest content in FAMCGS harvested on 17 May (6.89 mg/g), followed by that on 31 May (4.72 mg/g), 21 June (4.31 mg/g), and 13 July (4.27 mg/g). CK exhibited marked increased levels, approximately 3.66- to 6.31-fold after fermentation, showing the highest level in FAMCGS harvested on 17 May (4.00 mg/g), followed by that on 31 May (3.52 mg/g), 21 June (3.11 mg/g), and 13 July (2.84 mg/g). PC1 (59.52%) primarily captured fermentation-driven changes in composition, separating FAMCGS toward the positive and MCGS toward the negative side of PC1. PC2 (25.01%) explained harvest-related variations, with samples harvested on 17 May clustering higher than others (Figure 5A). The heatmap similarly demonstrated enrichment of Rg3 and CK in FAMCGS clusters, whereas Rb1, Re, and Rg1 were enriched in MCGS clusters, clearly illustrating the PPT-to-PPD conversion pattern highlighted in the PCA plots (Figure 5B).

The inclusion of multiple harvest periods allowed us to distinguish baseline developmental effects from fermentation-driven changes. Although fermentation exerted a more pronounced impact on overall functional enhancement, the higher total ginsenoside levels observed in early-harvested MCGS suggest that harvest timing influences the initial ginsenoside profile available for microbial biotransformation. Notably, both MCGS and FAMCGS harvested on 17 May exhibited the highest total and minor ginsenoside levels, respectively, suggesting that sprouts harvested on 17 May (the earliest collection point evaluated in the present study) provide physicochemical conditions favorable for ginsenoside accumulation and subsequent microbial biotransformation, consistent with previous observations [17]. Although the total ginsenoside contents decreased after fermentation, this reduction was accompanied by compositional shifts in individual ginsenosides. Microbial enzymatic activity, particularly glycosidases and other hydrolytic enzymes produced by LAB, likely facilitated the bioconversion of PPT-type ginsenosides into PPD-type compounds such as Rg3 and CK [2,11,12]. Consistent with our findings, increases in PPD-type ginsenosides following fermentation have been reported in previous studies, supporting the trends observed in the present work [41]. Although PPT-type ginsenosides such as Rb1, Rb2, Re, and Rg1 account for ~75% of the total ginsenoside content, they are known to exhibit relatively low bioavailability compared with their deglycosylated counterparts [4]. This bioconversion occurs through the enzymatic hydrolysis of glycosidic bonds by β-glucosidase and related hydrolases, which sequentially remove sugar moieties from PPT-type ginsenosides to yield deglycosylated PPD-type forms such as Rg3, Rh2, and CK [2]. Moreover, acidic and oxidative conditions generated during fermentation may improve cell wall permeability and enzyme accessibility, thereby promoting more efficient transformation [5,11,40]. PPD-type ginsenosides such as Rg3, Rh2, F2, and CK have been reported to exhibit higher biological activity and absorption rates in humans compared with major ginsenosides [3,8]. In particular, Rg3 and CK are widely recognized for their anticancer, anti-inflammatory, and neuroprotective effects [1,42]. The increase in minor ginsenosides after fermentation highlights the value of FAMCGS as a functional ingredient with enhanced bioactive properties.

3.6. TPC, TFC, and MRPs of MCGS Before and After Fermentation According to Harvest Periods

The results of the analysis of TPC, TFC, and MRPs of MCGS and FAMCGS according to harvest periods are presented in Figure 6. TPC showed no significant differences among MCGS samples, with the average content being 4.27 mg/g. However, it tended to increase after fermentation, with the FAMCGS samples showing an average TPC of 7.21 mg/g (Figure 6A). A similar trend was detected for TFC, which increased in FAMCGS samples compared with that in MCGS samples. The highest TFC was found in the FAMCGS sample harvested on 13 July (1.38 mg/g), although the difference was not statistically significant (Figure 6B). The levels of MRPs showed no significant differences between MCGS and FAMCGS samples across harvest periods; however, a gradually increasing trend was detected from MCGS (average: 2.02 OD_420nm) to FAMCGS (average: 2.84 OD_420nm) (Figure 6C).

The observed increase in TPC and TFC after fermentation in the present study may be attributed not only to enzymatic hydrolysis by microbial β-glucosidase but also to additional mechanisms, including acid-induced hydrolysis under reduced pH and microbial cell wall-degrading activities (e.g., cellulase and pectinase), which may facilitate the release of bound phenolics into more active aglycone forms [43]. Additionally, the generation of MRPs during fermentation may also contribute to increased TPC [44]. Consistent with the increases in TPC and TFC observed in this study, Hwang et al. [45] reported that both TPC and TFC increased after the thermal processing of ginseng into red ginseng, suggesting that heat-induced structural modification may enhance phenolic availability. Similarly, previous studies have demonstrated that LAB fermentation can increase TPC in plant-based substrates [46], supporting the role of microbial enzymatic activity in phenolic release. Furthermore, elevated levels of MRPs have been reported during thermal processing of fermented plant products compared with unfermented forms [47], indicating that combined thermal and fermentation processes can promote the formation of antioxidant-related compounds. Accordingly, the coordinated increase in TPC, TFC, and MRP levels observed in FAMCGS may partly explain the enhanced antioxidant activity measured in this study, highlighting the synergistic contribution of steaming-aging pretreatment and dual-strain fermentation to the functional improvement of FAMCGS [5].

3.7. Comparison of Antioxidant Activities and Enzyme Inhibitory Activities of MCGS Before and After Fermentation According to Harvest Periods

The antioxidant activities (DPPH, ABTS, ∙OH, and FRAP) and enzyme inhibitory activities (α-glucosidase and pancreatic lipase inhibition) of MCGS and FAMCGS according to harvest periods are illustrated in Figure 7. The DPPH radical scavenging activity of MCGS was highest in the harvest on 31 May (29.2%), followed by 17 May (28.1%), 21 June (22.4%), and 13 July (19.2%). In contrast, the FAMCGS samples demonstrated 41–47% scavenging activities, values that are approximately 1.72–2.75-fold higher than those of MCGS (Figure 7A). A similar trend was found for ABTS radical scavenging activity, which was higher in FAMCGS than MCGS, with no significant differences among harvest periods (Figure 7B). The hydroxyl radical scavenging activity also exhibited no significant differences according to harvest periods but tended to increase after fermentation of MCGS (average: 33.7%) to FAMCGS (average: 57.2%) (Figure 7C). The FRAP value exhibited an obvious increase after fermentation, with FAMCGS (average: 0.841 OD_593nm) showing higher values than MCGS (average: 0.527 OD_593nm), irrespective of harvest period (Figure 7D). The α-glucosidase inhibitory activity revealed no remarkable differences among harvest periods but increased after fermentation of MCGS (average: 4%) to FAMCGS (average: 35.75%), with the highest activity detected in the FAMCGS sample harvested on 17 May (37.4%) (Figure 7E). Similarly, pancreatic lipase inhibitory activity revealed no significant differences according to harvest periods but increased after fermentation of MCGS (average: 4%) to FAMCGS (average: 30%), with the highest activity found in the FAMCGS sample harvested on 17 May (33.6%) (Figure 7F).

Although the harvest period exerted little effect on antioxidant and enzyme inhibitory activities in this study, fermentation significantly improved both parameters, indicating that microbial biotransformation played a dominant role in overall activity enhancement, consistent with previous research [40]. The marked increases in antioxidant activities in FAMCGS may be closely associated with the elevated levels of TPC, TFC, and MRPs observed after steaming–aging and fermentation, as these compounds are known to act as hydrogen- or electron-donating antioxidants [14]. Furthermore, the increased levels of key minor ginsenosides such as Rg3 and CK may have further contributed to the enhanced antioxidant capacity, given their reported radical-scavenging and redox-modulating activities [48,49]. Nonetheless, Zhang et al. [50] reported higher DPPH and FRAP values in nonfermented red ginseng than in its fermented counterpart, which contrasts with the present findings and suggests that the impact of fermentation may depend on substrate characteristics and processing variables [51]. The increases in α-glucosidase and pancreatic lipase inhibitory activities after fermentation may be attributed to the generation of bioactive metabolites and the enhanced release or transformation of bound inhibitory compounds during microbial enzymatic activity [52]. Moreover, the various bioactive constituents naturally present in MCGS, such as ginsenosides, phenolic compounds, and amino acids, may have also contributed to the improved inhibitory effects [53]. Remarkably, the highest inhibitory activity was detected in the FAMCGS sample harvested on 17 May, suggesting that early-harvested MCGS, characterized by relatively higher baseline phytochemical levels, may provide a more favorable substrate for microbial biotransformation and subsequent activity enhancement. This interpretation is consistent with previous reports indicating that harvest timing significantly influences antioxidant capacity and phytochemical accumulation in plant materials [54,55]. To summarize, this study found that fermentation is an effective strategy to enhance the biological potential of MCGS. The significantly improved antioxidant and enzyme inhibitory activities suggest that FAMCGS may serve as a promising value-added food ingredient with enhanced in vitro efficacy [5,40]. However, the present study was limited to in vitro evaluations of antioxidant and enzyme inhibitory activities. In addition, bioactivities were assessed at a single concentration for comparative screening purposes, which does not allow precise determination of concentration-dependent effects or IC50 values. Therefore, the physiological relevance of these findings should be interpreted with caution. Future studies incorporating dose–response analyses and in vivo validation are necessary to provide a more comprehensive assessment of the bioactive potential of FAMCGS.

4. Conclusions

This study investigated the effects of harvest periods and fermentation using LAB on the bioactive properties of mountain-cultivated ginseng sprouts (MCGS). Fermentation substantially altered the compositional profile of MCGS, increasing the content of UFAs 1.07- to 1.21-fold, showing high mineral proportions of K and Ca (88.62%) and promoting substantial increases in the levels of key bioactive ginsenosides such as Rg3 (1.60- to 2.59-fold) and CK (3.66- to 6.31-fold). Furthermore, TPC, TFC, MRP levels, antioxidant capacity, ginsenoside conversion, and enzyme inhibitory activities consistently increased after fermentation, irrespective of the harvest period, thus confirming the pivotal role of fermentation in improving functional quality. Early-harvested MCGS (17 May) exhibited superior performance in several parameters, suggesting that the developmental stage modulates the extent of functional improvement. Hence, fermented and aged mountain-cultivated ginseng sprouts (FAMCGS) demonstrate potential as a value-added functional ingredient with enhanced bioactive properties. Nevertheless, the narrow range of fermentation conditions explored in this study is a limitation, and the effect of harvest periods on metabolite development requires further systematic investigation. Future studies should investigate diverse developmental stages and microbial communities, incorporate in vivo validation models, and explore the feasibility of industrial-scale fermentation to clarify the underlying mechanisms and practical applicability.