Open AccessArticle

Comparative Study on Selenium and Volatile Compounds in Selenium-Enriched Cardamine violifolia Pickles Fermented by Three Distinct Methods

Jue Gong

^1,2,

Shen Rao

^1,*

Xiaomeng Liu

Shuiyuan Cheng

¹,

Xin Cong

^1,2 and

Dingxiang Zhu

^1,2,*

National R&D Center for Se-Rich Agricultural Products Processing Technology, Wuhan Polytechnic University, Wuhan 430048, China

Enshi Se-Run Material Engineering Technology Co., Ltd., Enshi 445000, China

Authors to whom correspondence should be addressed.

Fermentation 2024, 10(12), 632; https://doi.org/10.3390/fermentation10120632

Submission received: 26 October 2024 / Revised: 7 December 2024 / Accepted: 8 December 2024 / Published: 11 December 2024

(This article belongs to the Section Fermentation for Food and Beverages)

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Abstract

Cardamine violifolia is a selenium (Se)-rich vegetable crop belonging to the Brassicaceae family. This study investigated the Se concentration and volatiles in the fresh (CK) C. violifolia, natural fermented (NF), Lactiplantibacillus plantarum (LP), and Leuconostoc mesenteroides (LM) fermented C. violifolia pickles. Results showed that fermentation promoted the levels of selenocysteine, methyl selenocysteine, and selenate. A total of 648 volatile compounds were found, including 119 terpenoids, 105 heterocyclic compounds, 103 esters, and 65 hydrocarbons. Differential analysis of volatiles indicated that fermentation induced the release of volatiles when compared to CK, whereas volatile profiles in LM and NF pickles showed notable differences from LP pickles. SeCys2, MeSeCys, and selenate significantly correlated to several volatile compounds, implying that Se metabolism may affect the formation of volatiles. Conclusively, fermentation promoted the release of aroma and bioactive volatiles and the degradation of unpleasant and harmful substances in C. violifolia pickles.

Keywords:

Cardamine violifolia; Leuconostoc mesenteroides; Lactiplantibacillus plantarum; natural fermentation; selenium

1. Introduction

Cardamine violifolia, an endemic cruciferous vegetable originating from Enshi, Hubei, exhibits remarkable selenium (Se) accumulation capabilities [1]. Grown in regions rich in Se deposits in Enshi, its aerial parts are capable of sequestering an impressive 3000 mg/kg or more of Se [2]. Notably, the National Health Commission of China officially recognized C. violifolia as a leafy vegetable in 2021, thereby authorizing its utilization as a legitimate food ingredient.

Selenium, an essential trace element vital for human health, has attracted increasing attention for its supplemental advantages [3,4]. C. violifolia distinguishes itself through its remarkable capacity to efficiently convert Se, transforming a substantial portion of absorbed inorganic Se into organic forms [5]. The primary organic Se compounds identified in this plant include selenocysteine (SeCys₂), selenomethionine (SeMet), and methyl selenocysteine (MeSeCys) [6], all of which play crucial roles in enhancing human health, particularly in cancer prevention and treatment [7]. In addition to its Se accumulation capabilities, C. violifolia, like other cruciferous plants, serves as a reservoir of numerous bioactive compounds such as flavonoids, glucosinolates, and phenolic acids [8,9]. Consequently, it assumes a critical role in the domain of Se-enriched foods and the natural extraction of organic Se, effectively addressing Se deficiency. This elevates C. violifolia to the status of a superior source for safe and quantifiable Se supplementation in humans, while also providing invaluable plant materials for related industries.

Pickling in China is a centuries-old tradition that involves the fermentation of pretreated vegetables to create a unique and flavorful food product [10]. Common examples include preserved mustard greens [11], sauerkraut [12], and Korean kimchi [13], all of which undergo a similar fermentation process. The key microorganisms responsible for this transformation are lactic acid bacteria [11]. Lactic acid bacteria comprise a diverse and abundant group of bacteria naturally found in the human intestine, many of which serve as essential probiotics that confer health benefits. Under anaerobic conditions, lactic acid bacteria thrive as the dominant flora, proliferating and producing various metabolic products, primarily organic acids [14]. Due to the relatively short fermentation period, pickled vegetables retain high concentrations of nutrients such as vitamins, carotenoids, amino acids, calcium, and iron, making them not only flavorful but also highly nutritious [13]. Moreover, these vegetables are known to enhance gastrointestinal function, boost human immunity, and significantly contribute to the overall health benefits associated with pickled foods [15]. The distinctive flavor of pickled vegetables is largely attributed to the fermentation process itself, particularly the activity of lactic acid bacteria [16]. These microorganisms generate a considerable amount of volatile aroma compounds, including thiols and esters, which are critical indicators for assessing the sensory quality of pickled vegetables.

As an emerging cruciferous vegetable, C. violifolia exhibits substantial biomass, rich nutritional content, and a distinctive taste and flavor, establishing it as an ideal raw material for pickle production. Nevertheless, there are currently limited reports regarding the utilization of C. violifolia in pickle making. Notably, its ability to accumulate Se and the alterations of Se during the fermentation process remain unexplored. This gap has hindered the development and utilization of C. violifolia. The objective of this study is to investigate the effects of various fermentation methods on the Se and flavor profile of C. violifolia pickles. In this study, C. violifolia pickles were prepared utilizing three fermentation methods: natural fermentation (NF), Lactiplantibacillus plantarum (LP) fermentation, and Leuconostoc mesenteroides (LM) fermentation. A comprehensive analysis of Se content, Se species, and volatile compounds was subsequently conducted. Through comparative analysis of the experimental results, this study aims to elucidate the impact of different fermentation methods on the changes of Se and volatile compounds, thereby providing a scientific foundation for the production and quality control of C. violifolia pickles.

2. Materials and Methods

2.1. Preparation of Lactic Acid Bacteria

Two lactic acid bacteria strains, Lactiplantibacillus plantarum (LP) and Leuconostoc mesenteroides subsp. mesenteroides (LM), were procured from the China Center of Industrial Culture Collection (https://m.china-cicc.org/ (accessed on 3 October 2024)) with strain numbers CICC20765 and CICC22184, respectively. Both strains were initially preserved at −80 °C in De Man, Rogosa, and Sharp medium (MRS) broth supplemented with 15% glycerol (v/v). After thawing, the strains were incubated at 30 °C for 24 h to achieve a final cell density of approximately 9 log CFU/mL. Following incubation, the cultures were centrifuged at 12,000× g for 20 min at 4 °C. The bacterial cells were then harvested, washed twice with 0.85% (w/v) sterile saline, and subsequently suspended in 0.85% (w/v) sterile saline at a concentration of 8 log CFU/mL for further experimentation.

2.2. Preparation of C. violifolia Pickles

Fresh C. violifolia leaves were harvested from the cultivation base of Enshi Se-Run Material Engineering Technology Co., Ltd., Enshi, China. The leaves were thoroughly rinsed with water and air-dried before being crushed using a domestic crusher (S18-LA181; Joyoung, Jinan, China) for 10 s. Subsequently, a 0.5% (w/w) salt brine and a 0.5% (w/w) vitamin C solution were added to the crushed material and mixed vigorously. The pH value was measured immediately before the mixture was transferred to autoclaved polyethylene vessels (10 L) and pressed to eliminate air in preparation for subsequent lactic acid fermentation. The initial pH value prior to fermentation was 6.43. Fermentation of the C. violifolia pickles was conducted under the following conditions: (I) natural fermentation (NF), (II) inoculated with L. plantarum (LP), and (III) inoculated with L. mesenteroides (LM). Additionally, the raw mixture that underwent no fermentation served as the control (CK). Both starter cultures received an approximate inoculation of 1.0 × 10⁶ CFU/g of C. violifolia. After fermentation for 12 h, the pH value of C. violifolia pickles was measured every 4 h, and the fermentation was terminated when it was no longer decreasing. After 20 h of fermentation, the pH value declined markedly to 4.80, 4.60, and 4.48 in NF, LM, and LP, respectively. There was no significant disparity between the pH values at 20 h and those at 24 h. Therefore, the fermentation process was eventually carried out for 24 h at 37 °C. All samples were subsequently collected and frozen at −40 °C for further analysis.

2.3. Determination of Total Se Content

The total Se content in the C. violifolia pickles was measured using a hydride generation atomic fluorescence spectrometry (AFS-8510; Haiguang Instruments, Beijing, China) according to the method described by Rao et al. [17]. In brief, 0.2 g of the sample was weighed and placed into a Teflon digestion tank. Subsequently, 8 mL of concentrated nitric acid was added to the tank, which was then placed in a microwave digestion furnace (M6, Preekem, Shanghai, China) for sample digestion. After digestion, the resulting sample solution was concentrated to 1 mL, and 5 mL of 50% hydrochloric acid (HCl) was added. The mixture was heated until it became clear and then transferred into a 500 mL volumetric flask. Ultrapure water was added to bring the solution to the final volume for Se determination. The instrument operating conditions were set as follows: negative high voltage at 260 V, Se lamp current at 80 mA, atomization temperature at 300 °C, auxiliary gas flow rate at 200 mL/min, and carrier gas flow rate at 600 mL/min.

2.4. Analysis of Se Species

Five Se species, including selenocysteine (SeCys₂), methyl selenocysteine (MeSeCys), selenomethionine (SeMet), selenite, and selenate, were quantified using liquid chromatography coupled with hydride generation atomic fluorescence spectrometry (HPLC-HG-AFS), following the methodology outlined by Rao et al. [8]. In this procedure, 0.1 g of crushed samples was placed into a 15 mL centrifuge tube containing 10 mL of pure water. The mixture was vortexed for 20 s, followed by ultrasonic shaking for 10 min at room temperature. After additional vortex shaking for 20 s, the sample was allowed to stand for 20 min before being centrifuged for 10 min at 9000 rpm. The resulting supernatant was filtered through a 0.22 μm filter membrane and used for Se speciation analysis via LC-HG-AFS (LC-AFS6500, Haiguang Instruments, Beijing, China). The working conditions of the LC-HG-AFS were as follows: chromatographic column, Hamilton PRP-X100 anion exchange column (250 mm × 4.1 mm, 10 μm), maintained at 25 °C; mobile phase A, 40 mmol/L ammonium phosphate dibasic (pH 6.0); mobile phase B, 100 mmol/L ammonium phosphate dibasic (pH 6.0); flow rate, 1.0 mL/min; injection volume, 100 μL. Standard compounds for these five Se species were obtained from the National Institute of Metrology of China, Beijing, China, which were used to create standard curves for quantification. The Se species in the extracts were identified by comparing their retention times with those of the standard compounds.

2.5. Volatile Analysis

Volatile metabolites were extracted using headspace solid-phase microextraction (SPME) and detected via gas chromatography–mass spectrometry (GC-MS, 8890B-7000D, Agilent, Palo Alto, CA, USA). The GC system was outfitted with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm, 5% phenyl-polymethylsiloxane). To prepare the samples, 0.500 g of pickle samples was accurately weighed and transferred into 20 mL headspace vials (Agilent, Palo Alto, CA, USA). Then, 2 mL of saturated sodium chloride solution was added to the vials to inhibit enzymatic activity. The vials were then sealed with crimp-top caps fitted with TFE–silicone headspace septa. Before extraction, the vials were preheated in the heating unit of an autosampler at 60 °C for 5 min. A 120 µm DVB/CWR/PDMS fiber (Agilent) was inserted into the vials and allowed to be exposed to the headspace of the samples for 15 min at 60 °C. Prior to this extraction, the fiber was conditioned at 250 °C for 5 min in a Fiber Conditioning Station.

Desorption of the volatiles from the coating of the fiber was accomplished in the injection port of the GC system at 250 °C for 5 min. The analyzing conditions of the GC-MS were as follows: carrier gas, helium with a linear velocity of 1.2 mL/min; column temperature program, held at 40 °C for 5 min, then increased at 10 °C/min to 100 °C, followed by 7 °C/min to 180 °C, 25 °C/min to 280 °C, and held for 5 min; MS electron impact (EI) ionization mode at 70 eV; ion source temperature, 230 °C; quadrupole mass detector temperature, 150 °C; interface temperature, 280 °C. The ion monitoring (SIM) mode was selected for the identification and quantification of volatile compounds, which contains the identified retention time and qualitative and quantitative ions. The ion detection mode was selected for accurate scanning, and each compound was selected with 1 quantitative ion and 2–3 qualitative ions. All the ions to be detected in each group were detected separately according to the order of peak appearance. If the retention time of the detected ions was consistent with the standard reference, and the selected ions appeared in the mass spectra of the samples after the deduction of the background, the substance was judged to be the substance. The quantitative ions were selected for integration and calibration to enhance the accuracy of the quantification.

The raw data generated by GC-MS were processed by Agilent MassHunter software (version 12.0). The volatile compounds were analyzed qualitatively and quantitatively based on a self-constructed database in Wuhan Metware Bio-tech. Co. (Wuhan, China). Principal component analysis (PCA) was performed by statistics function prompt within R. Pearson correlation coefficients between samples were calculated by the cor function in R and presented as heatmaps. Differential volatiles between treatment groups were determined by using the R package MetaboAnalystR (version 4.0) with the threshold of VIP ≥ 1 and |Log2FC| ≥ 1.0.

2.6. Statistical Analysis

Samples were biologically triplicated, with two technical replicates conducted for both total Se and Se species measurements. The data from these experiments were analyzed using SPSS (version 24, SPSS Inc., Chicago, IL, USA), employing one-way ANOVA followed by Duncan’s significant difference test at a significance level of p ≤ 0.05. For volatile detection, five replicates of each sample were utilized to ensure robustness in the measurements. Pearson correlation coefficients between Se and volatiles were calculated using Metware Cloud, a free online platform for data analysis (https://cloud.metware.cn (accessed on 3 October 2024)).

3. Results

3.1. Total Se and Se Species in C. violifolia Pickles

C. violifolia demonstrated a considerable accumulation of Se in the shoots. As shown in Table 1, the total Se concentrations in both fresh and fermented C. violifolia samples exceeded 200 μg/g DW (dry weight). Notably, fermentation had an insignificant effect on the total Se content in C. violifolia pickles when compared to the CK group. Interestingly, the total Se concentration in the NF pickles was significantly higher than that in the LP groups.

Three Se species, including SeCys₂, MeSeCys, and selenate, were detected in the C. violifolia pickles. However, only SeCys₂ was detected in the CK groups, and its concentration was significantly lower than that in the three fermented pickles. The highest SeCys₂ concentration was observed in the NF, followed by LM and LP. MeSeCys and selenate were not detected in the CK, but their concentrations were highest in the NF pickles and showed significantly elevated levels compared to those in the LM and LP groups.

3.2. Profiling of Volatile Compounds

A total of 648 volatile compounds were characterized by GC-MS in the present study (Supplementary Table S1). There were 600, 636, 636, and 638 volatile compounds detected in the CK, NF, LP, and LM samples, respectively. These volatile compounds were categorized into 15 classes (Figure 1A) with the four largest classes identified as follows: 119 terpenoids, 105 heterocyclic compounds, 103 esters, and 65 hydrocarbons. Additionally, notable quantities of other compound classes were observed: 21 acids, 49 alcohols, 40 aldehydes, 35 aromatics, and 54 ketones. In addition, some compounds, such as (R)-(+)-1-(p-tolyl)ethylamine, o-xylene, 2,4 dichlorophenol, styrene, and ethylbenzene, are artifacts, which may be the pollutants or compounds from fiber and column drain. PCA was performed to evaluate the variance in the four group samples. Results showed that PC1 and PC2 together accounted for 81.53% of the variation. Remarkable differences in PC1 were observed between the fermented C. violifolia pickles and the CK samples (Figure 1B). Distinct variation in PC2 was evident between the NF and LP pickles. Within each group, the samples exhibited minimal variation. Further analysis of correlations revealed high correlation coefficients among the fermented pickles, while low correlation coefficients were noted between the CK and the fermented C. violifolia samples (Supplementary Figure S1). These findings suggested significant differences in volatile profiles between the CK and the fermented samples. To visually represent the differences in volatile compounds, a heatmap was constructed (Figure 1C). It was evident that most volatiles were present at higher concentrations in the pickles compared to the CK. However, the concentration orders of alcohols and aldehydes among the four groups were CK > LP > LM > NF. Among the three pickle types, the LP group had the highest concentration of volatiles, surpassing those in both the NF and LM groups.

3.3. Overview of the Volatile Changes in the C. violifolia Samples

Differential analysis of volatile compounds was performed across the four sample groups (NF: natural fermentation; LP: inoculated with L. plantarum CICC20765; LM: inoculated with L. mesenteroides CICC22184; CK: control), resulting in the establishment of six comparison groups. This analysis identified a total of 358 differential volatile compounds (DVCs) that exhibited significant changes in abundance in at least one of the comparison groups. Notably, the number of DVCs observed in comparisons between fresh and fermented C. violifolia samples was markedly greater than those found in comparisons among the fermented samples. As illustrated in Figure 2A, the largest quantity of DVCs was noted in the comparison between the CK and LP pickles, followed by comparisons between CK vs. LM and CK vs. NF. The fewest DVCs were identified in the NF vs. LM comparison. These findings are aligned with the PCA results presented in Figure 1B. The 358 DVCs were categorized into 15 classes, with the three predominant classes being terpenoids, esters, and heterocyclic compounds (Figure 2B). Concentration analyses revealed that most DVCs had higher levels in the fermented C. violifolia samples compared to the CK (Figure 2C). Specifically, 47 of the DVCs were absent in the CK and emerged solely in the fermented C. violifolia samples, such as benzylamine, allyl isovalerate, and 3-methyl-phenol. Conversely, several volatile compounds experienced notable reductions in concentration in the fermented samples compared to the CK, for instance, 2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde and 5-methyl-2-(1-methylethylidene)-cyclohexanone in the terpenoids class, and 2,3-dihydroxy-benzoic acid and valproic acid in the acid class.

3.4. Differential Analysis of the Volatiles Between the Fresh and Fermented Samples

As previously stated, significant differences in volatile compounds between fresh and fermented C. violifolia samples were identified in the present study. A Venn diagram illustrated the overlap of 248 volatile compounds among the comparison groups CK vs. LP, CK vs. NF, and CK vs. LM (Figure 3A). These DVCs were divided into 14 classes (Figure 3B). Terpenoids, esters, and heterocyclic compounds were the dominant classes, followed by ketones and hydrocarbons, which also exhibited considerable counts. To further elucidate the changes in volatiles, the top altered volatile compounds with a fold change greater than 10 were screened in the CK vs. NF, CK vs. LP, and CK vs. LM comparison groups. Subsequently, an overlap of 48 DVCs was obtained among the three comparison groups (Figure 3C). These compounds were subjected to K-means cluster analysis. K-means analysis utilizes sample data similarity as a metric; the more similar the samples, the more likely they are to be grouped within a cluster. The 48 DVCs were classified into five subclasses (Figure 3D). Subclasses 1–4 displayed DVCs with higher relative concentrations in the three fermented samples compared to CK. In contrast, subclass 5 exhibited higher relative concentrations of DVCs in the CK. We extracted nine DVCs with higher relative concentrations in the CK and found that their concentrations in NF, LP, and LM were extremely low (Figure 3E). Conversely, the remaining 39 DVCs demonstrated significantly low concentrations in the CK. These comprised one acid, five alcohols, one aldehyde, two amines, one aromatic compound, six esters, four heterocyclic compounds, three hydrocarbons, four ketones, one nitrogen compound, four phenols, and seven terpenoids. Parts of these DVCs were visually represented in Figure 3F.

3.5. Differential Analysis of the Volatiles Among the Three Fermented C. violifolia Pickles

The counts of DVCs in the comparison groups of NF vs. LP and LM vs. LP were apparently more than that in the LM vs. NF (Figure 2A). The fermentation of C. violifolia with LP resulted in more substantial changes in volatiles than either NF or LM. Consequently, comparative analyses between NF vs. LP and LM vs. LP were conducted. A total of 103 DVCs were identified in NF vs. LP, while 48 DVCs were observed in LM vs. LP. An overlap of 40 DVCs was detected between NF vs. LP and LM vs. LP (Figure 4A). Of these, 14 exhibited higher concentration levels in the NF and LM samples, whereas the remaining 26 volatile compounds demonstrated higher concentrations in the LP (Figure 4B). The top 20 altered volatiles in the NF vs. LP and LM vs. LP comparison groups were analyzed, revealing that 11 of these volatiles were common to both groups (Figure 4C,D). Seven volatiles displayed markedly lower levels in the LP, while four volatiles showed significantly higher levels. For instance, the concentrations of methyl 5-hydroxynicotinate (XMW1398) and 3-methylbenzothiophene (XMW0533) were considerably higher in the NF (Figure 4C) and LM (Figure 4D) compared to those in the LP, whereas 4-hydroxy-benzeneethanol (NMW0193) exhibited a higher concentration in the LP.

3.6. Correlation Analysis Between Se and Volatile Compounds

Correlation analysis between Se and volatile compounds was carried out to explore the potential associations. The Pearson’s correlation coefficients between total Se, SeCys₂, MeSeCys, and selenate and the 358 DVCs were calculated. Here, 68 DVCs exhibited significant negative correlations, while 178 DVCs showed significant positive correlations with SeCys₂, MeSeCys, and selenate, using a threshold of |R| ≥ 0.8 and p < 0.05. Interestingly, total Se did not demonstrate any significant correlation with the DVCs. Subsequently, a correlation analysis with a more stringent threshold of |R| ≥ 0.9 and p < 0.01 was performed, revealing that 59 and 108 DVCs were negatively and positively correlated with SeCys₂, MeSeCys, and selenate. Further analysis using an even more rigorous criterion indicated that SeCys₂ significantly correlated with 19 volatile compounds at the evaluation standard of |R| > 0.95 and p < 0.001. The 19 volatile compounds included one alcohol, one amine, four aromatics, one ester, two heterocyclic compounds, three ketones, three phenols, three terpenoids, and one other compound (Figure 5A). The correlations between MeSeCys or selenate and DVCs were stronger than those observed for SeCys₂. Specifically, MeSeCys and selenate significantly correlated with 72 volatile compounds based on the judgment criterion of |R| > 0.99 and p < 0.001. These volatile compounds consisted of three acids, seven alcohols, five aldehydes, seven amines, four aromatics, twelve esters, ten heterocyclic compounds, six hydrocarbons, eight ketones, two nitrogen compounds, three phenols, one sulfur compound, and fourteen terpenoids (Figure 5B). Interestingly, we noted that several members among the former 19 compounds were also included in the latter 72 DVCs, indicating significant correlations with SeCys₂, MeSeCys, and selenate. To further elucidate the association between Se and the DVCs, both the within-group correlation of the 19 DVCs and the between-group correlation of these DVCs with Se were analyzed (Figure 5C). The results indicated that total Se correlated only with epizonarene. In contrast, SeCys₂, MeSeCys, and selenate exhibited significant correlations with the 19 DVCs. Additionally, significant correlations were noted among pairs of compounds; for instance, benzyl alcohol demonstrated a significant positive correlation with heptanoic acid methyl ester and a negative correlation with β-cyclocitral.

4. Discussion

Pickle is a traditional fermentation food popular in China, characterized by complex microbial successions that are highly sensitive to environmental changes [10]. This variability can lead to significant challenges in maintaining consistent product quality, especially within industrial manufacturing settings. The lengthy fermentation cycles further complicate efforts to achieve reliable outcomes. To address these issues, research has demonstrated the potential of artificial inoculation fermentation as an effective strategy for ensuring product stability and quality [12]. In the realm of pickle fermentation, two specific microorganisms, L. plantarum and L. mesenteroides, have emerged as promising candidates for artificial inoculation [11]. These strains are well-regarded for their contributions to the desirable characteristics of pickles, serving as a viable alternative to traditional natural fermentation approaches. By employing controlled inoculation with these strains, manufacturers can stabilize the quality of their pickle products while also potentially reducing the fermentation cycle duration, thereby enhancing overall production efficiency [10]. C. violifolia, a vegetable crop known for its remarkable ability to accumulate Se, presents unique opportunities for fortifying pickles with this essential nutrient. The present study found that different fermentation methods differently influenced the Se species in C. violifolia pickles. Our previous study has revealed that SeCys₂, MeSeCys, and selenate were the main Se species in fresh C. violifolia samples [17]. Fermentation using NF (natural fermentation), LP (inoculated with L. plantarum CICC20765), and LM (inoculated with L. mesenteroides CICC22184) significantly enhanced the biosynthesis of SeCys₂, MeSeCys, and selenate in comparison to the CK group. These findings suggest that microbial activities play a crucial role in the transformation of Se in C. violifolia pickles. The microorganisms involved are primarily derived from endophytes presented within C. violifolia. These endophytes establish a symbiotic relationship with the plant, and some possess the capability to metabolize Se. For example, a recent study revealed 14 endophytic Se-resistant strains from C. violifolia, including genera such as Oceanobacillus and Terribacillus [18]. The environmental conditions within the fermentation facility may be more conducive for microorganisms to metabolize Se and generate various Se species. However, the addition of LP and LM appeared to inhibit the formation of Se species when compared to NF pickles. This inhibition may result from LM and LP restricting the activities of certain Se-metabolizing microorganisms.

Previous studies have indicated that volatile flavor compounds are a critical factor influencing quality assessment and consumer acceptance of vegetable pickles [19]. The dynamic changes in the microbial community during the fermentation process can directly determine the metabolic reactions occurring within the plant matrix. This, in turn, significantly influences the production of aroma compounds [20]. In the present study, the diversity of volatile flavor compounds in fermented C. violifolia pickles increased, while the concentration of total volatile substances was observed to be nearly halved (Supplementary Table S1). Notably, the volatile changes in the NF group closely aligned with those of the two lactic bacteria inoculation groups, indicating that spontaneous fermentation may be driven by lactic endophytes residing in the leaves of C. violifolia.

Terpenoids are the predominant floral compounds and exhibit notable antioxidant and antimicrobial activity [21]. In all fermentation groups, the levels of these terpenoid compounds significantly increased. This enhancement could be attributed to glycosylases produced by microorganisms, which have the capability to cleave the bonds between terpenes and sugars, or potentially through their possible de novo production [22]. Interestingly, (-)-cis-carveol was found to have the highest abundance among the terpenoids. This compound has been shown to mitigate B-amyloid-peptide 1-42-(Aβ1-42-) induced memory impairment and oxidative stress in the rat hippocampus [23]. These findings are consistent with previous studies indicating that extracts from C. violifolia can alleviate metabolic disorders, memory dysfunction, and neuronal damage in the hippocampus resulting from oxidative stress [24]. Heterocyclic compounds, such as 2-hexanoylfuran, characterized by sweet, green, and beany notes [25], 2-methoxy-3-(2-methylpropyl-pyrazine) with the flavor of soil, grass, green, and pepper [26], furaneol, which imparts caramel attributes [27], were the primary aroma contributors to the flavor profiles of C. violifolia pickles, attributed to their lower odor threshold, which generally increased after fermentation. Additionally, (S)-3-(1-methyl-2-pyrrolidinyl)-pyridine, widely recognized as L-nicotine and the most recent among the five major classes of insecticides [28], was degraded largely by the fermentation process (Supplementary Table S1), suggesting that microbial activity may mitigate the residue of neonicotinoid pollutants in C. violifolia leaves. Esters are generated when alcohol and carboxylic acid functional groups react, contributing to the aroma and quality of pickles characterized by pleasant, fruity, and floral notes at lower thresholds [29,30]. Esters in the LP groups were higher than those in the CK groups, while the NF and the LM groups detected fewer esters, suggesting that fermentation inoculated with L. plantarum exhibited a richer ester-originated aroma.

Among the volatile acid compounds detected in this study, hexanedioic acid (charred bone) and decanoic acid (rancid, fatty), which were significantly reduced by the fermentation process, were two primary substances contributing to the pungent odor of C. violifolia raw leaves. These organic acids may interact with other volatile components, such as alcohols and aldehydes, resulting in the formation of numerous compounds [31]. Aldehydes (hexanal, dodecanal, and heptanal), characterized by sweet, fruity, leafy, nutty, and caramel-like odors, are derived from the oxidation of unsaturated fatty acids and the catabolism of amino acids and are regarded as key flavor compounds in fermented vegetables [32,33]. Notably, 4-hydroxybenzaldehyde, identified as a specific volatile compound in traditional Mexican curing of green vanilla beans, was the predominant aldehyde in C. violifolia leaves (Supplementary Table S1) and exhibited a significant decrease in fermented pickles due to its susceptibility to oxidation [34], leading to a reduction in the overall abundance of aldehydes. Alcohols possess a high aroma threshold, thus exerting a limited influence on the flavor of pickles [35]. Certain alcohols (e.g., 1-pentanol, 1-hexanol, 1-octanol, and 1-octen-3-ol) that increased during fermentation have been reported to exhibit a statistically significant correlation with the corresponding rising levels of aldehydes (e.g., pentanal, hexanal, or nonanal) [19,32]. This may elucidate why the concentration orders of alcohols and aldehydes among the four treatments in this study were identical (CK > LP > LM > NF).

In agreement with the study reported by Ma et al. [36], amines had high concentrations in fresh C. violifolia. Fermentation—regardless of inoculation—significantly reduced the concentration of amine, e.g., 4-methoxyformanilide, which is commonly regarded to have an unpleasant amine flavor. These results indicated that fermentation can reduce the unpleasant flavor of C. violifolia. Volatile sulfide compounds, including benzyl isothiocyanate, benzyl thiocyanate, and dimethyl trisulfide, were identified as the discriminant volatile substances in fresh C. violifolia leaves, consistent with previous findings [8] (Rao et al., 2021b). These sulfur compounds possess extremely low olfactory thresholds and are characterized by pungent sulfurous and spicy odors [37]. They are reported to be decomposition products of cysteine and methionine found in Brassicaceae [38]. Notably, benzyl isothiocyanate has been established as a representative component of benzyl glucosinolate hydrolyzates in cruciferous vegetables [39]. In this study, volatile sulfide compounds, particularly benzyl isothiocyanate, decreased dramatically in all three fermented groups (Supplementary Table S1).

5. Conclusions

Fermentation using NF (natural fermentation), LP (inoculated with L. plantarum CICC20765), and LM (inoculated with L. mesenteroides CICC22184) facilitated the generation of SeCys₂ and MeSeCys but did not significantly affect the total Se content. Fermentation enhanced the flavor and quality of C. violifolia pickles through the enrichment of aromatic and health-promoting compounds while simultaneously reducing the release of unpleasant and harmful volatiles. The LP group exhibited the highest abundance of terpenoids, heterocyclic compounds, and esters, including (-)-cis-carveol, 2-hexanoylfuran, 2-methoxy-3-(2-methylpropyl)-pyrazine, and furaneol. This group also recorded the lowest concentrations of volatile sulfur compounds, amines, and (S)-3-(1-methyl-2-pyrrolidinyl)-pyridine. These results indicate that fermentation greatly affects Se metabolism and volatile profile in C. violifolia pickles.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation10120632/s1, Figure S1: Correlation analysis of the samples. Table S1: Information on the volatile compounds detected in the present study.

Author Contributions

Conceptualization, S.R. and J.G.; methodology, J.G. and D.Z.; software, S.R.; validation, D.Z.; formal analysis, X.L.; investigation, J.G.; resources, X.C.; data curation, S.R.; writing—original draft preparation, J.G.; writing—review and editing, S.R. and X.L.; visualization, S.R.; supervision, S.C. and X.C.; project administration, S.C. and D.Z.; funding acquisition, X.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of Hubei Province (2024AFB398), the Special Program for Young Scientists and Technologists in Hubei Province (2023DJC206), the Science and Technology Major Program of Hubei Province (2024BBA002), and the Agricultural Science and Technology Achievement Transformation Fund Project (2024EBA010).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Materials, further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to thank Yue Zhang for the advice on designing the experiment.

Conflicts of Interest

Authors Jue Gong, Xin Cong and Dingxiang Zhu are employed by the Enshi Se-Run Material Engineering Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Overview of the volatile compounds in fermented C. violifolia pickles: (A) classification of the volatile compounds, (B) principal component analysis of the samples, and (C) concentration changes of the volatiles in each group. NF: natural fermentation; LP: inoculated with L. plantarum; LM: inoculated with L. mesenteroides; CK: control.

Figure 2. Comprehensive analysis of the DVCs in fermented C. violifolia pickles: (A) statistics of the DVCs in each comparison group, (B) classification of the DVCs, and (C) cluster analysis of the concentrations of DVCs. NF: natural fermentation; LP: inoculated with L. plantarum; LM: inoculated with L. mesenteroides; CK: control.

Figure 3. Analysis of the DVCs between CK and fermented C. violifolia pickles: (A) overlap of the three comparison groups; (B) classification of the 248 DVCs in the overlap; (C) overlap of the DVCs with a fold change greater than 10 in the three comparison groups; (D) K-means analysis of the 48 DVCs. Different color lines indicate different subclasses of compounds; (E) concentration changes of the nine DVCs in subclass 5 from the K-means analysis; and (F) concentration changes of the 13 top changed DVCs. NF: natural fermentation; LP: inoculated with L. plantarum; LM: inoculated with L. mesenteroides; CK: control.

Figure 4. Analysis of DVCs between the fermented C. violifolia pickles: (A) overlap of the two comparison groups; (B) concentration changes of the 40 DVCs in three pickles; (C) top changed DVCs in NF vs. LP comparison group; and (D) top changed DVCs in LM vs. LP comparison group. NF: natural fermentation; LP: inoculated with L. plantarum; LM: inoculated with L. mesenteroides; CK: control. XMW1398: methyl 5-hydroxynicotinate; XMW0533: 3-methylbenzothiophene; KMW0359: 3-ethyl-phenol; XMW0300: 3,5-dimethyl-phenol; KMW0469: 4-ethyl-2-methoxy-phenol; NMW0066: 2,4-dimethyl-benzenamine; D276: umbellulon; XMW0212: 1,4-benzodioxan-6-amine; NMW0193: 4-hydroxy-benzeneethanol; w21: 6-pentyl-2H-pyran-2-one; XMW0549: naphthalene.

Figure 5. Correlation analysis between Se and volatile compounds detected in C. violifolia pickles: (A) correlation between SeCys₂ and volatile compounds; (B) correlation between MeSeCys and selenate and volatile compounds; and (C) correlation between Se and the representative volatile compounds.

Table 1. Concentrations (ug/g DW) of the total Se and Se species in C. violifolia pickles.

Sample	Total Se	SeCys₂	MeSeCys	Selenate
CK	227.16 ± 2.46 ab	46.19 ± 0.67 d	ND	ND
NF	238.66 ± 7.32 a	71.73 ± 0.53 a	14.32 ± 0.60 a	4.46 ± 0.19 a
LM	230.40 ± 3.09 ab	64.66 ± 1.27 b	12.65 ± 0.32 b	3.42 ± 0.18 b
LP	216.66 ± 4.12 b	59.91 ± 1.11 c	12.58 ± 0.66 b	3.18 ± 0.09 b

Note: NF: natural fermentation; LP: inoculated with L. plantarum; LM: inoculated with L. mesenteroides; CK: control. Different letters (a–d) in the same column represent significant differences (p ≤ 0.05).

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Gong, J.; Rao, S.; Liu, X.; Cheng, S.; Cong, X.; Zhu, D. Comparative Study on Selenium and Volatile Compounds in Selenium-Enriched Cardamine violifolia Pickles Fermented by Three Distinct Methods. Fermentation 2024, 10, 632. https://doi.org/10.3390/fermentation10120632

AMA Style

Gong J, Rao S, Liu X, Cheng S, Cong X, Zhu D. Comparative Study on Selenium and Volatile Compounds in Selenium-Enriched Cardamine violifolia Pickles Fermented by Three Distinct Methods. Fermentation. 2024; 10(12):632. https://doi.org/10.3390/fermentation10120632

Chicago/Turabian Style

Gong, Jue, Shen Rao, Xiaomeng Liu, Shuiyuan Cheng, Xin Cong, and Dingxiang Zhu. 2024. "Comparative Study on Selenium and Volatile Compounds in Selenium-Enriched Cardamine violifolia Pickles Fermented by Three Distinct Methods" Fermentation 10, no. 12: 632. https://doi.org/10.3390/fermentation10120632

APA Style

Gong, J., Rao, S., Liu, X., Cheng, S., Cong, X., & Zhu, D. (2024). Comparative Study on Selenium and Volatile Compounds in Selenium-Enriched Cardamine violifolia Pickles Fermented by Three Distinct Methods. Fermentation, 10(12), 632. https://doi.org/10.3390/fermentation10120632

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