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ORIGINAL ARTICLE

Assessing the effects of homofermentative Lactiplantibacillus plantarum K25 and heterofermentative Limosilactobacillus fermentum 13-1 on the flavor and functional characteristics of fermented milk analyzed by metabolomics approach

Jingwei Wu1, Min Zhang2, Tianqi Luo1, Tongji Liu1, Qingxia Ren1, Zengjia Zhou1, Zhennai Yang1*, Tariq Aziz1*, Ashwag Shami3, Fahad Al-Asmari4, Khulood Fahad Alabbosh5, Doaa El-Ghareeb Keshek6,7

1Key Laboratory of Geriatric Nutrition and Health of Ministry of Education, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing, China;

2Key Laboratory of Agro-Products Primary Processing, Academy of Agricultural Planning and Engineering, MARA, Beijing, China;

3Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, 11671, Saudi Arabia;

4Department of Food and Nutrition Sciences, College of Agricultural and Food Sciences, King Faisal University, Al Ahsa 31982, Saudi Arabia

5Department of Biology, College of Science, University of Hail, Hail, Saudi Arabia

6Department of biology, Al-Jumum University College, Umm Al-Qura university, P.O Box7388, Makkah 21955, Sudia Arabia;

7Agriculture Genetic Engineering Research Institute (AGERI), Agriculture Research Centre, Giza, Egypt

These authors contributed equally to this work.

Abstract

Lactic acid bacteria (LAB) play a significant role in milk fermentation; however, the impact of different LAB fermentation types on the metabolic characteristics of fermented milk remains poorly understood. In this study, homofermentative Lactoplantibacillus plantarum K25 and heterofermentative Limosilactobacillus fermentum 13-1 were utilized in yogurt fermentation to produce fermented milk samples D-Lp and D-Lf, respectively, which were compared to the control yogurt sample (D) in terms of flavor and functional properties. Analysis revealed that strain K25 enhanced the production of milky aroma compounds such as 2,3-butanedione (8.82±1.13 ng/g) and hexanoic acid (28.94±1.39 ng/g), while strain 13-1 led to a decrease in 2,3-butanedione (0 ng/g) and acetoin (0 ng/g) levels along with an increase in the content of both acetic acid (23.58±1.99 ng/g) and ethanol (9.92±0.93 ng/g). Both strains demonstrated potential in reducing the bitterness of fermented milk by decreasing the levels of bitter amino acids and dipeptides in samples. Untargeted metabolomics analysis indicated that strain 13-1 induced more significant metabolic changes in fermented milk compared to strain K25. Both strains influenced various amino acid metabolisms, with strain K25 promoting lysine degradation and strain 13-1 enhancing tyrosine metabolism. Furthermore, an increase in different types of bioactive compounds was observed in different fermented milk samples. This study enhances our understanding of LAB strain metabolism in fermented milk.

Key words: fermented milk, flavor, functional characteristics, Lactoplantibacillus plantarum K25, Limosilactobacillus fermentum 13-1, metabolomics analysis

*Corresponding Authors: Zhennai Yang and Tariq Aziz, Key Laboratory of Geriatric Nutrition and Health of Ministry of Education, Beijing Engineering and Technology Research Center of Food Additives, Beijing Technology and Business University, Beijing 100048, China. Emails: [email protected]; [email protected]

Academic Editor: Prof. Valentina Alessandria, Università di Torino, Italy

Received: 5 September 2024; Accepted: 20 November 2024; Published: 1 January 2025

DOI: 10.15586/ijfs.v37i1.2779

© 2025 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/)

Introduction

Yogurt is a widely consumed dairy product globally; it is favored by consumers for its healthy nutritional value and smooth texture as well as characteristic flavors (Gu et al., 2020; Innocente et al., 2016; Tang et al., 2024). In recent years, the addition of probiotics or other functional lactic acid bacteria (LAB) to yogurt fermentation, along with the standard Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus (Nemati et al., 2023), has become a popular practice to enhance the yogurt’s flavor, texture, appearance, and probiotic properties (Aziz et al., 2024a; Aziz et al., 2024b). Commonly added LAB strains include homofermentative Lactobacillus acidophilus, Lacticaseibacillus casei, Lactoplantibacillus plantarum, etc., and heterofermentative Bifidobacterium animalis, Bifidobacterium longum, etc. (Adugna and Andualem, 2023). LAB strains are responsible for producing various metabolites, such as volatile compounds, peptides, and organic acids, during milk fermentation. These metabolites have diverse effects on the technological, nutritional, and sensory properties of the product ( Zhou et al., 2024a; Zhou et al., 2024b; Naveed et al., 2024; Naveed et al., 2023; .Isık et al., 2023; Aziz et al., 2021, Aziz et al., 2020).

Different types of LAB strains, such as homofermentative and heterofermentative, possess different carbohydrate metabolism and fermentation products, exhibiting different effects on the flavor and functional properties of yogurt (Gänzle, 2015). Homofermentative LAB metabolizes hexoses, typically glucose, primarily via the Embden–Meyerhof–Parnas (EMP) pathway, resulting in lactic acid as the sole product. On the other hand, heterofermentative LAB utilizes the phosphoketolase (PK) pathway to metabolize both hexoses and pentoses, resulting in a variety of metabolites (Zotta et al., 2018). Some homofermentative LAB strains are also capable of heterofermentation, and are referred to as facultative heterofermentative strains. Bifidobacterium undergoes a unique form of heterofermentation, producing lactic acid and acetic acid as end products. It was reported that homofermentative L. plantarum can improve the quality of silage (Wang et al., 2020b; Xu et al., 2019), while some heterofermentative LAB, such as Leuconostoc citreum GR1, performed well in kimchi fermentation (Moon et al., 2018). However, in the production of fermented milk, the traditional starters remain the obligately homofermentative Lactobacillus delbrueckii and Streptococcus thermophilus. The impact on flavor of fermented milk when supplementing these starters with specific facultatively heterofermentative and obligately heterofermentative strains for auxiliary fermentation is difficult to ascertain. This complexity arises because the intermediary metabolites generated through different fermentation pathways serve as precursors for crucial flavor compounds. For example, precursor of the highly significant 2,3-butanedione in yogurt originates from pyruvate within the EMP pathway. In recent years, assumptive attempts have been made to introduce a variety of strains with distinct fermentation types into yogurt fermentation research. The addition of homofermentative Lactobacillus casei and Lactobacillus acidophilus significantly impacted the levels of acetaldehyde and ketones in yogurt, respectively (Tian et al., 2017). The addition of heterofermentative Lactobacillus fermentum HY01 in yak milk increased its acetic acid content (Zhang et al., 2022), and heterofermentative Bifidobacterium CCFM5871 provided a rich taste and aroma to yogurt (Tian et al., 2022), although the obligate heterofermentative Lactobacillus brevis was not suitable for independent yogurt fermentation (Rönkä et al., 2003). However, the limited research data available are insufficient to elucidate the general patterns of the influence of fermentation types on yogurt flavor. Further studies are required to delve deeper into the mechanisms underlying the synthesis and metabolism of flavor compounds.

Metabolomics is a convenient tool used for high-throughput screening and analyzing metabolites in complex substrates by investigating dynamic changes in their contents and biological phenotypes (Han et al., 2024a). Metabolomics-based approaches are extensively used in scientific research on different fermented foods, such as red wine (Wang et al., 2023a), coffee (Chan et al., 2021), fermented pickle (Tomita et al., 2018), and sausages (Zhao et al., 2022b). Zhao et al. (2023) conducted a metabolomics study on the important differential metabolites in fermented milk with Lactobacillus plantarum NMGL2 during the storage period. Xia et al. (2023) revealed important metabolic pathways associated with the flavor of fermented milk produced by Lactococcus lactis subsp. lactis by a metabolomics approach. However, limited information is available on the metabolomics studies of functional metabolites and mechanism of the health promotion of probiotic fermentation in yogurt (Zha et al., 2021).

In order to better understand the effects of different fermentation types of strains on the flavor and metabolic pathway of fermented milk, a homofermentative Lp. plantarum K25 and a heterofermentative L. fermentum 13-1 were employed for co-fermentation with a commercial yogurt starter culture in our study. The effects of two bacterial strains on the changes in volatile flavor compounds in fermented milk were investigated using gas chromatography–mass spectrometry (GC-MS) technique. Metabolomics analysis was performed using ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC-Q-TOF-MS) to describe differences in metabolite profiles and relevant metabolic pathways by multivariate statistical methods. This study aimed to offer theoretical guidance for the potential application of different types of LAB strains in milk fermentation.

Materials and Methods

Materials and reagents

Bovine milk (protein 3.0%, fat 3.4%, w/v) was obtained from Beijing Sanyuan Food Co. Ltd (Beijing, China). Yogurt starter YO-MIX 300 LYO containing L. delbrueckii subsp. bulgaricus and S. thermophilus was purchased from Danisco (Denmark). LAB strains Lp. plantarum K25 and L. fermentum 13-1 were previously isolated from Kefir grains collected from Tibet and preserved in our laboratory (Aziz et al., 2022; Jiang et al., 2021). 2-Methyl-3-heptanone and normal alkanes (C10–C25) were acquired from Sigma-Aldrich® (Germany). Helium gas with a purity of 99.99% was supplied by Air Products and Chemicals Inc. (USA). HPLC-grade methanol, acetonitrile, isopropanol, and formic acid were sourced from Merck (Germany). L-2-chlorophenylalanine was purchased from Shanghai Macklin Biochemical Technology Co. Ltd (China).

Preparation of fermented milk

The milk was heated at 65°C for 30 min, followed by cooling to about 40°C and inoculation with the respective microbial strains. The inoculated milk samples were then placed in an incubator (Shanghai Yiheng, China) and incubated at 42°C till pH 4.5 was attained. After fermentation, the fermented milk samples were cooled and stored at 4°C for further analysis. Three experimental groups of samples were utilized in the experiment: control samples (D) inoculated only with yogurt starter (107 CFU/mL), group D-Lp samples inoculated with yogurt starter (107 CFU/mL) and Lp. plantarum K25 (107 CFU/mL), and group D-Lf samples inoculated with yogurt starter (107 CFU/mL) and L. fermentum 13-1 (107 CFU/mL). The experiment was performed in triplicate.

Determination of pH value

Dairy fermentation monitor (AMS Alliance, France) was used to monitor changes in pH values during fermentation.

Detection of volatile organic compounds (VOCs)

The methodology used by Han et al. (2024c) with slight modifications was followed. VOCs were extracted from a 10-g sample of fermented milk using solid-phase microextraction (SPME) and analyzed by GC-MS (7890A-7000; Agilent Technologies Inc., Santa Clara, CA, USA). The fermented milk was placed in a 30-mL extraction bottle and incubated with 1 μL of 2-methyl-3-heptanone (0.816 μg/μL) at 40°C for 30 min. A 2-cm long divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (50/30 μm; Supelco, Bellefonte, PA, USA) was inserted in the headspace bottle for volatile extraction for 30 min. The sampler was then inserted into a gas chromatography (GC) injector at 250°C for 5 min for desorption. Gas chromatography was carried out with a DB-Wax high-polarity polyethylene glycol chromatographic column (30 m × 0.25 mm, 0.25 μm) in the injector port in splitless mode using helium carrier gas with a flow rate of 1.2 mL/min. The temperature rise program was performed at 40°C for 3 min, then raised to 200°C (5°C/min), to a final temperature of 230°C (10°C/min, maintained for 3 min). Mass spectrometry (MS) was performed using an electronic ionization source with an electronic energy of 70 eV. The transfer line temperature was set at 280°C, the ion source temperature at 230°C, and the quadrupole temperature at 150°C. The mass scanning range was set from 40 m/z to 250 m/z. Identification of VOCs was performed using mass spectral databases (NIST 14 spectral library [https://chemdata.nist.gov]) and retention index (RI) comparison (actual and reference RIs), as described by Han et al. (2024b), enabling qualitative analysis. Quantitative analysis of the VOCs identified by GC-MS was performed using an internal standard semi-quantitative method. The concentrations of VOCs were calculated based on the ratio of the compound peak areas to the known concentration of internal standard. The actual RI was obtained by the calculated formula and the retention times of the series of n-alkanes (Qiu et al., 2024).

Untargeted metabolomics analysis

The fermented milk sample (100 μL) was subjected to extraction by mixing with 400 μL of extraction solvent (acetonitrile–methanol = 1:1, containing an internal standard of L-2-chlorophenylalanine at a concentration of 0.02 mg/mL). After 30 s of vortex, the mixture was subjected to ultrasound extraction for 30 min at 5°C with a frequency of 40 kHz. The treated samples were precipitated at -20°C for 30 min, followed by centrifugation for 15 min at 4°C and 13,000×g. The supernatant was dried by nitrogen, and further dissolved in 120 μL of a mixed solution of acetonitrile and water (1:1). The resulting solution was treated with the low-temperature ultrasound extraction at 40 kHz (5°C, 5 min). Finally, the solution was centrifuged for 5 min (4°C, 13,000×g). The supernatant was collected after filtration through a sterile membrane with a pore size of 0.22 μm and used for further analysis by using UPLC-Q-TOF-MS (Triple TOF5600, AB SCIEX, USA).

Chromatographic conditions: The separation was achieved on a BEH C18 column (100 × 2.1 mm, 1.8 µm) purchased from Waters Corporation (Milford, CT, USA). The mobile phase consisted of solvent A and solvent B. Solvent A was an aqueous solution containing 0.1% formic acid. Solvent B was a 1:1 mixture of acetonitrile and isopropanol, containing 0.1% formic acid. The flow rate was controlled at 0.40 mL/min and the column temperature was kept at 40°C.

Mass spectrometric conditions: The sample was acquired in positive and negative ionization modes within a mass scanning range of 50–1,000 m/z. The spray voltage was set at 5,000 V (positive) and 4,000 V (negative). The declustering potential was 80 V, and the spray gas pressure, auxiliary heating gas pressure, and curtain gas pressure were set at 50, 50, and 30 psi, respectively. The ion source heating temperature was 500°C, and collision energy was set at 20–60 V.

Data processing

Flavor data were analyzed using Excel 2019, and the Origin Pro 2022 software. Simca 14.1.0 was used to perform partial least squares discriminant analysis (PLS-DA) to visualize discrimination between samples. To indicate contribution of each variable to classification, the variable importance in projection (VIP) value of each variable was calculated.

Raw liquid chromatography–mass spectrometry (LC-MS) data were processed using the metabolomics software Progenesis QI (Waters Corporation). The MS values were matched against metabolite databases, Human Metabolome Database (HMDB) and METLIN metabolomics database, to obtain metabolite information. The preprocessed data were uploaded to the Majorbio Cloud platform (https://cloud.majorbio.com) for further data analysis, including principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), Student’s t-test, fold change analysis, and VIP analysis. The annotation of metabolic pathways and the classification of metabolites were carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and HMDB database, respectively.

Results and Discussion

Monitoring of pH changes

Figure 1 illustrates pH changes during milk fermentation with yogurt starter only (control D), together with Lp. plantarum K25 (D-Lp) and L. fermentum (D-Lf). The pH curves of the three groups exhibited a similar decreasing trend, but differences were observed in the time taken to reach the endpoint of fermentation (pH 4.5). Addition of Lp. plantarum K25 and L. fermentum 13-1 was found to promote milk fermentation with 180 min and 190 min, respectively, compared to the control with 228 min to reach pH 4.5. Tian et al. (2017) reported that the addition of probiotics accelerated decrease in pH during milk fermentation. Similar findings were obtained by Li et al. (2021), who observed that milk samples fermented with thermophilic Streptococci took longer time to reach the fermentation endpoint compared to the co-fermented samples with other LAB strains.

Figure 1. Changes in pH values during milk fermentation with yogurt starter (D), together with Lp. plantarum K25 (D-Lp) and L. fermentum 13-1 (D-Lf).

Identification of VOCs in fermented milk

In all, 20 VOCs were detected by SPME-GC-MS analysis in the three groups of fermented milk samples (D, D-Lp, and D-Lf), as presented in Table 1 and Figure 2. These compounds included two aldehydes, four ketones, six acids, four alcohols, and four other compounds. To investigate variation in flavor characteristics between the experimental groups, PLS-DA analysis was used to describe key VOCs. As shown in Figure 3A, the parallel processing of the same samples was clustered well with PC1 and PC2 being 41.3% and 24.9%, respectively. The cumulative variance contribution proportion of 66.2% was sufficient to reflect main variation of the original variable information. Based on the criteria of VIP > 1 and p < 0.05, 12 compounds were identified as key VOCs, which were hexanal, nonanal, 2,3-butanedione, acetoin, acetic acid, hexanoic acid, ethanol, 2-heptanol, 2,3-butanediol, dimethyl sulfone, and phenol (Figure 3C). These aroma compounds contributed different flavor characteristics to fermented milk with addition of homofermentative Lp. plantarum K25 and heterofermentative L. fermentum 13-1. In Table 1, data were expressed as mean ± standard deviation (SD) from replicate analyses (n = 3) of three replicate samples.

Table 1. Content of aroma volatile organic compounds in fermented milk samples.

Compounds CAS Content (ng/g) Actual RI Reference RI Method of identification
D D-Lp D-Lf
Hexanal 66-25-1 0.18±0.04a 1,079 1,084 MS-RI
Nonanal 124-19–6 0.87±0.01a 1,359 1,381 MS-RI
2,3-Butanedione 431-03-8 4.98±0.26b 8.82±1.13a 956 973 MS-RI
2-Heptanone 110-43-0 2.47±0.53a 2.06±0.13a,b 1.56±0.25b 1,194 1,180 MS-RI
Acetoin 513-86-0 6.88±1.11a 1,282 1,287 MS-RI
2-Nonanone 821-55-6 1.34±0.46a 1.32±0.30a 0.45±0.04b 1,394 1,387 MS-RI
Acetic acid 64-19-7 9.82±1.08b 11.75±0.74b 23.58±1.99a 1,440 1,441 MS-RI
Butyric acid 107-92-6 5.73±0.55a 4.88±0.42a 5.35±0.35a 1,615 1,628 MS-RI
Hexanoic acid 142-62-1 19.17±1.11c 28.94±1.39a 22.74±1.38b 1,918 1,846 MS-RI
Octanoic acid 124-07-2 14.30±1.78a 14.69±1.68a 16.78±1.51a 2,034 2,060 MS-RI
Decanoic acid 334-48-5 4.62±1.07a 5.41±0.11a 5.55±0.35a 2,174 2,290 MS-RI
Benzoic acid 65-85-0 4.49±0.73a 4.17±0.47a 4.69±0.60a 2,408 2,412 MS-RI
Ethanol 64-17-5 1.57±0.42b 9.92±0.93a 959 934 MS-RI
2-Heptanol 543-49-7 0.69±0.13a 1,318 1,316 MS-RI
2-Ethyl-1-hexanol 104-76-7 1.01±0.20a 1.32±0.55a 1.51±0.16a 1,485 1,474 MS-RI
2,3-Butanediol 513-85-9 3.54±0.24a 1,522 1,539 MS-RI
γ-Butyrolactone 96-48-0 0.61±0.13a 0.72±0.09a 0.68±0.03a 1,596 1,613 MS-RI
2-(2-Ethoxyethoxy)-ethanol 111-90-0 4.37±0.35a 4.36±0.26a 4.19±1.32a 1,604 1,622 MS-RI
Dimethyl sulfone 67-71-0 0.46±0.10a 0.24±0.05b 0.41±0.04a,b 1,930 1,912 MS-RI
Phenol 108-95-2 0.37±0.02a 0.24±0.05b 2,016 2,008 MS-RI

Different lowercase letters in each row indicate significant differences between samples (p< 0.05).

Symbol “-” means not found. D: control sample fermented with yogurt starter only; D-Lp: sample fermented with yogurt starter and Lp. plantarumK25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1; MS-RI: mass spectrometry–retention index.

Figure 2. Heatmap of the aroma compounds in the fermented milk samples D, D-Lp and D-Lf. D: control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

Figure 3. Analysis of the volatile compounds in the fermented milk samples by PLS score plot (A), PLS loading of the scatter plot of volatile compounds (B), and calculation of the variable importance in projection (VIP) values from the PLS (C). (D) control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

Aldehydes, such as hexanal and nonanal, are important components of odor, although their contents in fermented dairy products are usually low. The primary reasons for the low levels of aldehydes may be attributed to two aspects. On the one hand, aldehydes themselves are susceptible to reduction into alcohols or oxidation into acids (Chi et al., 2024); on the other hand, the inherent antioxidant background of yogurt (Shori et al., 2022) hinders the formation of aldehydes derived from the oxidative decomposition of fatty acids. Table 1 and Figure 3B show that hexanal and nonanal were only detected in the control sample.

Hexanal, typically derived from the auto-oxidation of linoleic acid (Zhao et al., 2022a), exhibits an aroma reminiscent of fresh grass (Wang et al., 2020a). Nonanal, primarily originating from unsaturated fatty acids through the formation of peroxide intermediates, serves to impart floral and citrus aroma characteristics to fermented milk (Zhang et al., 2024). The low levels or absence of these two aldehydes may be attributed to the yogurt environment itself. Relevant research indicates that the pH value is crucial for the successful neutralization of lipid oxidation in oil-in-water (O/W) emulsions containing antioxidants (Cantele et al., 2024). In acidic conditions, compared to neutral environments, the negative charge on oil droplets is weaker, making them less likely to attract cations, such as Fe3+, that initiate chain reactions, thereby inhibiting lipid oxidation. Consequently, antioxidants can more effectively retard the formation of hexanal and nonanal. In addition, the antioxidant activity of these compounds is also dependent on the prevailing pH during the reaction, often exhibiting enhanced performance in acidic environments (Bayram et al., 2023). Fermented milk can be approximated as an O/W emulsion, and its low pH environment delays fatty acid oxidation, thus inhibiting the formation of hexanal and nonanal.

Ketones, such as 2,3-butanedione and acetoin, are significant volatile compounds in yogurt, although the composition of ketones varied greatly among different fermented milk samples. The relative abundance of 2,3-butanedione and acetoin is strongly correlated with citrate metabolism, and other ketones can be formed through fatty acid oxidation, thermal degradation, amino acid degradation, and microbial metabolism (Gómez-Torres et al., 2016). 2,3-Butanedione gives the yogurt a buttery flavor (Innocente et al., 2016), but acetoin helps to provide a mild creamy aroma (Liu et al., 2022b). A study focusing on beef burgers (Botta et al., 2022) indicated that the accumulation of acetoin was primarily a consequence of homofermentative metabolism conducted by LAB, specifically Carnobacterium divergens. This finding underscored the intimate association between homofermentation and the formation of these buttery flavor compounds. Figure 3B shows that among the distributed dots representing each volatile compound, these two ketones are found to be near the points representing D and D-Lp. This suggested that the addition of homofermentative Lp. plantarum K25 promoted formation of ketones, specially 2,3-butanedione with significantly higher content (p < 0.05) in D-Lp than that in D. Moreover, neither of these two ketones was detected in the D-Lf samples, suggesting that the addition of heterofermentative L. fermentum 13-1 had a negative effect on the formation of these specific yogurt flavor ketones. Previously, Mukisa et al. (2017) also did not detect 2,3-butanedione and acetoin in the fermented milk produced by L. fermentum MNC34. Zhang et al. (2022) reported similar results using Lactobacillus fermentum HY01.

Acid is an essential component of fermented milk flavor, which mainly focuses on the formation of characteristic sour and fermentation odor (Tang et al., 2024). The content of acetic acid in the D-Lf sample was significantly higher (p < 0.05) than that in the D and D-Lp samples, probably because of the heterofermentative carbohydrate metabolism of L. fermentum. Ferrocino et al. (2018) reported that heterofermentative LAB strains could produce acetic acid through pyruvate metabolism, and L. sakei with high acetate kinase activity promoted the formation of acetic acid. However, too high concentration of acetic acid results in a vinegar flavor, which negatively affects the overall milk flavor. Furthermore, the content of hexanoic acid was highest in D-Lp sample, followed by D-Lf and D samples. Hexanoic acid appears in the fermentation and storage of dairy products, providing fatty, sweaty, and cheesy odor (Wang et al., 2023b).

Alcohols, which are mainly produced from oxidation of fats and reduction of aldehydes and ketones, contribute special flavors to fermented milk. Alcohols weaken the sour taste, although they have little effect on flavor because of high threshold and low concentration (Zhao et al., 2018). Compared to both D and D-Lp samples, the D-Lf sample contained significantly more alcohols (p < 0.05), especially ethanol with high content because of production from heterofermentative fermentation (Table 1 and Figure 2). In the study conducted by Botta et al. (2022), a positive correlation between ethanol and heterofermentative Leuconostoc gelidum was observed. Ethanol provides typical alcohol odor. 2-Heptanol may be related to lipolysis, offering green and floral aroma (Wang et al., 2023b). 2,3-Butanediol, derived from the reduction of acetoin, also contributes to creamy odor (Liu et al., 2022c). It is worth noting that although 2,3-butanediol was found in D-Lf sample, no acetoin or 2,3-butanedione was observed. The only speculation is that Lactobacillus fermentum has the ability to convert all acetoin into 2,3-butanediol.

In the present study, dimethyl sulfone and phenol were also identified as key volatile compounds in yogurt samples. Dimethyl sulfone, a type of sulfur-containing compound, was first noticed by Liu et al. (2022c). Heat treatment of milk prior to fermentation, microbial degradation of sulfur-containing amino acids, and catabolism of sulphydryl groups could produce sulfur-containing compounds (Pereda et al., 2008). Phenol was rarely identified as a key flavor compound and was occasionally discovered in dairy products, typically at low levels (Liu et al., 2022d; Wang et al., 2024).

Metabolomics analysis of the fermented milk

Metabolite clustering and multivariate analysis in D, D-Lp, and D-Lf samples

Metabolomics analysis of fermented milk samples demonstrated a total of 601 metabolites discovered in the three groups of samples, with 283 in cationic mode and 318 in anionic mode. The clustering and relative abundance of all metabolites are shown in Figure 4. Based on compound classification in HMDB, lipids and lipid-like molecules (322 species) and organic acids and derivatives (117 species) were found to be the predominant metabolites in yogurt samples. Clustering results showed that D and D-Lp clustered together, meaning that these two groups were more comparable in terms of the composition and content of metabolites, but D-Lf was different from these two groups.

Figure 4. Metabolite clustering heat map of the fermented milk samples (D, D-Lp and D-Lf), displaying compounds with abundance ranking in the top 300. D: control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

Principal component analysis was performed on D-Lp, D-Lf, and D samples, as shown in Figure 5A. The cumulative variance contribution rates in positive and negative modes were found to reach 72.0% and 66.4%, respectively, explaining changes in original variable information. OPLS-DA is a supervised recognition method that adopts orthogonalization to remove data irrelevant to categorizing information, and is considered a highly effective approach for the classification and establishment of discriminative model of samples (Du et al., 2021). In Figures 5B and 5C, the score plots of the D-Lp versus D model and the D-Lf versus D model clearly demonstrate that D-Lp is distinctly segregated into two classes from D, and D-Lf is separated into two classes from D, because of the considerable distances between different sample groups. In addition, the model was tested with 200 permutations to prevent over-fitting. The R2X, R2Y, and Q2 values for the D-Lp versus D model in positive and negative ion modes were 0.693, 0.998, and 0.986, and 0.637, 0.998, and 0.978, respectively. The R2X, R2Y, and Q2 values for the D-Lf versus D model in positive and negative ion modes were 0.678, 0.99, and 0.973, and 0.663, 0.969, and 0.944, respectively. R2X and R2Y reflect the ability to interpret information from X and Y matrices, and Q2 refers to the predictive power of the model. The closer these three values are to 1, the higher the credibility. Thus, both models exhibited good explanatory and predictive power.

Figure 5. (A) PCA scores plot of D-Lp, D-Lf, and D samples. (B) OPLS-DA scores plot of D-Lp vs D model. (C) OPLS-DA scores plot of D-Lf vs D model. D: control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

Analysis of metabolites that affect the taste of fermented milk

The flavor of fermented milk comprises two components: aroma and taste. Taste compounds are generally composed of hydrophilic small molecules or ions, including amino acids, peptides, organic acids, and sugars. Among them, amino acids and their derivatives, and peptides with taste activity are commonly found in various fermented foods, exerting a significant influence on food flavor (Zhao et al., 2016). Using a non-targeted metabolomics approach, a total of 53 amino acids, peptides, and the related amino acid derivatives were detected from the three groups of samples (Figure 6B). By establishing the PLS-DA model (Figures 6A and 6C), with the criteria of VIP >1 and p < 0.05, 10 key taste compounds were identified, including 3 amino acids: L-isoleucine (L-Ile), L-phenylalanine (L-Phe), and L-tryptophan (L-Try); 5 dipeptides: arginyl-proline (Arg-Pro), glutaminyl-proline (Gln-Pro), glycyl-phenylalanine (Gly-Phe), leucyl-glycine (Leu-Gly) and γ-glutamylarginine (γ-Glu-Arg); and 2 amino acid derivatives: 2-hepteneoylglycine and N-acetyl-L-glutamic acid. These taste compounds were primarily associated with bitterness and kokumi sensation as discussed below.

Figure 6. PLS biplot based on taste compounds (A), circle heatmap of taste compounds (B), VIP calculation based on the PLS model of taste compounds in the fermented milk samples (C). (D) control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

In fermented foods, taste compounds are generally derived from hydrolysis of proteins (Zhao et al., 2016). Free amino acids, such as L-Ile, L-Phe, and L-Try, as identified in this study, were reported to be bitter amino acids in some fermented foods, such as cheese and soy sauce (Zhao et al., 2016). In the samples added with Lp. plantarum K25 and L. fermentum 13-1, the richness of these three bitter amino acids was significantly decreased, suggesting the effect of reducing the bitterness of fermented milk by these two LAB strains. Furthermore, in the D-Lp sample, the content of bitter dipeptides, Gln-Pro and Leu-Gly, decreased significantly, while in the D-Lf sample, the bitter Arg-Pro and γ-Glu-Arg increased significantly (p < 0.05). The bitterness of these dipeptides was associated with their hydrophobic properties (Fu et al., 2018). Kim and Li-Chan (2006) found that in di- or tripeptides, large hydrophobic amino acids at the C-terminus contributed significantly to bitterness, and amino acids with highly hydrophobic side chains included Try, Ile, Tyr, Phe, Pro, Leu, and Val. When arginine was adjacent to proline, the bitterness of peptide was more stronger (Liu et al., 2022a). Bitter peptides had a much greater impact than bitter amino acids on the bitter taste of food. However, γ-glutamyl peptides were known as kokumi peptides that mainly contributed to the kokumi taste (Diez-Simon et al., 2020).

Analysis of functional metabolites in fermented milk and the related metabolic pathways

Using p < 0.05 and VIP > 1 as screening criteria, the two groups of fermented milk samples (D-Lp and D-Lf) were compared to the control sample (D) in a paired manner. Both D-Lp and D-Lf samples were found with 122 differential metabolites when each of them was compared with D sample (refer to the Supplementary Material for detailed information), and the cluster heatmaps of differential metabolites are shown in Figures 7A and 7B. The Venn diagram analysis showed 98 common differential metabolites for the two groups, with 24 specific ones that were different from each other (Figure 7C).

Figure 7. Cluster heatmaps of the differential metabolites in D-Lp (A, B), and Venn diagram of the differential metabolites in D-Lp vs D model and D-Lf vs D model (C). (D) control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

Based on the 122 differential metabolites in D-Lp compared to D, the pathway enrichment analysis of KEGG (Figure 8A) revealed that the addition of Lp. plantarum K25 led to changes in several metabolic pathways in fermented milk, particularly amino acid metabolism, including phenylalanine metabolism, valine, leucine, and isoleucine biosynthesis, cyanoamino acid metabolism, arginine and proline metabolism, and D-glutamine and D-glutamate metabolism. Sun et al. (2023) also found enrichment in arginine and proline metabolism, and alanine, aspartate, and glutamate metabolism in the co-fermented milk with Bifidobacterium adolescentis B8589 and Lacticaseibacillus paracasei PC-01. Lp. plantarum L3 was found to have a significant impact on phenylalanine metabolism in yogurt as analyzed by metabolomics method (Wang et al., 2023c). Amino acids and derivatives were shown to be closely related to yogurt flavor, for example, the volatile ketones produced from the degradation of amino acids (Zhang et al., 2023), and sulfides, such as dimethyl sulfone, produced from the decomposition of sulfur-containing amino acids (Liu et al., 2022d). Further analysis using the KEGG database revealed some differential metabolites to be explicitly involved in metabolic processes, including (R)-(+)-2-pyrrolidone-5-carboxylic acid, 2-hydroxycinnamic acid, linamarin, phenylacetic acid, L-proline, 2-phenylacetamide, pyroglutamic acid, 2-isopropylmalic acid, β-D-3-ribofuranosyluric acid, L-4-hydroxyglutamate semi-aldehyde, acetylcholine, and D-1-piperideine-2-carboxylic acid. Their relative horizontal changes are reflected in a volcano plot (Figure 9A).

Figure 8. Bubble chart of significantly enriched metabolic pathways in D-Lp (A) and D-Lf (B). The horizontal axis represents the enrichment significance p-value. The smaller the p-value, the more significant it is in statistics. Generally, a p-value less than 0.05 indicates a significant enrichment term for this function; The vertical axis represents the KEGG pathway. The size of the bubbles in the figure represents the amount of compound enriched in the metabolic concentration in this pathway. (D) control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

Figure 9. Volcano plot of metabolic products in D-Lp vs D model (A) and D-Lf vs D (B). FC stands for fold change. Red represents a significant increase, while blue represents a significant decrease. The larger the sample point, the larger the VIP. (D) control sample fermented with only the yogurt starter; D-Lp: sample fermented with yogurt starter and Lp. plantarum K25; D-Lf: sample fermented with yogurt starter and L. fermentum 13-1.

Figure 8B illustrates the metabolic pathway changes in D-Lf based on differential metabolites compared to D. Changes in amino acid metabolism in D-Lf were similar to those in D-Lp, but with different degrees of impact, and these pathways included phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, cyanoamino acid metabolism, and arginine and proline metabolism. Furthermore, the increased promotion of tyrosine metabolism in D-Lf was attributed to the significantly decreased levels of normetanephrine and maleic acid (p < 0.05) (Figures 8B and 9B). In contrast to D-Lp, the degradation of toluene and bisphenol was significantly promoted in D-Lf, as indicated by the discovery of 4-hydroxybenzaldehyde and P-salicylic acid. It was reported that 4-hydroxybenzaldehyde was present as a natural benzaldehyde in Gastrodia elata (Loh et al., 2022), and it possessed wound-healing properties and potential therapeutic effects in treating headaches (Chen et al., 2021). P-Salicylic acid, also known as 4-hydroxybenzoic acid, has antioxidant, antibacterial, and cardioprotective activities (Joshi et al., 2022). In the toluene degradation pathway, P-salicylic acid is directly converted from 4-hydroxybenzaldehyde by catalyzing with 4-hydroxybenzaldehyde dehydrogenase. N-acetyl-L-glutamate, derived from L-glutamate, was observed with a twice higher level in D-Lf than that in D (Figure 9B), and it exerted its primary biological activity by modulating the allosteric regulation of carbamoyl phosphate synthetase I (Harper et al., 2009).

As shown in Figure 10, a notable rise in phenylacetic acid and significantly decreased levels of 2-phenylacetamide and 2-hydroxycinnamic acid in D-Lp indicated the promoted phenylalanine metabolism (p < 0.05). Phenylacetic acid acted as an inhibitor of tyrosinase, a copper-containing enzyme catalyzing browning reaction in fruits and vegetables, and it also had antimicrobial activity (Zhu et al., 2011).

Figure 10. The metabolic pathway network of amino acids based on KEGG analysis. The colored metabolites indicate the identified metabolites, with red indicating increased abundance and green indicating decreased abundance. Dashed arrows represent multi-step conversion, while solid arrows represent single-step reactions, with adjacent enzymes in blue catalyzing this step.

The substantially decreased level of L-proline accompanied by the increased content of L-4-hydroxyglutamate semi-aldehyde (p < 0.05) reflected stimulation of proline metabolism. The level of pyroglutamic acid (pGlu) significantly increased (p < 0.05) due to the intramolecular dehydration and cyclization of L-glutamate in glutathione metabolism, and pGlu had antitumor, mitotic promotion, antidiabetic, and lipid-lowering effects (Aiello et al., 2022).

Moreover, (R)-(+)-2-pyrrolidone-5-carboxylic acid, linamarin, 2-isopropylmalic acid, and β-D-3-ribofuranosyluric acid were involved in several metabolism pathways, including D-amino acid metabolism, cyanogenic amino acid metabolism, and metabolism for the synthesis of valine, leucine, isoleucine, and purine. As a precursor for the biosynthesis of L-leucine and L-valine, 2-isopropylmalic acid had mild antioxidant activity and weak anti-pathogenic activity against some food-borne pathogens (Ricciutelli et al., 2020). Linamarin had neurotoxic effects, and it could induce motor disorders in mice (Rivadeneyra-Domínguez et al., 2013).

The significantly increased expression of D-1-piperideine-2-carboxylic acid (p < 0.05) indicated promoted lysine degradation by addition of Lp. plantarum K25. In addition, among the 24 specific differential metabolites in D-Lp, acetylcholine (Ach) was significantly up-regulated (Figure 8). ACh is a neurotransmitter widely present in the peripheral and central nervous systems, and it plays an important role in cognitive functions, encompassing thought processing, learning, and memory (Chen et al., 2023). As an oral flavonoid, poncirin is a flavonoid glycoside derivative with multiple pharma and other therapeutic effects (Yousof Ali et al., 2020), such as gastric protection (Lee et al., 2009), promotion of osteoblast differentiation (Yoon et al., 2011), and anti-inflammatory effects (Kang and Kim, 2016).

Conclusions

Addition of different fermentation types of LAB strains, such as the homofermentative Lp. plantarum K25 and heterofermentative L. fermentum 13-1, was shown to promote yogurt fermentation. Lp. plantarum K25 could increase the content of 2,3-butanedione and hexanoic acid whereas L. fermentum 13-1 caused a loss of 2,3-butanedione and acetoin, and greatly increased the content of acetic acid and ethanol in fermented milk. Both Lp. plantarum K25 and L. fermentum 13-1 could significantly reduce the content of bitter amino acids, such as L-Ile, L-Phe, and L-Try, bitter dipeptides, such as Gln-Pro and Leu-Gly in D-Lp sample, and bitter Arg-Pro in D-Lf sample, suggesting the potential of these two strains to reduce bitterness of fermented milk.

Further analysis of metabolomics revealed that L. fermentum 13-1 caused more metabolic changes in fermented milk than Lp. plantarum K25. Both strains had similar effects on several amino acid metabolism, including phenylalanine, valine, leucine, isoleucine, arginine, and proline, although Lp. plantarum K25 promoted lysine degradation, and L. fermentum 13-1 enhanced tyrosine metabolism. Moreover, a significant increase in bioactive compounds (p < 0.05) was found in fermented milk, for example, Ach associated with cognitive function and medicinal compound poncirin in D-Lp, suggesting the potential of these two strains to improve the functional characteristics of fermented milk. Future studies must focus on the possible synergistic effects of different flavor compounds and consumer perception-based sensory tests to evaluate changes in sensory characteristics. Further, future research should also concentrate on in vivo animal models to investigate the functional aspects of fermented milk by LAB strains of different fermentation types.

Conflicts of Interest

The authors declared no conflict of interest.

Funding

This work was supported by the National Natural Science Foundation of China (Project No. 32272296).

Acknowledgement

Authors are thankful to Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R31), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors also express their gratitude to the Deanship of Scientific Research (DSR) at King Faisal University under the Project number [KFU242509].

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Supplementary

D vs D-Lp

Metabolite Significant Regulate KEGGCompound ID Mode CAS ID M/Z Retention time Formula Library ID HMDB Superclass
Dihydrodigoxin yes up pos 09-10-5297 824.4849 3.619167 C41H66O14 HMDB0041879 Lipids and lipid-like molecules
Glutaminyl-Proline yes down pos - 244.129 0.801833 C10H17N3O4 HMDB0028805 Organic acids and derivatives
(R)-(+)-2-Pyrrolidone-5-carboxylic acid yes up C02237 pos - 130.0494 1.048317 C5H7NO3 HMDB0060262; HMDB0000805 Organic acids and derivatives
N-(1-Deoxy-1-fructosyl)leucine yes up pos 34393-18-5 294.1546 1.2893 C12H23NO7 HMDB0037840 Organic acids and derivatives
2-Hydroxycinnamic acid yes down C01772 pos 583-17-5 165.0543 0.822833 C9H8O3 HMDB0134028; HMDB0062655; HMDB0002641 Phenylpropanoids and polyketides
Nummularine A yes up C10011 pos 53947-95-8 648.3797 3.39985 C36H49N5O6 HMDB0029336 Organic acids and derivatives
PE(18:4(6Z,9Z,12Z,15Z)/22:6(4Z,7Z,10Z, 13Z,16Z,19Z)) yes up pos - 784.4895 3.671333 C45H70NO8P HMDB0009210; LMGP02010768 Lipids and lipid-like molecules
Poncirin yes up C09830 pos 14941-08-3 617.189 0.761 C28H34O14 LMPK12140333; HMDB0037487 Phenylpropanoids and polyketides
N-(1-Deoxy-1-fructosyl)phenylalanine yes up pos 87251-83-0 328.14 2.131817 C15H21NO7 HMDB0037846 Organic acids and derivatives
Tyrosyl-Valine yes down pos - 263.1389 3.036867 C14H20N2O4 HMDB0029118 Organic acids and derivatives
DL-o-Tyrosine yes down pos - 182.0807 1.109483 C9H11NO3 HMDB0006050 Organic acids and derivatives
Lysyl-Proline yes up pos - 226.1549 1.17065 C11H21N3O3 HMDB0028959 Organic acids and derivatives
Hydroxyprolyl-Alanine yes up pos - 185.0912 1.191133 C8H14N2O4 HMDB0028856 Organic acids and derivatives
3-[(3-Methylbutyl)nitrosoamino]-2-butanone yes up pos 71016-15-4 151.1225 1.817767 C9H18N2O2 HMDB0033553 Organooxygen compounds
Methionyl-Hydroxyproline yes up pos - 245.0955 2.0884 C10H18N2O4S HMDB0028974 Organic acids and derivatives
Prolyl-Alanine yes up pos - 169.0966 2.126433 C8H14N2O3 HMDB0029010 Organic acids and derivatives
1,4’-Bipiperidine-1’-carboxylic acid yes up C16836 pos - 195.1489 2.446067 C11H20N2O2 HMDB0060336 Organoheterocyclic compounds
Linamarin yes down C01594 pos 554-35-8 280.1375 2.780217 C10H17NO6 HMDB0005008; HMDB0033699 Organoheterocyclic compounds
Isoleucyl-Arginine yes up pos - 270.1922 2.831383 C12H25N5O3 HMDB0028901 Organic acids and derivatives
Prednisone yes up C07370 pos 02-03-1953 422.192 3.38985 C21H26O5 LMST02030180; HMDB0014773 Lipids and lipid-like molecules
7,8-Dihydrovomifoliol9-[apiosyl-(1->6)-glucoside] yes up pos 177261-70-0 562.289 3.5565 C24H40O12 HMDB0029771 Lipids and lipid-like molecules
Permetin A yes up pos 71888-70-5 562.3438 3.6925 C54H92N12O12 HMDB0030527 Organic acids and derivatives
Phenylalanyl-Gamma-glutamate yes up pos - 316.1288 3.765333 C14H19N3O4 HMDB0029009 Organic acids and derivatives
Norerythromycin yes down pos - 670.4146 3.806817 C35H63NO13 HMDB0061026 Organic oxygen compounds
CL(8:0/18:2(9Z,11Z)/18:2(9Z,11Z)/18:2 (9Z,11Z)) yes up pos - 679.4163 3.984317 C71H126O17P2 HMDB0123884 Lipids and lipid-like molecules
14alpha-Hydroxyixocarpanolide yes up pos 107221-65-8 521.3085 4.016317 C28H40O7 HMDB0034334 Lipids and lipid-like molecules
Amaranthussaponin II yes up pos 139742-10-2 497.2374 4.1308 C48H74O20 HMDB0041352 Lipids and lipid-like molecules
Dimethyl 3-methoxy-4-oxo-5-(8,11,14- pentadecatrienyl)-2-hexenedioate yes up pos - 904.5123 4.622617 C24H36O6 HMDB0032099 Organic acids and derivatives
Gibberellin A88 yes up pos 146959-87-7 394.1645 5.571217 C19H22O5 HMDB0039240 Lipids and lipid-like molecules
Ovalicin yes up C09674 pos 19683-98-8 360.1803 5.671717 C16H24O5 HMDB0038120 Organooxygen compounds
PE(17:1(9Z)/0:0) yes down pos - 466.2922 8.06525 C22H44NO7P LMGP02050008 -
CL(a-13:0/i-19:0/18:2(9Z,11Z)/i-20:0)[rac] yes up pos - 739.5023 8.918033 C79H150O17P2 HMDB0076304 Lipids and lipid-like molecules
CL(8:0/10:0/17:0/19:0) yes up pos - 618.4106 8.3409 C63H122O17P2 HMDB0117818 Lipids and lipid-like molecules
CL(18:0/22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,1 2Z)/22:5(4Z,7Z,10Z,13Z,16Z)) yes up pos - 788.5175 8.1769 C89H150O17P2 HMDB0057295 Lipids and lipid-like molecules
Persicaxanthin yes up pos 80952-82-5 385.2701 6.971483 C25H36O3 HMDB0034952 Lipids and lipid-like molecules
Cyclic 6-Hydroxymelatonin yes up pos - 211.0871 5.631383 C13H14N2O3 HMDB0060810 Organoheterocyclic compounds
Phenylacetic acid yes up C07086 pos 103-82-2 314.1391 5.631383 C8H8O2 HMDB0000209 Benzenoids
Ganglioside GD1a (d18:0/25:0) yes up pos - 991.0432 4.878433 C92H165N3O39 HMDB0011789 Lipids and lipid-like molecules
Tragopogonsaponin L yes up pos - 897.5081 4.694433 C50H74O15 HMDB0037926 Lipids and lipid-like molecules
Quinquenoside II yes down pos 208764-52-7 628.3416 4.48995 C62H104O24 HMDB0032815 Lipids and lipid-like molecules
Ganglioside GM2 (d18:0/24:0) yes up pos - 745.4557 4.05865 C74H134N2O26 HMDB0011904 Lipids and lipid-like molecules
Simonin IV yes up pos 151310-53-1 672.4087 3.84815 C68H120O24 HMDB0029977 Organic oxygen compounds
HMDB Class HMDB Subclass VIP_pred_ OPLS-DA VIP_PLS-DA FC(D_Lp/D) P_value FDR D-Lp1_1 D-Lp1_2 D-Lp1_3 D_1 D_2 D_3
Steroids and steroid derivatives Steroid lactones 1.4951 1.494 1.2553 8.77E-05 0.001111 4.3313 4.3489 4.4325 3.5544 3.4939 3.3975
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.3408 2.3399 0.2363 5.43E-07 4.85E-05 0.7066 0.6654 0.6386 2.8805 2.8529 2.7739
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.4963 1.4948 1.3741 4.43E-05 0.000708 3.2522 3.2372 3.2978 2.4339 2.398 2.2914
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.7263 1.7257 1.3951 1.38E-05 0.000325 4.1141 4.1526 4.2424 2.9601 3.041 2.9656
Cinnamic acids and derivatives Hydroxycinnamic acids and derivatives 2.2291 2.2203 0.324 0.01202 0.04243 0.0509 1.4303 1.5993 3.1903 3.2045 3.1166
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0392 2.0379 1.532 4.99E-06 0.00018 4.6926 4.7149 4.8024 3.1501 3.102 3.0239
Glycerophospholipids Glycerophosphoethanolamines 1.8972 1.8965 1.5055 3.66E-06 0.000155 4.2 4.2069 4.315 2.7988 2.8447 2.8068
Flavonoids Flavonoid glycosides 1.1407 1.1413 1.2053 0.001342 0.007784 3.1105 3.1326 3.1078 2.4655 2.5996 2.6933
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.4382 1.4366 1.2301 0.000389 0.003206 4.3998 4.3901 4.5064 3.6657 3.6712 3.4717
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.7645 1.7633 0.7294 3.52E-05 0.000607 3.2984 3.2576 3.43 4.6152 4.5707 4.5052
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.7156 1.7138 0.6847 9.17E-05 0.001143 2.4442 2.5274 2.6588 3.7319 3.7701 3.6388
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8727 1.8713 2.0637 1.47E-06 9.28E-05 2.6746 2.7462 2.6463 1.3044 1.3171 1.2886
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8938 1.894 2.2044 5.16E-05 0.000785 2.5769 2.5911 2.6626 1.0764 1.1525 1.3231
Carbonyl compounds Ketones 2.5545 2.5529 587.5256 1.51E-09 8.35E-06 2.5802 2.563 2.5985 0.0044 0.0045 0.0042
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8735 1.8715 3.5585 0.008182 0.03152 2.0931 1.7281 2.4227 0.3572 1.0492 0.3479
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8566 1.8549 1.8013 0.001687 0.009295 3.1523 3.0635 3.272 2.111 1.5796 1.5779
Piperidines Piperidinecarboxylic acids and derivatives 1.1555 1.156 1.2412 0.000284 0.002541 2.6704 2.7688 2.8208 2.2188 2.2321 2.2023
Benzimidazoles Sulfinylbenzimidazoles 1.8851 1.8837 0.5873 2.36E-05 0.000461 1.9646 1.9428 2.1045 3.4391 3.4591 3.3386
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8107 1.8078 2.5792 8.11E-05 0.001058 2.265 2.1197 1.9913 0.8248 0.8362 0.8107
Steroids and steroid derivatives Hydroxysteroids 2.2516 2.2504 2.9494 3.82E-07 4.10E-05 3.0042 2.9932 3.0907 1.0276 1.0397 1.0125
Fatty Acyls Fatty acyl glycosides 1.428 1.4298 1.8308 0.001606 0.008956 1.6462 1.8489 2.0256 1.0058 1.0179 0.9908
Peptidomimetics Depsipeptides 1.2005 1.2006 1.1825 0.00011 0.00128 3.6735 3.7074 3.797 3.1525 3.1658 3.1358
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.1883 2.1864 2.4985 0.001929 0.01025 3.2305 3.2527 3.3198 1.0467 1.8466 1.0316
Organooxygen compounds Carbohydrates and carbohydrate conjugates 1.413 1.4151 0.2907 0.006133 0.0253 0.2917 0.4591 0.2879 1.2729 1.3996 0.9014
Glycerophospholipids Glycerophosphoglycerophosphoglycerols 2.5503 2.5488 2.9164 7.33E-08 2.19E-05 3.8734 3.8933 3.9585 1.341 1.3537 1.3251
Steroids and steroid derivatives Steroid lactones 1.0286 1.0287 1.2667 0.001262 0.007441 2.0267 1.9677 2.1462 1.6173 1.6303 1.6011
Prenol lipids Terpene glycosides 1.1569 1.1573 1.2055 0.000388 0.0032 3.0862 3.1339 3.2484 2.6192 2.6325 2.6026
Keto acids and derivatives Beta-keto acids and derivatives 1.2255 1.2257 1.2669 0.000163 0.00168 2.784 2.9029 2.8711 2.2065 2.2943 2.2567
Prenol lipids Terpene lactones 1.7839 1.7846 2.8456 0.001967 0.01038 2.0561 2.0376 1.9449 0.784 0.3682 0.97
Carbonyl compounds Ketones 2.174 2.1719 4.6344 7.43E-06 0.000218 2.4002 2.342 2.4068 0.4961 0.6241 0.4223
- - 1.5309 1.5304 0.2716 0.006922 0.0277 0.6095 0.2584 0.2432 1.2715 1.1578 1.6637
Glycerophospholipids Glycerophosphoglycerophosphoglycerols 1.7646 1.7615 2.2528 0.00013 0.001432 2.3941 2.1624 2.1222 0.9891 1.0011 0.9742
Glycerophospholipids Glycerophosphoglycerophosphoglycerols 1.7846 1.7814 2.0059 0.007894 0.03061 3.0732 2.7288 2.2929 1.2645 1.1221 1.6469
Glycerophospholipids Glycerophosphoglycerophosphoglycerols 1.5002 1.495 1.4876 0.00622 0.02556 3.2098 2.8471 2.5924 1.9393 1.9525 1.9228
Prenol lipids Diterpenoids 1.3584 1.3603 2.6599 0.002628 0.01297 0.9969 1.3642 1.3091 0.4604 0.4692 0.4496
Indoles and derivatives Pyrroloindoles 1.9765 1.9762 10.6634 0.00054 0.00404 1.8253 1.7666 1.6171 0.0196 0.0202 0.4486
Benzene and substituted derivatives - 2.0235 2.0234 12.9736 8.60E-05 0.001097 1.6721 1.6595 1.9684 0.1364 0.1401 0.132
Sphingolipids Glycosphingolipids 1.4706 1.4711 1.4319 0.000751 0.005062 2.8036 2.9119 2.9888 1.8937 2.1623 2.0231
Prenol lipids Triterpenoids 1.0553 1.057 1.1272 0.003956 0.01784 4.0013 4.1884 4.2002 3.5863 3.7387 3.6678
Prenol lipids Triterpenoids 1.0947 1.0923 0.8787 0.003822 0.01734 3.488 3.6431 3.6443 3.9682 4.1866 4.1103
Sphingolipids Glycosphingolipids 2.6266 2.625 3.2408 2.36E-06 0.000118 3.8991 3.9289 4.0044 1.3379 1.1687 1.145
Organooxygen compounds Carbohydrates and carbohydrate conjugates 2.905 2.9039 4.0466 9.89E-07 7.21E-05 4.3792 4.4205 4.4922 0.9906 1.0998 1.1934
Metabolite Significant Regulate KEGGCompound ID Mode CAS ID M/Z Retention time Formula Library ID HMDB Superclass
L-Pyridosine yes up pos 31489-08-4 277.118 3.682333 C12H18N2O4 HMDB0029443 Organic acids and derivatives
Capsianside B yes down pos 121924-07-0 783.4093 3.661333 C76H124O33 HMDB0030733 Lipids and lipid-like molecules
Glutamylisoleucine yes up pos 5879-22-1 243.1336 3.5455 C11H20N2O5 HMDB0028822 Organic acids and derivatives
L-phenylalanyl-L-hydroxyproline yes up pos - 261.1231 3.473183 C14H18N2O4 HMDB0011176 Organic acids and derivatives
Schizonepetoside E yes up C17638 pos - 349.1846 3.462517 C16H28O8 - -
Methionyl-Proline yes up pos - 229.1001 3.27585 C10H18N2O3S HMDB0028981 Organic acids and derivatives
Isoleucyl-Hydroxyproline yes up pos - 227.1386 3.27585 C11H20N2O4 HMDB0028908 Organic acids and derivatives
Mabioside C yes down pos 156980-54-0 856.4407 3.24535 C42H64O14 HMDB0040653 Lipids and lipid-like molecules
Vinaginsenoside R1 yes up pos 156980-41-5 433.2516 3.172367 C44H74O15 HMDB0034624 Lipids and lipid-like molecules
54-Deoxyciguatoxin yes up pos 139341-09-6 559.2915 3.088367 C60H86O18 HMDB0029781 Phenylpropanoids and polyketides
Hoduloside X yes up pos 154971-14-9 547.2984 3.0677 C53H88O23 HMDB0040662 Lipids and lipid-like molecules
Tyrosyl-Proline yes down pos - 557.2608 2.780217 C14H18N2O4 HMDB0029113 Organic acids and derivatives
Uzarigenin 3-[xylosyl-(1->2)-rhamnoside] yes down pos 255861-29-1 338.1704 2.5584 C34H52O12 HMDB0036296 Lipids and lipid-like molecules
Hydroxyprolyl-Proline yes up pos - 211.1072 1.919267 C10H16N2O4 HMDB0028871 Organic acids and derivatives
L-Proline yes down C00148 pos 147-85-3 116.0703 1.807933 C5H9NO2 HMDB0000162 Organic acids and derivatives
Pyridinoline yes down pos 63800-01-1 429.1981 1.3408 C18H28N4O8 HMDB0000851 Organic acids and derivatives
L-Isoleucine yes down C00407 pos 73-32-5 132.1016 1.2998 C6H13NO2 HMDB0000172; HMDB0000557 Organic acids and derivatives
Glutamylalanine yes up pos 21064-18-6 201.0861 1.258967 C8H14N2O5 HMDB0003764 Organic acids and derivatives
2-Phenylacetamide yes down C02505 pos 103-81-1 136.0756 1.12015 C8H9NO HMDB0010715 Benzenoids
N-Benzylformamide yes down C15561 pos - 136.0755 0.833167 C8H9NO - -
Rac-4-Hydroxy-4-O-(beta-D-glucuronide)-all- trans-retinyl acetate yes down pos - 559.2347 0.811833 C28H40O9 HMDB0060141 Lipids and lipid-like molecules
Hydroxyprolyl-Hydroxyproline yes down pos - 245.1132 0.811833 C10H16N2O5 HMDB0028864 Organic acids and derivatives
Acetylcholine yes up C01996 pos 51-84-3 146.1172 0.750833 C7H15NO2 HMDB0000895 Organic nitrogen compounds
Vignatic acid A yes up neg 181485-19-8 552.2672 3.669033 C30H39N3O7 HMDB0033599 Organic acids and derivatives
Pyroglutamylvaline yes up neg 21282-10-0 227.1032 3.10265 C10H16N2O4 HMDB0094651 Organic acids and derivatives
Ganoderenic acid D yes up neg 100665-43-8 557.2579 3.834317 C30H40O7 HMDB0036059 Lipids and lipid-like molecules
D-(+)-3-Phenyllactic acid yes up neg - 165.056 3.985167 C9H10O3 - -
6-Acetyl-2,3-dihydro-2-(hydroxymethyl)- 4(1H)-pyridinone yes up neg 214218-63-0 190.0538 3.083317 C8H11NO3 HMDB0035178 Organoheterocyclic compounds
Austalide B yes up neg 81543-02-4 473.2039 4.85575 C26H34O8 HMDB0030004 Organoheterocyclic compounds
Pyroglutamic acid yes up C01879; C02237 neg 98-79-3 128.035 1.065783 C5H7NO3 HMDB0000267; HMDB0060262; HMDB0000805 Organic acids and derivatives
D-1-Piperideine-2-carboxylic acid yes up C04092 neg 2756-89-0 172.061 1.734133 C6H9NO2 HMDB0001084 Organoheterocyclic compounds
Megestrol yes up C07120 neg 3562-63-8 363.1749 1.792967 C21H28O3 LMST02030177; HMDB0014495 Lipids and lipid-like molecules
4-Oxo-13-cis-retinoate yes up neg - 349.1607 1.851983 C20H26O3 HMDB0012789 Lipids and lipid-like molecules
Jubanine A yes down neg 60375-07-7 716.3469 3.073417 C40H49N5O6 HMDB0030205 Organic acids and derivatives
Koryoginsenoside R1 yes down neg 171674-97-8 903.4839 3.092783 C46H76O15 HMDB0041351 Lipids and lipid-like molecules
Gamma-Glutamylphenylalanine yes up neg 7432-24-8 293.1145 3.131967 C14H18N2O5 HMDB0000594 Organic acids and derivatives
2-Isopropylmalic acid yes down C02504 neg 3237-44-3 175.0609 3.238817 C7H12O5 HMDB0000402 Lipids and lipid-like molecules
Licoricesaponin K2 yes down neg - 803.3805 3.248317 C42H62O16 HMDB0039250 Lipids and lipid-like molecules
Hoduloside VIII yes down neg 154971-12-7 951.469 3.53145 C46H76O18 HMDB0040660 Lipids and lipid-like molecules
Gamma-Glutamyltryptophan yes up neg 66471-20-3 314.114 3.786417 C16H19N3O5 HMDB0029160 Organic acids and derivatives
HMDB Class HMDB Subclass VIP_pred_ OPLS-DA VIP_PLS-DA FC(D_Lp/D) P_value FDR D-Lp1_1 D-Lp1_2 D-Lp1_3 D_1 D_2 D_3
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.5261 1.5255 1.4373 6.30E-06 0.000199 2.9934 3.005 3.0834 2.1066 2.1199 2.0901
Fatty Acyls Fatty acyl glycosides 1.5491 1.5475 0.7449 0.000205 0.002012 2.6947 2.7983 2.8982 3.8132 3.7849 3.6667
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.2674 2.2679 2.3573 0.000234 0.002181 3.5481 3.5641 3.6168 1.2293 1.5294 1.7938
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9651 1.9673 2.106 0.00129 0.007536 2.9429 2.9803 3.0795 1.3064 1.17 1.7998
- - 1.7584 1.758 2.158 7.05E-06 0.000211 2.2074 2.2943 2.3427 1.0582 1.0704 1.0431
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0574 2.0564 2.4481 3.22E-06 0.000142 2.7851 2.8296 2.8741 1.2281 1.1035 1.1356
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0222 2.0274 2.3894 0.01532 0.05015 3.0576 3.1386 3.1375 0.9466 0.774 2.1856
Prenol lipids Terpene glycosides 1.2574 1.2566 0.8618 0.000486 0.003768 3.9219 3.9259 4.0485 4.6603 4.6302 4.5129
Prenol lipids Terpene glycosides 1.378 1.3784 1.4067 0.007597 0.02973 2.7845 2.7358 2.8738 1.6971 2.2367 2.0333
Ciguatera toxins - 1.4321 1.4331 2.5644 0.00042 0.003401 1.2022 1.4093 1.4483 0.5283 0.5378 0.5167
Prenol lipids Triterpenoids 2.4091 2.4075 3.0194 2.91E-07 3.82E-05 3.4309 3.3733 3.4769 1.1272 1.166 1.1119
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0343 2.0343 0.4709 2.81E-05 0.000525 1.5017 1.4594 1.4262 3.2157 3.1345 2.9659
Steroids and steroid derivatives Steroid lactones 2.1551 2.1518 0.2026 0.004433 0.01947 0.1867 0.1719 1.1163 2.6634 2.2789 2.3379
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.6788 1.6804 2.0259 0.005983 0.02478 2.4004 1.984 2.6609 1.2944 0.9526 1.2304
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.3947 2.3933 0.1333 1.78E-08 1.43E-05 0.371 0.3464 0.3278 2.5927 2.631 2.6144
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8602 1.8583 0.6266 0.000257 0.002349 2.2253 2.2091 2.5279 3.7541 3.7445 3.6133
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.4068 1.4057 0.8133 0.000123 0.00138 3.3758 3.4156 3.5053 4.252 4.263 4.1463
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.5164 1.518 1.6589 0.001333 0.007739 2.2847 2.3482 2.4586 1.2261 1.4603 1.5883
Benzene and substituted derivatives Phenylacetamides 2.3969 2.3956 0.232 1.40E-05 0.000326 0.6373 0.8458 0.5784 3.0226 2.9691 2.8956
- - 1.9846 1.9842 0.4756 1.15E-05 0.000283 1.4654 1.4635 1.3152 3.0343 2.9564 2.935
Prenol lipids Terpene glycosides 1.7572 1.7565 0.6599 2.56E-06 0.000121 2.4144 2.3118 2.38 3.6049 3.5883 3.5768
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.5928 1.5918 0.7014 4.54E-06 0.000174 2.3243 2.3969 2.3539 3.3759 3.3883 3.3206
Organonitrogen compounds Quaternary ammonium salts 1.182 1.1815 1.1804 5.43E-05 0.000811 3.5876 3.6453 3.6604 3.1164 3.066 3.0454
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.1539 2.1509 3.0281 0.000109 0.001493 1.9426 2.0015 2.2346 0.6697 0.6585 0.7125
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0925 2.0979 1.7731 0.000166 0.001933 3.0025 2.9869 3.0594 1.7786 1.8108 1.5151
Prenol lipids Triterpenoids 1.8445 1.8457 1.4517 3.48E-05 0.000706 3.2031 3.2266 3.3186 2.3045 2.1851 2.2232
- - 1.3007 1.3024 1.1228 4.60E-06 0.000201 4.606 4.5772 4.5912 4.1138 4.0754 4.0784
Pyridines and derivatives Hydropyridines 1.3024 1.2825 1.3431 0.04999 0.1525 2.2709 2.3478 2.4048 1.3202 1.9816 1.9269
Benzopyrans 1-benzopyrans 1.3016 1.3002 1.2423 4.78E-05 0.000861 2.5889 2.5295 2.6044 2.0513 2.0652 2.0988
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.7331 2.7333 3.8747 1.94E-05 0.000479 3.091 2.838 3.0327 0.6956 0.8782 0.739
Pyridines and derivatives Hydropyridines 1.0683 1.0792 1.1829 0.03046 0.1017 2.5229 2.5638 2.7302 2.355 2.252 2.0007
Steroids and steroid derivatives Pregnane steroids 1.824 1.8213 1.8595 3.54E-05 0.000708 2.0618 2.1161 2.2148 1.1334 1.1201 1.1834
Prenol lipids Retinoids 2.5056 2.5028 8.6879 1.64E-05 0.000438 1.9498 2.1893 2.1589 0.2358 0.2298 0.2591
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.1374 1.1304 0.8434 0.03079 0.1027 2.3511 2.1949 2.5938 2.9147 2.8663 2.686
Prenol lipids Triterpenoids 1.0131 1.0056 0.9291 0.008124 0.03543 4.223 4.1691 4.3106 4.6447 4.5609 4.4659
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.2955 2.3037 2.6647 0.000493 0.004075 2.5054 2.553 2.6131 1.2602 0.7805 0.838
Fatty Acyls Fatty acids and conjugates 1.083 1.0786 0.9182 0.001071 0.00724 3.9437 3.9277 4.009 4.3661 4.3234 4.251
Prenol lipids Terpene glycosides 2.1922 2.1951 0.2725 0.000904 0.006305 0.3932 0.3805 0.8675 2.0387 1.9275 2.0546
Prenol lipids Triterpenoids 1.1057 1.0995 0.8672 0.01685 0.06335 2.5554 2.5212 2.7552 3.0885 3.0737 2.8704
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.8486 2.8472 3.6676 9.80E-08 2.90E-05 3.2724 3.2855 3.3278 0.8836 0.8711 0.9405
Metabolite Significant Regulate KEGGCompound ID Mode CAS ID M/Z Retention time Formula Library ID HMDB Superclass
5,9,11-trihydroxyprosta-6E,14Z-dien-1-oate yes up neg 80998-07-8 584.2476 3.824967 C30H37NO8 HMDB0062413 Lipids and lipid-like molecules
2-Hepteneoylglycine yes down neg - 206.0815 4.118383 C9H15NO3 HMDB0094728 Organic acids and derivatives
Lucidenic acid N yes up neg 364622-33-3 495.2539 4.539633 C27H40O6 HMDB0038352 Lipids and lipid-like molecules
Neuromedin N yes up C15868 neg 102577-25-3 654.3275 5.166483 C32H51N5O7 HMDB0013022 Organic acids and derivatives
Simvastatin yes up neg 79902-63-9 463.2665 5.389917 C25H38O5 HMDB0005007 Organoheterocyclic compounds
1-(beta-D-Glucopyranosyloxy)-3-octanone yes up neg 194919-40-9 341.1343 5.56715 C14H26O7 HMDB0031315 Lipids and lipid-like molecules
Homocapsaicin yes up C20215 neg 58493-48-4 364.2116 6.375883 C19H29NO3 HMDB0036329 Benzenoids
3-trans-Caffeoyltormentic acid yes up neg 144604-16-0 671.3502 7.393667 C39H54O8 HMDB0040650 Lipids and lipid-like molecules
PS(18:4(6Z,9Z,12Z,15Z)/18:4(6Z,9Z,1 2Z,15Z)) yes up neg - 796.4245 5.059 C42H66NO10P HMDB0112496; LMGP03010441 Lipids and lipid-like molecules
Pubesenolide yes up neg 98569-64-3 493.2765 5.01 C28H42O5 HMDB0033728 Lipids and lipid-like molecules
Neoporrigenin B yes up neg 196607-74-6 481.2757 4.980767 C27H42O5 HMDB0032680 Lipids and lipid-like molecules
Ganoderic acid C2 yes up neg 98296-48-1 555.2682 4.95125 C30H46O7 HMDB0035304 Lipids and lipid-like molecules
Esculentoside E yes up neg 65649-36-7 649.356 4.597833 C35H54O11 HMDB0034635 Lipids and lipid-like molecules
Dihydrozeatin-9-N-glucoside-O-glucoside yes up neg - 580.2053 4.386333 C22H35N5O11 HMDB0012212 Lipids and lipid-like molecules
Tyramine glucuronide yes up C03033 neg 27972-85-6 294.0979 4.17725 C14H19NO7 HMDB0010328 Organic oxygen compounds
3-hydroxyhexanoyl carnitine yes up neg - 334.1862 3.937833 C14H27NO5 HMDB0061633 Lipids and lipid-like molecules
Blasticidin S yes up C02010 neg 2079-00-7 403.1829 3.862633 C17H26N8O5 HMDB0030452 Organooxygen compounds
Acetyl-DL-Leucine yes up C02710 neg - 172.0976 3.824967 C8H15NO3 HMDB0011756 Organic acids and derivatives
N-(1-Deoxy-1-fructosyl)histidine yes up neg 25020-13-7 338.0981 3.688683 C12H19N3O7 HMDB0037839 Organic acids and derivatives
PS(MonoMe(11,3)/MonoMe(9,5)) yes down neg - 876.4386 3.669033 C44H74NO12P HMDB0061605 Lipids and lipid-like molecules
Ganosporelactone A yes down neg 138008-04-5 547.2438 3.610117 C30H40O7 HMDB0036406 Lipids and lipid-like molecules
Bilastine yes up neg 202189-78-4 498.2543 3.4824 C28H37N3O3 HMDB0240232 Organoheterocyclic compounds
Saponin E yes down neg 85191-73-7 817.4346 3.472767 C42H68O14 HMDB0035958 Lipids and lipid-like molecules
Digitoxin yes down C06955 neg 71-63-6 785.4064 3.364417 C41H64O13 HMDB0015468; LMST01120018 Lipids and lipid-like molecules
Benzoquinoneacetic acid yes down neg 10275-07-7 165.0189 3.3453 C8H6O4 HMDB0002334 Organic oxygen compounds
Testosterone sulfate yes up neg 651-45-6 413.1664 3.287133 C19H28O5S LMST05020032; HMDB0002833 Lipids and lipid-like molecules
Isosakuranin yes up neg 491-69-0 447.1327 3.277517 C22H24O10 LMPK12140336; HMDB0029481 Phenylpropanoids and polyketides
Glycinoeclepin B yes up neg 103847-17-2 593.2555 3.10265 C31H42O9 HMDB0037035 Lipids and lipid-like molecules
3,4’,5-Trihydroxy-3’,7-dimethoxyflavanone yes down neg 37971-67-8 331.085 3.005333 C17H16O7 HMDB0037503 Phenylpropanoids and polyketides
CDP-DG(16:0/20:4(8Z,11Z,14Z,17Z)) yes down neg - 982.4909 3.005333 C48H81N3O15P2 HMDB0006974; LMGP13010009 Lipids and lipid-like molecules
(4S,6R)-p-Mentha-1,8-diene-6,7-diol 7-glucoside yes up neg 402593-54-8 365.1343 2.900617 C16H26O7 HMDB0039056 Lipids and lipid-like molecules
Alliospiroside D yes down neg 114317-58-7 775.3863 2.880733 C39H62O14 HMDB0030915 Lipids and lipid-like molecules
(S)-Nerolidol 3-O-[a-L-rhamnopyranosyl-(1->2)-b-D-glucopyranoside] yes down neg 130466-30-7 575.3047 2.8711 C27H46O10 HMDB0040844 Lipids and lipid-like molecules
Methylprednisolone yes down neg 83-43-2 395.1809 2.790333 C22H30O5 HMDB0015094 Lipids and lipid-like molecules
Prolylhydroxyproline yes down neg 18684-24-7 273.1086 2.170683 C10H16N2O4 HMDB0006695; HMDB0029018 Organic acids and derivatives
Beta-D-3-Ribofuranosyluric acid yes down C05513 neg 2124-54-1 299.0627 2.0088 C10H12N4O7 HMDB0029920 Organoheterocyclic compounds
Blumenol C glucoside yes up neg 62512-23-6 393.1867 1.4248 C19H32O7 HMDB0040668 Lipids and lipid-like molecules
N-(1-Deoxy-1-fructosyl)isoleucine yes up neg 87304-79-8 292.1396 1.321317 C12H23NO7 HMDB0039780 Organic acids and derivatives
L-4-Hydroxyglutamate semialdehyde yes up C05938 neg - 128.0352 0.842117 C5H9NO4 HMDB0006556 Organic acids and derivatives
Neuraminic acid yes down C06469 neg 114-04-5 266.0873 0.690617 C9H17NO8 HMDB0000830 Organic oxygen compounds
HMDB Class HMDB Subclass VIP_pred_ OPLS-DA VIP_PLS-DA FC(D_Lp/D) P_value FDR D-Lp1_1 D-Lp1_2 D-Lp1_3 D_1 D_2 D_3
Prenol lipids Diterpenoids 1.4643 1.4619 1.3326 0.000529 0.004261 2.5539 2.4917 2.6945 1.9232 1.9091 1.9763
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.4209 1.4174 0.7634 9.60E-05 0.001387 1.9924 1.8785 1.9291 2.57 2.5094 2.5164
Prenol lipids Triterpenoids 1.6388 1.6405 1.4966 0.000135 0.001703 2.3381 2.4699 2.4516 1.6916 1.5852 1.5735
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.1004 2.0996 3.2233 1.62E-05 0.000436 1.9077 1.8016 1.9764 0.578 0.5675 0.6181
Lactones Delta valerolactones 1.455 1.4593 1.4331 0.000433 0.003715 2.1423 2.0349 2.1852 1.5246 1.5158 1.401
Fatty Acyls Fatty acyl glycosides 1.4073 1.412 1.6109 0.00037 0.003366 1.6446 1.4872 1.6031 1.0254 0.9778 0.9357
Benzene and substituted derivatives Phenols and derivatives 2.1982 2.2078 13.5415 0.000469 0.003903 1.8003 1.5042 1.4442 0.2895 0.0245 0.0367
Prenol lipids Triterpenoids 1.4254 1.4156 1.5181 0.002405 0.01325 1.7878 1.7775 1.8599 1.02 1.2552 1.2987
Glycerophospholipids Glycerophosphoserines 1.9715 1.9705 2.4226 1.19E-06 0.000104 1.9633 1.924 1.9809 0.7962 0.7842 0.8417
Steroids and steroid derivatives Steroid lactones 2.6952 2.7014 13.8826 8.65E-05 0.001302 2.3153 2.305 2.4063 0.4284 0.0103 0.0675
Steroids and steroid derivatives Steroidal glycosides 1.671 1.6688 1.4031 1.95E-05 0.000479 2.8433 2.8501 2.9323 2.0361 2.0219 2.0893
Prenol lipids Triterpenoids 1.0168 1.0158 1.1689 0.004287 0.02085 2.2285 2.1625 2.3128 1.8728 1.9799 1.884
Prenol lipids Triterpenoids 1.3948 1.3936 2.1993 0.01112 0.04515 0.9617 1.1297 1.3647 0.4995 0.67 0.4018
Fatty Acyls Fatty acyl glycosides 2.886 2.8852 1197471 2.89E-07 4.82E-05 2.4131 2.4388 2.5331 0 0 0
Organooxygen compounds Carbohydrates and carbohydrate conjugates 1.4016 1.3934 1.3821 0.001126 0.007448 2.1231 2.1737 2.1059 1.4223 1.5538 1.6562
Fatty Acyls Fatty acid esters 1.4319 1.434 1.9226 0.005216 0.02438 1.553 1.3166 1.1678 0.643 0.7027 0.7548
Carbohydrates and carbohydrate conjugates Sugar acids and derivatives 2.3564 2.3564 2.0505 1.43E-06 0.000114 3.1634 3.1813 3.2749 1.5853 1.5573 1.5484
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.7136 1.7158 1.4545 5.13E-05 0.000911 2.7937 2.7455 2.8619 1.9504 1.9661 1.8586
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.6615 1.6596 1.5237 2.72E-05 0.00059 2.3603 2.3325 2.4396 1.5471 1.5331 1.5993
Glycerophospholipids Glycerophosphoserines 1.7314 1.7307 0.7391 0.000448 0.003801 2.4338 2.5388 2.6877 3.4979 3.495 3.3695
Steroids and steroid derivatives Steroid lactones 1.0779 1.0751 0.8818 0.000322 0.003079 2.5717 2.5633 2.6296 2.9746 2.93 2.9002
Benzimidazoles - 1.51 1.5066 1.4619 0.000159 0.001892 2.0717 2.1399 2.2266 1.4555 1.4417 1.5074
Prenol lipids Terpene glycosides 1.2408 1.2348 0.884 0.003464 0.01771 3.5561 3.5715 3.6866 4.1567 4.1277 3.9504
Steroids and steroid derivatives Steroid lactones 1.4548 1.4556 0.6764 0.000221 0.002344 1.3395 1.3577 1.2773 1.977 1.876 2.0235
Organooxygen compounds Carbonyl compounds 1.3285 1.3175 0.7763 0.02584 0.08993 2.3755 1.8591 1.9169 2.7421 2.5801 2.6035
Steroids and steroid derivatives Sulfated steroids 1.9307 1.929 2.17 1.79E-05 0.00046 2.0267 1.9872 2.1286 0.9321 0.9195 0.9798
Flavonoids Flavonoid glycosides 1.8457 1.8499 1.6189 0.001425 0.00892 2.6704 2.7776 2.7436 1.6956 1.9058 1.4584
Prenol lipids Sesquiterpenoids 2.4735 2.4709 4.3932 9.77E-06 0.000315 2.2342 2.3639 2.4293 0.4865 0.5895 0.5234
Flavonoids O-methylated flavonoids 1.4637 1.4663 0.6884 0.002065 0.01166 1.5929 1.4174 1.3629 1.9946 2.1827 2.1764
Glycerophospholipids CDP-glycerols 1.2999 1.2964 0.8995 0.000659 0.004948 4.5024 4.5324 4.6071 5.1062 5.0879 4.9699
Prenol lipids Terpene glycosides 2.0815 2.0857 1.878 0.000252 0.002545 2.7043 2.7781 2.8783 1.6668 1.3602 1.4254
Steroids and steroid derivatives Steroidal glycosides 1.4143 1.4088 0.7734 0.006459 0.02927 2.0915 2.0064 2.3444 2.8794 2.7913 2.656
Fatty Acyls Fatty acyl glycosides 1.5405 1.5371 0.8351 0.000823 0.005881 3.5403 3.5937 3.7266 4.4044 4.3773 4.2238
Steroids and steroid derivatives Hydroxysteroids 2.4689 2.4661 0.1791 1.39E-06 0.000114 0.4451 0.3498 0.3826 2.2438 2.184 2.1479
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9978 2.004 0.4076 0.007823 0.03439 0.4302 0.895 1.3149 2.2082 2.1923 2.0758
Imidazopyrimidines Purines and purine derivatives 2.4684 2.4687 0.4457 0.001708 0.01019 1.4209 1.1131 1.9636 3.3853 3.3751 3.33
Fatty Acyls Fatty acyl glycosides 1.1048 1.104 1.2195 0.001114 0.007399 2.0926 2.0985 1.9777 1.6732 1.6591 1.7258
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.2313 1.2259 1.1846 0.002629 0.01421 2.8518 3.0778 3.0018 2.5004 2.4861 2.5539
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.7671 1.764 1.5088 0.002259 0.01259 2.6927 2.7827 3.0377 1.7889 2.0633 1.7907
Organooxygen compounds Carbohydrates and carbohydrate conjugates 1.3763 1.3707 0.7939 0.001492 0.009202 2.1582 2.284 2.1911 2.867 2.8274 2.6593
Metabolite Significant Regulate KEGGCompound ID Mode CAS ID M/Z Retention time Formula Library ID HMDB Superclass
Dihydrodigoxin yes up pos 09-10-5297 824.4849 3.619167 C41H66O14 HMDB0041879 Lipids and lipid-like molecules
Glutaminyl-Proline yes down pos - 244.129 0.801833 C10H17N3O4 HMDB0028805 Organic acids and derivatives
(R)-(+)-2-Pyrrolidone-5-carboxylic acid yes up C02237 pos - 130.0494 1.048317 C5H7NO3 HMDB0060262; HMDB0000805 Organic acids and derivatives
N-(1-Deoxy-1-fructosyl)leucine yes up pos 34393-18-5 294.1546 1.2893 C12H23NO7 HMDB0037840 Organic acids and derivatives
2-Hydroxycinnamic acid yes down C01772 pos 583-17-5 165.0543 0.822833 C9H8O3 HMDB0134028; HMDB0062655; HMDB0002641 Phenylpropanoids and polyketides
Nummularine A yes up C10011 pos 53947-95-8 648.3797 3.39985 C36H49N5O6 HMDB0029336 Organic acids and derivatives
PE(18:4(6Z,9Z,12Z,15Z)/22:6(4Z,7Z,10Z, 13Z,16Z,19Z)) yes up pos - 784.4895 3.671333 C45H70NO8P HMDB0009210; LMGP02010768 Lipids and lipid-like molecules
N-(1-Deoxy-1-fructosyl)phenylalanine yes up pos 87251-83-0 328.14 2.131817 C15H21NO7 HMDB0037846 Organic acids and derivatives
PG(18:1(9Z)/0:0) yes up pos - 533.2836 9.961467 C24H47O9P LMGP04050006 -
Tyrosyl-Valine yes down pos - 263.1389 3.036867 C14H20N2O4 HMDB0029118 Organic acids and derivatives
DL-o-Tyrosine yes down pos - 182.0807 1.109483 C9H11NO3 HMDB0006050 Organic acids and derivatives
Lysyl-Proline yes up pos - 226.1549 1.17065 C11H21N3O3 HMDB0028959 Organic acids and derivatives
Hydroxyprolyl-Alanine yes up pos - 185.0912 1.191133 C8H14N2O4 HMDB0028856 Organic acids and derivatives
3-[(3-Methylbutyl)nitrosoamino]-2-butanone yes up pos 71016-15-4 151.1225 1.817767 C9H18N2O2 HMDB0033553 Organooxygen compounds
Methionyl-Hydroxyproline yes up pos - 245.0955 2.0884 C10H18N2O4S HMDB0028974 Organic acids and derivatives
Prolyl-Alanine yes up pos - 169.0966 2.126433 C8H14N2O3 HMDB0029010 Organic acids and derivatives
1,4’-Bipiperidine-1’-carboxylic acid yes up C16836 pos - 195.1489 2.446067 C11H20N2O2 HMDB0060336 Organoheterocyclic compounds
Linamarin yes down C01594 pos 554-35-8 280.1375 2.780217 C10H17NO6 HMDB0005008; HMDB0033699 Organoheterocyclic compounds
Isoleucyl-Arginine yes up pos - 270.1922 2.831383 C12H25N5O3 HMDB0028901 Organic acids and derivatives
Prednisone yes up C07370 pos 02-03-1953 422.192 3.38985 C21H26O5 LMST02030180; HMDB0014773 Lipids and lipid-like molecules
7,8-Dihydrovomifoliol9-[apiosyl-(1->6)-glucoside] yes up pos 177261-70-0 562.289 3.5565 C24H40O12 HMDB0029771 Lipids and lipid-like molecules
Permetin A yes up pos 71888-70-5 562.3438 3.6925 C54H92N12O12 HMDB0030527 Organic acids and derivatives
Trigoneoside XIIIa yes up pos 290348-13-9 614.3097 3.7445 C57H94O28 HMDB0038313 Lipids and lipid-like molecules
Phenylalanyl-Gamma-glutamate yes up pos - 316.1288 3.765333 C14H19N3O4 HMDB0029009 Organic acids and derivatives
Ganglioside GM3 (d18:0/14:0) yes up pos - 575.3512 3.899983 C55H102N2O21 HMDB0011914 Lipids and lipid-like molecules
CL(8:0/18:2(9Z,11Z)/18:2(9Z,11Z)/18:2 (9Z,11Z)) yes up pos - 679.4163 3.984317 C71H126O17P2 HMDB0123884 Lipids and lipid-like molecules
Amaranthussaponin II yes up pos 139742-10-2 497.2374 4.1308 C48H74O20 HMDB0041352 Lipids and lipid-like molecules
Dimethyl 3-methoxy-4-oxo-5-(8,11,14- pentadecatrienyl)-2-hexenedioate yes up pos - 904.5123 4.622617 C24H36O6 HMDB0032099 Organic acids and derivatives
PA(22:5(4Z,7Z,10Z,13Z,16Z)/24:1(15Z)) yes down pos - 874.6345 4.673767 C49H85O8P HMDB0115375 Lipids and lipid-like molecules
Gibberellin A88 yes up pos 146959-87-7 394.1645 5.571217 C19H22O5 HMDB0039240 Lipids and lipid-like molecules
Ovalicin yes up C09674 pos 19683-98-8 360.1803 5.671717 C16H24O5 HMDB0038120 Organooxygen compounds
PE(17:1(9Z)/0:0) yes down pos - 466.2922 8.06525 C22H44NO7P LMGP02050008 -
CL(18:0/22:5(4Z,7Z,10Z,13Z,16Z)/18:2(9Z,1 2Z)/22:5(4Z,7Z,10Z,13Z,16Z)) yes up pos - 788.5175 8.1769 C89H150O17P2 HMDB0057295 Lipids and lipid-like molecules
Cyclic 6-Hydroxymelatonin yes up pos - 211.0871 5.631383 C13H14N2O3 HMDB0060810 Organoheterocyclic compounds
Phenylacetic acid yes up C07086 pos 103-82-2 314.1391 5.631383 C8H8O2 HMDB0000209 Benzenoids
Ganglioside GD1a (d18:0/25:0) yes up pos - 991.0432 4.878433 C92H165N3O39 HMDB0011789 Lipids and lipid-like molecules
Tragopogonsaponin L yes up pos - 897.5081 4.694433 C50H74O15 HMDB0037926 Lipids and lipid-like molecules
Quinquenoside II yes down pos 208764-52-7 628.3416 4.48995 C62H104O24 HMDB0032815 Lipids and lipid-like molecules
Ganglioside GM2 (d18:0/24:0) yes up pos - 745.4557 4.05865 C74H134N2O26 HMDB0011904 Lipids and lipid-like molecules
Simonin IV yes up pos 151310-53-1 672.4087 3.84815 C68H120O24 HMDB0029977 Organic oxygen compounds
L-Pyridosine yes up pos 31489-08-4 277.118 3.682333 C12H18N2O4 HMDB0029443 Organic acids and derivatives
Capsianside B yes down pos 121924-07-0 783.4093 3.661333 C76H124O33 HMDB0030733 Lipids and lipid-like molecules
HMDB Class HMDB Subclass VIP_pred_ OPLS-DA VIP_PLS-DA FC(D_Lf/D) P_value FDR D-Lf_1 D-Lf_2 D-Lf_3 D_1 D_2 D_3
Steroids and steroid derivatives Steroid lactones 1.5687 1.5618 1.2886 3.85E-05 0.000555 4.448 4.525 4.4879 3.5544 3.4939 3.3975
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.6562 1.6563 0.5956 0.000592 0.004155 1.6192 1.5408 1.9081 2.8805 2.8529 2.7739
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.4187 1.4088 1.3467 0.000186 0.001751 3.2306 3.1083 3.252 2.4339 2.398 2.2914
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.7862 1.7781 1.4343 3.22E-06 9.16E-05 4.241 4.2991 4.3197 2.9601 3.041 2.9656
Cinnamic acids and derivatives Hydroxycinnamic acids and derivatives 1.3972 1.3955 0.7454 0.000184 0.001746 2.4597 2.2724 2.3577 3.1903 3.2045 3.1166
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0635 2.0547 1.5602 1.58E-06 5.78E-05 4.7965 4.8367 4.8392 3.1501 3.102 3.0239
Glycerophospholipids Glycerophosphoethanolamines 1.9462 1.9389 1.547 5.41E-07 2.81E-05 4.3207 4.3991 4.3553 2.7988 2.8447 2.8068
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.491 1.4827 1.2528 0.000186 0.001751 4.4986 4.5544 4.4896 3.6657 3.6712 3.4717
- - 1.0575 1.054 1.1827 0.000746 0.004947 3.0061 3.0357 3.0085 2.649 2.5098 2.494
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.6757 1.6695 0.7487 3.98E-05 0.000566 3.3482 3.5118 3.3914 4.6152 4.5707 4.5052
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.332 1.3341 0.7989 0.000831 0.005389 3.0124 2.8239 3.0637 3.7319 3.7701 3.6388
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9358 1.927 2.1696 3.12E-06 8.96E-05 2.7768 2.7987 2.9067 1.3044 1.3171 1.2886
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.82 1.8132 2.1554 0.000474 0.00353 2.6488 2.3351 2.6725 1.0764 1.1525 1.3231
Carbonyl compounds Ketones 2.5453 2.5355 601.1837 7.30E-07 3.34E-05 2.549 2.6556 2.7177 0.0044 0.0045 0.0042
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8893 1.8717 3.5841 0.003625 0.01749 2.2559 2.0626 1.9687 0.3572 1.0492 0.3479
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9648 1.9585 1.9186 0.000927 0.005817 3.2751 3.4267 3.4047 2.111 1.5796 1.5779
Piperidines Piperidinecarboxylic acids and derivatives 1.2435 1.2369 1.2858 0.000177 0.001701 2.7749 2.8453 2.9352 2.2188 2.2321 2.2023
Benzimidazoles Sulfinylbenzimidazoles 1.8916 1.8853 0.5689 0.000218 0.001959 1.7524 2.1306 1.9412 3.4391 3.4591 3.3386
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9816 1.9759 2.9457 7.18E-06 0.000159 2.4134 2.5247 2.3441 0.8248 0.8362 0.8107
Steroids and steroid derivatives Hydroxysteroids 2.2282 2.2189 2.9649 8.95E-08 8.26E-06 3.0301 3.0179 3.0858 1.0276 1.0397 1.0125
Fatty Acyls Fatty acyl glycosides 1.8876 1.8806 2.4567 0.000153 0.001534 2.2604 2.5673 2.5807 1.0058 1.0179 0.9908
Peptidomimetics Depsipeptides 1.2467 1.2419 1.2009 1.68E-06 6.02E-05 3.7694 3.8073 3.7758 3.1525 3.1658 3.1358
Steroids and steroid derivatives Steroidal glycosides 1.2317 1.2284 1.2278 0.000898 0.005673 3.339 3.5031 3.4088 2.8844 2.762 2.7026
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.2083 2.1883 2.5535 0.001667 0.009325 3.3302 3.3134 3.375 1.0467 1.8466 1.0316
Sphingolipids Glycosphingolipids 1.006 0.9998 1.1405 8.31E-05 0.000975 3.3233 3.3179 3.3919 2.9327 2.946 2.9161
Glycerophospholipids Glycerophosphoglycerophosphoglycerols 2.5453 2.5355 2.9657 2.00E-09 9.85E-07 3.9593 3.9797 3.9841 1.341 1.3537 1.3251
Prenol lipids Terpene glycosides 1.2458 1.2401 1.2414 8.39E-06 0.000178 3.2126 3.2564 3.2809 2.6192 2.6325 2.6026
Keto acids and derivatives Beta-keto acids and derivatives 1.1605 1.153 1.2624 0.01026 0.04007 3.0514 2.614 2.864 2.2065 2.2943 2.2567
Glycerophospholipids Glycerophosphates 1.2621 1.2507 0.5382 0.03404 0.1045 0.9068 0.7412 1.0035 1.9788 1.21 1.737
Prenol lipids Terpene lactones 1.7262 1.7316 2.799 0.00218 0.01156 2.032 1.9106 1.9972 0.784 0.3682 0.97
Carbonyl compounds Ketones 2.151 2.1384 4.6558 1.49E-05 0.000278 2.4225 2.304 2.4556 0.4961 0.6241 0.4223
- - 1.3493 1.3236 0.3648 0.04196 0.1232 0.268 0.9984 0.2263 1.2715 1.1578 1.6637
Glycerophospholipids Glycerophosphoglycerophosphoglycerols 1.0107 1.0047 1.2549 0.04237 0.1242 2.707 2.1284 2.4599 1.9393 1.9525 1.9228
Indoles and derivatives Pyrroloindoles 1.7086 1.7091 8.7039 0.004103 0.01943 1.6261 1.1097 1.5138 0.0196 0.0202 0.4486
Benzene and substituted derivatives - 1.7705 1.7589 10.6608 0.0023 0.01209 1.6909 1.0757 1.589 0.1364 0.1401 0.132
Sphingolipids Glycosphingolipids 1.5333 1.5243 1.4793 0.00054 0.003871 2.8863 3.0328 3.0731 1.8937 2.1623 2.0231
Prenol lipids Triterpenoids 1.1136 1.1064 1.1416 0.001618 0.009134 4.0877 4.1938 4.2681 3.5863 3.7387 3.6678
Prenol lipids Triterpenoids 1.1483 1.1454 0.863 0.002665 0.01366 3.4274 3.6163 3.5408 3.9682 4.1866 4.1103
Sphingolipids Glycosphingolipids 2.629 2.6193 3.3131 1.46E-06 5.45E-05 4.0024 4.0516 4.0407 1.3379 1.1687 1.145
Organooxygen compounds Carbohydrates and carbohydrate conjugates 2.8747 2.865 4.0731 6.38E-07 3.14E-05 4.4283 4.4682 4.4823 0.9906 1.0998 1.1934
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.5222 1.5149 1.4482 1.89E-05 0.000331 2.9869 3.0407 3.121 2.1066 2.1199 2.0901
Fatty Acyls Fatty acyl glycosides 1.4321 1.4292 0.7747 0.00029 0.002416 2.8067 2.9978 2.9214 3.8132 3.7849 3.6667
Metabolite Significant Regulate KEGGCompound ID Mode CAS ID M/Z Retention time Formula Library ID HMDB Superclass
Glutamylisoleucine yes up pos 5879-22-1 243.1336 3.5455 C11H20N2O5 HMDB0028822 Organic acids and derivatives
L-phenylalanyl-L-hydroxyproline yes up pos - 261.1231 3.473183 C14H18N2O4 HMDB0011176 Organic acids and derivatives
Schizonepetoside E yes up C17638 pos - 349.1846 3.462517 C16H28O8 - -
Normetanephrine yes down C05589 pos 97-31-4 225.1229 3.316683 C9H13NO3 HMDB0000819 Benzenoids
Methionyl-Proline yes up pos - 229.1001 3.27585 C10H18N2O3S HMDB0028981 Organic acids and derivatives
Isoleucyl-Hydroxyproline yes up pos - 227.1386 3.27585 C11H20N2O4 HMDB0028908 Organic acids and derivatives
Mabioside C yes down pos 156980-54-0 856.4407 3.24535 C42H64O14 HMDB0040653 Lipids and lipid-like molecules
Vinaginsenoside R1 yes up pos 156980-41-5 433.2516 3.172367 C44H74O15 HMDB0034624 Lipids and lipid-like molecules
Mocimycin yes up pos 50935-71-2 819.4104 3.088367 C43H60N2O12 HMDB0030465 Organooxygen compounds
54-Deoxyciguatoxin yes up pos 139341-09-6 559.2915 3.088367 C60H86O18 HMDB0029781 Phenylpropanoids and polyketides
Hoduloside X yes up pos 154971-14-9 547.2984 3.0677 C53H88O23 HMDB0040662 Lipids and lipid-like molecules
Kinetensin 4-7 yes up pos 138482-56-1 604.3166 2.841383 C26H37N9O6 HMDB0012986 Organic acids and derivatives
Tyrosyl-Proline yes down pos - 557.2608 2.780217 C14H18N2O4 HMDB0029113 Organic acids and derivatives
Hydroxyprolyl-Proline yes up pos - 211.1072 1.919267 C10H16N2O4 HMDB0028871 Organic acids and derivatives
Gamma-Glutamylarginine yes up pos 31106-03-3 286.1512 1.8381 C11H21N5O5 HMDB0029143 Organic acids and derivatives
L-Proline yes down C00148 pos 147-85-3 116.0703 1.807933 C5H9NO2 HMDB0000162 Organic acids and derivatives
Pyridinoline yes down pos 63800-01-1 429.1981 1.3408 C18H28N4O8 HMDB0000851 Organic acids and derivatives
L-Isoleucine yes down C00407 pos 73-32-5 132.1016 1.2998 C6H13NO2 HMDB0000172; HMDB0000557 Organic acids and derivatives
Glutamylalanine yes up pos 21064-18-6 201.0861 1.258967 C8H14N2O5 HMDB0003764 Organic acids and derivatives
2-Phenylacetamide yes down C02505 pos 103-81-1 136.0756 1.12015 C8H9NO HMDB0010715 Benzenoids
N-Acetyl-L-glutamic acid yes up C00624 pos - 190.0706 1.109483 C7H11NO5 HMDB0001138 Organic acids and derivatives
N-Benzylformamide yes down C15561 pos - 136.0755 0.833167 C8H9NO - -
2-amino-6-hydroxyhexanoic acid yes up pos - 148.0963 0.833167 C6H13NO3 HMDB0142963 Organic acids and derivatives
Rac-4-Hydroxy-4-O-(beta-D-glucuronide)-all- trans-retinyl acetate yes down pos - 559.2347 0.811833 C28H40O9 HMDB0060141 Lipids and lipid-like molecules
Hydroxyprolyl-Hydroxyproline yes down pos - 245.1132 0.811833 C10H16N2O5 HMDB0028864 Organic acids and derivatives
Vignatic acid A yes up neg 181485-19-8 552.2672 3.669033 C30H39N3O7 HMDB0033599 Organic acids and derivatives
Pyroglutamylvaline yes up neg 21282-10-0 227.1032 3.10265 C10H16N2O4 HMDB0094651 Organic acids and derivatives
Ganoderenic acid D yes up neg 100665-43-8 557.2579 3.834317 C30H40O7 HMDB0036059 Lipids and lipid-like molecules
D-(+)-3-Phenyllactic acid yes up neg - 165.056 3.985167 C9H10O3 - -
Austalide B yes up neg 81543-02-4 473.2039 4.85575 C26H34O8 HMDB0030004 Organoheterocyclic compounds
Pyroglutamic acid yes up C01879; C02237 neg 98-79-3 128.035 1.065783 C5H7NO3 HMDB0000267; HMDB0060262; HMDB0000805 Organic acids and derivatives
4-Oxo-13-cis-retinoate yes up neg - 349.1607 1.851983 C20H26O3 HMDB0012789 Lipids and lipid-like molecules
Suxibuzone yes up neg 27470-51-5 473.151 2.4556 C24H26N2O6 HMDB0042019 Lipids and lipid-like molecules
Glaucarubolone15-O-beta-D-glucopyranoside yes up neg 89202-76-6 537.1949 2.7704 C26H36O13 HMDB0035036 Lipids and lipid-like molecules
Gamma-Glutamylphenylalanine yes up neg 7432-24-8 293.1145 3.131967 C14H18N2O5 HMDB0000594 Organic acids and derivatives
2-Isopropylmalic acid yes down C02504 neg 3237-44-3 175.0609 3.238817 C7H12O5 HMDB0000402 Lipids and lipid-like molecules
Licoricesaponin K2 yes down neg - 803.3805 3.248317 C42H62O16 HMDB0039250 Lipids and lipid-like molecules
Mevalonolactone yes down neg 503-48-0 129.056 3.502 C6H10O3 HMDB0006024 Organoheterocyclic compounds
Hoduloside VIII yes down neg 154971-12-7 951.469 3.53145 C46H76O18 HMDB0040660 Lipids and lipid-like molecules
4-Hydroxybenzaldehyde yes down C00633 neg 123-08-0 121.0296 3.61965 C7H6O2 HMDB0011718 Organic oxygen compounds
Gamma-Glutamyltryptophan yes up neg 66471-20-3 314.114 3.786417 C16H19N3O5 HMDB0029160 Organic acids and derivatives
HMDB Class HMDB Subclass VIP_pred_ OPLS-DA VIP_PLS-DA FC(D_Lf/D) P_value FDR D-Lf_1 D-Lf_2 D-Lf_3 D_1 D_2 D_3
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.2819 2.2778 2.4166 0.000194 0.001799 3.6565 3.6882 3.6522 1.2293 1.5294 1.7938
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9354 1.9393 2.1095 0.001225 0.007272 3.0347 2.9465 3.0376 1.3064 1.17 1.7998
- - 1.6865 1.6803 2.1041 0.000101 0.001126 2.0748 2.3059 2.2924 1.0582 1.0704 1.0431
Phenols Methoxyphenols 1.0815 1.0793 0.818 0.001876 0.0102 2.3509 2.1233 2.2009 2.7169 2.7015 2.741
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.042 2.0353 2.4697 1.77E-06 6.21E-05 2.8332 2.879 2.8532 1.2281 1.1035 1.1356
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0311 2.0497 2.4485 0.01324 0.04935 3.2063 3.1757 3.1828 0.9466 0.774 2.1856
Prenol lipids Terpene glycosides 1.2174 1.2164 0.8661 0.000531 0.003834 3.9061 4.0412 4.0089 4.6603 4.6302 4.5129
Prenol lipids Terpene glycosides 1.4252 1.4169 1.4449 0.007109 0.03019 2.7294 2.9819 2.9098 1.6971 2.2367 2.0333
Carbohydrates and carbohydrate conjugates Glycosyl compounds 1.1262 1.1334 1.4137 0.04956 0.1406 1.7616 2.5491 2.2721 1.5526 1.5656 1.5364
Ciguatera toxins - 1.9228 1.9147 3.8874 0.000273 0.00232 1.8072 2.1152 2.2307 0.5283 0.5378 0.5167
Prenol lipids Triterpenoids 2.4631 2.4536 3.1753 7.56E-07 3.43E-05 3.5183 3.661 3.6332 1.1272 1.166 1.1119
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.1672 1.1629 1.2254 0.009569 0.0379 3.0975 3.3782 3.3107 2.8044 2.7042 2.4761
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0463 2.0424 0.4467 4.68E-05 0.000634 1.4799 1.2928 1.3872 3.2157 3.1345 2.9659
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9417 1.9386 2.3425 0.000165 0.00163 2.6365 2.7206 2.7883 1.2944 0.9526 1.2304
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.8923 1.8828 3.2737 6.29E-05 0.000795 1.9625 2.1287 2.2268 0.6672 0.6837 0.5791
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.3036 2.2934 0.1731 8.07E-06 0.000173 0.5322 0.518 0.3068 2.5927 2.631 2.6144
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.6933 1.694 0.6768 0.00046 0.003449 2.4911 2.3357 2.6954 3.7541 3.7445 3.6133
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.7186 1.717 0.7121 6.36E-05 0.000799 3.0149 2.8978 3.1015 4.252 4.263 4.1463
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.3465 1.3418 1.5677 0.01368 0.05067 2.4353 1.9162 2.351 1.2261 1.4603 1.5883
Benzene and substituted derivatives Phenylacetamides 1.633 1.633 0.6232 0.000629 0.004366 1.9305 1.6305 1.9783 3.0226 2.9691 2.8956
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.631 1.6056 2.41 0.01933 0.06745 2.2523 1.695 2.1799 1.0977 1.1221 0.3221
- - 1.3665 1.3617 0.7439 3.28E-05 0.00049 2.1713 2.2433 2.2232 3.0343 2.9564 2.935
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.1557 1.1518 1.1912 1.43E-06 5.42E-05 3.3667 3.4041 3.397 2.851 2.8385 2.8455
Prenol lipids Terpene glycosides 1.8111 1.8049 0.627 1.11E-05 0.000224 2.348 2.1935 2.2116 3.6049 3.5883 3.5768
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.6115 1.607 0.6847 2.95E-06 8.67E-05 2.3396 2.2804 2.2859 3.3759 3.3883 3.3206
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.0649 2.0402 3.2383 7.20E-06 0.000225 2.2578 2.2442 2.1082 0.6697 0.6585 0.7125
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.929 1.9006 1.7866 0.000152 0.00167 3.0375 3.0051 3.073 1.7786 1.8108 1.5151
Prenol lipids Triterpenoids 1.7239 1.7013 1.4741 1.29E-05 0.000335 3.2925 3.2667 3.3367 2.3045 2.1851 2.2232
- - 1.3551 1.3399 1.1607 4.62E-05 0.000747 4.7904 4.6829 4.7657 4.1138 4.0754 4.0784
Benzopyrans 1-benzopyrans 1.1527 1.1476 1.2355 0.001507 0.009166 2.6528 2.4435 2.5826 2.0513 2.0652 2.0988
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.1922 2.1691 3.2456 0.00027 0.00253 2.6794 2.2428 2.5827 0.6956 0.8782 0.739
Prenol lipids Retinoids 2.142 2.1285 8.1043 0.004064 0.02031 2.2084 1.3807 2.285 0.2358 0.2298 0.2591
Fatty Acyls Fatty acid esters 1.2272 1.2086 1.3219 0.006088 0.02801 2.1568 2.5162 2.3745 1.7232 1.8042 1.8038
Prenol lipids Terpene lactones 1.7446 1.7234 2.7206 1.77E-06 9.26E-05 1.7452 1.7255 1.6788 0.6209 0.6101 0.6623
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.1557 2.127 2.7543 0.000383 0.003249 2.619 2.6278 2.6823 1.2602 0.7805 0.838
Fatty Acyls Fatty acids and conjugates 1.2521 1.2374 0.8697 8.07E-05 0.001057 3.7459 3.7432 3.7649 4.3661 4.3234 4.251
Prenol lipids Terpene glycosides 1.9483 1.9245 0.303 0.00132 0.008307 0.537 0.3556 0.9318 2.0387 1.9275 2.0546
Lactones Delta valerolactones 1.5081 1.4862 0.7055 3.34E-05 0.000607 2.0051 1.8958 1.9431 2.8084 2.7419 2.7325
Prenol lipids Triterpenoids 1.0521 1.0421 0.8602 0.005609 0.02641 2.6008 2.5283 2.6403 3.0885 3.0737 2.8704
Organooxygen compounds Carbonyl compounds 1.3255 1.3084 0.7602 0.0271 0.09294 2.547 1.9577 2.3894 3.2384 2.8274 3.0018
Carboxylic acids and derivatives Amino acids, peptides, and analogues 2.6016 2.5685 3.6821 1.37E-07 2.76E-05 3.3319 3.2671 3.3248 0.8836 0.8711 0.9405
Metabolite Significant Regulate KEGGCompound ID Mode CAS ID M/Z Retention time Formula Library ID HMDB Superclass
5,9,11-trihydroxyprosta-6E,14Z-dien-1-oate yes up neg 80998-07-8 584.2476 3.824967 C30H37NO8 HMDB0062413 Lipids and lipid-like molecules
2-Hepteneoylglycine yes down neg - 206.0815 4.118383 C9H15NO3 HMDB0094728 Organic acids and derivatives
Lucidenic acid N yes up neg 364622-33-3 495.2539 4.539633 C27H40O6 HMDB0038352 Lipids and lipid-like molecules
Neuromedin N yes up C15868 neg 102577-25-3 654.3275 5.166483 C32H51N5O7 HMDB0013022 Organic acids and derivatives
7alpha-Hydroxy-3-oxo-4-cholestenoate yes up C17337 neg 115538-85-7 465.28 5.379717 C27H42O4 HMDB0012458 Lipids and lipid-like molecules
Simvastatin yes up neg 79902-63-9 463.2665 5.389917 C25H38O5 HMDB0005007 Organoheterocyclic compounds
Homocapsaicin yes up C20215 neg 58493-48-4 364.2116 6.375883 C19H29NO3 HMDB0036329 Benzenoids
3-Oxohexadecanoic acid yes down neg - 315.2168 7.82605 C16H30O3 HMDB0010733 Lipids and lipid-like molecules
Prostaglandin F1a yes down C06475 neg 745-62-0 355.2475 5.8559 C20H36O5 HMDB0002685 Lipids and lipid-like molecules
2-(1-Pentenyl)furan yes up neg 81677-78-3 181.0865 5.729017 C9H12O HMDB0039782 Organoheterocyclic compounds
PS(18:4(6Z,9Z,12Z,15Z)/18:4(6Z,9Z,1 2Z,15Z)) yes up neg - 796.4245 5.059 C42H66NO10P HMDB0112496; LMGP03010441 Lipids and lipid-like molecules
Pubesenolide yes up neg 98569-64-3 493.2765 5.01 C28H42O5 HMDB0033728 Lipids and lipid-like molecules
Neoporrigenin B yes up neg 196607-74-6 481.2757 4.980767 C27H42O5 HMDB0032680 Lipids and lipid-like molecules
Ganoderic acid C2 yes up neg 98296-48-1 555.2682 4.95125 C30H46O7 HMDB0035304 Lipids and lipid-like molecules
P-Salicylic acid yes up C00156 neg - 137.0245 4.687783 C7H6O3 HMDB0000500 Benzenoids
Esculentoside E yes up neg 65649-36-7 649.356 4.597833 C35H54O11 HMDB0034635 Lipids and lipid-like molecules
PS(20:4(5Z,8Z,11Z,14Z)/22:6(4Z,7Z,10Z, 13Z,16Z,19Z)) yes up neg - 890.471 4.5786 C48H74NO10P LMGP03010978; HMDB0012439 Lipids and lipid-like molecules
Dihydrozeatin-9-N-glucoside-O-glucoside yes up neg - 580.2053 4.386333 C22H35N5O11 HMDB0012212 Lipids and lipid-like molecules
Durupcoside B yes up neg 121521-92-4 907.4613 4.051683 C47H74O18 HMDB0030976 Lipids and lipid-like molecules
Blasticidin S yes up C02010 neg 2079-00-7 403.1829 3.862633 C17H26N8O5 HMDB0030452 Organooxygen compounds
Acetyl-DL-Leucine yes up C02710 neg - 172.0976 3.824967 C8H15NO3 HMDB0011756 Organic acids and derivatives
N-(1-Deoxy-1-fructosyl)histidine yes up neg 25020-13-7 338.0981 3.688683 C12H19N3O7 HMDB0037839 Organic acids and derivatives
PS(MonoMe(11,3)/MonoMe(9,5)) yes down neg - 876.4386 3.669033 C44H74NO12P HMDB0061605 Lipids and lipid-like molecules
Bilastine yes up neg 202189-78-4 498.2543 3.4824 C28H37N3O3 HMDB0240232 Organoheterocyclic compounds
Digitoxin yes down C06955 neg 71-63-6 785.4064 3.364417 C41H64O13 HMDB0015468; LMST01120018 Lipids and lipid-like molecules
Benzoquinoneacetic acid yes down neg 10275-07-7 165.0189 3.3453 C8H6O4 HMDB0002334 Organic oxygen compounds
Testosterone sulfate yes up neg 651-45-6 413.1664 3.287133 C19H28O5S LMST05020032; HMDB0002833 Lipids and lipid-like molecules
Isosakuranin yes up neg 491-69-0 447.1327 3.277517 C22H24O10 LMPK12140336; HMDB0029481 Phenylpropanoids and polyketides
Glycinoeclepin B yes up neg 103847-17-2 593.2555 3.10265 C31H42O9 HMDB0037035 Lipids and lipid-like molecules
3,4’,5-Trihydroxy-3’,7-dimethoxyflavanone yes down neg 37971-67-8 331.085 3.005333 C17H16O7 HMDB0037503 Phenylpropanoids and polyketides
CDP-DG(16:0/20:4(8Z,11Z,14Z,17Z)) yes down neg - 982.4909 3.005333 C48H81N3O15P2 HMDB0006974; LMGP13010009 Lipids and lipid-like molecules
(4S,6R)-p-Mentha-1,8-diene-6,7-diol 7-glucoside yes up neg 402593-54-8 365.1343 2.900617 C16H26O7 HMDB0039056 Lipids and lipid-like molecules
Methylprednisolone yes down neg 83-43-2 395.1809 2.790333 C22H30O5 HMDB0015094 Lipids and lipid-like molecules
Prolylhydroxyproline yes down neg 18684-24-7 273.1086 2.170683 C10H16N2O4 HMDB0006695; HMDB0029018 Organic acids and derivatives
Beta-D-3-Ribofuranosyluric acid yes down C05513 neg 2124-54-1 299.0627 2.0088 C10H12N4O7 HMDB0029920 Organoheterocyclic compounds
Dicaffeoylputrescine yes down neg 60422-23-3 447.1347 0.955617 C22H24N2O6 HMDB0033467 Phenylpropanoids and polyketides
2-Hydroxyglutaric acid lactone yes up neg - 175.0239 0.85135 C5H6O4 HMDB0059743 Organoheterocyclic compounds
L-4-Hydroxyglutamate semialdehyde yes up C05938 neg - 128.0352 0.842117 C5H9NO4 HMDB0006556 Organic acids and derivatives
Maleic acid yes down C00122; C01384 neg 110-16-7 115.004 0.842117 C4H4O4 HMDB0000176 Organic acids and derivatives
HMDB Class HMDB Subclass VIP_pred_ OPLS-DA VIP_PLS-DA FC(D_Lf/D) P_value FDR D-Lf_1 D-Lf_2 D-Lf_3 D_1 D_2 D_3
Prenol lipids Diterpenoids 1.3882 1.3697 1.3554 9.46E-06 0.000265 2.5996 2.6253 2.6456 1.9232 1.9091 1.9763
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.0235 1.0074 0.8507 0.00029 0.002655 2.2031 2.1172 2.1429 2.57 2.5094 2.5164
Prenol lipids Triterpenoids 1.4169 1.4056 1.4527 0.00086 0.005967 2.4717 2.2203 2.3565 1.6916 1.5852 1.5735
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.9578 1.9304 3.322 2.51E-06 0.000117 1.8912 1.9824 1.9866 0.578 0.5675 0.6181
Steroids and steroid derivatives Bile acids, alcohols and derivatives 1.0294 1.0155 1.2705 8.82E-05 0.001121 1.7595 1.8004 1.7952 1.3924 1.3787 1.444
Lactones Delta valerolactones 1.3996 1.3784 1.4757 0.000123 0.001427 2.1358 2.2263 2.19 1.5246 1.5158 1.401
Benzene and substituted derivatives Phenols and derivatives 1.6455 1.6473 9.9487 0.008581 0.03704 1.525 0.8364 1.1277 0.2895 0.0245 0.0367
Fatty Acyls Fatty acids and conjugates 1.9667 1.9388 0.2236 0.01064 0.04431 0.864 0.2033 0.2284 1.7967 1.5795 2.4189
Fatty Acyls Eicosanoids 1.0805 1.0691 0.7159 0.0101 0.04219 1.1515 0.9971 1.3144 1.5355 1.6665 1.634
Heteroaromatic compounds - 1.065 1.0743 1.265 0.01324 0.05327 2.2351 1.9988 2.1537 1.6116 1.5973 1.8411
Glycerophospholipids Glycerophosphoserines 1.8578 1.8316 2.5229 7.10E-06 0.000225 1.9733 2.0385 2.1003 0.7962 0.7842 0.8417
Steroids and steroid derivatives Steroid lactones 2.4635 2.4322 13.9182 7.91E-05 0.00104 2.3806 2.3191 2.3432 0.4284 0.0103 0.0675
Steroids and steroid derivatives Steroidal glycosides 1.5647 1.5451 1.4261 3.77E-06 0.000142 2.9239 2.8969 2.9438 2.0361 2.0219 2.0893
Prenol lipids Triterpenoids 1.0299 1.012 1.2035 0.001504 0.009162 2.2272 2.3297 2.3448 1.8728 1.9799 1.884
Benzene and substituted derivatives Benzoic acids and derivatives 1.1946 1.1882 1.2394 0.001437 0.008838 2.7845 2.5965 2.7436 2.1262 2.1829 2.2463
Prenol lipids Triterpenoids 1.3328 1.3277 2.3769 0.02407 0.08546 1.444 0.8687 1.4219 0.4995 0.67 0.4018
Glycerophospholipids Glycerophosphoserines 1.0277 1.0109 1.2692 0.01434 0.0567 1.9324 1.973 1.9912 1.5875 1.6986 1.3623
Fatty Acyls Fatty acyl glycosides 2.6787 2.6425 1242218 4.91E-08 1.73E-05 2.5208 2.539 2.6011 0 0 0
Prenol lipids Terpene glycosides 1.2157 1.1969 1.7331 0.02833 0.09647 1.3637 1.5652 1.3987 0.4923 0.9823 1.0233
Carbohydrates and carbohydrate conjugates Sugar acids and derivatives 2.1481 2.1201 2.0505 1.37E-06 7.95E-05 3.2197 3.1422 3.2586 1.5853 1.5573 1.5484
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.5156 1.4949 1.4291 0.000244 0.002353 2.7711 2.6429 2.8398 1.9504 1.9661 1.8586
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.5808 1.5591 1.5718 8.85E-06 0.000252 2.4085 2.4904 2.4558 1.5471 1.5331 1.5993
Glycerophospholipids Glycerophosphoserines 1.2769 1.2676 0.8292 0.000395 0.003327 2.8039 2.919 2.8693 3.4979 3.495 3.3695
Benzimidazoles - 1.5093 1.4887 1.5538 2.71E-05 0.000537 2.2468 2.252 2.3448 1.4555 1.4417 1.5074
Steroids and steroid derivatives Steroid lactones 1.1367 1.1322 0.7417 0.01089 0.04503 1.2637 1.4719 1.6221 1.977 1.876 2.0235
Organooxygen compounds Carbonyl compounds 1.2997 1.2846 0.7676 0.000577 0.004429 1.9883 1.9973 2.0991 2.7421 2.5801 2.6035
Steroids and steroid derivatives Sulfated steroids 1.8157 1.7918 2.2484 1.17E-05 0.000315 2.1008 2.1994 2.0654 0.9321 0.9195 0.9798
Flavonoids Flavonoid glycosides 1.1811 1.1612 1.3242 0.01336 0.05367 2.2346 2.2227 2.245 1.6956 1.9058 1.4584
Prenol lipids Sesquiterpenoids 2.3161 2.2881 4.5883 8.51E-06 0.000247 2.5424 2.3392 2.4566 0.4865 0.5895 0.5234
Flavonoids O-methylated flavonoids 1.467 1.447 0.6322 0.000289 0.002655 1.3671 1.2949 1.3561 1.9946 2.1827 2.1764
Glycerophospholipids CDP-glycerols 1.1694 1.157 0.9021 0.000352 0.003074 4.554 4.5466 4.5794 5.1062 5.0879 4.9699
Prenol lipids Terpene glycosides 1.9028 1.8782 1.8787 0.000159 0.001716 2.7874 2.7639 2.8123 1.6668 1.3602 1.4254
Steroids and steroid derivatives Hydroxysteroids 2.2731 2.243 0.1605 4.70E-07 4.62E-05 0.3679 0.3263 0.3614 2.2438 2.184 2.1479
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.6294 1.6138 0.4854 0.0291 0.09831 1.3084 0.3904 1.4444 2.2082 2.1923 2.0758
Imidazopyrimidines Purines and purine derivatives 2.7684 2.7266 0.1719 0.000602 0.004549 1.0458 0.0682 0.6207 3.3853 3.3751 3.33
Cinnamic acids and derivatives Hydroxycinnamic acids and derivatives 1.5561 1.5372 0.7491 1.13E-06 7.23E-05 2.5478 2.5857 2.6073 3.4371 3.4525 3.441
Lactones Gamma butyrolactones 1.3513 1.329 1.437 0.001075 0.007088 2.095 2.2323 2.2429 1.5414 1.6227 1.4072
Carboxylic acids and derivatives Amino acids, peptides, and analogues 1.5835 1.5614 1.4843 0.000668 0.004962 2.846 2.7715 2.7571 1.7889 2.0633 1.7907
Carboxylic acids and derivatives Dicarboxylic acids and derivatives 1.3811 1.3644 0.7835 5.63E-05 0.00085 2.4717 2.4444 2.5106 3.1179 3.225 3.1378