Metabolomic analysis of garlic varieties revealed high cycloalliin content in fresh and black Kinkyou bulbs

Abstract

Garlic has unique aroma and functional components, exhibits anti-platelet aggregation activity, anti-allergic effects of ajoene, and so on, and is indispensable for many tasty dishes. To understand the characteristic metabolites, comprehensive metabolomic analysis of water-soluble components from garlic and black garlic (Kinkyou, Taisou, and Katei lines and Fukuchi White cultivar) was carried out using GC/MS. Kinkyou, which is cultivated as a new line in Kagawa, Japan, was assessed for physiologically important components in comparison with 3 popular garlic types. The loading values of principal component analysis of the characteristics of Kinkyou were mainly attributed to glutamic acid, threonine, cycloalliin, and so on. The amount of cycloalliin as a typical functional component of Kinkyou was determined to be about 327 mg/100 g dry wt. by HPLC analysis (2-fold that of the other 3 garlic). Additionally, the amount of cycloalliin in Kinkyou black garlic after processing was increased to 420 mg/100 g dry wt.

Introduction

Garlic (Allium sativum L.) has been widely used as a popular seasoning throughout the world. Garlic and black garlic are used as spices and seasonings, e.g., in olive oil, miso, pasta sauce, and soy sauce products in the case of fresh garlic, and curry sauce and direct consumption for black garlic. Many people enjoy the health benefits of garlic, e.g., antioxidative, antithrombotic, hypolipidemic, hypoglycemic, antihypertensive, and anti-Alzheimer's disease effects (Chauhan, 2005; Ray et al., 2011) as well as its good taste. Therefore, the development of new garlic cultivars that require minimal labor for watering and fertilization, are disease tolerant, and provide health benefits are currently anticipated.

Recently, fresh foods, such as vegetables and fruits, have been allowed to carry function claims and foods for function claims, adding value to products because Japanese consumers require healthy foods appropriate for an aging society (Takada, 2018).

Sulfides, such as diallyl sulfide, diallyl disulfide, and diallyl trisulfide, found in garlic and related processed products are reported to prevent colon cancer (Ngo et al., 2007). The antiallergic effects of ajoene (Usui and Suzuki, 1996) and the antiplatelet aggregation effects of cycloalliin (Agarwal et al., 1977) have been discussed. In addition, as processing of garlic into black garlic enhances the production of S-allyl-l-cysteine (SALC) and pyroglutamate, improvements of liver function (Kawasaki and Yagi-Tamura, 2017), antioxidant properties (Imai et al., 1994), and memory (Grioli et al., 1990) are highly expected, as well as enhanced sensory properties, such as taste and flavor (Zhang et al., 2016). As described above, it has been known that various ingredients are related to the functionality of garlic.

In recent years, simultaneous analysis of complex components, i.e., metabolomic analysis, has made it possible to evaluate trace amounts of various biological metabolites by semi-quantitative analyses (Wishart, 2008; Cevallos-Cevallos et al., 2009). Thus, metabolomic analyses using a GC/MS or GC/Tof-MS system for water-soluble components at milligram levels from soy sauce (Ohnishi et al., 2014), tea leaves (Pongsuwan et al., 2007), Chinese herbal medicine (Tianniam et al., 2008), and other samples have been successfully established for quality control (Ochi et al., 2012; Tamada et al., 2017) and to clarify the mechanisms responsible for component fluctuations during processing (Sumitani et al., 2014; Mabuchi et al., 2018).

Metabolomic analysis has begun to be widely applied to foods in order to clarify differences in a systematic manner, by comprehensively analyzing components rather than focusing on specific components.

To attain data for the selection/development of healthy and beneficial garlic products, GC/MS metabolomic analysis of fresh and processed garlic was applied. Target compounds were identified by metabolomic analysis and then quantitatively monitored by HPLC analysis. Finally, the physiological importance of components in fresh and processed garlic of the Kinkyou line is discussed.

In this study, Kinkyou, a new garlic line that requires minimal water and fertilizer inputs and is cultivated in Kagawa Prefecture, Japan, was assessed for the content of functional components in comparison with 3 other popular garlic, Shanghai Wase (Taisou and Katei lines) and Fukuchi White cultivars.

Materials and Methods

Garlic sample preparation    Four fresh garlic bulbs (Kinkyou, Taisou, Katei, Fukuchi White) produced in Kagawa and Aomori Prefectures at 2018 were obtained from Kagawa Prefectural Agricultural Experiment Station and a local market in Takamatsu, Kagawa (Table 1), and the garlic samples were peeled.

Table 1. Moisture contents of one cultivar and 3 lines of fresh garlic and black garlic.

Sample	Cultivation area	State	Moisture (%)	Number of samples
Kinkyou	Western Japan	Fresh	68.4	3
		Black garlic	68.3	3
Taisou	Western Japan	Fresh	58.8	3
		Black garlic	60.1	4
Katei	Western Japan	Fresh	57.8	5
		Black garlic	56.7	5
Fukuchi white	Northern Japan	Fresh	58.2	3
		Black garlic	62.4	4

Western Japan; Kagawa Prefecture, Northern Japan: Aomori Prefecture.

Black garlic sample preparation    Black garlic was prepared according to Molina-Calle et al., (2017) with minor modifications. Namely, four fresh garlic bulbs (Fukuchi White cultivar and Kinkyou, Taisou, and Katei lines) produced in Kagawa and Aomori Prefectures in 2018, as described above, were, respectively, sealed in polyethylene bags and heated at 72 °C for 2 weeks in a thermostatic chamber (IG 401; Yamato Scientific Co. Ltd., Tokyo, Japan) to make black garlic. After the heat treatment, the garlic was peeled.

Preparation of fresh garlic powders and black garlic pastes    The obtained garlic and black garlic (ca. 40–60 g) were sliced at 1.5 mm width and immediately frozen in liquid nitrogen. The frozen garlic samples were freeze-dried and ground with a food blender. Since the black garlic had a high sugar content, it became a paste after freeze-drying. In this study, therefore, freeze-dried paste (water content of 4 dried garlic samples was almost the same at 12 %) was used for the analysis. The garlic powders and black garlic pastes were individually sealed and stored at −35 °C until analyses.

GC/MS instrument    GC/MS analysis of garlic metabolites was performed on a GCMS-QP2010 Ultra (Shimadzu Co., Kyoto, Japan) equipped with a 30 m × 0.25 mm i.d. fused silica capillary column coated with 0.25 µm CP-SIL 8CB low bleed (Agilent Technologies Inc., CA, USA). The oven temperature was held at 80 °C for 2 min, then programmed to 330 °C at 15 °C/min, and held at the final temperature for 6 min. The carrier gas was helium (flow rate at 1 mL/min) and the split ratio was 1 : 25. The temperature at the injection port was set at 230 °C.

The operating conditions of the mass spectrometer were as follows: the transfer line and ion source temperatures were 250 °C and 200 °C; EI 70 eV; scanning rate of 10 000 u/sec (over a mass range from m/z 85–500).

Sample preparation for GC/MS analysis    Garlic extracts for GC/MS analysis were prepared according to the method described by Pongsuwan et al. (2007) with modifications. In brief, samples of dried garlic powder (15 mg) and black garlic paste (15 mg) in 2 mL Eppendorf tubes were frozen with liquid nitrogen, respectively, and homogenized with a Beads Crusher µT-12 (TAITEC Co., Saitama, Japan) and 3 mm φ zirconia beads. The treated garlic samples were then mixed with 1 mL MeOH:H₂O:CHCl₃ solution at a ratio of 5 : 2 : 2 (v/v/v) and added with 12 µg ribitol and 60 µL water. The individual mixtures were shaken with a thermomixer (Thermomixer Comfort; Eppendorf Co., Tokyo, Japan) at 37 °C for 30 min, and subsequently centrifuged at 13 000 rpm at 4 °C for 5 min. The supernatant (900 µL) was transferred to a 1.5 mL Eppendorf tube and added with 400 µL of water. Finally, the 100 µL upper layer (polar solution) was recovered to a 1.5 mL Eppendorf tube following brief vortexing and centrifugation at 13 000 rpm at 4 °C for 5 min. The extract was dried in a vacuum centrifuge dryer (CVE-100D; Tokyo Rikakikai Co., Tokyo, Japan) for 30 min to remove the volatile methanol and then the remaining aqueous solution was freeze-dried overnight. For oximation of monosaccharides and oligosaccharides (< trimer), 100 µL of 0.24 mM methoxyamine hydrochloride pyridine solution was added to the freeze-dried samples. The tubes were shaken with a thermomixer at 30 °C for 90 min. Finally, trimethylsilylation for GC/MS analysis was performed by adding 50 µL N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) to the methoxyamine hydrochloride pyridine solution, in which the freeze-dried samples were dissolved, followed by shaking with a thermomixer at 37 °C for 30 min.

Analysis of cycloalliin and isoalliin    To 200 mg of fresh garlic powders or 1 g of black garlic pastes in 15 mL centrifuge tubes was added 5 mL MeOH:H2O:HCO2H extraction solution at a ratio of 50 : 50 : 1 (v/v/v), and then ultrasonic treatment was carried out for 60 min for extraction. After centrifugation at 5 000 rpm for 10 min, the supernatant was collected in a 10 mL volumetric flask. The residue was re-extracted with 2 mL extraction solution, stirred for 1 min with a vortex mixer, centrifuged at 5 000 rpm for 10 min, and the supernatant was combined with the solution. The same treatment mentioned above was repeated and the supernatant was combined with the solution to fill up the 10 mL volumetric flask. A 2 or 5 mL aliquot of the adjusted solution mentioned above was loaded onto a bond elute SCX cartridge (500 mg; Agilent Technologies Inc.). To wash the cartridge, 5 mL of the extracting solvent was initially passed. Subsequently, 9.5 mL potassium dihydrogen phosphate solution (100 mM, pH 4.0) was passed and collected in a 10-mL volumetric flask, and the volume was adjusted to 10 mL. The solution was filtrated using a 0.45 µm membrane filter. Quantification of isoalliin and cycloalliin was performed by HPLC analysis. HPLC analysis of cycloalliin and isoalliin was performed on a Prominence UFLC (Shimadzu Co.), equipped with a 5 µm Capcell Pak SCX UG column (250 mm × 4.6 mm i.d., Osaka Soda Ltd., Osaka, Japan). The analytical conditions were as follows: mobile phase of 10 mM potassium dihydrogen phosphate (pH 2.5) at a constant flow rate (1.0 mL/min), injection volume of 10 µL, column temperature of 45 °C, and detection wavelength of 210 nm. The concentrations were calculated from the absolute calibration curve of each standard chemical obtained from Nagara Science Co., Ltd., (Gifu, Japan).

Amino acid analysis of fresh garlic    HPLC analysis of glutamic acid and cysteine was performed on an X-LC (JASCO Co., Tokyo, Japan) equipped with a 1.7 µm Accq-Tag ultra column (100 mm × 2.1 mm i.d., Waters Co., MA, USA). The extracts used for the analysis of cycloalliin and isoalliin were subjected to the following analytical conditions after derivatization using an AccQ-Tag kit. The mobile phase consisted of Waters AccQ-Tag Ultra Eluent A diluted 10-fold (A) and Waters AccQ-Tag Ultra Eluent B (B) with the following gradient conditions: 0 min, 99.9 % A; from 0 to 0.55 min, 99.9 % A; from 0.55 to 5.75 min, 90.9 % A; from 5.75 to 7.75 min, 78.8 % A; from 7.75 to 8.05 min, 40.4 % A; from 8.05 to 8.65 min, 40.4 % A; at a flow rate of 0.7 mL/min. The column temperature was 60 °C and the detection wavelength was 260 nm.

Heating test of isoalliin and cycloalliin    Cycloalliin and isoalliin solutions (50 ppm) were prepared using acetic acid buffer (pH 4.5, 10 mM), and 150 µL of each solution was individually sealed in a 500 µL microtube. Each microtube was heated at 72 °C for 32 hours in a thermostatic chamber. The heated samples were immediately frozen in a freezer and then stored at −35 °C until HPLC analysis.

Data preprocessing for metabolomic analysis    The CDF files obtained from mass spectral data were imported into Metalign software version 041012 (Lommen, 2009) to correct the baseline and to align all extracted mass peaks across all samples. Subsequently, the processed data was imported into AIoutput2 version 1.30 for 64 bit and Excel 2010 (Tsugawa et al., 2011) to identify and quantify the peaks, relatively. Lower limitation of total similarity score calculated from “difference in retention time (< 3 seconds)” and “matching score of the mass fragment” was set at 0.75 to ensure proper metabolite candidates for the identification. The GC retention times of components in garlic extracts were relatively adjusted using retention times of eight reference chemicals (alanine, fumaric acid, glutamic acid, lysine, malic acid, trehalose, tryptophan, and valine). The functional components specific to garlic (alliin, cycloalliin, S-allyl-l-cysteine, and S-methyl-l-cysteine) were analyzed by GC/MS after oximation and trimethylsilylation, and their retention times and fragment patterns were newly added to the library for AIoutput 2. The relative quantification was determined from the “relative quantification value”, which was calculated using the ratio of the peak area of the detected compound against the peak area of the internal standard, ribitol (12 µg/unit against 15 mg sample dry wt.), which is commonly used in metabolomic analysis using GC/MS (Ochi et al., 2012; Ohnishi et al., 2014; Tamada et al., 2017).

Statistical analysis    Principal component analysis was performed from relative quantitative values using metabolomic analysis software, AIoutput2 version 1.30 for 64 bit and Excel 2010.

A heat map was created by clustering using the Euclidean distance and Ward method with MetaboAnalyst 4.0 (Chong et al., 2018) and differences were expressed in grey scale gradation.

One-way analysis of variance and multiple comparison tests (Bonferroni - Dunn) were performed using statistical analysis software, “Statcel2” (OMS Ltd., Tokyo, Japan).

Results and Discussion

Metabolomic analysis of constituents among major garlic    Metabolomic analysis was applied to garlic of Fukuchi White cultivar (3 samples) and 3 lines of Chinese cultivars (Kinkyou: 3 samples, Taisou: 3 samples, Katei: 5 samples) in order to clarify the characteristics among the 4 fresh garlic. As a result, 89 components were identified and quantified by GC/MS analysis relative to ribitol intensity for metabolomic analysis. The amount of pyroglutamic acid could not be accurately distinguished from the amounts derived from glutamine and glutamic acid in the fresh garlic under the analytical conditions. Therefore, pyroglutamic acid was excluded from the 89 components analyzed. The heat map calculated from the relative quantitative values of the 88 water-soluble components identified (Table S-1) is shown in Fig. 1. Apparently, the Kinkyou line was allocated and characterized into a different and unique group in the heat map cluster from the cultivar (Fukuchi White) and 2 lines (Taisou, Katei). Principal component analysis of the relative quantitative values of the 88 components revealed differences among Kinkyou and the other cultivar and lines, with principal component 1 (PC1) and principal component 2 (PC2) accounting for 42 % and 16 % of the variance, respectively, as shown in Fig. 2. PC1 of Kinkyou was plotted in a different position from the other garlic samples, and PC2 (16 %) of Fukuchi White showed an obvious difference from Taisou and Katei. In order to statistically clarify the differences among the cultivar and lines, the loading values of each principal component were compared to one another (Table 2). Large loadings on PC1, such as l-glutamic acid (0.16), 2-aminobutyric acid (0.16), l-threonine (0.16), and cycloalliin (0.16), were positively correlated to the large PC1 value of Kinkyou. The loadings on PC2 of 4-aminobutyric acid (GABA) and S-allyl-l-cysteine (SALC) were −0.22 and 0.23, respectively, suggesting that GABA is dominant in Fukuchi White, and SALC is a characteristic compound in Kinkyou, Taisou, and Katei. Fukuchi White was obtained from a local market, and the post-harvest treatment is unknown. The high GABA content of Fukuchi White is not due to the difference in cultivar and lines, and the possibility of low temperature storage is also considered. As a consequence, Kinkyou was characterized by glutamic acid, 2-aminobutyric acid, threonine, and cycloalliin.

Table S-1 Relative quantification value of fresh garlic.

No.	Components	Relative quantification value
		Kinkyou			Taisou			Katei			Fukuchi white
		Average		SD	Average		SD	Average		SD	Average		SD
1	1-Kestose	0.00	±	0.00	0.00	±	0.00	0.06	±	0.13	0.00	±	0.00
2	2-Aminobutyric acid	0.05	±	0.00	0.00	±	0.00	0.00	±	0.00	0.01	±	0.01
3	2-Aminoethanol	0.21	±	0.01	0.12	±	0.01	0.14	±	0.01	0.14	±	0.02
4	2-Aminopimelic acid	2.51	±	0.20	1.42	±	0.11	1.45	±	0.21	1.78	±	0.08
5	2-Hydroxypyridine	0.08	±	0.01	0.07	±	0.01	0.06	±	0.01	0.08	±	0.05
6	2-Isopropylmalic acid	0.05	±	0.08	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
7	2-Picolinate	0.01	±	0.00	0.01	±	0.00	0.00	±	0.00	0.01	±	0.00
8	3-Methylglutarate	0.06	±	0.01	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
9	4-Aminobutyric acid	0.22	±	0.02	0.06	±	0.03	0.13	±	0.04	0.32	±	0.02
10	4-Hydroxyphenylpyruvate	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
11	4-Hydroxypyridine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.01	±	0.01
12	5-Amino-Levulinate	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00
13	5-Hydroxy-L-tryptophan	0.17	±	0.10	0.06	±	0.03	0.04	±	0.03	0.20	±	0.15
14	Aconitate	0.10	±	0.01	0.05	±	0.04	0.00	±	0.00	0.00	±	0.00
15	Alanine	2.35	±	0.24	0.46	±	0.07	0.53	±	0.19	0.62	±	0.35
16	Alliin	3.43	±	0.58	3.07	±	0.57	2.03	±	0.48	1.54	±	0.59
17	Arabitol	0.00	±	0.01	0.00	±	0.01	0.01	±	0.01	0.00	±	0.00
18	Asparagine	1.50	±	0.32	0.29	±	0.11	0.43	±	0.32	0.76	±	0.53
19	Aspartic acid	1.85	±	0.22	0.48	±	0.09	0.35	±	0.07	0.49	±	0.20
20	b-Alanine	0.05	±	0.01	0.01	±	0.00	0.01	±	0.01	0.01	±	0.01
21	b-Cyano-L-Alanine	0.01	±	0.00	0.00	±	0.00	0.00	±	0.01	0.00	±	0.00
22	b-N-Methyl-amino-L-alanine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.02	±	0.02
23	Caffeic acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.02	±	0.03
24	Citric acid + Isocitric acid	11.40	±	0.68	6.37	±	0.44	6.81	±	1.16	8.06	±	0.48
25	Citrulline	0.15	±	0.03	0.19	±	0.04	0.22	±	0.04	0.15	±	0.05
26	Phosphate	2.05	±	0.35	0.99	±	0.22	1.76	±	0.36	2.16	±	0.32
27	Cycloalliin	0.41	±	0.01	0.13	±	0.03	0.12	±	0.04	0.15	±	0.02
28	Fructose	14.60	±	0.63	7.81	±	0.70	9.94	±	0.79	13.00	±	0.78
29	Fumaric acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.01	±	0.01
30	Galactitol	0.09	±	0.01	0.03	±	0.01	0.09	±	0.01	0.08	±	0.01
31	Galacturonic acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.01
32	Glucose	0.05	±	0.01	0.05	±	0.04	0.05	±	0.02	0.15	±	0.01
33	Glutamic acid	2.51	±	0.09	0.58	±	0.11	0.56	±	0.15	0.77	±	0.20
34	Glutamine	3.11	±	0.41	0.69	±	0.17	0.58	±	0.26	1.39	±	0.14
35	Glyceric acid	0.02	±	0.00	0.00	±	0.01	0.00	±	0.00	0.00	±	0.00
36	Glycine	0.65	±	0.03	0.10	±	0.01	0.18	±	0.07	0.19	±	0.07
37	Glycyl-Glycine	0.12	±	0.03	0.14	±	0.03	0.16	±	0.06	0.13	±	0.06
38	Histidine	0.48	±	0.07	0.15	±	0.03	0.17	±	0.05	0.11	±	0.08
39	Indole-3-Acetaldehyde	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
40	Inositol	2.03	±	0.21	0.81	±	0.12	0.95	±	0.31	0.88	±	0.59
41	Isobutylamine	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.00	±	0.00
42	Isoleucine	0.33	±	0.01	0.13	±	0.01	0.17	±	0.04	0.22	±	0.04
43	Lactic acid	0.11	±	0.01	0.11	±	0.03	0.09	±	0.01	0.10	±	0.01
44	Lactitol	0.01	±	0.01	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
45	Lauric acid	0.00	±	0.00	0.01	±	0.01	0.00	±	0.00	0.00	±	0.00
46	Leucine	0.48	±	0.02	0.24	±	0.02	0.23	±	0.06	0.35	±	0.09
47	Lysine	1.27	±	0.09	0.88	±	0.16	0.82	±	0.25	0.72	±	0.22
48	Maleic acid	0.15	±	0.01	0.01	±	0.02	0.02	±	0.03	0.05	±	0.02
49	Malic acid	4.21	±	0.96	1.45	±	0.27	1.22	±	0.47	1.59	±	0.09
50	Melibiose	0.02	±	0.02	0.01	±	0.01	0.01	±	0.01	0.03	±	0.01
51	Methionine	0.08	±	0.01	0.06	±	0.00	0.10	±	0.02	0.09	±	0.02
52	Methionine sulfone	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
53	N-Acetyl-DL-Valine	0.03	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00
54	n-Butylamine	0.02	±	0.00	0.02	±	0.00	0.03	±	0.00	0.02	±	0.00
55	N-Carbamoyl-L-Aspartate	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.01
56	N-Methylethanolamine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
57	Octadecanoate	0.07	±	0.00	0.08	±	0.01	0.08	±	0.00	0.06	±	0.00
58	Ornithine	0.26	±	0.08	0.32	±	0.07	0.36	±	0.13	0.29	±	0.15
59	Oxalate	0.07	±	0.00	0.00	±	0.00	0.01	±	0.03	0.03	±	0.03
60	Phenylalanine	0.34	±	0.00	0.27	±	0.08	0.36	±	0.08	0.17	±	0.02
61	Phthalic acid	0.00	±	0.00	0.00	±	0.00	0.01	±	0.02	0.00	±	0.00
62	Pipecolic acid	0.14	±	0.01	0.16	±	0.05	0.07	±	0.04	0.03	±	0.02
63	Proline	2.25	±	0.42	1.89	±	0.10	2.21	±	0.45	2.17	±	0.75
64	Propyleneglycol	0.01	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
65	Putrescine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.02	±	0.01
66	Pyrogallol	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
67	Pyruvate+Oxalacetic acid	0.79	±	0.10	1.20	±	0.27	1.27	±	0.20	0.82	±	0.12
68	Quinolinic acid	0.22	±	0.04	0.20	±	0.05	0.13	±	0.03	0.05	±	0.08
69	Rhamnose	0.01	±	0.02	0.00	±	0.00	0.01	±	0.01	0.00	±	0.00
70	Ribose	0.00	±	0.01	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
71	S-Allyl-L-Cysteine	0.50	±	0.05	0.55	±	0.10	0.42	±	0.08	0.19	±	0.11
72	S-Benzyl-L-Cysteine	0.02	±	0.00	0.00	±	0.01	0.00	±	0.00	0.01	±	0.00
73	Serine	2.42	±	0.34	0.35	±	0.04	0.33	±	0.10	0.59	±	0.32
74	Shikimic acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
75	S-Methyl-L-Cysteine	0.16	±	0.01	0.06	±	0.01	0.03	±	0.01	0.02	±	0.01
76	Sorbose	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.01
77	Spermidine	0.02	±	0.00	0.01	±	0.00	0.01	±	0.00	0.02	±	0.00
78	Succinic acid	0.03	±	0.01	0.00	±	0.00	0.00	±	0.00	0.03	±	0.00
79	Sucrose	22.36	±	2.09	16.58	±	0.73	15.13	±	0.94	16.19	±	2.50
80	Tagatose	1.49	±	0.17	1.02	±	0.18	0.47	±	0.06	0.82	±	0.02
81	Threo-b-HydroxyAspartic acid	0.00	±	0.00	0.00	±	0.01	0.00	±	0.00	0.01	±	0.01
82	Threonine	0.50	±	0.02	0.09	±	0.02	0.09	±	0.03	0.11	±	0.07
83	Tryptophan	0.34	±	0.04	0.12	±	0.03	0.28	±	0.08	0.21	±	0.06
84	Tyramine	1.64	±	0.15	1.17	±	0.28	1.05	±	0.31	0.90	±	0.26
85	Tyrosine	1.25	±	0.12	0.33	±	0.05	0.56	±	0.13	0.44	±	0.03
86	Urea	0.01	±	0.01	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
87	Uridine	0.86	±	0.49	0.30	±	0.14	0.22	±	0.16	1.02	±	0.77
88	Valine	1.59	±	0.06	0.59	±	0.07	0.49	±	0.18	0.57	±	0.15

Data show in mean ± standard deviation

Values less than 0.005 are indicated as 0.00.

Fig. 1.

Heat map of 4 fresh garlic

Fig. 2.

Principal component analysis of identified compoundsin 4 fresh garlic with the score plots. Symbols: Kinkyou; ▲ Taisou; ■ Katei; ◆ Fukuchi white; ●

Table 2. Loading values of identified compounds in 4 fresh garlic.

Compound name	Loading value		Compound name	Loading value
	Principal component 1	Principal component 2		Principal component 1	Principal component 2
1-Kestose	−0.03	0.02	l-Leucine**	0.15	−0.07
2-Aminobutyric acid**	0.16	−0.01	l-Lysine*	0.13	0.10
2-Aminoethanol**	0.15	0.00	Maleic acid**	0.16	−0.02
2-Aminopimelic acid**	0.15	−0.05	Malic acid**	0.15	0.02
2-Hydroxypyridine	0.03	−0.06	Melibiose	0.06	−0.16
2-Isopropylmalic acid	0.09	−0.01	l-Methionine*	0.00	−0.03
2-Picolinate	0.09	0.03	Methionine sulfone	0.04	0.01
3-Methylglutarate**	0.16	0.02	N-Acetyl-l-valine**	0.16	0.02
4-Aminobutyric acid**	0.06	−0.22	N-Butylamine	−0.03	0.12
4-Hydroxyphenylpyruvate*	−0.03	−0.20	N-Carbamoyl-l-aspartate	0.01	−0.13
4-Hydroxypyridine	−0.01	−0.13	N-Methylethanolamine	0.01	−0.13
5-Amino-levulinate*	0.12	0.10	Octadecanoate**	0.01	0.23
5-Hydroxy-l-tryptophan	0.07	−0.15	l-Ornithine	−0.02	0.06
Aconitate**	0.13	0.09	Oxalate**	0.12	−0.07
l-Alanine**	0.16	0.02	l-Phenylalanine**	0.04	0.20
l-Alliin**	0.10	0.15	Phosphate**	0.08	−0.12
Arabitol	0.00	0.10	Phthalic acid	−0.02	0.06
l-Asparagine**	0.15	−0.04	Pipecolic acid**	0.06	0.19
l-Aspartic acid**	0.16	0.02	l-Proline	0.05	−0.01
β-Alanine**	0.16	0.03	Propyleneglycol**	0.14	0.03
β-Cyano-l-alanine	0.13	0.02	Putrescine**	−0.04	−0.24
β-N-Methyl-amino-l-alanine*	−0.01	−0.19	Pyrogallol	0.09	−0.13
Caffeic acid	0.01	−0.13	Pyruvate + Oxalacetic acid*	−0.10	0.14
Citric acid + Isocitric acid**	0.15	−0.04	Quinolinic acid**	0.09	0.18
l-Citrulline	−0.04	0.13	Rhamnose	0.03	0.07
Cycloalliin**	0.16	0.02	Ribose	0.09	−0.01
Fructose**	0.12	−0.14	S-Allyl-l-cysteine**	0.05	0.23
Fumaric acid**	−0.01	−0.23	S-Benzyl-l-cysteine**	0.15	−0.05
Galactitol**	0.07	−0.07	l-Serine**	0.16	0.01
Galacturonic acid	0.01	−0.13	Shikimic acid	0.09	0.03
Glucose**	−0.02	−0.22	S-Methyl-l-cysteine**	0.15	0.06
l-Glutamic acid**	0.16	0.01	Sorbose	−0.02	−0.13
l-Glutamine**	0.16	−0.04	Spermidine**	0.11	−0.14
Glyceric acid**	0.15	−0.01	Succinic acid**	0.12	−0.18
Glycine**	0.16	0.01	Sucrose**	0.15	0.02
Glycyl-glycine	−0.01	0.07	Tagatose or Psicose**	0.13	0.01
l-Histidine**	0.16	0.06	Threo-β-hydroxyaspartic acid	0.01	−0.11
Indole-3-α-Acetaldehyde	−0.02	−0.08	l-Threonine**	0.16	0.02
Inositol**	0.15	0.03	l-Tryptophan**	0.09	0.03
Isobutylamine	−0.02	0.13	Tyramine*	0.13	0.11
l-Isoleucine**	0.15	−0.05	l-Tyrosine**	0.15	0.03
Lactic acid	0.01	0.03	Urea	0.09	−0.01
Lactitol	0.09	0.03	Uridine	0.07	−0.15
Lauric acid	−0.04	0.03	l-Valine**	0.16	0.03

Asterisk indicates significant difference of relative quantification values of metabolites in each garlic

*  p < 0.05

**  p < 0.01

Evaluation of black garlic, focusing on functional ingredients    During processing of fresh garlic into black garlic, the components undergo complex changes to generally increase the antioxidant activity (Choi et al., 2014), and anticancer and antiobesity effects (Kimura et al., 2016). In order to characterize the processed black garlic of the cultivar and lines, metabolomic analysis of the prepared black garlic was carried out for the Fukuchi White cultivar (4 samples) and 3 lines from Chinese cultivars (Kinkyou: 3 samples, Taisou: 4 samples, Katei: 5 samples). As a result, 100 components were identified and quantified by GC/MS analysis relative to ribitol intensity for metabolomic analysis. The quantitative data of pyroglutamic acid was excluded from that of the identified compounds in the same manner as described above. Additionally, fructose was also excluded because the total ion intensity of fructose during GC/MS analysis showed scale out with the variation of the quantity caused by the hydrolysis of fructan and other fructo-oligosaccharides (Yuan et al., 2016; Zhang et al., 2016). The heat map calculated from the relative quantitative values of the 98 water-soluble components identified (Table S-2) is shown in Fig. 3. The pattern and density of the heat map (white and black) reflect the composition of individual compounds identified among the cultivar and lines. In Fig. 3, Kinkyou characteristically contained more components than the other cultivar and lines. Therefore, Kinkyou, which has a different heat map pattern and density, could be concluded to be a unique line, since it has more constituents of high content compared with the other cultivar and lines. Principal component analysis of the relative quantitative values of the 98 compounds from the 4 processed black garlic also revealed differences among Kinkyou and the other cultivar and lines, as well as the 4 kinds of fresh garlic, with principal component 1 (PC1) and principal component 2 (PC2) accounting for 23 % and 21 % of the variance, respectively, as shown in Fig. 4. PC1 could separate Fukuchi White and Kinkyou from Taisou. However, Katei could not be separated from any cultivar and lines. On the other hand, PC2 of Kinkyou was plotted in a clearly distinct position from the other garlic samples. In order to characterize the 4 garlic samples and identify important constituents, the loading value of each principal component was compared to one another (Table 3). Large loading values on PC2, such as l-glutamic acid (0.20), cycloalliin (0.20), and succinic acid (0.19), were positively correlated to the large PC2 value of Kinkyou. Glutamic acid and cycloalliin had high loading values for fresh garlic and black garlic, respectively. Therefore, these components are statistically important (p < 0.05) for characterizing fresh and processed garlic from the cultivar and 3 lines. As a consequence, it was found that l-glutamic acid and cycloalliin made a significant contribution to the discrimination of Kinkyou from the other samples for black garlic as well as fresh garlic.

Table S-2 Relative quantification value of black garlic.

No.	Components	Relative quantification value
		Kinkyou			Taisou			Katei			Fukuchi white
		Average		SD	Average		SD	Average		SD	Average		SD
1	1-Kestose	0.46	±	0.11	0.37	±	0.26	0.83	±	0.07	0.35	±	0.40
2	2-Aminobutyric acid	0.01	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
3	2-Aminoethanol	0.03	±	0.00	0.02	±	0.00	0.04	±	0.01	0.05	±	0.01
4	2-Aminopimelic acid	0.80	±	0.09	0.43	±	0.03	0.67	±	0.15	0.64	±	0.06
5	2-HydroxyButyrate	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
6	2-Hydroxypyridine	0.03	±	0.00	0.03	±	0.00	0.04	±	0.01	0.03	±	0.01
7	2-Picolinate	0.00	±	0.00	0.01	±	0.00	0.00	±	0.00	0.00	±	0.00
8	2-Thiouracil	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.01
9	3-Hydroxy-DL-Kynurenine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
10	3-Methylglutarate	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
11	4-Aminobutyric acid	0.21	±	0.02	0.16	±	0.01	0.27	±	0.04	0.39	±	0.04
12	4-Hydroxypyridine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
13	5-Aminovaleric acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
14	5-Hydroxy-L-tryptophan	0.88	±	0.03	1.33	±	0.23	1.18	±	0.33	0.86	±	0.17
15	5-Methylcytosine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
16	a,d-Diaminopimelate	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
17	Aconitate	0.06	±	0.01	0.08	±	0.02	0.07	±	0.03	0.06	±	0.02
18	Adenine	0.04	±	0.00	0.03	±	0.00	0.03	±	0.00	0.03	±	0.00
19	Alanine	1.23	±	0.14	0.87	±	0.06	0.89	±	0.12	1.02	±	0.17
20	Alanylalanine	0.20	±	0.17	0.08	±	0.15	0.00	±	0.00	0.00	±	0.00
21	Alliin	0.02	±	0.00	0.02	±	0.00	0.03	±	0.01	0.01	±	0.00
22	Allose	0.16	±	0.01	0.22	±	0.01	0.22	±	0.02	0.22	±	0.01
23	a-Phenylglycine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
24	Arabinose	0.09	±	0.00	0.07	±	0.02	0.06	±	0.02	0.05	±	0.01
25	Arabitol	0.00	±	0.01	0.00	±	0.01	0.01	±	0.00	0.00	±	0.00
26	Asparagine	0.21	±	0.04	0.05	±	0.03	0.14	±	0.11	0.24	±	0.24
27	Aspartic acid	0.40	±	0.05	0.13	±	0.02	0.14	±	0.04	0.18	±	0.10
28	b-N-Methyl-amino-L-alanine	0.01	±	0.00	0.02	±	0.00	0.01	±	0.00	0.01	±	0.00
29	Cadaverine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
30	Citric acid + Isocitric acid	3.84	±	0.58	2.10	±	0.14	3.22	±	0.70	3.04	±	0.28
31	Citrulline	0.03	±	0.01	0.04	±	0.01	0.06	±	0.03	0.07	±	0.03
32	Phosphate	3.56	±	0.28	3.78	±	0.27	4.57	±	0.47	4.40	±	0.55
33	Cycloalliin	0.10	±	0.01	0.03	±	0.00	0.04	±	0.01	0.04	±	0.01
34	Cystamine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
35	Cysteine+Cystine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
36	Dimethylbenzimidazole	0.01	±	0.01	0.01	±	0.00	0.00	±	0.00	0.00	±	0.00
37	Dopa	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
38	Fumaric acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
39	Galactitol	0.09	±	0.00	0.10	±	0.01	0.05	±	0.05	0.06	±	0.04
40	Galactosamine	0.27	±	0.02	0.34	±	0.03	0.25	±	0.03	0.25	±	0.03
41	Galacturonic acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
42	Glucosamine	1.13	±	0.07	1.49	±	0.06	1.15	±	0.14	1.06	±	0.10
43	Glucose	1.37	±	0.03	1.42	±	0.22	1.10	±	0.33	1.31	±	0.17
44	Glutamic acid	0.27	±	0.08	0.07	±	0.01	0.08	±	0.03	0.06	±	0.02
45	Glutamine	0.00	±	0.00	0.01	±	0.00	0.00	±	0.00	0.00	±	0.00
46	Glycine	0.36	±	0.04	0.25	±	0.02	0.18	±	0.02	0.21	±	0.06
47	Glycolic acid	1.10	±	0.07	0.81	±	0.11	0.79	±	0.09	0.55	±	0.02
48	Guanine	0.01	±	0.00	0.00	±	0.00	0.00	±	0.00	0.01	±	0.00
49	Histidine	0.02	±	0.00	0.00	±	0.00	0.01	±	0.01	0.01	±	0.01
50	Homocysteic acid	0.00	±	0.00	0.00	±	0.00	0.05	±	0.10	0.00	±	0.00
51	homoserine	0.01	±	0.00	0.01	±	0.00	0.01	±	0.01	0.02	±	0.00
52	Hydrocinnamate	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
53	Inositol	0.78	±	0.08	0.44	±	0.03	0.44	±	0.09	1.13	±	0.04
54	Isobutylamine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
55	Isoleucine	0.11	±	0.02	0.06	±	0.00	0.10	±	0.01	0.11	±	0.01
56	Kojic acid	0.01	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
57	Lactic acid	0.21	±	0.02	0.23	±	0.02	0.22	±	0.01	0.22	±	0.02
58	Leucine	0.17	±	0.01	0.12	±	0.01	0.17	±	0.02	0.25	±	0.02
59	Lyxose	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
60	Malic acid	1.12	±	0.16	0.61	±	0.03	0.70	±	0.16	1.02	±	0.11
61	Methionine	0.02	±	0.00	0.03	±	0.00	0.04	±	0.01	0.04	±	0.01
62	Methylsuccinic acid	0.37	±	0.03	0.29	±	0.05	0.26	±	0.04	0.20	±	0.03
63	N-a-Acetyl-L-Lysine	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00
64	N-a-Acetyl-L-Ornithine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
65	n-Butylamine	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00
66	N-Methylethanolamine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
67	Nonanoic acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
68	Octadecanoate	0.04	±	0.00	0.03	±	0.00	0.04	±	0.00	0.04	±	0.00
69	Ornithine	0.07	±	0.01	0.07	±	0.03	0.13	±	0.07	0.16	±	0.09
70	Oxalate	0.09	±	0.02	0.10	±	0.01	0.10	±	0.01	0.07	±	0.01
71	Phenylalanine	0.18	±	0.04	0.24	±	0.04	0.29	±	0.12	0.13	±	0.02
72	Pipecolic acid	0.03	±	0.01	0.03	±	0.01	0.03	±	0.02	0.02	±	0.01
73	Proline	0.67	±	0.13	0.63	±	0.11	0.95	±	0.29	0.64	±	0.18
74	Propyleneglycol	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00	0.01	±	0.00
75	Psicose	0.26	±	0.03	0.24	±	0.16	0.37	±	0.04	0.32	±	0.04
76	Putrescine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
77	Pyrogallol	0.00	±	0.00	0.00	±	0.00	0.01	±	0.00	0.00	±	0.00
78	Pyruvate+Oxalacetic acid	0.71	±	0.09	0.77	±	0.07	0.57	±	0.10	0.56	±	0.04
79	Rhamnose	0.03	±	0.00	0.02	±	0.00	0.02	±	0.01	0.02	±	0.01
80	Ribose	0.07	±	0.00	0.05	±	0.01	0.05	±	0.01	0.04	±	0.01
81	S-Allyl-L-Cysteine	0.22	±	0.03	0.51	±	0.06	0.39	±	0.09	0.45	±	0.16
82	S-Benzyl-L-Cysteine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
83	Serine	0.43	±	0.07	0.11	±	0.01	0.14	±	0.03	0.37	±	0.09
84	Shikimic acid	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
85	S-Methyl-L-Cysteine	0.09	±	0.02	0.06	±	0.01	0.03	±	0.01	0.07	±	0.06
86	Succinic acid	0.11	±	0.01	0.06	±	0.01	0.07	±	0.01	0.08	±	0.01
87	Theanine	0.01	±	0.00	0.01	±	0.00	0.01	±	0.01	0.01	±	0.01
88	Threo-b-HydroxyAspartic acid	0.01	±	0.00	0.00	±	0.00	0.01	±	0.01	0.00	±	0.00
89	Threonine	0.09	±	0.02	0.03	±	0.01	0.03	±	0.01	0.06	±	0.02
90	Thymine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
91	trans-4-Hydroxy-L-proline	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
92	Tryptamine	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
93	Tryptophan	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00
94	Tyramine	0.34	±	0.08	0.32	±	0.07	0.39	±	0.13	0.37	±	0.13
95	Tyrosine	0.22	±	0.05	0.10	±	0.01	0.20	±	0.04	0.11	±	0.02
96	Uridine	4.29	±	0.23	6.54	±	0.87	5.94	±	1.69	4.38	±	0.89
97	Valine	0.48	±	0.04	0.22	±	0.02	0.25	±	0.05	0.31	±	0.05
98	Xylose	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00	0.00	±	0.00

Data show in mean ± standard deviation

Values less than 0.005 are indicated as 0.00.

Fig. 3.

Heat map of 4 black garlic.

Fig. 4.

Principal component analysis of identified compoundsin 4 black garlic with the score plots. Symbols: Kinkyou; ▲ Taisou; ■ Katei; ◆ Fukuchi white; ●

Table 3. Loading values of identified compounds in 4 black garlic.

Compound name	Loading value		Compound name	Loading value
	Principal component 1	Principal component 2		Principal component 1	Principal component 2
1-Kestose*	0.02	−0.04	l-Homoserine**	−0.14	−0.01
2-Aminobutyric acid**	−0.04	0.19	Hydrocinnamate	0.07	−0.03
2-Aminoethanol**	−0.18	−0.07	Inositol**	−0.13	0.02
2-Aminopimelic acid**	−0.14	0.10	Isobutylamine	0.05	−0.03
2-Hydroxybutyrate	0.06	0.11	l-Isoleucine**	−0.17	0.04
2-Hydroxypyridine	0.01	−0.07	Kojic acid**	0.07	0.18
2-Picolinate	−0.03	−0.05	Lactic acid	0.09	−0.03
2-Thiouracil	−0.02	0.02	l-Leucine**	−0.16	−0.03
3-Hydroxy-l-kynurenine	0.01	0.11	Lyxose	−0.06	−0.03
3-Methylglutarate	0.01	0.11	Malic acid**	−0.15	0.12
4-Aminobutyric acid**	−0.15	−0.08	l-Methionine**	−0.10	−0.15
4-Hydroxypyridine	−0.03	−0.06	Methylsuccinic acid**	0.08	0.17
5-Aminovaleric acid	0.04	−0.06	N-α-Acetyl-l-lysine	−0.11	−0.06
5-Hydroxy-l-tryptophan*	0.19	−0.06	N-α-Acetyl-l-ornithine	0.07	−0.03
5-Methylcytosine	0.02	0.08	N-Butylamine	0.04	0.03
α,δ-Diaminopimelate	−0.04	0.11	N-Methylethanolamine*	0.03	0.15
Aconitate*	0.17	−0.01	Nonanoic acid	0.06	0.02
Adenine**	0.00	0.18	Octadecanoate*	−0.16	−0.04
l-Alanine*	−0.14	0.15	l-Ornithine	−0.18	−0.08
Alanylalanine	0.02	0.12	Oxalate*	0.13	0.04
l-Alliin**	0.10	−0.05	l-Phenylalanine*	0.00	−0.04
Allose**	0.08	−0.18	Phosphate*	−0.07	−0.12
α-Phenylglycine	−0.11	−0.06	Pipecolic acid	0.00	0.05
Arabinose*	0.14	0.14	l-Proline	−0.08	−0.04
Arabitol	0.02	−0.01	Propyleneglycol**	0.09	0.13
l-Asparagine	−0.17	0.04	Psicose	−0.08	−0.08
l-Aspartic acid**	−0.09	0.19	Putrescine	−0.13	−0.12
β-N-Methyl-amino-l-	0.09	−0.09	Pyrogallol	0.03	−0.05
Cadaverine	0.06	0.00	Pyruvate+Oxalacetic	0.11	0.08
Citric acid + Isocitric acid**	−0.14	0.11	Rhamnose	0.12	0.13
l-Citrulline	−0.17	−0.09	Ribose**	0.13	0.16
Cycloalliin**	−0.06	0.20	S-Allyl-l-cysteine*	−0.02	−0.15
Cystamine	0.14	−0.03	S-Benzyl-l-cysteine	0.01	0.07
Cysteine+Cystine	0.03	0.01	l-Serine**	−0.15	0.13
Dimethylbenzimidazole	0.05	0.08	Shikimic acid	−0.06	−0.03
Dopa	0.03	0.04	S-Methyl-l-cysteine	−0.10	0.09
Fumaric acid**	−0.02	0.18	Succinic acid**	−0.06	0.19
Galactitol	−0.01	0.05	Theanine	0.18	0.03
Galactosamine**	0.10	0.01	Threo-β-hydroxyaspartic*	−0.02	0
Galacturonic acid	−0.01	−0.04	l-Threonine**	−0.13	0.16
Glucosamine**	0.14	−0.03	Thymine	0.10	−0.06
Glucose	0.11	0.06	trans-4-Hydroxy-l-	−0.07	0.06
l-Glutamic acid**	−0.04	0.20	Tryptamine	−0.06	−0.04
l-Glutamine	−0.04	−0.12	l-Tryptophan	−0.14	−0.04
Glycine**	−0.01	0.19	Tyramine	−0.14	−0.03
Glycolic acid**	0.08	0.18	l-Tyrosine**	−0.04	0.12
Guanine	−0.17	−0.01	Uridine*	0.19	−0.07
l-Histidine*	−0.16	0.07	l-Valine**	−0.11	0.19
Homocysteic acid	0.07	−0.03	Xylose	−0.07	−0.07

Asterisk indicates significant difference of relative quantification values of metabolites in each garlic

*  p < 0.05

**  p < 0.01).

From the perspective of preventing and reducing the incidence of lifestyle diseases, cycloalliin appears to be more valuable than L-glutamic acid in light of the extensive evidence that cycloalliin accelerates antiplatelet aggregation activity (Agarwal et al., 1977) and reduces serum triglyceride levels (Yanagishita et al., 2003).

HPLC analyses of metabolites in fresh and processed black garlic    The relative quantitative value obtained by metabolomic analysis of cycloalliin by GC/MS may not be accurate, since the conditions of GC/MS analysis are not specific to cycloalliin. Thus, the accurate determination of cycloalliin and isoalliin by HPLC analysis, which can be directly determined without derivatization, is shown in Table 4. Cycloalliin and its precursor isoalliin of fresh garlic (0 weeks), intermediate garlic (1 week) and black garlic (2 weeks) were quantitatively compared to one another. Among the fresh garlic cultivar and 3 garlic lines, the amount of cycloalliin was in the range of about 107.1–327.0 mg/100 g dry wt. (Kinkyou, 327.0 mg/100 g dry wt.; Taisou, 107.1 mg/100 g dry wt.; Katei, 124.0 mg/100 g dry wt.; Fukuchi White, 140.7 mg/100 g dry wt.). Cycloalliin content was significantly higher in Kinkyou than the other cultivar and lines. Lawson et al. (1991) reported that isoalliin can be formed from N-(γ-l-glutamyl)-S-(E-1-propenyl)-l-cysteine (Glu-PEC), a precursor of isoalliin, under cold storage at 3 °C. Since Kinkyou and Taisou were grown in the same research field and treated according to the same method after harvest every year, the difference in cycloalliin content between Kinkyou (327.0 mg/100 g dry wt.) and Taisou (107.1 mg/100 g dry wt.) is likely not due to environmental factors. The glutamic acid and cysteine contents of fresh garlic are shown in Table 5. Kinkyou contained a significantly (p < 0.05) higher amount of cysteine, a source of sulfur, than the others. As isoalliin can be formed from γ-glutamylcysteine, which is synthesized from glutamic acid and cysteine, the abundant isoalliin content in Kinkyou can be attributed to the abundant cysteine content. Furthermore, among 14 fresh garlic cultivars grown in China, USA and Japan, Kinkyou was recognized to be in the highest group of cycloalliin content (Yamazaki et al. 2005; Yamazaki et al. 2011).

Table 4. Cycloalliin and isoalliin contents of one cultivar and 3 lines during the processing.

Sample	Contents (mg / 100 g dry wt.)						Conversion Rate (%)	Number of samples
	Cycloalliin			Isoalliin
0 week (Fresh garlic)	Average		SD	Average		SD
Kinkyou	327.0	±	22.3 a	190.6	±	13.7 a	−	3
Taisou	107.1	±	11.3 b	67.7	±	4.3 c	−	3
Katei	124.0	±	41.0 b	59.9	±	24.5 c	−	3
Fukuchi white	140.7	±	1.1 b	120.7	±	5.8 b	−	5
1 week	Average		SD	Average		SD
Kinkyou	389.1	±	41.3 a		N.D.		32.6	3
Taisou	148.1	±	10.3 b		N.D.		60.7	3
Katei	151.9	±	27.9 b		N.D.		46.6	3
Fukuchi white	212.5	±	16.2 b		N.D.		59.6	3
2 weeks (Black garlic)	Average		SD	Average		SD
Kinkyou	420.3	±	23.3 a		N.D.		49.0	4
Taisou	163.3	±	21.4 b		N.D.		82.8	3
Katei	146.7	±	32.4 b		N.D.		38.0	4
Fukuchi white	188.4	±	43.7 b		N.D.		39.6	5

Data show in mean ± standard deviation of HPLC analysis at 210 nm.

Different letters (a−c) indicate significant difference (p < 0.05).

Conversion rate (%) = (increasing the amount of cycloalliin / isoalliin content of fresh garlic) × 100.

Table 5. Glutamic acid and cysteine contents of one cultivar and 3 lines of fresh garlic.

Sample	Contents (mg / 100 g dry wt.)
	Glutamic acid			Cysteine
	Average		SD	Average		SD
Kinkyou	141.9	±	24.3 a	259.3	±	62.2 a
Taisou	111.3	±	11.4 ab	155.3	±	33.6 b
Katei	96.1	±	14.4 b	138.1	±	23.3 b
Fukuchi white	102.0	±	7.6 ab	97.5	±	28.4 b

Data show mean ± standard deviation (replication number was shown in Table 1)

Different letters (a, b) indicate significant difference (p < 0.05).

Cycloalliin contents among black garlic from the cultivar and 3 lines were increased during processing in the range of about 146.7–420.3 mg/100 g dry wt. (Kinkyou, 420.3 mg/100 g dry wt.; Taisou, 163.1 mg/100 g dry wt.; Katei, 146.7 mg/100 g dry wt.; Fukuchi White, 188.4 mg/100 g dry wt.). By processing to black garlic, cycloalliin contents were increased 1.3–1.5-fold compared to fresh garlic in every cultivar and line. In Kinkyou and Katei, the amount of cycloalliin was significantly (p < 0.05) increased after the 2-week heating period (Fig. 5). In addition, the content of isoalliin in fresh garlic was significantly higher in Kinkyou than other cultivar and lines, but it disappeared in the cultivar and lines after one-week processing. To understand the mechanism of cycloalliin formation during processing of black garlic, we focused on the decrease in isoalliin after one-week heating in comparison with the initial materials; and the formation mechanism of cycloalliin was proposed, as shown in Fig. 6. The decrease of isoalliin completely corresponded to the increase of cycloalliin during heat processing at 72 °C. The conversion rates from isoalliin to cycloalliin in the 4 black garlic samples after 2 weeks varied from 38.0 % to 82.3 % (Table 4). While the conversion rates of Kinkyou and Taisou increased from the first week to the second week, the conversion rates of Katei and Fukuchi White decreased. These results indicate that different phenomena, such as the formation or decomposition of cycloalliin, occur during the 2-week processing between Kinkyou and Taisou, and Katei and Fukuchi White. Decreases of cycloalliin in Katei and Fukuchi White may be caused by further conversion of cycloalliin to certain unknown compounds in the Maillard reaction and different enzyme activities; however, further detailed investigation is necessary to clarify the mechanism.

Fig. 5.

Changes in cycloalliin contents during heating.

Symbols: Kinkyou; ▲ Taisou; ■ Katei; ◆ Fukuchi white; ● Asterisk indicates significants difference of cycloalliin contents before and after treatment (*: p < 0.05; **: p < 0.01).

Fig. 6.

Conversion scheme from Glu-PEC to Cycloalliin

Heating test of isoalliin and cycloalliin    The conversion of isoalliin to cycloalliin under the conditions of black garlic formation was chemically confirmed with model compounds under model conditions (72 °C, pH 4.5). Under the model conditions, isoalliin was rapidly converted to cycloalliin, with a conversion efficiency of about 70 % during the treatment duration (Fig. 7a). It was also found that cycloalliin was stable throughout the reaction period (Fig. 7b). As the chemical conversion from isoalliin to cycloalliin was around 70 %, 80 % of cycloalliin in black garlic may be generated from isoalliin in fresh garlic through chemical reaction.

Fig. 7.

Conversion of isoalliin(a) and cycloalliin(b) in acetate buffer (72 °C, pH 4.5).

Symbols: Isoalliin; □ Cycloalliin; ○ in triplicated (n = 3).

In conclusion, metabolomic analysis of the water-soluble components, such as amino acids, especially sulfur-containing compounds, organic acids and so on, in fresh garlic and black garlic (one garlic cultivar and 3 lines) was an effective tool for the evaluation and selection of important and characteristic compounds found in fresh garlic and black garlic of the genus Allium. Finally, cycloalliin in the Kinkyou line could be detected and quantified as a functional component by HPLC analysis. A series of experiments with metabolomic analysis and heat-mapping technology enabled identification of differences according to garlic type, thereby providing an overall view of garlic components, while quantitative HPLC analysis of selected water-soluble components easily revealed the importance of cycloalliin. Cycloalliin, a physiologically important compound, was demonstrated to be contained at high levels in black garlic of the Kinkyou line in this paper. These findings may contribute to the utilization of Kinkyou black garlic as a value-added processed food.

Conflict of interest    There are no conflicts of interest to declare.

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