Changes in the antioxidant activity and metabolite analysis of black elephant garlic

doi:10.21203/rs.3.rs-2221036/v1

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Changes in the antioxidant activity and metabolite analysis of black elephant garlic

https://doi.org/10.21203/rs.3.rs-2221036/v1

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This study aimed to investigate the effects of the aging period on the black elephant garlic manufacturing process. Black elephant garlic is a processed elephant garlic product prepared by high temperature and high humidity treatment for 40 days. The proximate composition (moisture, crude lipid, crude protein, carbohydrate, and ash), minerals, color values, reducing sugars, pH, and antioxidant activities of elephant garlic and black elephant garlic were evaluated. The browning intensity of elephant garlic increased with the aging period, but the browning reaction terminated after aging for 30 days, showing the same browning level. Reducing sugars increased with increasing the aging period until 20 days, then decreased with the aging period, in contrast to the pH, which decreased from 6.47 to 3.68 with the increasing aging period. Antioxidant components, including the total polyphenol and total flavonoid contents of black elephant garlic, increased significantly until day 30 of aging. Through metabolite profiles from GC/MS analysis, it was confirmed that primary metabolites related to antioxidant components, such as lactic acid and 5-hydroxymethyl-2-furoic acid, were generated during the aging process of elephant garlic.

Elephant garlic

Black garlic

Antioxidant

Metabolite

Ageing period

Garlic (Allium sativum L.) is an important spice with a distinctive odor that has been widely consumed worldwide since ancient times and is particularly popular in Europe, Asia, and Africa[1]. Some of its many bioactive components are organic sulfides, saponins, phenolic compounds, and polysaccharides. Elephant garlic (EG) (Allium ampeloprasum L.) resembles leek (A. ampeloprasum var. porrum (L.) J. Gay) and common garlic in terms of shape and flavor, although it is three times larger than standard garlic[2, 3]. Also called giant garlic, Big Tex garlic, Tahiti garlic, or great-headed garlic, EG is large enough to weigh 450 g, has a milder flavor than garlic, and has a sweet taste, so it is used as a substitute for garlic in many countries[4]. The most important features of EG are its much lower contents of fiber and alliin, the sulfur-containing compound responsible for the aggressive taste of garlic, conferring this plant with a greater digestibility and a much more delicate taste than standard garlic. Black garlic has been recently introduced to the Asian market as a health product. Black garlic is formed by aging whole garlic at a high temperature and high humidity, causing the garlic to turn black because of browning compounds. Furthermore, black garlic does not exude a strong off-flavor like fresh garlic. During the heating process, various physicochemical changes occur, such as changes in color, texture, and flavor due to nonenzymatic browning reactions, such as the Maillard reaction and caramelization, which, in turn, alter the nutrient content.

In this study, BEG has produced from fresh EG by fermentation at 70℃ and 90% relative humidity for up to 40 days. Considering the various physicochemical changes that occur during the aging process, we hypothesized that there should be optimum aging conditions to maximize the antioxidant properties of BEG. In this regard, the objective of this study was to identify the physicochemical properties of BEG during 40 days of aging and determine the optimum aging period for maximum antioxidant properties. The results would contribute to further revealing the mechanism related to BEG formation and improving the quality of BEG.

Samples

EG was purchased from Gimpo Agricultural Association, South Korea in June 2020. EG was prepared based on the method described in [5]. EG was aged in a thermo-hygrostat chamber (KCL2000W, Tokyo Rikakikai Co., Ltd, Tokyo, Japan) at 70℃ in 90% relative humidity for 10, 20, 30, and 40 days. Elephant garlic and black elephant garlic (were freeze-dried (MCFD 8508, Ilshin Bio Base, Yangju, Korea) and used in the experiment.

Materials

Folin-Ciocalteu’s phenol reagent, DPPH, Methoxyamine hydrochloride, BSTFA, and pyridine (anhydrous 99.8%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluoranthene was purchased from Supelco (Bellefonte, PA, USA). All chemicals and reagents used in this study were of analytical grade.

Sample Preparation And Metabolites Analysis Gc/ms

About 1 g of freeze-dried black elephant garlic was mixed with 30 mL of 70% MeOH using sonication for 30 min. After filtration with 0.2 µm microporous membranes, 150 µL of the collected supernatant was transferred into a GC vial and dried using a speed vac. For the analysis of black elephant garlic compounds by GC-MS, dried extracts samples were redissolved and derivatized by the addition of 50 µL of 20 mg/mL methoxyamine hydrochloride in pyridine and incubated at 30℃ for 90 min for oxidation. To each sample add 50 µL BSTFA, for trimethylsilylation derivatization and 30 µL of fluoranthene (1,000 µg/mL in pyridine as an internal standard) and heat the mixture at 60℃ for 30 min. And then subjected to GC/MS analysis. Each 1.0 µL aliquot of derivatized sample was injected in a 30:1 split ratio into a Thermo scientific (Trace 1310/ISQ LT) gas chromatography-mass spectrometry (GC/MS) equipped with DB-5MS capillary column (60 mm × 0.25 mm × 0.25 µm, Agilent J & W Scientific, Santa Clara, CA, USA). Helium was used as the carrier gas with a constant flow rate of 1.5 ml/min. The temperature program was as follows: the initial temperature was 50℃ held for 2 min, raised to 180℃ at a rate of 5℃ /min and held for 8 min, and increased to 210℃ at a rate of 2.5℃/min, elevated to 325℃ at a rate of 5℃/min and was maintained for 10 min. The injector, transfer line, and ion source temperature were set to 300, 310, and 270℃, respectively. The mass range (35–650 m/z) in a full-scan mode for electron impact ionization (70eV) was applied. The solvent delay time was set to 14 min.

Identification And Quantification Of Metabolites

The identification of each metabolite was positively confirmed by comparison of retention time and mass spectral data with those of a NIST/EPA/NIH Mass Spectral Library (version 2.0, National Institute of Standards and Technology, Gaithersburg, MD, USA). All metabolites were identified by comparing mass fragments with the standard mass spectra on the commercial database NIST with a similarity of more than 70%. The calculated area of each compound was normalized by dividing the internal compound (fluoranthene) peak area to give semi-quantitative composition of components. Each compound was quantified against the internal standard by integrating the peak areas.

Determination Of Proximate Composition

General component analyses were conducted using AOAC method as the standard. Moisture was measured with a moisture meter (MB45, Ohaus Corporation, Zurich, Switzerland) using a 105℃ normal pressure and high-temperature drying method. Crude protein was measured using an automatic nitrogen distillation device (Kjeltec 2200 analyzer, Foss Co. Slangerupgade, Hillerod, Denmark) using the Micro-Kjeldahl nitrogen method and crude fat was measured by Soxhlet's extraction method using an automatic crude fat extractor (Soxhlet Avanti 2050, Foss Co., Slangerupgade, Hillerod, Denmark). Further, ash was quantified by a direct method at 550 ~ 600℃ using an electric furnace (Thermolyne F-48000, Bransted/Thermolyne Co., Dubuque, IA, USA). Carbohydrate content was determined by subtracting the content of water, crude protein, crude fat, and crude flour from 100 using the subtraction method.

Determination Of Mineral Contents

After adding distilled water to 1 g of the freeze-dried sample, it was filled to 30 mL, ultrasonically extracted for 1 h, and then ion components were extracted in an oven at 85℃ for 15 h. The extract was filtered through a 0.2 µm filter and analyzed using ion-chromatography (ICS-3000, Dionex, Sunnyvale, CA, USA). Among mineral nutrient, anion was quantified by referring to IonPac® AS20 Anion-Exchange Column and cation was quantified by referring to IonPac® CS16 Cation-Exchange Column.

Color Analysis

Surface color measurements of elephant garlic according to the aging period were determined using a Hunter color reader (CR-400, Minolta Co., Osaka, Japan) on cross-sections selected from five random locations of each sample. Lightness (L*-value), redness (a*-value), and yellowness (b*-value) values were recorded. A white standard plate with a lightness of 95.59, redness of -1.22, and yellowness of + 10.07 was used as a reference.

Ph, Reducing Sugars, And Browning Intensity Analysis

Add 10 g of elephant garlic to 10 times distilled water, homogenized for 3 min at 1,500 rpm with a homogenizer (PT-2100, Kinematica Ag, Littau, Switzerland), and then filter the supernatant to the sample. The pH was measured using a pH meter (PB 30, Sartorius, Goettingen, Germany) and the browning intensity was measured by UV-VIS spectrophotometer (T60UV, PG Instruments, Wibtoft, England) at an absorbance of 420 nm.

The determination of reducing sugar content was carried out using the DNS method[6]. The sample (1 mL) was mixed with 3 mL of the DNS reagent and shaken slightly. The mixture solution was heated in 75℃ water bath (WBT-10, Jeongbiotech co., Incheon, Korea) for 5 min. After heating, the mixture was cooled in running water for 10 min. The absorbance at 575 nm was determined. The standard curve used glucose and the reducing sugar content of the elephant garlic extract according to the aging period was expressed as the equivalent of D-glucose per 1 mL of the sample.

Extraction Of Elephant Garlic

After adding 20 times 70% ethanol to 40 g of each EG powder, the reflux extraction method by repeating 3 times for 2 h at 80°C in a water bath. The extract is filtered, concentrated under reduced pressure with a rotary vacuum evaporator (NVC-2100, EYELA, Tokyo, Japan) at 60°C, freeze-dried (MCFD 8508, Ilshin Bio Base, Yangju, Korea), and dried extracts were stored at − 40°C before analysis.

Determination Of Tpc

TPC was determined based on the method of Folin-Ciocalteu by Hillis & Swain[7]. Briefly, 150 µL of samples 2,400 µL of distilled water and 50 µL 2 N Folin-Ciocalteu reagent and these mixtures were reacted at room temperature for 3 min. Then, after 300 µL of 5% Na₂CO₃ was added, the mixtures were reacted in darkness for 2 h. The absorbance was measured using a UV/VS spectrophotometer at 725 nm. TPC results were expressed as mg GAE/g dry weight.

Determination Of Tfc

TFC was measured based on the method by Davis[8] with slight modification. Briefly, 1 mL of samples were mixed with 90% diethylene glycol (10 mL). The mixtures were added with 1 N NaOH (1 mL) and reacted at 37℃ for 1 h. The absorbance was measured at 420 nm. TFC results were expressed as mg RE/g dry weight.

Dpph Free Radical Scavenging Activity Assay

The DPPH radical-scavenging activity assay was performed by the method of Blios[9] with some modification. The 3 mL of samples were mixed to 1 mL of DPPH solution (1.5×10⁻⁴ M, in ethanol). The mixtures were incubated in darkness for 30 min at room temperature and measured at 517 nm to evaluate the absorbance using a UV/VIS spectrophotometer. The DPPH radical scavenging activity was calculated using the equation below:

DPPH radical scavenging activity (%) = (1-sample absorbance/control absorbance) × 100

Statistical Analysis

All experiments in this study were performed in triplicate replicates and the results are presented as the mean ± SD of three independent experiments. All experimental results were analyzed using the SPSS program (Statistical Analysis Program, version 25, IBM Co., Armonk, NY, USA). One-way analysis of variance (ANOVA) and Duncan’s multiple range test were used at the level of significance of p < 0.05. The partial least square (PLS) models were created using SIMCA software (Ver 17, Umetrics, Umea, Sweden) and bivariate correlation analyses were conducted using IBM SPSS Statistics ver 25.

Proximate Composition

The time-dependent changes in the proximate composition of the samples during aging at 70°C and 90% relative humidity are shown in Table 1. The moisture contents of EG, BEG1, BEG2, BEG3, and BEG4 were 6.28, 15.84, 16.95, 12.23, and 13.14 g/100 g, respectively(p < 0.001). In this experiment, moisture absorption occurs more actively than dehumidification at the beginning of aging, and the moisture content increases until 20 days. When the aging period exceeds 20 days, moisture is lost through the heat treatment process at a high temperature of 70°C, and the moisture absorption process decelerates, contributing to increasing the level of dehumidification. Therefore, the water content decreases after aging for 30 days because of the evaporation of the free water in the food, leaving only bound water.

Table 1

Proximate composition of elephant garlic powders according to the aging period
Aging period (days)	Proximate composition (%)
Aging period (days)	Moisture	Crude protein	Crude fat	Crude ash	Carbohydrate
0	6.28 ± 0.01^e	13.24 ± 0.04^a	0.24 ± 0.02^c	2.59 ± 0.03^d	77.90 ± 0.02^a
10	15.84 ± 0.01^b	10.96 ± 0.02^c	0.04 ± 0.01^e	2.85 ± 0.01^c	70.36 ± 0.00^d
20	16.95 ± 0.03^a	10.61 ± 0.08^e	0.10 ± 0.00^d	2.87 ± 0.01^c	69.58 ± 0.06^e
30	12.23 ± 0.03^d	11.29 ± 0.04^b	0.30 ± 0.03^b	3.14 ± 0.02^a	73.35 ± 0.04^b
40	13.14 ± 0.06^c	10.81 ± 0.05^d	0.64 ± 0.04^a	3.04 ± 0.01^b	73.02 ± 0.00^c
All values are mean ± SD(n = 3).
^a-e) Values with different letters within a column differ significantly by Duncan’s multiple range test(p < 0.05).

The crude protein content of the samples ranged from 10.61% in BEG2 to 13.24% in EG. The carbohydrate contents of EG, BEG1, BEG2, BEG3, and BEG4 were 77.90, 70.36, 69.58, 73.35, and 73.02 g/100 g, respectively(p < 0.001). Regarding the crude ash content, EG showed the lowest (2.59%) and BEG3 the highest (3.14%), indicating a tendency to increase with the aging period until 30 days of aging(p < 0.001). The crude fat content was in the range of 0.24−0.64%, and as the aging period increased, the crude fat content tended to increase(p < 0.001).

Mineral Contents

The mineral contents of the samples as a function of the aging period are shown in Table 2. Among minerals, the Potassium content was the highest in all samples, and the Potassium, Sulfate, and Phosphate content increased as the aging period elapsed(p < 0.001). EG had the lowest Potassium, Sodium, Magnesium, Calcium, Sulfate, and Phosphate before aging, but after aging Potassium, Sodium, Magnesium, Calcium, Sulfate, and Phosphate showed increase in mineral contents. Kim et al. also reported similar results of the highest Potassium and increased Sodium, Magnesium, Calcium, Sulfate and Phosphate in BEG after aging[10].

Table 2

Mineral contents of elephant garlic powders according to the aging period
Aging period (days)	Mineral contents (mg/100 g)
Aging period (days)	Sodium	Potassium	Magnesium	Calcium	Chlorine	Sulfate	Ammonium	Phosphate
0	1.93 ± 0.09^c	981.59 ± 0.37^e	31.74 ± 1.55^e	0.90 ± 0.20^e	78.49 ± 0.38^b	11.88 ± 0.33^d	149.01 ± 0.02^b	133.81 ± 1.39^e
10	1.78 ± 0.06^c	1184.41 ± 22.30^d	82.51 ± 0.77^a	8.12 ± 0.44^d	58.54 ± 1.65^d	30.84 ± 0.16^c	161.81 ± 1.23^a	672.73 ± 4.35^d
20	1.71 ± 0.05^c	1238.25 ± 1.96^c	65.61 ± 2.23^d	10.58 ± 0.09^c	62.95 ± 2.65^c	31.07 ± 1.06^c	118.66 ± 1.37^c	807.35 ± 3.28^c
30	9.82 ± 0.18^a	1290.54 ± 14.61^b	76.19 ± 1.14^c	21.17 ± 0.42^a	79.54 ± 1.43^b	44.15 ± 1.29^b	96.67 ± 1.62^d	934.04 ± 2.16^b
40	2.29 ± 0.23^b	1334.28 ± 8.16^a	80.84 ± 6.09^ab	17.80 ± 0.33^b	84.45 ± 1.47^a	57.73 ± 0.31^a	70.04 ± 0.56^e	1034.63 ± 2.76^a
All values are mean ± SD(n = 3).
^a-e) Values with different letters within a column differ significantly by Duncan’s multiple range test (p < 0.05).

Color Values And Browning Intensity

Color is one of the most important psychological properties affecting consumer perception toward food. The browning intensity of aged garlic is affected by temperature, moisture content, and reducing sugars[11]. The color values and browning intensity results of the BEG according to the aging period are shown in Table 3 and Fig. 1. The L*-value (lightness) was 95.33 for EG, which decreased sharply to 33.25−33.61 in BEG aged for 10 days(p < 0.001). Consistent with the development in the dark brown appearance of aged garlic during the aging period, the a*-value (redness) was the lowest in EG at -2.07 and in BEG1 at 7.37, showing an initial rapid increase in redness and tending to decrease with the prolonging of the aging period(p < 0.001). For the b*-value (yellowness), EG showed the highest value at 17.16, and in BEG1, BEG2, BEG3, and BEG4, it decreased to 11.22, 4.28, 2.86, and 2.03, respectively(p < 0.001).

Table 3

Color values, browning intensity, pH, and reducing sugar contents of elephant garlic according to the aging period
Aging period (days)	L value	a value	b value	Browning intensity (420 nm OD)	pH	Reducing sugar content (%)
0	95.33 ± 0.19^a	-2.07 ± 0.06^e	17.16 ± 0.11^a	0.40 ± 0.01^d	6.47 ± 0.03^a	0.37 ± 0.01^d
10	33.25 ± 0.81^b	7.37 ± 0.08^a	11.22 ± 0.52^b	1.19 ± 0.01^c	4.57 ± 0.01^b	7.16 ± 0.13^c
20	33.36 ± 0.64^b	6.10 ± 0.17^b	4.28 ± 0.74^c	2.83 ± 0.01^b	4.04 ± 0.02^c	9.67 ± 0.30^a
30	33.41 ± 0.45^b	3.66 ± 0.06^c	2.86 ± 0.41^d	3.00 ± 0.00^a	3.81 ± 0.01^d	9.23 ± 0.18^b
40	33.61 ± 0.50^b	2.35 ± 0.17^d	2.03 ± 0.31^d	3.00 ± 0.00^a	3.68 ± 0.01^e	9.11 ± 0.07^b
All values are mean ± SD(n = 3).
^a-e) Values with different letters within a column differ significantly by Duncan’s multiple range test(p < 0.05).

During the aging period, the browning intensity of the samples increased from 0 to 20 days and then plateaued, such that the final optical density value was 3.00(p < 0.001). Considering that polyphenol oxidase loses its activity at 50 ~ 70℃, it is reasoned that the degree of browning increased due to nonenzymatic browning reactions between amino acids of peptides with sugars, as well as between α-amino groups of proteins with sugars.

Ph

The changes in the pH of the samples during the aging period are shown in Table 3. The pH of EG was 6.47, but as the aging period increased, the pH decreased significantly, and it was confirmed that it was acidic(p < 0.001). Najman et al. reported a similar result that the pH of aged garlic (heated at 70°C, 80% relative humidity, 45 days) was around 2.1−2.5 pH units lower compared to fresh garlic[11]. The pH decrease in the heated garlic sample was partly associated with the browning development upon heat treatment during the black garlic manufacturing process. The formation of carboxylic acids, which are produced by the oxidation of aldehyde groups in aldohexoses, along with the formation of acidic compounds and the decrease in basic amino acids by combining with sugars, is reported to be responsible for the decrease in pH in the browning reaction[12, 13, 14]. In addition, it is thought the pH is lowered by the pyruvic acid produced by the decomposition of alliin in fresh garlic by the heat treatment process during the aging of garlic[15]. As a result of the low pH caused by the aging process, aged garlic has a comparatively long shelf life[16]. A low pH is beneficial for eliminating colonies of bacteria or fungi that cause food spoilage[17]. The decrease in pH value not only contributes to the acidic preservative action of black garlic but also produces a sour taste and mouthfeel.

Reducing Sugars

At high temperatures during garlic processing, numerous nonenzymatic browning reactions occur, such as the Maillard reaction, caramelization, and macromolecular degradation[18]. The changes in the reducing sugars of the elephant garlics during the aging period are shown in Table 3. The reducing sugars of EG, BEG1, BEG2, BEG3, and BEG4 were 0.37%, 7.16%, 9.67%, 9.23%, and 9.11%, respectively(p < 0.001). Although the reducing sugar content initially increased with the aging period, it showed a tendency to decrease after aging for 20 days. The decrease in pH during heat treatment promotes the decomposition of sucrose into glucose or fructose, thereby increasing the content of these saccharides during the maturation of black garlic[19]. This was exemplified in the study by Choi et al., who showed that the sugar content (e.g., glucose, fructose, sucrose, and maltose) increased in black garlic compared to fresh and steamed garlic[20, 21]. During the initial aging of garlic at 70℃, the rate of production of reducing sugar is faster than the rate of consumption in the Maillard reaction, which explains the increase in reducing sugar content until day 20 of aging. With further aging, reducing sugars due to consumption in the Maillard reaction decreases from day 30[22].

Profiling Of Metabolites In Eg According To The Aging Period

In this study, to identify the metabolites of major nutrients, including amino acids, organic acids, sugars, and sugar derivatives, in garlic according to the aging period, each sample was evaluated by GC/MS after derivatization. The results are shown in Table 4. In total, 41 metabolites were identified in the GC/MS data sets obtained from garlic according to the aging period. These compounds were found at various levels during the aging period and included 12 amino acids (alanine, leucine, isoleucine, valine, threonine, glycine, serine, aspartic acid, pyroglutamic acid, GABA, asparagine, and glutamic acid), 10 organic acids (propanoic acid, lactic acid, glycolic acid, oxalic acid, β-lactic acid, succinic acid, glyceric acid, malic acid, 2-deoxytetronic acid, and L-threonic acid), 13 sugars and sugar derivatives (D-erythro-pentofuranose, ribitol, 2-deoxy-D-erythro-pentofuranose, fructofuranose, sorbose, fructose, glucose, β-D-glucopyranose, sucrose, xylose, ribofuranose, fructopyranose, and 3-α-mannobiose), 6 others (phosphoric acid, 2-deoxypentonic acid, D-erythronic acid, 5-HMFA, 2-desoxy-pentos-3-ulose, and ribonic acid) (Table 4).

Table 4

Tentatively identified compounds of elephant garlic by GC-MS according to the aging period
Compounds	RT¹⁾ (min)	Treatment²⁾					TMS³⁾	Quantitative
Compounds	RT¹⁾ (min)	EG	BEG1	BEG2	BEG3	BEG4	TMS³⁾	Quantitative
Amino acids (12)
Alanine	17.79	521.19	70.45	322.18	199.19	227.72	2	116
Leucine	19.53	24.50	601.78	316.90	185.07	91.65	1	86
Isoleucine	20.18	N.A.⁴⁾	316.97	180.58	141.06	84.94	1	86
Valine	21.17	547.26	132.37	352.08	194.34	179.96	2	144
Threonine	23.44	20.88	134.10	62.73	27.29	10.51	2	117
Glycine	23.78	247.32	209.74	392.89	327.31	258.53	3	174
Serine	25.17	99.29	28.99	57.87	16.66	9.79	3	204
Aspartic acid	26.97	27.51	213.12	194.96	199.18	120.05	2	160
Pyroglutamic acid	29.55	2670.61	25623.1	34005.24	28167.81	24740.50	2	156
γ-Aminobutyric acid	29.74	411.97	317.87	511.51	205.84	103.26	3	174
Asparagine	31.84	811.77	713.83	209.12	82.64	N.A.	2	75
Glutamic acid	32.74	153.38	78.52	78.83	42.56	37.46	3	246
Organic acids (10)
Propanoic acid	16.15	49.65	532.77	328.54	187.20	143.59	2	147
Lactic acid	16.45	34.34	268.72	919.14	1236.65	1380.65	2	147
Glycolic acid	16.97	21.55	2020.76	3768.23	4811.96	5397.26	2	147
Oxalic acid	18.96	930.82	14.74	N.A.	N.A.	N.A.	2	147
β-Lactic acid	19.07	N.A.	315.19	1537.15	2640.01	3418.81	2	147
Succinic acid	24.00	34.68	230.71	307.44	438.10	547.67	2	147
Glyceric acid	24.35	12.87	624.24	1476.12	2246.77	2836.04	3	147
2-Deoxytetronic acid	27.05	N.A.	21.36	51.36	69.65	84.17	3	233
Malic acid	28.49	1928.60	5669.57	4980.11	5093.08	4800.89	3	147
L-Threonic acid	29.91	21.50	1144.63	1250.34	1419.17	1454.97	4	147
Sugars and sugar derivatives (13)
D-Erythro-pentofuranose	33.95	35.35	36.28	39.95	27.34	17.79	3	245
Ribitol	35.82	19.92	197.26	135.73	83.81	59.37	5	103
2-Deoxy-D-erythro-pentofuranose	39.02	23.35	43.66	181.76	237.03	300.97	3	129
Fructopyranose	40.93	N.A.	12546.04	79821.30	46575.95	40806.23	5	204
Fructose	43.50	1141.52	781178.8	824092.8	713242.3	741031.80	5	103
Sorbose	44.27	N.A.	1522.51	6331.34	5254.07	5251.97	5	103
Glucose	44.47	132.31	16917.33	80072.42	52372.73	48026.24	5	204
β-D-Glucopyranose	48.28	122.19	23690.27	109373.60	72818.34	68102.54	5	204
Sucrose	63.98	65372.61	138504.1	4686.93	113.43	N.A.	8	361
Xylose	64.31	N.A.	66.38	575.00	718.03	742.80	5	103
Ribofuranose	64.40	1109.61	28238.09	55670.27	49486.07	53381.66	4	217
Fructofuranose	64.72	79.16	45745.33	277550	187077.3	157197.2	5	217
3-α-Mannobiose	65.69	27.64	233.66	729.25	585.78	597.90	8	103
Others (6)
Phosphoric acid	22.76	396.49	19709.66	33735.43	39475.58	45228.11	3	299
2-Deoxypentonic acid	26.40	N.A.	37.91	103.05	96.86	99.32	2	103
D-Erythronic acid	27.09	N.A.	41.86	104.95	177.45	229.67	2	103
5-Hydroxymethyl-2-furoic acid	30.33	N.A.	10	114.01	186.82	211.84	2	271
2-Desoxy-pentos-3-ulose	37.99	N.A.	160.35	508.06	576.26	695.77	2	231
Ribonic acid	39.16	N.A.	170.71	253.27	265.49	281.77	5	292
¹⁾ RT: retention time.
²⁾ EG(elephant garlic), BEG1(black elephant garlic aged for 10 Day), BEG2(black elephant garlic aged for 20 Day), BEG3(black elephant garlic aged for 30 Day), BEG4(black elephant garlic aged for 40 Day).
³⁾ TMS: trimethylsilylation.
⁴⁾ N.A.: not available.
All values are concentration; fluoranthene equivalent ug/g, mean values (n = 3).

Among the amino acids detected, pyroglutamic acid displayed the highest content in all samples. Serine, asparagine, and glutamic acid tended to decrease as the aging period elapsed. Amino acid metabolites, such as leucine, isoleucine, threonine, glycine, aspartic acid, and pyroglutamic acid, showed the lowest content in EG, increased up to 10 or 20 days, and then decreased gradually with further aging. In addition, GABA, a biologically important non-protein amino acid, showed a tendency to decrease after 20 days. According to previous studies, GABA tends to decrease due to damage and involvement of amino acids in nonenzymic browning reactions during BEG production[23].

Among the organic acid metabolites detected, malic acid exhibited the highest content in all samples. Organic acids, such as lactic acid, glycolic acid, β-lactic acid, succinic acid, glyceric acid, 2-deoxytetronic acid, and L-threonic acid, showed a tendency to increase throughout the aging period. Conversely, oxalic acid, a toxic organic acid, was the only organic acid that decreased significantly after aging and was not detected in BEG2, BEG3, and BEG4. The increased organic acid contents in BEG result from the formation of carboxylic acids, produced by the oxidation of the aldehydes group in aldohexoses, and the decrease in basic amino acids by combining with sugars in the Maillard reaction[24, 25, 26].

Black garlic is rich in sugar and has a high total sugar content of 60−70% per mass[27]. During the aging process, garlic polysaccharides are broken down into monosaccharides, including glucose and fructose[28]. Fructose recorded the highest content of all the sugar and sugar derivative metabolites detected. Sugars 2-deoxy-D-erythro-pentofuranose and xylose tended to increase throughout aging. Most of the sugar and sugar derivative metabolites (D-erythro-pentofuranose, ribitol, fructopyranose, fructose, sorbose, glucose, β-D-glucopyranose, ribofuranose, fructofuranose, and 3-α-mannobiose) increased up to 20 days of aging and then decreased after that. Among the sugar metabolites, sucrose, a disaccharide, had the highest content in EG and was the only sugar metabolite to decrease as aging progressed.

As mentioned above, six metabolites classified as others were also detected. These six metabolites showed a tendency to increase with the aging period, and 2-deoxypentonic acid, D-erythronic acid, 5-HMFA, 2-desoxy-pentos-3-ulose, and ribonic acid were not detected in EG.

In a study on the antioxidant activity of the Maillard reaction products, most amino acids except cysteine and tryptophan and sugars, such as fructose and glucose, produce melanoidins, dark browning polymeric compounds, due to the Maillard reaction, which improve antioxidant activity[29]. In the present metabolite experiment, most of the sugars and amino acids required for the Maillard reaction increased up to 20 days of aging and decreased at 30 days.

Differences in metabolites appearing after the aging of foods are related to changes in the sensory properties of foods, such as color or flavor preference, as well as differences in antioxidant effects[30]. The sugar and amino acids of aged BEG were relatively high, so it is expected that the sweet and sour taste will be enhanced.

Total Polyphenol, Total Flavonoid Contents And Dpph Free Radical Scavenging Activity

Garlic is one of the richest sources of phenolic compounds among the common vegetables in the human diet. To clarify the antioxidant properties of black garlic during aging, we focused on the analysis of total polyphenol and total flavonoid contents (Table 5). The total polyphenol contents (11.84 to 27.08 mg GAE/g) and total flavonoid contents of BEG (2.48 to 8.75 mg RE/g) were not only significantly higher than those of EG (4.61 mg GAE/g and 0.86 mg RE/g) but also increased significantly until day 30 of aging, before decreasing after that (p < 0.001). According to Xu et al., heat treatment of the phenolic compounds increases the fraction of free phenolic acids but decreases the ester, glycoside, and ester-bound fractions, leading to an increase in free phenol forms[32].

Table 5

Antioxidants of elephant garlic according to the aging period
Aging period (days)	Total polyphenol content (mg GAE¹⁾/g)	Total flavonoid content (mg RE/g)	DPPH radical scavenging activity (%)
0	4.62 ± 0.48^b2)	0.86 ± 0.03^e	20.27 ± 0.13^e
10	11.84 ± 0.14^b	2.48 ± 0.05^d	50.49 ± 0.47^d
20	23.43 ± 0.41^a	7.27 ± 0.10^c	82.49 ± 0.26^c
30	27.08 ± 0.14^a	8.75 ± 0.21^a	90.98 ± 0.23^a
40	25.10 ± 0.71^a	8.39 ± 0.25^b	85.63 ± 0.24^b
All values are mean ± SD.
¹⁾ GAE: gallic acid equivalent, RE: rutin equivalent.
^a−e) Values with different letters within a column differ significantly by Duncan’s multiple range test(p < 0.05).

The DPPH radical scavenging activity ranged from 20.27% in EG to 90.98% in BEG3, indicating a tendency to increase with the aging period until 30 days(p < 0.001). In a previous study of black garlic, the DPPH activity increased intensively (about 2-fold) until day 21 of aging, then decreased slightly after that, showing a similar trend to the present study[20]. Based on the above results of antioxidant compounds and antioxidant activities, we propose that the optimum aging period for maximizing the antioxidant properties of BEG is 30 days.

Correlation Between Metabolite Variation and Antioxidant Activities of EG According to Aging Period.

PLS was performed to find out the correlation between the metabolic variation and antioxidant activity of EG according to the aging period. In the PLS biplot for EG aged for different times, PLS components 1 and 2 together accounted for 85.9% of the total variance: 64.1% and 21.8%, respectively. The parameters of the cross-validation modeling were PLS component 3, with R²X = 0.935, R²Y = 0.961, and Q² (cumulative) = 0.879. In this study, a single graphical representation, which combined the score and loading plots, was created, as shown in the PLS biplot. The PLS biplot shows the pair-wise correlation between all variables (X and Y), illustrating the association between antioxidant activities and metabolites. In addition, the chemical shift of the metabolites was represented by the X variables, and antioxidant activities were represented by the Y variables (Fig. 2). In Fig. 2, the PLS biplot showed a clear separation into three groups and strongly correlated with antioxidant activity. EG samples aged for 20, 30, and 40 days were located in the upper right of the PLS biplot, and antioxidant activity variables (total polyphenol content, total flavonoid content, and DPPH free radical scavenging activity) were found near these samples. This indicates that EG samples aged for 20, 30, and 40 days had higher antioxidant activity than samples aged for 0 and 10 days. These results suggest that some new antioxidant components might have been produced during the aging of EG.

When looking at a common metabolite with a correlation matrix of 0.8 or higher according to PLS, organic acids (lactic acid, glycolic acid, β-lactic acid, succinic acid, glyceric acid, and L-threonic acid), sugars and sugar derivative metabolites (2-deoxy-D-erythro-pentofuranose, glucose, xylose, ribofuranose, and fructofuranose), and other metabolites (2-deoxypentonic acid, D-erythronic acid, 2-desoxy-pentos-3-ulose, 5-HMFA, and ribonic acid) had a substantial influence on antioxidant activity.

In particular, lactic acid is the major organic acid in BEG, as found by metabolite analysis; therefore, lactic acid may be responsible for the unique flavor of black garlic. Furthermore, lactic acid is also a strong antioxidant, which could have contributed to the strong antioxidant capacity of black garlic[33].

In metabolite analysis, 5-HMFA, which is the main metabolite of 5-HMF, was not found in EG, whereas the amount of 5-HMFA increased during the processing of EG into BEG, reaching 10-fold the 5-HMF content at 40 days of the aging process compared with that aged for 10 days (Table 4). 5-HMF is a five-carbon-ring aromatic aldehyde that does not naturally occur in fresh foods but can be formed in sugar-containing foods during thermal treatments. Its formation derives from the catalytic dehydration of Amadori products during the Maillard reaction at high temperatures and the direct degradation of hexose sugars in an acidic environment. 5-HMF exhibits a range of biological and pharmacological properties, such as antioxidant[34], antitumor[35], and anti-inflammatory[36]. However, 5-HMF may be cytotoxic at high concentrations and causes irritation to tissues and internal organs of the human body, with mutagenicity and carcinogenicity in vivo[37, 38], although the results of epidemiological investigations have not yet confirmed the potential association of 5-HMF with cancer risks in humans[39]. 5-HMF affects not only the biological activity (one major antioxidant in black garlic) but also the sensory characteristics of black garlic (with bitterness at a relatively higher concentration)[40]. The formation of 5-HMF in foods is highly dependent on the processing and storage conditions, such as temperature and pH[41]. As a crucial intermediate in the Maillard reaction with a close correlation with the browning rate of food, 5-HMF can be an important criterion for black garlic in setting the aging period[40].

This study measured the content of quality indicators according to the aging period during the processing of EG. The aging period significantly affected the quality of BEG, including the antioxidant activity and content of various substances in garlic. Browning was shown to proceed with aging due to browning reactions, resulting in high antioxidant activity. In addition, the accumulation of reducing sugars is relatively high in aged BEG and the sweetness and umami taste are strengthened due to the increase in the sugars and amino acids. In addition, as the organic acids increased, the pH decreased, and expect to see improvement in storage properties. In the aging process of EG, compounds with strong antioxidant properties were formed due to nonenzymatic browning reactions, and the content of antioxidant components, including total polyphenols and total flavonoids, increased significantly up to 30 days. Thus, it was confirmed that the optimal ripening period for maximizing the antioxidant properties was 30 days. The PLS results confirmed the relationship between the antioxidant activity and metabolites of EG according to the aging period, showing that some metabolites, such as lactic acid, glycolic acid, 2-deoxy-D-erythro-pentofuranose, ribofuranose, 2-deoxypentonic acid, and 5-HMFA, were highly correlated with strong antioxidant activity. As such, we expect the difference in metabolites after aging was related to changes in the sensory characteristics of garlic, such as color and taste, and affected the antioxidant activity. These results can contribute to understanding the role of the appropriate processing period in shaping the quality of BEG. However, achieving quality in BEG production is a considerably complex process that is influenced by several factors besides aging period, such as temperature and relative humidity, during the aging process. Further effort to elucidate the effects of these factors on the quality of BEG products is required in future studies.

5-HMF 5-hydroxymethylfurfural

5-HMFA 5-hydroxymethyl-2-furoic acid

BEG black elephant garlic

BEG1 Elephant garlic aged for 10 days

BEG2 Elephant garlic aged for 20 days

BEG3 Elephant garlic aged for 30 days

BEG4 Elephant garlic aged for 40 days

BSTFA O-bis trimethylsilyl trifluoroacetamide

DNS 3,5-dinitrosalicylic acid

DPPH 2,2-diphenyl-1-picrylhydrazyl

EG Elephant garlic

GABA γ-aminobutyric acid

GAE Gallic acid equivalents

NIST National Institute of Standards and Technology

PCA Principal component analysis

PLS-DA Partial least square discrimination analysis

RE Rutin equivalents

TFC Total flavonoid content

TPC Total polyphenol content

Ethics Approval: Not applicable.

Consent to Participate: Not applicable.

Consent for Publication: Not applicable.

Competing Interests: Not applicable.

Funding: Not applicable.

Conflict of Interest: The authors state that there is no conflict of interest with respect to the objective, interpretation, and presentation of the results in this study.

Contributions: Young-Sil Han and Ki-Hyeon Sim contributed to the study conception and design. Se-Hyun Nam and Seung-Ok Yang performed the data analysis. Se-Hyun Nam wrote the main manuscript text and Myung-Hyun Kim edited the manuscript. All authors reviewed and approved the final version of the manuscript.

Data Availability: The data from the current study are available from the corresponding author and first author of the article on reasonable request.

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No competing interests reported.

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Changes in the antioxidant activity and metabolite analysis of black elephant garlic

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Abstract

Figures

Introduction

Material And Method

Samples

Materials

Sample Preparation And Metabolites Analysis Gc/ms

Identification And Quantification Of Metabolites

Determination Of Proximate Composition

Determination Of Mineral Contents

Color Analysis

Ph, Reducing Sugars, And Browning Intensity Analysis

Extraction Of Elephant Garlic

Determination Of Tpc

Determination Of Tfc

Dpph Free Radical Scavenging Activity Assay

Statistical Analysis

Results And Discussion

Proximate Composition

Mineral Contents

Color Values And Browning Intensity

Ph

Reducing Sugars

Profiling Of Metabolites In Eg According To The Aging Period

Total Polyphenol, Total Flavonoid Contents And Dpph Free Radical Scavenging Activity

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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