Abstract
The roasting treatment has been used to extend the shelf life of food and improves its quality, and eliminate or reduce toxic. In this study, we investigate the changes in the physicochemical properties and antioxidant activities of Ginkgo biloba seeds (GBS) according to the roasting temperature and duration. As the roasting temperature and duration increased, the pH (from 7.32 to 6.31) and total cyanide content (from 1.49 to 0.70 µg/g) decreased, whereas the titratable acidity (from 0.39 to 0.84%) increased. The antioxidant activities increased rapidly at 210 °C according to the increase in the roasting temperature and duration. The 4′-O-methylpyridoxine (MPN) content in the 210 °C heat treatment group decreased by more than 70% compared to the MPN content in the control group. These results suggest that heat-treated GBS could be used in food materials and medicines for decreasing cyanide and MPN contents as well as for increasing antioxidant compound contents.
Keywords: Ginkgo biloba seeds, Physicochemical properties, Antioxidant activities, Roasting conditions, 4′-O-methylpyridoxine
Introduction
Heat treatment extends the shelf life of food and improves its quality. But it has several drawbacks, such as the destruction of heat-sensitive nutrients and loss of active materials during the process. However, recent studies have reported that the contents of physiologically active substances increase as a result of various heat treatments, such as heating or blanching fruits and vegetables at approximately 100 °C, dry heat treatment including roasting at 150–200 °C, and wet heat treatment including autoclaving at 120 °C [1]. Therefore, various studies have been conducted on heat treatment processes. Among them, roasting has been reported to obtain a unique flavor and color by changing the ingredient and composition of products. It has been used to eliminate or reduce toxic and adverse effects to the human body, increase therapeutic effects, and increase the shelf life of materials [2].
Ginkgo biloba L. is a perennial plant endemic to Southeast Asia, including Korea, Japan, and China. Its seeds are drupes, with a smooth and odoriferous yellow outer seed coat and a hard and white inner seed coat having two cotyledons. The studies on G. biloba L. revealed flavonoids, diterpenes, sesquiterpenes, polyprenols, organic acids, and polysaccharides as its leaf components [3]. Studies on the flavonoid components of G. biloba leaves have been conducted to develop a drug that improves blood circulation to the brain and peripheral blood vessels by peripheral arterial dilation and platelet aggregation inhibition [4]. There have been several studies on G. biloba seeds (GBS). For example, in GBS, Wada et al. [5] identified 4′-O-methylpyridoxine (MPN), a food poisoning agent that causes vomiting, epilepsy, and convulsions by inhibiting pyridoxal kinase (a vitamin B6-activating enzyme). Leistner and Drewke [6] also described a dose of 11–50 mg/kg of the isolated MPN triggered symptoms of poisoning in the animals. The following aspects of GBS as a food material have been studied to date: (1) antioxidant activities of seeds of GBS [7]; (2) antimicrobial activity of extracts and fractions of G. biloba leaves, seeds, and the outer seed coat [8]; (3) use of desserts, grazed fruit, beverages or alcoholic drinks with added GBS [9]; and (4) Functional properties of protein isolates extracted from GBS [10]. As is evident, there has been little research regarding the transformation of GBS into high value-added food materials.
Therefore, in this study, we explored the possibility of developing G. biloba seeds as a functional and medicinal food by evaluating the changes in physicochemical quality, antioxidant activity, and MPN composition according to roasting temperature before and after heat treatment.
Materials and methods
Materials
Fresh GBS were obtained Yeongkwang in Jeollanam-do, Korea and prepared from washed seeds by removing the hulls. DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis-(2-ethylbenzothiazoline-6-sulfonate)), Folin–Ciocalteu’s phenol reagent, gallic acid, l-ascorbic acid, potassium cyanide, barbituric acid, isonicotinic acid, chloramine-T, and MPN were purchased from Sigma-Aldrich (St Louis, MO, USA). All organic solvents and other chemicals were of analytical grade or high performance liquid chromatography (HPLC) grade.
Sample preparation
The roasting condition was based on a method optimized by Jinap et al. [11]. Each 150 g sample was roasted at different temperatures (150, 180, and 210 °C) for different durations (0, 15, 30, 45, and 60 min), using a temperature-controlled roaster (MK-300, JC Company, Seoul, Korea). After cooling, the roasted seeds were freeze-dried using a freeze-dryer (TFD-8503, Ilshin Lab Co., Gyeonggi, Korea) and were vacuum-packed in polyethylene film. Each sample was ground in a coffee grinder (80350, Hamilton Beach Brands, Inc., VA, USA) and then sieved through mesh No. 40 to remove larger particles, and kept at − 30 °C before analysis. All procedures were conducted in triplicates.
Sample extraction
The lyophilizered sample (10 g) was extracted with distilled water and 70% ethanol (1:10, w/v) using an ultrasonic bath (JAC-3010, Kodo Co. Ltd., Gyeonggi, Korea) for 60 min. These solutions were then centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was filtered and concentrated using a rotary evaporator (CCA-111, Eyela, Tokyo, Japan), and then freeze-dried for performing various physicochemical analyses and measuring antioxidant activity.
Physicochemical analyses
The water content of GBS according to the roasting temperature and duration was determined by drying at 105 °C until insignificant consecutive weight changes were measured [12]. The pH values were measured using a pH meter (DOCU-pH meter; Sartorius, Bohemia, NY, USA) by putting 1.0 g of distilled water extract, which was dried using a freeze-drying method, in a stirrer (KMC-130SH, Vision Scientific Co., Ltd., Daejeon, Korea), and adding 10 mL of distilled water. At the same concentration, the titratable acidity was measured by a pH meter electrode method using AOAC [13]. The samples were neutralized with 0.1 N NaOH to pH 8.2. The used amount of 0.1 N NaOH was converted into citric acid content (%, w/w). The color was measured using a colorimeter (CR-300, Minolta, Osaka, Japan) for the Hunter L* (Lightness), a* (redness), b* (yellowness), and ΔE* (color difference) values. All samples were measured using a whiteboard for reference color (L* = 93.50, a* = 0.31, b* = 0.32). The measurements of water content, pH, titratable acidity, and chromaticity were repeated three times and the mean values were recorded.
Antioxidant compound analyses
The detection of amounts of total cyanide and colorimetric method based on the König reaction was proposed by Epstein [14]. Each lyophilizered sample (1.0 g) was homogenized with 40 mL of 0.1 M phosphoric acid and the solution was centrifuged for 20 min at 8000 rpm. A volume of 1 mL of the supernatant was transferred to a tight-capped vial and an equal amount of 4 M sulfuric acid was added. Hydrolysis was started by heating to 100 °C and continued for 50 min. The hydrolyzed sample was cooled in ice-cold water, with the stopper loosely in place, 3 mL of 3.0 M sodium hydroxide was added and after 5 min, 1 mL was added to 7 mL of 0.2 M acetate buffer at pH 5.0. Chloramine-T (0.4 mL) was added and approximately 5 min later, 1.6 mL of isonicotinic acid/barbituric acid was added. After 60 min, the absorbance was measured at 600 nm. A calibration curve was obtained using a standard solution of potassium cyanide. The amount of total cyanide present was obtained by linear extrapolation of the data to zero time.
The total phenolic content of the GBS was measured according to the method of Singleton and Rossi [15]. Folin–Ciocalteu reagent produces a spectrophotometrically determinable molybdenum V complex as a result of reduction of the reagent by polyphenol compounds. A total of 2 mL of a 2% Na2CO3 solution was added to 100 μL of ethanolic extract (1.0 mg/mL) from the GBS and allowed to stand at room temperature for 3 min, followed by addition of 0.1 mL of 50% Folin–Ciocalteu reagent. The resulting mixture was incubated for 30 min at room temperature, and the absorbance was measured at 750 nm. After calibration using a standard gallic acid curve as a reference, the total phenolic content was reported in µg of gallic acid equivalent per 1.0 mg (µg GAE/mg) of sample. Triplicate measurements were taken for all samples.
The reducing power was determined according to the method of Ouchemoukh et al. [16]. A volume of 2.5 mL of ethanolic extract (1.0 mg/mL) from GBS was mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6, Wako Pure Chemical Co., Osaka, Japan) and 2.5 mL of 1% potassium ferricyanide (Sigma), and the mixture was incubated at 50 °C for 20 min. After 2.5 mL of 10% trichloroacetic acid (w/v, Wako) was added, the mixture was centrifuged at 1000 rpm for 10 min. The upper layer (5 mL) was mixed with 5 mL of deionized water and 1 mL of 0.1% ferric chloride (Wako), and the absorbance was measured at 700 nm in a spectrophotometer (DU-800, Beckman Coulter Inc., CA, USA). A higher absorbance indicates a higher reducing power.
Antioxidant capacities
A DPPH solution for determination of the radical scavenging activity of G. biloba seeds was prepared using a modified method of Al et al. [17]. DPPH was diluted using 99.9% ethanol at 0.005 g/100 mL, followed by incubation in the dark for 2 h, which resulted in absorbance values of 1.5–1.7 at 520 nm. Additionally, 0.2 mL of an ethanolic extract (1.0 mg/mL) from GBS was added to 0.8 mL of the DPPH solution, agitated, and incubated at room temperature for 30 min, and the absorbance was measured at 520 nm. l-Ascorbic acid was added as a reference material. The DPPH radical scavenging activity was calculated as the percentage decrease in the absorbance at 520 nm relative to a blank. The positive control was 1.0% l-ascorbic acid. The percentage of the scavenging activity of the DPPH radical was calculated using the following equation: DPPH radical scavenging activity (%) = (A blank − A sample/A blank) × 100. In this equation, A blank and A sample are the absorbances of the control and extract, respectively. All samples were analyzed in triplicate.
The total antioxidant capacity of GBS with roasting was measured using the method of Re et al. [18] with ABTS cation decolorization assays. First, 7.4 mM ABTS and 2.6 mM potassium persulfate were mixed at a 1:1 ratio, agitated, and allowed to react in the dark for 12–16 h. This mixture was then diluted with distilled water using the molar absorption coefficient so that the absorbance value was 1.4–1.5 at 735 nm. Next, 50 μL of an ethanolic extract (1.0 mg/mL) from GBS was added to 1 mL of a diluted ABTS solution and allowed to react for 60 min, after which the absorbance was measured at 735 nm. The same amount of l-ascorbic acid was added for use as a reference material. The total antioxidant capacity was expressed as antioxidant capacity relevant to l-ascorbic acid (AEAC, mg AA eq/100 g). All samples were measured in triplicate.
4′-O-methylpyridoxine (MPN) content
The MPN content of GBS with roasting was determined by HPLC using a modified method of Suh et al. [19]. An Agilent HPLC system (1200 series, Agilent Technologies, Santa Clara, CA, USA) using a Capcell column (4.6 × 250 mm, 0.5 µm, Shisedo, Tokyo, Japan) equipped with autosampler, column heater and multiple-wavelength detector (G1365B, Shimadzu, Kyoto, Japan). The binary gradient consisted of 20 mM KH2PO4 in distilled water (A) and 20 mM KH2PO4 in acetonitrile (B). The gradient was applied as follows: 0–5 min, 0% B; 5–30 min, 50% B; 30–60 min, 100% B. The injection volume of the MPN extract was 20 µL. The column temperature was set to 30 °C and the flow rate was 1 mL/min.
Statistical analyses The data are expressed as the mean ± standard deviation (SD) and were subjected to analysis of variance (ANOVA) using the statistical package for the social sciences (SPSS, version 12.0, 2004; SPSS Inc., Chicago, IL, USA). Significant differences between sample means were determined at p < 0.05 using Duncan’s multiple comparison tests. Associations among antioxidative compound levels and activities in GBS with roasting were determined by using Pearson’s correlation analysis.
Results and discussion
Physicochemical analysis Table 1 and Fig. 1 show the water content, pH, and titratable acidity of GBS according to the roasting treatment temperature (150, 180, and 210 °C) and duration (0, 15, 30, 45, and 60 min) and their appearance. The GBS (Fig. 1) showed a darker shade of umber in the 210 °C heat treatment group than in the 150 and 180 °C heat treatment groups. In the 180 and 210 °C heat treatment groups, the groups that received heat treatment for 45 and 60 min showed a vivid brown color, thus indicating that the seeds of these groups were inedible. This change in appearance may have resulted from the rapid increase in brown pigment production at these roasting temperatures, which greatly support the caramelization reaction of monosaccharides and the Maillard reaction between amino components and reducing sugars.
Table 1.
Changes of water content, pH and titratable acidity in Ginkgo biloba seeds according to the roasting conditions
| Roasting temp. (°C) | Roasting time (min) | F value | |||||
|---|---|---|---|---|---|---|---|
| Control | 15 | 30 | 45 | 60 | |||
| Water content (%) | 150 | 52.12 ± 1.36a | 46.66 ± 2.78bA | 37.55 ± 2.77cA | 29.37 ± 2.59dA | 12.89 ± 1.55eA | 135.65*** |
| 180 | 52.12 ± 1.36a | 43.51 ± 1.88bAB | 30.18 ± 1.51cB | 25.26 ± 2.70dA | 10.09 ± 1.79eA | 220.24*** | |
| 210 | 52.12 ± 1.36a | 39.95 ± 1.75bB | 21.99 ± 1.54cC | 7.63 ± 1.98 dB | 2.62 ± 1.17eB | 691.23*** | |
| F value | – | 7.09* | 44.32*** | 80.18*** | 36.51*** | – | |
| pH | 150 | 5.63 ± 0.14b | 7.32 ± 0.14a | 7.27 ± 0.09a | 7.24 ± 0.10aA | 7.24 ± 0.12aA | 112.71*** |
| 180 | 5.63 ± 0.14c | 7.18 ± 0.11a | 7.13 ± 0.09a | 6.99 ± 0.07abB | 6.80 ± 0.13bB | 102.54*** | |
| 210 | 5.63 ± 0.14d | 7.15 ± 0.08a | 7.06 ± 0.12a | 6.53 ± 0.09bC | 6.31 ± 0.12bcC | 92.81*** | |
| F value | – | 1.92NS | 3.37NS | 50.65*** | 43.52*** | – | |
| Titratable acidity (%) | 150 | 1.70 ± 0.15a | 0.39 ± 0.08cB | 0.43 ± 0.08cB | 0.71 ± 0.11bAB | 0.75 ± 0.06b | 83.52*** |
| 180 | 1.70 ± 0.15a | 0.83 ± 0.08bA | 0.81 ± 0.07bA | 0.83 ± 0.07bA | 0.84 ± 0.08b | 51.97*** | |
| 210 | 1.70 ± 0.15a | 0.40 ± 0.08cB | 0.37 ± 0.08cB | 0.58 ± 0.08bB | 0.70 ± 0.07b | 95.45*** | |
| F value | – | 31.67*** | 33.13*** | 6.51* | 3.41NS | – | |
Control: Non-heated Ginkgo biloba seed
NS not significant
Values are mean ± SD
Different letters in the same row (a–e) and column (A–C) are significantly different at p < 0.05
Significant at *p < 0.05 and ***p < 0.001, respectively
Fig. 1.
Image of Ginkgo biloba seeds according to the roasting conditions. Ginkgo biloba seeds roasted at (A) 150 °C, (B) 180 °C, and (C) 210 °C
In the case of heat treatment for 60 min, the water contents depending on roasting heat treatment temperature were 12.89 ± 1.55, 10.09 ± 1.79 and 2.62 ± 1.17% in the 150, 180, and 210 °C heat treatment groups, respectively. Thus, compared to the control group that saw a decrease in water content of 52.12 ± 1.36%, a higher decrease of 75% was observed in this treatment. These results indicate that the water content decrease was more significantly affected by the temperature than by the duration. These results were similar to those obtained by Emily et al. [20] in the study using raw soybeans. Yoon and Kim [21] reported that as the heat treatment temperature increased, the elution of water-soluble components increased owing to the volume expansion resulting from the thermal contact-induced water evaporation increase. Thus, the higher the heat treatment temperature is, the stronger is the relationship between physicochemical and antioxidative component changes.
The pH value increased from 5.63 ± 0.14 before the heat treatment (control group) to 7.32 ± 0.14 after the heat treatment. However, in the case of the 60 min heat treatment, the pH values in the 180 and 210 °C heat treatment groups were 6.80 ± 0.13 and 6.31 ± 0.12, respectively, thus showing a decreasing trend (p < 0.05).
The titratable acidity decreased from 1.70 ± 0.15% before the heat treatment (control group) to 0.39 ± 0.08–0.83 ± 0.08% after the heat treatment; however, it was significantly increased (0.70 ± 0.07–0.84 ± 0.08%) from the 45 min heat treatment (p < 0.05), thus showing a contrasting trend with pH change.
Bae et al. [22] reported that the pH decreased in the hot water extract of Liriopis tubers and Korean red ginseng as the heat treatment temperature increased. Żyżelewicz et al. [23] also showed that the pH of roasted cocoa beans decreased; however, its acidity increased with increased heat treatment temperature. Their results demonstrated the same trend as the results of this study. As the heat treatment temperature and duration increased, the aldehyde functional group of reducing sugar (an organic acid precursor) was converted to the carbonyl functional group owing to the oxidation by heat. It is determined that as the basic amino acid bound to the sugar and was involved in the Maillard reaction, the soluble basic amino acid was gradually decreased, thus leading to the decreased pH and increased acidity [24].
Table 2 shows the chromaticity and color difference (ΔE*) of GBS depending on heat treatment temperature and duration. The L* value (Lightness) was the highest (95.59 ± 2.55) in the control group before heat treatment, but it significantly decreased (77.76 ± 1.97–86.30 ± 1.47) as the heat treatment temperature and duration increased (p < 0.05). In addition, as the heat treatment temperature and duration increased, the a* value (redness) was significantly increased and the b* value (yellowness) was significantly decreased (p < 0.05). Based on the degrees of color change (trace, 0.0–0.5; slight, 0.5–1.5; noticeable, 1.5–3.0; appreciable, 3.0–6.0; much, 6.0–12.0; very much, > 12.0) presented by the National Bureau of Standards’ regulations [25], all GBS showed color difference values (ΔE*) of 10 or more, thereby belonging to the “very much” category, after heat treatment, regardless of the heat treatment temperature and duration. These results were similar to those of Shakerardekani et al. [26] in terms of the soluble browning materials of pistachio kernels according to heat treatment temperature and duration. These previous studies showed that the L* and b* values decreased and the a* value increased with heat treatment. Therefore, as the heat treatment temperature and duration increased because of roasting, the chromaticity of GBS was more affected by redness than yellowness.
Table 2.
Changes of Hunter’s color L*, a*, b* and ΔE* value in Ginkgo biloba seeds according to the roasting conditions
| Roasting temp. (°C) | Hunter’s color value | Roasting time (min) | F value | ||||
|---|---|---|---|---|---|---|---|
| Control | 15 | 30 | 45 | 60 | |||
| L* | 150 | 95.59 ± 2.55a | 86.55 ± 1.25b | 88.46 ± 2.99b | 87.27 ± 1.04bA | 86.30 ± 1.47bA | 11.07** |
| 180 | 95.59 ± 2.55a | 84.69 ± 1.72bc | 85.51 ± 2.98b | 81.69 ± 1.69bcB | 81.32 ± 0.97cB | 22.68*** | |
| 210 | 95.59 ± 2.55a | 84.66 ± 2.03b | 83.69 ± 2.79b | 79.18 ± 1.17cB | 77.76 ± 1.97cC | 31.21*** | |
| F value | – | 1.23NS | 2.03NS | 29.11*** | 23.72*** | – | |
| a* | 150 | − 0.23 ± 0.09c | 3.25 ± 0.32aA | 2.74 ± 0.24bB | 3.07 ± 0.15abB | 2.84 ± 0.15bC | 145.72*** |
| 180 | − 0.23 ± 0.09d | 2.59 ± 0.22cB | 2.32 ± 0.25cB | 3.62 ± 0.49bB | 5.64 ± 0.15aB | 182.48*** | |
| 210 | − 0.23 ± 0.09e | 2.68 ± 0.25 dB | 4.22 ± 0.93cA | 6.13 ± 0.27bA | 7.19 ± 0.35aA | 114.01*** | |
| F value | – | 5.43* | 9.02* | 72.77*** | 262.81*** | – | |
| b* | 150 | 2.91 ± 1.07a | − 3.37 ± 1.29b | − 2.16 ± 1.47b | − 2.33 ± 1.40b | − 1.28 ± 0.55bA | 12.38*** |
| 180 | 2.91 ± 1.07a | − 1.58 ± 1.38b | − 1.16 ± 0.57b | − 1.01 ± 1.00b | − 2.06 ± 1.01bA | 11.09** | |
| 210 | 2.91 ± 1.07a | − 3.00 ± 1.01b | − 2.40 ± 0.32b | − 3.52 ± 0.77b | − 5.17 ± 0.41cB | 16.15*** | |
| F value | – | 1.76NS | 1.49NS | 4.00NS | 25.65*** | – | |
| ΔE* | 150 | – | 11.5 | 9.2 | 10.4 | 10.6 | – |
| 180 | – | 12.1 | 10.9 | 15.0 | 16.2 | – | |
| 210 | – | 12.8 | 13.8 | 18.7 | 20.9 | – | |
Control: Non-heated Ginkgo biloba seed
NS not significant
Values are mean ± SD
Different letters in the same row (a–e) and column (A–C) are significantly different at p < 0.05
Significant at *p < 0.05, **p < 0.01, and ***p < 0.001, respectively
Additionally, these results show that Maillard reaction and roasting progress was accelerated, thus increasing the brown pigment; the temperature and duration increased as well. Hodge [27] reported that dark brown polymers were produced by the condensation and polymerization of low-molecular-weight melanoidin over a long time at high temperatures.
Antioxidant compound analyses
Table 3 shows the total cyanide, total phenolic contents, and reducing power of GBS depending on the heat treatment temperature and duration. The total cyanide content significantly decreased with increasing temperature and duration (p < 0.05). Compared with the control, the 210 °C heat treatment group after a duration 30 min showed a significant reduction of over 50% (1.74 ± 0.04–0.78 ± 0.14 µg/g). In sorghum cultivars seeds, Ahmed et al. [28] reported that heat treatment of germinated seeds significantly reduced cyanide content and only traces were detected in the final product. Cardoso et al. [29] reported that heating cassava samples reduced the cyanide content by approximately 70%. Thus, the decreasing tendency of the cyanide content by heat treatment in these studies corroborates our results. However, Cho et al. [30] detected a small amount of cyanide content in GBS (0.25 ± 0.5 µg/g) using ion chromatography, thus showing a different result from ours in terms of the cyanide content. This may have resulted from the different measurement accuracies of the colorimetric method using acid hydrolysis and ion chromatography to measure cyanide.
Table 3.
Changes of total cyanide and total phenolic contents, reducing power and 4′-O-methylpyridoxine (MPN) content in Ginkgo biloba seeds according to the roasting conditions
| Roasting temp. (°C) | Roasting time (min) | F value | |||||
|---|---|---|---|---|---|---|---|
| Control | 15 | 30 | 45 | 60 | |||
| Total cyanide content (µg/g) | 150 | 1.74 ± 0.04a | 1.49 ± 0.11bA | 1.33 ± 0.07cA | 1.29 ± 0.09cA | 1.22 ± 0.06cA | 20.68*** |
| 180 | 1.74 ± 0.04a | 1.15 ± 0.04Bb | 1.16 ± 0.07bA | 1.16 ± 0.07bA | 1.17 ± 0.04bA | 68.53*** | |
| 210 | 1.74 ± 0.04a | 1.12 ± 0.14bb | 0.78 ± 0.14cB | 0.70 ± 0.17cB | 0.71 ± 0.14cB | 33.61*** | |
| F value | – | 11.60*** | 24.66*** | 21.14*** | 28.71*** | – | |
| Total phenolic content (µg GAE/mg) | 150 | 5.71 ± 0.34 | 5.59 ± 0.54 | 5.27 ± 0.34 | 5.36 ± 0.33B | 5.52 ± 0.31B | 0.63NS |
| 180 | 5.71 ± 0.34 | 5.71 ± 0.32 | 5.19 ± 0.32 | 4.98 ± 0.30B | 5.86 ± 0.32B | 4.34NS | |
| 210 | 5.71 ± 0.34c | 5.01 ± 0.30d | 4.82 ± 0.30d | 6.48 ± 0.30bA | 9.83 ± 0.32aA | 129.05*** | |
| F value | – | 2.53NS | 1.73NS | 18.70*** | 171.13*** | – | |
| Reducing power (A700nm) | 150 | 1.21 ± 0.13a | 0.41 ± 0.11cB | 0.44 ± 0.09cB | 0.77 ± 0.08bB | 0.92 ± 0.04bC | 38.55*** |
| 180 | 1.21 ± 0.13a | 0.68 ± 0.08cA | 0.71 ± 0.07cA | 0.91 ± 0.04bB | 1.35 ± 0.07aB | 39.11*** | |
| 210 | 1.21 ± 0.13b | 0.45 ± 0.14 dB | 0.89 ± 0.11cA | 1.56 ± 0.11aA | 1.68 ± 0.06aA | 59.87*** | |
| F value | – | 5.27* | 18.84*** | 82.09*** | 142.88*** | – | |
| MPN content (µg/g) | 150 | 198.05 ± 2.07a | 188.31 ± 2.67b | 185.96 ± 2.76b | 185.04 ± 1.97bA | 177.86 ± 2.14cA | 28.98*** |
| 180 | 198.05 ± 2.07a | 191.28 ± 1.69b | 188.06 ± 1.42b | 173.00 ± 1.60cB | 163.46 ± 2.46 dB | 169.13*** | |
| 210 | 198.05 ± 2.07a | 192.06 ± 1.75b | 183.81 ± 2.31c | 155.85 ± 3.08dC | 52.88 ± 2.77eC | 1816.86*** | |
| F value | – | 2.69NS | 2.72NS | 121.65*** | 2301.17*** | – | |
Control: Non-heated Ginkgo biloba seed
NS not significant
Values are mean ± SD
Different letters in the same row (a–e) and column (A–C) are significantly different at p < 0.05
Significant at *p < 0.05 and ***p < 0.001, respectively
The total phenolic content significantly increased in the 210 °C heat treatment group with increasing the heat treatment duration (p < 0.05). However, no significant difference was observed in the 150 and 180 °C heat treatment groups (p < 0.05). The total phenolic content with 60 min duration was significantly increased in the 210 °C heat treatment group (9.83 ± 0.32 µg GAE/mg) compared to the control group (5.71 ± 0.34 µg GAE/mg). Also, at high heat treatment temperature, the total phenolic content significantly increased after 45 min duration (p < 0.05). Mikašauskaitė et al. [31] reported the total phenolic content of GBS as 33.8 mg RE/g, which is different from that reported in the present study. Ellnain-Wojtaszek [32] reported that GBS contain phenolic acids, such as protocatechuic acid, vanillic acid, p-hydroxybenzoic acid, ferulic acid, caffeic acid, protocatechuic acid, and p-coumaric acid. The total phenolic content increase in the above results may have resulted from the conversion of a significant amount of polymer polyphenols, which were strongly ester-bonded to polysaccharides and oligosaccharides, into free polyphenols on heating [33]. Alternatively, it may have been due to the conversion of phenolic compounds of polymers into low-molecular-weight phenolic compounds because of heating at high temperatures [1].
The reducing power is the ability to donate electrons to reactive oxygen species and free radicals. The greater the reducing power and antioxidant activity are, the higher is the absorbance value. Table 3 shows the measurement results of the reducing power of GBS extracts (concentration, 1.0 mg/mL) depending on heat treatment temperature and duration. After heat treatment, the reducing power was in the absorbance range of 0.41 ± 0.11 A700–1.68 ± 0.06 A700, thus being significantly decreased compared to that of the control group (1.21 ± 0.13 A700; p < 0.05). Although the reducing power of the 180 and 210 °C heat treatment groups (1.35 ± 0.07 A700 and 1.68 ± 0.06 A700, respectively) during the 60 min heat treatment was significantly differ from that of the control group, it seemed that the reducing power was restored. The reducing power is reportedly related to the antioxidant activity and is associated with the presence of a reductone [34]. According to Terpinc et al. [35], the reducing power of camelina seed was 0.578 A740 before heat treatment and increased to 0.647 A740 after heat treatment, thus showing a similar trend to that of the present study.
The increased reducing power may have resulted from increased phenols and antioxidative components due to heat treatment. A comparison of these results from previous studies with the results of the total phenolic content and reducing power in this study indicate that the higher the treatment temperature is, the higher is the correlation with DPPH and ABTS radical activities.
Antioxidant capacities
The electron donating ability and ascorbic acid equivalent antioxidant activity (AEAC), which were measured in GBS using DPPH and ABTS radicals at the concentration of 1.0 mg/mL, increased with increasing temperature and duration of the heat treatment, as shown in Fig. 2 (p < 0.05). The DPPH radical scavenging activities were 34.03 ± 2.22 and 53.07 ± 2.37%, respectively, in the 180 and 210 °C heat treatment groups with 60 min duration, thus showing a significant increase compared to the activity in the control group (24.91 ± 2.34%; p < 0.05). The AEAC values significantly increased with increasing temperature and duration only in the 210 °C heat treatment group (38.48 ± 1.44 mg AA eq/100 g) relative to the control group values (30.40 ± 0.66 mg AA eq/100 g; p < 0.05). The antioxidant activities were high in the heat treatment groups with high phenol content, as shown by the total phenolic content (Table 3). These results suggest that phenolic compounds represent the antioxidative activity and that the antioxidative effect of phenolic compounds is increased by heat treatment. These results were consistent with the reports that the antioxidant activity was significantly increased after heat treatment of camelina seed [35]. Thus, the antioxidative effect was increased as free polyphenol compounds increased after heat treatment. In addition, the heat treatment may have converted active ingredients, which had been strongly bonded to the tissue, to free forms, thus leading to the increased antioxidant effects.
Fig. 2.
(A) DPPH and (B) ABTS radical scavenging activity of in Ginkgo biloba seeds according to the roasting conditions. Each bar represents the mean ± SD of triplicated measurement. Means with different letters in the roasting time (a–c) and roasting temperature (A–C) are significantly different at p < 0.05
4′-O-methylpyridoxine (MPN) content
Table 3 shows the content of MPN in GBS depending on heat treatment temperature and duration. As the heat treatment temperature and duration were increased, the MPN content significantly decreased compared to the control group before heat treatment (p < 0.05). The higher the temperature was, the greater was the decrease in the MPN content. Compared with the control group (198.05 ± 2.07 µg/g), in the 210 °C heat treatment group, the MPN content (52.88 ± 2.77 µg/g) decreased by over 70%. The MPN content showed a significant decrease in the 60 min heat treatment group at 150 °C, the 45 min heat treatment group at 180 °C, and the 30 min heat treatment group at 210 °C (p < 0.05), thus revealing that the MPN content decreased rapidly with increases in the heat treatment temperature. The MPN content of the control group before heat treatment reported in this study was similar to that reported by Yosimura et al. [36], whose study determined the MPN content of raw GBS to be 936 nmol/g (approximately 171.4 µg/g) and 1177 nmol/g (approximately 215.6 µg/g) using HPLC. In addition, Kobayasi et al. [37] reported that the MPN content of raw GBS significantly decreased in a 5 min boiled heat treatment group at 100 °C and the 45 s microwave heat treatment group, respectively. Thus, the decreases in the MPN content by heat treatment reported by them were similar to that reported in this study.
This may have resulted from the similarities between MPN and soluble vitamin B6 in terms of the molecular structure and metabolic pathways [6]. Moreover, MPN is synthesized by the methylation of pyridoxine (one of soluble vitamin B6) and pyridoxal-5′-phosphate derivatives [38]. Therefore, the MPN metabolic pathway may be similar to the metabolic pathway of vitamin B6 reduction by heat.
Correlation between total cyanide content, total phenolic content, and antioxidant activities
To compare the contribution of individual compounds to antioxidant activities, the Pearson’s correlation coefficient is showed in Table 4. The total phenolic content showed a positive correlation with the DPPH, as well as ABTS, only in GBS roasted at 210 °C. The DPPH radical scavenging activity had a positive correlation with ABTS in roasted GBS at 210 °C. These results indicate that the major compounds contributing to each radical scavenging activity and reducing power in GBS depending on heat treatment temperature are similar. Further studies are needed to elucidate the contribution of each compound to the antioxidant activity of GBS.
Table 4.
Pearson’s correlation coefficients between total cyanide content, total phenolic content and antioxidant activities of Ginkgo biloba seeds according to the roasting treatment temperature (150, 180 and 210 °C)
| Roasting temp. (°C) | Total cyanide content | Total phenolic content | Reducing power | DPPH | ABTS |
|---|---|---|---|---|---|
| 150 | |||||
| Total cyanide content | 1.0000 | ||||
| Total phenolic content | 0.7334 | 1.0000 | |||
| Reducing power | 0.4103 | 0.5876 | 1.0000 | ||
| DPPH | 0.3432 | 0.7034 | 0.8553 | 1.0000 | |
| ABTS | − 0.4517 | − 0.2913 | 0.5070 | 0.4574 | 1.0000 |
| 180 | |||||
| Total cyanide content | 1.0000 | ||||
| Total phenolic content | 0.3231 | 1.0000 | |||
| Reducing power | 0.4711 | 0.5209 | 1.0000 | ||
| DPPH | − 0.2435 | 0.4848 | 0.7189 | 1.0000 | |
| ABTS | − 0.2461 | − 0.0113 | 0.6674 | 0.8146 | 1.0000 |
| 210 | |||||
| Total cyanide content | 1.0000 | ||||
| Total phenolic content | − 0.3736 | 1.0000 | |||
| Reducing power | − 0.2855 | 0.7793 | 1.0000 | ||
| DPPH | − 0.5997 | 0.9494* | 0.8341 | 1.0000 | |
| ABTS | − 0.6142 | 0.9295* | 0.5804 | 0.9206* | 1.0000 |
Significant at *p < 0.05
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No. NRF-2017R1A6A3A11034208).
Abbreviations
- GBS
Ginkgo biloba seeds
- MPN
4′-O-methylpyridoxine
- DPPH
2,2-Diphenyl-1-picrylhydrazyl
- ABTS
2,2′-Azino-bis-(2-ethylbenzothiazoline-6-sulfonate)
- HPLC
High performance liquid chromatography
- AEAC
l-Ascorbic acid equivalent antioxidant capacity
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