Effect of Drying Conditions on Nutritional Quality and In Vitro Antioxidant Activity of Traditional Doenjang.

Doenjang, a major traditional Korean condiment, is often dried to reduce volume and thereby shipping cost while increasing shelf life. However, changes of nutritional and sensory properties of doenjang during processing have not been well understood. Therefore, this study aimed to evaluate how drying processes influence the nutritional and chemical properties of doenjang. When two drying methods, hot air drying and freeze drying were compared from the nutritional point of view, air-dried doenjang at 60°C or lower showed similar quality parameters including sensory scores, proximate composition, antioxidant capacity, amino acid composition, amino nitrogen, and acid value to freeze-dried doenjang. In contrast, the sample dried at 80°C and 100°C showed lower quality parameters than the freeze-dried one. Ferric reducing antioxidant potential (FRAP), total phenolics content, amino acid composition, and acid value were shown to reflect the sensory and physical properties of dried doenjang. In particular, the FRAP value of dried doenjang was sensitively responsive to drying temperatures and may be utilized as an early biomarker for quality deterioration of dried doenjang.

INTRODUCTION

Traditionally, fermented soybean products including doenjang (fermented soybean paste) and ganjang (soy sauce) are the base for various kinds of soups, sauces for Korean style salad, and a major seasoning in Korean dishes. Doenjang has traditionally been manufactured using fermented meju pre-soaked in brine (20% salt solution) for 30 days or longer. Meju, in turn, is manufactured by soaking soybeans in clean natural water, steaming, crushing, and subsequent molding into rectangular blocks which are being exposed to air so as to collect airborne microorganisms (mainly Aspergillus and Bacillus species) from the natural environment to ferment it. While commercial doenjang (equivalent to Japanese miso) manufactured by the fermentation of cooked soybeans with koji (or Aspergillus oryzae) is predominant in the market, traditional doenjang increasingly attracts consumers due to its unique flavor and taste (1,2).

Recently, Korean traditional doenjang received much attention due to its health-promoting benefits such as its antioxidant activity, anti-cancer effects, and associated anti-mutagenicity (3–9). While soybeans contains a variety of bioactive components, fungal fermentation could enhance the biological activities of bioactive compounds through enzymatic bioconversion. For instance, isoflavones present mostly in glycoside forms in raw soybeans could be converted, by fungal enzymes during fermentation, into aglycones, which are more bioavailable forms (10,11).

Doenjang contains approximately 60% of water and therefore has a relatively short shelf life. To improve its storage stability, it needs to be dried or processed to lower its water activity. However, drying at elevated temperatures could cause quality deterioration through chemical reactions. In particular, Maillard reactions may lead to darkening, increased acidity, generation of unique flavor and taste, while increasing the antioxidant potential of doenjang (12).

Thus, this study aims to investigate how drying conditions affect the nutritional properties and antioxidant potential of traditionally prepared doenjang.

MATERIALS AND METHODS

Materials

All chemicals and reagents used were of American Chemical Society grade. Gallic acid, Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,4,6-tripyridylstriazine (TPTZ) solution were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and isoflavones (genistein, daidzein, and glycitein) were obtained from Chengdu Biopurify Phytochemicals (Chengdu, China).

Drying of doenjang

Traditional doenjang aged for more than one year was obtained from Andong Jebiwon Agricultural Corp. (Andong, Gyeongbuk, Korea), and 100 g of the sampledried under different conditions including conventional hot-air drying at 40, 60, 80, and 100°C (HB-502S, Hanbaek Scientific Co., Ltd., Bucheon, Gyeonggi, Korea), and freeze-drying (FD 8512; Ilshin BioBase Co., Ltd., Yangju, Gyeonggi, Korea).

Preparation of doenjang extract for antioxidant activity assays

Dried doenjang samples were extracted with 10 volumes of 80% (v/v) ethanol, filtered, concentrated to a final concentration of 10 mg/mL, and filtered through a 0.2 μm sterile syringe filter (Sartorius, Göttingen, Germany) before assays.

FRAP assay

The FRAP of doenjang extract was determined as described previously (13). Briefly, 30 μL of H2O and 30 μL of ferrous sulfate as standard, or samples were incubated at room temperature with 1 mL of FRAP reagent, containing 300 mmol/L acetate buffer (pH 6.3), 10 mmol/L TPTZ solution, 20 mmol/L FeCl3 solution, and H2O. The absorbance at 593 nm was recorded after 4 min. FRAP values of unknowns were calculated by extrapolation of standard curves.

DPPH radical scavenging assay

The DPPH radical scavenging activity of doenjang extract was evaluated as previously described (14). Briefly, 50 μL of sample solution or dimethyl sulfoxide was added to 200 μL of 200 μM DPPH radical solution, which was freshly made. After 30 min of incubation at room temperature, the absorbance at 515 nm was measured. Synthetic antioxidant reagent, L-ascorbic acid, was used as a positive control, and all tests were conducted in triplicates.

ABTS radical cation decolorization assay

The ABTS solution (a mixture of 5 mL of 7 mM ABTS and 80 μL of 2.45 mM potassium persulfate) was allowed to react in the dark at room temperature for 12 h before use (15). The solution was diluted with ethanol so that its absorbance was adjusted to 0.7±0.02 at 734 nm. ABTS (1 mL) was mixed with a sample (50 μL) in a glass test tube by vortexing for 30 s. Absorbance was measured at 734 nm after 5 min. The percentage of radical scavenging activity was calculated by comparing the absorbance values of the control without samples. All determinations were triplicated (16).

Analyses of nutritional composition and chemical components

Proximate composition, pH, titratable acidity, acid value, salinity, amino nitrogen, total free amino acids, and biogenic amines of doenjang dried under different conditions were assayed according to AOAC methods (17). For the pH measurement of the samples, 25 mL of deionized distilled water was added to 5 g of doenjang samples and then homogenized and filtered with Whatman paper (No. 2, Advantec Toyo Kaisha Ltd., Tokyo, Japan). The pH of the sample was measured using a pH meter (MP220, Mettler-Toledo, Greifensee, Switzerland).

The total free amino acid composition was analyzed by an amino acid analyzer (Hitachi L-8900, Hitachi, Tokyo, Japan) after extracting amino acids from doenjang samples as previously described (16).

Determination of total phenolic, flavonoid, and total isoflavone contents

Total phenolics were determined using the Folin-Ciocalteu reagent (18). Briefly, 100 μL of extract was mixed with 50 μL of sodium bicarbonate solution [10% (w/v)], followed by the addition of 15 μL of Folin-Ciocalteu reagent (previously diluted 5-fold with distilled water). After 5 min at room temperature, the sample mixture was transferred to a 96-well microplate, and the absorbance at 655 nm was measured using a microplate reader (SunriseTM, Tecan Group Ltd., Männedorf, Switzerland). Results are expressed as gallic acid equivalents.

Total flavonoid content was determined by aluminum chloride using a colorimetric method previously described with slight modifications (19). Briefly, 25 μL of the sample was mixed with 75 μL of 95% methanol in a 96-well microplate. Then, 5 μL of 10% AlCl3·6H2O, 1 M potassium acetate, and 14 μL of distilled water were added, and the mixture was incubated for 40 min at room temperature. Absorbance readings were obtained at 415 nm with a microplate reader. The total flavonoid content of the samples was extrapolated from standard curves plotted with naringin at 0~50 μg/mL.

High-performance liquid chromatography (HPLC) analyses for total isoflavones including genistein, daidzein, and glycitein, were performed by the procedure described elsewhere (20). Briefly, 1 g of dried doenjang sample was placed in a glass test tube containing 10 mL of acetonitrile and 1 mL 0.1 N HCl. After shaking the test tube for 90 min at room temperature, the supernatant was filtered through a syringe filter (polyvinylidene difluoride membrane, 0.45 μm pore size, 13 mm diameter; WhatmanTM, Maidstone, UK). The filtrate (10 μL) was injected into a Jasco HPLC system equipped with a pump (PU-2089 Plus Quaternary Gradient Pump, Jasco International Co., Ltd., Tokyo, Japan) and a photodiode array detector (MD-2010 Plus Multi-wavelength Detector, Jasco International Co., Ltd.). The operating conditions of HPLC are specified in Table 1.

Table 1

Operating conditions for HPLC analysis of isoflavones

Description	Condition
Column	Gemini C18, 5 μm, 2.0×150 mm
Column oven temp.	25°C
Detector	Diode array detector, 254 nm
Mobile phase	A: 0.1 % acetic acid in waterB: 0.1 % acetic acid in acetonitrile
Flow rate	0.8 mL/min
Injection volume	10 μL
Composition of mobile phase	A:B=85:15 (0 min)→60:40 (30 min)→ 85:15 (40 min)

Surface color measurement

The color of dried powder of doenjang was measured using a chromatometer (CR 400, Minolta, Osaka, Japan), which provided CIE L*, a*, and b* values. Negative a* values indicate green and higher positive a* values represent red color. Higher positive b* values indicate a more yellow color (21).

Sensory analysis

The samples were organoleptically assessed by 10 panelists, using a sensory rating scale of 1~5 for color, odor, smooth taste, umami taste, bitterness, saltiness, and overall acceptability, as described previously (22). The organoleptic properties evaluated included: six attributes for color (5, bright; dark, 1), odor (5, like; 1, dislike), umami taste (5, strong; 1, weak), bitterness (5, strong; 1, weak), salty taste (5, strong; 1, weak), and overall acceptability (5, like; 1, dislike). The panel of assessors was an external panel of non-smokers who were very familiar with fermented soy products and were checked on the basis of sensory acuity and consistency. The protocol for sensory evaluation was approved by the Institutional Review Board (IRB) of Kyungpook National University (IRB #KNU-2018-0075).

Statistical analysis

Statistical significance of data was tested by analysis of variance, followed by Duncan’s multiple range test, using SPSS Statistics 22 software (SPSS Inc., Chicago, IL, USA). Data were presented as mean±standard deviation (SD). Mean values not sharing a common letter indicate statistically significant differences (P<0.05).

RESULTS

Drying curve for traditional doenjang at different drying temperatures

Korean traditional doenjang prepared at Andong Jebiwon Agricultural Corp. was subjected to conventional air drying using a convection oven at various temperatures. As shown in Fig. 1, doenjang with 60% moisture content showed typical drying curves. Total drying times at 45, 60, 80, and 100°C were approximately 36, 15, 9, and 6 h, respectively.

Fig. 1

Drying curve of traditional doenjang. Fifty gram of traditional doenjang obtained from Andong Jebiwon Agricultural Corp. was dried in convection oven at 45, 60, 80, and 100°C and weighed every 30 min until reached constant weight.

Antioxidant activity of doenjang dried under different conditions

The antioxidant capacity of doenjang was compared among the drying conditions, freeze drying and hot air drying at 45, 60, 80, and 100°C (Fig. 2). While doenjang dried under different conditions was not significantly different in DPPH radical scavenging activity (Fig. 2A), doenjang dried at relatively high temperatures showed a stronger antioxidant activity than the other drying conditions (P<0.05) as assayed by ABTS+ radical scavenging activity and FRAP (Fig. 2B and 2C). In particular, the extract prepared from doenjang dried in the convection oven at 100°C showed significantly higher antioxidant activity (P<0.05) than the samples dried at lower temperatures and a dose-dependent FRAP activity (Fig. 2C).

Fig. 2

Antioxidant activity of doenjang dried under different conditions. Doenjang dried under different conditions was extracted with 80% ethanol and then subjected to antioxidant activity assays such as DPPH (A) and ABTS+ (B) radical scavenging assays, and FRAP assay (C). Data are mean±SD (n=3). Bars not sharing a common letter (a–d) in same concentration group represent statistically significantly different values from each other (P<0.05). NS, not significant.

The content of total phenolics was significantly higher in doenjang dried at 100°C while the isoflavone content was higher in doenjang dried at 80 and 100°C than the samples dried at 45, 60°C, or freeze dried (Table 2).

Table 2

Total phenolics and flavonoids contents and isoflavone composition of dried doenjang (unit: μg/g db)

	Total phenolics1)	Total flavonoids2)	Isoflavones
			Genistein	Daidzein	Glycitein
Freeze dried	499.7±10.9b	1,493.2±8.4b	766.9±6.3ab	559.8±5.9ab	283.0±4.9b
Hot air drying
45°C	391.7±9.1d	1,457.2±70.6b	744.1±11.4ab	533.5±10.0b	297.6±29.5ab
60°C	428.3±13.8c	1,700.0±46.3a	704.9±19.5b	511.2±6.2b	274.2±25.3b
80°C	355.1±18.2e	1,529.7±33.5b	825.7±108.0a	602.9±71.8a	332.7±33.6a
100°C	579.9±15.7a	1,634.5±39.8a	808.6±15.7a	621.7±15.5a	329.6±15.4a

Total phenolic, total flavonoid, and isoflavone contents of doenjang dried under various conditions were assayed according to the protocols described in ‘MATERIALS AND METHODS’.

Data are mean±SD (n=3).

Values not sharing a common letter (a–e) are statistically significantly different from each other (P<0.05).

Gallic acid equivalent.

Naringin equivalent.

Proximate composition and quality parameters of doenjang dried under different conditions

The general composition of traditional doenjang dried at different temperatures or by freeze drying was not significantly different from each other. Doenjang dried in the convection oven at 100°C had significantly lower pH, high acidity, and acid value (P<0.05) (Table 3). However, the concentrations of biogenic amines and amino nitrogen were not significantly affected by drying temperature while those were slightly higher in freeze dried doenjang.

Table 3

Proximate composition and quality parameters of doenjang dried under different conditions

	Freeze dried	Hot air drying
		45°C	60°C	80°C	100°C
Ash (%)	12.9±0.0	12.9±0.0	12.9±0.0	12.9±0.0	12.9±0.0
Crude protein (%)	21.5±0.0b	25.5±0.0a	19.1±0.0c	17.6±0.0d	25.9±0.0a
Crude fat (%)	22.6±0.0a	17.4±0.0b	17.3±0.0b	16.4±0.1c	16.7±0.1c
Salt concentration (%)	60	62	60	56	60
pH	5.9	5.9	5.7	5.3	4.9
Total acids (%)	2.6	3.3	3.0	3.2	3.6
Acid value (mg KOH/g)	29.6±2.0bc	31.6±1.7ab	30.6±1.2b	27.4±0.0c	33.5±1.4a
Amino N (mg %)	865.8	799.4	772.7	798.5	739.4
Total free amino acids (mg/g)	83.4	83.8	84.2	88.6	87.6
2-Phenylethylamine (mg/g)	2.35	3.02	2.66	2.23	2.13
Histamine (mg/g)	0.06	0.05	0.02	0.05	0.03

Data are mean±SD (n=3).

Values not sharing a common letter (a–c) are statistically significantly different from each other (P<0.05).

The parameters other than proximate composition and acid value were values from a single measurement.

Free amino acid composition of traditional doenjang was compared according to the drying conditions (Table 4). The concentrations of most free amino acid including threonine, histidine, proline, and glutamic acid were increased in doenjang dried at 100°C, compared to the samples dried at lower temperatures or freeze-dried. A few amino acids such as aspartic acid, cystine, and arginine were reduced in doenjang dried at 100°C.

Table 4

Free amino acid composition of doenjang dried under different conditions (unit: μg/g)

Free amino acids	Freeze dried	Hot air dried
		45°C	60°C	80°C	100°C
Phosphoserine	ND	ND	ND	ND	ND
Taurine	ND	ND	ND	ND	ND
Phosphoethanol amine	ND	ND	ND	ND	ND
Urea	ND	ND	ND	ND	ND
Aspartic acid	2,104.63	2,128.95	2,058.92	2,077.93	2,042.94
Threonine	3,313.99	3,349.12	3,551.97	4,407.07	4,420.17
Serine	1,321.68	1,318.81	1,482.59	1,921.58	1,895.72
Glutamic acid	3,789.57	3,715.36	3,719.62	4,219.95	4,055.26
Sarcosine	15.04	17.22	16.27	12.83	29.04
α-Amino adipic acid	773.04	762.69	766.19	825.79	813.16
Glycine	4,398.86	4,390.67	4,228.85	4,273.12	4,236.02
Alanine	9,787.03	9,781.97	9,608.61	9,746.25	9,701.89
Citrulline	ND	ND	ND	ND	ND
α-Amino-n-butyric acid	1,989.76	2,119.67	1,385.04	972.48	1,019.12
Valine	5,993.62	6,125.71	6,137.41	6,360.48	6,096.60
Cystine	597.80	651.26	641.16	545.16	436.60
Methionine	2,232.14	2,190.96	2,141.32	2,168.67	2,106.26
Cystathionine	ND	ND	ND	ND	ND
Isoleucine	5,899.42	5,968.42	5,953.27	6,132.27	6,222.91
Leucine	11,232.13	11,331.96	11,224.81	11,299.74	11,391.40
Tyrosine	1,728.97	1,719.12	2,344.58	3,715.82	2,992.21
Phenylalanine	6,614.60	6,657.76	6,981.22	7,372.41	7,202.92
β-Alanine	183.94	283.71	338.16	321.29	214.88
β-Amino isobutyric acid	126.85	128.41	622.78	704.24	572.89
γ-Amino-n-butyric acid	12,777.12	12,926.22	12,609.79	13,031.51	12,720.77
Ethanolamine	132.62	133.42	126.18	129.82	126.75
Hydroxylysine	ND	ND	ND	ND	ND
Ornithine	2,183.28	2,156.84	2,075.23	2,204.57	2,164.97
Lysine	9,160.52	9,085.56	8,737.37	9,146.46	9,047.05
1-Methylhistidine	ND	ND	ND	ND	ND
Histidine	623.42	601.86	735.15	1,066.10	1,013.18
3-Methylhistidine	ND	ND	ND	ND	ND
Anserine	216.64	299.41	338.42	389.99	377.84
Carnosine	11.50	10.76	11.79	8.91	11.12
Arginine	204.87	205.73	205.71	186.41	187.80
Hydroxyl proline	ND	ND	ND	ND	1.48
Proline	5,313.56	5,389.03	5,512.10	5,230.70	6,301.28
Total free amino acid	92,726.61	93,180.60	93,554.52	98,471.56	97,402.23

ND, not detected.

Meanwhile, the air-dried sample at 100°C had a significantly lower L value and higher a value than the other samples, indicating darker color than the samples including freeze dried one (Table 5).

Table 5

Color of doenjang powder dried under different conditions

Color values	Drying method
	Freeze dried	Hot air dried
		45°C	60°C	80°C	100°C
L	71.33±0.24a	53.71±0.16c	52.74±0.03d	54.42±0.36b	49.29±0.12e
a	4.50±0.03e	7.39±0.03d	8.38±0.10c	8.66±0.06b	9.05±0.01a
b	21.36±0.14b	21.06±0.07c	19.09±0.07d	21.67±0.23a	18.28±0.08e
ΔE*ab	30.98±0.26c	43.85±0.17b	43.74±0.03b	44.13±0.38b	46.39±0.14a

Data are mean±SD (n=3).

Values not sharing a common alphabetical letter are statistically significantly different from each other (P<0.05).

Sensory evaluation by 10 trained panelists on doenjang dried at different drying temperatures showed that doenjang dried at 60°C had the highest overall acceptability among the sample groups (Table 6).

Table 6

Sensory characteristics of doenjang dried under different conditions

	Freeze dried	Hot air dried
		45°C	60°C	80°C	100°C
Color	2.9±1.2a	3.3±1.1a	3.3±1.3a	2.9±1.4a	2.5±1.0b
Odor	2.7±1.3NS	3.0±0.7	3.1±0.7	3.2±1.5	3.0±1.2
Umami taste	3.0±0.7a	2.9±1.0a	2.4±1.1b	2.1±1.2c	2.4±1.0c
Salty taste	3.0±1.0a	2.6±0.7a	3.0±0.9a	1.5±1.1b	2.4±1.0a
Bitterness	1.8±1.1a	1.9±0.9a	2.2±1.1a	2.7±1.2ab	2.9±1.0b
Overall acceptability	3.0±1.05a	2.5±0.9b	3.2±1.1a	2.4±1.4b	2.0±0.9c

Data are mean±SD (n=10).

Values not sharing a common letter (a–c) are statistically significantly different from each other (P<0.05).

The samples were organoleptically assessed by ten panelists, using a sensory rating scale of 1~5 for six parameters.

, not significant.

DISCUSSION

Doenjang, one of the most consumed Korean traditional seasonings and condiments, is traditionally manufactured by soaking meju in a salt solution for 2~3 months (9). Meju, a fermented rectangular block of crushed cooked soybeans, is made by allowing airborne microorganisms to grow on the surface of the cooked soybean block. The major microorganisms involved in meju fermentation are reported to be Bacillus subtilis and molds such as Rhizopus, Mucor, and Aspergillus species (1,9). Despite its high salt concentration, doenjang containing approximately 60% moisture is labile to microbial spoilage, so it is usually recommended to be stored at refrigerated temperature.

Removal of free water from doenjang by the drying process is one of the best ways to extend its shelf life and make a long distance shipping possible with the minimum quality change. However, the quality of doenjang is significantly affected by drying conditions including drying temperatures. Although the proximate composition of doenjang was little changed at drying temperatures lower than 100°C, some minor components such as free amino acids, amino nitrogen, and total phenolics showed significant change at drying temperatures as high as 100°C (Table 2 and 3). For instance, the total phenolic content was significantly increased in doenjang dried at 100°C (P<0.05), and the concentrations of free isoflavones such as genistein, daidzein, and glycitein were elevated, maybe due to increased chemical reactions at high temperatures. In addition, the acid value, which is a biomarker of lipid oxidation, was increased at drying temperatures of 100°C (Table 3). Interestingly, the content of total free amino acids was significantly increased in doenjang samples dried at 80 and 100°C, probably due to thermal degradation of proteins and peptides (Table 4).

In particular, doenjang samples dried at temperatures higher than 80°C showed elevated FRAP values, one of antioxidant capacity marker, while all samples dried under different drying conditions had similar DPPH and ABTS+ radical scavenging activities (Fig. 2). In contrast to the DPPH and ABTS+ assays, FRAP measures the absorbance change at 593 nm in test reaction mixtures with those containing ferrous ions of known concentrations. Ferric to ferrous ion reduction at low pH causes a colored ferrous-tripyridyltriazine complex to form (23). The results from this study imply that FRAP is a relatively sensitive biomarker for the quality change of doenjang by hot air drying.

Meanwhile, sensory evaluation by 10 trained panelists on doenjang dried under different conditions showed that doenjang powder prepared by drying at 60°C had the highest sensory score among all hot air drying temperatures used in the study, with similar overall sensory score for the freeze-dried samples (Table 6). Doenjang dried at 80 and 100°C was shown to have a burnt aroma, with concurrent changes in FRAP value, total free amino acid content, and acid value. In addition, the air-dried sample at 100°C had a darker color than the samples dried at lower temperatures of less than 80°C, while freeze-dried doenjang showed the brightest color (Table 5). The Maillard reaction is a mixture of chemical reactions between reducing sugars and amino groups of protein, peptides, or amino acids. The reaction rate is mainly affected by initial pH, temperature, time, and water activity (24,25). Among these factors, temperature is one of the most important parameters that affects the reaction rates and aroma characteristics of foods (26,27). Our study also demonstrated that drying at temperatures as high as 80°C and 100°C caused a significant decline in sensory score and changes in some components including total phenolics and free amino acids probably due to the Maillard reaction. In particular, doenjang powder prepared by drying at a high temperature of 100°C showed a significantly strong antioxidant activity and high FRAP values which can probably be mediated by Maillard reaction products.

In conclusion, hot air drying at 60°C or lower resulted in doenjang powder with similar sensory and nutritional qualities to that of freeze-drying. Also, the quality change of dried doenjang can be monitored most sensitively by the FRAP assay as well as sensory evaluation.