Comparison of Physicochemical Characteristics of Soy Sauces Made from Germinated Soybeans with Different Salt Concentrations ^†

Abstract

This research evaluated a soy sauce produced from germinated soybeans with varying salt concentrations. Soybeans were germinated over 48 and 72 h, then homogenized with roasted wheat and 0.1% of Aspergillus oryzae, and kept three days for koji fermentation. They were then homogenized with 18% and 22% salt, and then fermented for 210 days at ambient temperature for moromi fermentation. Moreover, 5 × 10⁷ CFU/mL of Zygosaccharomyces rouxii was used. The quality of soy sauce made from germinated soybeans over 48 h resulted in the best conditions regarding the physicochemical characteristics and sensory evaluation analysis.

1. Introduction

Soy sauce is a dark brown liquid, which has a light brown to black color with a salty and intense umami taste. It is a traditional sauce in China, Japan, Korea, and other Asian countries, due to its distinctive flavor and aroma, and has been gaining popularity in Western countries [1]. A variety of brands of soy sauce are imported from nearby nations. The amount of soy sauce produced locally is still rather small, due to the limitations of technology. Soy sauce is consumed not only because of its color and flavor but also for its nutritional value, which is paid attention to by consumers. Soy sauce exhibits antioxidant, antihyperuricemic, antimicrobial, anticarcinogenic anticataract, and antiplatelet activities. In addition, this product is rich in antioxidant substances, such as soy isoflavones, melanoidins, furan ketones, phenolic acids, organic acids, peptides, and β-carboline [2]. In a previous study, the bioactive compounds in soy sauce were improved by adding citrus peel in koji-making. Modifying the raw material composition during the koji-making process is a straightforward method that helps to raise the soy sauce’s quality. Studies have shown that commercial soy sauce quality is currently a little inconsistent [3].

Soybean is a renowned plant protein source with numerous health benefits, including lipids, vitamins, minerals, free sugar, isoflavones, flavonoids, and saponins, which has been used for soy sauce fermentation for a long period of time [4]. In addition, when soybeans were germinated, they were found to contain more nutrients, such as phenolics, L-ascorbic acid, and non-proteinogenic amino acids that exert a number of physiological functions, such as antioxidants and isoflavones, as well as gamma-aminobutyric acid (GABA), which can calm nerves, control blood pressure, and reduce excitement [5]. During moromi fermentation, one of the activities of germinated soybeans might be to alter the microbial diversity. Furthermore, the volatile substances in germinated soybean sauce might be slightly different from each other. In order to determine the ideal condition ratio of moromi fermentation and provide the highest-quality soy sauce available in Cambodian markets, this study compared soybeans fermented for 48 or 72 h. We aimed to evaluate the effect of germinated soybeans with different salt concentrations on the nutritional values and the physicochemical characteristics of soy sauce during 210 days of fermentation.

2. Materials and Methods

2.1. Materials

Soybeans were purchased from Samlout district, Battambang province, Cambodia. Wheat grains were bought from China, and the A. oryzae strain available in stock was used.

2.2. Experimental Design

The koji fermentation was prepared under three conditions with different materials and methods. Soybeans and germinated soybeans with varying times of germination and salt concentrations (18% and 22%), wheat grain, and 0.1% of A. oryzae powder [6] were used, which followed the Japanese style for making soy sauce (

Table 1. Different conditions during moromi fermentation.

Moromi Condition	A. oryzae (Spores)	Brine (%)	Z. rouxii (CFU/mL)	Condition Code
Soybean, wheat	0.5 × 108	18	5 × 107	NS18
Germinated soybeans 48 h, wheat	0.5 × 108	18	5 × 107	GF18
Germinated soybeans 72 h, wheat	0.5 × 108	18	5 × 107	GS18
Soybean, wheat	0.5 × 108	22	5 × 107	NS22
Germinated soybeans 48 h, wheat	0.5 × 108	22	5 × 107	GF22
Germinated soybeans 72 h, wheat	0.5 × 108	22	5 × 107	GS22

). Moreover, 5 × 10⁷ CFU/mL of Z. rouxii was used during moromi fermentation.

2.3. Methods

2.3.1. Koji Preparation

Soybeans were cleaned with tap water, soaked in a ratio 2:5 (w/v) with warm water at 40 °C for 2 h, and then placed in steel vessels. The soaked soybeans were stored in a dark place at room temperature and then germinated for 48 h and 72 h. The germinated soybeans were taken out, rinsed with tap water, and then autoclaved at 121 °C for 20 min to remove undesirable microorganisms. The whole wheat was rinsed with tap water, roasted at 150 °C for 40 min, and crushed into 4–5 pieces. The germinated soybeans and wheat were mixed in a ratio of 1:1 (w/w). Then, 0.1% (w/w) = 0.5 × 10⁸ spores of A. oryzae was inoculated for koji fermentation at room temperature for 3 days [7].

2.3.2. Moromi Preparation

The moromi fermentation was carried out in separate glass jars (3 L). The moromi fermentation was initiated by mixing the koji within a brine solution at 18% and 22% in a ratio of 1:1.5 (w/v). The fermentation was conducted at room temperature for 210 days. During fermentation, 5 × 10⁷ CFU/mL of Z. rouxii was poured into the moromi mash when the pH decreased to 5.2 [8].

2.3.3. Sampling

The koji samples were collected in amounts of 30 g, transferred into 50 mL Falcon tubes from each condition during fermentation, and then stored at 4 °C for further analysis. In addition, 50 mL of the moromi samples was collected on days 0, 30, 60, 90, 120, 150, 180, and 210 during moromi fermentation for further analysis of the physicochemical characteristics.

2.3.4. Physicochemical Analysis

The procedure to determine the protease activity was conducted by following Sigma’s Non-specific Protease Activity Assay—Casein as a Substrate, which is described by Cupp-Enyard [9]. The α-amylase activity of koji was determined by following a quantitative starch-iodine method [10]. The pH was measured by a pH meter model LAQUA F-72. The total amino acid nitrogen (AN) was measured by the formaldehyde titration procedure [11]. The 3,5-dinitrosalicylic acid (DNS) method was utilized to determine the content of the reducing sugars. The salt content of the sample was measured by using a salt meter model ES-421 made in Japan. The total phenolic content (TPC) was determined using the Folin–Ciocalteu reagent [12].

2.3.5. Sensory Evaluation

The consumer acceptance of soy sauce products was evaluated based on sensory evaluation using a hedonic test. There were 80 panelists, who evaluated 7 samples from commercial soy sauces, and 6 others evaluated soy sauce from conditions NS18, GF18, GS18, NS22, GF22, GS22, and CMS continuously, giving scores for Color, Odor, Aroma, Flavor, Texture, Sweetness, Saltiness, Sourness, Bitterness, and Overall Acceptability using a 9-point hedonic scale.

2.3.6. Statistical Analysis

All results were presented as the mean ± standard deviation (SD) from duplicate, separate, and independent measurements or testing. Statistical analysis was performed using SPSS 25. One-way ANOVA was used for calculating statistical significance and Duncan’s test for the (p < 0.05) significance level.

3. Results and Discussion

3.1. Physicochemical Characteristics of Soy Sauce

During the initial stage of koji fermentation, A. oryzae secretes a significant number of enzymes, resulting in the highest utilization rate of the raw materials. The higher activity from 24 h indicated that the protease activity increases as the soluble protein or peptide is hydrolyzed to amino acids. GF and GS dramatically increased to the maximum value of 2.28 ± 0.06 and 2.48 ± 0.09 U/g of the initial koji (Figure 1). Whereas, at 72 h of fermentation, the GF and GS were slightly decreased to 2.26 ± 0.00, 2.42 ± 0.06 U/g of the initial koji, respectively. The decline in neutral protease activity beyond 48 h of fermentation might be a consequence of the combined nutrient limitation and environmental pH fluctuations [13]. The α-amylase activity of conditions NS, GF and GS were increased rapidly at 48 h during fermentation, with values of 103 ± 0.13, 120 ± 1.62 and 132 ± 1.48 unit/g, respectively (Figure 1). This might be attributed to the production of significant quantities of the enzyme by A. oryzae during koji fermentation, even under low moisture conditions. The conditions GF and GS were significantly different (p < 0.05) from NS during 24 h and 72 h of koji fermentation, which might be influenced by the germination duration of the soybean raw material [14].

The pH was not significantly different (p > 0.05), decreasing from day 0 to day 15 and slightly decreasing from day 15 to day 210 of moromi fermentation (

Table 2. pH value of moromi fermentation over 210 days.

Duration (Days)	NS18	GF18	GS18	NS22	GF22	GS22
0	5.72 ± 0.13 a	5.72 ± 0.13 a	6.11 ± 0.00 b	6.04 ± 0.09 b	5.92 ± 0.17 a	6.19 ± 0.00 b
30	4.80 ± 0.01 b	4.64 ± 0.00 a	4.64 ± 0.00 a	4.88 ± 0.00 c	5.05 ± 0.00 e	4.96 ± 0.01 d
60	4.76 ± 0.00 b	4.64 ± 0.01 a	4.63 ± 0.01 a	4.86 ± 0.00 c	4.94 ± 0.00 d	4.94 ± 0.00 d
90	4.74 ± 0.00 c	4.64 ± 0.00 b	4.62 ± 0.00 a	4.83 ± 0.00 d	4.93 ± 0.00 e	4.93 ± 0.00 e
120	4.74 ± 0.01 c	4.64 ± 0.01 b	4.61 ± 0.01 a	4.81 ± 0.01 d	4.91 ± 0.01 e	4.91 ± 0.00 e
150	4.71 ± 0.01 b	4.62 ± 0.04 a	4.61 ± 0.01 a	4.77 ± 0.04 b	4.90 ± 0.01 c	4.86 ± 0.02 c
180	4.62 ± 0.06 b	4.52 ± 0.02 a	4.52 ± 0.03 a	4.73 ± 0.01 c	4.77 ± 0.02 c	4.81 ± 0.02 c
210	4.59 ± 0.01 b	4.51 ± 0.01 a	4.51 ± 0.01 a	4.71 ± 0.01 c	4.75 ± 0.02 d	4.76 ± 0.01 d

Note: Results are expressed as the mean ± SD (n = 2). Values with different letters are significantly different at p < 0.05.

). The higher pH values of NS22, GF22, and GS22 might be related to the higher salt concentration of the three samples. Conversely, high starch content typically leads to the increased production of lactic acid by lactic acid bacteria (LAB), which would normally acidify the environment and achieve sufficient lethality towards spoilage microorganisms [15].

The presence of AN during fermentation can be attributed to the activities of proteases and peptidases. The significantly (p ≥ 0.05) increased AN content (

Table 3. Amino nitrogen (g/100 mL) of moromi fermentation over 210 days.

Duration (Days)	NS18	GF18	GS18	NS22	GF22	GS22
0	0.04 ± 0.01 a	0.03 ± 0.00 a	0.02 ± 0.01 a	0.03 ± 0.01 a	0.04 ± 0.00 a	0.08 ± 0.01 b
30	0.32 ± 0.01 a	0.47 ± 0.03 b	0.50 ± 0.02 b	0.31 ± 0.03 a	0.47 ± 0.00 c	0.43 ± 0.00 b
60	0.66 ± 0.03 a	0.76 ± 0.01 b	0.78 ± 0.01 b	0.70 ± 0.01 a	0.72 ± 0.03 a	0.72 ± 0.04 a
90	0.80 ± 0.01 a	0.83 ± 0.01 a	0.83 ± 0.00 a	0.71 ± 0.01 a	0.78 ± 0.01 a	0.83 ± 0.08 a
120	0.82 ± 0.00 a	0.84 ± 0.02 a	0.84 ± 0.00 a	0.73 ± 0.02 a	0.79 ± 0.02 ab	0.84 ± 0.05 a
150	0.83 ± 0.04 a	0.85 ± 0.03 a	0.85 ± 0.01 a	0.74 ± 0.00 a	0.79 ± 0.01 b	0.84 ± 0.01 c
180	0.83 ± 0.04 a	0.86 ± 0.01 a	0.88 ± 0.01 a	0.75 ± 0.02 a	0.83 ± 0.01 b	0.86 ± 0.01 b
210	0.86 ± 0.04 a	0.88 ± 0.01 a	0.89 ± 0.01 a	0.83 ± 0.04 a	0.85 ± 0.01 a	0.86 ± 0.00 a

Note: Results are expressed as the mean ± SD (n = 2). Values with different letters are significantly different at p < 0.05.

) throughout the fermentation process coincides with the dominance of A. oryzae during this period [16]. Fermented soy sauce inoculated with aroma-producing yeasts achieved a final AN content exceeding 0.8 g/100 mL, thus conforming to the premium grade specifications established by China Standards. GF18 had a slightly higher AN, thereby increasing the protein decomposition [17].

The reducing sugar content increase was significantly different and then decreased rapidly from 45 days, which is generally attributed to the hydrolytic activity of fungal α-amylase on the starch present in the raw materials. According to the statistical analysis (p ≥ 0.05), the first stage of fermentation, RS, shown in Figure 2a, was not significantly different 2.23 ± 0.16, 2.43 ± 0.24, and 2.71 ± 0.16 g/100 mL in NS18, GF18 and GS18, respectively. NS22, GF22 and GS22, shown in Figure 2b, were also not significantly different in RS content, at 2.05 ± 0.18, 2.61 ± 0.09, and 2.17 ± 0.35 g/100 mL, respectively. In addition, yeast Z. rouxii was added at 45 days, resulting in a decrease. This might be due to the Maillard reaction, with substrate utilization by proliferating microbial strains and the inhibition of amylase activity caused by stress factors. The low RS content (<1.00 mg/100 mL) demonstrated some disadvantages of this soy sauce. Regarding the Institute of Standard of Cambodia [18], the standard range of reducing sugar content in soy sauce is 2–5 g/100 mL (CS 066:2011).

Salt plays a critical role in fermentation by influencing the growth of microorganisms, inhibiting fungal enzymatic activity. Comparing the two groups of salt concentration (18% and 22%), the p-value indicated those samples were significantly different (p < 0.05) (

Table 4. Salt content of moromi fermentation over 210 days.

Duration (Day)	NS18	GF18	GS18	NS22	GF22	GS22
0	17.45 ± 0.07 a	17.30 ± 0.00 a	17.65 ± 0.07 a	21.30 ± 0.42 b	21.30 ± 0.28 b	21.65 ± 0.07 b
30	16.65 ± 0.21 a	16.25 ± 0.21 a	16.00 ± 0.28 a	19.20 ± 0.42 b	18.75 ± 0.35 b	19.00 ± 0.28 b
60	15.60 ± 0.00 a	15.75 ± 0.07 a	15.55 ± 0.35 a	17.45 ± 0.49 b	17.65 ± 0.07 b	18.10 ± 0.42 b
90	14.80 ± 0.28 a	13.90 ± 0.42 a	13.60 ± 0.71 a	16.95 ± 0.21 b	16.45 ± 0.35 b	17.08 ± 0.03 b
120	14.30 ± 0.57 ab	12.95 ± 0.64 a	13.50 ± 0.42 a	16.00 ± 0.70 bc	16.20 ± 0.14 bc	16.60 ± 0.14 c
150	13.15 ± 0.35 a	12.90 ± 0.28 a	13.40 ± 0.28 a	16.15 ± 0.21 b	16.10 ± 0.57 b	16.40 ± 0.42 b
180	12.85 ± 0.07 a	12.75 ± 0.07 a	13.75 ± 0.07 b	16.15 ± 0.35 c	16.05 ± 0.35 c	16.45 ± 0.07 c
210	12.15 ± 0.07 a	12.35 ± 0.07 a	13.15 ± 0.07 a	15.80 ± 0.42 b	15.90 ± 0.28 b	15.95 ± 0.35 b

Note: Results are expressed as the mean ± SD (n = 2). Values with different letters are significantly different at p < 0.05.

). The decrease in salt at the early stage of fermentation can be attributed to the moisture-extracting function of the brine solution, while the high salt concentration inhibited the activity of various hydrolytic enzyme produced by A. oryzae. A good-quality soy sauce contains 16–18% NaCl [19].

Soybean-derived phenolic compounds and Maillard reaction products, generated via non-enzymatic browning pathways during fermentation, contribute to the overall antioxidant profile. The total phenolic count exhibited a significant increase throughout the moromi fermentation process. According to

Table 5. Total phenolic content (mg GAE/mL) of moromi fermentation over 210 days.

Duration (Day)	NS18	GF18	GS18	NS22	GF22	GS22
0	0.10 ± 0.00 a	0.14 ± 0.00 b	0.15 ± 0.00 b	0.15 ± 0.00 b	0.11 ± 0.00 a	0.27 ± 0.00 c
30	2.42 ± 0.01 a	2.93 ± 0.01 b	3.03 ± 0.01 c	3.08 ± 0.02 c	2.94 ± 0.02 b	2.33 ± 0.00 a
60	3.27 ± 0.15 a	4.14 ± 0.18 b	4.11 ± 0.03 b	3.05 ± 0.08 a	3.41 ± 0.05 b	3.44 ± 0.06 b
90	3.41 ± 0.13 a	4.20 ± 0.03 b	4.29 ± 0.00 b	3.45 ± 0.40 a	3.79 ± 0.01 a	4.19 ± 0.04 a
120	3.47 ± 0.06 a	5.10 ± 0.06 b	5.06 ± 0.11 b	3.59 ± 0.05 a	3.94 ± 0.26 a	4.92 ± 0.14 b
150	3.73 ± 0.21 a	5.62 ± 0.09 b	5.37 ± 0.03 b	3.62 ± 0.08 a	4.31 ± 0.02 b	5.02 ± 0.04 c
180	4.42 ± 0.33 a	5.62 ± 0.04 b	5.72 ± 0.15 b	5.26 ± 0.12 a	5.32 ± 0.16 a	5.22 ± 0.18 a
210	5.44 ± 0.15 a	6.40 ± 0.10 b	7.76 ± 0.25 a	5.56 ± 0.15 a	5.70 ± 0.30 a	5.71 ± 0.12 a

Note: Results are expressed as the mean ± SD (n = 2). Values with different letters are significantly different at p < 0.05.

, the concentration of these phenolics was significantly higher in germinated soybeans compared to non-germinated ones, potentially due to the influence of the raw material, whereby enzyme cellulase facilitates the breakdown of cellulose within the soybean sprout matrix [12].

3.2. Result of Sensory Evaluation

Sensory analysis, a scientific discipline distinct from statistical methods, is based on human sensory perception to evaluate food products. The conditions for NS18, NS22, GF22 and GS22 were not significantly different (p > 0.05), while they were significantly different (p < 0.05) from GF18, GS18 and CMS (commercial soy sauce). The commercial brand Kbal Ko (CMS) and GF18 were not significantly different, with the highest score being 7.44 ± 1.36 and 7.36 ± 0.96, respectively (Figure 3). GF18 obtained the highest score of all descriptors compared to other conditions, which might be related to the germination leading to a change in the chemical composition of soybean, which contributed to a more complex and appealing flavor in the resulting soy sauce. Furthermore, germinated soy sauce exhibits a distinct microbial community, which contributes to the overall sensory characteristics. Generally, a good quality soy sauce should have 6 months of moromi fermentation, while the soy sauce in this study had 7 months [20].

4. Conclusions

The germination time of the germinated soybeans had a significant impact on the physicochemical characteristics and sensory evaluation in this study. In particular, the koji fermentation made from germinated soybeans over 48 and 72 h had higher enzyme activity than non-germinated soybeans. Soy sauce made from germinated soybeans over 48 h with a salt concentration of 18% had the best quality in terms of the pH, reducing sugar, total phenolic content, amino acid nitrogen, enzyme activity, and sensory evaluation, which met the Cambodian Standard for soy sauce.