2024 Volume 30 Issue 3 Pages 343-352
Egg-koji, fermented eggs by koji-mold (Aspergillus oryzae, A. sojae, and A. luchuensis), contains various enzymes that are expected to change the flavor and components of eggs. Digestion of whole egg liquid with egg-koji resulted in an increase in amino acid content, aroma component and free fatty acid formation, and a decrease in triacylglycerol level. Egg-koji AO101 (A. oryzae) increased the umami taste while maintaining its “egg flavor.” Egg-koji AS309 (A. sojae) showed reduced saltiness, which was considered to be because of the large amount of amino acids and aromatic ingredients masking the saltiness. Egg-koji AL434 (A. luchuensis) had a strong bitter taste, which was likely owing to the large amount of fatty acids. The characteristics of these strains are expected to lead to the development of various food products.
Traditional fermented foods including sake, shochu, miso, soy sauce, and amazake are indispensable to the Japanese food habit “Washoku.” Koji is used when producing these fermented foods. Koji, which has been conventionally used, is a solid culture of koji-mold, typically Aspergillus fungi, produced from grain materials, such as rice, barley, and soybean. Koji secretes a wide variety of enzymes during the growth process (Ichishima, 1972). By decomposing and converting raw materials using the flavor, texture, and enzymes of koji and drawing out their nutrition, Japanese have created various fermented foods (Suzuki et al., 2021). In addition, the safety of koji-mold and koji is guaranteed by a long eating experience (Machida et al., 2008).
Eggs are a nutrient-rich food and have various recipes worldwide. The absorbability and nutritional functions of high-quality nutritional components (48.8 % protein, 40.8 % lipid, and 1.6 % carbohydrate in dry weight) (Ministry of Education, Culture, Sports, Science and Technology, 2020) of eggs can be further improved through enzymatic conversion by microorganisms. Since Japan has the second highest annual egg consumption per capita worldwide (Sugano, 2020), improvement in the flavor and nutrition of eggs has a pronounced social effect.
Previously, we developed “egg-koji” made by koji from egg materials (Nakagawa et al., 2023); when egg yolk liquid was digested using egg-koji, it had a satisfactory flavor difference from rice-koji (Miyamoto et al., 2019). There are approximately 12 000 genes in the A. oryzae RIB40 genome (Iwashita, 2009; Machida et al., 2005), of which 134 are proteolytic enzymes. As the enzymes of koji-mold have substrate specificity (Kusumoto et al., 2008; Yasui et al., 2020; Yoshii, 1973), growing koji-mold on eggs was expected to provide flavors that cannot be achieved by cooking, including decomposing egg proteins to produce amino acids to enhance umami or decomposing lipids to enhance aroma components.
The generation of “richness” of food involves flavor components, such as amino acids; aroma components; and texture-related structures and components (Nishimura and Egusa, 2016). In addition to the abovementioned components, thickness and persistence are required, and umami substances, fats, and oils are significant components. In this study, we focused on the components related to “richness” among the component changes caused by the fermentation of whole eggs with egg-koji and investigated their effects on sensory characteristics. Egg-koji and whole egg liquid were reacted in the presence of salt, and free amino acids, aroma components, and neutral lipids were measured in the digestive product. Furthermore, to investigate whether a difference in sensory characteristics was present, sensory evaluation of whole egg liquid digests (WELDs) was conducted.
Preparation of various raw materials for koji Egg-koji, rice-koji, barley-koji, and soy sauce-koji were prepared following the protocol of a previous study (Nakagawa et al., 2023) with slight modifications. Conidia of A. oryzae AO101, A. sojae AS309, or A. luchuensis AL434 (0.05 g each, 1/20 000 raw egg material weight) (Higuchi Matsunosuke Shoten Co., Ltd., Osaka, Japan) were inoculated into each of the steamed materials and incubated in a glass petri dish for 42 h at 30°C and 90 % humidity. Mixing was performed 18 and 24 h following inoculation, and the glass lid was removed and covered with filter paper for 18 h to prevent oxygen deficiency and drying.
Preparation of WELDs WELDs were prepared by digesting whole egg liquid (50 g) under 4 % NaCl at 60 °C for 24 h by koji (5 g). WELDs prepared with egg-koji AO101, AS309, and AL434 were denoted as E-101, E-309, and E-434, respectively. WELDs prepared with rice-koji AO101, barley-koji AO101, and soy sauce-koji AO101 were denoted as R-101, B-101, and S-101, respectively. Cooked egg powder (CEP), rice, barley, and soy sauce were prepared similar to the abovementioned process by adding only the material before making koji to the whole egg liquid. Control was made by adding only NaCl to the whole egg liquid. The pH level of WELDs was approximately 7.38–7.45.
Measurement of free amino acids in WELDs The crude protein in the sample was removed by adding 3.3 % trichloroacetic acid and filtering through a 0.45-µm PVDF filter. The amino acid composition of each filtered sample was measured using an auto amino acid analyzer (LA 8080, Hitachi High-Tech Corporation, Tokyo, Japan).
Measurement of aroma components in WELDs Samples (1.5 g) were diluted (3.0 g) and placed in a glass vial (10 mL) and heated at 40°C for 15 min. The released volatiles were adsorbed on a solid-phase microextraction (SPME) fiber (polydimethylsiloxane/carboxen/divinylbenzene, Stable Flex 50/30 µm, DVB/Carboxen/PDMS, Sigma-Aldrich, St. Louis, MO, USA) at 40 °C for 20 min. The volatiles adsorbed on SPME were separated using gas chromatography (7890B, Agilent Technologies, Santa Clara, CA, USA) with a capillary column (SOLGEL-WAX, 30 m × 0.25 mm × 0.25 µm; Trajan Scientific and Medical, Victoria, Australia). The volatiles isolated were analyzed using a mass spectrometer (5977A, Agilent Technologies). The following were the operating conditions: injector temperature, 250 °C; helium flow rate, 1.2 mL/min; and oven temperature, 35 °C for 5 min and subsequently programed to increase to 60 °C at a rate of 5 °C/min and 220 °C at a rate of 15 °C/min and held for 9.7 min. Mass spectra were obtained by electron ionization at 70 eV over 29–290 mass units, with an ion source temperature of 230 °C. Calibration of the SPME–GC/MS mass scale was regularly performed using perfluorotributylamine.
Lipid analysis of WELDs WELDs (100 mg) were vortexed with 1 mL of tert-butylmethylether/methanol (2:1, v/v) using a bead crusher (µT-12, Taitech, Aichi, Japan). Distilled water (500 µL) was added, and the mixed solutions were vortexed for 30 s using a vortex mixer. An organic layer was collected by centrifugation at 12 000 g for 10 min and transferred into a 2-mL vial bottle. Total lipids were analyzed using gas chromatography (7890A GC system, Agilent Technologies) equipped with a time-of-flight mass spectrometer (JMS-T100CGV, JEOL, Tokyo, Japan). The components were separated using a capillary column (DB-5HT, 0.25 mm I.D. × 30 m, Agilent Technologies). The column temperature was held for 2 min at 80 °C, subsequently increased to 330 °C at a rate of 15 °C/min and held for 6 min. The ionizing voltage was 70 V, and the ion chamber temperature was 280 °C. Helium was used as the carrier gas at a constant flow rate of 1.12 mL/min.
Sensory evaluation test of WELDs Sensory evaluation was performed according to the World Medical Association Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects, World Medical Association, Geneva, 2013. Seven trained panels comprising men and women aged 25–60 years evaluated the samples. A total of seven parameters of taste and aroma (saltiness, sweetness, umami taste, bitterness, egg flavor, thickness of flavor, and persistence of flavor) were evaluated in ten grades (1–10), and the averages were calculated. A. oryzae AO101 is a strain selected during egg-koji development (Nakagawa et al., 2023), and AO101 is used in the industrial production of aged egg yolk. The sensory characteristics of WELDs prepared with egg-koji and other grain-koji were compared using AO101 as a standard strain of egg-koji.
Amount of free amino acids in WELDs Compared with the control, the total amino acid content of each WELD was increased 13.9-fold (E-101), 18.1-fold (E-309), 2.7-fold (E-434), 2.0-fold (R-101), 3.6-fold (B-101), and 16.4-fold (S-101) (Fig. 1). The total amino acid content was positively correlated with the protease (pH, 6) activity of koji (R2 = 0.96) (Fig. 2). Different groups of enzymes produced according to the raw material being grown act on the proteins in the whole egg is one of the possibilities for the difference in total amino acid content between each raw material koji using AO101. Each free amino acid has its own taste (Ninomiya, 1968): Gly, Ala, Thr, Pro, and Ser have a sweet taste; Phe, Tyr, Arg, Ile, Leu, Val, Met, and Lys have a bitter taste; Glu and Asp have umami and sour tastes; and Gly, Ala, and Ser exhibit umami and sweetness at high concentrations. Although amino acids have various flavors, the composition of each amino acid is almost the same in all samples (Fig. 1); it is believed that the higher the total amount of amino acids, the more it affects the umami taste and the thickness of flavor, which leads to richness (Nishimura and Egusa, 2016).
Amount of free amino acids in whole egg liquid digests (WELDs).
Data are expressed as means ± SE (n = 3). Values with asterisk significantly differed compared with the control in Welch’s t-tests. **p < 0.01.
Protease activity (pH, 6) and total amount of amino acids in WELDs in each koji.
Data are expressed as means ± SE (n = 3).
Aroma components in WELDs The aroma components whose content significantly changed compared with that of the control and are related to richness and umami are shown in Fig. 3. 2-Methylbutanal and 3-methylbutanal are aldehydes that enhance the umami taste of the aftertaste (Inoue et al., 2018) and are formed by the degradation of isoleucine and leucine, respectively. 2-Methylbutanal was positively correlated with free isoleucine (Fig. 1) (R2 = 0.6034), and 3-methylbutanal was strongly positively correlated with leucine (R2 = 0.7909). These compounds were increased in all WELDs. 2-Ethylfuran is a furan compound used for improving the flavor of meat extracts (Fujimoto et al, 2008). This compound was significantly increased in E-101, E-309, and E-434, decreased in R-101, and unchanged in B-101 and S-101. Octanal, a citrus-flavored aldehyde (Inagaki, 2018), was significantly increased in E-101 and E-309. 1-Octen-3-ol, a mushroom-flavored alcohol (Inagaki, 2018; Kawabata, 2010; Hayase and Watanabe, 2014), is produced by the enzymatic degradation of linoleic acid, and adding 100 ppt of it enhances the rich taste of bonito and kelp soup (Hayase and Watanabe, 2014). It was increased in all WELDs except E-434. 1-Octen-3-one is a ketone compound with a mushroom-like or green-like flavor (Inagaki, 2018) that is formed during heating and aging. It was produced or increased by koji but was not generated in E-434. Methional, a cheese-flavored sulfide (Kawabata, 2010) that enhances the umami taste, was produced in E-101, E-309, E-434, and S-101, with the highest content in E-309. Furfural, an aldehyde in the almond flavor (Hayase and Watanabe, 2014) and contributes to the richness of miso, was significantly increased in E-101, E-309, E-434, R-101, and B-101 and decreased in S-101. Phenylacetaldehyde, a honey-flavored aldehyde that enhances richness (Inoue et al., 2018), was significantly increased in E-101, E-309, E-434, B-101, and S-101. Nonanal, a soapy-flavored aldehyde (Inagaki, 2018), was increased in all WELDs, except S-101. This compound in E-101 and S-101 was less than that in their respective raw materials. 2-Methylpyrazine is a pyrazine derivative of nutty aroma that is easily produced by heating sugars or amino acids, which is one of the characteristic flavors of eggs (Takano and Tanizawa, 2012). While 2-methylpyrazine was increased in all WELDs compared with that in the control, this compound in WELDs made with egg-koji was less than that in WELDs made with CEP, the raw material of egg-koji. One possible reason for the decrease in 2-methylpyrazine was that koji-mold was metabolized or degraded during growth on CEP. Hexanal, a green-flavored aldehyde (Migita et al., 2012), was significantly increased in E-101 and E-309, slightly increased in E-434 and S-101, and decreased in R-101 and B-101. As hexanal was generated from the oxidation of free fatty acids (FFAs), it was believed that E-101 and E-309, which had a large amount of FFAs (Table 1), were increased. Hexanal was abundant in soybean, the raw material of soy sauce, whereas it was scarce in S-101, suggesting that this component in soybean was significantly reduced by preparing koji. Benzaldehyde, a fruity-like or green-flavored aldehyde (Inoue et al., 2018), was increased in all koji. Conversely, benzaldehyde in S-101 was significantly reduced compared with that in the koji material. 1-Penten-3-ol, a green-flavored aldehyde that contributes to richness (Hartvigsen et al., 2000), was increased in E-309, unchanged in E-101, and decreased in the other compounds. The aroma components used in this study were divided into four types. The first type was an increase from the control, which was 2-metylbutanal, 3-metylbutanal, 2-ethylfuran, octanal, and 1-octen-3-ol. It was believed that the increase was because of the action of enzymes in koji and other plants. The second type was not included in the control and was a new occurrence of 1-octen-3-one, methional, furfural, and phenylacetaldehyde. The third type was increased more than the control but decreased compared with the koji material and was nonanal, methylpyrazine, hexanal, and benzaldehyde. Transformation and decomposition were believed to occur due to koji. The fourth type was unchanged or decreased from the control, which was 1-penten-3-ol. As the decrease was reduced with the addition of koji material, adsorption to the material was believed to occur.
Aroma components of WELDs.
Data are expressed as means ± SE (n = 3). Values with asterisk significantly differed compared with the control in Welch’s t-tests. *p < 0.05; **p < 0.01.
| WELDs | Comparatively (%) | |||
|---|---|---|---|---|
| FFAs | Diacylglycerol (DAG) | Triacylglycerol (TAG) | Cholesterol | |
| E-101 | 24.7 | 13.5 | 53.6 | 8.3 |
| E-309 | 28.6 | 15.9 | 47.6 | 8.0 |
| E-434 | 76.8 | 4.8 | 12.7 | 5.7 |
| R-101 | 0.48 | 16.6 | 75.8 | 7.2 |
| B-101 | 0.50 | 16.6 | 75.7 | 7.2 |
| S-101 | 2.20 | 17.5 | 72.2 | 8.1 |
| Control | 0.27 | 16.6 | 75.1 | 8.1 |
From these results, we expected that the increase in various aroma components in WELDs and their involvement with taste components including amino acids affected the “thickness of flavor” and “persistence of flavor,” which led to enhanced umami and richness.
Lipid analysis of WELDs The FFA content was increased in E-101 (24.7 %, Table 1), E-309 (28.6 %), and E-434 (76.9 %), which were egg-koji–treated WELDs, compared with that in the control (0.27 %). In addition, the triacylglycerol (TAG) content in E-101 (53.6 %), E-309 (47.6 %), and E-434 (12.7 %) was significantly lower than that in the control (75.1 %). As only trace amounts of FFAs were detected in R-101 (0.49 %), B-101 (0.49 %), and S-101(2.2 %), the egg-koji particularly egg-koji AL434 was believed to have a higher lipid-degrading capacity than the other koji. It has been reported that intracellular TAG lipase in A. oryzae functions in neutral lipid degradation, and it is considered that egg-koji secretes a large amount of lipase (Anantayanon et al., 2021). Therefore, we believe that measuring lipase activity will be necessary. The large amount of FFAs in E-101 and E309 may explain the increase in 1-penten-3-ol, 1-octen-3-one, and 1-octen-3-ol produced during FFA degradation (Fig. 3). As the decomposition products were not increased in E-434, egg-koji AL434 was believed to have a different FFA metabolism. FFAs are frequently at a sensory disadvantage owing to their bitterness and astringency. Conversely, diacylglycerol (DAG) and monoacylglycerol produced when FFAs are released from TAG act as emulsifiers and stabilizers (Takahashi, 2015). Moreover, it has been reported that FFAs have emulsifying power (Takahashi, 2015; Ota and Yuki, 1977). Therefore, when E-434 is diverted to food, the amount of emulsifier and stabilizer used may be reduced.
Sensory evaluation test of WELDs A. oryzae AO101 is a strain selected during egg-koji development (Nakagawa et al., 2023), and AO101 is used in the industrial production of aged egg yolk. The sensory characteristics of WELDs prepared with egg-koji and other grain-koji were compared using AO101 as the standard egg-koji strain.
Saltiness was significantly reduced in WELDs (Fig. 4), and it was believed to have been masked by amino acids (Fig. 1) and aroma components (Fig. 3) produced in WELDs. Umami was significantly elevated in each koji. One of the reasons was the increase in Glu, Asp (Fig. 1), 2-methylbutanal, 3-methylbutanal, and methional (Fig. 3) levels related to umami. The egg flavor was increased in E-101 and decreased in R-101, B-101, and S-101. 2-Methylpyrazine is an aroma component that forms the egg flavor. However, no significant difference in the amount of this compound was noted between E-101 and S-101 (Fig. 3), suggesting the presence of other components involved in the egg flavor. The thickness and persistence of flavor were significantly increased in each koji. The increase in these flavors was assumed to be because of the increase in the levels of amino acids and aroma compounds for the same reason as the increase in umami. No WELDs exhibited significant sweetness compared with the control. Glucose, produced by carbohydrate digestion, is a sweetening ingredient in amazake, a koji-digested rice. Conversely, the carbohydrate content of eggs was very low (1.6 %), suggesting that less glucose is produced and that there was little change in sweetness. No significant difference in bitterness was observed among the WELDs. Bitter compounds include alkaloids, terpenes, amino acids, peptides, and organic acids (Hagiwara, 1980). Phe, Tyr, Arg, Ile, Leu, Val, Met, and Lys are amino acids that exhibit a bitter taste (Ninomiya, 1968), and no significant difference in the amount of these amino acids was noted among the WELDs (Fig. 1), which may be one reason for the lack of a significant difference in bitterness. E-101 was the only WELD that enhanced the umami and richness without losing its egg flavor. Additives such as monosodium glutamate and nucleic acid are added to foods to enhance the umami taste; however, by using WELDs such as E-101, these seasonings and additives can be reduced.
Sensory evaluation test of WELDs prepared with egg-koji AO101 and grain-koji.
Data are expressed as means ± SE (n = 7). Values with asterisk significantly differed compared with the control in Welch’s t-tests. *p < 0.05; **p < 0.01.
Next, to determine the effect of different strains of koji-mold used in the preparation of egg-koji on sensory characteristics, sensory tests were performed on E-101, E-309, and E-434 (Fig. 5). Saltiness, sweetness, umami, egg flavor, thickness of flavor, and persistence of flavor were not significantly different among E-101, E-309, and E-434. Umami and richness were enhanced while maintaining the egg flavor. E-309 had almost the same rating as E-101; however, the saltiness rating was lower than that of E-101, and it was believed that salt habituation progressed. As E-309 had a higher total amino acid content than E-101 (Fig. 1) and had more aroma components including methional, 1-octen-3-ol, and phenylacetaldehyde (Fig. 3), it was considered to be a composite of their total components (Nikkuni, 1997). By improving the reaction conditions between E-309 and whole egg liquid, developing products with enhanced umami taste compared with E-101 may be possible. Although E-434 contains a large amount of FFAs (Fig. 3) with a negative bitter taste, FFAs as well as DAG and monoacylglycerol generated from TAG have strong emulsifying power. In the case of egg foods, maintaining the emulsified state is important (Takahashi, 2015), and emulsifiers such as lecithin and fatty acid esterified with glycerin, propylene glycol, and sorbitan are frequently added, emphasizing the usefulness of E-434 as an emulsifier adjuster.
Sensory evaluation test of WELDs prepared with egg-koji.
Data are expressed as means ± SE (n = 7). Values with asterisk significantly differed compared with the control in Welch’s t-tests. *p < 0.05; **p < 0.01.
Finally, egg white accounts for approximately 60 % of the edible part of whole egg (Ministry of Education, Culture, Sports, Science and Technology, 2020). Therefore, it is expected to reduce food loss by using whole egg liquid instead of egg yolk liquid. This is an advantage toward the realization of a sustainable society.
Using egg-koji prepared from different Aspergillus strains, WELDs with enhanced umami and richness while retaining the egg flavor of whole eggs could be prepared. Different characteristics of each egg-koji were shown, including AS309 producing more free amino acids and aroma components and E-434 having the potential to take the emulsifying power of FFAs, suggesting that seasonings and additives can be reduced in food design. These outcomes have unfolded the possibility of developing novel egg-related foods.
Conflict of interest There are no conflicts of interest to declare.
