Increase in the Activity of α-amylase Produced by Aspergillus oryzae in Dried Unripe Apple Pulp Culture on Addition of NaNO₃ and MgSO₄

Abstract

Unripe apples contain large quantities of starch and represent an unused organic resource. Starch hydrolysis in unripe apples may provide an effective method to utilize this resource as a food material. Aspergillus oryzae can be grown using unripe apples. However, the activity level of α-amylase produced by A. oryzae grown in dried unripe apple pulp remains unconfirmed. Therefore, we here examined various culture conditions that might increase this α-amylase activity. Our results suggested that for culturing A. oryzae in dried unripe apple pulp using the shaking culture method, the addition of NaNO₃ and MgSO₄·7H₂O as nitrogen and magnesium and/or sulfur sources, respectively, was essential. Thus, α-amylase production by A. oryzae required high oxygen levels and additional nitrogen and magnesium and/or sulfur sources, both of which occur in low quantities in dried unripe apple pulp.

Introduction

During the annual Japanese apple cultivation season, high-quality apples are abundant and unripe apples are discarded. These unripe apples contain large quantities of starch and represent an unused organic resource (Singh et al., 2005). Plants containing high amounts of starch can be used as a staple food as well as a component of various processed foods after starch hydrolysis using Aspergillus oryzae (koji mold). Thus, starch hydrolysis in unripe apples may provide an effective method to utilize this resource as a novel food material. In Japan, several types of koji molds can be purchased from specialized shops as microbial starters. In our previous study, koji molds were screened for their starch hydrolyzing ability in dried unripe apple pulp, and the koji mold A. oryzae AOK 139 was selected (Izawa et al., 2014). A. oryzae AOK 139, which is mainly used in miso production, demonstrates antimutagenic activity and produces free fatty acids, fatty acid ethyl esters, lipase, and cellulase (Watanabe et al., 2004). As the cellulase from A. oryzae AOK 139 digests the cell wall of unripe apple, this koji mold may have access to the intercellular starch.

The α-amylase produced by A. oryzae saccharifies starch, and has been used to produce various processed foods. Nevertheless, for practical use in the processed food industry, high α-amylase activity is necessary. Although A. oryzae AOK 139 can be grown using unripe apples (Izawa et al., 2014), its α-amylase activity when cultured on dried unripe apple pulp remains unconfirmed. Therefore, in the present study, we examined various culture conditions to increase the α-amylase activity of A. oryzae AOK 139. In this study, demonstration of the feasibility of producing processed foods from unripe apple starch using koji mold is expected. Although the functions of polyphenols in unripe apples have been extensively studied (Kanda et al., 1998, Tokura et al., 2005), research on the utilization of starch from unripe apples is scarce. Identification of the ability of koji mold to utilize starch in unripe apples might lead to the effective utilization of this unused resource.

Materials and Methods

Samples    Unripe apples (Malus domestica Borkh. cv Fuji) were used in this study. Apples were harvested from a private orchard in Hirosaki, Aomori, Japan, at the end of May 2010. They were stored in a warehouse at the orchard at room temperature for approximately 2–4 days and subsequently transferred to the laboratory for experimentation. The dry koji mold A. oryzae AOK 139 was purchased from Akita Konno Co. (Akita, Japan). Rice was purchased from a local grocery store and used as the control.

Static culture    For solid culture, chopped unripe apples were employed as the sample. Rice that had absorbed a sufficient amount of water was employed as the control. For liquid culture, unripe apple juice was employed as the sample. Samples weighing 100 g were added to Erlenmeyer flasks, sealed with breathable silicone rubber stoppers, and autoclaved. After cooling on a clean bench, the samples were inoculated with dry koji mold (0.1% of the sample weight) and incubated at 40°C with 98% humidity for 72 h in a Humidity Chamber IG400 (Yamato Scientific Co., Ltd., Tokyo, Japan). The cultures were stirred twice daily using a sterilized glass bar. Subsequently, the static cultures were stored in sterilized 1% NaCl solution at 4°C overnight. The next day, the cultures were filtered through filter paper and the filtrates were used as crude extracts for the assay of α-amylase activity.

Shaking culture    Dried unripe apple pulp that had absorbed a sufficient amount of water, and in which mixed minerals were added, was employed as the sample. The mixed minerals in Czapek-Dox medium (mainly used for culturing fungi) were comprised of 0.3% NaNO₃, 0.1% KH₂PO₄, 0.05% MgSO₄·7H₂O, 0.05% KCl, and 0.01% FeSO₄·7H₂O (Masuda et al., 2009). Rice that had absorbed a sufficient amount of water was employed as the control. Samples weighing 10 g and 190 mL of distilled water were added to Erlenmeyer flasks, sealed with paper stoppers, and autoclaved. After cooling on a clean bench, the samples were inoculated with dry koji mold (0.1% of the sample weight) and incubated at 30°C for 72 h in a shaking incubator. After 72-h incubation, the cultures were stored at 4°C overnight. The next day, the cultures were filtered through filter paper and the filtrates were used as crude extracts for the assay of α-amylase activity.

Measurement of α-amylase activity    The α-amylase activity of the crude extract was measured using an α-amylase assay kit (Kikkoman Corp., Tokyo, Japan).

Measurement of nitrogen content    The nitrogen contents in dried unripe apple pulp and rice (control) were measured using the Kjeldahl method.

Statistical analysis    Data are expressed as means ± SEM (n = 3), and were analyzed using one-way ANOVA with Tukey's (Table 1 and Fig. 3) or Dunnett's (Fig. 2) multiple comparison test and a Student's t-test (Fig. 1 and Table 2). Differences were regarded as significant at p < 0.05. All statistical analyses were performed using IBM SPSS Statistics version 21 software (IBM Japan, Tokyo, Japan).

Table 1. The activities of α-amylase produced by Aspergillus oryzae AOK 139 in static solid culture

Substrate	Stopper	α-amylase activity (U/g of koji)
Unripe apple	Silicone rubber	0.01 ± 0.01a
	Paper	0.04 ± 0.01a
Rice	Silicone rubber	14.22 ± 0.40b
	Paper	28.23 ± 0.50c

Enzyme activity was measured at the end of the 72-h incubation period. Data are expressed as means ± SEM (n = 3). Means not sharing a letter differ, P < 0.05.

Fig. 3.

α-Amylase activity of Aspergillus oryzae AOK 139 on liquid shaking culture using unripe apple pulp added NaNO₃ and / or MgSO₄·7H₂O.

Closed circles: mix minerals (0.3% NaNO₃, 0.1% KH₂PO₄, 0.05% MgSO₄·7H₂O, 0.05% KCl and 0.01% FeSO₄·7H₂O), open circles: 0.3% NaNO₃ and 0.05% MgSO₄·7H₂O, open squares: 0.3% NaNO₃, open triangles: 0.05% MgSO₄·7H₂O. Data are expressed as means ± SEM (n = 3). * P < 0.05 compared with mix minerals.

Fig. 2.

The activities of α-amylase produced by Aspergillus oryzae AOK 139 on liquid shaking culture using unripe apple pulp supplemented with mixed minerals without a certain mineral

Closed circles: mixed minerals (0.3% NaNO₃, 0.1% KH₂PO₄, 0.05% MgSO₄·7H₂O, 0.05% KCl, and 0.01% FeSO₄·7H₂O), closed squares: mixed minerals without NaNO₃, closed triangles: mix minerals without KH₂PO₄, open circles: mixed minerals without MgSO₄·7H₂O, open squares: mix minerals without KCl, and open triangles: mix minerals without FeSO₄·7H₂O. Paper stoppers were used to seal cultures. Data are expressed as means ± SEM (n = 3). * P < 0.05 when compared with mixed minerals.

Fig. 1.

The activities of α-amylase produced by Aspergillus oryzae AOK 139 on liquid shaking culture.

Open circles: unripe apple, closed circles: unripe apple with added mixed minerals, open squares: rice. Paper stoppers were used to seal cultures. Data are expressed as means ± SEM (n = 3). * P < 0.05 when compared with rice and unripe apple.

Table 2. Nitrogen content of dried unripe apple pulp

	Nitrogen content (%)
Unripe apple	0.71 ± 0.01
Rice	1.03 ± 0.00*

Data are expressed as means ± SEM (n = 3).

*  P < 0.05.

Results

In the present study, A. oryzae was cultured in dried unripe apple pulp using the same method as that employed for cultivating rice koji. Table 1 shows the activities of α-amylase produced by koji mold in solid static cultures. The α-amylase activities in unripe apple koji mold cultures sealed with paper and silicone rubber stoppers were very low. In contrast, the α-amylase activities were significantly higher in rice koji mold cultures sealed with paper stoppers than in rice koji mold cultures sealed with silicone rubber stoppers. Moreover, the α-amylase activities were significantly higher in rice koji mold cultures than in unripe apple koji mold cultures.

As shown in Fig. 1, in shaking cultures, the α-amylase activities in both unripe apple as well as rice koji mold cultures sealed with paper stoppers were very low. In contrast, the α-amylase activities were significantly higher in unripe apple koji mold cultures with added mixed minerals than in unripe apple or rice koji mold cultures without mixed minerals. Subsequently, the maximum α-amylase activity in unripe apple koji mold cultures containing mixed minerals was evaluated over a 72-h incubation period.

To determine which minerals contributed to the increased α-amylase activity, the shaking cultures of A. oryzae in dried unripe apple pulp with added mixed minerals and sealed with paper stoppers were tested after excluding one mineral at a time. As presented in Fig. 2, the α-amylase activities were significantly lower in cultures without NaNO₃ or MgSO₄·7H₂O than in the other cultures. Consequently, we examined whether the α-amylase activity increased with the addition of NaNO₃ and/or MgSO₄·7H₂O to the medium. As shown in Fig. 3, the α-amylase activities were significantly lower in shaking cultures supplemented with NaNO₃ and/or MgSO₄·7H₂O and sealed with paper stoppers than in cultures containing mixed minerals. However, the α-amylase activities were significantly higher in cultures containing both NaNO₃ and MgSO₄·7H₂O than in cultures containing either NaNO₃ or MgSO₄·7H₂O, and were not significantly different from those in cultures containing mixed minerals (96-h incubation). Subsequently, we compared the nitrogen contents in dried unripe apple pulp and rice (Table 2) and found that the nitrogen content was significantly lower in dried unripe apple pulp than in rice.

Discussion

Rice koji is generally cultivated using solid static culture. In the present study, the α-amylase activity in solid static rice koji mold cultures was measurable, whereas that in solid static unripe apple koji mold cultures was very low, suggesting that the solid static culture is unsuitable for cultivating apple koji. In addition, the α-amylase activities were higher in rice koji mold cultures sealed with paper stoppers than in cultures sealed with silicone rubber stoppers. Silicone rubber stoppers are generally used for sealing flasks/tubes containing microbial cultures. However, owing to the poor gas permeability of silicone rubber stoppers compared with that of paper stoppers, they are unsuitable for sealing mold cultures. The rate of α-amylase production of A. oryzae is reduced under very low oxygen conditions (Rahardjo et al., 2005). Thus, the α-amylase production and activity in culture flasks sealed with rubber stoppers may have been suppressed owing to the low oxygen conditions.

The α-amylase activity was considerably lower in static unripe apple koji mold cultures sealed with silicone rubber stoppers than in rice koji mold cultures sealed with silicone rubber stoppers. In general, the enzyme activities of molds are higher in solid culture than in liquid culture and are less affected when the culture conditions are modified (Shoji et al., 2007, Masuda et al., 2009). Therefore, we investigated the effects of using liquid shaking culture sealed with paper stoppers on the α-amylase activity. Our results indicated that the α-amylase activity was significantly lower in unripe apple koji mold cultures than in rice koji mold cultures. The production of starch-hydrolyzing enzymes is reduced in the presence of glucose in koji cultures (Ruijter et al., 1997, Shoji et al., 2007). However, in the present study, the glucose concentration in unripe apple koji mold cultures was not measured. It is thought that the amount of glucose in unripe apples is low and that glucose accumulation occurs when the apples mature. Therefore, the low α-amylase activity observed in unripe apple koji mold cultures may not be associated with glucose concentration.

Although the nutrients required for A. oryzae growth are present in sufficient quantities in rice, they may not be present in unripe apples. Nevertheless, starch, the carbon source necessary for A. oryzae growth is present in both unripe apples and rice. Therefore, we attempted to culture koji mold in unripe apple pulp supplemented with various minerals used for cultivating general molds. Our results revealed that the α-amylase activity was significantly higher in both cultures supplemented with minerals than in cultures without added minerals, suggesting that some of the minerals added may have contributed to A. oryzae growth, consequently enhancing its α-amylase activity. Investigation of the contribution of minerals to the α-amylase activity revealed that the α-amylase activity was significantly lower in cultures without NaNO₃ or MgSO₄·7H₂O than in those with added mixed minerals, indicating that NaNO₃ and MgSO₄ are necessary for koji mold growth in unripe apples. NaNO₃ may be utilized as a nitrogen source for koji mold growth. However, as the nitrogen content is lower in unripe apples than in rice, the α-amylase activity was significantly lower in unripe apple koji mold cultures without NaNO₃ than in cultures supplemented with NaNO₃. In contrast, the α-amylase activity in unripe apple koji mold cultures containing both NaNO₃ and MgSO₄·7H₂O was not significantly different from that in cultures containing all the added mixed minerals (96-h incubation) and was significantly higher than that in cultures containing either NaNO₃ or MgSO₄·7H₂O. Furthermore, the nitrogen content in unripe apples was found to be only 70% of that in rice. These results indicated that unripe apples contain insufficient amounts of nitrogen for A. oryzae growth, and thus, NaNO₃ supplementation improved the growth and α-amylase activity of A. oryzae.

When MgSO₄·7H₂O was added along with NaNO₃ to the medium, the α-amylase activity was higher, suggesting that MgSO₄·7H₂O, as a magnesium and/or sulfur source, is needed for koji mold growth and that unripe apples may contain insufficient quantities of magnesium and/or sulfur. It must be noted that the quantity of each added mineral employed in the present study was the same and that the α-amylase activity of unripe apple koji mold might increase with the increasing dose of NaNO₃ and MgSO₄·7H₂O.

Our results suggested the feasibility of producing processed foods from unripe apple starch using koji mold. However, there are a number of factors that remain to be studied, such as the usage of enzyme preparations and/or other microbes. In conclusion, our findings indicate that the addition of NaNO₃ as a nitrogen source and MgSO₄·7H₂O as a magnesium and/or sulfur source to unripe apple koji mold shaking cultures is essential for enhancing the α-amylase activity.

Acknowledgments    This research was funded in part by the Adaptable and Seamless Technology Transfer Program through Target-driven R&D Grant Number AS232Z00823E, the Japan Science and Technology Agency.

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