Effects of sound waves on the enzyme activity of rice-koji

Rice-koji is an important agent for the saccharification of fermented food production in Japan. In kojimaking, temperature, humidity, light and oxygen are important environmental factors. However, there is no report of koji-making controlled by sound wave. In this study, focusing on the enzyme activity of rice-koji, we confirmed the relationship between enzyme activity and frequency of sound waves. Here, the enzyme activities of rice-koji cultured with no sound and sound waves at seven different frequencies (1.0, 2.5, 6.3, 8.0, 10.0, 12.5 and 16.0 kHz) were compared. Using two types of polished rice from different production areas, the activities were investigated. In this study, we investigated the enzyme activity of four types of rice-koji and found frequencies where the enzyme activity was significantly different to activity with no sound. In particular, the frequency of significant difference was investigated to confirm both rice. In the results, due to the irradiation frequency of 6.3 kHz, glucoamylase activity of both ricekoji decreased significantly (P<0.01) compared to the silent control. Further, due to the irradiation frequency of 1.0 kHz, acid protease activity confirmed a significant difference between the silent control and a frequency of 1.0 kHz (P<0.01). That is, as the activity of one of rice-koji increased, that of another rice-koji decreased. However, the activity of α-amylase and acid carboxypeptidase was not affected at any frequency. These results suggest that sound waves, particularly at frequencies of 1.0 and 6.3 kHz, have marked effects on the specific enzymatic activity of rice-koji.


INTRODUCTION
In the production of fermented food in the Orient region, rice-koji, which is prepared by culturing fungal conidia on cooked rice, is an important saccharifying agent.During the production of rice-koji, temperature, humidity and oxygen are key factors affecting quality (Murakami, 1988).Yanagiuchi et al. (1993) reported that the supply of carbon dioxide affected the fatty acid composition, color of koji and components of koji extract.On the other hand, Iemura et al. (1996) reported that koji-making sealed in toko period, that is, incubation period, was higher in the activities of α-amylase and glucoamylase, while, lower in the activities of acid protease and acid *Corresponding author.E-mail: noriaki@bio.sojo-u.ac.jp.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License carboxypeptidase than koji-making not sealed up in toko period.Furthermore, it was recently confirmed that the germination of conidia is affected by light and the possibility of using light to control the production of ricekoji was demonstrated (Hatakeyama and Kitamoto, 2008).Yan et al. (2014) reported the optimum conditions for glucoamylase, α-amylase and acid protease of Aspergillus oryzae koji.Glucoamylase and α-amylase activity could reach maximum under the following conditions; inoculation temperature at 35°C, 0.25% inoculation quality, culture humidity at 75%, culture temperature at 37°C and 56 h culture time.On the other hand, acid protease could reach maximum when the culture temperature was 33°C.Zhang and Gao (2011) reported that the optimum condition for amino peptidase activity in wheat bran koji making were water ratio 100%, raw material cooking for 21 min and koji making for 44 h.However, there are no reports for utilization of sound waves during the rice-koji making process.
In Japan, sound waves have been used in various processes, including the aging of spirits, particularly shochu, cultivation of fruit and vegetables and even the breeding of cattle.For shochu, acoustic aging of black sugarshochu has been reported.For vegetables, growth promotion has been reported.For livestock, relaxation effects have been reported.However, there are no reports as paper.It is only been empirically applied.The reason for this is that elucidation of scientific mechanism of sound effects is thought to be difficult.The mechanism is not understood although it was been suggested that sound waves could affect the rate of proton transfer in proteins (Barnes et al., 1985).Furthermore, it was reported that sound waves at a frequency of 2.0 kHz have been shown to affect the sugar composition of plants (Satoh and Tanisu, 2013).Based on these findings, we speculated that the production of rice-koji could be optimized using sound waves and thus attempted to determine the critical sound wave frequencies that would specifically impact the enzyme activity of rice-koji.

Materials
Rice-koji was prepared using commercially purchased polished rice (Koshihikari) produced in Niigata and Miyagi Prefectures, Japan.Hereafter, the former was expressed as N-rice, the latter was expressed M-rice.Tane-koji (Aspergillus oryzae) was purchased from Kawachi Genichiro Shoten Co., Ltd., Kagoshima, Japan.

Rice-koji making
Rice-koji was prepared according to the method of Saigusa and Ohba (2007).Briefly polished rice (200 g) was soaked in water at 15°C for 20 min and the water was then drained over a 2 h period at 15°C.The rice was then steamed for 40 min in a pot-type steamer, removed, and allowed to stand until the temperature decreased to 40°C.Next, 0.2 g of Tane-koji spores was inoculated onto the steamed rice, which was then mixed to uniformly disperse the spores.Except for the steaming process, the cultivation procedure was performed under sterile conditions.After inoculation, the steamed rice was transferred to a Petri dish of 152 mm in diameter and compacted with a spoon.A piece of filter paper was inserted inside the cover, and the plate was incubated at 30°C for 20 h with no sound waves.A further 20 h to irradiate sound wave, rice-koji was moved to a tray which covered gauze and containing water in bottom.Incubation was performed in incubator built in speaker that is connected to the audio generator MINIRATOR MR2 (NIT Japan Co., Ltd, Tokyo, Japan) and instrument for amplification.The ricekoji was incubated in the presence of sound waves with frequencies ranging from 0 to 16.0 kHz.The sound power level of audio generator was keep -50 dBv.To keep moisture, making-koji was performed in plastic bag.The effects of sound wave frequency on the enzyme activity of glucoamylase, -amylase, acid protease, and acid carboxypeptidase as main enzymes of rice-koji was then investigated.Rice -koji was prepared one each time.After extraction of enzymes from three places at random from both ricekoji, enzyme activity was measured.

Acid carboxypeptidase
Acid carboxypeptidase produces amino acids from proteins and oligopeptides.The acid carboxypeptidase activity was measured with Cbz-Tyr-Ala (carbobenzoxy-L-tyrosine-L-alanine) as a substrate using a commercially available kit (Kikkoman Co., Ltd.).

Acid protease
Acid protease produces oligosaccharides from proteins.The acid protease activity was measured with casein as a substrate according to the method described by the National Tax Administration (1993).

Moisture content
The moisture content of steamed rice was measured using an FD-600 Infrared Moisture Determination Balance (Kyushu Kett Co. Ltd., Fukuoka, Japan).

RESULTS AND DISCUSSION
In this study, we aimed to prove that sound waves are one of the important environmental factors influencing the enzyme activities of rice-koji.To identify the critical frequencies affecting enzyme activity, rice-koji was produced in acoustic culture conditions using one of eight frequencies and the enzyme activities of the resulting rice-koji were compared.
For glucoamylase activity, significant difference was observed between no sound and a frequency of 6.3 kHz (Scheffe's F test).Also, glucoamylase activity of both rice-koji decreased significantly (P<0.01)(Figure 1A).On the other hand, for acid protease activity, significant difference was observed between no sound and a frequency of 1.0 kHz (Scheffe's F test).That is, as the activity of one of the rice-koji increased, another rice-koji decreased significantly (P<0.01)(Figure 1B).For amylase and acid carboxypeptidase activities, there were no frequencies at which significant differences were observed in both rice-koji (Scheffe's F test) (Figure 2A,  B).In this experiment, three statistical analyses were carried as multiple comparison test.Results of significant different test in glucoamylase and acid protease activity were same results gotten for three methods, Scheffe's F test, Tukey and Bonferroni/Dunn.Furthermore, from these analyses, we were unable to determine the cause and/or mechanism of the impact of frequency on rice-koji enzymes.To date, no studies have examined the effects of frequency on rice-koji enzymes.However, our findings do suggest that sound waves may be an important environmental factor influencing the rice-koji making process.In addition, we identified two sound wave frequencies (1.0 and 6.3 kHz) that might affect the enzyme activity of rice-koji.We also observed that the rice-koji made from the two different sources responded differently to frequency of 1.0, 6.3 kHz.To determine the cause for this difference, the moisture content of rice-koji made from Niigata and Miyagi Prefectures was compared.Results show that the moisture content of cooked rice produced in Niigata Prefecture was 33.8%, while that of rice produced in Miyagi Prefecture was 40.7% (Table 1).Considering from the view point of vibration, it may be in close relationship between water and sound wave.In generally, the moisture content of steamed rice used in sake brewing is 27-32%.Furthermore, the more moisture content of steamed rice, enzyme activity decreases (Okazaki et al., 1979).However, in this study, it was confirmed that even when water content is high, enzyme activity increased significantly (P<0.01), by irradiating sound wave such as  in glucoamylase (1.0, 12.5 kHz), acid protease (16.0 kHz), -Amylase (1.0 kHz) and acid carboxypeptidase (1.0 kHz).Mishiro et al. (2000) reported that glucoamylase activity tends to increase according to the integration temperature of ginjo koji, that is starter for specially brewed Japanese sake.Okazaki et al. (1979) also reported the relationship between enzyme production and temperature.In that paper, they reported that -Amylase and glucoamylase were produced well in optimum temperature of koji growth (37.5°C), while acid carboxypeptidase and acid protease were produced well under 35°C.In this study, in spite of constant temperature during koji-making, the difference of enzyme activity was observed by irradiation sound wave of various frequencies.
From these results, a possibility was suggested that a particular frequency might affect the enzyme production of rice-koji.In particular, with regard to 6.3 kHz, reduction of glucoamylase activity in the two types of rice-koji was also confirmed.Although the execution of repeat experiments is necessary, by the frequency of 6.3 kHz, a possibility of inhibition of the enzyme production or production of the active inhibitor is consider.Although the mechanism is not currently known about the effects of waves, overall impact of various factors, such as acoustic conditions, humidity, temperature and rice components (from the difference of water absorption rate), are considered.
As a result, enzyme activity does not depend at all on the high and low frequency.But, it was confirmed that each feature rice-koji was made with each frequency.From these results, the possibility was suggested that sound wave irradiation was one of the important environment condition for rice-koji making.
To further optimize the use of sound waves during the production of rice-koji, the underlying mechanism of frequency on enzyme production will need to be conclusively determined.

Figure 1 .
Figure 1.Effects of frequency of sound wave on enzyme activity of rice-koji.A. Glucoamylase activity.B. Acid protease activity.White bar showed N-rice, and black bar showed M-rice.Symbol (**) shows the significant difference (P<0.01).Especially, symbol (**) shown on the bar, indicated that there was significant difference between sound and no sound (0 kHz) condition (Scheffe's F test).Only the frequency of the indicated symbol (**) on the bar in both white and black, shows the frequency that affect the enzyme activity of rice-koji.Each value are presented as the mean ± S.D. (n=3).

Figure 2 .
Figure 2. Effects of frequency of sound wave on enzyme activity of rice-koji.A. α-Amylase activity.B. Acid carboxypeptidase activity.White bar showed N-rice, and black bar showed M-rice.Symbol (**) showed the significant difference (P<0.01).Especially, symbol (**) shown on the bar, indicated that there was significant difference between sound and no sound (0 kHz) condition (Scheffe's F test).Only the frequency of the indicated symbol (**) on the bar in both white and black, shows the frequency that affect the enzyme activity of rice-koji.Each value are presented as the mean ± S.D. (n=3).

Table 1 .
Water absorption and moisture content of rice used for rice-koji.Absorption rate of water in raw rice.Calculation was performed as followed.[(weight of rice after drainweight of rice before soak) / weight of rice before soak]×100.*2 Absorption rate of water in steamed rice.Calculation was performed as followed.[(weight of rice after steamweight of rice before soak) / weight of rice before soak]×100.*3 Moisture content of rice after steam.Analysis method was described in the text.Both rice were produced in Sep.2009 and purchased commercially in May 2010. *1