Combined use of rice-koji cultured with Aspergillus oryzae and Aspergillus luchuensis for enhanced dephosphorylation of phytate to myo-inositol in brown rice-koji-amazake, a sweet brown rice beverage

Junichiro Marui; Yohei Shiraishi; Mio Takeura; Sirinan Shompoosang; Patthinan Varichanan; Sayvisene Boulom

doi:10.3136/fstr.FSTR-D-24-00170

Abstract

Koji-amazake is a non-alcoholic sweet rice beverage popular in Japan, and commercial products are now available in Southeast Asian countries such as Thailand. It is made from boiled rice and rice-koji, a Japanese traditional fermentation starter using Aspergillus oryzae. The citrate-producing Aspergillus luchuensis is also used for the fermentation. Unpolished brown rice containing various nutrients is a promising ingredient; however, reduced phytate in the rice bran layer is desirable, as its strong chelating action may inhibit mineral absorption. Rice-koji made with A. luchuensis was found to be superior to that made with A. oryzae in dephosphorylation of phytate to myo-inositol in brown rice-koji-amazake. As citrate from A. luchuensis affects the flavor and degree of saccharification of the product, the combined use of rice-koji made with those two species should be considered to optimize the flavor and nutritional benefits of the product.

Introduction

Koji-amazake is a non-alcoholic sweet rice beverage popular in Japan since ancient times (Kurahashi, 2021), and commercial products are now available in Southeast Asian countries such as Thailand. It is made by mixing boiled rice and rice-koji, a Japanese traditional fungal fermentation starter with various hydrolytic enzyme activities, such as amylase and protease. These are secreted by the food industrial Aspergillus fungus, which is grown on steamed rice surfaces (Yamashita, 2021). Koji-amazake fermentation is conducted for several hours at 50 to 60 °C to facilitate the enzymatic saccharification of rice starch, the most abundant carbohydrate in rice, to about 20 % glucose and a certain percentage of oligosaccharides. These include prebiotic isomalto-oligosaccharides, known to stimulate beneficial colonic microbiota and improve host health (Kurahashi, 2021, Sorndech et al., 2018). Generally, in koji-amazake production, polished white rice is saccharified using rice-koji made from Aspergillus oryzae strains commonly used for brewing other Japanese traditional fermented foods (Yamashita, 2021). In recent years, koji-amazake products made with rice-koji produced using Aspergillus luchuensis species, which characteristically secrete large amounts of citrate (Futagami, 2022), have appeared on the market.

Koji-amazake contains various nutrients such as carbohydrates, protein, free amino acids, fatty acids, and B vitamins (Kurahashi, 2021), although these contents vary depending on the degree of rice polishing used. In this respect, if unpolished brown rice is used as an ingredient for koji-amazake instead of polished white rice, the macro- and micronutrients as well as dietary fibers, antioxidants, and bioactive compounds contained in the bran layer (Saleh et al., 2019) could be incorporated into the product. This approach could also be applied to Southeast Asian countries, such as Thailand and Laos, that produce rice as a major agricultural product and staple food.

Phytate (myo-inositol hexakisphosphate) is a principal storage form of phosphorus found in the aleurone layer of bran in cereal grains (O’Dell et al., 1972). Dietary phytate has received attention as an antinutrient that strongly chelates minerals to form insoluble salts, leading to decreased mineral bioavailability in the human gut (Kumar et al., 2010). However, recent research has also revealed its health-promoting activities including antioxidant, diabetes prevention, anti-inflammatory, and colon cancer regulation (Cheng and Xu, 2023). To maximize the health benefits of phytate while enhancing the bioavailability of micronutrients, it is desirable to combine a balanced, diverse diet with appropriate food processing techniques that reduce excess phytate in food grains such as brown rice. Therefore, the present study focuses on the enzymatic reduction of phytate in brown rice used in koji-amazake production.

Phytases, enzymes that catalyze the release of inorganic phosphorous from phytate, are found in plants and microorganisms including fungi (Wodzinski and Ullah, 1996). Fungal phytases catalyze dephosphorylation of phytate to myo-inositol monophosphate (Wyss et al., 1999). The combination of a phytase and acid phosphatase that exhibit different substrate specificity can act synergistically for complete dephosphorylation of phytate to myo-inositol (Wyss et al., 1999). myo-inositol, a known component of breast milk, is a recommended additive for preterm and term infant formula because of its important role in lung development (Cavalli et al., 2006; Koletzko et al., 2005), and it has also been studied for its potential health benefits (Chhetri, 2019). Overall, optimizing production of brown rice-koji-amazake (hereafter BR-koji-amazake) to reduce phytate and increase inositol is crucial, owing to the action of phytases and acid phosphatases in the rice-koji, although details of the mode of enzymatic action for phytases and acid phosphatases in rice-koji made with A. oryzae and A. luchuensis species remain to be clarified. To date, eight phytase and five acid phosphatase genes orthologous to Aspergillus niger phytase and Penicillium chrysogenum acid phosphatase, respectively, have been studied at the transcriptional level in rice-koji (Marui et al., 2012; Marui et al., 2013), however, the involvement of those enzymes in phytate dephosphorylation in BR-koji-amazake is unclear.

In this study, the reduction of phytate and subsequent generation of myo-inositol was demonstrated quantitatively in laboratory-scale BR-koji-amazake production. The effects of using rice-koji made with different combinations of A. oryzae and A. luchuensis species were investigated, focusing on the enhancement of phytate dephosphorylation in the products as well as the saccharification of brown rice material, to clarify the optimal mixing ratio for rice-koji made with these different fungal species.

Materials and Methods

Preparation of BR-koji-amazake Unpolished brown rice of a Japanese sticky rice variety, Himenomochi, and dried rice-koji products made with industrial strains of Aspergillus oryzae or Aspergillus luchuensis (Kojiya Sanzaemon Co., Toyohashi, Aichi, Japan), hereafter referred to as Ao-rice-koji and Al-rice-koji, respectively, were used in this study. Five grams of raw brown rice was soaked overnight in 10 mL of reverse osmosis (RO) water at 4 °C in a 100-mL glass vial, then boiled at 100 °C for 30 min using an autoclave sterilizer LSX-500 (TOMY, Tokyo, Japan). It was then cooled to about 55 °C and mixed with 3 g of Ao- and Al-rice-koji at various mixing ratios, followed by incubation at 55 °C. After 8 h of incubation, the BR-koji-amazake products in the glass vials were heated in boiling water for 20 min and stored at −20 °C until use. As a non-fermented control, 3 g of Ao-rice-koji inactivated by heat treatment at 121 °C for 15 min using an LSX-500 autoclave sterilizer (TOMY) was mixed with the boiled brown rice, and the mixture was immediately heated in boiling water for 20 min and stored at −20 °C until use.

Measurement of phytate Phytate was measured using high-performance liquid chromatography (HPLC). Each BR-koji-amazake sample was homogenized for 1 min with four volumes of RO water using a food processor (Silent Millser, Iwatani, Osaka, Japan). Subsequently, 1.25 g of the homogenized sample was mixed with 1.25 mL of 1.28 N HCl in a 5-mL microcentrifuge tube and stirred at 2 400 rpm for 1 h at 4 °C using a multi-tube vortex BSR-MTV100 (Bio Medical Science, Tokyo, Japan), followed by centrifugation at 18 000 × g for 15 min at 4 °C. A 150-µL aliquot of the clear supernatant was mixed with the same volume of ultrapure water in a 1.5-mL microcentrifuge tube and heated at 100 °C for 20 min, then centrifuged at 18 000 × g for 15 min at 4 °C. The clear supernatant was filtered with a 0.22-µm membrane filter and subjected to HPLC as described below.

For each sample, chromatography was performed on a binary HPLC system (Shimadzu, Kyoto, Japan) equipped with a TSKgel DEAE-5PW anion-exchange column (7.5 mm φ × 75 mm, Tosoh, Tokyo, Japan). A 100-µL injected sample of phytate was eluted over 40 min with a linear gradient of 0.01 M HNO₃ to 0.4 M NaNO₃, with 0.01M HNO₃ at a flow rate of 1.0 mL/min. The eluted phytate was reacted in a post column tube with Wade reagent (0.06 % FeCl₃·6H₂O, 0.3 % sulfosalicylic acid) at a flow rate of 0.5 mL/min and the decrease in absorbance at 500 nm was measured. Phytate concentrations of the samples were calculated by using standard solutions at concentrations of 0.1, 0.2, 0.3, and 0.5 mg/mL, respectively.

Measurement of myo-inositol myo-inositol content of BR-koji-amazake was measured by microbiological methods. The BR-koji-amazake sample was homogenized in the same manner as described for the phytate measurement, and centrifuged at 18 000 × g for 15 min at 4 °C. The clear supernatant was further diluted with RO water in a range of 40–160 times so that the measured value was within the range of the standard curve. After adjusting the pH of the diluted solution to around 8, it was heated at 100 °C for 30 min in a 1.5-mL microcentrifuge tube for sterilization. A 160-µL aliquot of the sample solution was mixed in a sterilized 1.5-mL microcentrifuge tube with the same volume of inositol assay medium ((NH₄)₂SO₄ 7.5 g/L, KH₂PO₄ 1.1 g/L, KCl 850 mg/L, CaCl₂•2H₂O 250 mg/L, MgSO₄•7H₂O 250 mg/L, MnSO₄ 5 mg/L, FeCl₃ 5 mg/L, glucose 100 g/L, citrate 2 g/L, potassium citrate 10 g/L, acid hydrolysate casein 10 g/L, thiamine hydrochloride 500 µg/L, pyridoxine hydrochloride 50 µg/L, calcium pantothenate 500 µg/L, biotin 5 mg/L) to which Saccharomyces cerevisiae NBRC0565, precultured in YM liquid medium (yeast extract 5 g/L, malt extract 3 g/L, peptone 5 g/L, glucose 10 g/L), was added (as an indicator microorganism) so that the optical density at 600 nm wavelength (OD600 nm) would be 0.0005. After 20 to 21 h of incubation at 30 °C, 300 µL of the culture was transferred to a microtiter well for measuring OD600 nm by a microplate reader Enspire (PerkinElmer, Waltham, MA, USA). myo-inositol concentration in each sample was calculated by a calibration curve generated using the standard solutions at concentrations of 0.125, 0.25, 0.5, 0.75, and 1.25 mg/mL, respectively.

Additional methods Brix percentage and pH of the supernatant of the centrifuged BR-koji-amazake samples were analyzed using a pocket refractometer PAL-S (ATAGO, Tokyo, Japan) and compact pH meter LAQUAtwin-pH-22B (Horiba, Kyoto, Japan), respectively. Citrate levels in the BR-koji-amazake samples were measured with a F-kit citric acid (JK International, Tokyo, Japan) according to the manufacturer’s protocol. Mechanical taste sensor measurements were conducted with a TS-5000Z taste sensing system (Intelligent Sensor Technology, Inc., Atsugi, Japan) according to the manufacturer’s instructions. To assess the significance of differences in the phytate and myo-inositol contents between experimental conditions in this study, Welch’s t-test was performed using Excel (Microsoft Corp., Redmond, WA, USA).

Results and Discussion

Citrate content, pH, and Brix percentage of BR-koji-amazake prepared using rice-koji made with A. oryzae and A. luchuensis In this study, an unpolished brown rice variety of Japanese glutinous rice was employed as a model for the ingredients used in preparing BR-koji-amazake, as glutinous rice varieties are commonly consumed in Japan and Southeast Asian countries such as Thailand and Laos. We hypothesized that the use of rice-koji made with A. luchuensis (Al-rice-koji) in the production of BR-koji-amazake would produce a product with unique flavor and ingredient characteristics that differ from the conventional rice-koji made with A. oryzae (Ao-rice-koji), attributable to the effect of citrate produced by A. luchuensis and the actions of hydrolytic enzymes (from both Aspergillus species) exhibiting different behaviors at acidic pH conditions. While citrate was below the detection limit in the conventional BR-koji-amazake product with a pH value of 5.98 (Table 1), it increased in a stepwise manner from 0.18 to 0.56 % as the proportion of Al-rice-koji increased to 25, 50, 75, and 100 % (Table 1). Additionally, the product pH decreased from 4.74 to 3.68 with an increasing proportion of citrate (Table 1). Such an acidic pH would be advantageous in reducing the risk of spoilage bacteria growing in the product. Brix values of the products decreased from 37.1 to 35.4 % as the proportion of Al-rice-koji increased from 0 to 100 %, (Table 1), suggesting the efficiency of saccharification in the preparation of BR-koji-amazake was higher with the Ao-rice-koji than with the Al-rice-koji examined in this study. It was previously reported that the α-amylase activity of rice-koji made with A. oryzae was about eight times higher than that made with A. luchuensis; however, it was unstable compared with the activity of A. luchuensis under acidic conditions (pH value of 4.0 or lower) and the activity decreased to about 50 % at pH 3.5 compared with the optimum conditions at pH 5.0 (Iwano and Mikami, 1988). Therefore, when combining Ao- and Al-rice-koji for making BR-koji-amazake via relatively short-term saccharification, it is desirable to adjust the ratio so that the pH is maintained at higher than 4.0 to ensure a sufficient level of rice starch saccharification in the product, although the effect of the acidity of citrate on the product taste should also be considered.

Table 1. Citrate content, pH, and Brix percentage of BR-koji-amazake prepared by combinatorial use of rice-koji made with Aspergillus oryzae (Ao) and Aspergillus luchuensis (Al) species.

Proportion (%) of rice-koji^a
Ao-rice-koji	Al-rice-koji	Citrate : %^b	pH ^b	Brix: % ^b
100	0	—^c	5.98 (0.04)	37.2 (0.17)
75	25	0.18 (0.01)	4.74 (0.02)	36.7 (0.24)
50	50	0.26 (0.04)	4.17 (0.03)	36.0 (0.12)
25	75	0.44 (0.04)	3.88 (0.05)	35.6 (0.19)
0	100	0.56 (0.01)	3.68 (0.05)	35.4 (0.42)

^a Proportion (%) of Ao- and Al-rice-koji to the total amount of rice-koji used for each BR-koji-amazake sample.

^b Mean values with standard errors in parentheses from triplicate measurements.

^c —, undetectable level.

Mechanical sensory evaluation of BR-koji-amazake products In this study, mechanical sensory evaluation of the BR-koji-amazake products was conducted with a TS-5000Z taste sensor (Intelligent Sensor Technology, Inc.) equipped with multichannel electrodes using a lipid polymer membrane. This sensor can quantify the five basic tastes of sourness, saltiness, sweetness, bitterness, and umami, as well as astringency, and shows good correlation with human sensory perception (Wu et al., 2020). Figure 1 shows the evaluation results using BR-koji-amazake prepared with Ao-rice koji alone as a reference. In the BR-koji-amazake samples produced using Al-rice koji, the increase in sourness was approximately proportional to the rise in citric acid content, while sweetness, bitterness, and umami decreased as the proportion of Al-rice koji increased (Fig. 1). No change in astringency was observed (data not shown), and while an increase in saltiness was detected in the products made with Al-rice koji (data not shown), it is believed to be an artifact caused by the sensor’s reaction to citrate. The use of Al-rice koji not only imparts sourness to BR-koji-amazake but also attenuates starch saccharification and proteolysis, which release amino acids responsible for umami and bitterness. This may be attributable to the acidification of the fermentation environment and changes in the composition of hydrolytic enzymes.

Fig. 1

Radar chart of the relative difference in sweetness, sourness, bitterness, and umami of BR-koji-amazake prepared with varied proportions of Ao- and Al-rice-koji.

BR-koji-amazake prepared only with Ao-rice-koji was used as a reference with taste value set to 0. Proportions (%) of Ao-rice-koji: Al-rice-koji set for each sample is as follows: filled circle, 100 : 0; filled triangle, 75 : 25; open triangle, 50 : 50; filled square, 25 : 75; open square, 0 : 100.

Reduction of phytate in BR-koji-amazake products The dephosphorylation of phytate to myo-inositol by enzymes in rice-koji was expected to be an important factor in the production of BR-koji-amazake with enhanced nutritional value and health benefits. In BR-koji-amazake made using only Ao-rice-koji as the fermentation starter, phytate was decreased by approximately 20 % compared with the non-fermented control (Table 2). Substituting 25 % of the rice-koji with Al-rice-koji resulted in an approximate 60 % reduction of phytate in the product (Table 2). These differences were confirmed to be significant by Welch’s t-test (p-values < 1 %). In the product that incorporated the same proportion of Al-rice-koji but with enzymes inactivated by heat treatment at 121 °C for 15 min, a phytate reduction nearly identical to that produced with Ao-rice-koji alone was observed (data not shown). This suggests that phytases from Al-rice-koji are primarily responsible for the enhanced degradation of phytate in the BR-koji-amazake product in this study. The decrease in phytate was enhanced as the proportion of Al-rice-koji increased in the products. Increasing the Al-rice-koji proportion to 75 and 100 % resulted in a phytate decomposition rate of about 75 %. Citrate is thought to enhance mineral bioavailability. As supporting evidence, the addition of citrate was reported to significantly increase iron absorption from rice meals in humans (Gillooly et al., 1983, Ballot et al., 1987). Furthermore, a combination of citrate addition, dephytinization, and iron supplementation has been shown to significantly increase iron absorption in oat-based beverages (Zhang et al., 2007). The reduction of phytate in BR-koji-amazake using Al-rice-koji containing citrate, as demonstrated in this study, could be a beneficial processing method for brown rice, as it enhances the intake of brown rice micronutrients, including minerals that would otherwise not be as readily absorbed because of the absorption-inhibiting phytate.

Table 2. Phytate and myo-inositol contents of BR-koji-amazake products prepared by combined use of rice-koji made with Aspergillus oryzae (Ao) and Aspergillus luchuensis (Al) species.

Proportion (%) of rice-koji^a
Ao-rice-koji	Al-rice-koji	Phytate (mg/100 g)^c	myo-inositol (mg/100 g)^c
100 (inactivated)^b	0	652 (6.13)	2.15 (0.05)
100	0	516 (31.0)	9.26 (1.60)
75	25	257 (43.9)	34.5 (1.36)
50	50	183 (24.8)	42.3 (2.23)
25	75	143 (51.2)	49.0 (1.61)
0	100	158 (11.6)	47.9 (1.47)

^a Proportion (%) of Ao- and Al-rice-koji to the total amount of rice-koji used for each BR-koji-amazake sample.

^b Ao-rice-koji inactivated by heat treatment was used as a non-fermented control as described in Materials and Methods.

^c Mean values with standard errors in parentheses from triplicate measurements.

Production of myo-inositol in BR-koji-amazake products Considering the potential health benefits of myo-inositol and its important role in infant growth, optimization of manufacturing methods to increase myo-inositol is also desirable for developing BR-koji-amazake. Both Ao- and Al-rice-koji used in this study possessed enzymes including phytases and acid phosphatases that function in the complete dephosphorylation of phytate to myo-inositol (Table 2). It was also clear that the use of Al-rice-koji markedly increased inositol contents in the BR-koji-amazake products. Substituting 25 % of the rice-koji with Al-rice-koji resulted in an approximate 3.7-fold increase of myo-inositol in the product, reaching its maximum (49.0 mg/100 g) when the proportion of Al-rice-koji used was increased to 75 % of the total (Table 2). These increases were confirmed to be significant by Welch’s t-test, with p-values of less than 1 %. Similarly to phytate reduction, the use of heat-inactivated Al-rice-koji did not promote inositol production (data not shown). Taken together, under the experimental conditions in this study, enzymes derived from Al-rice-koji were more actively involved in phytate dephosphorylation than those of Ao-rice-koji in the BR-koji-amazake. This is probably due to differences in the optimum pH of the enzymes between the two Aspergillus strains, as well as in their stability at the fermentation temperature (55 °C) and the acidic fermentation environment generated by the citrate from Al-rice-koji. A. oryzae AphA is currently the sole phytase from this species with both an identified gene and reported enzymatic properties (Yoshino-Yasuda et al., 2012). However, this enzyme may have little effect on phytate dephosphorylation in BR-koji-amazake production, as it rapidly lost activity at temperatures exceeding 35 °C, although it was stable over a wide pH range from 3.0 to 7.0 (optimum pH of 4.0) (Yoshino-Yasuda et al., 2012). Enzymatic properties such as optimum pH and substrate specificity of the phytases / acid phosphatases of A. oryzae and A. luchuensis species expressed in rice-koji are of considerable interest for identifying the key enzymes in both species responsible for enhancing dephosphorylation of phytate to inositol in BR-koji-amazake. Considering the manufacturing conditions, it is desirable to identify the genes for phytases and acid phosphatases that can act stably even at high temperatures of 55 to 60 °C and acidic environments with a pH of about 4.5. To achieve this, investigating differences in the degree of phytate dephosphorylation in BR-koji-amazake as well as the related gene expression profiles among rice-koji cultured with diverse industrial A. oryzae and A. luchuensis strains should also be considered. The series of analysis methods demonstrated in this study are easily applicable to the evaluation of rice-koji samples cultured with various Aspergillus strains.

Brown rice-based diets are attracting worldwide attention because of their high nutritional value and health functions. The BR-koji-amazake demonstrated in this study can be made by using Japanese rice-koji and local rice varieties in a relatively simple manner, offering a practical approach to its utilization as a daily nutritional food. Nutritional challenges such as malnutrition, low household food diversity, and food insecurity persist in remote communities, such as those in the mountainous areas of Laos (Boulom et al., 2020). Thus, phytate dephosphorylation in BR-koji-amazake, seen in terms of reducing the risk of inhibited mineral absorption, is an important advantage in the global utilization of koji-amazake. The combined use of rice-koji cultured with A. oryzae and A. luchuensis demonstrated in this study is a promising approach for enhanced dephosphorylation of phytate to myo-inositol in BR-koji-amazake. The taste preference characteristics of interregional consumers, particularly regarding sweetness and sourness, are of considerable interest for further study. Such information should be useful for optimizing the combination of Ao- and Al-rice-koji, accounting for both the efficient dephosphorylation of phytate to myo-inositol and the taste preferences of international consumers.

Acknowledgements The authors would like to thank Dr. Yutaka Wagu for his thoughtful guidance and Ms. Mari Momma (JIRCAS) for technical assistance. This research was funded by the JIRCAS research program “Indigenous crops and food design”.

Conflict of interest There are no conflicts of interest to declare.

References

責任著者(Corresponding author)

訂正情報

J-STAGEへの登録はこちら（無料）