Notes
Effect of light exposure on enzyme activity in wheat bran solid-state fermentation by Aspergillus oryzae
2023 Volume 29 Issue 6 Pages 475-479
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2023 Volume 29 Issue 6 Pages 475-479
The regulation of enzyme activity in the production of various fermented foods using koji mold has been optimized in many ways, and few additional refinements are apparent. This study examined light as a modifiable factor with which to adjust the enzyme activities of koji. A previous study has shown improved saccharification of wheat bran with Aspergillus oryzae RIB40 under at least 8 h of darkness. In the work reported here, light exposure had no significant effect on the saccharification activity of A. oryzae RIB1187. However, light exposure for 8 h or more continuously decreased protease-specific activity in RIB1187, as was seen previously in RIB40. This information may help manufacturers to improve fungal enzyme production, processes, and facilities.
Koji mold is used in the traditional fermentation industry in Japan to produce large quantities of various digestive enzymes when grown on cereal grains. Koji, a fermentation product of koji mold, is used as an enzyme source in food processing. Koji mold includes three species of Aspergillus, i.e., A. oryzae, A. sojae, and A. luchuensisi). The options and proportions of enzyme activities needed to produce the desired final product are managed by selecting the appropriate koji mold strains and controlling the temperature during the fermentation process. For example, koji for brewing sake uses a strain with high glycolytic enzyme activity to saccharify the starch of rice at ≥42 °C. Koji for brewing soy sauce, in which >90% of the soybean protein is decomposed to amino acids, uses a strain with high protease activity at around 35 °C.
The traditional arts of managing the enzyme activities of koji are well developed and have been refined to optimize the production conditions in each brewery; little latitude remains to further improve the options of seed koji and temperature control. However, lighting treatment may affect enzyme activities. Some genomes in the genus Aspergillus have homologs of the near-ultraviolet and blue photoreceptor cryptochromes, the blue photoreceptor white-color complex, the green photoreceptor opsin, and the red photoreceptor phytochrome (Rodriguez-Romero et al., 2010). Laboratory strains of A. oryzae RIB40 conserve the machinery for response to light (Hatakeyama et al., 2007); for example, the presence or absence of light determines the activity of A. oryzae F6, which is derived from RIB40, acid proteases (Murthy et al. 2015). In addition, light exposure can increase or decrease the expression of genes for glycolytic enzymes, proteolytic enzymes, lipases, and other lipid metabolism-related enzymes (which are essential for brewing), as seen in transcriptome analysis in fluid culture (Suzuki and Kusumoto, 2020). A. oryzae RIB40 is a standard laboratory strain for the study of A. oryzae in molecular biology and most photoresponse studies of A. oryzae have been performed using RIB40. We found that the photoresponses of A. oryzae RIB1187 were opposite to strain RIB40 in a previous study (Murthy et al., 2018). Here, we studied the effect of light exposure on the enzyme activities of wheat bran solid-state fermentation by strain RIB1187. The aim of this study was to explore the manipulation of light exposure to control enzyme activities so as to improve productivity in the fermentation industry.
Strains Aspergillus oryzae strain RIB1187 was obtained from the National Research Institute of Brewing, Japan.
Media and Culture Methods Potato-dextrose agar (DIFCO, Tokyo, Japan) was used for maintenance and conidiogenesis of Aspergillus. The wheat bran culture for solid-state fermentation was prepared by adding 2 g wheat bran and 1 mL distilled water to a 100-mL Erlenmeyer flask, autoclave sterilization, and addition of 2 mL of A. oryzae conidial suspension; the moisture content of the wheat bran culture was 60 %. The thickness of the wheat bran was approximately 2–5 mm with several crevices. The solid-state fermentation experiments were performed in triplicate with three independent cultures. All flasks were incubated at 30 °C for 48 h.
Light exposure Flasks were statically incubated in darkness interrupted by periods of light without agitation. Light was supplied by a 4-W white fluorescent lamp (satellite F4T5D) installed in the incubator and controlled by an electric timer. Three flasks were arranged in a semicircle around the white fluorescent lamp. The distance between the center of the lamp tube and the center of the bottom of the flask was 15 cm. The flasks were exposed to one of eight lighting treatments, i.e., for 0 h (dark culture), light exposure only during hours 0–8, 8–16, 16–24, 24–32, 32–40, or 40–48 of the incubation period, or continuously light exposure for 48 h. The photon flux density was measured by a photon sensor (IKS-27/101, Koito, Yokohama, Japan). The mean photon flux density on the surface of the media was 10 μmol m−2 s−1 under light.
Enzyme solution preparation The fermented wheat bran from a flask was suspended in 16 mL sterilized Milli Q water and allowed to stand at 4 °C overnight. Enzymes dissolved in the resulting suspensions were extracted by filtration through Miracloth (Calbiochem, San Diego, CA, USA), and the filtrate was adjusted to 25 mL with sterilized Milli Q water. The enzymes were partially purified by 30 %-95 % (w/v) ammonium sulfate fractionation. The precipitate from the 95% ammonium sulfate precipitation was re-dissolved in 2.5 mL McIlvaine buffer (pH 5.7) and desalted with PD-10 (Cytiva Japan, Tokyo, Japan). The total protein concentration of the enzyme solution was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.
Assay of saccharification activity The saccharification activity of the enzyme solutions was measured by a Kikkoman saccharification activity measurement kit (Kikkoman, Noda, Japan), according to the manufacturer's instructions. The G2-β-PNP provided in the kit was converted to G1-β-PNP by glucoamylase and alpha-glucosidase in the samples of enzyme solution prepared above. The beta-glucosidase provided in the kit then liberated p-nitro phenol from G1-β-PNP. One unit of enzyme activity was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per min under the assay conditions. Specific activity was defined as the resulting unit values, standardized by the total protein amount, in U mg−1.
Assay of protease activity The protease activity of the enzyme solutions was measured according to the method of Maeda et al. (2015) with slight modification. The substrate stock solution was prepared by dissolving 1.25 % (w/v) azocasein in 100 mM TrisHCl buffer (pH 7.5) containing 1 mM CaCl2. Samples of enzyme solution were mixed with substrate stock solution (40 or 160 μL) and incubated at 37 °C for 20 h. The reaction was terminated by addition of 200 μL of 10 % (w/v) trichloroacetic acid, and the sample was centrifuged at 21 600 × g at 4 °C for 10 min. The supernatant (350 μL) was collected and an equal volume of 0.75 M NaOH was added. The optical density was measured at 440 nm on a U-1900 spectrophotometer (Hitachi High-Tech, Hitachi, Japan). One unit of enzyme activity was defined as an increase of one absorption unit per min at 440 nm under the assay conditions. Specific activity was defined as the resulting unit values standardized by the total protein amount, in U mg−1.
Statistics All experiments were performed in triplicate. Statistical significance was tested with Student's t-test and ANOVA in MS Excel software.
Saccharification activity during 48-h solid-state culture of A. oryzae on wheat bran. A, strain RIB1187. B, strain RIB40 (from Suzuki, 2021). X-axis values indicate the times during which the darkness was interrupted by a lighted period, i.e., not at all (dark), or during hours 0 to 8, or 8 to 16, or 16 to 24, etc., or throughout the culture period (light). *P < 0.001.
The saccharification activity with wheat bran solid-state fermentation using A. oryzae RIB1187 was not significantly affected by any of the light exposure treatments (Fig. 1A). Interestingly, the photoresponses of RIB1187 are considered opposite to those of strain RIB40 (Murthy et al., 2018). In a previous identical experiment using A. oryzae RIB40 (Suzuki, 2021), saccharification activity was significantly reduced under exposure to light (Fig. 1B). The inhibitory effect of light exposure at any timing on the saccharifying activity of RIB40 is canceled by a dark period (Suzuki, 2021). This suggests that expression of the genes for the principal enzymes for saccharification is temporarily and reversibly suppressed only under light exposure; the genes appear to be expressed during darkness in both RIB1187 and RIB40 (Fig. 2). Suzuki and Kusumoto (2020) found that as little as 10 min of light exposure is sufficient to suppress the expression of genes for alpha-amylase and glucoamylase in liquid culture of A. oryzae RIB40. Here, we had hypothesized that either 48 h of darkness (total darkness treatment) or 40 h of darkness (as in the six short light exposure treatments) would be sufficient for expression of the principal enzymes for saccharification, and once expressed, these enzymes would remain active at least until the enzyme activity was measured at 48 h. However, in RIB1187, there were no significant differences in the saccharification activity among all treatments (Fig. 1A). This suggests that light exposure had no inhibitory effect on the expression of genes for the principal enzymes for saccharification in RIB1187.
Schematic model of light influence on saccharification activity.
Among other responses, light exposure inhibits growth, conidiogenesis (Suzuki and Kusumoto, 2013), and amylase gene expression (Suzuki and Kusumoto, 2020) in liquid culture of RIB40, whereas it promotes all three in RIB1187 (Suzuki and Kusumoto, 2020). There may be a common system among these responses that is regulated by light (Fig. 2), but this is not yet clear.
Protease-specific activity was significantly suppressed by light exposure of at least 8 h at any period to approximately half its rate in total darkness in RIB1187 (Fig 3A). The same phenomenon was previously observed in the case of RIB40 (Suzuki, 2021; Fig. 3B). This suggests that the inhibitory effect of light exposure was persistent, and as early as the first 8-h light exposure was sufficient to suppress the protease-specific activity up to 48 h. In RIB1187, the protease-specific activities decreased significantly more in the light treatments applied after 24 h of culture than in those applied before 24 h (Fig. 3A). In the 24–32-, 32–40-, and 40–48-h light treatments, suppression of protease-specific activity was equal to that observed in the continuous light treatment in RIB1187 (Fig. 3A). In the case of RIB40, the protease activity did not significantly differ after 8-h light treatment at any period (Suzuki, 2021; Fig. 3B). Namely, the inhibitory effect of 8-h light treatment on protease-specific activity continued until 48 h in RIB40 (Suzuki, 2021; Fig. 3B). In RIB 1187, the inhibitory effect continued until 24 h, then protease-specific activity was partially restored (Fig. 3A). This suggests that the protease polypeptides expressed in the first 24–40 h disappeared during the 8-h light exposure in RIB1187. This may be partly attributable to catabolism of the already formed protease polypeptides in the fungal cells during light exposure, in addition to suppression of gene expression of the proteases (Fig. 4).
Protease activity of during 48-h solid-state culture of A. oryzae on wheat bran. A, strain RIB1187. B, strain RIB40 (from Suzuki, 2021). X-axis values as in Fig. 1. *p < 0.001.
Schematic model of light influence on protease activity.
Light upregulates the most highly expressed protease gene in RIB1187, that for aspartyl protease 2, but downregulates the second most highly expressed one, that for metallopeptidase 2 (Suzuki and Kusumoto, 2020). However, the advantages of these responses are not clear from the transcriptome data. Nevertheless, it is interesting to note that protease-specific activity in RIB1187 differed significantly between before and after 24 h (Fig. 3A); i.e., the inhibitory effect of light exposure on protease-specific activity appeared to be stronger after 24 h of culture. RIB1187 was originally purified from soy sauce koji. We may be able to recommend that the brewing or fermentation of some foods that require high protease activities, such as soy sauce, should occur under complete darkness from beginning to end (“Tanetsuke” meaning spore inoculation to “Dekoji” meaning the end of the koji production process). In the commercial enzyme industry, it is also recommended that proteases be produced under total darkness. It is unclear why proteases would be unnecessary for Aspergillus growth in light-exposed areas such as terrestrial environments. However, if the soil is assumed to be in constant darkness, decomposing protein-based nutrients may enable Aspergillus to grow there.
In conclusion, light exposure can inhibit both saccharification activity and protease activity of wheat bran solid-state fermentation by A. oryzae. The detailed mechanism of the response to light exposure differed among the strains. Light exposure suppressed protease activities to approximately half the rate observed in total darkness. This information may be helpful to manufacturers of fungal enzymes to improve production processes and facilities.
Acknowledgements This research was supported by grants from the Tojuro Iijima Foundation for Food Science and Technology. K.-I. K. was supported by the Institute for Fermentation, Osaka, Japan (grant number K-2021-008). We thank Yuriko Yamamoto for her great help in all experiments in this study.
Conflict of interest There are no conflicts of interest to declare.
