Study of haze-komi and metabolite distribution relation in rice koji by the combination of metabolomics and imaging techniques

Laboratory of Bioresource Engineering (Fukusaki Lab)

Adinda Putri Wisman

Chapter 1: General Introduction

Rice koji, made by inoculating Aspergillus oryzae on steamed rice, is an important material involved in the production of various fermented products, such as sake, mirin, miso, and others. As such, the examination of rice koji features is essential for the overall production process. One feature of rice koji is the presence of haze, the white areas on the surface of rice that represents the existence of fungal mycelia. The degree of mycelial penetration, also known as haze-komi, is thought to have a high correlation with koji quality. However, no studies have been done based on the observation of exhaustive metabolites level. Hence, metabolite level studies related to haze-komi is a topic of interest in the brewing industry. To facilitate the required study, it is necessary to improve the conventional visualization of haze-komi profile. In addition, a correlation study between haze-komi profile and the component profile inside rice koji would be expected.

Thus, the objective of this study is to observe the trend of haze-komi and metabolites alteration during rice koji-making. Time-course analysis of rice koji using the combination of metabolomics and imaging techniques was planned to achieve this objective. First, β-glucuronidase (GUS) reporter system was used for haze-komi visualization. Then, metabolic profiling using GC-MS and LC-MS was conducted to obtain information on the metabolic changes during the rice koji-making process. Finally, mass spectrometry imaging (MSI) was utilized to observe the distribution profile of metabolites selected by time-course metabolome analysis of rice koji during the process.

Chapter 2: Mapping of haze-komi using GUS histochemical staining

To determine whether the rice koji is ready for the following processes, the surface of steamed rice is inspected for the signs of haze-komi. Some research has described the effort on whole rice haze-komi observation through several methods, such as transmitted lights or fluorescence-based imaging. However, obtaining a clear image requires the use of confocal laser scanning microscopy (CLSM) as the autofluorescence by rice cells itself may produce a background signal that hinders the observation of true mycelial penetration.

In the first step of this study, an alternative for mycelia visualization was presented through the use of GUS histochemical staining. An A. oryzae strain that can express GUS was utilized to make rice koji, which in turn was used for the observation of haze-komi during the rice koji-making process. With the optimization of the preparation method, clear visualization was achieved for the observation of haze-komi on a cross-section of rice koji. The method was applied to time-course samples of rice koji, revealing the extent of mycelial penetration at specific time points during the rice koji-making process.

Chapter 3: Time course metabolite profiling of rice koji made by GUS strain Aspergillus oryzae

The untargeted metabolic profiling of rice koji has been reported by several studies. These studies focused on the effects of materials involved, such as the fungal strain used or the polishing rate of rice. Some reported the changes during a time course observation, in which different classes of metabolites such as sugars, amino acids, and organic acids fluctuated during the process. Nevertheless, the discussions are more focused on the effects of materials rather than the time-related changes. Additionally, comprehensive metabolite profiling of rice koji with more attention on amino acids and other amine metabolites is still missing.

In this chapter, the rice koji used during chapter 2 study was subjected to untargeted metabolite profiling using GC-MS as well as amino acids and other amines profiling by LC-MS. The time course profiling revealed the changes of sugars, sugar alcohols, amino acids, organic acids, polyamines, and other metabolites during the rice koji-making process. Most of the metabolites were significantly changed during the koji-making, with several sugars, amino acids, and other amine metabolites showing the highest relative intensity in different timing. The results obtained confirmed the effects of A. oryzae inoculation towards metabolite production, as well as adding another perspective through the analysis of other amines such as polyamines during rice-koji making.

Chapter 4: Visualization of metabolites in rice koji made by GUS strain Aspergillus oryzae

The visualization of metabolites on foodstuffs has been a topic of interest in the food industry. The use of MSI is especially appealing as it is able to visualize multiple compounds without the use of any labeling technique. The application of MSI for food imaging has been done for various purposes, such as characterization and quality evaluation of food materials. This technique is suitable for investigating the effect of haze-komi towards the metabolite changes during rice koji-making, as visual data is required to compare with the profile of mycelial penetration.

As such, the same samples from previous chapters were used for the imaging of metabolites in rice koji. Sugars, sugar alcohols, amino acids, and other amines were focused in this study, as these metabolites showed the most changes between classes. The imaging results were compared with GUS histochemically-stained rice koji images. Sugars and sugar alcohols were not shown to be affected by the extent of mycelial penetration, while several amino acids and other amines seem to be correlated with haze-komi. From these results, it was revealed that haze-komi progression is related to the distribution of specific metabolites inside rice koji to some degree.

Chapter 5: Conclusions and perspectives

The combination of metabolomics and imaging techniques successfully accumulated knowledge about the trend of haze-komi and metabolites alteration during the rice koji-making process. This is the first report that correlated the degree of haze-komi with the profile of metabolites inside during the rice koji-making process. The presented method is applicable for further investigation on factors related to haze-komi, such as the moisture content or the polishing rate of rice.

List of Publications:

1. Wisman, A. P., Tamada, Y., Hirohata, S., Gomi, K., Fukusaki, E., & Shimma, S. (2020). Mapping haze-komi on rice koji grains using β-glucuronidase expressing Aspergillus oryzae and mass spectrometry imaging. Journal of Bioscience and Bioengineering, 129(3), 296–301. https://doi.org/10.1016/j.jbiosc.2019.09.016

2. Wisman, A. P., Tamada, Y., Hirohata, S., Fukusaki, E., & Shimma, S. (2020). Metabolic visualization reveals the distinct distribution of sugars and amino acids in rice koji. Mass Spectrometry, 9(1), A0089. https://doi.org/10.5702/massspectrometry.A0089