Visualization of Phospholipids and Glucose in Rice Koji Using Microscopic Mass Spectrometry Imaging

Significance of the Topic

Rice koji supplies essential digestive enzymes that drive starch and protein breakdown in sake brewing and greatly influences taste and aroma. Conventional quality assessment is largely empirical. Mass spectrometry imaging offers spatially resolved chemical maps that can improve scientific understanding of rice koji composition and support consistent product quality.

Objectives and Study Overview

This study aimed to apply microscopic MSI to map phospholipid and glucose distributions in rice koji and to develop a method for visualizing hyphal invasion (hazekomi) by combining a GUS reporter system with MSI.

Methodology

Samples included raw, steamed, and rice koji prepared from Yamada Nishiki and Hakutsuru Nishiki varieties with 70% polishing rate. Sections of 20 µm thickness were prepared by cryomicrotome after embedding in 4% carboxymethyl cellulose and mounted on ITO coated glass. Phospholipid matrix (CHCA) was deposited by vapor and spray using an iMLayer system. NEDC matrix was applied and recrystallized for glucose detection. MSI was performed on a Shimadzu iMScope in positive mode (m/z 400–800, 25 µm resolution) and negative mode (m/z 180–230, 50 µm resolution). Data were processed with Imaging MS Solution and IMAGEREVEAL MS.

Instrumentation Used

• Shimadzu iMScope QT imaging mass microscope with iMLayer vapor deposition
• Cryomicrotome and adhesive cryofilm for sample sectioning
• ITO coated glass slides and conductive tape for mounting
• Imaging MS Solution and IMAGEREVEAL MS software suites

Main Results and Discussion

Choline signal was concentrated in the germ of raw grain, decreased after steaming, and reappeared in inner regions of koji. Lysophosphatidylcholines LPC(16:0) and LPC(18:2) and other phospholipids showed heterogeneous distributions aligned with hyphal growth. A spray method applying X-Gluc to GUS expressing koji sections preserved morphology and revealed depth of hyphal invasion. MSI mapping of [M+Cl]– glucose ion demonstrated increasing glucose from outer to inner regions over 24–43 h of fermentation, correlating with hazekomi progression. Spatial heterogeneity in glucose accumulation highlighted variable enzyme activity within grains.

Benefits and Practical Applications

Spatial chemical imaging supports quality control and process optimization in rice koji production. Insights into enzyme penetration and metabolite gradients can guide improvements in steaming and inoculation. The combined GUS-MSI approach offers a tool for studying fungal growth and metabolic distribution in fermentation products.

Future Trends and Potential Applications

Next generation MSI platforms with higher mass resolution, sensitivity, and wider mass range will enable mapping of additional metabolites. Non-targeted analysis tools may uncover new quality markers. The methodology can be extended to other fermented foods such as miso and soy sauce for improved scientific control of fermentation.

Conclusion

Microscopic MSI combined with a GUS reporter system provides novel spatial insights into rice koji fermentation. The approach quantifies key phospholipids and glucose distributions and visualizes hyphal invasion. These findings offer a scientific basis for consistent quality and process optimization in sake brewing. Future instrument advances will expand capabilities for comprehensive metabolite imaging.

Reference

1 Miyoshi K, Enomoto Y, Fukusaki E, Shimma S Application Note No. 57 Shimadzu
2 Shimma S, Sagawa T Application Note No. 63 Shimadzu
3 Shimma S et al J Mass Spectrom 2013 48 1285
4 Zaima N et al Rapid Commun Mass Spectrom 2010 24 2723
5 Wisman AP et al J Biosci Bioeng 2020 129 296
6 Wisman AP et al J Brew Soc Japan In press
7 Yoshii M, Aramaki I J Brew Soc Japan 2001 96 806
8 Wang J et al Anal Chem 2015 87 422