Metabolomic-based study for the investigation of the effect of the environmental condition during fermentation and raw materials on the end product of tempe

Laboratory of Bioresource Engineering (Fukusaki Lab)

Adinda Darwati Kadar

Chapter 1: General introduction

Tempe (also known as tempeh) is an Indonesian fermented soybean food. Tempe is also known as white soybean cake. The white color of tempe comes from Rhizopus spp. mycelia which bound the beans together into a compact soycake. Currently, tempe industries ranging from home to large-scale industry are in demand of 70% of the total Indonesian soybean supply. Currently, there are more than 100,000 active tempe producers in Indonesia. This makes tempe a very important local product with high cultural as well as economic value for Indonesian.

Tempe has been gaining international attention since the early 19^th century (Shurtleff and Aoyagi, 2020). It is worth noting that tempe recognition does not parallel with the progression in the effort to understand the industrial fermentation of tempe, particularly from a scientific standpoint. Currently, available information provides detailed but fragmented knowledge about tempe fermentation. Metabolomics approach is a very useful technology to evaluate the quality of the food product based on the metabolite profiles of the end products. Therefore, the present study aims to conduct GC/MS-based metabolomics to the study of tempe. This study pursues the understanding of the effect of factors related to the production processes to the end product of tempe as seen through the metabolite profiles.

Chapter 2: Untargeted metabolomics for the classification of tempe from different regions and production processes in Indonesia

Differences in the production process and unique fermentation conditions in different regions result in varieties of tempe. Despite its high cultural and economic values, there are very few studies on the characterization of tempe based on the differences in the production process and geographical origin. In this chapter, untargeted metabolomics is applied for the study of Indonesian tempe obtained from various regions and different production processes. Seventeen samples were collected from six different cities in Java Island, Indonesia which were produced by local tempe workshop, semi-modern industry, and more modernized industry. Untargeted metabolomics by gas chromatography coupled with mass spectrometry (GC/MS) was conducted to discriminate various kinds of tempe and identify metabolites that are associated with these differences. Results showed that tempe produced in different regions clustered together according to the cities and their categories. Sugars and amino acids groups were found to be primary compounds that contributed to this result. These differences are, hypothetically, related to the environmental condition of each region origin i.e. average temperature and humidity. Furthermore, a sensory evaluation involving 75 semi-trained panelists was done to evaluate the preference of consumers towards the representative samples. This is the first report that addresses the metabolic differences between different varieties of tempe from different regions and production processes.

Chapter 3: Metabolomics-based study of the effect of raw materials on the end product of tempe

In the previous chapter, it was suggested that the metabolite profiles of tempe were affected by factors related to the region of origin. In this chapter, we applied GC/MS-based metabolite profiling to investigate the effect of region-related factors that might affect the final products of tempe. This includes environmental factors, raw materials, and starter cultures. This study was divided into three sections. The first section was the analysis of the effect of environmental conditions. In this section, a method called ‘cross fermentation’ was employed. Three different regions in Indonesia (Bogor, Ngawi, Gunung Kidul) were involved. Here, metabolite profiling was applied to tempe samples that underwent pre-fungal fermentation (PFF) stage and fungal fermentation (FF) stage in different places. The results showed that environmental conditions during PFF affect the metabolite profile of tempe more than during FF. It was also suggested that the average humidity during PFF might play a greater role than the average temperature at the same stage. The second section was to investigate the effect of the raw materials (soybean, water, and commercial starter culture) on the metabolite profile of tempe. The results implied that each tested factor affects the metabolome profiles of tempe. The last section was conducted to confirm the findings. In this section, 17 unbiased industrial samples were collected from the market place in Japan and Indonesia. Interestingly, the results showed that one Japanese sample (coded JP-8) was clustered together with Indonesian samples in principal component analysis (PCA). The JP-8 sample, later on, was known to use commercial starter made in Indonesia. This discovery suggested the influence of starter culture is stronger than soybean, water, and environmental condition in determining the metabolite profile, hence attribute, of tempe.

Chapter 4: Conclusions and future perspectives

Metabolomic-based study of tempe had enabled us to understand the effect of the controllable factors during tempe fermentation. As has been demonstrated in Chapter 2 and Chapter 3, environmental conditions, soybean, water, and starter culture play roles in determining metabolite profiles. The results have also been suggested that such factors showed different degrees of importance in affecting the metabolite profiles. The validation set demonstrated that the commercial starter cultures, or starter culture in general, influence metabolite profiles more than other tested factors. The finding of this study will be useful for larger-scale industries to maintain or improve the quality of tempe for delicate purposes (pharmaceutical grade) and or common purposes (staple food).

List of publications:

1. Kadar, A. D.; Aditiawati, P.; Astawan, M.; Putri, S. P.; Fukusaki, E. Gas Chromatography Coupled with Mass Spectrometry-Based Metabolomics for the Classification of Tempe from Different Regions and Production Processes in Indonesia. J. Biosci. Bioeng. 2018, 126 (3), 411–416. https://doi.org/10.1016/j.jbiosc.2018.03.020.

2. Kadar, A. D.; Astawan, M.; Putri, S. P.; Fukusaki, E. Metabolomics-based study of the effect of raw materials to the end product of tempe-an Indonesian fermented soybean. Metabolites. 2020 (submitted)

Reference: Shurtleff, W. Aoyagi A. (2020) History of Tempeh and Tempeh Products (1815-2020): Extensively Annotated Bibliography and Sourcebook. California: Soyinfo Center