Name （ RIFQI AHMAD RIYANTO ）
Metabolomics-based Study for Investigating the Effect of Microbial Interventions in Tempe Soaking Step
(微生物介入がテンペの浸漬段階に及ぼす影響を調査するためのメタボロミクス研究)
Abstract of Thesis
Tempe is a traditional Indonesian food produced through the solid-state fermentation of legumes, primarily soybeans, using Rhizopus spp. mold. It is a staple source of protein and is recognized for its functional health benefits. The production process of tempe involves several steps: soaking, dehulling, cooking, inoculating with a starter culture, packaging, and allowing for fungal fermentation. Variations in tempe production mainly arise during the soaking and cooking stages. Soaking is essential, as it hydrates the soybeans, removes anti-nutritional factors, enhances protein bioavailability, inhibits harmful microorganisms, and supports the growth of the tempe starter culture. However, due to variations in climate and processing methods, natural acid fermentation may not consistently occur, particularly in temperate climates. To address this issue, several chemical and microbial modifications have been implemented during the soaking step to ensure effective acidification. Despite this, comprehensive information regarding the metabolite profile of tempe produced with these modifications during soaking is still limited. Consequently, this study aims to investigate the impact of microbial intervention during the tempe soaking step on its metabolome. Prior to tempe fungal fermentation, soybeans were soaked with Lactiplantibacillus plantarum NBRC 101978 and Pichia burtonii NBRC 0844. Tempe samples were subjected to comprehensive analysis using a widely targeted gas chromatography-mass spectrometry (GC-MS) metabolomics approach. A total of 100 metabolites of sugars, amino acids, fatty acids, and organic acids were annotated in all samples. Principal component analysis (PCA) showed that microbial interventions led to alterations in the metabolome of all samples, including the accumulation of amino acids in lactic acid bacteria (LAB) soaked-soybean tempe and biogenic amine, tyramine, in yeast soaked-soybean tempe. Unlike chemically added soaked-soybean tempe, microbial intervention significantly reduced the relative intensity levels of several sugars, including antinutrient raffinose, by more than two-fold. Furthermore, microbial interventions in the tempe soaking step significantly elevated the levels of several bioactive metabolites more than two-fold. Further study seeks to investigate the effect of different lactic acid bacteria species (Limosilactobacillus fermentum and Lactiplantibacillus plantarum) and inoculum sizes (2.5, 5, 10% v/v) applied in tempe soaking on the metabolome of tempe. Tempe samples underwent thorough analysis using a widely targeted GC-MS metabolomics approach. A total of 86 metabolites, including sugars, amino acids, fatty acids, and organic acids, were annotated in all the samples. PCA showed that different LAB led to alterations in the metabolome of tempe with sugars being more abundant in L. fermentum treatment while L. plantarum treatment was dominated by amino acids and others. Additionally, PCA indicated that variations in inoculum size influenced the metabolite profile of tempe, with the metabolites contributing to the separation were from organic acid, amino acid, and sugar groups. The differentiation of lactic acid bacteria species significantly influences the metabolome of tempe, specifically affecting the antinutrient and bioactive compounds. In contrast, variations in inoculum size do not demonstrate a notable impact in terms of isoflavones and biogenic amine reduction. This research highlights the potential of microbial interventions during soybean soaking, benefiting other soy-based fermented foods.