Metabolite Profiling And Anti-Aging Activity Of Rice Koji Fermented With Aspergillus Oryzae And Aspergillus Cristatus: A Comparative Study

Abstract: Rice koji, used as a starter for maximizing fermentation benefits, produces versatile end products depending on the inoculum microbes used. Here, we performed metabolite profiling to compare rice koji fermented with two important filamentous fungi, Aspergillus oryzae and A. cristatus, for 8 days. The multivariate analyses showed distinct patterns of primary and secondary metabolites in the two koji. The rice koji fermented with A. oryzae (RAO) showed increased α-glucosidase activity and higher contents of sugar derivatives than the one fermented with A. cristatus (RAC). RAC showed enhanced β-glucosidase activity and increased contents of flavonoids and lysophospholipids, compared to RAO. Overall, at the final fermentation stage (8 days), the antioxidant activities and anti-aging effects were higher in RAC than in RAO, corresponding to the increased metabolites such as flavonoids and auroglaucin derivatives in RAC. This comparative metabolomic approach can be applied in production optimization and quality control analyses of koji products.

Keywords: rice koji; microbe; solid-state fermentation; anti-aging effect; antioxidant activity

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1. Introduction

Fermentation, which has a history of thousands of years, is increasingly being recognized as a method of enhancing the nutrition and bioactivities of food products, in addition to processing and preserving them [1]. Rice koji is made by solid-state fermentation using steamed rice grains inoculated with microorganisms to secrete enzymes and produces beneficial metabolites. In recent years, various attempts to create delicate fermentation conditions have led to advanced fermentation efficacy and better food palatability [2,3]. Due to its advantages, rice koji finds applications in industrial fields such as fermented foods and beverages and cosmetics [4–6]. 

Reactive oxygen species (ROS) are generated under conditions of oxidative stress and are by-products of aerobic metabolism. These free radicals can induce the degradation of biomolecules, resulting in oxidative damages, such as inflflammation and acceleration of the skin aging process [7]. To develop a balance between ROS production and elimination, ROS scavengers, known as antioxidants, play an important role in alleviating oxidative stress and are mainly obtained from natural sources [8]. These free radicals are involved in the aging process, and scavenging them through the intake of antioxidants from natural sources is crucial in delaying aging [9]. In recent years, many studies have reported that rice koji can enhance the potential antioxidant activities of raw materials by improvement of the fermentation substrate [10,11].

The skin’s extracellular matrix (ECM) consists of collagen and elastin fibers, which promote the elasticity of the skin to restore and maintain its original shape and state [12]. The destruction of the dermal ECM is an indicator of aging. It occurs due to the upregulation of collagen-degrading matrix metalloproteinase-1 (MMP-1), also known as collagenase. Therefore, studies of various phytochemicals that can slow down the skin aging process by stimulating collagen and elastin synthesis and inhibiting MMP-1 are increasing [13–16]. Seo et al. showed that fermented rice bran affects skin fibroblast collagen, inflflammatory factor (IL-a), and MMP-1 [17]. Hence, various compounds found in rice, such as flavonoids and phenolic acids have antioxidant activity, and fermented rice koji has the potential to ameliorate skin photoaging by UV radiation [18]. Aspergillus, a fifilamentous fungus, is a typical inoculum microbe for producing many beneficial metabolites such as simple sugars, fatty acids, and amino acids from koji in Asia. In particular, Aspergillus oryzae is the most common microorganism used in the production of koji because of its ensured safety and various enzymes, such as amylase, protease, and peptidase [19].

Aspergillus cristatus is used in tea fermentation, such as Fuzhuan brick tea, which has probiotics and protects against UVB-induced photoaging [20,21]. It has also been reported to enhance the antioxidant activity of various other raw materials [22,23]. Currently, efforts are increasingly being dedicated to improving the quality of fermentation starters [4,24]. Previous studies have shown a comparative metabolic study of Aspergillus and Bacillus, widely used in rice koji [25]. However, there is a scarcity of information about the metabolomic differences between the same genera but different species of fungi. To select optimal microbes that can be introduced in the market for health with nutraceutical and cosmeceutical applications, there is a need for a comprehensive understanding of the metabolism of different inoculum microbes by comparing their bioactivity and metabolites. 

In this study, we profiled the metabolites of rice koji fermented with different Aspergillus spp. (A. cristatus and A. oryzae) in terms of metabolomics to compare the metabolism of the two fifilamentous fungi. We also measured enzyme activity, antioxidant activity, and RNA expression of skin anti-aging factors (collagen, elastin, and MMP-1) to compare the two koji. Furthermore, we conducted correlation analysis to suggest potential candidate metabolites that contribute to antioxidant activity and skin anti-aging effects. A comprehensive analysis of MS-based metabolite profiling for comparing the two koji inoculums established a relationship between enzyme activities, metabolomes, and bioactivities. Here, we present a blueprint of the overall metabolic state, correlated with the bioactivities of the two different koji inoculums.

2. Results

2.1. Metabolic Profiling for Rice Koji Fermented with Different Aspergillus spp. 

Different metabolomes of rice koji samples inoculated with A. cristatus or A. oryzae were compared using multivariate analysis according to the GC–MS and LC–MS data sets. The principal component analysis (PCA) score plot obtained from the UHPLC–LTQ–OrbitrapMS/MS and GC–TOF–MS revealed a total variance of 40.9% (PC1, 22.01%; PC2, 18.89%) and 52.88% (PC1, 34.70%; PC2, 18.18%), respectively (Figure 1A,B). Both PCA results indicated that the starting point of the fermentation was assembled, but consequently distinguished by different inoculation fungi according to different fermentation times. Partial least squares discriminant analysis (PLS-DA) elucidated statistical patterns same to the metabolite distribution in PCA (Supplementary Figure S1A,B). 

As shown in the PCA obtained from the UHPLC–LTQ–Orbitrap–MS/MS analyses (Figure 1A), there are signifificant differences in the eighth day, and both eight-day samples were subjected to an orthogonal partial least square discriminant analysis (OPLS-DA), which showed a clear separation by OPLS component 1, accounting for 86.11% of the variance in data (Supplementary Figure S1C). The 31 metabolites were selected from UHPLC–LTQ–Orbitrap–MS/MS data, which is considered a major contributor to the discrepancy in eighth-day rice kojis fermented with two different inoculum microbes based on their variable importance in projection values (VIP > 1.0) and p-values (p < 0.05) from OPLS-DA analysis (Supplementary Table S1). These metabolites included 2 carboxylic acids, 5 phenolic acids, 7 flavonoids, 2 long-chain fatty acids, 11 lysophospholipids, and anc4 hydroquinones. The metabolites were tentatively identified by comparing published literature (molecular weight, molecular formula, retention time, mass fragment patterns, and UV absorbances) and data from an in-house library.

Figure 1. Principal component analysis (PCA) score plot from the (A) UHPLC-LTO-Orbitrap-MS/MS and (B) GC-TOF-MSdata sets of rice koi fermented with Aspergillus cristatus or A. oryzne.(filled symbols, A. cristatus; unfilled symbols, A. oryzne, O, 0 day;  , , 2 day; V, V, 4 day;  6 day;  , 8 day).

2.1.1. Temporal Metabolomes for Rice Koji with Different Aspergillus spp. InoculationAccording to Fermentation Time

The metabolic pathways of rice koji dependent on different inoculation microbes were represented by a heat map to visualize metabolite change patterns in accordance with fermentation times (Figure 2). The color on a blue-to-red gradient represents the mean normalized relative abundance of each metabolite under each experimental condition. The trends of most metabolites in rice koji fermented with A. cristatus (RAC) and A. oryzaeRAO) showed a gradually increasing pattern with fermentation time. The metabolites associated with carbohydrate metabolism mostly represented an increasing pattern except for glucose, xylose, sucrose, and maltose, which are sugars. In addition, phenolic acid flavonoids, and hydroquinone contents were enhanced with fermentation time, except for ferulic acid. Among the fatty acids, most metabolites showed an increasing pattern while pimelic acid showed a decrease. Lysophospholipids presented disparate patterns with different fermentation times and inoculation fungi.

Figure 2. Scheme of the metabolic pathway and relative levels of metabolites in rice koji fermented with Aspergillus cristatus or A. oryzae. The pathway was adapted from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and modified. The colored squares represent the fold changes (blue to red) normalized by the average of all values for each metabolite. 

2.1.2. Relative Disparity in the Level of Discriminant Metabolites in Rice Koji Fermented byA. cristatus or A. oryzae

As shown in Figure 2, the contents of primary and secondary metabolites exhibited different patterns in accordance with different inoculation fungi. In the case of glucose which is the center of carbohydrate metabolism, the patterns of A. cristatus koji showed a decrease, whereas A. one koji showed decreasing patterns at the initial fermentation point but gradually increased until the final fermentation point. Furthermore, the sugar alcohols were higher in RAO than in RAC. Particularly, auroglaucin derivatives were enhanced significantly only in RAC because they are a unique pigment compound produced by A. cristatus. In addition, most flavonoids were increased significantly in RAC compared to RAO, except for 3,8-dimethylherbacetin. Among the phenolic acids, ferulic acid and benzoic acid were increased in both samples, but dihydroxybenzoic acid, caffeoylquinic acid and vanillic acid were increased only in RAC. Lysophospholipids increased in RAC, but contrasting tendency was observed in RAO. Fatty acids showed greater patterns of increased RAO than in AC

2.2. Comparison of Enzymatic Production and Bioactivity in Rice Koji Fermented with DifferentMicroorganisms

To compare the phenotypes of RAC and RAO, we evaluated the enzyme activity and anti-aging effects on skin cells, antioxidant activity, total flavonoid contents (TFC), and total phenolic contents (IPC) (Figure 3). Enzyme production of both the koji increased with fermentation time, except for a-amylase in RAO. Interestingly, the a-glucosidase-content was twice higher in RAO than in RAC with 10.12 and 3.52 units respectively; in contrast, B-glucosidase content was forth higher in RAC than in RAO with 19.05 units and5.49 unit respectively in accordance with fermentation times. The functional phenotype of both the koji (antioxidant activity and skin anti-aging factor) indicated that the rice koji with A. cristatus had higher antioxidant activities in ABTS, DPPH, and FRAP at the final fermentation time (8 days) with 1.05, 0.40, 0.66 TEAC (Trolox equivalent antioxidant capacity) respectively. Additionally, the content of flavonoid was higher in RAC than RAO with 0.07 NE (naringin equivalent) and 0.01 NE respectively. Whereas the content of total phenol was higher in RAO than RAC with 0.32 EGA (equivalent gallic acid) and 0.28 EGArespectively. The results of skin anti-aging factors (elastin, collagen, and MMP-1) indicated hat at the termination of the fermentation. ACRNA expression level with 7.77 and 13.76 and lower relative MMP-1 RNA expression level with 2.35 compared to B-actin. Meanwhile, RAO showed a gradual increase in RNA expression of elastin and collagen following fermentation.

Figure 3. Comparison of enzyme production (A), skin ant-aging factor (B) and antioxidant activity, total flavonoid contents), and total phenolic contents (IPC) (C) in rice koji fermented with different Aspergillus spp. (black color, A. cristatus white color, A. oryzne). The enzymatic activities are a-amylase activity, B-glucosidase activity, and a-glucosidase activity(A). The relative mRNA expression level is measured for the following: collagen (COL1A1), elastin (ELN), and matrix metalloproteinase-1 (MMP-1) (B). The antioxidant activities depicted are ABTS, DPPH radical scavenging, FRAP, totalMavonoid content, and total phenolic content (C). Significant differences between different inoculation microbes were identified by t-test (* p < 0.05, ** p < 0.01).

To determine the metabolites that potentially contributed to bioactivity, a correlation analysis between fermented koji metabolites and bioactivities was conducted (Supplement.tary Figure S2). Overall, Pearson's correlation coefficient map showed the RAC higher correlation with bioactivities than RAO. In RAC, organic acids, flavonoids, lysophospholipids, fatty acids, hydroquinone, sugar derivatives showed a high positive correlation with bioactivities. For RAO, organic acids, flavonoids and fatty acids, and sugar derivatives indicated a positive correlation with bioactivities. The metabolites that had a Pearson correlation coefficient value higher than 0.5 are represented in a network map (Figure 4)In both the koji products, organic acids, fatty acids, flavonoids, and sugar derivatives were potential contributors of bioactivities. RNA expression of elastin was associated with metabolites of RAC, whereas RNA expression of collagen was associated with metabolites of RAO. In addition, TFC showed a correlation with RAC. Furthermore, lysophospholipids and hydroquinone were strong antioxidant activity contributors to RAC

Figure 4, The metabolites that have a Pearson's correlation coefficient value higher than 0.5 are represented by a network map in rice koji fermented with (A) Aspergillus cristatus or (B) A. oryzne. The box symbols represent bioactivities (gray color, antioxidant activity TPC and TFC; black color, skin anti-aging effect on cell) and the colored symbols indicate the metabolites (same series were distinguished by different color and shape: o, hydroquinone: , organic acids: , fatty acids, flavonoids;, lysophospholipids; o, sugar and sugar derivatives; unknown).

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