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

3. Discussion

Different parts of rice, such as husk, bran, embryo, and endosperm, from the surface to the interior, have different chemical compositions (26). In particular, rice bran contains various phenolic acids and flavonoids, which are known to exhibit antioxidant activity. In addition, the rice cell wall is composed of an arabinoxylan structure that includes xylose, arabinose, ferulic acid, and ferulic acid (27]. The rice cell wall is generally hard to penetrate, and rice koji offers the advantage of easy penetration of the rice cell wall by various enzymes such as protease and glucosidase from inoculum microbes (24. Therefore rice koii shows higher levels of tyrosinase inhibitory activity and antioxidant activities than its raw materials because it contains valuable enriched compounds (28.

Click Here To Get More Info About Cistanche Antioxidant Activity

We followed the metabolomics approach for rice koji fermented with two different filamentous fungi, which elucidated significant differences in enzyme activity, production of metabolites, and bioactivities. Activities of various enzymes such as a-amylase, &glucosidase, and -glucosidase produced by the inoculated A. cristatus and A. oryzae increased with fermentation time (Figure 3). Because these enzymes break down the arabinoxylan structure, diverse phenolic acids were separated from the rice cell wall in both samples, as shown in Figure 2. These phenolic acids are potential antioxidants that alleviate oxidative stress 291. Thus, antioxidant activities and TPC assay increased with increasing fermentation time as the phenolic acid content was enhanced (Figures 2 and 4)In particular, RAC has a higher content of flavonoids than RAO because it has a higher level of -glucosidase, which hydrolyzes the -glycosidic linkage from the rice cell wall during the growth of A. cristatus in rice koji. In addition to detaching from the rice cell wall-glucosidase hydrolyzes the flavonoid glucoside form to an aglycon form that possesses higher antioxidant activity 30]. The increased flavonoid glucoside form and aglycon form increase antioxidant activities such as ABTS, DPPH FRAP, and TFC, which may affect the antioxidant activity of RAC, as shown in the correlation network map (Figure 4). This phenomenon was also observed in a previous study that showed biotransformation of glucoside isoflavones to aglycones and increasing patterns in antioxidant activity according to mentation time in soybean fermented with. cristatus [311.

RAO has a higher level of a-glucosidase activity that cleaves a-glycosidic linkages and autogenerates higher content of glucose. Besides the fact that glucose is the main carbon source for fungus, in RAC, the glucose level decreased following fermentation because itwas used for the synthesis of secondary metabolites such as auroglaucin derivatives, which are distinctive pigment compounds produced by A. cristatus, and not A. oryzne. Previous studies have reported that auroglaucin derivatives have activity in DPPH and are assumed as the potential antioxidant compounds [32]. Furthermore, the collapsed rice cell wall could allow enzymes to penetrate into the innermost parts of the rice [24]. Hence, more and more metabolites could be extracted freely without interruption from the rice outer wall.

In the correlation network map between bioactivities and metabolites of both RAO and RAC (Figure 4), the common tendencies were that flavonoids, organic acids, sugar derivatives, and fatty acids were suggested as potential contributors to bioactivities. Flavonoids and phenolic acids are renowned antioxidants and have many advantages with respect to various functions. Due to their ability to alleviate oxidative stress, they are used to enhance food quality and ameliorate skin aging [33]. Additionally, a previous study has reported that fatty acids and antioxidants could create a synergistic effect for the prevention and management of skin aging [34].

On the other hand, auroglaucin and lysophospholipid derivatives serve as additional contributors to metabolites in RAC [35]. The auroglaucin derivatives have antioxidant activities, as stated above, and therefore, we assume that they have the potential to terminate free radical chain reactions to relieve skin stresses. Yahagi et al. demonstrated that lysophospholipids could maintain skin moisturization by enhancing the expression of factors associated with the skin barrier and hydration functions in the skin [36]. Moisturization is a vital factor for healthy skin because dryness induces skin impairment that is characterized by roughness, scaly skin and fine wrinkles [37,38]. We estimate that auroglaucin and lysophospholipids have better skin anti-aging effects at the final fermentation stage in RAC than in RAO. Zhao et al. demonstrated that Fuzhuan brick tea, which contains the dominant fungus A. cristatus, can inhibit photoaging via quenching of ROS and triggering of Nrf2 signaling cascades [21]. Therefore, we assume that RAC offers a higher anti-aging potential than RAO by acting through indirect routes such as establishing better skin conditions for abundant moisture and relieving free radical stress.

Overall, we believe that the enhanced fatty acids, phenolic acids, flavonoids, lysophospholipids, and hydroquinones may increase antioxidant activities and improve RNA expression of elastin and collagen, as well as suppress RNA expression of MMP-1, at the end of fermentation. These compounds exhibited different patterns of change in metabolites according to the inoculum fungus and affected various bioactivities. This study elucidated the difference in overall metabolism between different species of the same Aspergillus genus by using a metabolomics approach. In addition, different enzyme activities influenced the production of different metabolites and induced different bioactivities in RAO and RAC

4. Materials and Methods

4.1. Chemicals and Reagents

4.2. Sample Preparation and Extraction

The koji molds A. oryzae KCCM 11372 (Korean Culture Center of Microorganism,KCCM; Republic of Korea) and A. cristatus (Aspergillus cristatus Cosmax-GF from Cosmax BTI R&I center; Seongnam, Korea) were used for fermentation of rice and separately inoculated. Each microorganism was maintained on malt extract agar (malt extract, 20 g; glucose, 20 g; peptone, 1 g; agar, 20 g/L) at 28 ◦C. The bioprocess of fermentation steps for koji production was adapted from Lee et al. [11]. The rice koji samples fermented with A. oryzae and A. cristatus were harvested every 2 days (from day 0 to day 8) and stored at deep freezing conditions (−80 ◦C) until further analyses. All samples were prepared with two biological replicates.

The method of extraction of rice koji sample was adapted from Lee et al. with slight modifications [11]. Briefly, the pulverized freeze-dried rice koji samples (5 g) were extracted by adding 80% aqueous ethanol (40 mL) and agitating on an orbital shaker (200 rpm for 24 h) at room temperature. After centrifugation of the samples at 10,000 rpm for 5 min at 4 ◦C, the supernatants were filtered with a 0.22 µm Millex GP filter (Merck Millipore, Billerica, MA, USA). The filtered sample extracts were dried using a speed vacuum concentrator (Hanil, Seoul, Korea) and the dry weight was measured to evaluate the extraction yield. 

4.3. GC–TOF–MS Analysis 

The derivatization steps of extracted rice koji samples were as described by Lee et al. [11]. GC–TOF–MS analysis was conducted on an Agilent 7890A GC system (Santa Clara, CA, USA) with a Pegasus HT TOF-MS (Leco Corporation, St. Joseph, MI, USA). The carrier gas (helium) was used with an RTx-5MS (30 m length × 0.25 mm inner diameter, J&W Scientific, Folsom, CA, USA) at a constant flow rate of 1.5 mL/min. The temperatures of the injector and ion source were maintained at 250 and 230 ◦C, respectively. The oven temperature was maintained at 75 ◦C for 2 min and then increased to 300 ◦C at 15 ◦C/min, which was sustained for 3 min. Then, 1 µL of the sample was injected with a mass scan range of m/z 50–800. All sample analyses were performed with three analytical replicates. 

4.4. UHPLC–LTQ–Orbitrap–MS Analysis

The extracted rice koji samples were analyzed for secondary metabolites using ultrahighperformance liquid chromatography linear trap quadrupole orbitrap tandem mass spectrometry (UHPLC–LTQ–Orbitrap–MS/MS) using the protocols described by Kwon et al. [39]. Each sample was separated using a Phenomenex KINETEX® C18 column (100 mm 2.1 mm, 1.7 m particle size; Torrance, CA, USA). The mass spectra and photodiode array range in both positive and negative ion modes were tuned for m/z 100−1000 and 200−600 nm, respectively. 

4.5. Data Processing and Statistical Analysis

The raw GC–TOF–MS and UHPLC–LTQ–Orbitrap–MS/MS data were transformed to netCDF (*.cdf) format using Leco ChromaTOF and Thermo Xcalibur software, respectively. The respective net CDF (*.cdf) files were subjected to MetAlign (accessed on 13 July 2021)) software-mediated data processing using the protocols described by Lee et al. [11,24]. The mass spectrometric data, which represent the suitable peak mass (m/z), retention times (min), and peak area information as variables, were evaluated using SIMCA-P+ 12.0 software (Umetrics, Umea, Sweden) for multivariate statistical analysis. Before principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least square discriminant analysis (OPLS-DA), the data sets were log-transformed, and unit variance was scaled to compare the rice koji fermented with different fungi. PASW Statistics 18 (SPSS, Inc., Chicago, IL, USA) was used to test for signifificant differences (p-value of < 0.05) by one-way analysis of variance and to calculate the correlation coefficient values for a correlation map. The correlation network map between metabolites that have a Pearson’s correlation coefficient value higher than 0.5 and bioactivities was constructed with the Cytoscape software (https://www.cytoscape.org/ (accessed on 13 July 2021)). The identifification of tentative metabolites was carried out by matching the molecular weights and molecular composition, retention time, mass fragment 

patterns, and absorbance of ultraviolet (UV) data from the literature and our in-house library

4.6. Determination of Enzymatic Activities 

Enzymatic activity assays for α-amylase, β-glucosidase, and α-glucosidase were performed according to previous studies [25,40,41]. A 10 g quantity of each rice koji sample was extracted in 90 mL of water by shaking on an orbital shaker at 120 pm and 25 ◦C for 1 h. After filtering the samples, the supernatants were used to evaluate enzyme activities. 

4.7. Determination of Antioxidant Activities and Total Phenolic and Flavonoid Contents

To determine the antioxidant activity of the rice koji samples, ABTS, DPPH, ferric reducing antioxidant power (FRAP), total phenolic contents (TPC), and total flavonoid contents (TFC) assays were conducted in triplicate 

The ABTS and FRAP assays were performed using the method described by Lee et al. [24]. In brief, the ABTS stock solution diluted with distilled water to achieve a final absorbance of 0.7 ± 0.02 at 750 nm (180 µL) was added to each sample extract (20 µL) in a 96-well plate. The reaction was allowed to take place for 6 min in the dark at room temperature. The absorbance was measured at 750 nm using a spectrophotometer. For the FRAP assay, a mixture of 300 mM acetate buffer (pH 3.6), 20 mM iron (III) chloride, and 10 mM 2,4,6-tripyridyl-S-triazine (TPTZ) solution in 40 mM HCl (10:1:1, v/v/v) were prepared. The sample (10 µL) was mixed with 300 µL of FRAP reagent and incubated at room temperature for 6 min. The absorbance was measured at 570 nm. The DPPH assay was carried out following the method adapted from Won et al. [42], where 180 µL of the DPPH stock solution (0.2 mM in ethanol) was mixed with 20 µL of the rice koji with two different fungal extracts in 96-well plates and allowed to react for 20 min at room temperature in the dark. The free radical absorbance by DPPH was measured at 515 nm. The results of ABTS, FRAP, and DPPH are represented as the Trolox equivalent antioxidant capacity (TEAC) concentration (mM) per milligram of koji. The standard concentration curves ranged from 0.0078 mM to 1 mM TEAC.

For the TFC and TPC assays, a method used by Lee et al. [25] was followed. For the TFC assay, 20 µL of each rice koji sample was mixed with 20 µL of 1 N NaOH and 180 µL of 90% diethylene glycol in a 96-well plate. After incubation of the mixture for 60 min at room temperature, the absorbance was measured at 405 nm. TFC is presented as the naringin equivalent (NE) concentration (mM) per milligram of koji. The standard concentration curve was linear between 0.0027 and 0.3445 mM NE. For the analysis of the TPC assay, 20 µL of each sample was incubated with 100 µL of 0.2 N Folin–Ciocalteu reagent in 96-well plates at room temperature for 6 min. Then, 80 µL of 7.5% sodium carbonate (Na2CO3) solution was added to the mixture and allowed to react for 60 min at room temperature. Finally, the absorbance was evaluated at 750 nm. The results are indicated as gallic acid equivalent (GE) concentrations (mM) per milligram of koji in a standard concentration range of 0.0230–2.9391 mM GE.

4.8. Cell Cultures

4.9. Real-Time Polymerase Chain Reaction

To isolate and quantify the total RNA from the cell pellets, Trizol reagent was used, and the analysis was done using a spectrophotometer. The synthesis of cDNA was carried out in a total reaction volume of 20 µL; the reaction mixture consisted of 2 µg of total RNA, oligo (dT), and reverse transcription premix under the following reaction conditions: 45 ◦C for 45 min, followed by 95 ◦C for 5 min. RT-PCR was used for the quantification of gene expression, and the results were subsequently analyzed using the StepOne PlusTM system software (Applied Biosystems, Foster City, CA, USA). RT-PCR amplifications were conducted using SYBR Green PCR Master Mix with premixed ROX (Applied Biosystems, Foster City, CA, USA) and primers (Bioneer, Daejeon, Korea) in an ABI 7300 instrument following the manufacturer’s protocol. The reaction conditions were as follows: initiation at 95 ◦C for 10 min, followed by cycling conditions of 95 ◦C for 15 s, 60 ◦C for 30 s, and 72 ◦C for 30 s for 40 cycles. β-actin was used as an internal control. 

In conclusion, rice koji showed the production of different metabolites and bioactivities according to different Aspergillus species used. The higher levels of flavonoids and auroglaucin derivatives in RAC resulted in higher antioxidant activity than in RAO. In addition, the synergistic effects of fatty acid and antioxidant compounds found in both the koji were associated with the RNA expression of the skin anti-aging factor. Auroglaucin derivatives and lysophospholipids found in RAC were also candidates that could be associated with RNA expression of skin anti-aging factors. Therefore, even though rice koji is fermented using members of the same genus (Aspergillus), there are signifificant differences in enzyme activities and metabolites for the different species, and they affect bioactivities such as antioxidant and anti-aging activities. Hence, this study provides comprehensive insight, as well as logic for a rational choice of inoculation microbes, with respect to

metabolomics, to improve the quality of commercial production of koji.

Supplementary Materials: The following are available online at, Figure S1: The PLS-DA score plot (A,B) and OPLS-DA score plot (C) for rice koji fermented with Aspergillus cristatus or A. oryzae were obtained from UHPLC–LTQ–OrbitrapMS/MS (A,C) and GC–TOF–MS (B)., Table S1: List of signifificantly distinct metabolites from rice koji with different Aspergillus spp. during fermentation identified by UHPLC–LTQ–Orbitrap-MS/MS, Table S2: List of signifificantly distinct metabolites from rice koji with different Aspergillus spp. during fermentation identified by GC-TOF-MS, Figure S2: Correlation map of bioactivities (skin cell effect and antioxidant activity) and rice koji fermented with Aspergillus cristatus or A. oryzae metabolites according to Pearson’s correlation coefficient. Each square indicates Pearson’s correlation coefficient values (r).

Author Contributions: Conceptualization, C.L. and S.L.; methodology, H.L., S.L., S.K. (Seoyeon Kyung) and J.R.; validation, H.L., S.L. and S.K. (Seoyeon Kyung); formal analysis, H.L. and S.K. (Seoyeon Kyung); investigation, H.L. and S.L.; resources, J.R., S.K. (Seunghyun Kang) and M.P.; writing—original draft preparation, H.L.; writing—review and editing, H.L. and S.L.; visualization, H.L.; supervision, S.L. and C.L.; project administration, S.K. (Seunghyun Kang), M.P., and C.L.; funding acquisition, C.L. All authors have read and agreed to the published version of the manuscript. 

Funding: This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry(IPET) through the Agricultural Microbiome R&D Program(The Strategic Initiative for Microbiomes in Agriculture and Food), funded by the Ministry of Agriculture, Food and Rural Affairs(MAFRA) (Grant number 918011-04-3-HD020). Additionally, this work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry(IPET) through the High Value-added Food Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs(MAFRA) (grant number 318027-04-3-HD030)

Data Availability Statement: The data presented in this study are available on request from the corresponding author

Acknowledgments: This research was supported by the Konkuk University Researcher Fund in 2020.

References 

1. Sanlier, N.; Gokcen, B.B.; Sezgin, A.C. Health benefits of fermented foods. Crit. Rev. Food Sci. Nutr. 2019, 59, 506–527. [CrossRef] 

2. Yu, K.-W.; Lee, S.-E.; Choi, H.-S.; Suh, H.J.; Ra, K.S.; Choi, J.W.; Hwang, J.-H. Optimization for rice koji preparation using Aspergillus oryzae CJCM-4 isolated from a Korean traditional meju. Food Sci. Biotechnol. 2012, 21, 129–135. [CrossRef]

3. Yang, Y.; Xia, Y.; Lin, X.; Wang, G.; Zhang, H.; Xiong, Z.; Yu, H.; Yu, J.; Ai, L. Improvement of flavor profiles in Chinese rice wine by creating fermenting yeast with superior ethanol tolerance and fermentation activity. Food Res. Int. 2018, 108, 83–92. [CrossRef] [PubMed] 

4. Ichikawa, E.; Hirata, S.; Hata, Y.; Yazawa, H.; Tamura, H.; Kaneoke, M.; Iwashita, K.; Hirata, D. Effect of koji starter on metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei. Biosci. Biotechnol. Biochem. 2020, 84, 1714–1723. [CrossRef] 

5. Phetpornpaisan, P.; Tippayawat, P.; Jay, M.; Sutthanut, K. A local Thai cultivar glutinous black rice bran: A source of functional compounds in immunomodulation, cell viability and collagen synthesis, and matrix metalloproteinase-2 and-9 inhibition. J. Funct. Foods 2014, 7, 650–661. [CrossRef] 

6. Kim, A.J.; Choi, J.N.; Kim, J.; Kim, H.Y.; Park, S.B.; Yeo, S.H.; Choi, J.H.; Liu, K.H.; Lee, C.H. Metabolite profiling and bioactivity of rice koji fermented by Aspergillus strains. J. Microbiol. Biotechnol. 2012, 22, 100–106. [CrossRef] [PubMed] 

7. Ames, B.N.; Shigenaga, M.K.; Hagen, T.M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. USA 1993, 90, 7915–7922. [CrossRef] [PubMed]

8. Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M.T.; Mazur, M.; Telser, J. Free radicals, and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 2007, 39, 44–84. [CrossRef] 

9. Uttara, B.; Singh, A.V.; Zamboni, P.; Mahajan, R.T. Oxidative stress and neurodegenerative diseases: A review of upstream and downstream antioxidant therapeutic options. Curr. Neuropharmacol. 2009, 7, 65–74. [CrossRef] 

10. Yen, G.-C.; Chang, Y.-C.; Su, S.-W. Antioxidant activity and active compounds of rice koji fermented with Aspergillus candidus. Food Chem. 2003, 83, 49–54. [CrossRef]

11. Lee, D.E.; Lee, S.; Singh, D.; Jang, E.S.; Shin, H.W.; Moon, B.S.; Lee, C.H. Time-resolved comparative metabolomes for Koji fermentation with brown-, white-, and giant embryo-rice. Food Chem. 2017, 231, 258–266. [CrossRef] [PubMed] 

12. Jarrar, M.; Behl, S.; Shaheen, N.; Fatima, A.; Nasab, R. Anti-Aging Effects of Retinol and Alpha Hydroxy Acid on Elastin Fibers of Artificially Photo-Aged Human Dermal Fibroblast Cell Lines. Int. J. Med Health Biomed. Pharm. Eng. 2015, 7, 328.

13. Bolla, S.R.; Al-Subaie, A.M.; Al-Jindan, R.Y.; Balakrishna, J.P.; Ravi, P.K.; Veeraraghavan, V.P.; Pillai, A.A.; Gollapalli, S.S.R.; Joseph, J.P.; Surapaneni, K.M. In vitro wound healing potency of methanolic leaf extract of Aristolochia saccata is possibly mediated by its stimulatory effect on collagen-1 expression. Heliyon 2019, 5, e01648. [CrossRef] [PubMed]

14. Meinke, M.C.; Nowbary, C.K.; Schanzer, S.; Vollert, H.; Lademann, J.; Darvin, M.E. Inflfluences of Orally Taken Carotenoid-Rich Curly Kale Extract on Collagen I/Elastin Index of the Skin. Nutrients 2017, 9, 775. [CrossRef] [PubMed] 

15. Majeed, M.; Bhat, B.; Anand, S.; Sivakumar, A.; Paliwal, P.; Geetha, K.G. Inhibition of UV-induced ROS and collagen damage by Phyllanthus emblica extract in normal human dermal fibroblasts. J. Cosmet. Sci. 2011, 62, 49–56. [PubMed] 

16. Masuda, M.; Murata, K.; Naruto, S.; Uwaya, A.; Isami, F.; Matsuda, H. Matrix metalloproteinase-1 inhibitory activities of Morinda citrifolia seed extract and its constituents in UVA-irradiated human dermal fibroblasts. Biol. Pharm. Bull. 2012, 35, 210–215. [CrossRef] [PubMed] 

17. Seo, Y.-K.; Jung, S.-H.; Song, K.-Y.; Park, J.-K.; Park, C.-S. Anti-photoaging effect of fermented rice bran extract on UV-induced normal skin fibroblasts. Eur. Food Res. Technol. 2010, 231, 163–169. [CrossRef] 

18. Goufo, P.; Trindade, H. Rice antioxidants: Phenolic acids, flavonoids, anthocyanins, proanthocyanidins, tocopherols, tocotrienols, γ-oryzanol, and phytic acid. Food Sci. Nutr. 2014, 2, 75–104. [CrossRef] 

19. Bechman, A.; Phillips, R.D.; Chen, J. Changes in selected physical property and enzyme activity of rice and barley koji during fermentation and storage. J. Food. Sci. 2012, 77, M318–M322. [CrossRef] 

20. Kang, D.; Su, M.; Duan, Y.; Huang, Y. Eurotium cristatum, a potential probiotic fungus from Fuzhuan brick tea, alleviated obesity in mice by modulating gut microbiota. Food Funct. 2019, 10, 5032–5045. [CrossRef]

21. Zhao, P.; Alam, M.B.; Lee, S.H. Protection of UVB-Induced Photoaging by Fuzhuan-Brick Tea Aqueous Extract via MAPKs/Nrf2- Mediated Down-Regulation of MMP-1. Nutrients 2018, 11, 60. [CrossRef] [PubMed]

22. Hur, S.J.; Lee, S.Y.; Kim, Y.C.; Choi, I.; Kim, G.B. Effect of fermentation on the antioxidant activity in plant-based foods. Food Chem. 2014, 160, 346–356. [CrossRef] [PubMed] 

23. Zhou, S.-D.; Xu, X.; Lin, Y.-F.; Xia, H.-Y.; Huang, L.; Dong, M.-S. Online screening and identifification of free radical scavenging compounds in Angelica dahurica fermented with Eurotium cristatum using an HPLC-PDA-Triple-TOF-MS/MS-ABTS system. Food Chem. 2019, 272, 670–678. [CrossRef] [PubMed] 

24. Lee, S.; Lee, D.E.; Singh, D.; Lee, C.H. Metabolomics Reveal Optimal Grain Preprocessing (Milling) toward Rice Koji Fermentation. J. Agric. Food Chem. 2018, 66, 2694–2703. [CrossRef]

Ask for more:

Email:wallence.suen@wecistanche.com whatsapp:+86 15292862950