Fermentation Microbiology | Applied Microbiology
Microbiol. Biotechnol. Lett. 2022; 50(4): 522-532
https://doi.org/10.48022/mbl.2208.08003
Jae Ho Choi1, Jiyon Kim2, Taekyun Shin3, Myeong Seon Ryu4, Hee-Jong Yang4, Do-Youn Jeong4, Hong-Seok Son5*, and Tatsuya Unno1,2*
1Subtropical/Tropical Organism Gene Bank, Jeju National University, Jeju 63243, Republic of Korea
2Faculty of Biotechnology, College of Applied Life Sciences, SARI, Jeju National University, Jeju 63243, Republic of Korea
3Department of Veterinary Anatomy, College of Veterinary Medicine and Veterinary Medical Research Institute, Jeju National University, Jeju 63243, Republic of Korea
4Department of Research and Development, Microbial Institute for Fermentation Industry (MIFI), Sunchang 56048, Republic of Korea
5Department of Food Biosciences and Technology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea
Correspondence to :
Hong-Seok Son, sonhs@korea.ac.kr
Tatsuya Unno, tatsu@jejunu.ac.kr
Cheonggukjang is a traditional fermented food in Korea, which is known to exert beneficial effects on health. In this study, we evaluated the effects of cheonggukjang fermented by Bacillus subtilis SCGB 574 (B574) on high fat diet (HFD)-deteriorated large intestinal health. Rats were fed with HFD or HFD supplemented with 10.1% cheonggukjang (B574). Fecal microbiota was analyzed based on 16S rRNA gene sequences, and the fecal and serum metabolome were measured using GC-MS. Our results showed that SCGB574 intake significantly reduced body weight, restored tight junction components, and ameliorated inflammatory cell infiltration. SCGB574 also shifted gut microbiota by increasing the abundance of short chain fatty acid producers such as Alistipes and Flintibacter, although it decreased the abundance of Lactobacillus. Serum and fecal metabolome analyses showed significantly different metabolic profiles between the groups. The top five metabolites increased by SCGB574 were i) arginine biosynthesis, ii) alanine, aspartate, and glutamate metabolism; iii) starch and sucrose metabolism; iv) neomycin, kanamycin, and gentamicin biosynthesis; and v) galactose metabolism. These results showed that cheonggukjang fermented by SCGB574 ameliorates adverse effects of HFD through improving intestinal health.
Keywords: Bacillus subtilis SCGB574, Cheonggukjang, gut microbiota, short chain fatty acids, tight junction
Soybean is a plant that contains various physiologically active substances, such as proteins, oligosaccharides, dietary fibers, isoflavones, saponins, lecithin, and protease inhibitors. It is known for its beneficial effects in various diseases and health aspects, such as weight control, anti-inflammation, and digestive diseases [1]. Soybeans produce short-chain fatty acids (SCFA), such as acetic acid, propionic acid, and butyric acid, which are the main energy sources for gut microbiota, and beneficial bacteria in the intestine [2, 3]. Insoluble dietary fiber, such as cellulose and lignin, promotes digestion and absorption and enhances intestinal peristalsis to improve constipation [4, 5].
Changes in the intestinal environment due to modifications in dietary habits cause changes in the gut microbiota colonies, resulting in the occurrence of inflammatory bowel disease and metabolic diseases such as autoimmune diseases, obesity, and diabetes, which have recently emerged as social problems. In particular, a high-fat diet reduces the binding force of the intestinal epithelial barrier due to imbalances in induction, change, and chronicization of gut microbiota present in the body, and intestinal leaky syndrome (leaky gut) caused by increased infection by harmful gut microbiota, as shown in an animal model. Despite the clinical significance associated with obesity-induced intestinal disease, the role of the intestinal cell binding force (tight junctions) in obese patients is still unclear.
Because of the various effects of soybeans, multiple studies have been conducted on various functional materials such as food, cosmetics, and pharmaceuticals, but there are not many studies on the tight junction function in intestinal health. Fermented soybean is a functional food that exhibits excellent biological activities, such as anti-inflammatory and immunomodulatory functions, and is a rich source of nutrients, phytochemicals, bioactive compounds, and probiotics [6]. Recent research data showed that natto and miso contain large amounts of physiologically active compounds such as nattokinase, bacilopeptidase F, vitamin K2, dipicolinic acid, γ-polyglutamic acid, isoflavones, vanillic acid and syric acid, which suggested health-promoting effects of these fermented bean products [7]. Cheonggukjang contains compounds such as isoflavones, peptides, aglycones, and dietary fiber and is a food rich in polygamma-glutamic acid. The consumption of cheonggukjang improves type 2 diabetes by lowering insulin sensitivity and enhancing insulin secretion in animal models [8]. Cheonggukjang along with doenjang is one of the important fermented foods consumed in Korea and exhibits strong antimutagenic activities against several carcinogens/mutagens such as aflatoxin B1 [9]. In addition, several reports have shown that increased intake of cheonggukjang has anti-obesity and anti-diabetic effects [10, 11], cardiovascular disease prevention and anti-aging effects [12−16].
A previous study showed that treatment with Cheonggukjang fermented by
Ethanol were obtained from Sigma-Aldrich (USA). Nuclease-free water was obtained from Invitrogen (USA). RNAiso Plus reagent was obtained from Takara Korea Biomedical Inc. (Korea). RT kit and oligo dT were obtained from BioFACT Inc., Korea.
Soybeans used in the production of Cheonggukjang were purchased from Sunchang, Jeollabuk-do, and fermented using
SPF 6-week-old male Sprague Dawley (SD) rats were purchased from DBL Co., Ltd. (Korea). The rats were allowed free access to a rodent chow diet (Orientbio, Korea) and tap water. Rats were grown in a conditioned environment at 22 ± 2℃ and 50 ± 5% relative humidity with a 12-h dark/light cycle. The composition and formulation of the 60% high-fat diet (HFD; DooYeol Biotech, Korea) are detailed in Table 1. Cheonggukjang powder comprised approximately 10.1% of the diet, which is an average portion of the previous studies using this cheonggukjang high-fat diet [8, 19]. Rats were randomly divided into the following three groups (n = 8 rats/group): (1) normal diet group (ND), (2) HFD group (HFD), and (3) HFD +
Table 1 . The compositions and formulas of high fat diet.
Class description | Ingredients | HFD | B574 | ||||
---|---|---|---|---|---|---|---|
g/Kg | % | Kcal | g/Kg | % | Kcal | ||
Protein | Casein | 265.0 | 28.4 | 1,060.0 | 224.2 | 23.7 | 896.9 |
Protein | L-Cystine | 4.0 | 0.4 | 16.0 | 4.0 | 0.4 | 16.0 |
Carbohydrate | Maltodextrin | 160.0 | 17.1 | 640.0 | 142.8 | 15.1 | 571.2 |
Carbohydrate | Sucrose | 90.0 | 9.6 | 360.0 | 80.3 | 8.5 | 321.3 |
Fat | Lard | 310.0 | 33.2 | 2,790.0 | 291.4 | 30.8 | 2,623.0 |
Fat | Soybean Oil | 30.0 | 3.2 | 270.0 | 28.2 | 3.0 | 253.8 |
Fiber | Cellulose | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 | 0.0 |
Mineral | Mineral Mix | 48.0 | 5.1 | 0.0 | 48.0 | 5.1 | 0.0 |
Vitamin | Vitamin Mix | 21.0 | 2.2 | 84.0 | 21.0 | 2.2 | 84.0 |
Vitamin | Choline Bitartrate | 3.0 | 0.3 | 0.0 | 3.0 | 0.3 | 0.0 |
Food additive | Calcium Phosphate | 3.4 | 0.4 | 0.0 | 3.4 | 0.4 | 0.0 |
Food additive | B574 | 0.0 | 0.0 | 0.0 | 100 | 10.6 | 453.8 |
Total | 934.4 | 100.0 | 5,220.0 | 946.4 | 100.0 | 5,220.0 |
The tissues of the large intestine were dissected and fixated in 10% neutral buffered formalin, prepared with paraffin, and stained with hematoxylin and eosin (H&E) (Histoire, Korea). Histopathological observation of each section was observed at 100 × magnification under a microscope at the College of Veterinary Medicine, Jeju National University.
The large intestinal total RNA was extracted using RNAiso Plus reagent (Takara Korea Biomedical Inc., Korea) and a spectrophotometer DS-11 plus (Denovix Inc., USA) was used to measure its concentration. Complementary DNA (cDNA) was synthesized from 1 μg of RNA using the BioFACT™ RT Kit (BioFACT Inc., Korea) with oligo dT primers for reverse transcription. Zonula occludens-1 (ZO-1), Claudin-1, Occludin-1, and β-actin genes were targeted for quantitative realtime polymerase chain reaction (q-PCR) using TB Green™ Premix Ex Taq™ (Takara Korea Biomedical Inc.) to measure quantitative gene expression related to cellular tight junctions. The primer sequences used in this study are listed in Table 2. PCR reactions were performed in triplicate with the Thermal Cycler Dice® Real Time System Lite (Takara Bio Inc., Japan).
Table 2 . Primer sequences for qPCR.
Gene | Sequences | NCBI Number |
---|---|---|
ZO-1 | F CTGCCTCGAACCTCTACTC | NM_001106266.1 |
R TAACTTCGTGGGTACTGGTCAA | ||
Claudin-1 | F TGCAGCTTCTGGGTTTCA | NM_031699.3 |
R AAACGCAGGACATCCACA | ||
Occludin-1 | F ATCCTGTCTATGCTCGTCA | NM_031329.3 |
R GTAACCTCCGAAGCCACC | ||
β-actin | F TGGCACCACCATGTACC | NM_031144.3 |
R CCACCAATCCACACAGAGT |
The cecal DNA was extracted from approximately 200 mg of ceca using a PowerFecal DNA extraction kit (Qiagen, Germany). MiSeq library was constructed using the Two-step PCR according to the manufacturers’ instruction. The V3 and V4 regions of the 16S rRNA gene was amplified using the 341F (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3) and 806R (5-GTCTCGTGGGCTCGGAGA TGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3) primer sets. PCR amplicons were sent to Macrogen Inc. (Korea) to perform Illumina sequencing using MiSeq. MOTHUR software was used as described (https://mothur.org/wiki/miseq_sop/). Sequences were aligned against Silva.nr_v138, and the taxonomic classification was performed using RDP trainset version 18. Opti. clust algorithm was applied to assign operational taxonomic units (OTUs) at a sequence dissimilarity of 97%. PICRUSt2 was used to predict metabolic activities. Alpha diversity (Chao and Shannon) was calculated using the MOTHUR software. Non-metric multidimensional scaling (NMDS) was also performed using the MOTHUR software. Taxonomic compositions were visualized in a heatmap using the pheatmap function in R.
Serum and fecal metabolites were extracted by adding 1 ml of protein precipitant (cold methanol, 70% v/v) to 30 μl of serum in 250 μl Eppendorf tubes. The tubes were vortexed and incubated at 37℃ for 30 min, followed by centrifugation at 13,572 g for 5 min at 4℃ to remove the precipitated protein. Quality control (QC) samples were prepared by pooling equal volumes (approximately 10 μl) of each sample prior to derivatization. Ten microliters of ribitol (0.5 mg/l) was added as an internal standard (IS). Finally, the collected supernatant of each sample was concentrated to dryness using an Eppendorf vacuum centrifuge for 3 h at 45℃. After drying, 100 μl of O-methoxyamine hydrochloride in pyridine solution (20mg/ml) was added to each sample. After vortex-mixing each sample for 30 s, all samples were incubated at 30℃ for 90 min in the dark. Silylation was performed by adding 50 μl of N-methyl-N-trimethylsilyl-trifluoroacetamide containing 1% trimethylchlorosilane. Following vortexing each sample for 30 s, the samples were incubated at 37℃ for 30 min. The samples were then centrifuged at 15,928 g for 10 min, and the supernatant was subjected to GC-MS analysis. To measure the performance and stability of the system together with the reproducibility of the sample treatment procedure, QC samples were analyzed every 30 samples throughout the run.
GC-MS (QP2020, Shimadzu, Japan) was used to analyze the derivatized samples. Metabolites were separated using Rtx-5MS with a fused silica capillary column (30 m × 0.25 mm ID, J&W Scientific, USA). The front inlet temperature was set at 230℃. The column temperature was isothermally maintained at 80℃ for 2 min and then raised by 15℃/min to 330℃ then maintained for 6 min. The transfer line was 250℃ and ion source temperature was 200℃. A 70 eV electron beam was used to achieve ionization. The helium gas flow rate was set to 1 ml/min. A mass range of 85−500 m/z was recorded in 20 scans per second. A Shimadzu GC solution (Shimadzu) was used to obtain chromatograms and mass spectra. GC-MS data were extracted from Shimadzu GC-MS Postrun Analysis to netCDF format file and then processed with MetAlign software to detect peaks and alignments [20]. The resulting CSV-format file was imported into AIoutput software to identify and predict peaks [21]. SIMCA-P 15.0 (Umetrics, Sweden) was used to visualize the results of principal component analysis (PCA) and OPLS-DA of GC-MS data. Permutation test was repeated 200 times to cross-validate. Metabolites with VIP > 1.0, and significance set at
Statistical significance was evaluated using the Tukey-Kramer test through one-way analysis of variance (ANOVA), with significance set at
The beneficial effects of soybeans and their modified products have been scientifically shown through studies using various animal models. In previous studies, fermented soybean products attenuated diet-induced body and fat weight in an obese model.
At baseline, no significant differences in body weights were observed. After 10 weeks of HFD intake, the HFD significantly increased body weight. Our results showed that Cheonggukjang supplementation significantly reduced the enhanced body weight gain and decreased fat accumulation compared to the control group (Tables 3 and 4). A recent study reported that Cheonggukjang, 4.5% soybean products fermented by
Table 3 . Effects of Cheonggukjang on the HFD-induced body weight gain in rat.
Group | Initial Body Weight (g) | Final Body Weight (g) | Body Weight Change (g) |
---|---|---|---|
ND | 205.3 ± 5.13 | 256.6 ± 10.27 | 51.3 ± 11.26 |
HFD | 204.1 ± 4.27 | 429.9 ± 12.45 ### | 225.8 ± 13.95 ### |
B574 | 209.9 ± 9.82 | 381.1 ± 7.51 *** | 171.3 ± 16.58 *** |
1Results were indicated as mean ± standard deviation (N = 8). Compared with ND, ###
Table 4 . Effects of Cheonggukjang on the HFD-induced fat weight in rat.
Group | Fat Weight (g) |
---|---|
ND | 5.2 ± 0.42 |
HFD | 27.9 ± 1.57 ### |
B574 | 20.2 ± 1.55 *** |
1Results were indicated as mean ± standard deviation (N = 8). Compared with ND, ###
Consumption of HFD in rodents leads to weight gain, increases adipose tissue weight, and promotes hyperlipidemia and hyperglycemia [22, 23]. In this study, we showed that the weight gain increase was significantly higher in the HFD group than in the ND group. Interestingly, B574 group showed no difference in feed intake compared with the other groups but showed lower weight increase compared to the HFD group. These results are consistent with previous findings, which were known to prevent weight gain from HFD in animals fed fermented soy food.
The intestine not only absorbs essential nutrients, but also protects the host from various ingested toxins and gut microbes [24]. The intestinal barrier system consists of a mucous layer, intestinal epithelial cells, and tight junctions, all of which are susceptible to external factors such as dietary fat intake [25]. When the components of the intestinal barrier system are destroyed, intestinal permeability increases, leading to intestinal diseases such as inflammatory bowel disease, necrotizing enteritis, and celiac disease [26]. Several studies have reported that excessive intake of fat destroys the intestinal epithelial barrier and reduces the tightness of tight junctions. To functionally assess the degree of avidity of the intestinal epithelial barrier associated with obesity, we explored the messenger RNA (mRNA) expression of tight junction markers in the large intestine tissue. The mRNA expression of tight junctional components, including ZO-1, Claudin-1, and Occludin-1, was decreased in the HFD group, while
Mucus is secreted by goblet cells to provide the mucus layer in the normal colon tissue. MUC2 is a gel-forming mucus and a major structural component of the protective mucus layer in the colon tissue [27]. It has been reported that MUC2 expression decreases with the progression of colitis in various animal models [28]. In models of IBD, it often causes loss of the colonic mucus layer and goblet cells. Moreover, it impairs epithelial barrier function by reducing tight junction proteins, including transmembrane barrier protein (occludin) and cytoplasmic scaffolding protein (ZO-1) in the IBD model [29]. These histological changes were improved by ingestion of cheonggukjang.
We also performed histopathological analysis to evaluate HFD-induced destruction of the large intestinal epithelial tissue. We found that HFD intake caused infiltration of inflammatory cells and loss of epithelial cells in the large intestine epithelial tissue. These histopathological changes were improved by supplementation with Chunggukjang (Fig. 3). Our results are consistent with those of previous studies showing that consumption of fermented soybean products improves the integrity of the large intestinal epithelial barrier and infiltration of inflammatory cells. Colonic tissue under normal conditions consists of epithelial layer, mucosal layer, and mucosal matrix. However, inflammatory responses of colonic tissue induce histological changes such as epithelial cell disruption, goblet cell reduction, and inflammatory cell infiltration [30]. Ingestion of DSS depletes goblet cells and increases inflammatory cell infiltration in colon tissues. These histological changes were significantly improved by cheonggukjang treatments. Taken together, we suggest that the consumption of fermented soybean product, cheonggukjang, may confer beneficial effects on large intestinal health.
In this study, we obtained a total of 1,623,405 reads that were rarefied to 10,000 reads per sample for downstream analysis. Among the ND groups, two samples were removed due to sequencing error. Results from Fig. 4 and Table S1 show that the distribution of gut microbiota in all treatment groups were significantly different from each other (
Bacterial composition analysis showed that the effects of diet were clear at the family level, while the effects of Cheonggukjang appeared to be clear at the genus level (Fig. S2). Therefore, a differential abundance test was performed to identify the biomarkers at the genus level. The results in Fig. 5 show 35 genera (17 increased and 18 decreased) and nine genera (four decreased and five increased) by HFD and Cheonggukjang, respectively. Abundance of genera decreased by HFD includes probiotics such as
It should be noted that
PCA was used to determine the intrinsic similarity of the spectral profiles of the GC-MS (Fig. 6). A clear separation among the groups was observed in the score plot from the serum and feces samples, suggesting that metabolites of the serum and feces were altered by HFD and
Metabolite set enrichment analysis (MSEA) was performed to identify the metabolic pathways affected by
From the present study, we conclude that Cheonggukjang fermented by
This research was supported, in part, by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1A6A1A03012862) and the Traditional Culture Convergence Research Program through the NRF funded by the Ministry of Science and Information and Communications Technology (ICT) (NRF-2016M3C1B5907152), Republic of Korea. We are grateful to Sustainable Agriculture Research Institute (SARI) in Jeju National University for providing the experimental facilities.
Conceptualization, J.H.C. and T.U.; methodology, J.H.C., J.Y.K., T.S., H.S., and T.U.; software, H.S. and T.U.; validation, J.H.C., H.S., and T.U.; formal analysis, J.H.C., H.S., and T.U.; investigation, J.H.C., J.Y.K., H.S., and T.U.; resources, M.R.S., H.Y., and D.J.; data curation, J.H.C., H.S., and T.U.; writing-original draft preparation, J.H.C., H.S., and T.U.; writing-review and editing, J.H.C., H.S., and T.U.; visualization, J.H.C., H.S., and T.U.; supervision, T.U.; project administration, T.U.; funding acquisition, T.U. All authors have read and agreed to the published version of the manuscript.
The authors have no financial conflicts of interest to declare.
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