Changes in Microbiota and Short-Chain Fatty Acids Following 3-Month Pilot Intervention Study Feeding Brown Rice Ball (Omusubi) to Healthy Volunteers

*Shaw Watanabe
Asia Pacific Clinical Nutrition Society, Visiting Professor, Tokyo University Of Agriculture, Tokyo, Japan

*Corresponding Author:
Shaw Watanabe
Asia Pacific Clinical Nutrition Society, Visiting Professor, Tokyo University Of Agriculture, Tokyo, Japan
Email:watashaw@lifescience.or.jp

Published on: 2020-11-18

Abstract

Background: The health effects of brown rice are still in debate. From September to December 2019, 30 employees in the Ministry of Agriculture, Forestry, and Fisheries conducted the pilot intervention study on bowel movement, intestinal microbiota, fecal short-chain fatty acids, and inflammatory biomarkers to see the health effects.
Subjects and Methods: Brown rice genmai onigiri (rice cake) was provided 5/week as a business lunch for 12 weeks. Participants practiced the pre-and post-questionnaires, daily life records, monthly blood pressure measurements, and body composition. Before and after the intervention, the fecal samples were used for the simultaneous measurement of intestinal microbiota and short-chain fatty acids. Biochemical data involving IL-6, CRP, and TNFa were obtained for correlation analysis with microbiota changes.
Results and Discussion: The body weight decreased in about half the participants, and bowel movements and stool status improved significantly. Dominant microbiota were Firmicutes (around 65%), Actinobacteria (15-17%), Bacteroidetes (5-7%), and less than 1% of Proteobacteria, Verrucomicrobia, and Fusobacteria. Significant microbiota change was an increase in Actinobacteria and a decrease in Proteobacteria. Verrucomicrobia and Fusobacteria also tended to decrease. In short-chain fatty acids, acetate and propionate grew to decline, while n-butyrate and i-valerate slightly increased.
Acetate, propionate positively correlated with IL-6, and n-butyrate, and n-valerate showed a positive correlation with IL6 and CRP. Isobutyrate and isovalerate negatively correlated TNFα. The upper tertial of genmai eaters showed beneficial effects.
Conclusion: Replacement of one meal per day to brown rice omusubi showed health benefits in more than half of the participants. The relationship between bacterial species and short-chain fatty acids suggested the holistic control of SCFA and inflammatory biomarkers.

Keywords

Brown Rice; Intervention; Microbiota; Blautia; Short-Chain Fatty Acid; IL-6; CRP

Introduction

Current epidemiological data are inconclusive about the effects of traditional brown rice on health [1,2]. We previously carried out a cross-sectional study of 1100 participants who consumed brown rice daily [2]. Brown rice eaters preferred to eat Japanese foods and traditional vegetables, avoiding meat, dairy products, and western foods [3]. Brown rice eaters showed a lower BMI in men and women of all ages. We found that eating brown rice (Genmai) is beneficial for maintaining proper body weight. It was challenging to find habitual genmai eaters in the previous epidemiological studies because white rice is softer and tastier and fits with any side dish.

Kenzo Futaki founded the Japan Society of Integrative Medicine in 1953, advocated the "20 virtues of Genmai," and tried to spread the brown rice diet. Dietary fiber, which brown rice is rich in, positively influences bowel movement and creates a beneficial intestinal environment by maintaining bacterial flora [4-7]. Recent intestinal bacterial research has found that people who ingest dietary fiber show increased bacteria producing citric acid, propionic acid, and butyric acid. Bifidobacteria are predominant in the microbiota of these people. Blautia wexlerae and Blautia luti were also common in Japanese [8]. The bowel movements of those on a brown rice diet can induce an excellent intestinal environment, as indicated by banana-shaped stools and defecation more than once a day.

The Medical Rice Association of Japan advocates brown rice consumption based on accumulating scientific evidence about its health benefits. As part of the "Brown Rice Taste Contest G1 Grand Prix", sponsored by the medical rice association, 54 kinds of organic brown rice were proven to contain dietary fiber, γ-oryzanol, high antioxidant activity, GABA, vitamins, and minerals [9]. These ingredients are the functional basis of brown rice.

Volunteer members of The Ministry of Agriculture, Forestry, and Fisheries attempted to confirm the relationship between brown rice eating and health by eating genmai omusubi for lunch for 12 weeks [10]. Previous cross-sectional studies have strongly suggested that eating brown rice improves bowel movement through the ideal symbiosis with intestinal microbiota and the production of short-chain fatty acids (SCFAs). These cause various health effects on obesity, blood pressure, and healthy feeling [11,12].

This project investigated simultaneous changes in the gut microbiota and short-chain fatty acids before and after brown rice ball (omusubi) intervention.

Ethical Issues

The Ethics Review Board in the Life Science Promoting Association approved this study (No. 003 in 2018). Each participant was free to withdraw at any time during the course. The research group stored confidential information separately in a locked container, and the analysis was done using anonymous data.

Subjects and Method

Subjects

Thirty participants were recruited from the Ministry of Agriculture, Forestry, and Fisheries employees and voluntarily participated in the study, providing written informed consent before the examination. Eighteen men and 12 women participated in the study [10]. They were all healthy workers except for seven males who showed mild hypertension. They completed pre- and post-intervention questionnaires, daily records of genmai (brown rice) consumption and bowel movements, monthly health check-ups, and collected feces before and after the intervention.

Questionnaire

Each participant completed an 8-page questionnaire before the intervention [10]. It included questions about age, height, current weight, BMI, weight at 20 years old, maximal weight and age in the past, kind of staple rice, food preferences and eating habits, dietary awareness, food intake status (sFFQ), meal sketches, lifestyle and habits, current health condition, bowel movements, and stool features, health history of themselves and their parents and siblings, chief complaints, changes in health condition since the previous year, healthy habits, liquor, tobacco, eating out, supplements, fasting experience, physical activities, lifestyle, occupations, education, income, stress, and life creed (religion). In women's health, we also asked questions about menarche, menstruation, hormone use, delivery history, childcare history, etc. After the intervention, participants completed a shorter, four-page questionnaire to discover diet, health condition, and feeling changes.

Intervention

The intervention was carried out from October 2019 to December 2019 [10]. The participants ate a rice ball lunch box made from brown rice "omusubi" on weekdays for at least four days a week. Twenty-five participants ate brown rice 45 or more times in the three months, and 7 participants ate brown rice 57 times or more. Bread and noodle intake decreased under increasing rice consumption. Before and after the survey, stools were collected to analyze the intestinal flora and SCFAs simultaneously. Biochemical data were obtained from 9 ml of blood samples collected at the end of the study. During the intervention, the participants measured body weight, blood pressure, and body composition using impedance body composition monitors. Participants reported the number of bowel movements and stool shapes (modified Bristol stool scale) every day. There was no intervention for breakfast, dinner, and snacks.

Intestinal Bacteria and Short-Chain Fatty Acids

Thirty participants provided stool samples in dry tubes. Fresh fecal samples were collected from 3 points on the stools and immediately frozen in a home refrigerator. Fecal samples (approximately 50-100 mg) were sent to Techno Suruga Laboratory, Shizuoka, in a dry ice container for sequence amplicon analysis [12-14]. The stools were separated for microbiota analysis and short-chain fatty acids measurement.

An 0.8 ml sample of the suspension was homogenized with zirconia beads using a FastPrep24 Instrument (MP Biomedicals, Santa Ana, CA). DNA was extracted from the suspension using an extractor (Precision System Science, Chiba, Japan). MagDEA DNA 200 (GC) (Precision System Science) was used for automatic nucleic acid extraction. The V3-V4 region of 16S rDNA was amplified using a mixture of the forward primer and reverse primer.

PCR was performed using the GeneAmp PCR system 9700 (ABI, Foster City, CA). The PCR reaction and preparation of the amplicon pool were performed.

More than 30,000 determined 16S rDNA sequences from each sample were subjected to a homology search using Metagenome@Kim software (World Fusion Co., Ltd., Tokyo, Japan) against the Techno-Suruga Lab Microbial Identification Database DB-BA10.0 (Techno-Suruga Laboratory).

 Short-chain fatty acids (SCFAs) in the feces were determined by a modified method, as previously described by García-Villalba R, et al. (2012) [15]. For the determination of SCFA, 0.1 g of feces was put in a 2.0 ml tube with zirconia beads and suspended with 0.9 mL 0.5% phosphoric acid. Each sample was heated at 85°C for 15 min, vortexed at 5 m/s for 45 s using FastPrep 24 (MP Biomedicals, CA, USA), and centrifuged at 14,000 rpm for 10 min. Then, 0.4 ml of the supernatant was transferred to a 1.5 ml tube, mixed with 0.4 ml ethyl acetate, shaken for 30 min, and centrifuged at 14,000 rpm for 10 min. Finally, 0.2 ml of the supernatant was mixed with 1 mM 4-methyl valeric acid as an internal standard.

SCFAs were measured by gas chromatography with a flame ionization detector (7890B, Agilent Technologies, USA) and a capillary column DB-WAXetr (30 m, 0.25 mm id, 0.25 μm film thickness, Agilent Technologies, USA). Helium was used as the carrier gas at 1.2 mL/min. The detector temperature was kept at 250°C. The oven temperature program was as follows: 50°C; then 10°C/min to 90°C; 15°C /min to 150°C; 5°C /min to 170°C; 20°C/min to a final temperature of 250°C, held for 4 min. One microliter of the extract was injected in the splitless mode. Acetate, propionate, n-butyrate, i-butyrate, n-valerate, i-valerate, and n-caproate were measured.

Statistical Analysis

All data were stored in an Excel database and transferred to IBM-SPSS ver. 24 for statistical analyses [16]. Parametric analysis for continuous variables and non-parametric analysis for categorical data were carried out. Data were examined using an unpaired t-test or the two-sided Mann-Whitney test and Fisher's X2 test for categorical variables. Spearman's correlation analysis was carried out between microbiota profiles at the phylum level and species level. A paired t-test was used to examine the changes between pre- and post-intervention. The principal component analysis was performed to detect groups of coexistence and interaction. Bacterial profiles of more than 0.1% of the total composition on average or max profiles of more than 1.0% were selected for analysis. Most tables listed mean, sd, or median, because the distribution of bacilli was irregular, mostly where the population was small, and the profile was variable.

P values less than 0.05 were marked and p < 0.05 was considered significant. The statistical significance was shown as * p < 0.05, ** p < 0.01, *** p < 0.001.

Discussion

There have been many reports on the health effects of Genmai (brown rice), but the mechanism has not been clarified yet [1,2]. Dietary fiber and g-oryzanol in genmai are candidates for its functional factors [6,9, and 18]. Dietary fibers, mostly indigestible, water-insoluble fibers, stimulate fermentation by the microbiota in the colon. It produces SCFAs, such as acetic acid, propionic acid, butyric acid, and valeric acid, causing various health effects [7,19]. Different dietary fibers led to different responses. Generally, brown rice eaters' intestinal bacterial profiles maintained their diversity and balance of bacteria [13].

We found that genmai eating helped maintain healthy body weight, BMI, and right bowel movements [2]. As an additional benefit, brown rice eaters preferred to eat plant-based Japanese foods, avoiding meat and dairy products [3]. They disliked oily and spicy flavors, and their selection was based upon factors such as whether the food was fresh, organic, without additives or genetically modified foodstuffs, and domestically produced.

In animal experiments using experimental mice, γ-oryzanol works on the brain through the blood-brain barrier to suppress animal fat intake [6].

Numerous animal models and human studies have consistently demonstrated that gut microbiota can modulate host health [4,7].

There is no research on whether brown rice should be eaten in every meal or replaced some of it. To understand the dose-effect of brown rice eating, we adopted the method of merely changing lunch to brown rice balls (omusubi) 5 days a week. Most participants ate genmai only for lunch, but it caused specific microbiota profiles and improved the intestinal environment. They significantly improved bowel movements and stool conditions.

When the number of meals with brown rice for lunch was divided into three, and the composition of intestinal bacteria in each was examined, the top one third had higher Firmicutes and Actinobacteria levels, Bacterioidetes and Proteobacteria decreased.

Short-chain fatty acids implicate the maintenance of the host's ecological homeostasis not only as an energy source but also through G protein-coupled receptors on the cell membrane, such as GRP41, GRP43, GRP109, and Olfr78 [17,20-25]. Butyrate is the ligand of GFP109, which suppresses the expression of IL-6 from macrophages and dendritic cells in the large intestine, enhances the production of IL-10 and retinoic acid, which allows the maintenance of regulatory T cell (Treg) homeostasis. In particular, butyrate enhances histone acetylation in the gene promoter region and enhancer region of Foxp3, the master gene of Treg, by inhibiting histone deacetylase and causing differentiation of naive T cells to Treg cells. The increase of Treg cells reduces the excess intestinal inflammatory response.

The above phenomenon contributes to host eco-regulation as a whole. The two most dominant bacterial species in the human colon with a significant contribution to butyrate production are Faecalibacterium prausnitzii and Eubacterium rectale [26,27]. In the present study, genus Blautia and Ruminococcus increased, but Faecalibacterium and Bifidobacteria were decreased or slightly increased. Brown rice eaters showed additional benefits, such as the low prevalence of Fusobacterium.

In about 200 microbiota profiles, 8 out of 30 regression factors showed a significant correlation with SCFAs. Each component factor had a 1:1 correspondence with a specific SCFA, and some showed a correlation with a few SCFAs. Each bacterial species was positive or negative concerning a specific SCFA. Acetate has a negative relationship with most bacteria, indicating that it may inhibit many bacteria. Blautia wexlerae showed a positive relationship with acetate but a negative relationship with i-butyrate and i-valerate. Large populations like Blautia may have a strong influence, but a deficient proportion of minor bacteria at a 0.01% level could also influence the network.

We used to believe that the more SCFAs, the better immunity of the subject. However, a negative correlation seemed to be more important to maintain a stable environment [28,29]. A pathogenic bacterium, like Escherichia coli/Shigera was suppressed to very low concentration under the predominant growth of Firmicutes.

All of these components contribute to stabilizing the innate immunity of a subject.

Acetate, propionate, n-butyrate, and n-valerate were all significantly correlated with IL-6 and CRP inflammatory biomarkers. Those who consumed brown rice at a high rate showed higher levels of Blautia wexlerae and low acetate, propionate, and n-valerate levels. They also tended to show low IL-6 and CRP levels. Páez A, et al. (2020) [29] suggested that the depletion of B. luti and B. wexlerae species in the gut ecosystem may occur in obesity cases and contribute to metabolic inflammation leading to insulin resistance. Changes in the microbiota caused by brown rice eating should strengthen innate immunity by suppressing SCFAs. The negative relationship between COVID-19 pneumonia and rice consumption by countries may reflect such innate immunity [30].

Our study's weakness is that our study was a comparison of pre- and post-intervention data and not a randomized clinical study. However, in nutritional intervention studies for healthy people, individual variation in the control population is always a problem. Even though a crossover design was planned, it could not exclude the effect of waiting time. Therefore, pre- and post-intervention comparison seemed to be the most cost-effective design to determine the complex microbiota and SCFA profiles' alterations. High adherence to the monthly interview and the writing of daily records of consumption of genmai omusubi and bowel movements by participants guaranteed the high reliability of the data. However, the possibility of selection bias nevertheless remained. Although fecal samples may not represent all intestinal events, the microbiota compositions in this study were suitable for comparison between individuals. Changes in the microbiota and SCFAs were limited to what could be detected in the feces, but this may still reflect the colonic environment. The interaction of the intestinal flora-composing bacteria group involves various factors such as nutrition, cross-feeding, optimal environment (pH, acidity, etc.), and antibacterial substances. In the intestinal flora, an autonomous balance-maintaining mechanism of the intestinal microbial ecosystem has been advocated, and our research was also conducted in that category.

Repeating this intervention study with other populations could allow generalization of the results.

In summary, implementing a brown rice diet for lunch yielded benefits by changing the microbiota and SCFAs to improve bowel movement and shape of stools. Blautia wexlerae was the dominant bacterium found at the species level. There were special groups of microbiotas that maintained the levels of SCFAs. The group that consumed more significant amounts of genmai lunch showed a high prevalence of Firmicutes, increased Actinobacteria, and decreased Proteobacteria and Fusobacteria. These changes lowered SCFA levels, and inflammatory markers, such as IL-6 and CPR, suggested an inverse correlation with the intake number of genmai lunches. Proliferation and suppression of microbiota to maintain the gut ecosystem seemed essential for anti-inflammation activity, in which Blautia wexlerae would play a vital role.

Acknowledgment

The authors thank the 30 employees of the Ministry of Agriculture, Forestry, and Fisheries who participated in this project as research volunteers. We also extend our appreciation to the "Food and Health" practical verification project team; to Mr. Toyohisa Aoyama, Secretary-General of Research, Genmai Rice Box Lunch.

The authors sincerely appreciate Drs. Philippe Calain for his honest discussion and editing of this manuscript.

Declaration of Interest

None.

Credit Roles

S.W, K.K: Conceptualization, Formal analysis, Project administration, Writing review & editing,

K.K, M.M, T.E: Recruit of participants, interview and clinical measurement, original draft,

S.M: Statistical calculation and judgment,

T.H, T.K, A.M: Measure of microbiota and SCFA, and data processing.

Funding

The Lifescience Promoting Association supported the research. A part of this work was supported by the Ministry of Agriculture, Forestry, and Fisheries and the grant from the Project of the NARO Bio-oriented Technology Research Advancement Institution (#01026C). 

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