H OH metabolites OH Article Fiber-Rich Barley Increases Butyric Acid-Producing Bacteria in the Human Gut Microbiota Shohei Akagawa 1 , Yuko Akagawa 1 , Yoko Nakai 1, Mitsuru Yamagishi 1, Sohsaku Yamanouchi 1, Takahisa Kimata 1, Kazushige Chino 2, Taiga Tamiya 3 , Masaki Hashiyada 4, Atsushi Akane 4 , Shoji Tsuji 1 and Kazunari Kaneko 1,* 1 Department of Pediatrics, Kansai Medical University, 2-5-1 Shinmachi, Hirakata 573-1010, Japan; [email protected] (S.A.); [email protected] (Y.A.); [email protected] (Y.N.); [email protected] (M.Y.); [email protected] (S.Y.); [email protected] (T.K.); [email protected] (S.T.) 2 Healthcare New Business Division, TEIJIN Limited, 3-2-1 Kasumigaseki, Tokyo 100-8585, Japan; [email protected] 3 Bio Palette Co., Ltd., 1-1 Rokkodai-cho, Kobe 650-0047, Japan; [email protected] 4 Department of Legal Medicine, Kansai Medical University, 2-5-1 Shinmachi, Hirakata 573-1010, Japan; [email protected] (M.H.); [email protected] (A.A.) * Correspondence: [email protected]; Tel.: +81-72-804-0101 Abstract: Butyric acid produced in the intestine by butyric acid-producing bacteria (BAPB) is known to suppress excessive inflammatory response and may prevent chronic disease development. We evaluated whether fiber-rich barley intake increases BAPB in the gut and concomitantly butyric acid in feces. Eighteen healthy adults received granola containing functional barley (BARLEYmax®) once daily for four weeks. Fecal DNA before intake, after intake, and one month after intake was analyzed Citation: Akagawa, S.; Akagawa, Y.; using 16S rRNA gene sequencing to assess microbial diversity, microbial composition at the order Nakai, Y.; Yamagishi, M.; level, and the proportion of BAPB. Fecal butyric acid concentration was also measured. There were Yamanouchi, S.; Kimata, T.; Chino, K.; Tamiya, T.; Hashiyada, M.; Akane, no significant differences in diversities and microbial composition between samples. The proportion A.; et al. Fiber-Rich Barley Increases of BAPB increased significantly after the intake (from 5.9% to 8.2%). However, one month after Butyric Acid-Producing Bacteria in stopping the intake, the proportion of BAPB returned to the original value (5.4%). Fecal butyric acid the Human Gut Microbiota. concentration increased significantly from 0.99 mg/g feces before intake to 1.43 mg/g after intake Metabolites 2021, 11, 559. https:// (p = 0.028), which decreased significantly to 0.87 mg/g after stopping intake (p = 0.008). As BAPB doi.org/10.3390/metabo11080559 produce butyric acid by degrading dietary fiber, functional barley may act as a prebiotic, increasing BAPB and consequently butyric acid in the intestine. Academic Editor: Cholsoon Jang Keywords: microbiota; butyric acid-producing bacteria; 16S rRNA gene sequencing; dietary fiber; prebiotics Received: 2 August 2021 Accepted: 19 August 2021 Published: 22 August 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in More than 100 trillion bacteria, including 1000 different species, form the gut mi- published maps and institutional affil- crobiota in the human intestine, greatly affecting human health [1]. Butyric acid, one of iations. the short chain fatty acids (SCFA) produced by butyric acid-producing bacteria (BAPB) (e.g., portion of Alistipes and Porphyromonas (order Bacteroidales); Clostridium, Eubacterium, Feacalibacterium, and Roseburia (order Clostridiales)) in the intestine, has garnered attention owing to its role in the maturation of regulatory T cells in the intestine, which suppress excessive immune responses [2,3]. A decrease in BAPB in the intestine and in fecal butyric Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. acid concentration has been reported in patients with idiopathic nephrotic syndrome [4,5], This article is an open access article food allergy [6], cerebrovascular disease [7], and type 2 diabetes [8,9]. Therefore, increasing distributed under the terms and BAPB in the gut microbiota may aid in the prevention and treatment of various chronic conditions of the Creative Commons diseases. Although probiotics have been employed extensively to correct gut microbiota Attribution (CC BY) license (https:// imbalance (dysbiosis), their effects are limited, as most probiotics only pass through the creativecommons.org/licenses/by/ intestine without residing in and modulating the gut microbial composition [10,11]. In- 4.0/). stead, the efficacy of prebiotics has been receiving attention recently. According to the Metabolites 2021, 11, 559. https://doi.org/10.3390/metabo11080559 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, 559 2 of 9 International Scientific Association for Probiotics and Prebiotics, prebiotics are defined as “a substrate that is selectively utilized by host microorganisms conferring a health benefit” and include dietary fibers such as β-glucan, fructan, resistant starch, and oligosaccha- rides [12]. Whole-grain products using corn, wheat, and barley are known to regulate the gut microbiota owing to their high content of dietary fiber [13–15]. BARLEYmax®, a line of non-genetically modified barley developed by the Commonwealth Scientific and Industrial Research Organization, contains twice the amount of dietary fiber and four times the resistant starch compared to regular barley and is reported to elevate the fecal SCFA concentration [16]. However, no study has analyzed its effect on the human gut microbiota, especially focusing on butyric acid. Therefore, we evaluated whether the intake of fiber-rich barley, BARLEYmax, elevates fecal butyric acid concentration by increasing BAPB in the gut of healthy adults. 2. Results 2.1. Participants’ Characteristics A total of the 20 participants took part in this study; however, the analysis was performed on 18 participants after excluding two participants (owing to antibiotic use during the study period). The median age of the subjects, including 12 males (67%), was 35.9 years (IQR, 33.8–41.6 years). Seventeen participants (94%) were born vaginally. Median body mass index was 22.0 (19.6–24.6). Fifteen participants had allergic diseases, mostly allergic rhinitis (14 participants). Three participants were taking anti-histamine drugs for allergic rhinitis and one participant was taking magnesium oxide for constipation. Twelve participants (67%) were taking probiotics constantly. The compliance rate for BARLEYmax (the percentage of days for which participants had BARLEYmax during the 28-day study period) was 78% (64–93%) (Table1). Table 1. Characteristics of participants. n = 18 Sex, male (%) 12 (67%) Age (years) 35.9 (33.8–41.6) Body mass index 22.0 (19.6–24.6) Mode of delivery, vaginal delivery (%) 17 (94%) Allergic disease (%) 15 (83%) Allergic rhinitis (%) 14 (78%) Atopic dermatitis (%) 1 (6%) Daily use of prescription drugs (%) 4 (22%) Antihistamine (%) 3 (17%) Magnesium oxide (%) 1 (6%) Daily use of probiotics (%) † 12 (66%) Compliance rate (%) ‡ 79% (64–93%) Consumed BARLEYmax granola BARLEYmax alone 6 (33%) With Milk 8 (44%) With Yogurt 3 (17%) With Soy milk 1 (6%) Data are expressed as number (%) or median (IQR). † Four or more times per week of eating yogurt, cheese, miso (fermented soybean paste), pickles, probiotic drink, or probiotic supplements. ‡ Percentage of days for which participants took granola during the 28-day study period. 2.2. Alpha Diversity There was no significant difference in the numbers of observed species between the pre, post, and 1M groups (46 [IQR, 38–50], 45 [42–50], and 44 [38–50], respectively, p > 0.05). Moreover, there were no significant differences in Shannon and Simpson indices (Shannon Metabolites 2021, 11, 559 3 of 9 2.2. Alpha Diversity Metabolites 2021, 11, 559 There was no significant difference in the numbers of observed species between3 the of 9 pre, post, and 1M groups (46 [IQR, 38–50], 45 [42–50], and 44 [38–50], respectively, p > 0.05). Moreover, there were no significant differences in Shannon and Simpson indices (Shannon index: 4.10 [3.96–4.41], 4.01 [3.81–4.14], and 4.15 [3.53–4.23], respectively, p > 0.05;index: Simpson 4.10 [3.96–4.41], index: 0.91 4.01 [0.88–0.93], [3.81–4.14], 0.89 and [0.87–0.92], 4.15 [3.53–4.23], and 0.91 respectively, [0.85–0.92],p > respectively, 0.05; Simpson p >index: 0.05) 0.91(Figure [0.88–0.93], 1A). 0.89 [0.87–0.92], and 0.91 [0.85–0.92], respectively, p > 0.05) (Figure1A). Figure 1. Changes in gut microbiota microbiota diversity and and composition composition due due to to the the intake intake of of BARLEYmax: BARLEYmax:( A) numbers of species and Shannon and Simpson indices. (B) principal coordinates analysis plot of Bray–Curtis dissimilarity. Each point repre- and Shannon and Simpson indices. (B) principal coordinates analysis plot of Bray–Curtis dissimilarity. Each point represents sents a sample. The circular, triangular, and square points represent the samples from the pre, post, and one-month a sample. The circular, triangular, and square points represent the samples from the pre, post, and one-month groups, groups, respectively. (C) Compositions of the gut microbiota at the order level. Each bar represents an individual sample (left)respectively. or a group (C) (right). Compositions of the gut microbiota at the order level. Each bar represents an individual sample (left) or a group (right). 2.3. Beta Diversity 2.3. Beta Diversity To evaluate the differences in gut microbiota between the pre, post, and one-month To evaluate the differences in gut microbiota between the pre, post, and one-month groups, a principal coordinates analysis plot of Bray–Curtis dissimilarity was generated, groups, a principal coordinates analysis plot of Bray–Curtis dissimilarity was generated, and the sample was characterized in two dimensions. No apparent clusters were observed and the sample was characterized in two dimensions.