Key Microbiota Identification Using Functional Gene Analysis during Pepper (Piper nigrum L.) Peeling

RESEARCH ARTICLE Key Microbiota Identification Using Functional Gene Analysis during Pepper (Piper nigrum L.) Peeling Jiachao Zhang1☯, Qisong Hu1☯, Chuanbiao Xu1, Sixin Liu2*, Congfa Li1* 1 College of Food Science and Technology, Hainan University, Haikou, 570228, P. R. China, 2 College of Materials and Chemical Engineering, Hainan University, Haikou, 570228, P. R. China ☯ These authors contributed equally to this work. * (CL); (SL) a11111 Abstract OPEN ACCESS Citation: Zhang J, Hu Q, Xu C, Liu S, Li C (2016) Key Microbiota Identification Using Functional Gene Analysis during Pepper (Piper nigrum L.) Peeling. PLoS ONE 11(10): e0165206. doi:10.1371/journal.pone.0165206 Editor: Seon-Woo Lee, Dong-A University, REPUBLIC OF KOREA Received: May 29, 2016 Accepted: October 7, 2016 Pepper pericarp microbiota plays an important role in the pepper peeling process for the production of white pepper. We collected pepper samples at different peeling time points from Hainan Province, China, and used a metagenomic approach to identify changes in the pericarp microbiota based on functional gene analysis. UniFrac distance-based principal coordinates analysis revealed significant changes in the pericarp microbiota structure during peeling, which were attributed to increases in bacteria from the genera Selenomonas and Prevotella. We identified 28 core operational taxonomic units at each time point, mainly belonging to Selenomonas, Prevotella, Megasphaera, Anaerovibrio, and Clostridium genera. The results were confirmed by quantitative polymerase chain reaction. At the functional level, we observed significant increases in microbial features related to acetyl xylan esterase and pectinesterase for pericarp degradation during peeling. These findings offer a new insight into biodegradation for pepper peeling and will promote the development of the white pepper industry. Published: October 21, 2016 Copyright: © 2016 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The sequence data reported in this paper have been deposited in the NCBI database (SRA: SRR2976395). Funding: This research was supported by the National Natural Science Foundation of China (no. 31160339), Natural Science Foundation of Hainan province (no. 20163042) and Scientific Research Foundation of Hainan University (no. KYQD1548). Competing Interests: The authors have declared that no competing interests exist. Introduction Pepper (Piper nigrum L.), one of the most famous spices in the world, is an important member of the family Piperaceae. It is native to India, and is mostly cultivated in tropical and subtropical regions [1]. Pepper fruits contain 1.0%–2.5% volatile oil and 5%–9% alkaloids, mainly piperine, chavicine, piperidine, piperetine, and resin [2]. Pepper alkaloids exhibit a wide variety of biological effects, including immunomodulatory, anti-carcinogenic, anti-asthmatic, stimulatory, hepatoprotective, anti-inflammatory, anti-microbial, and anti-ulcer [3, 4]. Pepper is therefore also widely used in medicine and health care [5, 6]. China is one of the largest pepper producers in the world, with an estimated annual production of 27,210 tons. Hainan Province produces more than 90% of the country’s pepper crop, with more than 80% of the product being white pepper [7]. The traditional method for pepper peeling is known as retting. In this process, ripe pepper fruits are separated from the stalk, PLOS ONE | DOI:10.1371/journal.pone.0165206 October 21, 2016 1 / 12 Key Microbiota and Functional Genes tightly packed into jute bags, and steeped in still or flowing water for 7–14 days. During this procedure, the pericarp decays, and the pepper is kneaded until the pericarp is removed. The method is still used by most pepper farmers because of its simplicity. However, it is time-consuming and produces an unpleasant skatole smell [8, 9]. It therefore makes sense to improve on the current pepper peeling technology. In recent years, biodegradation has been considered as a new approach for pepper peeling [10, 11]. Pectinase produced by various pericarp microbes plays a key role in pericarp degradation and peeling. Compared with traditional peeling technology, biodegradation is advantageous in terms of time consumption, product quality, and level of environmental pollution [11]. However, previous research showed that use of a single microbe for peeling resulted in a poor quality of white pepper [12]. Therefore, we suggested that the peeling process required a complex enzyme system produced by various interacting microbes rather than a single microorganism [13, 14]. Accordingly, it is necessary to explore the functional genes of key microbiota to develop the biodegradation method of pepper peeling. With the development of next-generation sequencing (NGS), the high-throughput sequencing-based metagenomic approach has been widely applied in microbiology [15, 16] to reveal the dynamic changes in the structure of microbiota and their functional genes [17]. In the present study, pepper samples were collected from Hainan Province, China, at different peeling time points. The metagenomic approach was used to explore core microbes and functional genes related to pericarp degradation during pepper peeling. Materials and Methods Experimental design and pepper sample collection A longitudinal study design was used to investigate core microbiotas and their functional genes during pepper peeling. Pepper samples were collected in triplicate at the pepper farms in different peeling time points from Qionghai city (Group 1) and Wanning city (Group 2) of Hainan Province, China. When peeling, the pepper fruit (containing pericarp, pulpa and one pepper kernel) was tightly packed into jute bags, and retting in still or flowing retting in the water for 6 days, then the peeled pepper was obtained. Detailed sample information is listed in Table 1. The pepper samples were collected on private pepper farm in Qionghai and Wanning. After Table 1. Sample information and α diversity in present study. Group Group 1 Qionghai City Group 2 Wanning City Sample Time Point Reads OTU number Shannon index Pep3 (n = 3) 0 day 19082±1225 126±22 2.55±0.11 Pep5 (n = 3) 1 day 18644±2782 178±15 3.25±0.08 Pep7 (n = 3) 2 day 19725±955 285±38 4.22±0.23 Pep9 (n = 3) 3 day 19921±842 247±17 3.97±0.14 Pep11 (n = 3) 4 day 20251±3046 191±8 3.63±0.21 Pep13 (n = 3) 5 day 19836±1958 201±24 3.71±0.16 Pep15 (n = 3) 6 day 18882±495 199±16 2.42±0.16 Pep2 (n = 3) 0 day 20031±795 135±7 2.68±0.09 Pep4 (n = 3) 1 day 19968±1034 166±14 2.98±0.17 Pep6 (n = 3) 2 day 19845±1684 277±19 4.18±0.26 Pep8 (n = 3) 3 day 18964±2131 235±51 3.86±0.51 Pep10 (n = 3) 4 day 19047±3017 175±21 3.22±0.14 Pep12 (n = (...truncated)