Journal Home page : www.jeb.co.in « E-mail : [email protected] Original Research TM Journal of Environmental Biology JEBTM p-ISSN: 0254-8704 DOI : http://doi.org/10.22438/jeb/39/2/MRN-450 e-ISSN: 2394-0379 CODEN: JEBIDP Analysis of microbial communities in local cultivars of astringent persimmon (Diospyros kaki ) fruits grown in Gyeongnam Province of Korea White Smoke Just write. PDlagiarism etector Abstract Authors Info J.E. Choi1,2 , S.H. Choi 3, J.S. Lee 4, K.C. Aim : The objective of the present study was to characterize the microbial communities within the stalks of Lee 4556, S.M. Kang , J.I. Kim , M.S. Choi , astringent persimmons fruits grown locally in Korea and two commercial herbal products (kaki calyx- H.G. Kim 671,W.T. Seo , K.Y.Lee , B.C.Moon 1Korean & kaki calyx-Chinese, using pyrosequencing based on 16S and 18S genes. and Y. M. Kang1,2 * 12K-herb Research Center, UST, Korean Methodology : 'Gojongsi' sample was Medicine Life Science, collected from Sancheong province, Korea Institute of Oriental Medicine 'Danseongsi' from Sancheong province (KIOM), 1672 Yuseong-daero, Yuseong-gu, Daejeon-34054, Republic of Korea and 'Bansi' from Miryang province during different seasons (October to 3AtoGen Co., Ltd. 11-8, Techno 1-ro, Yuseong-gu, Daejeon-34015, Republic December) from Korea Forest of Korea Environment Research Institute at 4Korea Research Institute of Bioscience Gyeongsangnam-do. Thirteen samples and Biotechnology (KRIBB), 181 Ipsin-gil, were divided into four groups A, B, C and Jeongeup-si, Jeollabuk-do-56212, Republic D. Group A consisted of three kinds of of Korea undried stalks collected in October. 5 Forest Research Department, Group B consisted of three kinds of dried Gyeongsangnam-do Forest Environment Research Institute, Jinju-52615, Republic stalks and surfaces of persimmons of Korea collected in December. Group C 6Department of Forest Environmental consisted of two Korean herbal Resources, Gyeongsang National medicines (Kaki Calyx) sold in Korea University, JinJu-52828, Republic of Korea which are commercial products. Group 7Department of Food Science, Gyeongnam D consisted of two Chinese herbal National University of Science and medicines (Kaki Calyx) sold in China. Technology, Jinju-52725, Republic of Korea Thirteen samples cultured in Luria- Bertani broth and potato dextrose broth *Corresponding Author Email : under specific conditions were collected [email protected] using sterile streaking sticks and placed into sterile plastic conical tubes. Key words Results : Metschnikowia sp. was the Diospyros kaki Online Copy dominant microorganism in groups A Metschnikowia sp. and B compared with those of groups C Pyrosequencing and D on the hit map.Metschnikowia sp. Species diversity were detected in group A and B (stalks of herbal medicines without the Publication Info manufacturing process).Metschnikowia sp. was amplified for identification. The results of sequence analysis of 18S rDNA gene showed that there were no matches greater than 97%. Therefore, the Paper received : 29.12.2016 Revised received : 23.04.2017 Metschnikowia sp. identified in this study may be a novel species. Re-revised received : 12.05.2017 Interpretation : The present study provides information on various microorganisms isolated from Accepted : 28.06.2017 nature's fruits and provides a list of microorganisms that can be experimented later. © Triveni Enterprises, Lucknow (India) Journal of Environmental Biology March 2018 Vol. 39 237-246 238 J.E. Choi et al. Introduction time by profiling large numbers of samples simultaneously (Chun et al., 2010). Indeed, in previous studies pyrosequencing has The genus of Persimmon (Diospyros ) belongs to several been used to identify and profile microorganisms from astringent plant family Ebenaceae which consists of about 400 species that persimmons grown under different conditions (Jeonet al ., 2013; have been cultivated for many years in Asian counties, including Kimet al ., 2011b). Korea, China and Japan (Hidalgoet al ., 2012; Guo et al ., 2006). Diospyros kaki Thunb. is one of the cultivated species and is The objective of the present study was to characterize the classified into four types; pollination constant non astringent microbial communities within the stalks of astringent persimmon (PCNA), pollination variant non astringent (PVNA), pollination fruits grown locally in Korea and two commercial herbal products variant astringent (PVA) and pollination constant astringent (PCA) (kaki calyx-Korean and kaki calyx-Chinese), using pyrosequencing (Parfittet al ., 2015). The astringent persimmon is edible after basedon16Sand18Sgenes. reducing its astringency which occurs with ripening of the fruits or Materials and Methods after removal of its high water-soluble tannin content artificially (Li et al., 2011;Arnal et al ., 2003). Sample collection : 'Gojongsi'sample was collected from Sancheong province, 'Danseongsi' from Sancheong province Persimmons have various chemical constituents such as and 'Bansi' from Miryang province collected during different carotenoids, tannins, flavonoids, sugars, hydrocarbons, lipids, seasons (October and December) from Korea Forest hydrocarbons, aromatics, terpenoids and steroids (Mallavadhani Environment Research Institute at Gyeongsangnam-do. In the et al., 1998).Astringent persimmons contain water-soluble tannin present study, thirteen samples were divided into four groups. components has a bitter taste and tannic acid which has been Group A consisted of three kinds of undried stalks collected in shown to have anti-aging, anticancer and herbivory defense October. Group B consisted of three kinds of dried stalks and effects (Parket al ., 2004; Parfitt et al ., 2015; Kim et al ., 2011a). surfaces of persimmons collected in December. Group C consisted of two Korean herbal medicines (Kaki Calyx) sold in Kaki Calyx is a type of traditional Korean medicine Korea. Group D consisted of two Chinese herbal medicines (Kaki containing persimmons (Nanjing Zhong yi yao da xueet al ., Calyx) sold in China. Details of the samples has been previously 2006). In fact, according to Korean medical documents Dong- described (Choiet al ., 2016). uibogam [https://en.wikipedia.org/wiki/Dongui_Bogam] and Ben Cao Gang Mu [https://en.wikipedia.org/wiki/ Compendium_ of_ Microorganism preparation and cultures : Thirteen samples Materia_Medica], the stalks of astringent persimmons are known were collected into stalks and peels, placed in sterile plastic to be effective in the treatment of bed-wetting, vomiting. Further conical tubes, and cultured in 15 ml of Luria-Bertani broth and studies showed that persimmons have been used to treat potato dextrose broth at 25 oC laboratory conditions to compare hiccups, flu, and atherosclerosis and to prevent heart disease differences among microorganisms grown in two culture media. (Herbology Editorial Committee of Korean Medicine Schoolset al , Cultured suspensions (200 μl) were transferred to Luria-Bertani 2004; Heoet al ., 2001). Persimmons have also been reported to -1 -1 -1 have effects on blood vessels, strengthening of the lungs and agar (LBA; 10.0 g l tryptone, 5.0 gl yeast extract, 10.0 g l sodium chloride, 10 g l-1 agar; Difco) and potato dextrose agar alleviation of bronchial diseases (Herbology Editorial Committee -1 -1 -1 of Korean Medicine Schools, 2004; Heoet al ., 2001; Chinese (PDA; 4.0 g l potato starch, 20.0 g l dextrose, 15 g l agar; Difco) Pharmacopoeia Commission, 2010). Although genetic diversity plates at 25°C for 24 hrs. and pharmacological studies of astringent persimmons have recently been reported from native provinces (Hwanget al ., 2010; DNA extraction, polymerase chain reaction (PCR) and Choiet al ., 2016), few studies have attempted to analyze the pyrosequencing : Pyrosequencing was carried out following the diversity of microorganisms on astringent persimmons. Thus, this standard protocol (Bueeet al ., 2009; Serkebaeva et al. , 2013). study characterized and identified the microorganisms found on Colonies on agar plates were collected using sterile streaking the native cultivars of astringent persimmon using sticks and placed in sterile plastic conical tubes for identification pyrosequencing-a meta-genomic analysis technique. by pyrosequencing. The samples were grinded in a Precellys Grinder (Bertin Technologies, France), centrifuged and washed Onlinetwice Copy with 1× phosphate-buffered saline (PBS; Biowhittaker, Astringent persimmons are processed typically to remove its astringency to make it consumable. The level of maturity of Walkersville, MD, USA). Pellets were stored at -20°C in 1.5-ml E- astringent persimmons may differ according to the processing tubes before DNAisolation. environment, and microbiological changes can occur owing to variations in moisture, humidity and light (Candiret al ., 2009). Genomic DNA was extracted from the colonies using a Therefore, pyrosequencing is a new high-throughput DNA MagListo Genomic DNA Extraction kit (Bioneer, Korea). The sequencing technique can be used for microbial genome extracted DNA was quantified at 220 nm using a NanoDrop sequencing and discrimination of microorganism strains collected 1000 spectrophotometer (NanoDrop, USA). The DNAextracted under different conditions (Cummingset al ., 2013). The high- from the samples was confirmed by running DNA extracts on throughput nature of pyrosequencing makes it possible to save 1% agarose gels. Bacterial 16S DNA universal primers 27F (5′- Journal of Environmental Biology, March 2018 Microbial community analysis of D. kaki