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Isolation and Identification of Microorganisms from the  Materials Express 2158-5849/2021/11/428/006 Copyright © 2021 by American Scientific Publishers All rights reserved. doi:10.1166/mex.2021.1913 Printed in the United States of America www.aspbs.com/mex Isolation and identification of microorganisms from the “Dongli” pear and optimization of pear juice fermentation conditions Jean Pierre Ndayambaje1, Ping Zhao1, Xiao Liu1, Zhenji Tian2,∗, Haining He3, and Guangrui Yang3 1School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, Gansu, PR China 2School of Science, Lanzhou University of Technology, Lanzhou 730050, Gansu, PR China 3Gansu Zhongshang Food Quality Test and Detection Company Limited, Lanzhou 730010, Gansu, PR China ABSTRACT The present study attempted to isolate and identify microorganisms that can be used as fermentation agents from the “Dongli” pear cultivar.IP: Out 192.168.39.210 of 19 microorganisms On: Fri, identified 24 Sep 2021 from “Dongli”,18:54:39 18 were bacteria and 1 fungus (phlebiopsis gigantea). All of thoseCopyright: microorganisms American do Scientific not have Publishers the capacity of fermenting pear juice. Using Delivered by Ingenta commercial yeast (Saccharomyces cerevisiae), the fermentation was carried out at 22 C, 25 C, 28 C, and 30 C, and the fermentation at 30 C proved to be the fastest. The alcohol by volume of the final wine after Article fermentation was 17.5%. Keywords: Dongli, Isolation, Identification, DNA Extraction, Fermentation. 1. INTRODUCTION varieties in northern and central China [2, 9]. Accord- “Dongli”, which translates to “frozen pear”, is one of ing to Bassil and Postman (2010), pears can be divided the ussurian pear cultivars that grow wild in Gansu into two groups: European pear (Pyrus communis)and province, China. Over 150 cultivars of ussurian pear Asian pear (Pyrus pyrifolia). European pear (P. commu- (Pyrus ussuriensis) are found in central and northeast- nis) is widely cultivated in Europe, North and South ern China [1, 2]. Ussurian pear (P. ussuriensis) is one America, Africa and Australia, while Asian pear (P. p y ri- of the cultivars of Chinese pear. Based on the origin of folia) is found in East Asia [10–12]. Teng et al. [13] production, pears are classified into Japanese pear (Pyrus reported that four major commercial varieties of Asian pyrifolia), Chinese pear (Pyrus bretschneideri; Pyrus pear cultivated in China are Chinese white pear (Pyrus ussuriensis) and European pear (Pyrus communis) [3, 4]. pyrifolia Nakai), Chinese sand pear (Pyrus bretschnei- China is currently the largest producer of pear with deri Rehder), ussurian pear (Pyrus ussuriensis Maxim), 68% (16.2 million tons) of world production [5]. Pear and Xinjiang pear (Pyrus sinkangensis). Chinese white is the third most widely cultivated fruit in China, after pear (P. pyrifolia Nakai) is mostly grown in the Yangtze apple and orange [6, 7]. The most cultivated pear species River Valley that extends in the provinces of Gansu, in China are P. ussuriensis, P. bretschneideri, P. p y ri- Liaoning and Shanxi in China [14, 15], while Chinese folia, P. communis, P. sinkiangensis,andP. pashia [8]. sand pear (P. bretschneideri Rehder) is native to north- Chinese sand pear (Pyrus bretschneideri Rehder) and ern China, mainly in Hebei, Shandong, Liaoning, Shaanxi, P. ussurien- ussurian pear (Pyrus ussuriensis Maxim.) are the major Guangdong, and Jiangxi [15]. Ussurian pear ( sis Maxim.) is found in central and northern China in the provinces of Gansu and Qinhai [16], while Xinjiang pear ∗Author to whom correspondence should be addressed. (P. sinkiangensis) is mainly distributed in north-western 428 Mater. Express, Vol. 11, No. 3, 2021 Isolation and identification of microorganisms from the “Dongli” pear Materials Express Ndayambaje et al. China in the province of Xinjiang [17]. The trees of 2.2. Sample Preparation “Dongli” are able to resist freezing temperatures for a long The samples were thoroughly washed, disinfected with time and have adapted to the harsh winter conditions of ethanol (85%) and then rinsed in three different changes of the northern and central China. Cao et al. [1] reported that distilled water. They were cut with a sterilized knife and ussurian pear varieties are able to grow at temperatures of a small piece of the pulp (1 g) was put in a test tube con- −45 Cto−52 C. taining 9 ml of distilled water and homogenized using a Microbial investigations conducted on different fruits sterile glass rod. The homogenate was then serially diluted −4 including pears have isolated not only pathogenic bacte- down to 10 . ria on the surface of fruits [18, 19], but also endophytic microorganisms from the inner part of fruits [20, 21]. 2.3. Microbial Isolation and Identification Endophytic microorganisms are microorganisms that live Plates of already prepared potato dextrose agar (PDA) between plant tissues [21, 22]. They pose no negative were inoculated with 0.1ml aliquots of serially diluted samples and incubated at ambient room temperature (25– effect on plants [23], and are reportedly involved in plants 30 C) for 7 days. After 7 days, microbial colonies pre- growth and development [23, 24], and protection against senting different morphologies from the PDA agar plates pathogen attack [20, 25]. Because of their antifungal and were purified by re-plating them on PDA agar plates sev- antimicrobial capabilities, researches have been conducted eral times, and purified colonies were isolated into slant to isolate those microorganisms from plants, and use them test tubes for further identification. as a safer and eco-friendly way of preservation by prevent- Total genomic DNA was extracted from mycelium ing pathogen invasion in highly perishable crops such as obtained from pure cultures. The mycelium was removed fruits and vegetables. Previous studies suggested that once from each culture using a sterile transfer needle and isolated, endophytic microorganisms can be used as bio- placed in a sterile 1.5 mL microcentrifuge tube contain- logical control agents [20], and fermentation agents [26]. ing 300 L of extraction buffer (0.2 M Tris-HCl, 0.25 M Strobel [27] pointed out that isolated endophytic fungi can NaCl, 25 mM EDTA, and 2% Sodium Dodecyl Sulfate, Article be used as fermentation agents during alcoholic fermen- pH 8.5). Uncapped tubes were then placed in a boiling tation. Even if most microorganismsIP: 192.168.39.210 are generally inacti- On: Fri,water 24 Sep bath 2021 for 5 18:54:39 minutes, and cooled to 25 C; then 200 L vated at freezing temperatures, they becomeCopyright: active American again Scientificof phenol Publishers that was equilibrated with extraction buffer once the produce is brought back to ambient temperatures.Delivered by(vol/vol), Ingenta and 200 L of chloroform were added. The This study intends to isolate and identify from “Dongli” tubes were vortexed for 4 minutes and then centrifuged at some of those endophytic microorganisms that can be used 12.000 rpm for 5 minutes. The supernatant was pipetted to a new sterile 1.5 mL tube and 200 L of chloroform was as fermentation agents. added; the mixture was vortexed for 30 seconds and then Fermentation is one of the oldest forms of food preser- centrifuged at 12.000 rpm for 15 minutes. The supernatant vation technologies in the world [28–30]. It is an easy and was again transferred to a new 1.5 mL tube and 200 L environmentally friendly technique that has been employed of isopropanol was added; the capped tube was inverted for generations to preserve fruits in the form of drinks [31]. several times to adequately mix and precipitate DNA and Fermentation involves the conversion of carbohydrates then centrifuged at 12.000 rpm for 15 minutes. to alcohols and Carbon Dioxide or organic acids using The supernatant was discarded, and the nucleic acid microorganisms such as yeasts and bacteria [32]. Home- pellet was washed in 400 L of 70% ethanol and cen- made wine production has been practiced with apple and trifuged at 12.000 rpm for 5 minutes. Again, the super- pear [33] as a way of preservation. “Perry” or pear cider natant was discarded, and the nucleic acid pellet was is the common name of a product obtained by fermenting air-dried for 10 minutes, resuspended in 50 Loflow- pear juice [34]. During this study, “Dongli” juice fermen- TE buffer (10 mM Tris-HCl and 0.1 mM EDTA, pH 8.5), tation was carried out at various temperatures to deter- and gently agitated to dissolve the DNA. DNA was treated minee the optimum fermentation temperature suitable for with Ribonuclease A for a final concentration of 10 gof “Dongli” wine processing. RNase/mL for 1 hour at 37 C. 2.4. Pear Juice Fermentation 2. MATERIALS AND METHODS 2.4.1. Juice Preparation 2.1. Sample Collection After cleaning and washing, 1 kg of pear fruits was pressed “Dongli” were randomly collected in separate batches of with 500 mL of pure water added to get the juice out, and 3 from orchards and local markets in Lanzhou, China. 10 g of Sodium erythrobate was added to protect the color. A clean polyethylene bag was used to carry fruits to the 2.5 g of pectinase was added into the juice while citric laboratory. The fruits were stored at 4 C and then ana- acid was used to adjust the value of pH to 5.4, which was lyzed within 24 hours of acquisition. bathed at 50 C for 60 min to improve the juice yield, Mater. Express, Vol. 11, pp. 428–433, 2021 429 Materials Express Isolation and identification of microorganisms from the “Dongli” pear Ndayambaje et al. followed by pH adjustment to 6.5 by NaOH, then filtered burner was then set to a low flame and the fermented with 200 mesh sieves. About 400 g of sugar was added to pear wine sample was brought to start boiling.
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