Isolation and Identification of Microorganisms from the Â

Materials Express

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. The dis- the juice to increase the concentration to 26.5% Brix. The tillation was carried out until approximately 140 mL of previous sugar Brix was 10%. clear distillate was collected in the graduated cylinder. The graduated cylinder was removed from the apparatus and 2.4.2. Yeast Culture its contents allowed to cool to room temperature. Dis- 0.5gofyeast(Saccharomyces cerevisiae) was inoculated tilled water was then carefully added to the distillate until into 250 mL flask containing 100mL sterile water. Cul- 150 mL volume, the same volume as the original wine −1 tured for 30 min at 30 C, and the yeast culture (0.2 mL ) sample. was used for alcohol fermentation. A short-range hydrometer with a range between 0.96– 1.00 was used to measure the specific gravity (SG) of the 2.4.3. Yeast Inoculation distillate at room temperature. After getting the specific The fermentation was carried out following the method of gravity, the conversion of the specific gravity to % Alco- Song et al. [35]. 50 mL pear juice was poured into 250 mL hol by volume (ABV) was carried out using a look up flask sealed with an airtight sealing film, after heating at table [36]. 105 C for 5 min, 2 mL of the yeast culture was inoculated andculturedat22C, 25 C, 28 C and 30 C with static fermentation for about 4 days. 3. RESULTS AND DISCUSSION 3.1. Microbial Isolation and Identification 2.4.4. Fermented Juice Alcohol Analysis After 7 days incubation period, colonies on the PDA plates A combination of distillation and hydrometer reading was were counted and recorded in colony forming unit per mil- used to test the alcohol by volume of the final wine. Since limeter (CFU/mL) (Table I). wine consists of other compounds in addition to water and The colonies observed during this study ranged from ethanol, distillation separates ethanol and water from those 5 colonies (experiment 2 plate B) as the lowest CFU/mL less volatile constituents of wine, and hydrometer reading × 4 (5 10 ) to 43 colonies (experiment 1 plate B2)asthe measures the specific gravity (SG)IP: 192.168.39.210 of the water-ethanol On: Fri,highest 24 Sep CFU/mL 2021 18:54:39 (43 × 104). Most plates carried 10 to 20 mixture. The specific gravity of the distillateCopyright: is recordedAmerican Scientificcolonies Publishers with few going beyond 20 colonies per plate. Delivered by Ingenta and compared to the lookup table used to estimate the Understandably, there were few colonies per plate because alcohol by volume of the distillate. the sample was serially diluted up to 10−4. Microorgan- 150 mL of fermented pear wine was poured into a Article isms are often found on the surface of fruits but few stud- 250 mL graduated cylinder. The content of the gradu- ies have found that they can naturally be inside the fruits ated cylinder was then transferred to the 250 mL boil- as well [37, 38]. In this study, the growth of microorgan- ing flask and 50 mL of distilled water was used to rinse down walls of the graduated cylinder. This helps to cap- isms on the PDA medium illustrated that there are indeed ture all of the alcohol still remaining in the graduated microorganisms present inside “Dongli”. The samples used −4 cylinder and that extra volume of water also prevent the were serially diluted up to 10 and still significant growth flask from boiling dry. A few boiling stones were added of microorganisms was observed. to the boiling flask to prevent violent bumping while boil- 19 strains were prepared from isolated pure cultures, ing and the boiling flask was joined to the foam trap and and used for DNA extraction identification. Genomic DNA cold-water intake was turned on to keep the condenser was extracted from these 19 strains by SDS/CTAB method. cool. A 250 mL graduated cylinder was placed under the The results of the 1% agarose gel electrophoresis were as condenser’s output to collect the distillate. The Bunsen follow (Fig. 1).

Table I. Number of colonies and CFU/mL in 3 different experiments.

Plates Experiment 1 CFU/mL Experiment 2 CFU/mL Experiment 3 CFU/mL

A 22 colonies 22 × 104 14 colonies 14 × 104 16 colonies 16 × 104 B 9 colonies 9 × 104 5 colonies 5 × 104 10 colonies 10 × 104 C 27 colonies 27 × 104 18 colonies 18 × 104 9 colonies 9 × 104 A1 17 colonies 17 × 104 12 colonies 12 × 104 17 colonies 17 × 104 B1 21 colonies 21 × 104 15 colonies 15 × 104 13 colonies 13 × 104 C1 22 colonies 22 × 104 20 colonies 20 × 104 34 colonies 34 × 104 A2 14 colonies 14 × 104 11 colonies 11 × 104 8 colonies 8 × 104 B2 43 colonies 43 × 104 17 colonies 17 × 104 15 colonies 15 × 104 C2 14 colonies 14 × 104 7 colonies 7 × 104 19 colonies 19 × 104

430 Mater. Express, Vol. 11, pp. 428–433, 2021 Isolation and identiﬁcation of microorganisms from the “Dongli” pear Materials Express Ndayambaje et al.

Fig. 1. Successful genomic DNA extraction.

Table II. Summary of identiﬁcation results.

Sequence number Numbering Identiﬁed species

Z1 Ur-1-18-2/r-1-18-1 Curtobacterium citreum Z2 Ur-1-18-1-4 Frigoribacterium faeni Z3 Ur-1-18-3-8 Curtobacterium flaccumfaciens Z4 Ur-1-18-5-9 Frigoribacterium faeni Z5 r-1-29-1-2-3 Brevibacterium frigoritolerans Z6 V-1-18-5 Bacillus subtilis Z7 P-3 Bacillus megaterium Z8 L-u-1 Streptomyces pseudogriseolus Fig. 2. Chart illustrating the evolution of SG of pear juice undergoing Z9 L-u-2 Frigoribacterium faeni Z10 L-e-1-2 Frigoribacterium faeni fermentation at 22 C. Z11 Ur-1-18-7 Microbacterium foliorum Z12 P-1 Bacillus subtilis fermentation was undergoing showed that it was fast in Z13 V-1-18-4 Bacillus megaterium Z14 Ur-1-18-11 Bacillus cereus fermentation at higher temperature (28 C and 30 C) than Z15 F-L Amycolatopsis marina at lower temperature (22 C and 25 C). The juice fermen- Z16 V-1-18-2 Acetobacter pasteurianus tation at 30 C was the fastest to reach the final gravity Z17 V-1-18-1 Acetobacter pasteurianus (FG) because it only took 8 days to complete considering Z18 P-P-2 Bacillus subtilis Article Z19 L-e-3 Phlebiopsis gigantea the 4 days of fermentation prior to recording the daily specific gravity of the fermenting juice (Fig. 5). The FG of IP: 192.168.39.210 On: Fri, 24 Sep 2021 18:54:39 Copyright: American Scientificthe juice Publishers fermenting at 28 C was recorded after 14 days After DNA extraction, all 19 strains have beenDelivered iden- by(Fig. Ingenta 4). At 25 C, the juice fermented slowly and the tified and the microorganisms identified are presented in completion of fermentation was observed after 18 days Table II. (Fig. 3). The slowest fermentation rate was observed at The results of DNA extraction illustrated that 18 strains 22 C, which was the room temperature by the time of out of 19 are bacteria (Table II). The only fungus was the experiment because it was the beginning of winter, and identified as Phlebiopsis gigantea. Surprisingly, all of the the indoor heating system was making inside a bit warmer identified microorganisms cannot be used as fermentation compared to freezing temperatures that were outside. The agents. fermentation took more than 50 days to finally record constant SG (Fig. 2). 3.2. Pear Juice Fermentation The prepared juice original (OG) gravity (see Table III) was 1.098 before undergoing fermentation. When the juice undergoes fermentation, yeasts convert sugar in the juice into alcohol and carbon dioxide and this conversion decreases the specific gravity because the more the alcohol, the lower the specific gravity. After 4 days of fermentation, the specific gravity (SG) was recorded daily until an almost constant SG is recorded for 3 consecutive days. After 4 days, the original gravity (OG) decreased indicating that the conversion of sugar into alcohol by yeasts was taking place. However, the rate of which the

Table III. Initial pH, OG and Brix of juice before fermentation.

Extracted juice + sugar pH 6.5 (adjusted) Original gravity (OG) 1.098 Brix 26.5 (adjusted) Fig. 3. Chart illustrating the evolution of SG of pear juice undergoing fermentation at 25 C.

Mater. Express, Vol. 11, pp. 428–433, 2021 431 Materials Express Isolation and identiﬁcation of microorganisms from the “Dongli” pear Ndayambaje et al.

The recorded ﬁnal gravity (FG) was 0.978, 0.976, 0.977 and 0.976 for wines fermented at 22 C, 25 C, 28 C and 30 C respectively. Comparing these FG to the lookup table [36] used in this study, the results showed that the wine produced after fermentation no matter what time it took to complete was around 17.5% ABV. This is a high concentration for wines but considering that we added sugar to the juice to adjust the concentration to 26.5% Brix, it is understandable that the sugar to the juice con- tributed to the higher alcohol content of the wine.

4. CONCLUSION Our work intended to isolate and identify microorganisms from the “Dongli” pulp that can be used as fermentation agents. 19 microorganisms were identified, and 18 out of 19 were bacteria, with the last one being a fungus Fig. 4. Chart illustrating the evolution of SG of pear juice undergoing (Phlebiopsis gigantea). Since all identified microorgan- fermentation at 28 C. isms cannot be used as fermentation agents, fermentation was carried out using Saccharomyces cerevisiae,andthe Microorganisms in general have different preference fermentation at 30 C proved to be the fastest. Our current when it comes to temperature of the environment in which findings have led us to the conclusion that the microorgan- they are growing. Based on their growth temperature isms naturally present in the pulp of “Dongli” are mostly range, microorganisms constitute psychrotrophic microor- bacteria that cannot be used as fermentation agents. To ganisms, which can grow at refrigeration temperature, ferment “Dongli” pear juice, commercial yeasts (Saccha- mesophilic microorganisms that grow at room temperature romyces cerevisiae) can be purchased and used, and we and thermophilic microorganismsIP: that 192.168.39.210 grow at highertem- On: Fri,recommend 24 Sep 2021 to carry18:54:39 out fermentation at the highest room Copyright: American Scientifictemperature Publishers possible to provide the best growth environ- perature [39]. Fermentation yeasts are mesophilicDelivered microor- by Ingenta ganisms that thrive better at room temperature [40]. ment for yeasts and hence accelerate the conversion of However, the rate of their growth increases with the sugar into alcohol. Pear production is increasing not only

Article increase of temperature. As long as they are still in their in China, but also all over the world, yet pear fruits are range of growth, fermentation yeasts, namely Saccha- less studied compared to other fruits of the Rosaceae fam- romyces cerevisiae, will grow but the rate of growth at ily such as apples. More researches on “Dongli” and other 30 C is bigger than that at 22 C even if it is still under the less studied pear varieties are needed to be able to under- range of temperature at which mesophilic microorganisms stand very well pear fruits. thrive. Conﬂicts of Interest There are no conﬂicts to declare.

Funding The preparation of this paper did not receive any direct ﬁnancial support.

References and Notes 1. Cao, Y., Tian, L., Gao Y. and Liu, F., 2012. Genetic diversity of cultivated and wild Ussurian Pear (Pyrus ussuriensis Maxim.) in China evaluated with M13-tailed SSR markers. Genetic Resources and Crop Evolution, 59(1), pp.9–17. 2. Katayama, H., Amo, H., Wuyun, T., Uematsu, C. and Iketani, H., 2016. Genetic structure and diversity of the wild Ussurian pear in East Asia. Breeding Science, 66(1), pp.90–99. 3. Kaur, R. and Arya, V., 2012. Ethnomedicinal and phytochemical perspectives of Pyrus communis Linn. Journal of Pharmacognosy and Phytochemistry, 1(2), pp.14–19. 4. Challice, J.S. and Westwood, M.N., 2010. Numerical taxonomic Fig. 5. Chart illustrating the evolution of SG of pear juice undergoing studies of the genus pyrus using both chemical and botanical char- fermentation at 30 C. acters. Botanical Journal of the Linnean Society, 2, pp.121–148.

432 Mater. Express, Vol. 11, pp. 428–433, 2021 Isolation and identiﬁcation of microorganisms from the “Dongli” pear Materials Express Ndayambaje et al.

5. Casadei, E. and Albert, J., 2003. Food and agriculture organization considerations for deﬁning functioning of microbial endophytes. of the united nations. Encyclopedia of Food Sciences & Nutrition, Microbiology and Molecular Biology Reviews, 79(3), pp.293–320. 1(2), pp.2587–2593. 24. Abadias, M., Altisent, R., Usall, J., Torres, R., Oliveira, M. and 6. Jun, W. and Hongsheng, G., 2002. The production of Asian pears in Viñas, I., 2014. Biopreservation of fresh-cut melon using the strain China. Acta Horticulturae, 587, pp.71–80. Pseudomonas graminis CPA-7. Postharvest Biology and Technol- 7. Yuanwen and Teng, 2011. The pear industry and research in China. ogy, 96, pp.69–77. Acta Horticulturae, 909, pp.161–170. 25. Lugtenberg, B.J.J., Caradus, J.R. and Johnson, L.J., 2016. Fungal 8. Saito, T., Fang, C., Shin, S., Hwang, H.S. and Sharifani, M., endophytes for sustainable crop production. FEMS Microbiology 2005. Status of the pear industry in Asia. Acta Horticulturae, 671, Ecology, 92(12), pp.1–37. pp.57–63. 26. Alegre, I., Viñas, I., Usall, J., Anguera, M., Figge, M.J. and 9. Qin, G., Tao, S., Cao, Y., Wu, J., Zhang, H., Huang, W. and Abadias, M., 2012.AnEnterobacteriaceae species isolated from Zhang, S., 2012. Evaluation of the volatile proﬁle of 33 Pyrus apples controls foodborne pathogens on fresh-cut apples and ussuriensis cultivars by HS-SPME with GC-MS. Food Chem- peaches. Postharvest Biology and Technology, 74, pp.118–124. istry, 134(4), pp.2367–2382. 27. Strobel, G., 2018. The emergence of endophytic microbes and their 10. Bell, R.L. and Janick, J., 1990. Quantitative genetic analysis of fruit biological promise. Journal of Fungi, 4(2), p.57. quality in pear. Journal of the American Society for Horticultural 28. Thorat, P.P., Sadawarte, S.K., Sawate, A.R. and Machewad, G.M., Science, 115(5), pp.829–834. 2017. Studies on effect of fermentation on physicochemical proper- 11. Monte-Corvo, L., Cabrita, L., Oliveira, C. and Leitão, J., 2000. ties of vegetables and preparation of sauce. International Journal of Assessment of genetic relationships among pyrus species and culti- Current Microbiology and Applied Sciences, 6(8), pp.3537–3545. vars using AFLP and RAPD markers. Genetic Resources and Crop 29. Swain, M.R., Anandharaj, M., Ray, R.C. and Parveen Rani, R., 2014. Evolution, 47(3), pp.257–265. Fermented fruits and vegetables of asia: A potential source of pro- 12. Faoro, I.D. and Orth, A.I., 2015. Flower visiting insects during the biotics. Biotechnology Research International, 2014, pp.1–19. bloom period of Japanese pear orchards in Brazil. Acta Horticul- 30. Paul Ross, R., Morgan, S. and Hill, C., 2002. Preservation and fer- turae, 1094, pp.275–279. mentation: Past, present and future. International Journal of Food 13. Teng, Y., Li, G., Jia, H. and Wu, R., 2013. Changes in volatile Microbiology, 79(1–2), pp.3–16. organic compound composition during the ripening of “Nanguoli” 31. Saranraj, P., Sivasakthivelan, P. and Naveen, M., 2017. Fermentation pears (Pyrus ussuriensis Maxim) harvested at different growing loca- of fruit wine and its quality analysis: A review. Australian Journal tions. Journal of Horticultural Science and Biotechnology, 88(5), of Science and Technology, 1(2), pp.85–97.

pp.563–570. 32. D’Souza, P.A., Rao, S.C., Naik, P.A., Vyas, S., Palan, A.M., Article 14. Shen, T., 1980. Pears in China. Hort Science, 15(1), pp.13–17. Cornelio, B., Shet, V.B. and Rao, C.V., 2016. Production of fer- 15. Bassil, N. and Postman, J.D., 2010. Identification of european and mented fruit juice using unconventional seasonal fruits through batch asian pears using est-ssrs from pyrus.IP:Genetic 192.168.39.210 Resources & CropOn: Fri, 24fermentation. Sep 2021Research 18:54:39 Journal of Chemical and Environmental Sci- Evolution, 57(3), pp.357–370. Copyright: American Scientificences, Publishers4, pp.1–5. 16. Bao, L., Chen, K., Zhang, D., Cao, Y., Yamamoto, T. andDelivered Teng, Y., by33. IngentaAjit, E.J., Dominic, D., Farook, F., Promod, A. and Kumar, B.M.B., 2007. Genetic diversity and similarity of pear (Pyrus L.) cultivars 2018. Preparation of wine from fruits of Musa accuminata and native to East Asia revealed by SSR (simple sequence repeat) mark- Ananas comosus; its physicochemical analyses and sensory evalua- ers. Genetic Resources and Crop Evolution, 54(5), pp.959–971. tion. Integrative Food, Nutrition and Metabolism, 5(6), pp.1–5. 17. Jiang, S., Zheng, X., Yu, P., Yue, X., Ahmed, M., Cai, D. and 34. Obaedo, M.E. and Ikenebomeh, M.J., 2009. Microbiology and pro- Teng, Y., 2016. Primitive genepools of Asian pears and their duction of banana (Musa sapientum)wine.Nigerian Journal of complexhybrid origins inferred from fluorescent sequence-specific Microbiology, 23, pp.1886–1891. amplification polymorphism (SSAP) markers based on LTR retro- 35. Song, S., Pan, L. and Wu, X., 2015. Production of dangshan pear transposons. PLoS One, 11(2), p.e0149192. wine by liquid fermentation based on orthogonal design optimal 18. Battcock, M. and Azam-Ali, S., 1998. Fermented fruits and vegeta- experiments. Journal of Food Engineering and Technology, 2(4), bles. A global perspective. Fao Agricultural Services Bulletin, p.134. pp.17–23. 19. Khatri, P.K. and Sharma, S., 2018. Microbial examination for spoil 36. Amerine, B.M.A. and Ough, C.S., 1988. Methods for Analysis of fruits and vegetables and its isolation, identification, and antimicro- Musts and Wines. J. Wiley. bial sensitivity pattern. International Journal of Current Microbiol- 37. Barth, M., Hankinson, T.R., Zhuang, H. and Breidt, F., 2009.Micro- ogy and Applied Sciences, 7(12), pp.2671–2679. biological spoilage of fruits and vegetables. Compendium of the 20. Prasad, M.P. and Dagar, S., 2014. Identification and characteriza- Microbiological Spoilage of Foods and Beverages. Food Micro- tion of Endophytic bacteria from fruits like Avacado and Black biology and Food Safety, edited by W. Sperber and M. Doyle, grapes. International Journal of Current Microbiology and Applied pp.135–183. Sciences, 3(8), pp.937–947. 38. Sardella, D., Muscat, A., Brincat, J.P., Gatt, R., Decelis, S. 21. Gouda, S., Das, G., Sen, S.K., Shin, H.S. and Patra, J.K., 2016. and Valdramidis, V., 2016. A comprehensive review of the pear Endophytes: A treasure house of bioactive compounds of medicinal fungal diseases. International Journal of Fruit Science, 16(4), importance. Frontiers in Microbiology, 7, pp.1–8. pp.351–377. 22. Khare, E., Mishra, J. and Arora, N.K., 2018. Multifaceted interac- 39. Maria, T., Pedrosa, S. and Clerici, 2013. Ronald E. Wrolstad, food tions between endophytes and plant: Developments and prospects. carbohydrate chemistry. Food Research International, 50(1), pp.307– Frontiers in Microbiology, 9, pp.1–12. 307. 23. Hardoim, P.R., van Overbeek, L.S., Berg, G., Pirttilä, A.M., 40. Rotundifolius, S., Equipment and Assays, Fruits N.C., Ngú, N.L. Compant, S., Campisano, A., Döring, M. and Sessitsch, A., 2015. and Nghiép, N., 2013. United states department of agriculture food The hidden world within plants: Ecological and evolutionary safety and inspection service. Tailieu Vn.

Received: 8 May 2020. Accepted: 9 June 2020.

Mater. Express, Vol. 11, pp. 428–433, 2021 433