PaPer Ital. J. Food Sci., vol. 28 - 2016 9 - Keywords: lactic acid bacteria, xi-gua-mian, fermented watermelon, Taiwan - IsolatIon and characterIzatIon of lactIc acId bacterIa from xi-gua-mian (fermented watermelon), a tradItIonal fermented food In taIwan YI-sheng chena,*, huI-chung wua, chI-rong Yua, zIh-YIn chena, YI-chen lua and fujItoshI YanagIdab aDepartment of Biotechnology, Ming Chuan University, No. 5 De-Ming Road, Gui-Shan, Taoyuan 333, Taiwan b The Institute of Enology and Viticulture, Yamanashi University, 1-13-1 Kitashin, Kofu, Yamanashi 400-0005, Japan *Corresponding author: Tel. +886 33507001 ext. 3540, Fax +886 33593878, email: yisheng@mail.mcu.edu.tw AbstrAct Young watermelon fruit was peeled and pickled for fermentation to produce a unique ferment- ed food named xi-gua-mian (fermented watermelon) in taiwan. In this study, we investigated the LAb microflora in xi-gua-mian. A total of 176 LAb isolates were identified; 118 cultures were iso- lated from the xi-gua-mian sample collected from three different farmers markets and 58 from six young watermelon fruit samples. these isolates were characterized phenotypically and then divid- ed into seven groups (A to G) by restriction fragment length polymorphism analysis, sequencing of 16s ribosomal DNA and other genotypic analysis. Lactobacillus plantarum was the most abun- dant LAb found in xi-gua-mian samples collected in southern taiwan, tainan city and Pediococ- cus pentosaceus was the most abundant LAb in northern taiwan, taoyuan county. We found that LAb stains are similar in samples collected in the same geographic region but significant var- iations were observed between samples collected among different regions. On the other hand, a greater LAb diversity was observed in the young watermelon fruit samples. In addition, 10 Lacto- coccus lactis subsp. lactis showed inhibitory activity against the indicator strain L. sakei subsp. sakei JcM 1157t. this is the first report describing the distribution and varieties of LAb existing in the xi-gua-mian and the young watermelon fruits. mailto:yisheng@mail.mcu.edu.tw 10 Ital. J. Food Sci., vol. 28 - 2016 INtrODuctION Watermelon (Citrullus lanatus) is a popular fruit in taiwan. the farming area dedicated to watermelon production in taiwan is reported to be largest among all fruits (LIN et al., 2009). to have a better harvest, surplus fruits are elim- inated and only one fruit is retained for every stock in the early phase of watermelon culti- vation. In taiwan, farmers use the eliminated young watermelon fruits to produce a unique fermented food named xi-gua-mian (fermented watermelon). these immature watermelon fruits are peeled, cut, mixed with salt (Nacl) and then placed in a bucket. salt is added to a final concentration of approximately 3-6%, and the bucket is sealed with heavy stones placed on the top of the cover. this process usually continues for 3 days and then the exuded water is drained. the bucket is sealed again with heavy stones and the fermen- tation process continues for at least 2 weeks at room temperature. because of the contribution of the lactic acid bacteria (LAb), it has a special sour and sweet flavor. Xi-gua-mian is usually applied as a seasoning for various pork, sea- food and poultry dishes in order to add a slightly acidic taste. Although the product is very popu- lar, it has not been studied in detail. Lactic acid bacteria (LAb) has been frequent- ly found in various taiwanese fermented foods such as yan-tsai-shin (fermented broccoli stems), yan-jiang (fermented ginger), jiang-sun (ferment- ed bamboo shoot), suan-tsai (fermented mus- tard), dochi (fermented black beans), jiang-gua (fermented cucumbers), yan-dong-gua (ferment- ed wax gourd) and pobuzihi (fermented cum- mingcordia) (chANG et al., 2011; cheN et al., 2006a, 2006b, 2010, 2012, 2013a, 2013b; LAN et al., 2009). In these cited studies, various LAb species, such as Enterococcus faecium, Lactoba- cillus plantarum, Lactococcus lactis subsp. lac- tis, Weissella cibaria and W. paramesenteroides, were frequently found in the taiwanese ferment- ed products. however, there has been very lit- tle research reported on LAb distribution in fer- mented watermelon (xi-gua-mian). One important attribute of LAb is the bacte- riocin-producing abilities to inhibit food spoil- age bacteria and many LAb strains isolated from the taiwanese fermented foods were found to produce various bacteriocins. some bacterioc- ins produced by these strains were further iden- tified as novel bacteriocins in the later studies such as enterocin tW21, weissellicin L and en- terocin t (chANG et al., 2013; cheN et al., 2013c; LIANG et al., 2013). the objectives of this study were to isolate LAb from the xi-gua-mian, to identify the isolates to the species level and to detect the antibacteri- 16S rDNA RFLP groups A B C D E F G Sample No. Location pH Salt con. (g/L) Viable acid-pro- ducing cells (log CFU/mL)α Lactic acid (g/L) L. p la nt ar um L. p en to su s P. p en to sa ce us Lc . l ac tis s ub sp . l ac tis Le u. m es en te ro id es W . p ar am es en te ro id es E. c as sl ifl av us Fermented watermelon S1 Tainan 4.6 3.8 7.36±0.18 35.5 30 8 S2 Tainan 3.9 3.8 6.77±0.17 95.0 36 4 S3 Taoyuan 4.1 6.0 8.00±0.05 80.0 40 Fresh watermelon W1 Hualien – – 1.84±0.09 – 7 (1β) 1 2 W2 Hualien – – 3.77±0.01 – 2 1 7 (7) 2 1 W3 Hualien – – 3.25±0.03 – 3 1 3 (2) 1 2 W4 Tainan – – 3.04±0.06 – 10 W5 Tainan – – 3.51±0.08 – 12 W6 Chiayi – – 1.48±0.03 – 3 Total 71 14 40 20 4 3 24 α The data are expressed as the mean±SD (n=3). β Number of BLIS-producing strains. Abbreviations: L., Lactobacillus; P., Pediococcus; Lc., Lactococcus; Leu., Leuconostoc; W., Weissella; E., Enterococcus. table 1 - Analysis results and characteristics of isolates. Ital. J. Food Sci., vol. 28 - 2016 11 al activities of the isolates. Our results provide an example to understand the rich resources of LAb strains in the traditional taiwanese fer- mented food. MAterIALs AND MethODs Xi-gua-mian and the young watermelon fruit samples A total of 3 xi-gua-mian samples (s1-s3) were collected at three traditional farmers markets lo- cated in tainan city and taoyuan county (ta- ble 1, Fig. 1A). In addition, six young watermel- on fruit samples (W1-W6, approximately 8-15 cm in size) were collected from hualien coun- ty, tainan city and chiayi county (table 1, Fig. 1b). samples were stored at 4°c and analyzed within 24 h of acquisition from the markets and the watermelon fields. the salt concentration and ph of xi-gua-mian juice was measured by using a model sK-5s salt meter (sato Keiryoki, tokyo, Japan) and a model b-112 compact ph meter (horiba, Kyoto, Japan), respectively. Lac- tic acid in each xi-gua-mian samples was deter- mined with a D-/L-Lactic Acid test kit (r-biop- harm AG, Darmstadt, Germany), according to the manufacturer’s instructions. Isolation of LAB An initial analysis results showed that the xi- gua-mian samples s1 and s2 contained 3.8 % Nacl and sample s3 contained 6 % (table 1). therefore, Mrs agar (DifcotM Lactobacilli Mrs broth; sparks, MD, usA) containing 3 % Nacl was used for the isolation of LAb from xi-gua-mi- an samples s1 and s2. On the other hand, Mrs agar containing 6 % Nacl was used for isolation from sample s3 and Mrs agar without adding Nacl was used for isolation from young water- melon fruit samples. to distinguish acid-produc- ing bacteria from other bacteria, 1% cacO 3 was added to the Mrs agar, and only colonies with a clear zone around them were selected (KOzAKI et al., 1992). 0.5 g of crushed young watermel- on fruit samples, and 0.5-mL aliquot of each xi- gua-mian juice samples were taken for LAb iso- lation. the isolation procedures of LAb were per- formed according to the methods described by cheN et al. (2013a). RFLP and sequence analysis of 16S rDNA rFLP and sequence analysis of 16s rDNA were used to classify and identify the bacteri- al isolates. A colony Pcr method described by sheu et al. (2000) was performed in this study. Pcr reactions were carried out using a Genom- ics Taq gene amplification Pcr kit (Genomics, taipei, taiwan) and performed on a Gene Amp Pcr system 9700 (Perkinelmer corp., boston, MA, usA) under the following conditions: 95°c for 3 min, 30 cycles of 95°c for 30 s, 60°c for 30 s and 72°c for 90 s, a final extension of 72°c for 10 min, and completion at 4°c (cheN et al., 2013b). 16s rDNA gene was amplified using the 16s rDNA universal primers 27F (5’- AGAGttt - GAtcctGGctcAG -3’) and 1492r (5’- GGttAc- cttGttAcGActt-3’) (cheN et al., 2013b). rFLP analysis of 16s rDNA was also performed, as described by cheN et al., (2013b). In this study, three restriction enzymes, AccII (cG/cG), HaeIII (GG/cc) and AluI (AG/ct) (chen et al., 2013b), were mainly used for grouping. For sequence analysis of 16s rDNA, the Pcr products were purified and then sequenced with the following primer: 5´-GtcAAttcctttGAGttt-3´ (920r). sequence homologies were examined by com- paring the obtained sequences with those in the DNA Data bank of Japan (DDbJ; http://www. ddbj.nig.ac.jp/) using bLAst. Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum A multiplex Pcr assay with recA gene-derived primers was performed using the methods and conditions described by tOrrIANI et al. (2001). Fig. 1 - Pictures of (A) xi-gua-mian and (b) young watermelon fruit. http://www.ddbj.nig.ac.jp http://www.ddbj.nig.ac.jp 12 Ital. J. Food Sci., vol. 28 - 2016 Differentiation of Leuconostoc mesenter- oides and Leu. pseudomesenteroides A rapid identification method described by JANG et al. (2003) was used to distinguish Leu- conostoc mesenteroides and Leu. pseudomesen- teroides isolates. briefly, a Pcr product of the isolate was amplified by using Leuconostoc-spe- cific primers and then digested by using the re- striction enzyme Tsp509I (/AAtt) (JANG et al., 2003). restriction fragments were visualized on a 2% agarose gel in 1× tAe. Differentiation of W. paramesenteroides and W. hellenica In this study, isolate which showed high se- quence homology to W. paramesenteroides and W. hellenica was further confirmed by using the restriction enzyme HhaI (GcG/c) described by cheN et al. (2012). Effect of NaCl on growth of isolates effect of Nacl on growth of isolates was as- sessed, as described by KOzAKI et al. (1992), by testing isolates for growth in Mrs broth contain- ing 0, 3 and 6% Nacl. Detection of antibacterial activity the agar well diffusion method as described by srIONNuAL et al. (2007) was used to detect and determine the antibacterial activities of isolates. Lactobacillus sakei subsp. sakei JcM 1157t was used as the indicator strain in this study. Anti- bacterial activity was further confirmed by ph adjustment and proteinase K treatment (srION- NuAL et al., 2007). to determine whether nisin is the antibacteri- al substance, a Pcr assay with the nisin-specif- ic Pcr primers, NIsL: 5’-cGAGcAtAAtAAAcG- Gc-3’ and NIsr: 5’-GGAtAGtAtccAtGtct - GAAc-3’, described by VILLANI et al. (2001), were used for amplification in this study. In addition, a nisin z producing strain, Lc. lactis subsp. lac- tis c101910 (YANAGIDA et al., 2006) was used as the positive control and a non-bLIs (bacteri- ocin-like inhibitory substance) producing strain was used as the negative control. resuLts In the xi-gua-mian samples collected from dif- ferent markets, analyses of xi-gua-mian juice revealed different salt concentrations from 3.8 to 6.0% and lactic acid concentrations from 35.5 to 95.0 g/L (table 1). the average number of viable acid-producing cells was 7.36±0.18, 6.77±0.17 and 8.00±0.05 log cFu/mL from the xi-gua-mian samples s1, s2 and s3, respec- tively (table 1). the detailed analysis values of each sample are shown in table 1 and a total of 118 acid-producing bacteria were isolated from these samples. On the other hand, a total of 58 acid-produc- ing bacteria were isolated from the young wa- termelon fruit samples. the number of viable acid producing cells on the six different young watermelon fruit samples was listed in table 1. the total 176 isolates were initially divided into six groups (r1-r6) according to cell mor- phology and the results of the 16s rDNA rFLP analysis. Of these isolated strains, 85 were placed in group r1, 40 in group r2, 20 in group r3, 4 in group r4, 3 in group r5, and 24 in group r6, according to rFLP patterns observed following digestion of their DNA with AccII, HaeI- II, and AluI. to identify the isolates, represent- ative strains were randomly selected from each group, and 16s rDNA sequencing analysis was performed. the results identified group r1 iso- lates as Lactobacillus plantarum-related species, group r2 as Pediococcus pentosaceus, group r3 as Lactococcus lactis subsp. lactis, group r4 as Leuconostoc mesenteroides, group r5 as Weis- sella paramesenteroides, and group r6 as Ente- rococcus cassliflavus. the identification of group r1 isolates was further verified using a multiplex Pcr assay with recA gene-derived primers (tOrrIANI et al., 2001). An expected amplification band lo- cated at 318 bp and one at 218 bp (Fig. 2, lane 1 and 2) was respectively obtained from 71 and 14 isolates. seventy-one isolates were therefore identified as L. plantarum and re-classified into group A. the remaining 14 isolates were identi- fied as L. pentosus and re-classified into group b. All 4 isolates in group r4 were confirmed as Leu. mesenteroides based on Tsp509I digested fragments of the Pcr product of Leuconostoc- specific primers and re-classified into group e (JANG et al., 2003) (Fig. 2, lane 3; table 1). Iso- lates in group r5 were further verified based on Hha I digested fragments of their 16s Pcr prod- uct (cheN et al., 2012). All 3 strains were iden- tified as W. paramesenteroides and re-classified into group F (Fig. 2, lane 4; table 1). Following the re-classification of groups r1, r4 and r5, isolates in the remaining groups were also re- classified with a new code. the detailed distri- butions of LAb species are shown in table 1. effect of Nacl on growth of all 176 isolates was estimated. All P. pentosaceus, E. cassliflavus, L. plantarum, L. pentosus, W. paramesenteroides and Lc. lactis subsp. lactis isolates grew well in Mrs broth containing 0, 3 and 6 % Nacl except Leu. mesenteroides isolates. Growth of Leu. mes- enteroides isolates was observed neither in 3 nor 6 % Nacl Mrs broth. ten isolated Lc. lactis subsp. lactis strains showed antibacterial activity against L. sakei subsp. sakei JcM 1157t (table 1). the bLIs pro- duced by all 10 strains maintained their anti- bacterial activities after neutralization (ph 6.5) Ital. J. Food Sci., vol. 28 - 2016 13 but lost their antibacterial activities completely after treatment with proteinase K. In addition, nisin-specific primers were used to amplify a Pcr fragment and identify the bLIs from these 10 strains. An expected amplification band lo- cated at 320 bp (Fig. 2, lane 5) was obtained from all Lc. lactis subsp. lactis isolates and the nisin z producing strain, Lc. lactis subsp. lac- tis c101910 (YANAGIDA et al., 2006). No am- plification band was observed from the nega- tive control strain. DIscussION In this study, LAb diversity in xi-gua-mian samples collected from different farmers markets and young watermelon fruits were studied. the final concentration of lactic acid and low ph val- ues determined in the xi-gua-mian samples sug- gested that LAb contributed to the aroma and flavor development in xi-gua-mian. the experimental data were treated accord- ing to critical values of student’s t-test. the vi- able acid-producing cell numbers between xi- gua-mian and fresh watermelon was significant- ly different (p<0.0002). We also found that the viable acid-producing cell numbers within ge- ographical areas were different, but the statis- tical difference (standard deviation within xi- gua-mian group and fresh watermelon group was 0.61 and 0.93, respectively) was less than that between xi-gua-mian and fresh watermel- on groups (4.56). In addition, halotolerance of all isolates were assessed. All isolated strains grew well in Mrs broth containing 3% and 6 % Nacl except Leu. mesenteroides isolates. Presence of Nacl in xi- gua-mian and isolation medium therefore might limited the growth of Leu. mesenteroides. Pres- ence of Leu. mesenteroides was only observed in fresh watermelon fruits but not in xi-gua-mi- an. It is therefore considered that salt concen- tration has effect on diversity of LAb in the xi- gua-mian. similar influence of Nacl concentra- tions on diversity of LAb in fermented foods has also been found in our previous studies (cheN et al., 2006a; 2006b). compared to the isolation results of xi-gua- mian, fewer viable acid-producing cell number were observed from the young watermelon fruit samples. It is presumably because the raw ma- terial always presents a lower number or mi- croorganisms or the absence of salt in substra- tum used for the isolation from young water- melon fruit samples does not allow the selec- tion of LAb. As in the case of xi-gua-mian sam- ples, LAb stains are similar in samples collect- ed in the same geographic region and diversities were observed between samples collected among different regions in the young watermelon fruit samples (table 1). Different climate conditions were considered as the main factor, which may affect the distribution of LAb. Although xi-gua-mian samples s1 and s2 were collected at different traditional farmers markets located in tainan city, L. plantarum and L. pento- sus were the most abundant LAb found in these two samples (table 1). Different to the isolation results obtained in the tainan city, P. pentosa- ceus was the most abundant LAb found in the sample collected in taoyuan county (table 1). Geographically, tainan city is located in south- ern part of taiwan that belongs to the tropics, while taoyuan county is in northern subtropical regions. It is therefore considered that regional factors, such as climate conditions, raw materi- als for fermentation and fermentation methods, may affect the distribution of LAb. Lactobacillus plantarum has been identified elsewhere as one of the most abundant LAb found in several taiwanese fermented vegeta- bles such as fermented bamboo shoots (jiang- sun), fermented cucumbers (jiang-gua), ferment- ed broccoli stems (yan-tsai-shin) and fermented cummingcordia (pobuzihi) (cheN et al., 2010b, 2012, 2013a, 2013b). As well as L. plantarum, P. pentosaceus also have been previously found Fig. 2 - 16s rDNA rFLP patterns of AccII, HaeIII and AluI digests from Groups A to G. Lane M, size marker; A, AccII digested patterns; h, HaeIII digested patterns; u, AluI digested patterns; 1, amplification products obtained from the recA multiplex assay of L. plantarum isolates; 2, amplification products obtained from the recA multiplex assay of L. pentosus isolates; 3, Tsp509I digested patterns of Leuconostoc-specific Pcr products from Group e isolates; 4, HhaI digested patterns of Group F isolates; 5, Pcr products using nisin-specific primers. 14 Ital. J. Food Sci., vol. 28 - 2016 as the most abundant LAb in the fermented mustard (suan-tsai) (cheN et al., 2006a). In ad- dition, L. plantarum was found both in the par- tial samples of xi-gua-mian and the young wa- termelon fruits. It is therefore considered that L. plantarum found in xi-gua-mian may origi- nate from the young watermelon fruits. to clar- ify these points, advanced analysis on more xi- gua-mian and young watermelon fruit samples will be necessary in the future. the results of the antibacterial activity assay indicated that total 10 Lc. lactis subsp. lactis iso- lates showed inhibitory activities against L. sakei subsp. sakei JcM 1157t. complete inactivation of these bLIs produced by all 10 strains were observed after treating the cell-free supernatant with proteinase K, which indicates the proteina- ceous nature of the active agents. When ampli- fied with nisin-specific primers, the amplification band located at 320 bp indicated the existence of nisin-producing genes and bLIs from these 10 Lc. lactis subsp. lactis could be nisin-related variants (VILLANI et al., 2001; zeNDO et al., 2003). how- ever, detailed information such as heat stability, their effect on enzymes, inhibition spectra, accu- rate molecular mass and amino acid sequences were not established in the current study. Although LAb have been widely found in var- ious fresh fruits, vegetables and various plant pickles, little information on the diversity of LAb associated with fermented watermelon or young watermelon fruits was obtained from previous studies. Future studies in our laboratory will characterize and identify the nisin-like bLIs, and we anticipate that the bLIs of LAb will be useful as food preservatives. the authors also hope that the results of this study can offer use- ful information for the improvement of xi-gua- mian production. 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