Curr Microbiol (2014) 68:440–447 DOI 10.1007/s00284-013-0495-2
Diversity of Lactic Acid Bacteria Associated with Fresh Coffee Cherries in Taiwan Kun-hon Leong • Yi-sheng Chen • Shwu-fen Pan • Jen-jye Chen • Hui-chung Wu • Yu-chung Chang • Fujitoshi Yanagida
Received: 6 August 2013 / Accepted: 13 October 2013 / Published online: 1 December 2013 Ó Springer Science+Business Media New York 2013
Abstract A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substanceproducing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.
Kun-hon Leong and Yi-sheng Chen have contributed equally to this work. K. Leong Y. Chen (&) S. Pan J. Chen H. Wu Y. Chang Department of Biotechnology, Ming Chuan University, No. 5 De-Ming Road, Gui-Shan Township, Taoyuan 333, Taiwan e-mail: [email protected] F. Yanagida The Institute of Enology and Viticulture, Yamanashi University, 1-13-1 Kitashin, Kofu, Yamanashi 400-0005, Japan
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Introduction Brazil, Paraguay, Venezuela, Colombia, Indonesia, Ethiopia, India, and Mexico are the main coffee producers in the world. Coffea arabica (arabica) and C. canephora (robusta) are the most common coffee species on the world market [19]. As teatime has thrived in Taiwan in recent years, the consumer market for coffee has grown accordingly [20]. Therefore, more local farmers have begun cultivating coffee in Taiwan, with C. arabica L. (arabica) being the main coffee species cultivated. Diversity of lactic acid bacteria (LAB) associated with coffee fermentation,, and coffee pulp silage has been previously studied [1, 6, 7, 9, 17]. Species such as Leuconostoc mesenteroides, and Lactobacillus plantarum were frequently found in most of these studies [1, 2, 7, 17]. However, studies on LAB distribution in fresh coffee cherries remain scarce. Many fungi produce dangerous chemical compounds known as mycotoxins [8]. Food-borne fungi such as Aspergillus and Penicillium have been frequently found in coffee beans. Species of Aspergillus are the main producers of mycotoxins, such as aflatoxin and ochratoxin A (OTA), in coffee [3, 7, 12, 14]. Previous studies have proposed the possibility of applying LAB as potential antagonists of food-borne fungi [5, 7, 13]. Antifungal LAB could be applied to coffee production and are, therefore, attracting special interest. The objectives of this study were to isolate LAB from fresh coffee cherries, identify the isolates to the species level, and determine their antibacterial, and, and antifungal activities. Materials and Methods Coffee Cherry Samples A total of three coffee cherry samples (Coffea arabica L.) were collected at different coffee farms located in the
1
1 1 (1) 1
1 2 10 30 a
4.0 9 101 1,200–1,300
Number of BLIS-producing strains
44 Chiayi F3
44 700–800
700–800
Altitude (m)
7
6
2 6 7 (7a) 1
5
9
25
Pingtung F2
2.1 9 105
Miaoli F1
Total
Enterococcus faecalis Enterococcus sp. Weissella thailandensis Leuconostoc citreum Lactococcus lactis subsp. lactis Weissella confusa Location Coffee farm
Viable acidproducing cells (CFU/g)
A
Sequence analysis of 16S rDNA was used to identify the bacterial isolates. PCR reactions were carried out using the methods described previously. The PCR products were purified with a Clean/Gel Extraction Kit (BioKit, Miaoli, Taiwan) and then sequenced with the following primer:
16S rDNA RFLP group
Sequence Analysis of 16S rDNA
Table 1 Analyses results and characteristics of isolates
16S rDNA RFLP analysis was used to classify the bacterial isolates. PCR colony identification was performed using the method described by Sheu et al. [18]. PCR reactions were carried out using a Genomics Taq gene amplification PCR kit (Genomics, Taipei, Taiwan) and performed on a Gene Amp PCR System 9700 (PerkinElmer Corp., Boston, MA, USA), following the methods described by Chen et al. [4]. Three different restriction enzymes, AccII (CG/CG), HaeIII (GG/CC), and AluI (AG/CT), were used to generate the main groups in this study [4]. The PCR products were visualized on a 2 % agarose gel in 1 9 TAE.
B
16S rDNA RFLP Analysis
2.6 9 102
C
D
E
F
G
MRS agar (DifcoTM Lactobacilli MRS Broth; Sparks, MD, USA) was used for the isolation of LAB. Coffee cherry samples collected from each farm were mixed together and then crushed. A 0.5 g sample of crushed coffee cherries was mixed with 4.5 mL of a 0.75 % NaCl solution. The resultant solution was diluted (10 to 1,000-fold) and then spread directly onto the surface of MRS agar plates. Plates were incubated under anaerobic conditions (Mitsubishi AnaeroPakTM System, Pack-Anaero; Mitsubishi Gas Chemicals, Tokyo, Japan) at 30 °C for 3–5 days. To distinguish the acid-producing bacteria from other bacteria, 1 % CaCO3 was previously added to the MRS agar, and only colonies with a surrounding clear zone were selected. Colonies of acid-producing bacteria were randomly selected from MRS plates and purified by replating on MRS agar plates. Colonies were reselected and initially examined for Gram staining and the production of catalase. Only Gram-positive, catalase-negative strains were selected. The selected strains were stored at -80 °C in 10 % skim milk broth.
Lactobacillus plantarum
Isolation of LAB
Leuconostoc pseudomesenteroides
H
I
following cities: Miaoli County (F1, at an approximate altitude in the range of 700–800 m), Pingtung County (F2, at an approximate altitude in the range of 700–800 m), and Chiayi County (F3, at an approximate altitude in the range of 1,200–1,300 m) (Table 1). More than 100 coffee cherries were randomly sampled at each coffee farm and then collected in sterile bags. Samples were analyzed within 24 h of acquisition from the coffee farms.
1
441 Enterococcus sp.
K. Leong et al.: Diversity of Lactic Acid Bacteria
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442
50 -GTCAATTCCTTTGAGTTT-30 (920R). DNA sequencing was performed using an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Partial sequencing, approximately 900 bp ahead, was performed and manually aligned with Genetyx-Win version 5.1 (Genetyx Corp., Tokyo, Japan) software. Sequence similarities were examined by comparing the obtained sequences with those in the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/) using BLAST. Sequence Analysis of the Housekeeping Gene pheS To confirm the identification results, representative strains were randomly selected from each group, and pheS gene sequencing analysis was performed. Amplification and sequencing were performed using forward primer pheS-21-F (50 -CAYCCNGCHCGYGAYATGC-30 ), and reverse primer pheS-22-R (50 -CCWARVCCRAARGCAAARCC-30 ) or pheS-23-R (50 -GGRTGRACCATVCCNGCHCC-30 ), which were previously designed and reported by Naser et al. [15]. Sequence homologies were assessed by comparing the obtained sequences with those in the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/) using BLAST.
K. Leong et al.: Diversity of Lactic Acid Bacteria
brief, potato dextrose agar plates were overlaid with the MRS soft agar (0.7 %) seeded with 104 spores/mL of the fungal culture. The cultured MRS broth of each LAB strain was mixed well by vortexing, and then streaked onto the surface of the fungal pre-seeded MRS soft agar plates. The streak dimension was approximately 3 9 7 mm. The plates were incubated aerobically at 30 °C for 72 h. Clear zones of inhibition were recorded by determining the distance from the bacterial streak to the edge of visible fungal hyphae. The results were scored as follows: no visible inhibition; w, weak inhibition (inhibition area B 1 mm); ?, inhibition area from 1 to 2 mm; and ??, inhibition area [ 2 mm. Detection of Antibacterial Activity The agar well diffusion method described by Yanagida et al. [24] was used to detect and determine the antibacterial activities of isolates. Lactobacillus sakei JCM 1157T was used as the indicator strain in this study. Antibacterial activity was further confirmed by pH adjustment, and proteinase K treatment [24].
Results Differentiation of Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum A multiplex PCR assay with recA gene-derived primers was performed using the methods and conditions described by Torriani et al. [23]. The PCR products were visualized on a 2 % agarose gel in 1 9 TAE. Physiological Analysis The effects of low initial pH, and temperature on growth were assessed in this study. The experiments were carried out by inoculating an overnight-cultured colony of each isolate into 200 lL of MRS broth adjusted to pH 5.0, and 4.5. Growths at 10, 25, 30, 37, and 45 °C were assessed using the method described by Kozaki et al. [11]. Cell growths of two replicates were turbidometrically determined at 600 nm by means of a SpectraMax 190 Microplate reader (Molecular Devices, Sunnyvale, CA, USA). Uninoculated MRS broth was used as a negative control. Screening for Antifungal Activity of LAB Isolates The coffee cherries LAB isolates were screened for antifungal activity using the agar streak assay described by Lan et al. [13]. The fungal strain Aspergillus flavus ATCC 32592 was used as the indicator strain in this study. In
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A total of 102 acid-producing bacteria were isolated in the current study; 26 were from the coffee farm F1, 66 were from the coffee farm F2, and, and 10 were from the coffee farm F3. Cell viability was assessed using MRS plates and is listed in Table 1. According to cell morphology and the results of the 16S rDNA RFLP analysis, the 102 isolates were divided into nine groups (A to I; Fig. 1). To identify the isolates, partial 16S rDNA and pheS gene sequencing analyses were performed. A total of 44 isolates in group A were identified as Weissella confusa, 30 isolates in group B as Leuconostoc pseudomesenteroides, 10 isolates in group C as Lactobacillus plantarum-related species, 7 isolates in group D as Lactococcus lactis subsp. lactis, 6 isolates in group E as Leuconostoc citreum, 2 isolates in group F as Weissella thailandensis, 1 isolate in group G (strain no. ALS12) as Enterococcus sp., 1 isolate in group H as Enterococcus faecalis, and 1 isolate in group I (strain no. ALS3) as Enterococcus sp. (Table 1). The sequences determined in this study have been deposited in the DDBJ database with accession numbers from AB854169 to AB854277. The 10 Lactobacillus plantarum-related isolates were further verified using a multiplex PCR assay with recA gene-derived primers [23]. An expected amplification band located at 318 bp was obtained from all 10 isolates (Fig. 1). The 10 isolates were therefore identified as Lactobacillus plantarum.
K. Leong et al.: Diversity of Lactic Acid Bacteria
The unidentified enterococcal strain ALS12 showed high 16S rRNA gene sequence similarity (99.4 %) to Enterococcus casseliflavus CECT 969T (AJ420804). However, strain ALS12 showed the highest sequence homology, up to 96.2 %, with the pheS gene sequences (AB854276) of Enterococcus casseliflavus CECT 969T (AJ843470). Strain ALS12 was therefore identified as Enterococcus sp. in the current study. On the other hand, strain no. ALS3 showed the highest sequence similarity, up to 98.6 %, with the 16S rRNA gene sequences (AB807774) of Enterococcus pallens ATCC BAA-351T (DQ411812). However, no identical result could be obtained for strain ALS3 when comparing the 16S
443
rDNA sequence to that of other known Enterococcus species. Strain ALS3 was further verified using rpoA and pheS gene sequencing analyses [15]. Strain ALS3 showed the highest sequence homology, up to 90.3 %, with the rpoA gene sequences (AB807776) of Enterococcus avium ATCC 14025T (AJ843518) and 81.1 % with the pheS gene sequences (AB807775) of Enterococcus faecalis LMG 7937T (AJ843387). Based on the results obtained in the current study, strain ALS3 was therefore identified as Enterococcus sp. Sequences of the representative strains of each group were aligned using CLUSTAL W software [22], and distances were calculated, according to Kimura’s two-
Fig. 1 16S rDNA RFLP patterns of AccII, HaeIII, and AluI digests from Groups A to I. 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
Fig. 2 Neighbor-joining tree of isolates and related type strains based on 16S rRNA sequences. Bootstrap values are indicated at branch points based on 1,000 iterations. Bacillus subtilis NCDO 1769T was used as an outgroup. Bar 0.02 substitutions per nucleotide position
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444
K. Leong et al.: Diversity of Lactic Acid Bacteria
Fig. 3 Neighbor-joining tree of isolates and related type strains based on pheS gene sequences. Bootstrap values are indicated at branch points based on 1,000 iterations. Streptococcus gordonii LMG 14516T was used as an outgroup. Bar 0.05 substitutions per nucleotide position
parameter model [10]. Phylogenetic trees were then constructed using the neighbor-joining method [16], with bootstrap analysis based on 1,000 iterations. The MEGA 5.05 package [21] was used for all analyses. Phylogenetic analyses of the 16S rRNA and pheS gene sequences obtained in this study are shown in Figs. 2 and 3, respectively. Cell growth at different initial pH and temperatures was determined, and the results are listed in Table 2. Inhibitory activities against A. flavus ATCC 32592 were observed with 9 Leuconostoc pseudomesenteroides, 2 Leuconostoc citreum, 5 Lactobacillus plantarum, 17 Weissella confusa, and, and 1 Enterococcus faecalis strains. Specifically, inhibitory abilities against A. flavus ATCC 32592 of one Lactobacillus plantarum (strain PD101) , and 3 Weissella confusa (strains PD103, PD104, and, and PD106) were scored as ‘‘??’’; six Leuconostoc pseudomesenteroides (strains CF113, CF115, CF119, CF120, CF122, and CF123), 1 Lactobacillus plantarum (strain PD113), and, and 12 Weissella confusa (strains PD107, PD110, PD111, PD114, PD116, PD202, PD203,
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PD205, PD208, PD414, PD415, and, and PD416) were scored as ‘‘?’’; the remaining strains were scored as ‘‘w’’ (strains CF104, CF124, CF125, CF126, PD108, PD109, PD102, PD112, PD212, PD115, and ALS13). In addition, seven Lactococcus lactis subsp. lactis, and, and one Enterococcus faecalis strains showed the antibacterial activities against the indicator strain Lactobacillus sakei JCM 1157T (Table 1). The bacteriocin-like inhibitory substances (BLISs) produced by all the eight strains maintained their antibacterial activities after neutralization (pH 6.8); however, activities were completely lost after treatment with Proteinase K.
Discussion Schillinger et al. [17] reported that various Leuconostoc, and Weissella species were isolated from coffee fermentation; and Leuconostoc mesenteroides, Leuconostoc citreum,and Leuconostoc pseudomesenteroides were the most abundant LAB found in this study. Similar results were
0 0 1 0 0 1 0 0 1 2 0 0 0 0 6 0 W., Weissella; Leu., Leuconostoc; L., Lactobacillus; Lc., Lactococcus; Ent., Enterococcus
33
? OD value [ 0.5; w OD value C 0.2; - OD value \ 0.2
0
445
1 6 0 0 10 5 2 23 3
1
Growth at pH 4.5
8
0
0 0
1 0
0 0
0 1
1 1
0 0
0 0
1 2
0 2
0 0
0 3
0 0
3 0
6 0
7 0
0 7
0 2
0 0
0 8
10 5
26 1
2 23
3 0
3 38 Growth at pH 5.0
0 44 Growth at 45 °C
3
0
0 0
0 1
1 0
0 0
0 1
1 0
0 0
0 1
1 0
0 0
0 2
2 0
0 0
0 6
6 0
0 3
0 7
4 0
0 0
0 10
10 0
16 1
0 30
13 0
0
44 Growth at 37 °C
0
44 Growth at 30 °C
0
1
0 0
0 0
1 0
1 0
0 1
0 0
0 0
0 1
1 0
0 0
0 2
0 3
0 0
3 0
6 0
7 0
0 7
0 6
0 0
3 1
10 0
18 8
11 19
4 16
0 2 Growth at 25 °C
23 5
42
Growth at 10 °C
w ? w ? w ? ? Physiological analysis
w
-
?
w
-
?
w
-
?
w
-
6 7 10 30 44 Total number
Leu. citreum Lc. lactis subsp. lactis Leu. pseudomesenteroides W. confusa
L. plantarum
B A 16S rDNA RFLP group
Table 2 Phenotypic characteristics of isolates
C
D
E
-
?
w
-
?
w
-
1 1 2
Ent. faecalis Enterococcus sp. W. thailandensis
F
G
H
-
1
Enterococcus sp.
I
K. Leong et al.: Diversity of Lactic Acid Bacteria
found in the studies of Avallone et al. [1, 2], in which Leuconostoc mesenteroides was the predominant species found in coffee (C. arabica L.) fermentation. In this study, Leuconostoc, and Weissella species were also found in the fresh coffee cherries. These findings indicate that the LAB present in coffee fermentation probably originated from the fresh coffee cherries. Besides, Leuconostoc and Weissella species, and Lactobacillus plantarum were also found in the silage of fresh coffee pulp [7]. In the current study, both Leuconostoc pseudomesenteroides and Lactobacillus plantarum were found in the coffee cherries of farms F1 and F2, which were located at a similar altitude in the range of 700–800 m (Table 1). Nevertheless, differences in LAB diversity were also observed in coffee farms F1 and F2. Isolates of Weissella confusa, and Leuconostoc citreum were found only at the coffee farm F2, where Weissella confusa was the most abundant LAB found. Regional similarities and differences in diversity were observed in the current study. The LAB species found at coffee farms F1 and F2 were similar to the findings of previous studies [1, 2, 7]. However, a different LAB diversity was observed at the coffee farm F3. Lactococcus lactis subsp. lactis was the most abundant LAB in F3 samples, and was not found in the coffee cherry samples collected from farms F1 and F2. In addition, the remaining enterococcal cultures were also found only at the coffee farm F3 (Table 1). Altitude was the major difference between coffee farm F3 and the other two farms. It is therefore thought that geographical factors, especially the altitude and climate, may affect the distribution of LAB. To confirm this, further experiments are necessary in the future. The obtained results of growth temperature analysis indicated that the most isolates grew well at 25 and 30 °C. Only a small number of isolates grew at 10 and 45 °C. In addition, the most isolates grew well at low initial pH of 5.0 and 4.5, except for Weissella thailandensis and a few of the Weissella confusa, and Leuconostoc pseudomesenteroides isolates (Table 2). However, a relationship between these factors and LAB distribution was not observed in the current study. The antifungal activities of LAB isolates were determined in this study. The results suggested that the differences in antifungal activities can be identified at the strain level. Similar to the previous studies of Djossou et al. [7] and Lan et al. [13], some differences in antifungal ability between strains were also observed. Here, for example, strains PD101, CF104, and CF105 have been identified as Lactobacillus plantarum cultures. It is proposed that strains with high antifungal ability may have utility in decreasing the numbers of mycotoxin-producing fungi during coffee fermentation. Besides antifungal activity, a total of seven Lactococcus lactis subsp. lactis, and one Enterococcus faecalis strains
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were found to have BLIS-producing abilities (Table 1). However, the detailed characterization of BLISs, including heat stability, their effect on enzymes, and their inhibition spectra, was not established in the current study. Future studies in our laboratory will characterize and identify the BLISs, and we anticipate that the BLISs of coffee cherryisolated LAB will be useful as food preservatives. Furthermore, it was found that pheS or rpoA gene sequences provided more efficient and accurate information than 16S rRNA gene sequences and phenotypic characteristics in the identification procedures. For example, isolates in group B showed the high 16S rRNA sequence similarities to both Leuconostoc pseudomesenteroides (99.9 %), and Leuconostoc mesenteroides (99.6 %) species. However, the pheS gene of group B isolates showed high sequence similarities to Leuconostoc pseudomesenteroides LMG 11482T (up to 98.0 %), but low similarities to Leuconostoc mesenteroides LMG 6893T (*82.1 %). Isolates in group B were therefore identified as Leuconostoc pseudomesenteroides. Similar results were also observed in strains ALS12, and ALS3. The results of genetic analyses of strains ALS12 and ALS3 suggest the possibility of being two potential novel species or subspecies of Enterococcus. To clarify this, additional genetic and physiological data, such as GC content, saccharide fermentation ability,, and DNA–DNA relatedness values between strains and the closest type strains, are necessary. In conclusion, this is the first report describing the distribution and varieties of LAB that exist in fresh coffee cherries in Taiwan. Numerous cultures with antifungal abilities or BLIS-producing abilities were found. Two potential novel species or subspecies of LAB were also found in the current study. Future studies in our laboratory will characterize and identify the antifungal substances and BLISs, and we anticipate that the antifungal substances or BLISs of coffee cherry-isolated LAB will be useful in food preservation. Application of antifungal LAB in coffee cultivation may reduce pecuniary loss in coffee production. In addition, some isolates found in the fresh coffee cherries may be applicable as starters in coffee fermentation. We expect that the results of this study can offer useful information for the improvement of coffee production. Acknowledgments The authors would like to thank the National Science Council Taiwan for financially supporting this study, under Contract No. NSC 101-2313-B-130-001 granted to Shwu-fen Pan, and Contract No. NSC 102-2313-B-130-001 granted to Yi-sheng Chen.
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K. Leong et al.: Diversity of Lactic Acid Bacteria 2. Avallone S, Brillouet JM, Guyot B, Olguin E, Guiraud JP (2002) Involvement of pectolytic micro-organisms in coffee fermentation. Int J Food Sci Technol 37:191–198 3. Batista LR, Chalfoun SM, Prado G, Schwan RF, Wheals AE (2003) Toxigenic fungi associated with processed (green) coffee beans (Coffea arabica L.). Int J Food Microbiol 85:293–300 4. Chen YS, Wu HC, Pan SF, Lin BG, Lin YH, Tung WC, Li YL, Chiang CM, Yanagida F (2013) Isolation and characterization of lactic acid bacteria from yan-taozih (pickled peaches) in Taiwan. Ann Microbiol 63:607–614 5. Crowley S, Mahony J, van Sinderen D (2013) Broad-spectrum antifungal-producing lactic acid bacteria and their application in fruit models. Folia Microbiol 58:291–299 6. De Bruyne K, Schillinger U, Caroline L, Boehringer B, Cleenwerck I, Vancanneyt M, De Vuyst L, Franz CM, Vandamme P (2007) Leuconostoc holzapfelii sp. nov., isolated from Ethiopian coffee fermentation and assessment of sequence analysis of housekeeping genes for delineation of Leuconostoc species. Int J Syst Evol Microbiol 57:2952–2959 7. Djossou O, Perraud-Gaime I, Mirleau FL, Rodriguez-Serrano G, Karou G, Niamke S, Ouzari I, Boudabous A, Roussos S (2011) Robusta coffee beans post-harvest microflora: Lactobacillus plantarum sp. as potential antagonist of Aspergillus carbonarius. Anaerobe 17:267–272 ´ (2012) Presence of Aspergillus and other 8. Gamboa-Gaita´n MA fungal symbionts in coffee beans from Colombia. Acta Biol Colomb 17:39–50 9. Guzma´n-Lo´pez O, Loera O, Parada JL, Castillo-Morales A, Martı´nez-Ramı´rez C, Augur C, Gaime-Perraud I, Saucedo-Castan˜eda G (2009) Microcultures of lactic acid bacteria: characterization and selection of strains, optimization of nutrients and gallic acid concentration. J Ind Microbiol Biotechnol 36:11–20 10. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120 11. Kozaki M, Uchimura T, Okada S (1992) Experimental manual of lactic acid bacteria. Asakurasyoten, Tokyo, pp 29–72 12. Kumar S, Kunwar A, Gautam S, Sharma A (2012) Inactivation of A. ochraceus spores and detoxification of ochratoxin A in coffee beans by gamma irradiation. J Food Sci 77:T44–T51 13. Lan WT, Chen YS, Wu HC, Yanagida F (2012) Bio-protective potential of lactic acid bacteria isolated from fermented wax gourd. Folia Microbiol 57:99–105 14. Magnani M, Fernandes T, Prete CEC, Homechim M, Ono EYS, Vilas-Boas LA, Sartori D, Furlaneto MC, Fungaro MHP (2005) Molecular identification of Aspergillus spp. isolated from coffee beans. Sci Agric 62:45–49 15. Naser SM, Thompson FL, Hoste B, Gevers D, Dawyndt P, Vancanneyt M, Swings J (2005) Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151:2141–2150 16. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 17. Schillinger U, Boehringer B, Wallbaum S, Caroline L, Gonfa A, Kostinek MHN, Holzapfel WH, Franz CM (2008) A genus-specific PCR method for differentiation between Leuconostoc and Weissella and its application in identification of heterofermentative lactic acid bacteria from coffee fermentation. FEMS Microbiol Lett 286:222–226 18. Sheu DS, Wang YT, Lee CY (2000) Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR. Microbiology 146:2019–2025 19. Silva CF, Batista LR, Abreu LM, Dias ES, Schwan RF (2008) Succession of bacterial and fungal communities during natural
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Diversity of Lactic Acid Bacteria Associated with Fresh Coffee Cherries in Taiwan Kun-hon Leong • Yi-sheng Chen • Shwu-fen Pan • Jen-jye Chen • Hui-chung Wu • Yu-chung Chang • Fujitoshi Yanagida
Received: 6 August 2013 / Accepted: 13 October 2013 / Published online: 1 December 2013 Ó Springer Science+Business Media New York 2013
Abstract A total of 102 lactic acid bacteria (LAB) were isolated from three different coffee farms in Taiwan. These isolates were classified and identified by the restriction fragment length polymorphism analysis and sequencing of 16S ribosomal DNA. Heterofermentative Leuconostoc, and Weissella species were the most common LAB found in two farms located at an approximate altitude of 800 m. Lactococcus lactis subsp. lactis was the most common LAB found in the remaining farm was located at an approximate altitude of 1,200 m. It is therefore suggested that the altitude and climate may affect the distribution of LAB. On the basis of phylogenetic analysis, two strains included in the genera Enterococcus were considered as two potential novel species or subspecies. In addition, a total of 34 isolates showed the antifungal activity against Aspergillus flavus. Moreover, seven Lactococcus lactis subsp. lactis strains and one Enterococcus faecalis strain were found to have bacteriocin-like inhibitory substanceproducing capability. These results suggest that various LAB are associated with fresh coffee cherries in Taiwan. Some of the isolates found in this study showed potential as antifungal agents.
Kun-hon Leong and Yi-sheng Chen have contributed equally to this work. K. Leong Y. Chen (&) S. Pan J. Chen H. Wu Y. Chang Department of Biotechnology, Ming Chuan University, No. 5 De-Ming Road, Gui-Shan Township, Taoyuan 333, Taiwan e-mail: [email protected] F. Yanagida The Institute of Enology and Viticulture, Yamanashi University, 1-13-1 Kitashin, Kofu, Yamanashi 400-0005, Japan
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Introduction Brazil, Paraguay, Venezuela, Colombia, Indonesia, Ethiopia, India, and Mexico are the main coffee producers in the world. Coffea arabica (arabica) and C. canephora (robusta) are the most common coffee species on the world market [19]. As teatime has thrived in Taiwan in recent years, the consumer market for coffee has grown accordingly [20]. Therefore, more local farmers have begun cultivating coffee in Taiwan, with C. arabica L. (arabica) being the main coffee species cultivated. Diversity of lactic acid bacteria (LAB) associated with coffee fermentation,, and coffee pulp silage has been previously studied [1, 6, 7, 9, 17]. Species such as Leuconostoc mesenteroides, and Lactobacillus plantarum were frequently found in most of these studies [1, 2, 7, 17]. However, studies on LAB distribution in fresh coffee cherries remain scarce. Many fungi produce dangerous chemical compounds known as mycotoxins [8]. Food-borne fungi such as Aspergillus and Penicillium have been frequently found in coffee beans. Species of Aspergillus are the main producers of mycotoxins, such as aflatoxin and ochratoxin A (OTA), in coffee [3, 7, 12, 14]. Previous studies have proposed the possibility of applying LAB as potential antagonists of food-borne fungi [5, 7, 13]. Antifungal LAB could be applied to coffee production and are, therefore, attracting special interest. The objectives of this study were to isolate LAB from fresh coffee cherries, identify the isolates to the species level, and determine their antibacterial, and, and antifungal activities. Materials and Methods Coffee Cherry Samples A total of three coffee cherry samples (Coffea arabica L.) were collected at different coffee farms located in the
1
1 1 (1) 1
1 2 10 30 a
4.0 9 101 1,200–1,300
Number of BLIS-producing strains
44 Chiayi F3
44 700–800
700–800
Altitude (m)
7
6
2 6 7 (7a) 1
5
9
25
Pingtung F2
2.1 9 105
Miaoli F1
Total
Enterococcus faecalis Enterococcus sp. Weissella thailandensis Leuconostoc citreum Lactococcus lactis subsp. lactis Weissella confusa Location Coffee farm
Viable acidproducing cells (CFU/g)
A
Sequence analysis of 16S rDNA was used to identify the bacterial isolates. PCR reactions were carried out using the methods described previously. The PCR products were purified with a Clean/Gel Extraction Kit (BioKit, Miaoli, Taiwan) and then sequenced with the following primer:
16S rDNA RFLP group
Sequence Analysis of 16S rDNA
Table 1 Analyses results and characteristics of isolates
16S rDNA RFLP analysis was used to classify the bacterial isolates. PCR colony identification was performed using the method described by Sheu et al. [18]. PCR reactions were carried out using a Genomics Taq gene amplification PCR kit (Genomics, Taipei, Taiwan) and performed on a Gene Amp PCR System 9700 (PerkinElmer Corp., Boston, MA, USA), following the methods described by Chen et al. [4]. Three different restriction enzymes, AccII (CG/CG), HaeIII (GG/CC), and AluI (AG/CT), were used to generate the main groups in this study [4]. The PCR products were visualized on a 2 % agarose gel in 1 9 TAE.
B
16S rDNA RFLP Analysis
2.6 9 102
C
D
E
F
G
MRS agar (DifcoTM Lactobacilli MRS Broth; Sparks, MD, USA) was used for the isolation of LAB. Coffee cherry samples collected from each farm were mixed together and then crushed. A 0.5 g sample of crushed coffee cherries was mixed with 4.5 mL of a 0.75 % NaCl solution. The resultant solution was diluted (10 to 1,000-fold) and then spread directly onto the surface of MRS agar plates. Plates were incubated under anaerobic conditions (Mitsubishi AnaeroPakTM System, Pack-Anaero; Mitsubishi Gas Chemicals, Tokyo, Japan) at 30 °C for 3–5 days. To distinguish the acid-producing bacteria from other bacteria, 1 % CaCO3 was previously added to the MRS agar, and only colonies with a surrounding clear zone were selected. Colonies of acid-producing bacteria were randomly selected from MRS plates and purified by replating on MRS agar plates. Colonies were reselected and initially examined for Gram staining and the production of catalase. Only Gram-positive, catalase-negative strains were selected. The selected strains were stored at -80 °C in 10 % skim milk broth.
Lactobacillus plantarum
Isolation of LAB
Leuconostoc pseudomesenteroides
H
I
following cities: Miaoli County (F1, at an approximate altitude in the range of 700–800 m), Pingtung County (F2, at an approximate altitude in the range of 700–800 m), and Chiayi County (F3, at an approximate altitude in the range of 1,200–1,300 m) (Table 1). More than 100 coffee cherries were randomly sampled at each coffee farm and then collected in sterile bags. Samples were analyzed within 24 h of acquisition from the coffee farms.
1
441 Enterococcus sp.
K. Leong et al.: Diversity of Lactic Acid Bacteria
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442
50 -GTCAATTCCTTTGAGTTT-30 (920R). DNA sequencing was performed using an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Partial sequencing, approximately 900 bp ahead, was performed and manually aligned with Genetyx-Win version 5.1 (Genetyx Corp., Tokyo, Japan) software. Sequence similarities were examined by comparing the obtained sequences with those in the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/) using BLAST. Sequence Analysis of the Housekeeping Gene pheS To confirm the identification results, representative strains were randomly selected from each group, and pheS gene sequencing analysis was performed. Amplification and sequencing were performed using forward primer pheS-21-F (50 -CAYCCNGCHCGYGAYATGC-30 ), and reverse primer pheS-22-R (50 -CCWARVCCRAARGCAAARCC-30 ) or pheS-23-R (50 -GGRTGRACCATVCCNGCHCC-30 ), which were previously designed and reported by Naser et al. [15]. Sequence homologies were assessed by comparing the obtained sequences with those in the DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/) using BLAST.
K. Leong et al.: Diversity of Lactic Acid Bacteria
brief, potato dextrose agar plates were overlaid with the MRS soft agar (0.7 %) seeded with 104 spores/mL of the fungal culture. The cultured MRS broth of each LAB strain was mixed well by vortexing, and then streaked onto the surface of the fungal pre-seeded MRS soft agar plates. The streak dimension was approximately 3 9 7 mm. The plates were incubated aerobically at 30 °C for 72 h. Clear zones of inhibition were recorded by determining the distance from the bacterial streak to the edge of visible fungal hyphae. The results were scored as follows: no visible inhibition; w, weak inhibition (inhibition area B 1 mm); ?, inhibition area from 1 to 2 mm; and ??, inhibition area [ 2 mm. Detection of Antibacterial Activity The agar well diffusion method described by Yanagida et al. [24] was used to detect and determine the antibacterial activities of isolates. Lactobacillus sakei JCM 1157T was used as the indicator strain in this study. Antibacterial activity was further confirmed by pH adjustment, and proteinase K treatment [24].
Results Differentiation of Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum A multiplex PCR assay with recA gene-derived primers was performed using the methods and conditions described by Torriani et al. [23]. The PCR products were visualized on a 2 % agarose gel in 1 9 TAE. Physiological Analysis The effects of low initial pH, and temperature on growth were assessed in this study. The experiments were carried out by inoculating an overnight-cultured colony of each isolate into 200 lL of MRS broth adjusted to pH 5.0, and 4.5. Growths at 10, 25, 30, 37, and 45 °C were assessed using the method described by Kozaki et al. [11]. Cell growths of two replicates were turbidometrically determined at 600 nm by means of a SpectraMax 190 Microplate reader (Molecular Devices, Sunnyvale, CA, USA). Uninoculated MRS broth was used as a negative control. Screening for Antifungal Activity of LAB Isolates The coffee cherries LAB isolates were screened for antifungal activity using the agar streak assay described by Lan et al. [13]. The fungal strain Aspergillus flavus ATCC 32592 was used as the indicator strain in this study. In
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A total of 102 acid-producing bacteria were isolated in the current study; 26 were from the coffee farm F1, 66 were from the coffee farm F2, and, and 10 were from the coffee farm F3. Cell viability was assessed using MRS plates and is listed in Table 1. According to cell morphology and the results of the 16S rDNA RFLP analysis, the 102 isolates were divided into nine groups (A to I; Fig. 1). To identify the isolates, partial 16S rDNA and pheS gene sequencing analyses were performed. A total of 44 isolates in group A were identified as Weissella confusa, 30 isolates in group B as Leuconostoc pseudomesenteroides, 10 isolates in group C as Lactobacillus plantarum-related species, 7 isolates in group D as Lactococcus lactis subsp. lactis, 6 isolates in group E as Leuconostoc citreum, 2 isolates in group F as Weissella thailandensis, 1 isolate in group G (strain no. ALS12) as Enterococcus sp., 1 isolate in group H as Enterococcus faecalis, and 1 isolate in group I (strain no. ALS3) as Enterococcus sp. (Table 1). The sequences determined in this study have been deposited in the DDBJ database with accession numbers from AB854169 to AB854277. The 10 Lactobacillus plantarum-related isolates were further verified using a multiplex PCR assay with recA gene-derived primers [23]. An expected amplification band located at 318 bp was obtained from all 10 isolates (Fig. 1). The 10 isolates were therefore identified as Lactobacillus plantarum.
K. Leong et al.: Diversity of Lactic Acid Bacteria
The unidentified enterococcal strain ALS12 showed high 16S rRNA gene sequence similarity (99.4 %) to Enterococcus casseliflavus CECT 969T (AJ420804). However, strain ALS12 showed the highest sequence homology, up to 96.2 %, with the pheS gene sequences (AB854276) of Enterococcus casseliflavus CECT 969T (AJ843470). Strain ALS12 was therefore identified as Enterococcus sp. in the current study. On the other hand, strain no. ALS3 showed the highest sequence similarity, up to 98.6 %, with the 16S rRNA gene sequences (AB807774) of Enterococcus pallens ATCC BAA-351T (DQ411812). However, no identical result could be obtained for strain ALS3 when comparing the 16S
443
rDNA sequence to that of other known Enterococcus species. Strain ALS3 was further verified using rpoA and pheS gene sequencing analyses [15]. Strain ALS3 showed the highest sequence homology, up to 90.3 %, with the rpoA gene sequences (AB807776) of Enterococcus avium ATCC 14025T (AJ843518) and 81.1 % with the pheS gene sequences (AB807775) of Enterococcus faecalis LMG 7937T (AJ843387). Based on the results obtained in the current study, strain ALS3 was therefore identified as Enterococcus sp. Sequences of the representative strains of each group were aligned using CLUSTAL W software [22], and distances were calculated, according to Kimura’s two-
Fig. 1 16S rDNA RFLP patterns of AccII, HaeIII, and AluI digests from Groups A to I. 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
Fig. 2 Neighbor-joining tree of isolates and related type strains based on 16S rRNA sequences. Bootstrap values are indicated at branch points based on 1,000 iterations. Bacillus subtilis NCDO 1769T was used as an outgroup. Bar 0.02 substitutions per nucleotide position
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K. Leong et al.: Diversity of Lactic Acid Bacteria
Fig. 3 Neighbor-joining tree of isolates and related type strains based on pheS gene sequences. Bootstrap values are indicated at branch points based on 1,000 iterations. Streptococcus gordonii LMG 14516T was used as an outgroup. Bar 0.05 substitutions per nucleotide position
parameter model [10]. Phylogenetic trees were then constructed using the neighbor-joining method [16], with bootstrap analysis based on 1,000 iterations. The MEGA 5.05 package [21] was used for all analyses. Phylogenetic analyses of the 16S rRNA and pheS gene sequences obtained in this study are shown in Figs. 2 and 3, respectively. Cell growth at different initial pH and temperatures was determined, and the results are listed in Table 2. Inhibitory activities against A. flavus ATCC 32592 were observed with 9 Leuconostoc pseudomesenteroides, 2 Leuconostoc citreum, 5 Lactobacillus plantarum, 17 Weissella confusa, and, and 1 Enterococcus faecalis strains. Specifically, inhibitory abilities against A. flavus ATCC 32592 of one Lactobacillus plantarum (strain PD101) , and 3 Weissella confusa (strains PD103, PD104, and, and PD106) were scored as ‘‘??’’; six Leuconostoc pseudomesenteroides (strains CF113, CF115, CF119, CF120, CF122, and CF123), 1 Lactobacillus plantarum (strain PD113), and, and 12 Weissella confusa (strains PD107, PD110, PD111, PD114, PD116, PD202, PD203,
123
PD205, PD208, PD414, PD415, and, and PD416) were scored as ‘‘?’’; the remaining strains were scored as ‘‘w’’ (strains CF104, CF124, CF125, CF126, PD108, PD109, PD102, PD112, PD212, PD115, and ALS13). In addition, seven Lactococcus lactis subsp. lactis, and, and one Enterococcus faecalis strains showed the antibacterial activities against the indicator strain Lactobacillus sakei JCM 1157T (Table 1). The bacteriocin-like inhibitory substances (BLISs) produced by all the eight strains maintained their antibacterial activities after neutralization (pH 6.8); however, activities were completely lost after treatment with Proteinase K.
Discussion Schillinger et al. [17] reported that various Leuconostoc, and Weissella species were isolated from coffee fermentation; and Leuconostoc mesenteroides, Leuconostoc citreum,and Leuconostoc pseudomesenteroides were the most abundant LAB found in this study. Similar results were
0 0 1 0 0 1 0 0 1 2 0 0 0 0 6 0 W., Weissella; Leu., Leuconostoc; L., Lactobacillus; Lc., Lactococcus; Ent., Enterococcus
33
? OD value [ 0.5; w OD value C 0.2; - OD value \ 0.2
0
445
1 6 0 0 10 5 2 23 3
1
Growth at pH 4.5
8
0
0 0
1 0
0 0
0 1
1 1
0 0
0 0
1 2
0 2
0 0
0 3
0 0
3 0
6 0
7 0
0 7
0 2
0 0
0 8
10 5
26 1
2 23
3 0
3 38 Growth at pH 5.0
0 44 Growth at 45 °C
3
0
0 0
0 1
1 0
0 0
0 1
1 0
0 0
0 1
1 0
0 0
0 2
2 0
0 0
0 6
6 0
0 3
0 7
4 0
0 0
0 10
10 0
16 1
0 30
13 0
0
44 Growth at 37 °C
0
44 Growth at 30 °C
0
1
0 0
0 0
1 0
1 0
0 1
0 0
0 0
0 1
1 0
0 0
0 2
0 3
0 0
3 0
6 0
7 0
0 7
0 6
0 0
3 1
10 0
18 8
11 19
4 16
0 2 Growth at 25 °C
23 5
42
Growth at 10 °C
w ? w ? w ? ? Physiological analysis
w
-
?
w
-
?
w
-
?
w
-
6 7 10 30 44 Total number
Leu. citreum Lc. lactis subsp. lactis Leu. pseudomesenteroides W. confusa
L. plantarum
B A 16S rDNA RFLP group
Table 2 Phenotypic characteristics of isolates
C
D
E
-
?
w
-
?
w
-
1 1 2
Ent. faecalis Enterococcus sp. W. thailandensis
F
G
H
-
1
Enterococcus sp.
I
K. Leong et al.: Diversity of Lactic Acid Bacteria
found in the studies of Avallone et al. [1, 2], in which Leuconostoc mesenteroides was the predominant species found in coffee (C. arabica L.) fermentation. In this study, Leuconostoc, and Weissella species were also found in the fresh coffee cherries. These findings indicate that the LAB present in coffee fermentation probably originated from the fresh coffee cherries. Besides, Leuconostoc and Weissella species, and Lactobacillus plantarum were also found in the silage of fresh coffee pulp [7]. In the current study, both Leuconostoc pseudomesenteroides and Lactobacillus plantarum were found in the coffee cherries of farms F1 and F2, which were located at a similar altitude in the range of 700–800 m (Table 1). Nevertheless, differences in LAB diversity were also observed in coffee farms F1 and F2. Isolates of Weissella confusa, and Leuconostoc citreum were found only at the coffee farm F2, where Weissella confusa was the most abundant LAB found. Regional similarities and differences in diversity were observed in the current study. The LAB species found at coffee farms F1 and F2 were similar to the findings of previous studies [1, 2, 7]. However, a different LAB diversity was observed at the coffee farm F3. Lactococcus lactis subsp. lactis was the most abundant LAB in F3 samples, and was not found in the coffee cherry samples collected from farms F1 and F2. In addition, the remaining enterococcal cultures were also found only at the coffee farm F3 (Table 1). Altitude was the major difference between coffee farm F3 and the other two farms. It is therefore thought that geographical factors, especially the altitude and climate, may affect the distribution of LAB. To confirm this, further experiments are necessary in the future. The obtained results of growth temperature analysis indicated that the most isolates grew well at 25 and 30 °C. Only a small number of isolates grew at 10 and 45 °C. In addition, the most isolates grew well at low initial pH of 5.0 and 4.5, except for Weissella thailandensis and a few of the Weissella confusa, and Leuconostoc pseudomesenteroides isolates (Table 2). However, a relationship between these factors and LAB distribution was not observed in the current study. The antifungal activities of LAB isolates were determined in this study. The results suggested that the differences in antifungal activities can be identified at the strain level. Similar to the previous studies of Djossou et al. [7] and Lan et al. [13], some differences in antifungal ability between strains were also observed. Here, for example, strains PD101, CF104, and CF105 have been identified as Lactobacillus plantarum cultures. It is proposed that strains with high antifungal ability may have utility in decreasing the numbers of mycotoxin-producing fungi during coffee fermentation. Besides antifungal activity, a total of seven Lactococcus lactis subsp. lactis, and one Enterococcus faecalis strains
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446
were found to have BLIS-producing abilities (Table 1). However, the detailed characterization of BLISs, including heat stability, their effect on enzymes, and their inhibition spectra, was not established in the current study. Future studies in our laboratory will characterize and identify the BLISs, and we anticipate that the BLISs of coffee cherryisolated LAB will be useful as food preservatives. Furthermore, it was found that pheS or rpoA gene sequences provided more efficient and accurate information than 16S rRNA gene sequences and phenotypic characteristics in the identification procedures. For example, isolates in group B showed the high 16S rRNA sequence similarities to both Leuconostoc pseudomesenteroides (99.9 %), and Leuconostoc mesenteroides (99.6 %) species. However, the pheS gene of group B isolates showed high sequence similarities to Leuconostoc pseudomesenteroides LMG 11482T (up to 98.0 %), but low similarities to Leuconostoc mesenteroides LMG 6893T (*82.1 %). Isolates in group B were therefore identified as Leuconostoc pseudomesenteroides. Similar results were also observed in strains ALS12, and ALS3. The results of genetic analyses of strains ALS12 and ALS3 suggest the possibility of being two potential novel species or subspecies of Enterococcus. To clarify this, additional genetic and physiological data, such as GC content, saccharide fermentation ability,, and DNA–DNA relatedness values between strains and the closest type strains, are necessary. In conclusion, this is the first report describing the distribution and varieties of LAB that exist in fresh coffee cherries in Taiwan. Numerous cultures with antifungal abilities or BLIS-producing abilities were found. Two potential novel species or subspecies of LAB were also found in the current study. Future studies in our laboratory will characterize and identify the antifungal substances and BLISs, and we anticipate that the antifungal substances or BLISs of coffee cherry-isolated LAB will be useful in food preservation. Application of antifungal LAB in coffee cultivation may reduce pecuniary loss in coffee production. In addition, some isolates found in the fresh coffee cherries may be applicable as starters in coffee fermentation. We expect that the results of this study can offer useful information for the improvement of coffee production. Acknowledgments The authors would like to thank the National Science Council Taiwan for financially supporting this study, under Contract No. NSC 101-2313-B-130-001 granted to Shwu-fen Pan, and Contract No. NSC 102-2313-B-130-001 granted to Yi-sheng Chen.
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