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Research article
Identification of LAB and Fungi in Laru, a Fermentation Starter, by PCR-DGGE, SDS-PAGE, and MALDI-TOF MS
Department of Food Science and Biotechnology and Institute of Life Sciences and Resources, Kyung Hee University, Yongin 17104, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2018; 28(1): 32-39
Published January 28, 2018 https://doi.org/10.4014/jmb.1705.05044
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
Laru is a dry, dusty, flattened starter that is used to prepare a fermented rice dish called tapai. Laru is known by various names according to the region in which it is made: laru (Brunei Darussalam and Sabah) [1], ragi tapai (Malaysia) [2], ragi tapé (Indonesia) [3, 4], and look-pang (Thailand) [5, 6]. Traditionally, laru is prepared by mixing rice flour with dry, ground spices such as garlic (
The majority of the starters are small (3-6 cm), round, flattened cakes of rice flour that are air- or sun-dried. Since tapai starters are mainly manufactured aseptically by villagers, different combinations of ingredients are used and are typically not disclosed [1]. The microbiota composition also differs according to the combination of ingredients and the country from which the ingredients were obtained. According to Atmodjo [9], a good ragi must be able to inhibit the growth of undesirable microbes.
Both culture-independent (polymerase chain reaction-denaturant gradient gel electrophoresis (PCR-DGGE)) and culture-dependent methods (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)) have been used to detect LAB and fungi in laru samples. PCR-DGGE, which is able to detect both live and dead unculturable cells, enables the rapid analysis of species and changes in microbial communities [15, 16]. In contrast, SDS-PAGE analysis of whole-cell protein extracts is an easy and quick method to identify large numbers of strains with sufficient taxonomic resolution at both the species and subspecies levels if conducted under highly standardized conditions [17]. MALDI-TOF MS, a chemotaxonomic method in which LAB species are identified on the basis of mass spectral patterns of ribosomal proteins, can also be used to confirm 16S rRNA and 18S rRNA gene sequencing results derived from SDS-PAGE protein band groupings [18-20].
We aimed to implement both culture-independent and culture-dependent methods (PCR-DGGE, SDS-PAGE, and MALDI-TOF MS) for the identification of LAB and fungal communities from randomly selected laru samples collected from the upper parts of Borneo Island (Brunei Darussalam, Sabah, and Sarawak). Since the laru samples analyzed in this study were prepared traditionally, we predicted a high diversity of bacteria and fungi from the natural environments. The data obtained from this study will be useful for understanding the microbiological content of traditionally prepared laru for tapai fermentation and for determining how differences in microbiological content account for differences in time until consumption, quality, shelf life, alcohol content, and sugar content in tapai products. Thus, our results will also be useful for the development of refined starters for uniform tapai production.
Materials and Methods
Isolation of LAB and Fungi from Laru Samples
Seventeen laru samples were purchased from randomly chosen markets along the upper coast of Borneo Island in 2015. The samples were manufactured in the following regional groups: region K (samples 2, 6, and 11: Kota Belud, Sabah, Malaysia), region L (sample 8: Lawas, Sarawak, Malaysia), region E (sample 10: Beaufort, Sabah, Malaysia), region T (samples 12 and 15: Tawau, Sabah, Malaysia), region P (sample 1: Penampang, Sabah, Malaysia), region D (sample 4: Donggongon, Sabah, Malaysia), and region B (samples 3, 5, 7, 9, 13, 14, 16, and 17: Labi, Belait, Brunei).
Ten grams of each homogenized sample was aseptically weighed and transferred to a sterile stomacher filter bag (BA6141/STR; Seward, UK). Next, 90 ml of sterile water was added, and the suspension was mixed in a stomacher apparatus (Circular Stomacher 400; Seward, USA) for 60 sec. Appropriate serial dilutions (101 –108) were plated in duplicate on Man, Rogosa, and Sharpe agar (MRS) (Difco, USA) and incubated at 30°C for 48 h under anaerobic conditions using an Anaeropack instrument (Mitsubishi Gas Chemical, Japan). Colonies were also grown on yeast extract-glucose-chloramphenicol agar (YGC) (MBcell, Korea) at 30°C for 48 h under aerobic conditions. LAB and fungal colonies were randomly subcultured in MRS and Sabouraud broth (MBcell), respectively. Each isolate was mixed with 80% (v/v) glycerol at a 7:3 (isolate:glycerol) ratio and stored at -80°C for further use.
DNA Extraction
Each homogenized sample was filtered through two layers of cheesecloth prior to DNA extraction. Filtrates were centrifuged at 16,200 ×
PCR-DGGE Analysis
The PCR products were analyzed on a 2% agarose gel before DGGE analysis. PCR-DGGE analysis was conducted according to the protocols described by Kim
-
Table 1 . Sequences of the primers used in this study
Target Primer Sequence (5’à3’) Reference Bacteria First PCR 27F (F) AGAGTTTGATCCTGGCTCAG [21] 1492 (R) GGCTACCTTGTTACGACTT Nested PCR GC-338f (F) CGCCCGCCGCGCGGCGGGCGGGGCGGGGGACGGGGGACTCCTACGGGAGGCAGCAG [22] 518r (R) ATTACCGCGGCTGCTGG Fungi First PCR NS1 (F) GTAGTCATATGCTTGTCTC [23] FR1 (R) AICCATTCAATCGGTAIT Nested PCR NS3 (F) CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGGCAAGTCTGGTGCCAGCAGCC [24] YM951r (R) TTGGCAAATGCTTTCGC
The PCR amplifications were performed in a Mastercycler instrument (Eppendorf, Germany) in a final volume of 25 μl consisting of 5 μl of template, 2.5 μl of 10× PCR buffer, 2 μl of dNTP mixture (2.5 mM each), 0.1 μl of Taq polymerase (5 U μ/l; Takara Biotechnology, Japan), and 0.4 pM of each primer. The PCR products were analyzed on a 2% agarose gel before DGGE analysis.
The resultant amplicons were mixed with 5 μl of 6× loading dye and directly loaded onto 80 g/l polyacrylamide gels with a denaturing gradient of 20% to 50% urea-formamide. The gels were processed in 1× TAE buffer (40 mM Tris, 20 mM acetate, 1 mM EDTA, pH 8.0) on a Dcode Universal Mutation Detection system (Bio-Rad, USA) for 30 min at 40 V and 15.5 h at 60 V. The gels were then stained with ethidium bromide for 30 min, after which images were captured using a Quantum ST4 1100 system (ST4V16.07; Vilber Lourmat, France).
Sterile blades were used to excise bands of interest from the gels. The gel slices were incubated overnight at 4°C in ultra-filtered water to allow passive diffusion of the DNA from the polyacrylamide matrix into the water for use as template for re-amplification using the DGGE primers GC-338F/518R and GC-NS3/YM951R. The resultant PCR products were run again on polyacrylamide gels to improve the band purity for sequencing, after which the excised gel slices were incubated overnight at 4°C.
Sequencing of DGGE Bands
The eluted DNA was amplified using the same primer pairs, but without the GC clamp. The PCR products were purified using a QIAquick PCR purification kit (Qiagen, USA). Sequences were determined using an automated DNA sequencer (Jenotech, Korea). Partial ribosomal DNA sequences from the laru samples were searched against the GenBank database using BLAST [25] to identify the closest known relatives.
SDS-PAGE Whole-Cell Protein Extract Analysis and 16S rRNA/18S rRNA Gene Sequencing
Proteins in whole-cell extracts from strains cultured in MRS and YGC media were denatured before SDS-PAGE was performed according to the protocols described by Jung
Identification of LAB and Fungi Using MALDI-TOF MS
To identify LAB isolates, a single colony isolated on MRS medium (30°C, 24 h) was spotted on a MSP 96-target polished steel plate (Bruker Daltonik GmbH, Germany). Each colony spot was overlaid with 1 μl of matrix solution (α-cyano-4-hydroxycinnamic acid (g/l) in acetonitrile:water:trifluoracetic acid (50:47.5:2.5)). The matrix-sample mixture was crystallized by air-drying at room temperature for 5 min, loaded in a mass spectrometer, and subjected to MALDI-TOF MS analysis.
Identification of fungal isolates was performed as described by Pavlovic
A Microflex LT Biotyper MALDI-TOF MS system (Bruker Daltonik) equipped with a 337 nm nitrogen cartridge laser (MNL106; Bruker Daltonik) was used for all analyses. The Biotyper instrument system was controlled using Flexcontrol ver. 3.4 software. Ions were accelerated in a 20 kV electric field through a grid and separated according to mass to charge (
Results and Discussion
PCR-DGGE Analysis
We used PCR-DGGE, a culture-independent method that utilizes nested PCR products, to detect the presence of LAB and fungi in the laru samples. DGGE images were used to compare bacterial (Fig. 1A) and fungal (Fig. 1B) communities in the 17 laru samples. The corresponding sequencing results are listed in Table 2.
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Table 2 . Identification of bacterial and fungal species in the laru samples by sequencing the 16S V3 rRNA and 18S rRNA fragments excised from PCR-DGGE gels
DGGE Band no. Species identification Homology (%) Accession No. Bacteria a Weissella cibaria 100 JN851745.1 b Leuconostoc citreum 97 LC096222.1 c Leuconostoc mesenteroides 99 JN863609.1 d Staphylococcus haemolyticus 99 KT026096.1 e Weissella paramesenteroides 99 KP189212.1 f Lactobacillus brevis 99 JN863616.1 g Pediococcus pentosaceus 100 JN851781.1 h Staphylococcus kloosii 99 KF233801.1 i Lactococcus lactis 99 EF204360.1 Fungi a Saccharomycopsis fibuligera 100 KP119822.1 b Hyphopichia burtonii 99 JQ698903.1 c Rhizopus oryzae /Amylomyces rouxii 100 KJ408539.1/KJ588788.1 d Mucor indicus 99 KM527229.1 e Kodamaea ohmeri 99 KM006493.1 f Candida intermedia 100 EF408189.1
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Fig. 1. DGGE patterns of 16S V3 rRNA gene sequences (A) and 18S rRNA gene sequences (B) in the laru samples (1-17).
The identified LAB included
The most predominant LAB was
The LAB and fungal profiles displayed by the PCR-DGGE bands (Figs. 1A and 1B, respectively) were next used to determine whether or not the samples originated from the same source or region. Samples 2, 6, and 11 originated from region K and shared
SDS-PAGE Whole-Cell Protein Extract Analysis and 16S rRNA/18S rRNA Gene Sequencing
The patterns resulting from SDS-PAGE analysis of whole-cell protein extracts were compared and classified into four groups (1-4, 2-10, 1-7, and 1-9) of LAB strains and seven groups (4-6y, 6-2y, 1-1y, 9-7y, 4-12y, 1-7y, and 4-3y) of fungus strains (Fig. 2). Isolates were further analyzed by 16S rRNA and 18S rRNA gene sequencing. The identities of the LAB isolates and fungal isolates from the SDS-PAGE patterns and their corresponding NCBI accession numbers are listed in Table 3. Among the four LAB groups,
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Table 3 . Identification of LAB and fungal isolates selected from SDS-PAGE patterns of whole-cell protein extracts by 16S rRNA and 18S rRNA gene sequencing analyses.
Isolate no. of SDS-PAGE patterna Species identification (NCBI Accession No.) Homologyc (%) LAB 1-7b Lactobacillus fermentum (JF903803.1)99 1-4 Lactobacillus plantarum (KR336551.1)100 2-10 Pediococcus pentosaceus (KJ477378.1)100 1-9 Lactobacillus brevis (AB969780.1)99 Fungi 1-7y Saccharomyces cerevisiae (GQ458028.1)99 4-6y Pichia anomala (EF427893.1)99 6-2y Saccharomycopsis fibuligera (KP119820.1)99 9-7y Hyphopichia burtonii (JQ698903.1)99 1-1y Candida parapsilosis (KT229545.1)99 4-12y Kodamaea ohmeri (HQ412607.1)99 4-3y Candida orthopsilosis (AY 520277.1)99 aIsolate No. designation refers to that in Fig. 2.
bLaru samples 1-17.
c16S/18S rRNA gene sequences of the LAB and fungal strains were searched against the NCBI sequence database.
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Fig. 2. SDS-PAGE whole-cell protein patterns of lactic acid bacteria (A) and fungi (B) representative of the four LAB and seven fungi groups isolated from laru samples. (A) Lanes: M, protein Mw markers (kDa); 1, 1-4; 2, 2-10; 3, 1-7; 4, 1-9. (B) Lanes: M, protein Mw markers (kDa); 1, 4-6y; 2, 6-2y; 3, 1-1y; 4, 9-7y; 5, 4- 12y; 6, 1-7y; 7, 4-3y.
Identification of LAB and Fungal Isolates Using MALDI-TOF MS
In MALDI-TOF MS analysis, ribosomal proteins produce a fingerprint in the form of a mass spectrum. We selected a number of the identified isolates for MALDI-TOF MS analysis. The LAB and fungi results obtained from MALDI-TOF MS analysis were consistent with those based on whole-cell protein extract patterns and 16S/18S rRNA gene sequence analyses (Table 4), except
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Table 4 . Comparison of the 16S and 18S rRNA gene sequencing and MALDI-TOF MS results.
rRNA gene sequencing MALDI-TOF MS (Score value) 16S Lactobacillus fermentum L. fermentum DSM 20391 DSM (1.864)Lactobacillus plantarum L. plantarum DSM 12028 DSM (2.045)Pediococcus pentosaceus P. pentosaceus DSM 20206 DSM (1.899)Lactobacillus brevis L. brevis DSM 20054T DSM (1.951)18S Saccharomyces cerevisiae S. cerevisiae isolate LGL Muenchen (1.817)Pichia anomala P. anomala DSM 70260 (2.079)Hyphopichia burtonii H. burtonii MY00872_06 ERL (1.764)Candida parapsilosis C. parapsilosis ATCC 22019 THL (2.269)Kodamaea (Pichia) ohmeriPichia ohmeri MY970_1_09 ERL (1.999)Candida orthopsilosis C. orthopsilosis P3119_8_37 HAC (2.059)Saccharomycopsis fibuligera No reliable identification
In general, the culture-independent method showed that all 17 laru samples contained at least one LAB species (Fig. 1A), whereas the culture-dependent methods detected LAB in only three samples (Table 3). The LAB species detected by the culture-independent method were
Overall, the culture-independent method identified seven LAB and six fungal species, whereas the culture-dependent methods identified only four LAB and seven fungal species. Both types of method were able to detect the presence of
Our data indicate that each method favors the identification of certain species. In this regard, the culture-independent method is most suitable for monitoring the general distribution of LAB and fungal communities. This method could be used to predict whether multiple samples originated from the same region or have similar functions, although minor inconsistencies in the detected species will invariably be present. On the other hand, the culture-dependent methods are not as suitable for determining tapai origin. This is due to the nature of uncontrolled tapai preparation, which results in inconsistencies in the presence of detected microorganisms and in the number of isolates, even when the samples are manufactured from the same company or originate from the region. In addition, our data clearly indicate that laru preparation does not always take place under well-controlled and standardized conditions, as demonstrated by the findings from region B (samples 3, 5, 6, 7, 9, 13, 14, 16, and 17), region K (samples 2, 6, and 11), and region T (samples 12 and 15). However, the combination of both approaches can provide an overall view of the laru microbiota.
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Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2018; 28(1): 32-39
Published online January 28, 2018 https://doi.org/10.4014/jmb.1705.05044
Copyright © The Korean Society for Microbiology and Biotechnology.
Identification of LAB and Fungi in Laru, a Fermentation Starter, by PCR-DGGE, SDS-PAGE, and MALDI-TOF MS
Lenny S. F. Ahmadsah 1, Eiseul Kim 1, Youn-Sik Jung 1 and Hae-Yeong Kim 1*
Department of Food Science and Biotechnology and Institute of Life Sciences and Resources, Kyung Hee University, Yongin 17104, Republic of Korea
Correspondence to:Hae-Yeong Kim
hykim@khu.ac.kr
Abstract
Samples of Laru (a fermentation starter) obtained from the upper part of Borneo Island were analyzed for their lactic acid bacteria (LAB) and fungal diversity using both a cultureindependent method (PCR-DGGE) and culture-dependent methods (SDS-PAGE and MALDITOF MS). Pediococcus pentosaceus, Lactobacillus brevis, Saccharomycopsis fibuligera, Hyphopichia burtonii, and Kodamaea ohmeri were detected by all three methods. In addition, Weissella cibaria, Weissella paramesenteroides, Leuconostoc citreum, Leuconostoc mesenteroides, Lactococcus lactis, Rhizopus oryzae/Amylomyces rouxii, Mucor indicus, and Candida intermedia were detected by PCR-DGGE. In contrast, Lactobacillus fermentum, Lactobacillus plantarum, Pichia anomala, Candida parapsilosis, and Candida orthopsilosis were detected only by the culture-dependent methods. Our results indicate that the culture-independent method can be used to determine whether multiple laru samples originated from the same manufacturing region; however, using the culture-independent and the two culture-dependent approaches in combination provides a more comprehensive overview of the laru microbiota.
Keywords: Laru, ragi, lactic acid bacteria, PCR-DGGE, SDS-PAGE
Introduction
Laru is a dry, dusty, flattened starter that is used to prepare a fermented rice dish called tapai. Laru is known by various names according to the region in which it is made: laru (Brunei Darussalam and Sabah) [1], ragi tapai (Malaysia) [2], ragi tapé (Indonesia) [3, 4], and look-pang (Thailand) [5, 6]. Traditionally, laru is prepared by mixing rice flour with dry, ground spices such as garlic (
The majority of the starters are small (3-6 cm), round, flattened cakes of rice flour that are air- or sun-dried. Since tapai starters are mainly manufactured aseptically by villagers, different combinations of ingredients are used and are typically not disclosed [1]. The microbiota composition also differs according to the combination of ingredients and the country from which the ingredients were obtained. According to Atmodjo [9], a good ragi must be able to inhibit the growth of undesirable microbes.
Both culture-independent (polymerase chain reaction-denaturant gradient gel electrophoresis (PCR-DGGE)) and culture-dependent methods (sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)) have been used to detect LAB and fungi in laru samples. PCR-DGGE, which is able to detect both live and dead unculturable cells, enables the rapid analysis of species and changes in microbial communities [15, 16]. In contrast, SDS-PAGE analysis of whole-cell protein extracts is an easy and quick method to identify large numbers of strains with sufficient taxonomic resolution at both the species and subspecies levels if conducted under highly standardized conditions [17]. MALDI-TOF MS, a chemotaxonomic method in which LAB species are identified on the basis of mass spectral patterns of ribosomal proteins, can also be used to confirm 16S rRNA and 18S rRNA gene sequencing results derived from SDS-PAGE protein band groupings [18-20].
We aimed to implement both culture-independent and culture-dependent methods (PCR-DGGE, SDS-PAGE, and MALDI-TOF MS) for the identification of LAB and fungal communities from randomly selected laru samples collected from the upper parts of Borneo Island (Brunei Darussalam, Sabah, and Sarawak). Since the laru samples analyzed in this study were prepared traditionally, we predicted a high diversity of bacteria and fungi from the natural environments. The data obtained from this study will be useful for understanding the microbiological content of traditionally prepared laru for tapai fermentation and for determining how differences in microbiological content account for differences in time until consumption, quality, shelf life, alcohol content, and sugar content in tapai products. Thus, our results will also be useful for the development of refined starters for uniform tapai production.
Materials and Methods
Isolation of LAB and Fungi from Laru Samples
Seventeen laru samples were purchased from randomly chosen markets along the upper coast of Borneo Island in 2015. The samples were manufactured in the following regional groups: region K (samples 2, 6, and 11: Kota Belud, Sabah, Malaysia), region L (sample 8: Lawas, Sarawak, Malaysia), region E (sample 10: Beaufort, Sabah, Malaysia), region T (samples 12 and 15: Tawau, Sabah, Malaysia), region P (sample 1: Penampang, Sabah, Malaysia), region D (sample 4: Donggongon, Sabah, Malaysia), and region B (samples 3, 5, 7, 9, 13, 14, 16, and 17: Labi, Belait, Brunei).
Ten grams of each homogenized sample was aseptically weighed and transferred to a sterile stomacher filter bag (BA6141/STR; Seward, UK). Next, 90 ml of sterile water was added, and the suspension was mixed in a stomacher apparatus (Circular Stomacher 400; Seward, USA) for 60 sec. Appropriate serial dilutions (101 –108) were plated in duplicate on Man, Rogosa, and Sharpe agar (MRS) (Difco, USA) and incubated at 30°C for 48 h under anaerobic conditions using an Anaeropack instrument (Mitsubishi Gas Chemical, Japan). Colonies were also grown on yeast extract-glucose-chloramphenicol agar (YGC) (MBcell, Korea) at 30°C for 48 h under aerobic conditions. LAB and fungal colonies were randomly subcultured in MRS and Sabouraud broth (MBcell), respectively. Each isolate was mixed with 80% (v/v) glycerol at a 7:3 (isolate:glycerol) ratio and stored at -80°C for further use.
DNA Extraction
Each homogenized sample was filtered through two layers of cheesecloth prior to DNA extraction. Filtrates were centrifuged at 16,200 ×
PCR-DGGE Analysis
The PCR products were analyzed on a 2% agarose gel before DGGE analysis. PCR-DGGE analysis was conducted according to the protocols described by Kim
-
Table 1 . Sequences of the primers used in this study.
Target Primer Sequence (5’à3’) Reference Bacteria First PCR 27F (F) AGAGTTTGATCCTGGCTCAG [21] 1492 (R) GGCTACCTTGTTACGACTT Nested PCR GC-338f (F) CGCCCGCCGCGCGGCGGGCGGGGCGGGGGACGGGGGACTCCTACGGGAGGCAGCAG [22] 518r (R) ATTACCGCGGCTGCTGG Fungi First PCR NS1 (F) GTAGTCATATGCTTGTCTC [23] FR1 (R) AICCATTCAATCGGTAIT Nested PCR NS3 (F) CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGGCAAGTCTGGTGCCAGCAGCC [24] YM951r (R) TTGGCAAATGCTTTCGC
The PCR amplifications were performed in a Mastercycler instrument (Eppendorf, Germany) in a final volume of 25 μl consisting of 5 μl of template, 2.5 μl of 10× PCR buffer, 2 μl of dNTP mixture (2.5 mM each), 0.1 μl of Taq polymerase (5 U μ/l; Takara Biotechnology, Japan), and 0.4 pM of each primer. The PCR products were analyzed on a 2% agarose gel before DGGE analysis.
The resultant amplicons were mixed with 5 μl of 6× loading dye and directly loaded onto 80 g/l polyacrylamide gels with a denaturing gradient of 20% to 50% urea-formamide. The gels were processed in 1× TAE buffer (40 mM Tris, 20 mM acetate, 1 mM EDTA, pH 8.0) on a Dcode Universal Mutation Detection system (Bio-Rad, USA) for 30 min at 40 V and 15.5 h at 60 V. The gels were then stained with ethidium bromide for 30 min, after which images were captured using a Quantum ST4 1100 system (ST4V16.07; Vilber Lourmat, France).
Sterile blades were used to excise bands of interest from the gels. The gel slices were incubated overnight at 4°C in ultra-filtered water to allow passive diffusion of the DNA from the polyacrylamide matrix into the water for use as template for re-amplification using the DGGE primers GC-338F/518R and GC-NS3/YM951R. The resultant PCR products were run again on polyacrylamide gels to improve the band purity for sequencing, after which the excised gel slices were incubated overnight at 4°C.
Sequencing of DGGE Bands
The eluted DNA was amplified using the same primer pairs, but without the GC clamp. The PCR products were purified using a QIAquick PCR purification kit (Qiagen, USA). Sequences were determined using an automated DNA sequencer (Jenotech, Korea). Partial ribosomal DNA sequences from the laru samples were searched against the GenBank database using BLAST [25] to identify the closest known relatives.
SDS-PAGE Whole-Cell Protein Extract Analysis and 16S rRNA/18S rRNA Gene Sequencing
Proteins in whole-cell extracts from strains cultured in MRS and YGC media were denatured before SDS-PAGE was performed according to the protocols described by Jung
Identification of LAB and Fungi Using MALDI-TOF MS
To identify LAB isolates, a single colony isolated on MRS medium (30°C, 24 h) was spotted on a MSP 96-target polished steel plate (Bruker Daltonik GmbH, Germany). Each colony spot was overlaid with 1 μl of matrix solution (α-cyano-4-hydroxycinnamic acid (g/l) in acetonitrile:water:trifluoracetic acid (50:47.5:2.5)). The matrix-sample mixture was crystallized by air-drying at room temperature for 5 min, loaded in a mass spectrometer, and subjected to MALDI-TOF MS analysis.
Identification of fungal isolates was performed as described by Pavlovic
A Microflex LT Biotyper MALDI-TOF MS system (Bruker Daltonik) equipped with a 337 nm nitrogen cartridge laser (MNL106; Bruker Daltonik) was used for all analyses. The Biotyper instrument system was controlled using Flexcontrol ver. 3.4 software. Ions were accelerated in a 20 kV electric field through a grid and separated according to mass to charge (
Results and Discussion
PCR-DGGE Analysis
We used PCR-DGGE, a culture-independent method that utilizes nested PCR products, to detect the presence of LAB and fungi in the laru samples. DGGE images were used to compare bacterial (Fig. 1A) and fungal (Fig. 1B) communities in the 17 laru samples. The corresponding sequencing results are listed in Table 2.
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Table 2 . Identification of bacterial and fungal species in the laru samples by sequencing the 16S V3 rRNA and 18S rRNA fragments excised from PCR-DGGE gels.
DGGE Band no. Species identification Homology (%) Accession No. Bacteria a Weissella cibaria 100 JN851745.1 b Leuconostoc citreum 97 LC096222.1 c Leuconostoc mesenteroides 99 JN863609.1 d Staphylococcus haemolyticus 99 KT026096.1 e Weissella paramesenteroides 99 KP189212.1 f Lactobacillus brevis 99 JN863616.1 g Pediococcus pentosaceus 100 JN851781.1 h Staphylococcus kloosii 99 KF233801.1 i Lactococcus lactis 99 EF204360.1 Fungi a Saccharomycopsis fibuligera 100 KP119822.1 b Hyphopichia burtonii 99 JQ698903.1 c Rhizopus oryzae /Amylomyces rouxii 100 KJ408539.1/KJ588788.1 d Mucor indicus 99 KM527229.1 e Kodamaea ohmeri 99 KM006493.1 f Candida intermedia 100 EF408189.1
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Figure 1. DGGE patterns of 16S V3 rRNA gene sequences (A) and 18S rRNA gene sequences (B) in the laru samples (1-17).
The identified LAB included
The most predominant LAB was
The LAB and fungal profiles displayed by the PCR-DGGE bands (Figs. 1A and 1B, respectively) were next used to determine whether or not the samples originated from the same source or region. Samples 2, 6, and 11 originated from region K and shared
SDS-PAGE Whole-Cell Protein Extract Analysis and 16S rRNA/18S rRNA Gene Sequencing
The patterns resulting from SDS-PAGE analysis of whole-cell protein extracts were compared and classified into four groups (1-4, 2-10, 1-7, and 1-9) of LAB strains and seven groups (4-6y, 6-2y, 1-1y, 9-7y, 4-12y, 1-7y, and 4-3y) of fungus strains (Fig. 2). Isolates were further analyzed by 16S rRNA and 18S rRNA gene sequencing. The identities of the LAB isolates and fungal isolates from the SDS-PAGE patterns and their corresponding NCBI accession numbers are listed in Table 3. Among the four LAB groups,
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Table 3 . Identification of LAB and fungal isolates selected from SDS-PAGE patterns of whole-cell protein extracts by 16S rRNA and 18S rRNA gene sequencing analyses..
Isolate no. of SDS-PAGE patterna Species identification (NCBI Accession No.) Homologyc (%) LAB 1-7b Lactobacillus fermentum (JF903803.1)99 1-4 Lactobacillus plantarum (KR336551.1)100 2-10 Pediococcus pentosaceus (KJ477378.1)100 1-9 Lactobacillus brevis (AB969780.1)99 Fungi 1-7y Saccharomyces cerevisiae (GQ458028.1)99 4-6y Pichia anomala (EF427893.1)99 6-2y Saccharomycopsis fibuligera (KP119820.1)99 9-7y Hyphopichia burtonii (JQ698903.1)99 1-1y Candida parapsilosis (KT229545.1)99 4-12y Kodamaea ohmeri (HQ412607.1)99 4-3y Candida orthopsilosis (AY 520277.1)99 aIsolate No. designation refers to that in Fig. 2..
bLaru samples 1-17..
c16S/18S rRNA gene sequences of the LAB and fungal strains were searched against the NCBI sequence database..
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Figure 2. SDS-PAGE whole-cell protein patterns of lactic acid bacteria (A) and fungi (B) representative of the four LAB and seven fungi groups isolated from laru samples. (A) Lanes: M, protein Mw markers (kDa); 1, 1-4; 2, 2-10; 3, 1-7; 4, 1-9. (B) Lanes: M, protein Mw markers (kDa); 1, 4-6y; 2, 6-2y; 3, 1-1y; 4, 9-7y; 5, 4- 12y; 6, 1-7y; 7, 4-3y.
Identification of LAB and Fungal Isolates Using MALDI-TOF MS
In MALDI-TOF MS analysis, ribosomal proteins produce a fingerprint in the form of a mass spectrum. We selected a number of the identified isolates for MALDI-TOF MS analysis. The LAB and fungi results obtained from MALDI-TOF MS analysis were consistent with those based on whole-cell protein extract patterns and 16S/18S rRNA gene sequence analyses (Table 4), except
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Table 4 . Comparison of the 16S and 18S rRNA gene sequencing and MALDI-TOF MS results..
rRNA gene sequencing MALDI-TOF MS (Score value) 16S Lactobacillus fermentum L. fermentum DSM 20391 DSM (1.864)Lactobacillus plantarum L. plantarum DSM 12028 DSM (2.045)Pediococcus pentosaceus P. pentosaceus DSM 20206 DSM (1.899)Lactobacillus brevis L. brevis DSM 20054T DSM (1.951)18S Saccharomyces cerevisiae S. cerevisiae isolate LGL Muenchen (1.817)Pichia anomala P. anomala DSM 70260 (2.079)Hyphopichia burtonii H. burtonii MY00872_06 ERL (1.764)Candida parapsilosis C. parapsilosis ATCC 22019 THL (2.269)Kodamaea (Pichia) ohmeriPichia ohmeri MY970_1_09 ERL (1.999)Candida orthopsilosis C. orthopsilosis P3119_8_37 HAC (2.059)Saccharomycopsis fibuligera No reliable identification
In general, the culture-independent method showed that all 17 laru samples contained at least one LAB species (Fig. 1A), whereas the culture-dependent methods detected LAB in only three samples (Table 3). The LAB species detected by the culture-independent method were
Overall, the culture-independent method identified seven LAB and six fungal species, whereas the culture-dependent methods identified only four LAB and seven fungal species. Both types of method were able to detect the presence of
Our data indicate that each method favors the identification of certain species. In this regard, the culture-independent method is most suitable for monitoring the general distribution of LAB and fungal communities. This method could be used to predict whether multiple samples originated from the same region or have similar functions, although minor inconsistencies in the detected species will invariably be present. On the other hand, the culture-dependent methods are not as suitable for determining tapai origin. This is due to the nature of uncontrolled tapai preparation, which results in inconsistencies in the presence of detected microorganisms and in the number of isolates, even when the samples are manufactured from the same company or originate from the region. In addition, our data clearly indicate that laru preparation does not always take place under well-controlled and standardized conditions, as demonstrated by the findings from region B (samples 3, 5, 6, 7, 9, 13, 14, 16, and 17), region K (samples 2, 6, and 11), and region T (samples 12 and 15). However, the combination of both approaches can provide an overall view of the laru microbiota.
Fig 1.
Fig 2.
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Table 1 . Sequences of the primers used in this study.
Target Primer Sequence (5’à3’) Reference Bacteria First PCR 27F (F) AGAGTTTGATCCTGGCTCAG [21] 1492 (R) GGCTACCTTGTTACGACTT Nested PCR GC-338f (F) CGCCCGCCGCGCGGCGGGCGGGGCGGGGGACGGGGGACTCCTACGGGAGGCAGCAG [22] 518r (R) ATTACCGCGGCTGCTGG Fungi First PCR NS1 (F) GTAGTCATATGCTTGTCTC [23] FR1 (R) AICCATTCAATCGGTAIT Nested PCR NS3 (F) CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGGCAAGTCTGGTGCCAGCAGCC [24] YM951r (R) TTGGCAAATGCTTTCGC
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Table 2 . Identification of bacterial and fungal species in the laru samples by sequencing the 16S V3 rRNA and 18S rRNA fragments excised from PCR-DGGE gels.
DGGE Band no. Species identification Homology (%) Accession No. Bacteria a Weissella cibaria 100 JN851745.1 b Leuconostoc citreum 97 LC096222.1 c Leuconostoc mesenteroides 99 JN863609.1 d Staphylococcus haemolyticus 99 KT026096.1 e Weissella paramesenteroides 99 KP189212.1 f Lactobacillus brevis 99 JN863616.1 g Pediococcus pentosaceus 100 JN851781.1 h Staphylococcus kloosii 99 KF233801.1 i Lactococcus lactis 99 EF204360.1 Fungi a Saccharomycopsis fibuligera 100 KP119822.1 b Hyphopichia burtonii 99 JQ698903.1 c Rhizopus oryzae /Amylomyces rouxii 100 KJ408539.1/KJ588788.1 d Mucor indicus 99 KM527229.1 e Kodamaea ohmeri 99 KM006493.1 f Candida intermedia 100 EF408189.1
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Table 3 . Identification of LAB and fungal isolates selected from SDS-PAGE patterns of whole-cell protein extracts by 16S rRNA and 18S rRNA gene sequencing analyses..
Isolate no. of SDS-PAGE patterna Species identification (NCBI Accession No.) Homologyc (%) LAB 1-7b Lactobacillus fermentum (JF903803.1)99 1-4 Lactobacillus plantarum (KR336551.1)100 2-10 Pediococcus pentosaceus (KJ477378.1)100 1-9 Lactobacillus brevis (AB969780.1)99 Fungi 1-7y Saccharomyces cerevisiae (GQ458028.1)99 4-6y Pichia anomala (EF427893.1)99 6-2y Saccharomycopsis fibuligera (KP119820.1)99 9-7y Hyphopichia burtonii (JQ698903.1)99 1-1y Candida parapsilosis (KT229545.1)99 4-12y Kodamaea ohmeri (HQ412607.1)99 4-3y Candida orthopsilosis (AY 520277.1)99 aIsolate No. designation refers to that in Fig. 2..
bLaru samples 1-17..
c16S/18S rRNA gene sequences of the LAB and fungal strains were searched against the NCBI sequence database..
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Table 4 . Comparison of the 16S and 18S rRNA gene sequencing and MALDI-TOF MS results..
rRNA gene sequencing MALDI-TOF MS (Score value) 16S Lactobacillus fermentum L. fermentum DSM 20391 DSM (1.864)Lactobacillus plantarum L. plantarum DSM 12028 DSM (2.045)Pediococcus pentosaceus P. pentosaceus DSM 20206 DSM (1.899)Lactobacillus brevis L. brevis DSM 20054T DSM (1.951)18S Saccharomyces cerevisiae S. cerevisiae isolate LGL Muenchen (1.817)Pichia anomala P. anomala DSM 70260 (2.079)Hyphopichia burtonii H. burtonii MY00872_06 ERL (1.764)Candida parapsilosis C. parapsilosis ATCC 22019 THL (2.269)Kodamaea (Pichia) ohmeriPichia ohmeri MY970_1_09 ERL (1.999)Candida orthopsilosis C. orthopsilosis P3119_8_37 HAC (2.059)Saccharomycopsis fibuligera No reliable identification
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