Molecular identification and phylogenetic analysis of GABA-producing lactic acid bacteria isolated from indigenous dadih of West Sumatera, Indonesia

Background: Dadih (fermented buffalo milk) is a traditional Indonesian food originating from West Sumatra province. The fermentation process is carried out by lactic acid bacteria (LAB), which are naturally present in buffalo milk. Lactic acid bacteria have been reported as one of potential producers of γ-aminobutyric acid (GABA). GABA acts as a neurotransmitter inhibitor of the central nervous system. Methods: In this study, molecular identification and phylogenetic analysis of GABA producing LAB isolated from indigenous dadih of West Sumatera were determined. Identification of the GABA-producing LAB DS15 was based on conventional polymerase chain reaction. 16S rRNA gene sequence analysis was used to identify LAB DS15. Results: PCR of the 16S rRNA gene sequence of LAB DS15 gave an approximately 1400 bp amplicon. Phylogenetic analysis showed that LAB DS15 was Pediococcus acidilactici, with high similarity of 99% at 100% query coverage to Pediococcus acidilactici strain DSM 20284. Conclusions: It can be concluded that GABA producing LAB isolated from indigenous dadih was Pediococcus acidilactici.


Introduction
The non-proteinogenic amino acid γ-aminobutyric acid (GABA) is widely found in bacteria, animals, plants, and fungi (Dhakal et al., 2012;Nonaka et al., 2017). GABA acts as a neurotransmitter inhibitor of the central nervous system (Olsen & Li, 2012). It is formed by decarboxylation of L-glutamate, a reaction catalyzed by an enzyme that depends on the peridoxal phosphate of decarboxylated L-glutamate (Murray et al., 2003). Lactic acid bacteria (LAB) have been reported as a potential producer of GABA (Seo et al., 2013;Wu & Shah, 2017). LAB are generally regarded as safe and non-pathogenic microbes, and has been referred to as 'generally recognized as safe'. Therefore, GABA-producing LAB can be used directly in functional foods (Zhao et al., 2017). Some LAB can be found in the dairy industry for the production of cheese, yogurt, and other fermented milk products (Yamada et al., 2018).
Dadih (fermented buffalo milk) is an Indonesian traditional food originating from West Sumatra Province; it is an extremely popular dairy product in Bukittinggi, Padangpanjang, Solok, Lima Puluh Kota, and Tanah Datar, Indonesia (Surono, 2015). Dadih is made from buffalo milk which is fermented in bamboo for 24-48 hours. The fermentation process is carried out by LAB which are naturally present in buffalo milk (Rizqiati et al., 2015) and the environment (Wirawati et al., 2017). Studies have found that, the LAB strains present in dadih are generally Lactobacillus, Streptococcus, Leuconostoc and Lactococcus (Collado et al., 2007;Surono, 2003).
Extraction of DNA is a basic principle in molecular analysis and it is one of the success factors in DNA amplification that is used in the analysis of genetic characters (Mustafa et al., 2016). Polymerase chain reaction (PCR) and phylogenetic analysis based on 16S rRNA gene sequences have been used for successful identification of isolates from various fermented food products (Malik et al., 2015). These molecular approaches have allowed Lactobacillus species to be reliably identified (Henry et al., 2015). This research was conducted to identify and to characterize GABA producing LAB isolated from indigenous dadih of West Sumatera based on 16 S rRNA gene sequence analysis.

Sample
This study used lactic acid bacteria (LAB) DS15, a GABAproducing LAB isolated from dadih of West Sumatera origin. This bacterium was isolated previously according to the method described by Ali et al. (2009). The experiment was carried out at the Feed Technology Industry Laboratory, Faculty of Animal Science, Andalas University, West Sumatra, Indonesia. LAB DS15 was grown anaerobically in MRS medium (Merck, Darmstadt, Germany) at 30°C and stored for further analysis.
Isolation of bacterial genomic DNA Isolation of the total genome of LAB DS15 was done using Genomic DNA Mini Kit purchased from Invitrogen (Pure-LinkTM, USA) by following the manufacturer's instructions. We used Lysozyme (PureLinkTM, USA) at a concentration of 20 mg/ml to break down the bacterial cell wall to improve protein or nucleic acid extraction efficiency.
16S rRNA gene amplification Genomic DNA of LAB DS15 was used for amplification of 16S rRNA gene. Amplification was done using forward primer 63F (5'-CAG GCC TAA CAC ATG CAA GTC-3') and reverse primer 1387R (5'-GGG CGG GGT GTA CAA GGC-3'). of Laboratory of Medical Molecular Biology and Diagnostic, Indonesian Institute of Sciences. The reaction was carried out in a volume of 50 μl. The PCR mixture contained 22 μl of MQ, 25 μl DreamTaq Green DNA Polymerase (Thermo Fisher Scientific, USA), 1 μl of each forward and reverse primer (10 μM each, IDT synthesized) and 1 μl template. Amplification conditions were 5 minutes of preheating at 95°C, 30 seconds denaturation at 95°C, 30 seconds of primer annealing at 58°C, 1 minute extension step at 72°C and post cycling extension of 5 minutes at 72°C for 35 cycles. The reactions were carried out in a thermal cycler (Biometra's T-Personal Thermal Cycler, USA).

Electrophoresis
PCR products were stored at 4°C for further examination using 1% (w/v) agarose electrophoresis in 1x TAE, 100 V for 30 minutes. The DNA bands formed from electrophoresis process was visualized using UV transluminator. The marker used was 1 Kb Plus DNA ladder (ThermoFisher Scientific).
Sequence alignment of the 16S rRNA gene Sequencing of the 16S rRNA gene was performed at the Laboratory of Medical Molecular Biology and Diagnostic, Indonesian Institute of Sciences, Jakarta. Sequencing results were edited (contig and peak chromatogram verification) using the SeqMan TM II program. Analysis of 16S rRNA sequences of LAB DS15 was performed using NCBI BLAST. Multiple alignment was done using the ClustalX 2.1 program. BioEdit version 7.2.5 in edit mode to see the absence of an inverted sequence and align the sequence length. Kinship visualization was done using the combined phylogenetic tree of the MEGA 7.0.20 program with the Neighbor-Joining hood method (Saitou & Nei, 1987).

Results and discussion
The identification of LAB DS15 to determine the strain was done based on 16S rRNA gene. The first step was amplification using PCR method, with the electrophoresis image shown in Supplementary File 1. The amplification process was carried out to obtain more copies of the 16S rRNA gene for the sequencing process. Analysis of sequencing results begun by aligning the base sequence of the 63F forward sequence and reverse 138R using the SegMan program. PCR of the 16S rRNA gene of LAB DS15 gave an approximately 1400 bp amplicon ( Figure 1). DNA sequencing results were analyzed using NCBI BLAST. According to Willey et al. (2009), 16S rRNA sequencing looks at the similarity of isolates to those already available in GenBank; this is one molecular detection method that is ideal enough to know the kinship relationship between bacteria because the 16S rRNA sequence is a gene found in all microbes and is indispensable in maintain life. The 16S rRNA gene sequencing identified the LAB DS15 to belong to the genus Pediococcus, forming a well-defined cluster with Pediococcus acidilactici. This cluster was recovered in 100% of bootstrap analysis. Pediococcus spp. are widely described as probiotics (Porto et al., 2017). Abbasiliasi et al. (2012) also found Pediococcus acidilactici in fermented milk products. Pediococcus acidilactici are important LAB which have been used as starter cultures in meat, vegetable and dairy fermentation causing characteristic flavor changes, improving hygiene and extending the shelf life of these products (Mora et al., 1997;Porto et al., 2017).
A phylogenetic tree ( Figure 2) was constructed to determine the kinship relationship of LAB DS15. The phylogenetic tree is known to show a high consistency of the relationships between organisms. In this study, the isolate showed similarity of 99% at 100% query coverage to Pediococcus acidilactici strain DSM 20284. A value of 99% indicates that the isolate can be considered as the same species with Pediococcus acidilactici strain DSM 20284. The sequence of homology levels was high, as shown by the red color with a score of ≥200 (Figure 3). Saitou & Nei (1987) indicated that the evolutionary history of organisms can be known using the neighbour-joining method. Organisms within the same taxa are normally clustered together in the phylogenetic tree and have better bootstrap values (Felsenstein, 1985). In this study, we drew a phylogenetic tree to scale and determined the evolutionary distances using the p-distance method. A total of 26 nucleotide sequences and codon positions 1st + 2nd + and 3rd + noncoding were considered, using MEGA 7.0 as reported by Kumar et al. (2016) for evolutionary analyses.  From the results of this homology it can be concluded that the two sequences are the same and have an evolutionary relationship.
The next closest species for which a sequence alignment of at least 100% query coverage was observed were Pediococcus pentosaceus strain DSM 20336, Pediococcus acidilactici strain NGRI 0510Q and Pediococcus argentini strain CRL 776 at 98% similarity to the DS15 isolate. Pediococcus stilesi strain FAIR-E 180 showed 98% similarity with 99% query coverage. An alignment query result of 100% indicates a significant alignment, which means the search sequence in this study was identical with the identified genus, even at the species level.

Conclusion
The PCR of 16S rRNA gene sequence gave an approximately 1400 bp amplicon for LAB DS15, isolated from indigenous dadih of West Sumatera. Phylogenetic analysis showed that LAB DS15 was Pediococcus acidilactici, with 99% similarity to Pediococcus acidilactici strain DSM 20284.

Supplementary File 1. Electrophoresis image of the PCR amplification product.
Click here to access the data.

Open Peer Review Current Peer Review Status:
Version 3

INTRODUCTION
In general, the introduction is relatively good but needs to be added by the results of research from

Hosono et al 1989 and Wirawati et al., 2019 about micflora in dadih
In the introduction, the author needs to explain in more detail the role of GABA produced by LAB and other organisms METHODS An explanation should be added as to why only to choose the DS15 strain producing GABA in this study.
It is necessary to add the reference methods used in the 16S rRNA gene amplification analysis and electrophoresis process

RESULTS AND DISCUSSION
The results are well presented and discussed systematically because the authors used only one strain.

REFERENCES
The author needs to add references as a follow-up to suggestions for improvement in the introduction and method of this paper

The strength of this paper
The strength of study is the first research to report on GABA-producing LAB from dadih and local fermented milk products from Indonesia The weakness of this paper One of the weaknesses of this study is that the authors only used one LAB dadih isolate (strain DS15) so that no comparative data were produced and the discussion was relatively limited.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.

Competing Interests:
I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.
1. Introduction -fairly good and can be improved i. Dadih from Indonesia has-, , and How did they Lactobacillus Streptococcus Leuconostoc Lactococcusidentify the bacteria? Biochemical tests or using molecular approaches? ii. Is there any data on dadih from Malaysia as well for comparison. 2.4 Electrophoresis 1% change to 1% (w/v) in 1x change to1x TAE, 100 V The marker should be mentioned in this section, 1 Kb ladder? What kind of dye did you used? Red dye, syber green, ethidium bromide? State their brand as well Results and discussion Good -due to a single strain/isolate was studied, thus, the explanation is straight forward.

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
No competing interests were disclosed.
ii. Is there any data on dadih from Malaysia as well for comparison.
No, we don't have data on dadih from Malaysia.
2. Methods -can be improved 2.1 Sample -Subtopic sample suggested to change -Bacterial strain The month and year of the bacterium should be mentioned for ex: in June 2009. Why too long to continue the partial sequence 16s rRNA analysis?
Bacterial strains isolated in July 2017. We did this isolation based on the method of Ali (2009), not isolates from the author. et al.,

2 Isolation of bacterial genomic DNA
We used lysozyme-change to "Twenty (20) mg/ml of lysozyme was used to break down ...." Please state where did you keep the genomic DNA. Example in -20 C freezer or 4 C refrigerator prior Analysis We keep the genomic DNA in 4 C refrigerator.

16S rRNA gene amplification Please state the after forward and reverse primers is mentioned. reference
We got reference for forward primer 63F (5'-CAG GCC TAA CAC ATG CAA GTC-3') and reverse primer 1387R (5'-GGG CGG GGT GTA CAA GGC-3') from Laboratory of Medical Molecular Biology and Diagnostic, Indonesian Institute of Sciences, Jakarta, Indonesia.
2.4 Electrophoresis 1% change to 1% (w/v) Ok we will change in 1x change to1x TAE, 100 V Ok we will change The marker should be mentioned in this section, 1 Kb ladder? What kind of dye did you used? Red dye, syber green, ethidium bromide? State their brand as well We used 1 Kb Plus DNA Ladder (ThermoFisher Scientific) Results and discussion Good -due to a single strain/isolate was studied, thus, the explanation is straight forward. Thank You.
No competing interests were disclosed. Exploration of isolates from dadih has been carried out, but no studies have used these isolates as GABA producers. In this study we obtained DS15 isolate from the isolation of various fermented foods which had the highest GABA production. We did this isolation based on the method of Ali et FAIR-E 180 shows 98% similarity with 99% query coverage. P. stilesi 3. As emphasized by the authors, they should elaborate, about characterization and GABA production potential of the strain.
We have carried out quantitative screening on some of the isolates we obtained from dadih, and we found that DS15 isolates produced the highest amount of GABA. Data and discussion are used in another publications.
4. Authors should elaborate on the novelty of the study.
The novelty of this study was the use of bacterial isolates from dadih as a GABA producer.
No competing interests were disclosed.
There is no massive bacterial isolation from dadih; neither no microbial community profiling for dadih, nor pathway identification of GABA production for microbial community of dadih; so, one isolate from dadih does not mean anything. In this study, we isolated bacteria from various fermented food products (dadih, ikan budu, asam durian and tape singkong), determined their GABA producing ability and selected the isolate with the highest GABA production for further identification. The results of isolation and characterization are explained in other articles. The distribution of LAB isolates from the indigenous West Sumatera fermented food (dadih only) is; Origin from Aiadingin area. No competing interests were disclosed.

Competing Interests:
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com