The Genome of the DY46 strain
The whole genome of the L. plantarum DY46 strain composed of a circular chromosome of 3,332,827 bp with a GC ratio of 44.3395%, a total of 3.219 genes, comprising of 3.054 protein-coding sequences, 61 tRNAs, 2 rRNAs, 4 non-coding RNAs and 98 pseudogenes (Fig. 1. and Table 1. ). As a result of the KEGG orthology screening, the encoded proteins identified on the genome of the DY46 strain were classified in 20 different functional categories and summarized in Table S22. The Ortho ANI values of the DY46 and other Lb. plantarum strains were displayed in Fig. 2. Based on Ortho ANI results, Lb. plantarum DY46 showed a high level of identical genetic reciprocal similarity of 99.36%, 99.05%, 99.03%, 99.02% for Lb. plantarum ATCC 8014, WCFS1, ATCC 14917 and ST-III respectively. ATCC 8014 and 14917 were isolated from pickled cabbage, while the well-known probiotic strain WCFS1 was isolated from human saliva (Kleerebezem et al. 2003; Siezen et al. 2010). The strains of ATCC 8014, WCFS1 and ATCC 14917 are commercial strains and considered probiotics (Gaudana et al. 2010; Liu et al. 2015). The ST-III strain was originated from a Korean fermented vegetable called “kimchi”(Wang et al. 2011). On the other side, the strains RI-113 (98.99%), Y44(98.97%) and LL441(98.7%) showed the lowest genetic similarity to the DY46 among the studied L. plantarum strains. As expected, LL441 (cheese) and RI-113 (salami) showed a greater genetic distance than other strains due to their isolation sources (Gonzalez et al. 1994; Flórez and Mayo 2018; Inglin and Meile 2020). L. plantarum can be found in many different environments and shares its ecological niche with L. pentosus and L. paraplantarum and other facultative heterofermentative members of the genus Lactobacillus (Stiles and Holzapfel 1997). Besides, L. plantarum, L. pentosus and L. paraplantarum display very close phenotypes and are genotypically similar due to their rRNA have as same as sequence identity (> 99%). Therefore, these species cannot be discriminated from each other using 16s rDNA sequence analysis (Parente et al. 2010). According to Ortho ANI results which were shown in Fig. 2. L. paraplantarum and L. pentosus exhibit 85.89% and 79.93% similarity with the DY46 strain, respectively. It is usually reported that the ANI value should be above 95–96% to consider that the genomes of the two species are the same (Lee et al. 2016). This confirms that the DY46 strain belongs to the Lb. plantarum species.
Table 1
Genomic features of Lactiplantibacillus plantarum DY46
Item | Complete Genome |
Size (bp) | 3.332.827 |
GC content (%) | 44.3395 |
Genes (total) | 3.219 |
Protein coding sequences | 3.054 |
tRNA | 61 |
rRNA | 2 |
Non-coding RNA | 4 |
Pseudogenes | 98 |
Carbohydrate Fermentation
The lactobacilli can derive ATP from heterofermentative and/or heterofermentative carbohydrate fermentation based on each species/strain preference of sugar utilization. Uncovering carbohydrate fermentation patterns has been widely applied to determine phenotypic traits. Per API 50 CHL test results DY46 can metabolize 22 different carbohydrates out of 49 being tested (Table S21). It is important to note that DY46 cannot metabolize D-Sorbitol, although its genome has sorbitol-6-phosphate 2-dehydrogenase (srlD) and the glucitol/sorbitol phosphotransferase system (srlB, srlE and srl A ) genes. This might be due to the lack of expression of the abovementioned genes encoding specific enzymes required to metabolize sorbitol (Buron-Moles et al. 2019). Lactiplantibacillus plantarum is a protean and resilient species that can grow on a wide range of carbohydrate sources. This phenotypic character is associated with genes involved in carbohydrate metabolism and transport. Most of the transporters involved in carbohydrate metabolism are located in the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) (Gänzle and Follador 2012; Gao et al. 2020). The entire PTS of DY46 strain that was encoded by its genome, comprises PTS System Enzyme I (general enzyme gene, ptsI), phosphocarrier protein HPr gene (ptsH), 26 complete/incomplete substrate-specific enzyme II (EII) complexes genes (Table S24). In the genome of the DY46 strain, the genes of glucose-glucoside, fructose-mannose-sorbose, glucitol, Lactose-N, N’-diacetylchitobiose-β-glucosides and N-Acetylglucoseamine EII complex families were observed as multiple copies, while L-ascorbate, sorbose, mannitol and galactitol EII complex genes were found as single copies. Additionally, the DY46 possesses several other carbohydrate transporter encoding genes on its genome. However, their substrate specificity is unknown and could not be predicted. According to Kleerebezem et al. (2003) various sugar transporter systems are known to import more than one substrate. Lb. plantarum is classified into the facultative heterofermentative Lactobacillus species, which utilize the sugars by way of glycolysis (Embden-Meyerhof-Parnas (EMP) pathway) or the phosphoketolase (PK) pathway, leading to homolactic or heterolactic fermentation routes, respectively (Liu et al. 2015). DY46 genome carries 6-phosphofructokinase 1, fructose-bisphosphate aldolase, glucose-6-phosphate isomerase, transketolase and phosphoketolase genes, which are encoding the key enzymes of EMP and PK pathways (Eiteman and Ramalingam 2015). The genes encoding enzymes related in the intact EMP and PK pathways were predicted in the genome of the DY46 strain and listed in Table S23. Furthermore, 1-phosphofructokinase enzyme encoding fruK gene was detected which is used as the key gene for differentiation of hetero- and homofermentative lactobacilli species (Orita et al. 2008; Zheng et al. 2015). On the other hand, Buron-Moles et al. (2019) reported that obligate heterofermentative strains lacked the 1-phosphofructokinase (PFK) enzyme, in addition to PFK absence, both obligately and facultative heterofermentative species were specifically characterized by the presence of L-arabinose isomerase, L-ribulose kinase and ribulose phosphate epimerase enzymes, respectively (Buron-Moles et al. 2019). Although these enzymes are not found in the genome of DY46, it could be predicted that DY46 has a facultative heterofermentative carbohydrate metabolism like other Lb. plantarum strains.
DY46 also possesses six L-Lactate dehydrogenase (ldhL) genes in the genome of DY46 which is smaller than what Liu et al. (2015) and Gao et al. (2020) reported. The DY 46 appears to be capable of synthesizing only a single isomer of lactate, which may be due to a stable chromosomal deletion (Ferain et al. 1994). Apart from the lactate dehydrogenase genes, the DY46 genome has several other pyruvate depletive enzymes which are responsible for the synthesis of other flavor compounds, such as acetaldehyde, acetoin, oxaloacetate, acetoin and ethanol (Papagianni 2012; Gao et al. 2020). Moreover, the D-lactose and D-galactose fermenting capacity of the DY46 confirmed by both in vitro and in silico analysis. The API results revealed that DY 46 was able to utilize glucose and lactose while, the BlastKOALA scanning results revealed that all enzymes of the Leloir metabolic pathway were present, together with 2 copies of the beta-galactosidase gene in the genome of the DY46.
The Leloir pathway enzyme encoding genes in the DY46 genome consist of one copy of galactokinase gene, one copy of UDP-glucose-hexose-1-phosphate-uridylyltransferase gene, four copies of UDP-glucose-4-epimerase and three copies of aldose-1-epimerase genes. The presence of genes encoding major enzymes of galactose metabolism and in vitro test results are hallmarks of the potential utilization of the DY46 as a dairy starter culture. Similar to Lb. plantarum strains of WCFS1(Kleerebezem et al. 2003), 5 − 2(Liu et al. 2015), Y44 (Gao et al. 2020) and ZJ316 (Li et al. 2016), the Lb. plantarum DY46 genome did not encode all of the tricarboxylic acid cycle-related proteins, although certain genes were found and shown in Table S23. According to KEGG mapper results, the genome of the DY46 strain encloses 203 genes that are related to central and other carbohydrate metabolism. The composition of those gene pools are as follows: 23 glycolysis/gluconeogenesis related genes, 8 TCA cycle-associated genes, 15 pentose phosphate pathway genes, 4 pentose and glucuronate interconversions related genes, 19 fructose and mannose metabolism genes, 19 galactose metabolism genes, 3 ascorbate and aldarate metabolism genes, 22 starch and sucrose metabolism genes, 27 amino sugar and nucleotide sugar metabolism genes, 28 pyruvate metabolism genes, 11 glyoxylate and dicarboxylate metabolism genes, 11 propanoate metabolism genes, 8 Butanoate metabolism genes, 3 C5-branched dibasic acid metabolism genes, 3 inositol phosphate metabolism genes. The gene number associated with carbohydrate metabolism is the same as Lb. plantarum Y44 (Gao et al. 2020). The number of carbohydrate-active enzyme genes of Lb. plantarum ZLP001 (Zhang et al. 2018) and Lb. plantarum KDLS1.0391 (Jia et al. 2017) were reported relatively low (119 and 190 genes, respectively) than the DY46 strain. However, there was no evidence of carbohydrate fermentation patterns that were found to be able to compare with the DY46 strain. In addition, as shown in table 3, Y44 (24 sugars) and ATCC14917 (25 sugars) were able to metabolize a higher number of sugars than DY46. These differences may arise from the physiological and genetic adaptation of the strains to the ecological niches in which they were being isolated.
Bacteriocin biosynthesis
According to the results of the whole genome search of the DY46 strain against the BAGEL database, the gene cluster responsible for bacteriocin biosynthesis consists of 26 genes and its total length is approximately 24.2 kb (Fig. 3). In this gene cluster, transport-related genes, immunity protein and plantaricin precursor genes and several core genes (Pln E, Pln F and pln K) are encoded and all protein sequences confirmed by protein BLAST (Table S1). L. plantarum DY46 strain was found to have the same core genes encoding Class II bacteriocins as ATCC8014 (Yu et al. 2020). The plnEF locus is widely distributed among L. plantarum strains isolated from various ecological niches. The well-studied plnEF loci have also been reported in L. plantarum WCFS1, NC8, JDM1, C11, V90, J51 and J23 strains, respectively (Tai et al. 2015). On the other hand, the isoelectric points (pI) and amino acid lengths of the pln E and pln F mature peptides that do not have the GG leader sequence that we detected (Table S2) are identical to pln E and pln F bacteriocins that were previously reported in WCFS1, NC8, J23, J51, C11 and V90 strains (Diep et al. 2009). Normally, plnJ and pln K peptides, which are subunits of plantaricin JK, are encoded in the same gene cluster/operon and more effective together, whereas in the present study only the pln K peptide was detected with 95.35% percent identity (Todorov 2009). Moreover, the pI (8.59) and length of the mature pln K peptide (28aa) detected have differed significantly from the mature plnK peptides (pI:10.52; 32aa) as previously reported in C11, NC8, V90 and WCFS1 (Diep et al. 2009). Apart from the core genes, the presence of secretion genes pln H (HlyD, accessory protein for ABC-transporter; (Accession no: WP_027821501.1), pln G (LanT, Bacteriocin ABC-transporter; WP_027821502.1) have been verified in the plantaricin gene cluster. Bacteriocin ABC transporters are involved in the transport of the mature peptide through the cell membrane, which is formed by deleting the leader peptide sequence from prebacteriocin (Havarstein et al. 1995). The accessory protein (also called the accessory factor) is another necessary component for the ABC transporter system-dependent translocation process (Nes et al. 1996). Another gene cluster member identified is the putative Na+/H+ antiporter protein (orf00033: AFM80194.1), which sustainably maintains intracellular proton balance and leading to the enabling of ATP required for ABC transporters (Jia et al. 2017). In addition, the bacteriocin gene cluster contains genes encoding orf00020 (CAAX amino terminal protease family protein; EFK30757.1) and orf 00028 (bacteriocin immunity protein; WP_127526380.1) immunity proteins that play a role in protecting bacteria from their mature bacteriocins (Todorov 2009). The other members of the bacteriocin biosynthetic gene cluster were listed in supplemental table S1.
Antibiotic resistance
Antibiotic susceptibility of the DY46 strain was evaluated as reported by the Clinical and Laboratory Standards Institute’s performance standards. The zone of inhibition (ZOI) values of fourteen antibiotics tested against the DY46 strain was shown in Table S3 with resistome search match results. DY46 was found to be resistant (ZOI ≤ 14mm) to methicillin (5 µg), oxacillin (1 µg), streptomycin (10 µg), vancomycin (30 µg), amikacin (30 µg), kanamycin (30 µg), azithromycin (15 µg), tetracycline (30 µg) and rifampacin (5 µg). Apart from these, DY46 was sensitive (ZOI ≥ 20mm) to ampicillin (10 µg), carbenicillin (100 µg) and amoxycillin (25 µg), while it showed intermediate sensitivity (ZOI ~ 15 to 19 mm) to Penicillin G (10 U) and Erythromycin (10 µg). The antibiotic resistance profile of the DY46 strain showed partial similarity with the previously reported L. plantarum profiles (Sharma et al. 2016, 2017; Klarin et al. 2019). There are no antibiotic resistance genes that have been found in both the ResFinder 4.1 and CARD databases. However, twenty-one antibiotic resistance genes which were shown in Table S3 have been detected with PATRIC 3.6.8 and KEGG databases. The identified genes were found to be related to β-Lactams (9), Streptomycin (2), Vancomycin (7), Macrolides (1), Tetracyclines (1) and Rifampacin (1). It is commonly accepted that Lactobacillus species have very high resistance to aminoglycosides. Lb. plantarum is known to be resistant to vancomycin due to its intrinsic peptidoglycan precursors consisting of D-lactate instead of D-alanine at the C-terminus (Gueimonde et al. 2013; Álvarez-Cisneros and Ponce-Alquicira 2018; Campedelli et al. 2019). Besides, the VanX gene is highly specific for hydrolyzing D-ala-D-ala dipeptides and a significant precursor of the cell wall (Liu et al. 2015). The vancomycin resistance genes were identified in the genome of DY46 consists of phospho-N-acetylmuramoyl-pentapeptide-transferase (mraY), alanine racemase (alr), D-alanyl-D-alanine ligase (ddl), UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (murF), UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase (murG), D-Ala-D-Ala carboxypeptidase (vanY) and D-Ala-D-Ala dipeptidase (vanX). In addition, streptomycin resistance responsible gibD and S12p genes were detected. However, no specific genes were found for amikacin and kanamycin, even if resistance was present. This is because genotype and phenotype do not overlap completely (Zhang et al. 2012). Apart from these, RlmA (II) (23S rRNA (guanine(748)-N(1))-methyltransferase), S10p (SSU ribosomal protein S10p (S20e)), rpoB (DNA-directed RNA polymerase beta subunit) genes detected which were related with macrolides, tetrcyclines and rifamycins, respectively. Moreover, mecA (penicillin-binding protein 1A), pbp2a (penicillin-binding protein 2A) and PenP (beta-lactamase class A) major genes responsible for beta-lactam resistance were detected on the DY46 genome. Methicillin and oxacillin resistance is known to be associated with penicillin-binding proteins. Interestingly, resistance to Penicillin G was observed in DY46, although Lactobacillus are known to be susceptible. Some authors have reported Penicillin G-resistance in recent years in some strains of Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus plantarum, which also confirms our study (Abriouel et al. 2015; Zheng et al. 2017). Because of the growing concern that foods and/or common bacteria may serve as potential reservoirs for antimicrobial resistance genes, probiotics must not carry transferable antimicrobial resistance genes to be used for humans or animals (Zhang et al. 2012). Because many Lactobacillus species have intrinsic resistance to many antimicrobial compounds, and such resistance is known not to be associated with any particular safety concerns. However, the intrinsic antibiotic resistance genes on the chromosome should not be flanked by integrases and/or transposases. As a result of Protein BLAST screening for antibiotic resistance genes detected in this study, no evidence of horizontal gene transfer was found (Table S20).
Prophages and horizontal gene transfer
Prophage search results display 9 prophage regions (three intact, two questionable and four incomplete) found in the genome of the DY46 strain and summarized in Table 2. One of the three intact prophage regions showed similarity with Lactob_Sha1_NC_019489 (48.8Kb), (region 1), and the other two like Lactob_phig1e_NC_004305 (39.9Kb) and Staphy_SPbeta_like_NC_029119 (29.6Kb), region 2 and region9, respectively (Fig S1.). It was determined that Lactob_Sha_1 and Lactob_phig1e showed the highest protein matching among the identified prophages. These are the most common temperate prophages ever described in L. plantarum strains (Pei et al. 2020). All the prophage regions have attL/attR sequences and integrase except for region 6 (Paenib_PBL1c) and 7 (Bacill_vB_BtS_BMBtp14). In bacterial genomes, integrases are functional identifiers for phages, pathogenicity islands and integrative plasmids (Juhas et al. 2009; Liu et al. 2015). Three integrases (PP_00611(region 1), PP_01193(region 2) and PP_03267 (region 9)) were determined in the identified intact regions. The location of attL/attR sequences varies within intact phages. The attL sequences in regions 2 and 9 are located upstream of the integrase, while the attR sequence of region 1 was found downstream of the integrase. Additionally, attL and attR sequences of phage 1 and phage 9 were identical, but the attL and attR sequences of phage 2 are different from them. The Intact phage region 1 extends from 558,178 bp to 607,042 bp of the genome and includes 58 protein-coding sequences containing all prophage components from PP_00554 (transposase) to PP_00611 (phage integrase). The intact region 2 extends from 1,217,186 bp to 1,257,125 bp of the genome and consists of 49 protein-coding sequences containing prophage components from PP_01193 (phage integrase) to PP_01241. Moreover, the intact region 3 (9) is located between 3,302,034 bp to 3,331,708 bp of the genome and consists of 33 protein-coding sequences containing prophage components from PP_03263 (protease) to PP_03295. Unlike previously reported for L. plantarum WCFS1(Ventura et al. 2003) and 5 − 2 strains (Liu et al. 2015), only the two intact phages (Sha1 and phig1e) were found to contain all packaging/head/tail gene clusters, DNA packaging genes and the lysis cassette. All components of the identified prophage elements have been listed in Table S4-S12.
Table 2
The predicted prophage regions of Lactiplantibacillus plantarum DY46 genome
Region | Length | Completeness | Score | Region Position (bp) | Total Proteins | Most Common Phage (Number of matching proteins) | GC% | attL/attR sites | Integrase ORF start-stop |
1 | 48.8Kb | Intact | 150 | 558178–607042 | 58 | Lactob_Sha1_NC_019489 (27) | 41.1 | + | 605879–607042 |
2 | 39.9Kb | Intact | 150 | 1217186–1257125 | 49 | Lactob_phig1e_NC_004305 (20) | 41.2 | + | 1217352–1218560 |
3 | 27.2Kb | Questionable | 70 | 2511806–2539007 | 21 | Lactob_Sha1_NC_019489 (3) | 42.4 | + | 2537850–2539007 |
4 | 24.6Kb | Incomplete | 30 | 2969719–2994363 | 9 | Entero_IME_EF3_NC_023595 (2) | 45.2 | + | 2981712–2982866 |
5 | 17.8Kb | Incomplete | 60 | 3002777–3020641 | 11 | Bacill_Waukesha92_NC_025424 (2) | 41.6 | + | 3018894–3019580 |
6 | 11.3Kb | Questionable | 80 | 3034765–3046067 | 21 | Paenib_PBL1c_NC_048689 (2) | 39.7 | - | - |
7 | 5.7Kb | Incomplete | 30 | 3136597–3142320 | 9 | Bacill_vB_BtS_BMBtp14_NC_048640 (2) | 38.7 | - | - |
8 | 11.7Kb | Incomplete | 50 | 3249993–3261724 | 15 | Escher_ESCO5_NC_047776 (4) | 38.9 | + | 3261137–3261724 |
9 | 29.6Kb | Intact | 150 | 3302034–3331708 | 33 | Staphy_SPbeta_like_NC_029119 (4) | 39.3 | + | 3313269–3314018 |
Horizontal gene transfer between bacteria can usually occur by natural competence or bacteriophage infection (Kleerebezem et al. 2003; Liu et al. 2015). The result of sequence homology screening revealed that most of the genes of the DY46 genome were homologous to Lactiplantibacillus plantarum genes and only 1.52% (49 genes ) of total genes in the genome may have been gained thru horizontal gene transfer from other bacteria, e.g. Pediococcus acidilactici, Pediococcus pentosaceus, Lactiplantibacillus pentosus, Lactiplantibacillus paraplantarum, Leuconostoc sp., Pediococcus damnosus, Paucilactobacillus suebicus, Liquorilactobacillus hordei, Fructilactobacillus lindneri, Lacticaseibacillus paracasei, Loigolactobacillus coryniformis, Lactobacillus kefiranofaciens, Weissella jogaejeotgali, Lactobacillus sanfranciscensis, Lactiplantibacillus argentoratensis, Companilactobacillus paralimentarius, Companilactobacillus sp., Companilactobacillus bobalius, Levilactobacillus brevis, Levilactobacillus fujinensis, Lentilactobacillus buchneri, Lactobacillus crispatus, Levilactobacillus paucivorans, Lactococcus lactis subsp. lactis bv. diacetylactis, Lactiplantibacillus daowaiensis, Companilactobacillus nodensis, Levilactobacillus lindianensis, Latilactobacillus sakei, Bifidobacterium longum, Lapidilactobacillus mulanensis, Limosilactobacillus fermentum, Lactobacillus diolivorans. It was determined that most of the vertically transferred genes originated from Lactobacillus species are found in the microbiota of fermented vegetables. Among these 49 genes, 12 transposase genes (PP_02973, PP_02974, PP_02980, PP_02981, PP_03087, PP_03207, PP_03208, PP_03275, PP_03281, PP_03285, PP_03288, PP_03295) which were derived from recombination, repair and replication. Moreover, all the genes considered horizontally transferred were phage related and summarized in Table S13-19.
Probiotic properties
When new probiotic strains are discovered, certain characterization tests are required to confirm probiotic properties. Therefore, probiotic characterization tests were performed to confirm the probiotic properties of the DY46. The β-haemolysis test results showed that DY46 does not have β-hemolytic activity. The cell surface hydrophobicity of DY46 characterized by using xylene. As shown in Fig. 4A, the cell surface hydrophobicity of DY46 appears to increase in direct proportion to the bile salt concentration. Cell surface hydrophobicity of the DY46 was determined as 33%, 38.5% and 46.1% at 3, 5 and 10g/L bile salt concentrations, respectively. The cell surface hydrophobicity of the control sample was found to be at 4.38%. However, similar to present work, it has been reported in previous studies that some lactobacilli, including L. acidophilus and L. johnsonii strains displayed surface hydrophobicity as low as 2–5% (Rijnaarts et al. 1993; Schillinger et al. 2005). Kaushik et al. (2009) reported that such large differences in cell surface hydrophobicity could occur due to differences in the expression level of cell surface proteins, depending on environmental conditions and/or bacterial strain.
Auto-aggregation is an important bacterial characteristic in different ecological niches, especially in human and animal mucosa where probiotics confer health benefits. The auto-aggregation capacity is an important factor for maintaining sufficient numbers of probiotic strains under adverse conditions of the oral cavity and gastrointestinal tract. The cellular auto-aggregation test results presented in Fig. 4B revealed that there is an inverse correlation between auto-aggregation and bile salt concentrations. The auto-aggregation ability of DY46 was determined as 85.7% for the control sample, while it was determined as 84.69%, 79.05% and 51.35% for bile salt concentrations of 3, 5 and 10 g/L, respectively. (Li et al. 2015) reported auto-aggregation ranged from 0.86 to 65.15% in different Lb. fermentum strains isolated from various Chinese fermented foods. Ramos et al. (2013) reported an auto-aggregation value between 18.08 to 20.94% in cocoa originated Lb. plantarum strains. Moreover, Goel et al. (2020) reported 52.91% and 72.84% auto-aggregation ability for two different Lb. plantarum strain isolated from Indian fermented foods. Similar to the present study, Aslim et al. (2007) observed a reduction of auto-aggregation capacity in the presence of bile in Lb. delbrueckii subsp. bulgaricus strains. The fact that the DY46 showed an auto-aggregation capacity of 51.35% at a bile salt concentration close to the real gastrointestinal tract (GIT) environment is significant to prove its probiotic property. Based on the antimicrobial activity test results, the DY46 displayed an apparent zone of inhibition (> 5 mm) against K. pneumoniae (ATCC 13883), P. vulgaris (ATCC 8427) and S. Typhimurium (ATCC 14028) whereas, there is no inhibition zone observed against other test strains. The lack of Pln J peptide in the genome of DY46 strain brings confounding factor of whether inhibiton zones achieved against test pathogens might also be due to the ogranic acids produced by DY46.
It is necessary to test probiotic candidates against acid and bile salts to determine their resistance under inevitable conditions of the human gastrointestinal tract (GIT) (Angmo et al. 2016). The growth kinetics of DY46 at different pH (2, 3, 4, 5 and 7) conditions at 30 and 37ºC are shown in Figures S2 and S3. No or ignorable level of growths seen at pH = 2 and pH = 3 conditions for both 30 and 37C. Similar findings were also reported that cell density of Lactobacilli are significantly influenced by low pH, especially at 1.5 and 2 (Guo et al. 2010; Ortakci and Sert 2012; Ortakci et al. 2012; Angmo et al. 2016)
The duration of lag phase achieved was pH = 7 < pH = 5 < pH = 4 in both 30 and 37ºC. The maximum specific growth rates achieved at both temperatures were as follows pH = 7 > pH = 5 > pH = 4. Similarly, final cell turbidities achieved were pH = 7 > pH = 5 > pH = 4. The µmax levels achieved at 30 and 37ºC were slightly different with the latter providing better growth performance. This perhaps relates to the probiotic potential of DY46 that can proliferate at a body temperature of 37ºC. The decreasing µmax values and poor or no growth seen at lower pH values are perhaps due to the cellular machinery effort to maintain relatively neutral pH values in the cytoplasm at the expenditure of ATP to push thru H+ cations across the outside of the membrane (Axelsson 2004).
The growth kinetics of DY46 at different bile (0.3, 0.5, 1% and control) conditions at 30 and 37ºC are shown in Figures S4 and S5. A significantly lower lag phase duration observed at 37ºC vs 30ºC. The µmax values achieved at 37ºC were as follows Control (No bile) > 0.3% bile > 0.5% bile > 1% bile. Final cell densities achieved at 37ºC were Control (No bile) > 0.3% bile > 0.5% bile = 1% bile. We speculate that the bile salt exerts certain stress on the cell and is triggered by temperature. Nevertheless, DY46 can still proliferate to remarkable cell concentrations as measured with microplate reader across all bile levels even at 37ºC. It was interesting to note that although longer lag phases achieved for all bile treatments at 30ºC, the cells further caught up with higher µmax values achieved compared to 37ºC. This shows DY46 perhaps could better tolerate bile salt stress at a lower temperature which can be supported by the observation of similar final cell densities across all bile concentrations at 30ºC.
Overall, better growth kinetics achieved at 37ºC for pH conditions tested though lower pH values reduced or decreased growth completely. Although a shorter lag phase seen at 37ºC with all bile concentrations evaluated, DY46 cells better-tolerated bile salt at 30ºC with higher µmax and final cell concentrations obtained. The DY46 can be resisting and proliferating under adverse conditions with moderately lower pH values and bile concentration mimicking the human GIT.
In conclusion, whole-genome sequencing and physiological characterization of Lb. plantarum DY46 isolated from Shalgam has been performed to determine probiotic properties of this novel strain. Genome analysis revealed this strain follows a facultative heterofermentative sugar metabolism where hexoses are cleaved thru glycolysis versus pentoses are hydrolyzed via the pentose phosphate pathway. Genome evidence predicted DY46 could biosynthesize Plantaricin-E, Plantaricin-F, Plantaricin-K showing the antimicrobial potential of DY46 which was confirmed by in vitro antagonistic activity test that supernatants of DY46 culture provided inhibition zones against K. pneumonia ATCC 13883, P. vulgaris ATCC 8427, S. Thypimirium ATCC 14028. Also, DY46 is tolerant to acid and bile concentrations mimicking human gastrointestinal conditions. Overall, Lb. plantarum DY46 is a promising bacterium possessing certain probiotic traits confirmed by in vitro analysis and perhaps a potential dietary supplement candidate that might provide therapeutic benefits to the host.