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Whole Genome Sequence of Lactiplantibacillus plantarum HOM3204 and Its Antioxidant Effect on D-Galactose-Induced Aging in Mice
1Coree Beijing Co., Ltd., No. A-7 Tianzhu West Rd., Tianzhu Airport Industrial Zone A, Shunyi District, Beijing 101312, P.R. China
2Dx&Vx Co., Ltd., Seoul 13201, Republic of Korea
3Health Food Function Testing Center, College of Applied Arts and Science, Beijing Union University, Beijing 100101, P.R. China
J. Microbiol. Biotechnol. 2023; 33(8): 1030-1038
Published August 28, 2023 https://doi.org/10.4014/jmb.2209.09021
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Probiotics are live microorganisms that confer health benefits to the host when administered in adequate amounts [1].
The requirement of whole-genome sequencing (WGS) analysis of probiotic candidates to assess their food safety was proposed by the European Food Safety Authority in 2019 [10]. Accordingly, genes encoding antimicrobial resistance, virulence, and toxigenicity were subjected to extensive assessments [10]. Whole genome sequences of many
Reactive oxygen species (ROS), including hydroxyl radicals, superoxide anions, and hydrogen peroxide, are produced via oxygen metabolism and balanced by the rate of oxidant formation and elimination [14, 15]. Oxidative stress, caused by an imbalance between the generation of ROS and antioxidant defense systems, is associated with the natural aging process and pathogenesis of many diseases [16]. Accumulating evidence demonstrates that probiotics are effective against oxidative stress via enzymatic antioxidant defenses, including SOD, GSH-Px, and glutathione reductase (GR), and antioxidant metabolites, such as GSH, butyrate, and folate [17, 18]. Several
The D-galactose-induced aging mouse model, which mimics natural aging, is one of the most commonly used models for oxidative stress studies [21]. Researchers often employ this model to determine the anti-aging activities and antioxidant effects of probiotics [21-23]. D-Galactose injection increases oxidative stress by increasing the malonaldehyde (MDA) levels and decreasing the activity of antioxidant enzymes in mice [24, 25]. MDA is the principal and most studied product of polyunsaturated fatty acid peroxidation [26]. Some studies have assessed MDA to quantify the level of oxidative stress in vitro and in vivo [26].
In the present study, we conducted WGS analysis of
Materials and Methods
Genomic DNA Extraction, Genome Sequencing, Assembly, and Annotation
The whole genome of
The genome was sequenced using the PacBio Sequel (Pacific Biosciences, USA) and Illumina HiSeq platforms (Illumina Inc., USA) [27]. Low-quality reads were filtered out using the single-molecule, real-time sequencing technology (SMRT, v2.3.0) and the high-quality filtered reads were assembled to generate one contig without any gaps [28]. The paired-end strategy was used in the Illumina sequencing platform. Falcon (v0.3.0) was used for sub-read self-correction and three-generation sequence assembly [29]. Sub-reads were then processed to generate consensus sequences using Quiver (v2.2.2) [28]. A single-pass read accuracy improver (Sprai, v0.9.9.23) was used to correct the sequencing errors in single-pass reads [30]. Contigs were circularized using Circlator [31]. The assembled genome was annotated to identify the protein-coding and RNA genes using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline [32].
Gene prediction of the assembled genome was conducted using Prodigal (v2.6.3) [33]. Functions of the predicted protein-coding genes were annotated using the Clusters of Orthologous Groups (COG) database annotations based on protein alignment using the Diamond software (e-value < 1e-5) [34]. Prophages were predicted using PhiSpy (v2.3) [35]. Pathogen–host interactions and the Comprehensive Antibiotic Resistance Database (CARD) were used for pathogenicity and drug resistance analyses, respectively [36, 37]. Carbohydrate-Active Enzymes (CAZy) analysis was performed using the CAZy database [38]. tRNA and rRNA genes were predicted using tRNAscan-SE (v1.3.1) [39] and rRNAmmer (v1.2) [40], respectively. Finally, sRNAs were predicted using BLAST against the Rfam database [41], and the circular genome graph was created using Circos (v0.69) [42].
Comparative Analysis
Ten reference
Evaluation of Antioxidant Activity of Lpb. plantarum Strains In Vitro
T-AOC and hydroxyl radical scavenging, SOD, GSH-Px, and GSH activities were determined using A015, A018-1-1, A001-2, A005, and A006-1-1 assay kits, respectively (China). Following this, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was determined, according to a modified method of Lin and Chang [46]. Briefly, 2 ml of intracellular cell-free extract was mixed with 2 ml of DPPH ethanol solution (0.2 mmol/l). The mixed solution was placed in the dark for 30 min at 25°C and centrifuged at 11,000 ×
DPPH scavenging activity (%) = [1 – (Ai – Aj)/A0] ×100%.
All in vitro assays were performed in triplicates.
Antioxidant Effects of Lpb. plantarum HOM3204 on D-Galactose-Induced Aging in Mice
Freeze-dried
Statistical Analysis
Data are presented as the mean ± standard error of the mean. Data analysis was conducted using one-way analysis of variance, followed by Tukey’s multiple comparisons test with the SPSS software (version 25, IBM, Corp., USA). Values were considered statistically significant at
Results
Genome Features
As shown in Fig. 1 and Table 1, the complete genome of
-
Table 1 . Genome features of
Lactiplantibacillus plantarum HOM3204.Attribute Chromosome Plasmid 1 Plasmid 2 Genome size (bp) 3,232,697 48,573 17,060 DNA GC content (%) 44.61 39.04 40.57 Protein-coding genes 2,247 35 7 rRNA genes 16 0 0 tRNA genes 68 0 0 sRNA genes 38 3 1
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Fig. 1. Circular genome graph of
Lactiplantibacillus plantarum HOM3204. Circles, from inside to outside, represent the genome size, GC skew, GC contents, coding sequence (CDS) in the reverse strand, tRNA and rRNA genes in reverse strand, tRNA and rRNA genes in forward strand, and CDS in forward strand. A–Z, respectively, indicate the functional classification of CDS genes on the chromosome and plasmids using the Clusters of Orthologous Groups (COG) database. Circos (v0.69) software was used to create a genomic map with the given information.
One prophage in plasmid 1 was identified using PhiSpy. No drug resistance and virulence genes were found according to the minimum cutoff of 90% nucleotide identity over a minimum coverage length of 60% [47, 48] using CARD and VFDB, respectively.
On the chromosome, 2,247 genes (74.2%) were classified into COG functional categories (Fig. 2). Two hundred and fifty-one genes (11.17%) belonged to amino acid transport and metabolism, 282 genes (12.55%) belonged to carbohydrate transport and metabolism, 266 genes (11.84%) belonged to transcription, and 381 genes (16.96%) belonged to general function prediction only.
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Fig. 2. Functional categorization of all predicted open reading frames (ORFs) in the
Lpb. plantarum HOM3204 genome using the COG database. Diamond (E-value < 1e-5) was used for protein alignment.
Comparison of Lpb. plantarum Strains
To understand the evolutionary relationship between the strains, ML and ANI trees were constructed. The results are shown in Figs. 3 and 4, respectively. According to the analysis of the ML tree, 10 strains, namely ST-III, 299v, WCFS1, ZJ316, LPL-1, J26, 16, KDLS1.0391, P-8, and LP3, were not grouped together with HOM3204, suggesting that
-
Fig. 3. ML tree analysis of
Lpb. plantarum HOM3204 with 10 available complete genome sequences ofLpb. plantarum . ParaAT (V2.0) was used as a parallel tool for constructing multiple protein-coding DNA alignments. The maximum likelihood (ML) phylogenetic tree was constructed using RAxML. Numbers above the branches indicate the bootstrap supports from 500 replicates. The higher the bootstrap value, the more reliable is the evolution tree.
-
Fig. 4. Average nucleotide identity (ANI) tree analysis of
Lpb. plantarum HOM3204 and 10 available genome sequences ofLpb. plantarum strains. ANI tree was constructed using Pyani software.
Genome features of the ten
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Table 2 . Comparison of the chromosomal properties of different
Lpb. plantarum strains.Strain HOM3204 WCFS1 LP3 ST-III 299v J26 LPL-1 16 P-8 ZJ316 KLDS1.0391 Genome size (bp) 3,232,697 3,308,273 3,259,858 3,254,376 3,302,055 3,096,468 3,186,859 3,044,678 3,035,719 3,203,964 2,886,607 No. of plasmids 2 3 2 1 0 4 1 10 7 3 3 GC content (%) 44.61 44.47 44.50 44.58 44.40 44.80 44.65 44.74 44.80 44.65 44.80 Annotated genes 3,064 3,116 3,077 3,071 3,153 3,043 3.049 2,874 2,956 3,043 2,891 tRNA genes 72 72 73 70 57 70 67 68 71 63 52 rRNA genes 16 16 16 15 3 16 16 16 16 15 13 ANI (%) 100% 99.31% 99.22% 99.16% 99.14% 99.06% 99.03% 98.98% 98.98% 98.94% 98.93%
Oxidative Stress-Related Proteins
We identified the oxidative stress-related proteins encoded in the genome of
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Table 3 . Oxidative stress-related proteins of
Lpb. plantarum HOM3204.Oxidative stress-related protein Locus tag Database Glutathione peroxidase Chr-gene 0184 GO:0004602/COG0386 Removal of superoxide radicals Chr-gene 0584 GO:0019430 Glutathione-disulfide reductase Chr-gene 0323, Chr-gene 0991, Chr-gene 1464, Chr-gene 2697 GO:0004362 Response to the oxygen radical Chr-gene 1464, Chr-gene 2697 GO:0000305 Catalytic activity Chr-gene 0102, Chr-gene 1140, Chr-gene 1357, Chr-gene 1915, Chr-gene 1931, Chr-gene 2678 GO:0003824 Catalase Chr-gene 2929 GO:0004096 Flavin reductase (NADH) Chr-gene 0045, Chr-gene 1116, Chr-gene 2249, Chr-gene 2250, Chr-gene 2680, GO:0036382 Thioredoxin-disulfide reductase Chr-gene 0584, Chr-gene 1888, Chr-gene 2175, Chr-gene 2817 GO:0004791 Thioredoxin peroxidase Chr-gene 1928 GO:0008379 Thioredoxin reductase Chr-gene 0584, Chr-gene 2138 COG:0492 DNA-binding ferritin-like protein Plasmid 2-gene 0005 COG:0783
In Vitro Antioxidant Activity
In this study, six indices (T-AOC, hydroxyl radical, DPPH radical, SOD, GSH-Px, and GSH) were chosen to evaluate the antioxidant activity of
-
Table 4 . Antioxidant activities of different
Lpb. plantarum strains in vitro.Strain T-AOC (U/ml) ·OH scavenging (%) DPPH scavenging (%) SOD (U/ml) GSH-Px (U/ml) GSH (mg/l) HOM3204 23.84 ± 2.44 76.84 ± 0.36 94.18 ± 0.45 28.89 ± 0.30 20.57 ± 2.73 37.56 ± 2.81 ST-Ⅲ 4.13 ± 0.44** 76.57 ± 0.36 93.00 ± 0.65 27.34 ± 0.52* 24.29 ± 3.18 23.72 ± 3.98** Lp-115 15.48 ± 1.13** 77.16 ± 0.60 91.03 ± 0.53** 24.63 ± 2.11* 15.60 ± 2.06 59.85 ± 5.57** Vitamin C 16.90 ± 1.42** 36.70 ± 1.33** 96.14 ± 0.08** 34.32 ± 0.36** ND ND Comparison of the
Lpb. plantarum HOM3204 strain with other strains: *p < 0.05, **p < 0.01. ·OH, hydroxyl radical scavenging.ND, not determined.
Antioxidant Effect of Lpb. plantarum HOM3204 on D-Galactose-Induced Aging in Mice
D-Galactose-induced aging mice showed a significant increase in the level of MDA compared to the control group (D-galactose vs. control, 6.31 ± 0.85 vs. 7.77 ± 1.11 nmol/ml,
The effects of
-
Table 5 . Effects of
Lpb. plantarum HOM3204 on malonaldehyde (MDA), protein carbonyl, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH) levels in the D-galactoseinduced oxidative injury mouse model.Group MDA (nmol/ml) Protein carbonyl (nmol/mgprot) SOD (U/ml) GSH-Px (U/ml) GSH (mgGSH/gprot) Model group 7.81 ± 1.43 7.02 ± 1.36 213 ± 38 390 ± 83 6.17 ± 0.79 HOM3204 7.59 ± 1.19 6.49 ± 1.98 230 ± 48 469 ± 68* 7.35 ± 1.47* Comparison of the
Lpb. plantarum HOM3204 group with the model group: *p < 0.05.
Discussion
WGS is generally used to study the information and potential functions of genes. Functional genomics research helps to better understand the molecular mechanisms of action of probiotics. [49]. Currently, 682 genome datasets of
We used the ML and ANI trees for the comparative analysis of
Hydroxyl radical, DPPH radical, T-AOC, SOD, GSH-Px, and GSH have been widely used as evaluation indices for ROS-related antioxidant activity [19, 20]. Strains with strong antioxidant activity can cope with oxidative stress. In this study,
Oxidative stress-related proteins, particularly SOD and GSH-Px, were identified in the genome of
In this study, we proved that
Data Availability
The complete nucleotide sequence of
Authors Contributions
D.Z., S.Z. contributed to the experiment design and interpreted all the results. D.Z. performed probiotic characterization in vitro tests. T.W. prepared the probiotics powders. Y.Z. performed animal related experiments. D.Z. performed statistical analysis and wrote the manuscript. S.Z., S.L. and C.L. edited the manuscript. All authors read and approved the final manuscript.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2023; 33(8): 1030-1038
Published online August 28, 2023 https://doi.org/10.4014/jmb.2209.09021
Copyright © The Korean Society for Microbiology and Biotechnology.
Whole Genome Sequence of Lactiplantibacillus plantarum HOM3204 and Its Antioxidant Effect on D-Galactose-Induced Aging in Mice
Di Zhang1, Heesung Shin2, Tingting Wang1, Yaxin Zhao3, Suwon Lee1,2, Chongyoon Lim1,2, and Shiqi Zhang1*
1Coree Beijing Co., Ltd., No. A-7 Tianzhu West Rd., Tianzhu Airport Industrial Zone A, Shunyi District, Beijing 101312, P.R. China
2Dx&Vx Co., Ltd., Seoul 13201, Republic of Korea
3Health Food Function Testing Center, College of Applied Arts and Science, Beijing Union University, Beijing 100101, P.R. China
Correspondence to:Shiqi Zhang, zsqkevin@163.com
Abstract
Lactiplantibacillus plantarum, previously named Lactobacillus plantarum, is a facultative, homofermentative lactic acid bacterium widely distributed in nature. Several Lpb. plantarum strains have been demonstrated to possess good probiotic properties, and Lpb. plantarum HOM3204 is a potential probiotic strain isolated from homemade pickled cabbage plants. In this study, whole-genome sequencing was performed to acquire genetic information and predict the function of HOM3204, which has a circular chromosome of 3,232,697 bp and two plasmids of 48,573 and 17,060 bp, respectively. Moreover, various oxidative stress-related genes were identified in the strain, and its antioxidant activity was evaluated in vitro and in vivo. Compared to reference strains, the intracellular cell-free extracts of Lpb. plantarum HOM3204 at a dose of 1010 colony-forming units (CFU)/ml in vitro exhibited stronger antioxidant properties, such as total antioxidant activity, 2,2-diphenyl-1-picrylhydrazyl radical scavenging rate, superoxide dismutase activity, and glutathione (GSH) content. Daily administration of 109 CFU Lpb. plantarum HOM3204 for 45 days significantly improved the antioxidant function by increasing the glutathione peroxidase activity in the whole blood and GSH concentration in the livers of D-galactose-induced aging mice. These results suggest that Lpb. plantarum HOM3204 can potentially be used as a food ingredient with good antioxidant properties.
Keywords: Lactiplantibacillus plantarum, whole genome sequence, antioxidant activity, D-galactose-induced aging, oxidative stress
Introduction
Probiotics are live microorganisms that confer health benefits to the host when administered in adequate amounts [1].
The requirement of whole-genome sequencing (WGS) analysis of probiotic candidates to assess their food safety was proposed by the European Food Safety Authority in 2019 [10]. Accordingly, genes encoding antimicrobial resistance, virulence, and toxigenicity were subjected to extensive assessments [10]. Whole genome sequences of many
Reactive oxygen species (ROS), including hydroxyl radicals, superoxide anions, and hydrogen peroxide, are produced via oxygen metabolism and balanced by the rate of oxidant formation and elimination [14, 15]. Oxidative stress, caused by an imbalance between the generation of ROS and antioxidant defense systems, is associated with the natural aging process and pathogenesis of many diseases [16]. Accumulating evidence demonstrates that probiotics are effective against oxidative stress via enzymatic antioxidant defenses, including SOD, GSH-Px, and glutathione reductase (GR), and antioxidant metabolites, such as GSH, butyrate, and folate [17, 18]. Several
The D-galactose-induced aging mouse model, which mimics natural aging, is one of the most commonly used models for oxidative stress studies [21]. Researchers often employ this model to determine the anti-aging activities and antioxidant effects of probiotics [21-23]. D-Galactose injection increases oxidative stress by increasing the malonaldehyde (MDA) levels and decreasing the activity of antioxidant enzymes in mice [24, 25]. MDA is the principal and most studied product of polyunsaturated fatty acid peroxidation [26]. Some studies have assessed MDA to quantify the level of oxidative stress in vitro and in vivo [26].
In the present study, we conducted WGS analysis of
Materials and Methods
Genomic DNA Extraction, Genome Sequencing, Assembly, and Annotation
The whole genome of
The genome was sequenced using the PacBio Sequel (Pacific Biosciences, USA) and Illumina HiSeq platforms (Illumina Inc., USA) [27]. Low-quality reads were filtered out using the single-molecule, real-time sequencing technology (SMRT, v2.3.0) and the high-quality filtered reads were assembled to generate one contig without any gaps [28]. The paired-end strategy was used in the Illumina sequencing platform. Falcon (v0.3.0) was used for sub-read self-correction and three-generation sequence assembly [29]. Sub-reads were then processed to generate consensus sequences using Quiver (v2.2.2) [28]. A single-pass read accuracy improver (Sprai, v0.9.9.23) was used to correct the sequencing errors in single-pass reads [30]. Contigs were circularized using Circlator [31]. The assembled genome was annotated to identify the protein-coding and RNA genes using the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline [32].
Gene prediction of the assembled genome was conducted using Prodigal (v2.6.3) [33]. Functions of the predicted protein-coding genes were annotated using the Clusters of Orthologous Groups (COG) database annotations based on protein alignment using the Diamond software (e-value < 1e-5) [34]. Prophages were predicted using PhiSpy (v2.3) [35]. Pathogen–host interactions and the Comprehensive Antibiotic Resistance Database (CARD) were used for pathogenicity and drug resistance analyses, respectively [36, 37]. Carbohydrate-Active Enzymes (CAZy) analysis was performed using the CAZy database [38]. tRNA and rRNA genes were predicted using tRNAscan-SE (v1.3.1) [39] and rRNAmmer (v1.2) [40], respectively. Finally, sRNAs were predicted using BLAST against the Rfam database [41], and the circular genome graph was created using Circos (v0.69) [42].
Comparative Analysis
Ten reference
Evaluation of Antioxidant Activity of Lpb. plantarum Strains In Vitro
T-AOC and hydroxyl radical scavenging, SOD, GSH-Px, and GSH activities were determined using A015, A018-1-1, A001-2, A005, and A006-1-1 assay kits, respectively (China). Following this, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was determined, according to a modified method of Lin and Chang [46]. Briefly, 2 ml of intracellular cell-free extract was mixed with 2 ml of DPPH ethanol solution (0.2 mmol/l). The mixed solution was placed in the dark for 30 min at 25°C and centrifuged at 11,000 ×
DPPH scavenging activity (%) = [1 – (Ai – Aj)/A0] ×100%.
All in vitro assays were performed in triplicates.
Antioxidant Effects of Lpb. plantarum HOM3204 on D-Galactose-Induced Aging in Mice
Freeze-dried
Statistical Analysis
Data are presented as the mean ± standard error of the mean. Data analysis was conducted using one-way analysis of variance, followed by Tukey’s multiple comparisons test with the SPSS software (version 25, IBM, Corp., USA). Values were considered statistically significant at
Results
Genome Features
As shown in Fig. 1 and Table 1, the complete genome of
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Table 1 . Genome features of
Lactiplantibacillus plantarum HOM3204..Attribute Chromosome Plasmid 1 Plasmid 2 Genome size (bp) 3,232,697 48,573 17,060 DNA GC content (%) 44.61 39.04 40.57 Protein-coding genes 2,247 35 7 rRNA genes 16 0 0 tRNA genes 68 0 0 sRNA genes 38 3 1
-
Figure 1. Circular genome graph of
Lactiplantibacillus plantarum HOM3204. Circles, from inside to outside, represent the genome size, GC skew, GC contents, coding sequence (CDS) in the reverse strand, tRNA and rRNA genes in reverse strand, tRNA and rRNA genes in forward strand, and CDS in forward strand. A–Z, respectively, indicate the functional classification of CDS genes on the chromosome and plasmids using the Clusters of Orthologous Groups (COG) database. Circos (v0.69) software was used to create a genomic map with the given information.
One prophage in plasmid 1 was identified using PhiSpy. No drug resistance and virulence genes were found according to the minimum cutoff of 90% nucleotide identity over a minimum coverage length of 60% [47, 48] using CARD and VFDB, respectively.
On the chromosome, 2,247 genes (74.2%) were classified into COG functional categories (Fig. 2). Two hundred and fifty-one genes (11.17%) belonged to amino acid transport and metabolism, 282 genes (12.55%) belonged to carbohydrate transport and metabolism, 266 genes (11.84%) belonged to transcription, and 381 genes (16.96%) belonged to general function prediction only.
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Figure 2. Functional categorization of all predicted open reading frames (ORFs) in the
Lpb. plantarum HOM3204 genome using the COG database. Diamond (E-value < 1e-5) was used for protein alignment.
Comparison of Lpb. plantarum Strains
To understand the evolutionary relationship between the strains, ML and ANI trees were constructed. The results are shown in Figs. 3 and 4, respectively. According to the analysis of the ML tree, 10 strains, namely ST-III, 299v, WCFS1, ZJ316, LPL-1, J26, 16, KDLS1.0391, P-8, and LP3, were not grouped together with HOM3204, suggesting that
-
Figure 3. ML tree analysis of
Lpb. plantarum HOM3204 with 10 available complete genome sequences ofLpb. plantarum . ParaAT (V2.0) was used as a parallel tool for constructing multiple protein-coding DNA alignments. The maximum likelihood (ML) phylogenetic tree was constructed using RAxML. Numbers above the branches indicate the bootstrap supports from 500 replicates. The higher the bootstrap value, the more reliable is the evolution tree.
-
Figure 4. Average nucleotide identity (ANI) tree analysis of
Lpb. plantarum HOM3204 and 10 available genome sequences ofLpb. plantarum strains. ANI tree was constructed using Pyani software.
Genome features of the ten
-
Table 2 . Comparison of the chromosomal properties of different
Lpb. plantarum strains..Strain HOM3204 WCFS1 LP3 ST-III 299v J26 LPL-1 16 P-8 ZJ316 KLDS1.0391 Genome size (bp) 3,232,697 3,308,273 3,259,858 3,254,376 3,302,055 3,096,468 3,186,859 3,044,678 3,035,719 3,203,964 2,886,607 No. of plasmids 2 3 2 1 0 4 1 10 7 3 3 GC content (%) 44.61 44.47 44.50 44.58 44.40 44.80 44.65 44.74 44.80 44.65 44.80 Annotated genes 3,064 3,116 3,077 3,071 3,153 3,043 3.049 2,874 2,956 3,043 2,891 tRNA genes 72 72 73 70 57 70 67 68 71 63 52 rRNA genes 16 16 16 15 3 16 16 16 16 15 13 ANI (%) 100% 99.31% 99.22% 99.16% 99.14% 99.06% 99.03% 98.98% 98.98% 98.94% 98.93%
Oxidative Stress-Related Proteins
We identified the oxidative stress-related proteins encoded in the genome of
-
Table 3 . Oxidative stress-related proteins of
Lpb. plantarum HOM3204..Oxidative stress-related protein Locus tag Database Glutathione peroxidase Chr-gene 0184 GO:0004602/COG0386 Removal of superoxide radicals Chr-gene 0584 GO:0019430 Glutathione-disulfide reductase Chr-gene 0323, Chr-gene 0991, Chr-gene 1464, Chr-gene 2697 GO:0004362 Response to the oxygen radical Chr-gene 1464, Chr-gene 2697 GO:0000305 Catalytic activity Chr-gene 0102, Chr-gene 1140, Chr-gene 1357, Chr-gene 1915, Chr-gene 1931, Chr-gene 2678 GO:0003824 Catalase Chr-gene 2929 GO:0004096 Flavin reductase (NADH) Chr-gene 0045, Chr-gene 1116, Chr-gene 2249, Chr-gene 2250, Chr-gene 2680, GO:0036382 Thioredoxin-disulfide reductase Chr-gene 0584, Chr-gene 1888, Chr-gene 2175, Chr-gene 2817 GO:0004791 Thioredoxin peroxidase Chr-gene 1928 GO:0008379 Thioredoxin reductase Chr-gene 0584, Chr-gene 2138 COG:0492 DNA-binding ferritin-like protein Plasmid 2-gene 0005 COG:0783
In Vitro Antioxidant Activity
In this study, six indices (T-AOC, hydroxyl radical, DPPH radical, SOD, GSH-Px, and GSH) were chosen to evaluate the antioxidant activity of
-
Table 4 . Antioxidant activities of different
Lpb. plantarum strains in vitro..Strain T-AOC (U/ml) ·OH scavenging (%) DPPH scavenging (%) SOD (U/ml) GSH-Px (U/ml) GSH (mg/l) HOM3204 23.84 ± 2.44 76.84 ± 0.36 94.18 ± 0.45 28.89 ± 0.30 20.57 ± 2.73 37.56 ± 2.81 ST-Ⅲ 4.13 ± 0.44** 76.57 ± 0.36 93.00 ± 0.65 27.34 ± 0.52* 24.29 ± 3.18 23.72 ± 3.98** Lp-115 15.48 ± 1.13** 77.16 ± 0.60 91.03 ± 0.53** 24.63 ± 2.11* 15.60 ± 2.06 59.85 ± 5.57** Vitamin C 16.90 ± 1.42** 36.70 ± 1.33** 96.14 ± 0.08** 34.32 ± 0.36** ND ND Comparison of the
Lpb. plantarum HOM3204 strain with other strains: *p < 0.05, **p < 0.01. ·OH, hydroxyl radical scavenging..ND, not determined..
Antioxidant Effect of Lpb. plantarum HOM3204 on D-Galactose-Induced Aging in Mice
D-Galactose-induced aging mice showed a significant increase in the level of MDA compared to the control group (D-galactose vs. control, 6.31 ± 0.85 vs. 7.77 ± 1.11 nmol/ml,
The effects of
-
Table 5 . Effects of
Lpb. plantarum HOM3204 on malonaldehyde (MDA), protein carbonyl, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH) levels in the D-galactoseinduced oxidative injury mouse model..Group MDA (nmol/ml) Protein carbonyl (nmol/mgprot) SOD (U/ml) GSH-Px (U/ml) GSH (mgGSH/gprot) Model group 7.81 ± 1.43 7.02 ± 1.36 213 ± 38 390 ± 83 6.17 ± 0.79 HOM3204 7.59 ± 1.19 6.49 ± 1.98 230 ± 48 469 ± 68* 7.35 ± 1.47* Comparison of the
Lpb. plantarum HOM3204 group with the model group: *p < 0.05..
Discussion
WGS is generally used to study the information and potential functions of genes. Functional genomics research helps to better understand the molecular mechanisms of action of probiotics. [49]. Currently, 682 genome datasets of
We used the ML and ANI trees for the comparative analysis of
Hydroxyl radical, DPPH radical, T-AOC, SOD, GSH-Px, and GSH have been widely used as evaluation indices for ROS-related antioxidant activity [19, 20]. Strains with strong antioxidant activity can cope with oxidative stress. In this study,
Oxidative stress-related proteins, particularly SOD and GSH-Px, were identified in the genome of
In this study, we proved that
Data Availability
The complete nucleotide sequence of
Authors Contributions
D.Z., S.Z. contributed to the experiment design and interpreted all the results. D.Z. performed probiotic characterization in vitro tests. T.W. prepared the probiotics powders. Y.Z. performed animal related experiments. D.Z. performed statistical analysis and wrote the manuscript. S.Z., S.L. and C.L. edited the manuscript. All authors read and approved the final manuscript.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

-
Table 1 . Genome features of
Lactiplantibacillus plantarum HOM3204..Attribute Chromosome Plasmid 1 Plasmid 2 Genome size (bp) 3,232,697 48,573 17,060 DNA GC content (%) 44.61 39.04 40.57 Protein-coding genes 2,247 35 7 rRNA genes 16 0 0 tRNA genes 68 0 0 sRNA genes 38 3 1
-
Table 2 . Comparison of the chromosomal properties of different
Lpb. plantarum strains..Strain HOM3204 WCFS1 LP3 ST-III 299v J26 LPL-1 16 P-8 ZJ316 KLDS1.0391 Genome size (bp) 3,232,697 3,308,273 3,259,858 3,254,376 3,302,055 3,096,468 3,186,859 3,044,678 3,035,719 3,203,964 2,886,607 No. of plasmids 2 3 2 1 0 4 1 10 7 3 3 GC content (%) 44.61 44.47 44.50 44.58 44.40 44.80 44.65 44.74 44.80 44.65 44.80 Annotated genes 3,064 3,116 3,077 3,071 3,153 3,043 3.049 2,874 2,956 3,043 2,891 tRNA genes 72 72 73 70 57 70 67 68 71 63 52 rRNA genes 16 16 16 15 3 16 16 16 16 15 13 ANI (%) 100% 99.31% 99.22% 99.16% 99.14% 99.06% 99.03% 98.98% 98.98% 98.94% 98.93%
-
Table 3 . Oxidative stress-related proteins of
Lpb. plantarum HOM3204..Oxidative stress-related protein Locus tag Database Glutathione peroxidase Chr-gene 0184 GO:0004602/COG0386 Removal of superoxide radicals Chr-gene 0584 GO:0019430 Glutathione-disulfide reductase Chr-gene 0323, Chr-gene 0991, Chr-gene 1464, Chr-gene 2697 GO:0004362 Response to the oxygen radical Chr-gene 1464, Chr-gene 2697 GO:0000305 Catalytic activity Chr-gene 0102, Chr-gene 1140, Chr-gene 1357, Chr-gene 1915, Chr-gene 1931, Chr-gene 2678 GO:0003824 Catalase Chr-gene 2929 GO:0004096 Flavin reductase (NADH) Chr-gene 0045, Chr-gene 1116, Chr-gene 2249, Chr-gene 2250, Chr-gene 2680, GO:0036382 Thioredoxin-disulfide reductase Chr-gene 0584, Chr-gene 1888, Chr-gene 2175, Chr-gene 2817 GO:0004791 Thioredoxin peroxidase Chr-gene 1928 GO:0008379 Thioredoxin reductase Chr-gene 0584, Chr-gene 2138 COG:0492 DNA-binding ferritin-like protein Plasmid 2-gene 0005 COG:0783
-
Table 4 . Antioxidant activities of different
Lpb. plantarum strains in vitro..Strain T-AOC (U/ml) ·OH scavenging (%) DPPH scavenging (%) SOD (U/ml) GSH-Px (U/ml) GSH (mg/l) HOM3204 23.84 ± 2.44 76.84 ± 0.36 94.18 ± 0.45 28.89 ± 0.30 20.57 ± 2.73 37.56 ± 2.81 ST-Ⅲ 4.13 ± 0.44** 76.57 ± 0.36 93.00 ± 0.65 27.34 ± 0.52* 24.29 ± 3.18 23.72 ± 3.98** Lp-115 15.48 ± 1.13** 77.16 ± 0.60 91.03 ± 0.53** 24.63 ± 2.11* 15.60 ± 2.06 59.85 ± 5.57** Vitamin C 16.90 ± 1.42** 36.70 ± 1.33** 96.14 ± 0.08** 34.32 ± 0.36** ND ND Comparison of the
Lpb. plantarum HOM3204 strain with other strains: *p < 0.05, **p < 0.01. ·OH, hydroxyl radical scavenging..ND, not determined..
-
Table 5 . Effects of
Lpb. plantarum HOM3204 on malonaldehyde (MDA), protein carbonyl, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH) levels in the D-galactoseinduced oxidative injury mouse model..Group MDA (nmol/ml) Protein carbonyl (nmol/mgprot) SOD (U/ml) GSH-Px (U/ml) GSH (mgGSH/gprot) Model group 7.81 ± 1.43 7.02 ± 1.36 213 ± 38 390 ± 83 6.17 ± 0.79 HOM3204 7.59 ± 1.19 6.49 ± 1.98 230 ± 48 469 ± 68* 7.35 ± 1.47* Comparison of the
Lpb. plantarum HOM3204 group with the model group: *p < 0.05..
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