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Melanin Bleaching and Melanogenesis Inhibition Effects of Pediococcus acidilactici PMC48 Isolated from Korean Perilla Leaf Kimchi
1Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea 2Probiotics Microbiome Convergence Center, Asan 31538, Republic of Korea 3Emory university, Institute for Quantitative Theory and Methods (QuanTM), GA 30322, USA
Correspondence to:J. Microbiol. Biotechnol. 2020; 30(7): 1051-1059
Published July 28, 2020 https://doi.org/10.4014/jmb.2003.03007
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract

Introduction
Melanin is primarily an indole derivative of L-dihydroxyphenylalanine. It is highly oxidative in nature. It is the major pigment present in surface structures of vertebrates [1]. The name “melanin” comes from the ancient Greek melanos, meaning “dark”. The origin of its name is currently unclear. However, it is usually attributed to a Swedish chemist Berzelius (1840) [2]. Human skin coloration is dependent almost exclusively on the concentration and spatial distribution of chromophores melanin and haemoglobin, where melanin plays a dominant role in driving constitutive coloration [3]. Skin darkening is due to the presence of a chemically inert and stable pigment known as melanin that is produced deep inside the skin but is displayed as a mosaic at the surface of the body [4].
It has been traditionally believed that skin pigmentation is the most important photoprotective factor because melanin not only functions as a broadband UV absorbent, but also possesses antioxidant and radical scavenging properties. Although moderate amounts of melanin have good effects on the human body, deposition by excessive synthesis can cause pigmentary disorders such as lentigo, naevus, freckles, age spots, chloasma, and melanoma [5]. An increased amount of melanin in the skin is called hypermelanosis or melanoderma [6]. Hypermelanosis in the epidermis is caused by an increase in melanin in basal and suprabasal layers of the skin associated with a normal or elevated amount of melanocytes. This is a common dermatologic problem that may have substantial impacts on the patient since it affects the appearance and quality of life [7], Treatment of hypermelanosis involves the use of topical hypopigmenting agents such as hydroquinone, tretinoin, kojic acid, azelaic acid, and arbutin that can inhibit novel synthesis of melanin in melanocytes [8]. Most previous treatment options for these disorders remain unsatisfactory [9]. Therefore, it is necessary to develop a hypermelanosis treatment agent having a new mechanism of action different from existing treatments.
An alternative way of skin lightening is by decolouring melanin pigment. Although melanins are very stable compounds, under special conditions chemical or photochemical degradation and biodegradation by fungi are possible [10]. It has been reported that
This research is about the development of a new microorganism that can directly degrade melanin that has already been synthesized and deposited while having the function of inhibiting melanin synthesis. The ultimate purpose of this study is to develop medicines and cosmetics using the new microorganism. Therefore, microorganisms derived from traditional Korean fermented foods were screened. Such foods have been consumed in Korea for a long time with secured safety.
Materials and Methods
Isolation of Melanin Degrading Microorganisms from Korean Traditional Fermented Foods
Twenty kinds of traditional fermented foods were obtained from various parts of Korea and microorganisms were isolated. Abalone and sea urchin sauce, conche & ghee sauce, cured cheese, cured kimchi, cuttle fish sauce, kimchi (fresh), kimchi (old), seasoning soybean paste, mustard pickles, soypaste mixed with red peppers, perilla leaf kimchi, and soybean sauce were used for isolating different kinds of microorganisms. Several kinds of media were used and aerobic/anaerobic condition were given for each medium to isolate 252 types of isolates. Brain Heart Infusion (BD, 211065), M17 (Kisanbio, MB-M1192), Tos-MUP (Kisanbio, MB-T0892), and MRS (BD, 288210) agar were used for this experiment.
Agar Well Diffusion Method
The agar well diffusion method was applied to measure melanin degradation. Briefly, 100 μl of culture of
Melanin degradation assay in broth. The tube broth method was applied to measure melanin degradation. Briefly, 100 μl of culture and culture filtrate of
Tyrosinase Inhibition Test Using Tyrosinase as a Substrate
In order to assay the inhibitory effect of
Tyrosinase Inhibition Test based on L-DOPA
In order to assay the inhibitory effect of
DPPH Radical Scavenging Effect Test
The purple color of DPPH solution fades rapidly after interaction with proton-radical scavengers. The radical scavenging activity of
Melanin Content Measurement
B16F10 (
API 50 CHL Test
Fermentation of carbohydrates was determined using API 50 CHL, a standardized system consisting of 50 biochemical tests for the study of carbohydrate metabolism by microorganisms. Pure water (10 ml) was dispensed into the incubation box with the strip placed in the incubation box after bacterial cultures were introduced into the API 50 CHL system in API 50 CHL medium (5 ml) in concentration 2 McFarland. The set-up system was then incubated at 37°C for 48 h after wells were filled with bacterial suspensions by the line mark with the addition of mineral oil. Identification tables were prepared as (+/-) according to color change in evaluation of results of API strips reaction. Numerical profiles of strains were identified adding positive values in indicative table. Species designations were identified by evaluating with an identification software apiwebTM.
Whole Genome Sequencing
Genomic DNA of
Cell Cytotoxicity
Cell viability assay of B16F10 was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [30]. Briefly, 1* 10^4 cells/well was seeded into a 96-well plate. These cells were exposed to
Results and Discussion
Isolation of Melanin Degrading Microorganisms from Korean Traditional Fermented Foods
Twenty kinds of traditional fermented foods were obtained from various parts of Korea and microorganisms were isolated. A total of 252 types of isolates were grown using MRS agar and 49 kinds of microorganisms were harvested, except for overlapping isolates by product and morphology. Microorganisms that could degrade melanin were screened using the agar well diffusion method with melanin-containing agar media. Among 49 kinds of microorganisms, isolate PMC48 originated from perilla leaf kimchi degraded melanin around its pellets to form a clear zone. Genome analysis and melanin degradation or biosynthesis inhibition effects of the isolated microorganism, PMC48, was further performed or analyzed in depth.
Biochemical Characteristics of the Isolated Bacterial Strain
Current methods for characterizing and identifying bacterial isolates include a variety of routine phenotypic, biochemical, enzymatic, and molecular tests. The use of phenotypic and biochemical tests for identification has been the traditional standard for many years [13]. Biochemical characterization of isolated bacterial strains was carried out for identification and phenotypic characterizations of bacteria (Table 1). Based on biochemical and morphological tests according to the Bergey’s manual [14], PMC48 isolate was identified as
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Table 1 . Ability of melanin degrading isolate PMC48 to form acid from different carbohydrates.
No Type of test 24h 48h No Type of test 24h 48h 0 Control + + 25 Esculine + + 1 Glycerol - - 26 - Salicin + + 2 Erythritol - - 27 D-Cellibiose + + 3 D-arabinose - - 28 D-Maltose + + 4 L-arabinose + + 29 D-Lactose - + 5 D-ribose + + 30 D-Melibiose - - 6 D-xylose + + 31 D-Sacharose + + 7 L-xylose - - 32 D-Trehalose + + 8 D-adonitel - - 33 Inulin - - 9 Methyl-βD-xylopyranoside - - 34 D-Melezitose - - 10 D-galactose + + 35 D-Raffinose - - 11 D-glucose + + 36 Amidon - - 12 D-fructose + + 37 Glycogen - - 13 D-mannose + + 38 Xylitol - - 14 L-sorbose - - 39 Gentibiose + + 15 L-rhamnose - - 40 D-Turanose - - 16 Dulcitol - - 41 D-Lyxose - - 17 Inocitol - - 42 D-Tagatose + + 18 D-mannitol - + 43 D-Fucose - - 19 D-sorbitol - + 44 L-Fucose - - 20 Methyl-αD-mannopyranoside - - 45 D-arabitol - - 21 Methyl-αD-glucopyranoside - - 46 L-arabitol - - 22 N-acetylglucosamine + + 47 Potassium gluconate - + 23 Amygdaline + + 48 Potassium 2 ketogluconate - - 24 Arbutine + + 49 Potassium 5 ketogluconate - - (+): positive reaction (yellow), no. 25 (black); (-): negative reaction (violet)
Identification of Isolated Bacterial Strains based on 16S rRNA Gene Sequence Analysis
The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker used [16]. Therefore, 16S ribosomal RNA sequences have been used extensively in the classification and identification of bacteria [17]. The comparison of almost complete 16S rRNA gene sequences has been widely used to establish taxonomic relationships between prokaryotic strains. Sequence similarity of 98.65% is currently recognized as the cutoff for delineating species [17]. PMC48, a melanin- degrading isolate, was identified taxonomically by robust method of 16S rRNA gene sequencing (Table 2). By comparing its 16S rRNA sequence with those deposited at The National Center for Biotechnology Information (NCBI) reference sequence database, the isolated strain was found to belong to
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Table 2 . Identification of isolated bacterial strain, PMC48, based on 16S rRNA gene sequence analysis and their close relative published in DNA databases.
NCBI Reference Organism Length Score Identities Gaps E Value NR_042057.1 Pediococcus acidilactici DSM 202841569 2732 bits (1479) 1497/1505 (99%) 4/1505 (0%) 0.0 NR_042058.1 Pediococcus pentosaceus DSM 203361569 2632 bits (1425) 1481/1508 (98%) 4/1508 (0%) 0.0 NR_041640.1 Pediococcus acidilactici NGRI 0510Q1540 2573 bits (1393) 1450/1474 (98%) 18/1474 (1%) 0.0 NR_042401.1 Pediococcus stilesii strain FAIR-E 1801529 2571 bits (1392) 1461/1496 (98%) 2/1496 (0%) 0.0 NR_075029.1 Pediococcus claussenii strain ATCC BAA-3441567 2518 bits (1363) 1460/1507 (97%) 6/1507 (0%) 0.0 NR_042623.1 Pediococcus argentinicus strain CRL 7761492 2484 bits (1345) 1445/1494 (97%) 6/1494 (0%) 0.0 NR_042232.1 Pediococcus claussenii strain P061472 2423 bits (1312) 1419/1471 (96%) 5/1471 (0%) 0.0 NR_113922.1 Pediococcus parvulus strain NBRC 1006731501 2386 bits (1292) 1429/1496 (96%) 7/1496 (0%) 0.0 NR_043290.1 Pediococcus cellicola strain Z-81542 2377 bits (1287) 1432/1503 (95%) 5/1503 (0%) 0.0 NR_025388.1 Pediococcus inopinatus strain DSM 202851551 2366 bits (1281) 1430/1503 (95%) 5/1503 (0%) 0.0 NR_042087.1 Pediococcus damnosus strain DSM 203311561 2351 bits (1273) 1428/1503 (95%) 10/1503 (1%) 0.0 NR_043291.2 Pediococcus ethanolidurans strain Z-91501 2344 bits (1269) 1404/1470 (96%) 6/1470 (0%) 0.0 NR_029136.1 Pediococcus parvulus strain S-1821436 2302 bits (1246) 1374/1437 (96%) 6/1437 (0%) 0.0 NR_125575.1 Lactobacillus brantae DSM 23927 strain SL11081545 2289 bits (1239) 1417/1502 (94%) 16/1502 (1%) 0.0 NR_115654.1 Pediococcus damnosus strain JCM 58861497 2281 bits (1235) 1411/1499 (94%) 11/1499 (1%) 0.0 NR_109538.1 Lactobacillus curieae strain S1L191540 2265 bits (1226) 1417/1510 (94%) 9/1510 (1%) 0.0 NR_113290.1 Lactobacillus senioris DSM 24302 = JCM 17472 strain YIT 123641562 2265 bits (1226) 1416/1509 (94%) 8/1509 (1%) 0.0 NR_113289.1 Lactobacillus saniviri JCM 17471 = DSM 243011558 2263 bits (1225) 1420/1513 (94%) 18/1513 (1%) 0.0 NR_116411.1 Lactobacillus kimchicus JCM 15530 strain DCY511499 2235 bits (1210) 1409/1504 (94%) 17/1504 (1%) 0.0 NR_042442.1 Lactobacillus malefermentans strain DSM 57051556 2233 bits (1209) 1410/1506 (94%) 17/1506 (1%) 0.0
Study of Genome Properties and Comparative Analysis of the Selected Isolate, PMC48
Primary features of the genome of strain PMC48 are presented in Fig. 1. Strain PMC 48 contained a single, circular chromosome of 2,043,929 bp, with an average GC content of 42.2%. We detected 2,026 coding sequences (CDSs) in the genome, with an average length of 870.5bp (Fig. 1A). As shown in Fig. 1B, predicted CDSs were grouped by Clusters of Orthologous Groups (COG) functional categorizations. Among these CDSs, 1,892 proteins were assigned to COG families [18]. Biological functions could be defined for 1,351 (66.7%) of predicted proteins, while 541 CDSs (26.7%) were homologous to conserved proteins with unknown functions in other organisms. The remaining 134 hypothetical proteins (6.6%) had no match with any known proteins in the database. Furthermore, 57 tRNA and 15 rRNA genes were predicted.
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Fig. 1.
High-throughput genome sequencing of (Pediococcus acidilactici strain PMC48.A ) Circularmap ofPediococcus acidilactici PMC48 strain genome. Antisense and sense strands (colored according to COG categories) and RNA genes (red, tRNA; blue, rRNA) are shown from the outer periphery to the center. Inner circles show the GC skew, with yellow and blue indicating positive and negative values, respectively, and the GC content is indicated in red and green. This genome map was visualized using CLgenomics. (B ) Relative abundance of cluster of orthologous groups (COG) functional categories of genes.
OrthoANI provides a more robust and faster means of calculating average nucleotide identity for taxonomic purposes [19]. Using whole genome sequence data of PMC48 strain, similarity analysis was performed using the OrthoANI method with strains that shared high similarities in 16S rRNA analyses (Fig. 2). When OrthoANI analysis was performed to compare the isolate identified from this study to all publicly available
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Fig. 2.
Phylogenomic tree and OrthoANI result calculated with available genomes of Values bigger than 96% indicate that strains belong to the same species. The results between two strains are given in the junction point of the diagonals departing from each strain,Pediococcus andLactobacillus species.i.e. , OrthoANI value betweenPediococcus acidilactici PMC48 andLactobacillus delbrueckii subsp. bulgaricus ATCC 11842 is 74.0%. (2-column fitting image).
We then compared
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Table 3 . Comparison of the chromosomal properties of
Pediococcus acidilactici strains.Strain PMC48 LPBC161 S1 MA18/5M K3 NGRI 0510QT Sources Sesame leaf kimchi Mature coffee cherry Makgeolli Pasture Gramineae Nuruk Ryegrass Silage Genome size (bp) 2,043,929 1,960,506 1,980,172 1,992,928 1,991,399 2,047,078 G+C content (%) 42.2 42.2 42 42.1 42.1 41.2 Predicted CDS 2,026 2,019 1,525 1,967 1,525 2,154 Number of rRNA genes 15 6 7 NC 8 2 Number of tRNA genes 57 52 40 NC 50 54 NC: not confirmed.
Melanolytic Activity of P. acidilactici PMC48
Melanin-degrading activity of
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Fig. 3.
Degradation profiles of melanin by PMC48 strain. The agar well diffusion method (A -D ) and the tube broth method (E -H ) were applied to measure melanin degradation. The melanin degradation effect of the broth culture (A ,B ,E ) or cell free culture filtrate (F ) of PMC48 was measured. PMC48 culture of late exponential phase was used, and the filtrate was prepared by filtering the supernatant from which cells were removed by centrifugation of the culture medium. Control drugs, arbutin (C ,G ) and hydroquinone (D ,H ) were tested under the same conditions. In the agar well diffusion method, agar medium containing 1 mg/ml of melanin was used, and 100 μl of culture medium of PMC48 or same amount of control drugs (10 mg/ml of stock solution) were added and they were incubated for 24 h. In the tube broth method, a 10 ml liquid medium containing 1 mg/ml of melanin was used, and 100 ul of PMC48 culture solution and culture filtrate or final control 1 mM control drugs were added to the tube and after 24 h incubation and melanin was extracted.
Inhibitory Effect of P. acidilactici PMC48 on Melanin Biosynthesis
The main method of treating hypermelanosis so far is by using an agent that can inhibit melanin synthesis in melanocytes [26, 27]. Therefore, we investigated the inhibitory effect of PMC48 on tyrosinase, a melanin synthase, to determine whether this PMC48 strain might be compatible with the existing melanin synthesis technology in addition to its direct melanin degradation effect (Fig. 4). Tyrosinase inhibitory effects of PMC48 strains were measured by comparing two tyrosinase inhibitory tests using tyrosine and 3,4-dihydroxyphenylalanine (L- DOPA) with representative tyrosinase inhibitors hydroquinone and arbutin. In the tyrosinase inhibition test using tyrosine as a substrate, arbutin and hydroquinone had great inhibitory effects, whereas PMC48 culture was ineffective (Fig. 4A). In the tyrosinase inhibition test based on L-DOPA, PMC48 cultures also had inhibitory effects, similar to inhibitory effects of arbutin and hydroquinone (Fig. 4B).
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Fig. 4.
Inhibitory effect of tyrosinase and dopa oxidation of Arbutin and kojic acid were used as positive standards in the above assay (Pediococcus acidilactici PMC48 culture filtrate.A ,B ). Antioxidative capacity ofP. acidilactici PMC48 culture filtrate was evaluated by determination of 2,2-diphenyl-1-picryl-hydrazyl scavenging capacity. Vitamin C was used as positive standards in the above assay (C ). Data are mean ± SD of three separate experiments. Values are significantly different by comparison with the control. *p < 0.05; **p < 0.01; ***p < 0.001.
When exposed to ultraviolet radiation, the human skin produces profuse reactive oxygen species (ROS) which in turn activate tyrosinase by mobilizing α-melanocyte-stimulating hormone in the epidermis and finally stimulates melanocytes to produce melanin [28]. In this regard, a strategy for developing agents having both tyrosinase-suppressing and antioxidant effects has recently emerged [28]. Therefore, this study also tested the effect of PMC48 along with vitamin C as a representative antioxidant. Results confirmed that the PMC48 culture solution had DPPH radical scavenging effect of 18.5% (
Whitening Activity of P. acidilactici PMC48 in B16F10 Murine Melanoma Cells
Based on results of in vitro direct melanin-degrading and melanin synthesis-inhibiting effects of PMC48 shown above, its whitening effect on melanocytes was tested (Fig. 5). The whitening effect test of PMC48 strain on melanocytes B16F10 activated by α-melanocyte-stimulating hormone (α-MSH) showed a significant decrease in the amount of melanin (Fig. 5A). This effect of PMC48 culture was quantified with optical absorbance method (Fig. 5B) and enzyme-linked immuno-sorbent assay (ELISA) (Fig. 5C).
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Fig. 5.
Inhibitory effects of B16F10 cells were cultured in the presence or absence ofPediococcus acidilactici PMC48 culture filtrate on melanogenesis.P. acidilactici PMC48 for 72 h after treatment with α-melanocyte-stimulating hormone (α-MSH). Cells were harvested in a microcentrifuge tube (A ), and the optical density was determined (B ). Results are represented as percentages of control, and the data are presented as mean ± SD of three separate experiments. Values are significantly different by comparison with the control. *p < 0.05; **p < 0.01; ***p < 0.001.
Safety Properties of P. acidilactici PMC48
The safety of the PMC48 strain in the development of hypermelanosis treatment was evaluated. Cytotoxicity experiments using B16F10 cells showed no toxic effects under all conditions (Fig. 6).
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Fig. 6.
Cell viability assay. B16F10 cells were treated with various concentration of bacterial culture filtrate (1.0, 2.5, 5.0, 7.5, and 10%; v/v) for 24 h and the cell viability was measured by MTT assay. Results are expressed as percentage of cell viability relative to control. Values are significantly different by comparison with the control. *p < 0.05; **p < 0.01; ***p < 0.001.
In conclusion, PMC48, a new strain of
Acknowledgments
This research was financially supported by the Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the “Regional industry based organization support program” (reference number P0001942) supervised by the Korea Institute for Advancement of Technology (KIAT). This study was also supported by Soonchunhyang University Research Fund.
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. 2020; 30(7): 1051-1059
Published online July 28, 2020 https://doi.org/10.4014/jmb.2003.03007
Copyright © The Korean Society for Microbiology and Biotechnology.
Melanin Bleaching and Melanogenesis Inhibition Effects of Pediococcus acidilactici PMC48 Isolated from Korean Perilla Leaf Kimchi
Sukyung Kim 1, 2+, Hoonhee Seo 1, 2+, Hafij Al Mahmud 1, 2, Md Imtiazul Islam 1, 2, Omme Fatema Sultana 1, 2, Youngkyoung Lee 1, 2, Minhee Kim 3 and Ho-Yeon Song 1, 2*
1Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan 31151, Republic of Korea 2Probiotics Microbiome Convergence Center, Asan 31538, Republic of Korea 3Emory university, Institute for Quantitative Theory and Methods (QuanTM), GA 30322, USA
Correspondence to:Ho-Yeon Song
songmic@sch.ac.kr
Abstract
Overproduction and accumulation of melanin in the skin will darken the skin and cause skin disorders. So far, components that can inhibit tyrosinase, a melanin synthase of melanocytes, have been developed and used as ingredients of cosmetics or pharmaceutical products. However, most of existing substances can only inhibit the biosynthesis of melanin while melanin that is already synthesized and deposited is not directly decomposed. Thus, their effects in decreasing melanin concentration in the skin are weak. To overcome the limitation of existing therapeutic agents, we started to develop a substance that could directly biodegrade melanin. We screened traditional fermented food microorganisms for their abilities to direct biodegrade melanin. As a result, we found that a kimchi-derived Pediococcus acidilactici PMC48 had a direct melanin-degrading effect. This PMC48 strain is a new strain, different from P. acidilactici strains reported so far. It not only directly degrades melanin, but also has tyrosinase-inhibiting effect. It has a direct melanindecomposition effect. It exceeds existing melanin synthesis-inhibiting technology. It is expected to be of high value as a raw material for melanin degradation drugs and cosmetics.
Keywords: Pediococcus acidilactici PMC48, melanin, tyrosinase, perilla leaf kimchi
Introduction
Melanin is primarily an indole derivative of L-dihydroxyphenylalanine. It is highly oxidative in nature. It is the major pigment present in surface structures of vertebrates [1]. The name “melanin” comes from the ancient Greek melanos, meaning “dark”. The origin of its name is currently unclear. However, it is usually attributed to a Swedish chemist Berzelius (1840) [2]. Human skin coloration is dependent almost exclusively on the concentration and spatial distribution of chromophores melanin and haemoglobin, where melanin plays a dominant role in driving constitutive coloration [3]. Skin darkening is due to the presence of a chemically inert and stable pigment known as melanin that is produced deep inside the skin but is displayed as a mosaic at the surface of the body [4].
It has been traditionally believed that skin pigmentation is the most important photoprotective factor because melanin not only functions as a broadband UV absorbent, but also possesses antioxidant and radical scavenging properties. Although moderate amounts of melanin have good effects on the human body, deposition by excessive synthesis can cause pigmentary disorders such as lentigo, naevus, freckles, age spots, chloasma, and melanoma [5]. An increased amount of melanin in the skin is called hypermelanosis or melanoderma [6]. Hypermelanosis in the epidermis is caused by an increase in melanin in basal and suprabasal layers of the skin associated with a normal or elevated amount of melanocytes. This is a common dermatologic problem that may have substantial impacts on the patient since it affects the appearance and quality of life [7], Treatment of hypermelanosis involves the use of topical hypopigmenting agents such as hydroquinone, tretinoin, kojic acid, azelaic acid, and arbutin that can inhibit novel synthesis of melanin in melanocytes [8]. Most previous treatment options for these disorders remain unsatisfactory [9]. Therefore, it is necessary to develop a hypermelanosis treatment agent having a new mechanism of action different from existing treatments.
An alternative way of skin lightening is by decolouring melanin pigment. Although melanins are very stable compounds, under special conditions chemical or photochemical degradation and biodegradation by fungi are possible [10]. It has been reported that
This research is about the development of a new microorganism that can directly degrade melanin that has already been synthesized and deposited while having the function of inhibiting melanin synthesis. The ultimate purpose of this study is to develop medicines and cosmetics using the new microorganism. Therefore, microorganisms derived from traditional Korean fermented foods were screened. Such foods have been consumed in Korea for a long time with secured safety.
Materials and Methods
Isolation of Melanin Degrading Microorganisms from Korean Traditional Fermented Foods
Twenty kinds of traditional fermented foods were obtained from various parts of Korea and microorganisms were isolated. Abalone and sea urchin sauce, conche & ghee sauce, cured cheese, cured kimchi, cuttle fish sauce, kimchi (fresh), kimchi (old), seasoning soybean paste, mustard pickles, soypaste mixed with red peppers, perilla leaf kimchi, and soybean sauce were used for isolating different kinds of microorganisms. Several kinds of media were used and aerobic/anaerobic condition were given for each medium to isolate 252 types of isolates. Brain Heart Infusion (BD, 211065), M17 (Kisanbio, MB-M1192), Tos-MUP (Kisanbio, MB-T0892), and MRS (BD, 288210) agar were used for this experiment.
Agar Well Diffusion Method
The agar well diffusion method was applied to measure melanin degradation. Briefly, 100 μl of culture of
Melanin degradation assay in broth. The tube broth method was applied to measure melanin degradation. Briefly, 100 μl of culture and culture filtrate of
Tyrosinase Inhibition Test Using Tyrosinase as a Substrate
In order to assay the inhibitory effect of
Tyrosinase Inhibition Test based on L-DOPA
In order to assay the inhibitory effect of
DPPH Radical Scavenging Effect Test
The purple color of DPPH solution fades rapidly after interaction with proton-radical scavengers. The radical scavenging activity of
Melanin Content Measurement
B16F10 (
API 50 CHL Test
Fermentation of carbohydrates was determined using API 50 CHL, a standardized system consisting of 50 biochemical tests for the study of carbohydrate metabolism by microorganisms. Pure water (10 ml) was dispensed into the incubation box with the strip placed in the incubation box after bacterial cultures were introduced into the API 50 CHL system in API 50 CHL medium (5 ml) in concentration 2 McFarland. The set-up system was then incubated at 37°C for 48 h after wells were filled with bacterial suspensions by the line mark with the addition of mineral oil. Identification tables were prepared as (+/-) according to color change in evaluation of results of API strips reaction. Numerical profiles of strains were identified adding positive values in indicative table. Species designations were identified by evaluating with an identification software apiwebTM.
Whole Genome Sequencing
Genomic DNA of
Cell Cytotoxicity
Cell viability assay of B16F10 was performed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [30]. Briefly, 1* 10^4 cells/well was seeded into a 96-well plate. These cells were exposed to
Results and Discussion
Isolation of Melanin Degrading Microorganisms from Korean Traditional Fermented Foods
Twenty kinds of traditional fermented foods were obtained from various parts of Korea and microorganisms were isolated. A total of 252 types of isolates were grown using MRS agar and 49 kinds of microorganisms were harvested, except for overlapping isolates by product and morphology. Microorganisms that could degrade melanin were screened using the agar well diffusion method with melanin-containing agar media. Among 49 kinds of microorganisms, isolate PMC48 originated from perilla leaf kimchi degraded melanin around its pellets to form a clear zone. Genome analysis and melanin degradation or biosynthesis inhibition effects of the isolated microorganism, PMC48, was further performed or analyzed in depth.
Biochemical Characteristics of the Isolated Bacterial Strain
Current methods for characterizing and identifying bacterial isolates include a variety of routine phenotypic, biochemical, enzymatic, and molecular tests. The use of phenotypic and biochemical tests for identification has been the traditional standard for many years [13]. Biochemical characterization of isolated bacterial strains was carried out for identification and phenotypic characterizations of bacteria (Table 1). Based on biochemical and morphological tests according to the Bergey’s manual [14], PMC48 isolate was identified as
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Table 1 . Ability of melanin degrading isolate PMC48 to form acid from different carbohydrates..
No Type of test 24h 48h No Type of test 24h 48h 0 Control + + 25 Esculine + + 1 Glycerol - - 26 - Salicin + + 2 Erythritol - - 27 D-Cellibiose + + 3 D-arabinose - - 28 D-Maltose + + 4 L-arabinose + + 29 D-Lactose - + 5 D-ribose + + 30 D-Melibiose - - 6 D-xylose + + 31 D-Sacharose + + 7 L-xylose - - 32 D-Trehalose + + 8 D-adonitel - - 33 Inulin - - 9 Methyl-βD-xylopyranoside - - 34 D-Melezitose - - 10 D-galactose + + 35 D-Raffinose - - 11 D-glucose + + 36 Amidon - - 12 D-fructose + + 37 Glycogen - - 13 D-mannose + + 38 Xylitol - - 14 L-sorbose - - 39 Gentibiose + + 15 L-rhamnose - - 40 D-Turanose - - 16 Dulcitol - - 41 D-Lyxose - - 17 Inocitol - - 42 D-Tagatose + + 18 D-mannitol - + 43 D-Fucose - - 19 D-sorbitol - + 44 L-Fucose - - 20 Methyl-αD-mannopyranoside - - 45 D-arabitol - - 21 Methyl-αD-glucopyranoside - - 46 L-arabitol - - 22 N-acetylglucosamine + + 47 Potassium gluconate - + 23 Amygdaline + + 48 Potassium 2 ketogluconate - - 24 Arbutine + + 49 Potassium 5 ketogluconate - - (+): positive reaction (yellow), no. 25 (black); (-): negative reaction (violet).
Identification of Isolated Bacterial Strains based on 16S rRNA Gene Sequence Analysis
The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker used [16]. Therefore, 16S ribosomal RNA sequences have been used extensively in the classification and identification of bacteria [17]. The comparison of almost complete 16S rRNA gene sequences has been widely used to establish taxonomic relationships between prokaryotic strains. Sequence similarity of 98.65% is currently recognized as the cutoff for delineating species [17]. PMC48, a melanin- degrading isolate, was identified taxonomically by robust method of 16S rRNA gene sequencing (Table 2). By comparing its 16S rRNA sequence with those deposited at The National Center for Biotechnology Information (NCBI) reference sequence database, the isolated strain was found to belong to
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Table 2 . Identification of isolated bacterial strain, PMC48, based on 16S rRNA gene sequence analysis and their close relative published in DNA databases..
NCBI Reference Organism Length Score Identities Gaps E Value NR_042057.1 Pediococcus acidilactici DSM 202841569 2732 bits (1479) 1497/1505 (99%) 4/1505 (0%) 0.0 NR_042058.1 Pediococcus pentosaceus DSM 203361569 2632 bits (1425) 1481/1508 (98%) 4/1508 (0%) 0.0 NR_041640.1 Pediococcus acidilactici NGRI 0510Q1540 2573 bits (1393) 1450/1474 (98%) 18/1474 (1%) 0.0 NR_042401.1 Pediococcus stilesii strain FAIR-E 1801529 2571 bits (1392) 1461/1496 (98%) 2/1496 (0%) 0.0 NR_075029.1 Pediococcus claussenii strain ATCC BAA-3441567 2518 bits (1363) 1460/1507 (97%) 6/1507 (0%) 0.0 NR_042623.1 Pediococcus argentinicus strain CRL 7761492 2484 bits (1345) 1445/1494 (97%) 6/1494 (0%) 0.0 NR_042232.1 Pediococcus claussenii strain P061472 2423 bits (1312) 1419/1471 (96%) 5/1471 (0%) 0.0 NR_113922.1 Pediococcus parvulus strain NBRC 1006731501 2386 bits (1292) 1429/1496 (96%) 7/1496 (0%) 0.0 NR_043290.1 Pediococcus cellicola strain Z-81542 2377 bits (1287) 1432/1503 (95%) 5/1503 (0%) 0.0 NR_025388.1 Pediococcus inopinatus strain DSM 202851551 2366 bits (1281) 1430/1503 (95%) 5/1503 (0%) 0.0 NR_042087.1 Pediococcus damnosus strain DSM 203311561 2351 bits (1273) 1428/1503 (95%) 10/1503 (1%) 0.0 NR_043291.2 Pediococcus ethanolidurans strain Z-91501 2344 bits (1269) 1404/1470 (96%) 6/1470 (0%) 0.0 NR_029136.1 Pediococcus parvulus strain S-1821436 2302 bits (1246) 1374/1437 (96%) 6/1437 (0%) 0.0 NR_125575.1 Lactobacillus brantae DSM 23927 strain SL11081545 2289 bits (1239) 1417/1502 (94%) 16/1502 (1%) 0.0 NR_115654.1 Pediococcus damnosus strain JCM 58861497 2281 bits (1235) 1411/1499 (94%) 11/1499 (1%) 0.0 NR_109538.1 Lactobacillus curieae strain S1L191540 2265 bits (1226) 1417/1510 (94%) 9/1510 (1%) 0.0 NR_113290.1 Lactobacillus senioris DSM 24302 = JCM 17472 strain YIT 123641562 2265 bits (1226) 1416/1509 (94%) 8/1509 (1%) 0.0 NR_113289.1 Lactobacillus saniviri JCM 17471 = DSM 243011558 2263 bits (1225) 1420/1513 (94%) 18/1513 (1%) 0.0 NR_116411.1 Lactobacillus kimchicus JCM 15530 strain DCY511499 2235 bits (1210) 1409/1504 (94%) 17/1504 (1%) 0.0 NR_042442.1 Lactobacillus malefermentans strain DSM 57051556 2233 bits (1209) 1410/1506 (94%) 17/1506 (1%) 0.0
Study of Genome Properties and Comparative Analysis of the Selected Isolate, PMC48
Primary features of the genome of strain PMC48 are presented in Fig. 1. Strain PMC 48 contained a single, circular chromosome of 2,043,929 bp, with an average GC content of 42.2%. We detected 2,026 coding sequences (CDSs) in the genome, with an average length of 870.5bp (Fig. 1A). As shown in Fig. 1B, predicted CDSs were grouped by Clusters of Orthologous Groups (COG) functional categorizations. Among these CDSs, 1,892 proteins were assigned to COG families [18]. Biological functions could be defined for 1,351 (66.7%) of predicted proteins, while 541 CDSs (26.7%) were homologous to conserved proteins with unknown functions in other organisms. The remaining 134 hypothetical proteins (6.6%) had no match with any known proteins in the database. Furthermore, 57 tRNA and 15 rRNA genes were predicted.
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Figure 1.
High-throughput genome sequencing of (Pediococcus acidilactici strain PMC48.A ) Circularmap ofPediococcus acidilactici PMC48 strain genome. Antisense and sense strands (colored according to COG categories) and RNA genes (red, tRNA; blue, rRNA) are shown from the outer periphery to the center. Inner circles show the GC skew, with yellow and blue indicating positive and negative values, respectively, and the GC content is indicated in red and green. This genome map was visualized using CLgenomics. (B ) Relative abundance of cluster of orthologous groups (COG) functional categories of genes.
OrthoANI provides a more robust and faster means of calculating average nucleotide identity for taxonomic purposes [19]. Using whole genome sequence data of PMC48 strain, similarity analysis was performed using the OrthoANI method with strains that shared high similarities in 16S rRNA analyses (Fig. 2). When OrthoANI analysis was performed to compare the isolate identified from this study to all publicly available
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Figure 2.
Phylogenomic tree and OrthoANI result calculated with available genomes of Values bigger than 96% indicate that strains belong to the same species. The results between two strains are given in the junction point of the diagonals departing from each strain,Pediococcus andLactobacillus species.i.e. , OrthoANI value betweenPediococcus acidilactici PMC48 andLactobacillus delbrueckii subsp. bulgaricus ATCC 11842 is 74.0%. (2-column fitting image).
We then compared
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Table 3 . Comparison of the chromosomal properties of
Pediococcus acidilactici strains..Strain PMC48 LPBC161 S1 MA18/5M K3 NGRI 0510QT Sources Sesame leaf kimchi Mature coffee cherry Makgeolli Pasture Gramineae Nuruk Ryegrass Silage Genome size (bp) 2,043,929 1,960,506 1,980,172 1,992,928 1,991,399 2,047,078 G+C content (%) 42.2 42.2 42 42.1 42.1 41.2 Predicted CDS 2,026 2,019 1,525 1,967 1,525 2,154 Number of rRNA genes 15 6 7 NC 8 2 Number of tRNA genes 57 52 40 NC 50 54 NC: not confirmed..
Melanolytic Activity of P. acidilactici PMC48
Melanin-degrading activity of
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Figure 3.
Degradation profiles of melanin by PMC48 strain. The agar well diffusion method (A -D ) and the tube broth method (E -H ) were applied to measure melanin degradation. The melanin degradation effect of the broth culture (A ,B ,E ) or cell free culture filtrate (F ) of PMC48 was measured. PMC48 culture of late exponential phase was used, and the filtrate was prepared by filtering the supernatant from which cells were removed by centrifugation of the culture medium. Control drugs, arbutin (C ,G ) and hydroquinone (D ,H ) were tested under the same conditions. In the agar well diffusion method, agar medium containing 1 mg/ml of melanin was used, and 100 μl of culture medium of PMC48 or same amount of control drugs (10 mg/ml of stock solution) were added and they were incubated for 24 h. In the tube broth method, a 10 ml liquid medium containing 1 mg/ml of melanin was used, and 100 ul of PMC48 culture solution and culture filtrate or final control 1 mM control drugs were added to the tube and after 24 h incubation and melanin was extracted.
Inhibitory Effect of P. acidilactici PMC48 on Melanin Biosynthesis
The main method of treating hypermelanosis so far is by using an agent that can inhibit melanin synthesis in melanocytes [26, 27]. Therefore, we investigated the inhibitory effect of PMC48 on tyrosinase, a melanin synthase, to determine whether this PMC48 strain might be compatible with the existing melanin synthesis technology in addition to its direct melanin degradation effect (Fig. 4). Tyrosinase inhibitory effects of PMC48 strains were measured by comparing two tyrosinase inhibitory tests using tyrosine and 3,4-dihydroxyphenylalanine (L- DOPA) with representative tyrosinase inhibitors hydroquinone and arbutin. In the tyrosinase inhibition test using tyrosine as a substrate, arbutin and hydroquinone had great inhibitory effects, whereas PMC48 culture was ineffective (Fig. 4A). In the tyrosinase inhibition test based on L-DOPA, PMC48 cultures also had inhibitory effects, similar to inhibitory effects of arbutin and hydroquinone (Fig. 4B).
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Figure 4.
Inhibitory effect of tyrosinase and dopa oxidation of Arbutin and kojic acid were used as positive standards in the above assay (Pediococcus acidilactici PMC48 culture filtrate.A ,B ). Antioxidative capacity ofP. acidilactici PMC48 culture filtrate was evaluated by determination of 2,2-diphenyl-1-picryl-hydrazyl scavenging capacity. Vitamin C was used as positive standards in the above assay (C ). Data are mean ± SD of three separate experiments. Values are significantly different by comparison with the control. *p < 0.05; **p < 0.01; ***p < 0.001.
When exposed to ultraviolet radiation, the human skin produces profuse reactive oxygen species (ROS) which in turn activate tyrosinase by mobilizing α-melanocyte-stimulating hormone in the epidermis and finally stimulates melanocytes to produce melanin [28]. In this regard, a strategy for developing agents having both tyrosinase-suppressing and antioxidant effects has recently emerged [28]. Therefore, this study also tested the effect of PMC48 along with vitamin C as a representative antioxidant. Results confirmed that the PMC48 culture solution had DPPH radical scavenging effect of 18.5% (
Whitening Activity of P. acidilactici PMC48 in B16F10 Murine Melanoma Cells
Based on results of in vitro direct melanin-degrading and melanin synthesis-inhibiting effects of PMC48 shown above, its whitening effect on melanocytes was tested (Fig. 5). The whitening effect test of PMC48 strain on melanocytes B16F10 activated by α-melanocyte-stimulating hormone (α-MSH) showed a significant decrease in the amount of melanin (Fig. 5A). This effect of PMC48 culture was quantified with optical absorbance method (Fig. 5B) and enzyme-linked immuno-sorbent assay (ELISA) (Fig. 5C).
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Figure 5.
Inhibitory effects of B16F10 cells were cultured in the presence or absence ofPediococcus acidilactici PMC48 culture filtrate on melanogenesis.P. acidilactici PMC48 for 72 h after treatment with α-melanocyte-stimulating hormone (α-MSH). Cells were harvested in a microcentrifuge tube (A ), and the optical density was determined (B ). Results are represented as percentages of control, and the data are presented as mean ± SD of three separate experiments. Values are significantly different by comparison with the control. *p < 0.05; **p < 0.01; ***p < 0.001.
Safety Properties of P. acidilactici PMC48
The safety of the PMC48 strain in the development of hypermelanosis treatment was evaluated. Cytotoxicity experiments using B16F10 cells showed no toxic effects under all conditions (Fig. 6).
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Figure 6.
Cell viability assay. B16F10 cells were treated with various concentration of bacterial culture filtrate (1.0, 2.5, 5.0, 7.5, and 10%; v/v) for 24 h and the cell viability was measured by MTT assay. Results are expressed as percentage of cell viability relative to control. Values are significantly different by comparison with the control. *p < 0.05; **p < 0.01; ***p < 0.001.
In conclusion, PMC48, a new strain of
Acknowledgments
This research was financially supported by the Ministry of Trade, Industry, and Energy (MOTIE), Korea, under the “Regional industry based organization support program” (reference number P0001942) supervised by the Korea Institute for Advancement of Technology (KIAT). This study was also supported by Soonchunhyang University Research Fund.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Fig 6.

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Table 1 . Ability of melanin degrading isolate PMC48 to form acid from different carbohydrates..
No Type of test 24h 48h No Type of test 24h 48h 0 Control + + 25 Esculine + + 1 Glycerol - - 26 - Salicin + + 2 Erythritol - - 27 D-Cellibiose + + 3 D-arabinose - - 28 D-Maltose + + 4 L-arabinose + + 29 D-Lactose - + 5 D-ribose + + 30 D-Melibiose - - 6 D-xylose + + 31 D-Sacharose + + 7 L-xylose - - 32 D-Trehalose + + 8 D-adonitel - - 33 Inulin - - 9 Methyl-βD-xylopyranoside - - 34 D-Melezitose - - 10 D-galactose + + 35 D-Raffinose - - 11 D-glucose + + 36 Amidon - - 12 D-fructose + + 37 Glycogen - - 13 D-mannose + + 38 Xylitol - - 14 L-sorbose - - 39 Gentibiose + + 15 L-rhamnose - - 40 D-Turanose - - 16 Dulcitol - - 41 D-Lyxose - - 17 Inocitol - - 42 D-Tagatose + + 18 D-mannitol - + 43 D-Fucose - - 19 D-sorbitol - + 44 L-Fucose - - 20 Methyl-αD-mannopyranoside - - 45 D-arabitol - - 21 Methyl-αD-glucopyranoside - - 46 L-arabitol - - 22 N-acetylglucosamine + + 47 Potassium gluconate - + 23 Amygdaline + + 48 Potassium 2 ketogluconate - - 24 Arbutine + + 49 Potassium 5 ketogluconate - - (+): positive reaction (yellow), no. 25 (black); (-): negative reaction (violet).
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Table 2 . Identification of isolated bacterial strain, PMC48, based on 16S rRNA gene sequence analysis and their close relative published in DNA databases..
NCBI Reference Organism Length Score Identities Gaps E Value NR_042057.1 Pediococcus acidilactici DSM 202841569 2732 bits (1479) 1497/1505 (99%) 4/1505 (0%) 0.0 NR_042058.1 Pediococcus pentosaceus DSM 203361569 2632 bits (1425) 1481/1508 (98%) 4/1508 (0%) 0.0 NR_041640.1 Pediococcus acidilactici NGRI 0510Q1540 2573 bits (1393) 1450/1474 (98%) 18/1474 (1%) 0.0 NR_042401.1 Pediococcus stilesii strain FAIR-E 1801529 2571 bits (1392) 1461/1496 (98%) 2/1496 (0%) 0.0 NR_075029.1 Pediococcus claussenii strain ATCC BAA-3441567 2518 bits (1363) 1460/1507 (97%) 6/1507 (0%) 0.0 NR_042623.1 Pediococcus argentinicus strain CRL 7761492 2484 bits (1345) 1445/1494 (97%) 6/1494 (0%) 0.0 NR_042232.1 Pediococcus claussenii strain P061472 2423 bits (1312) 1419/1471 (96%) 5/1471 (0%) 0.0 NR_113922.1 Pediococcus parvulus strain NBRC 1006731501 2386 bits (1292) 1429/1496 (96%) 7/1496 (0%) 0.0 NR_043290.1 Pediococcus cellicola strain Z-81542 2377 bits (1287) 1432/1503 (95%) 5/1503 (0%) 0.0 NR_025388.1 Pediococcus inopinatus strain DSM 202851551 2366 bits (1281) 1430/1503 (95%) 5/1503 (0%) 0.0 NR_042087.1 Pediococcus damnosus strain DSM 203311561 2351 bits (1273) 1428/1503 (95%) 10/1503 (1%) 0.0 NR_043291.2 Pediococcus ethanolidurans strain Z-91501 2344 bits (1269) 1404/1470 (96%) 6/1470 (0%) 0.0 NR_029136.1 Pediococcus parvulus strain S-1821436 2302 bits (1246) 1374/1437 (96%) 6/1437 (0%) 0.0 NR_125575.1 Lactobacillus brantae DSM 23927 strain SL11081545 2289 bits (1239) 1417/1502 (94%) 16/1502 (1%) 0.0 NR_115654.1 Pediococcus damnosus strain JCM 58861497 2281 bits (1235) 1411/1499 (94%) 11/1499 (1%) 0.0 NR_109538.1 Lactobacillus curieae strain S1L191540 2265 bits (1226) 1417/1510 (94%) 9/1510 (1%) 0.0 NR_113290.1 Lactobacillus senioris DSM 24302 = JCM 17472 strain YIT 123641562 2265 bits (1226) 1416/1509 (94%) 8/1509 (1%) 0.0 NR_113289.1 Lactobacillus saniviri JCM 17471 = DSM 243011558 2263 bits (1225) 1420/1513 (94%) 18/1513 (1%) 0.0 NR_116411.1 Lactobacillus kimchicus JCM 15530 strain DCY511499 2235 bits (1210) 1409/1504 (94%) 17/1504 (1%) 0.0 NR_042442.1 Lactobacillus malefermentans strain DSM 57051556 2233 bits (1209) 1410/1506 (94%) 17/1506 (1%) 0.0
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Table 3 . Comparison of the chromosomal properties of
Pediococcus acidilactici strains..Strain PMC48 LPBC161 S1 MA18/5M K3 NGRI 0510QT Sources Sesame leaf kimchi Mature coffee cherry Makgeolli Pasture Gramineae Nuruk Ryegrass Silage Genome size (bp) 2,043,929 1,960,506 1,980,172 1,992,928 1,991,399 2,047,078 G+C content (%) 42.2 42.2 42 42.1 42.1 41.2 Predicted CDS 2,026 2,019 1,525 1,967 1,525 2,154 Number of rRNA genes 15 6 7 NC 8 2 Number of tRNA genes 57 52 40 NC 50 54 NC: not confirmed..
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