Scholarly article on topic 'Evaluation of ginsenoside bioconversion of lactic acid bacteria isolated from kimchi'

Evaluation of ginsenoside bioconversion of lactic acid bacteria isolated from kimchi Academic research paper on "Animal and dairy science"

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{"ginsenoside bioconversion" / "ginsenoside Rg3" / "ginsenoside Rg5" / LC-MS/MS}

Abstract of research paper on Animal and dairy science, author of scientific article — Boyeon Park, Hyelyeon Hwang, Jina Lee, Sung-Oh Sohn, Se Hee Lee, et al.

Abstract Background Panax ginseng is a physiologically active plant widely used in traditional medicine that is characterized by the presence of ginsenosides. Rb1, a major ginsenoside, is used as the starting material for producing ginsenoside derivatives with enhanced pharmaceutical potentials through chemical, enzymatic, or microbial transformation. Methods To investigate the bioconversion of ginsenoside Rb1, we prepared kimchi originated bacterial strains Leuconostoc mensenteroides WiKim19, Pediococcus pentosaceus WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49 and analyzed bioconversion products using LC-MS/MS mass spectrometer. Results L. mesenteroides WiKim19 and Pediococcus pentosaceus WiKim20 converted ginsenoside Rb1 into the ginsenoside Rg3 approximately five times more than Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49. L mesenteroides WIKim19 showed positive correlation with β-glucosidase activity and higher transformation ability of ginsenoside Rb1 into Rg3 than the other strains whereas, P. pentosaceus WiKim20 showed an elevated production of Rb3 even with lack of β-glucosidase activity but have the highest acidity among the five lactic acid bacteria (LAB). Conclusion Ginsenoside Rg5 concentration of five LABs have ranged from ∼2.6 μg/mL to 6.5 μg/mL and increased in accordance with the incubation periods. Our results indicate that the enzymatic activity along with acidic condition contribute to the production of minor ginsenoside from lactic acid bacteria.

Academic research paper on topic "Evaluation of ginsenoside bioconversion of lactic acid bacteria isolated from kimchi"

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Contents lists available at ScienceDirect

Journal of Ginseng Research

journal homepage: http://www.ginsengres.org

Research article

Evaluation of ginsenoside bioconversion of lactic acid bacteria isolated from kimchi

Boyeon Parkq, Hyelyeon Hwang q, Jina Lee, Sung-Oh Sohn, Se Hee Lee, Min Young Jung, Hyeong In Lim, Hae Woong Park, Jong-Hee Lee*

World Institute of Kimchi, Gwangu, Korea

ARTICLE INFO

Article history: Received 27 January 2016 Received in Revised form 30 September 2016 Accepted 5 October 2016 Available online xxx

Keywords:

acidity

b-glucosidase

bioconversion

ginsenoside Rg3

LC-MS/MS

ABSTRACT

Background: Panax ginseng is a physiologically active plant widely used in traditional medicine that is characterized by the presence of ginsenosides. Rb1, a major ginsenoside, is used as the starting material for producing ginsenoside derivatives with enhanced pharmaceutical potentials through chemical, enzymatic, or microbial transformation.

Methods: To investigate the bioconversion of ginsenoside Rb1, we prepared kimchi originated bacterial strains Leuconostoc mensenteroides WiKim19, Pediococcus pentosaceus WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49 and analyzed bioconversion products using LC-MS/MS mass spectrometer.

Results: L. mesenteroides WiKim19 and Pediococcus pentosaceus WiKim20 converted ginsenoside Rb1 into the ginsenoside Rg3 approximately five times more than Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and Lactobacillus sakei WiKim49. L mesenteroides WIKim19 showed positive correlation with b-glucosidase activity and higher transformation ability of ginsenoside Rb1 into Rg3 than the other strains whereas, P. pentosaceus WiKim20 showed an elevated production of Rb3 even with lack of b-glucosidase activity but have the highest acidity among the five lactic acid bacteria (LAB). Conclusion: Ginsenoside Rg5 concentration of five LABs have ranged from ~2.6 mg/mL to 6.5 mg/mL and increased in accordance with the incubation periods. Our results indicate that the enzymatic activity along with acidic condition contribute to the production of minor ginsenoside from lactic acid bacteria.

Copyright © 2016, The Korean Society of Ginseng, Published by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Q6 Lactic acid bacteria (LABs) have been used as probiotics and are present in many fermented foods (cheese, yogurt, butter, and kimchi), where they influence the taste and preservation by producing lactic acid and/or alcohol. Some enzymes produced by LABs can efficiently utilize ingested nutrients to benefit the host, i.e., linoleic acid isomerase from Lactobacillus acidophilus produces conjugated linoleic acid, which has biological properties, from linoleic acid [1], and b-glucosidase from Lactobacillus para-plantarum converts isoflavone glucosides, which are not absorbed by enterocytes [2], to absorbable aglycones [3]. Recent studies reported the bioconversion of ginsenosides using Lactobacillus pen-tosus and Leuconostoc citreum isolated from fermented foods due to b-glucosidase activity [4,5].

The major commercial ginsengs such as Panax ginseng Meyer (Korean Red Ginseng), Panax quinquifolium (American ginseng), and Panax notoginseng (Burk.) F.H. Chen (Notoginseng) have been widely used as traditional herbal medicines [6]. Ginsenosides (ginseng saponins) are the major pharmacological constituents of ginseng, and over 100 ginsenosides have been identified [5,7]. Major ginsenosides (80% of the ginsenosides) are composed of Rb1, Rb2, Rc, Rd, Re, and Rg1; minor ginsenosides are their deglycosy-lated forms and composed of Rg3, Rh2, Rh1, F2, C-K, Rg2, Rh1, Rg5, and F1 [8].

Minor ginsenosides are known to have a greater pharmaceutical potential than major ginsenosides [9—14]. However, naturally occurring minor ginsenosides are present at very low concentrations. Therefore, hydrolysis of sugar moieties from abundant major ginsenosides are needed to produce minor ginsenosides. Gut

* Corresponding author. World Institute of Kimchi, Gwangju 61755, Korea. q E-mail address: leejonghee@wikim.re.kr (J.-H. Lee). These authors contributed equally to this paper.

http://dx.doi.org/10.1016/j.jgr.2016.10.003

p1226-8453 e2093-4947/$ — see front matter Copyright © 2016, The Korean Society of Ginseng, Published by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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1 microbiota metabolize orally administered ginseng and help

2 transport across the epithelial membrane [15] and human intesti-

3 nal microbiota convert major ginsenosides to minor ginsenosides

4 [10,16,17]. However, ginsenoside metabolism varies between in-

5 dividuals depending on the population of gut microbiota, such as

6 Ruminococcus spp., Bacteroides spp., and Bifidobacterium spp. [17].

7 Acidic environments as well as intestinal microbiota have impor-

8 tant influences on the bioconversion of ginsenoside and the low pH

9 of gastrointestinal environment could activate the deglycosylation

10 of ginsenoside by acidic hydrolysis response [18—20].

11 Kimchi is a traditional Korean food that is fermented vegetables

12 including cabbage and various seasonings. Kimchi has antioxidative

13 and antidiabetic properties and bacteria isolated from kimchi

14 produce beneficial enzymes [21—23]. Various LABs play important

15 roles during kimchi fermentation: Lactobacillus and Leuconostoc are

16 the predominant genera of the kimchi microbiome in the kimchi

17 fermentation [24]. Lactobacillus species have neuroprotective,

18 antifungal, and anticolitic properties [25—27] and Leuconostoc

19 species play key roles in decreasing foodborne pathogen growth,

20 viral activity, and the effects of lipid profiles [28—30]. Recent

21 studies have suggested that LABs from kimchi produce hydrolytic

22 enzymes that catalyze ginsenoside bioconversion by removing the

23 glycosyl group of major ginsenosides [4,5].

24 In this study, we isolated LABs associated with kimchi fermen-

25 tation from homemade kimchi, and compared availability for gin-

26 senoside bioconversion of five LAB strains such as Leuconostoc

27 mensenteroides WiKim19, Pediococcus pentosaceus WiKim20,

28 Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48, and

29 Lactobacillus sakei WiKim49 by quantitating transformed ginseno-

30 side using a sensitive and reliable LC-MS/MS method.

32 2. Materials and methods

34 Q7 2.1. Materials

36 Leuconostoc mensenteroides WiKim19, Pediococcus pentosaceus

37 WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis

38 WiKim48, and Lactobacillus sakei WiKim49 were isolated from

39 homemade kimchi using de Man, Rogosa, and Sharpe (MRS) media.

40 MRS broth was purchased from Difco (Miller, Becton Dickinson, and

41 Co., Sparks, MD, USA). Ginsenosides Rb1, Rg3, digoxin (internal

42 standard), and b-glucosidase activity assay kit were purchased from

43 Sigma Aldrich (St. Louis, MO, USA). Ginsenoside -F2, -Rg1, -Rf, -Ro,

44 -Rg2, -R1, -Ra1, -Rb2, -Rb3, -F1, -Rd, -Rg5, -compound K; -Rh2, -Rh4,

45 and gypenoside XVII were purchased from Ambo Institute (Dae-

46 jeon, Korea). API 50 CH and inoculating fluid was purchased from

47 Q8 bioMerieux (Lyon, France).

49 2.2. Determination of 16S rRNA gene sequences and phylogenetic

50 analysis

52 To identify the isolates using 16S rRNA sequencing, the isolates

53 Q9 were sent to Macrogen Inc., Korea sequencing service (www.

54 macrogen.com). The obtained sequences were compared with

55 available 16S rRNA sequences in the EzTaxon Server [26] to evaluate

56 sequence similarity. Multiple sequence alignment of the 16S rRNA

57 sequences from five lactic acid bacteria and these related species

58 were performed with CLUSTAL W [27]. The phylogenetic trees were

59 constructed using MEGA6 [28] with neighbor-joining [29] based on

60 1,000 random bootstrap replicates for each.

62 2.3. Assay of ginsenoside Rb1 bioconversion by lactic acid bacteria

64 L. mensenteroides WiKim19, P. pentosaceus WiKim20, L. brevis

65 WiKim47, L. lactis WiKim48, and L. sakei WiKim49 were inoculated

in MRS broth, until absorbance reached 600 nm of 1.0. The strains 66

were cultured at 30° C for 1 d, 3 d, and 7 d with ginsenoside Rb1 (a 67

final concentration of 200p,M) dissolved in MeOH. After centrifu- 68

gation at 5,000g for 10 min, 2 mg/mL digoxin as an internal stan- 69

dard was added and purified using Sep-Pak Light C18 cartridges 70

(Waters, Milford, MA, USA) and then dissolved in MeOH. 71

2.4. Assay of ginsenosides by LC-MS/MS 73

Ginsenosides Rb1 and minor ginsenosides in the reactions 75

were analyzed by using UPLC (Waters), coupled to a TripleTOF 76

5600 plus system with electrospray ionization (ESI; AB SCIEX, 77

Framingham, MA, USA). To investigate and separate the precursor 78

and fragmentation ions of ginsenosides Rb1, minor ginsenosides, 79

an Acquity UPLC BEH C18 column (2.1 mm x 100 mm, 1.7 mm 80

particle size) from Waters was used at a flow rate of 0.5 mL/min. 81

UPLC conditions were as follows: solvent A, water containing 82

10mM ammonium acetate; solvent B, acetonitrile containing 83

10mM ammonium acetate; gradient, 0-0.5 min (5% B), 0.5- 84

14.5 min (5-30% B), 14.5-15.5 min (30-32% B), 15.5-16.5 min 85

(32-40% B), 16.5-17 min (40-55% B), 17-19 min (55% B), 19- 86

25 min (90% B), and 25-30 min (5% B). Two microliters of each 87

sample were injected for the UPLC analysis, and peaks were 88

identified by comparing their retention times and fragment ion 89

with that of reference compound. 90

The mass spectrometry conditions were optimized under 91

the negative ion mode as follows: curtain gas, 30; collision 92

energy, -30; declustering potential, -80; nebulizer gas (Gas 1), 40 93

at MRM mode; heater gas (Gas 2), 50. The ion spray voltage 94

was -4,500 V. Ginsenosides in all reaction mixtures were quanti- 95

fied with multiple reaction monitoring (MRM) using selected 96

transitions as follows: Rb1, m/z 1,107/945; Rg3, F2, m/z 783 /621; 97

Rg5, m/z 765/603; digoxin, m/z 779/649, Rg1, Rf m/z 799/637; 98

Ro, m/z 955 / 793; Rg2, m/z 783/637; R1, m/z 931 /769; Ra1, m/z 99

1,209/1,077; Rb2, Rb3, m/z 1,077 / 945; F1, m/z 637/475; Rd, m/ 100

z 945/783; XVII, m/z 945/323; compound K, m/z 621 /459; Rh2, 101

m/z 621 /459; and Rh4 m/z 619/161. 102

Data acquisition and processing were carried out using Analyst 103

TF 1.6 and PeakVeiw 1.2 software (AB SCIEX), respectively. The data 104

obtained from multiple reaction monitoring (MRM) mode were 105

quantitated using MultiQuant software (AB SCIEX). The standard 106

solutions containing 10-200p,M were injected into the UPLC with 107

2 mg/mL digoxin. The linear calibration curve for peak area ratio 108

(ginsenoside/digoxin) was obtained for the quantification of gin- 109

senoside. The amounts of the ginsenosides in each sample were 110

determined from corresponding calibration curves. 111

2.5. Assay of ß-glucosidase activities using cell lysates 113

L. mensenteroides WiKim19, P. pentosaceus WiKim20, L. brevis 115

WiKim47, L. lactis WiKim48, and L. sakei WiKim49 were cultured 116

for 1 d in MRS broth at 30°C. The supernatant was removed after 117

centrifugation at 12,000g for 10 min, and cell lysates including 118

intracellular ß-glucosidase were prepared by bead beating in 119

50mM sodium phosphate buffer (pH 7.0). Protein concentrations 120

of cell lysates were determined using a Pierce BCA Protein Assay 121

Kit (Thermo, Rockford, IL, USA). The proteins were diluted to the 122

concentration of ~0.5 mg/mL to assay enzyme activity. The 123

enzyme activity was determined using ß-glucosidase activity 124

assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the 125

manufacturer's protocol. The release of p-nitrophenol was 126

measured at 405 nm (SPECTROstar Nano, BMG Labtech, Orten- 127

berg, Germany). Analysis was performed in duplicate for each 128

strain. One unit of ß-glucosidase is the amount of enzyme that 129

catalyzes the hydrolysis of 1.0 mmole substrate per min at pH 7.0. 130

B. Park et al / Lactic acid bacteria isolated from kimchi

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-Escherichia coli ATCC 11775T (JMST01000030)

Leuconostoc lactis WiKim48

■Leuconostoc lactis KCTC 3528T (AEOR01001150) 9S-Leuconostoc holzapfelii BFE 7000т (AM600682) -Leuconostoc citreum ATCC 49370T (AF111948) I—Leuconostoc palmae TMW 2.694T (AM940225) rLeuconostoc mesenteroides subsp. suionicum LMG 8159т (HM443957) Leuconostoc mesenteroides WiKim19

Leuconostoc mesenteroides subsp. dextranicum NRIC 1539т (AB023246) Pediococcus acidilactici DSM 20284T (GL397069) Pediococcus lolii NGRI 0510QT (BANK01000051) —Pediococcus stilesii LMG 23082T (AJ973157) —Pediococcus pentosaceus DSM 20336T (AJ305321) —Pediococcus pentosaceus WiKim20 —Lactobacillus senmaizukei L13T (AB297927)

100 100

^Lactobacillus parabrevis DCY50T (AM158249) actobacillus koreensis DCY50T (FJ904277) -Lactobacillus hammesii TMW 1.1236T (AJ632219) Lactobacillus brevis ATCC 14869T (KI271266) Lactobacillus brevis Wikim47 actobacillus fuchuensis JCM 11249T (BAMJ01000063) ¡-Lactobacillus curvatus LMG 9198T (AJ621550) Lactobacillus graminis DSM 20719т (AM113778) Lactobacillus sakei WiKim22

Lactobacillus sakei subsp. sakei JCM 115Г (BALW01000030) \-Lactobacillus sakei subsp. camosus CCUG 31331T (AY204892)

Fig. 1. Phylogenetic trees constructed from 16S rRNA gene sequences. The phylogenetic relationships of Leuconostoc mensenteroides WiKim19, Pediococcus pentosaceus WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48 and Lactobacillus sakei WiKim49 with other species are shown. Trees were constructed using the neighbor-joining method. The numbers at the nodes represent bootstrap values (> 60%) are expressed as percentages of 1,000 replicates. Escherichia coli ATCC 11775 T was used as an outgroup. Bar, 0.01 accumulated changes per nucleotide.

The results were reported as mean ± standard deviation. Statistical analyses were performed using GraphPad Prism 6 (Graph-Pad software, La Jolla, CA, USA). One-tailed Student t test for unpaired samples were used to assess significance of the differences between the three LAB strains. Differences with p values < 0.05 were considered statistically significant.

2.6. Measurement of pH and acidity

The LABs were cultivated in the MRS broth at a temperature of 30° C for 0 d and 3 d. The pH about 10 mL of each culture broth was

measured using electrode pH meter (Mettler Toledo). Acidity was measured by titration against 0.1N NaOH to a phenolphthalein endpoint, pH 8.3.

3. Results and discussion

Although minor ginsenosides have various beneficial effects, only small percentages are found in ginseng. Therefore, many studies have investigated methods such as heating, mild acid hydrolysis, and enzymatic transformation for conversion of major ginsenosides [35—37].

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LAB strains were isolated from kimchi previously and the strains were identified by 16S rRNA sequencing. The identified 16S rRNA sequences were deposited on NCBI GenBank under accession No. KT759681 (L. mesenteroides WiKiml9), KX890131 (P. pentosaceus WiKim20), KX886794 (L. brevis WiKim47), KX886799 (L. lactis WiKim48), and KX886806 (L. sakei WiKim49). The constructed phylogenetic trees clustered WiKim 19, 20, 47, 48, and WiKim 49 with the Lactobacillus, Leuconostoc, and Pediococcus genera and well matched with reference strains (Fig. 1).

The API 50 CH (bioMerieux) was used to determine the carbohydrate assimilation profile of five LABs. The test preparations were incubated at 30°, and readings were made after 48 h. The biochemical characteristics are listed in Table 1. L. mesenteroides WiKim19 showed the utilization of diverse a (1/4), ß (1/4), a (1 /6), a (1 /2) linkage. L. brevis WiKim47 could not use maltose, lactose, inulin, and cellobiose which have a (1/4) and diverse ß (1 /4) linkage. P. pentosaceus WiKim20 showed the similar carbon utilizing profile with L. mesenteroides WiKim19. Both could use inulin and gentiobiose, which have ß (1 /2) and ß (1 /4) linkage, respectively. However, P. pentosaceus WiKim20 did not use lactose, which has galactopyranosyl ß (1 /4) linkage.

To investigate the bacterial bioconversion of ginsenoside Rb1 into minor ginsenosides, ginsenoside bioconversion was performed by incubation of five LABs with 200p,M of Rb1 and measured the 15 minor ginsenoside components (ginsenoside -Rg3, -F2, -Rg5, -Rg1, -Rf, -Ro, -Rg2, -R1, -Ra1, -Rb2, -Rb3, -F1, -Rd, -compound K, -Rh2, -Rh4, gypenoside XVII).

In the results, five LABs did not produce the other minor ginsenosides except ginsenoside Rg3 and Rg5 (Figs. 1A and 1B). The

content of ginsenoside Rg3 increased in L. mesenteroides WiKiml9 (5.2 ± l.l mg/mL), P. pentosaceus WiKim20 (4.5 ± 0.9 mg/mL) and other lactic acid bacteria produced from ~0.8 ± 0.4 mg/mL to l.l ± 0.6 mg/mL. Interestingly, we detected the ginsenoside Rg5 from five LABs ranged from 2.6 ± 0.7 mg/mL to 6.5 ± 3.0 mg/mL concentration at 7 d of incubation. L. mesenteroides WiKiml9 (6.5 ± 3.0 mg/mL) and P. pentosaceus WiKim20 (4.6 ± 0.8 mg/mL) showed the higher ginsenoside Rg5 content among the five LAB strains (Fig. 2B).

It is reported that bacterial ß-glucosidase activity has been particularly involved among many glycosyl hydrolases to transform ginsenoside Rbl, which removes two glucose molecules at 20-C of protopanaxadiol into ginsenoside Rg3 [38]. To determine whether ß-glucosidase influences the bioconversion of ginsenoside, we tested the ß-glucosidase enzyme activity. L. mesenteroides WiKiml9, which used diverse glycosidic linkage substrates (Table l ), showed the highest ß-glucosidase activity supporting the maximum bioconversion capacity. L. lactis WiKim48 and L. sakei WiKim49 exhibited the reduced productions of Rg3 along with the low ß-glucosidase activities. Interestingly P. pentosaceus WiKim20 showed an elevated production of Rb3 in spite of lack of ß-gluco-sidase activity (Fig. 3).

A recent study reported that the conversion of ginsenoside Rbl into Rg3 and Rg5 with organic acid such as D-, L-tartaric acid, citric acid, and acetic acid [39]. Under acidic conditions, the low pH environment enhances the deglycosylation of ginsenoside by acidic hydrolysis response. Lactic acid bacteria are well known for their organic acid production, which lowered the pH during the fermentation and help the long-term storage by preventing

Table 1

Distinctive features of the carbohydrate fermentation profiles of five LAB strains determined using API 50 CH (bioMerieux, Lyon, France)

Characteristic Lactobacillus brevis WiKim47 Pediococcus pentosaceus WiKim20 Leuconostoc Lactis WiKim48 Lactobacillus sakei WiKim49 Leuconostoc mesenteroides WiKim19

Glycerol - w - - -

Erythritol - w - - -

-D-arabinose - w - - -

L-Arabinose + + + + +

D-Ribose + + + + +

D-Xylose + + + - +

D-Galactose + + + + +

D-Glucose + + + + +

D-Fructose + + + + +

D-Mannose + + + + +

D-Mannitol w - - - +

D-Sorbitol - - - - +

Methyl-a D-glucopyranoside + - + - +

N-Acetylglucosamine + + + + +

Amygdalin - + - - +

Arbutin - + + - +

Esculin ferric citrate - + + + +

Salicin - + + + +

D-Cellobiose - + + + +

D-Maltose + + + - +

D-Lactose (bovine origin) + - - + +

D-Melibiose + + + + +

D-Saccharose (sucrose) + + + + +

D-Trehalose - + + + +

Inulin - + - - +

D-Melezitose - - - - +

D-Raffinose + + + - +

Amidon (starch) - - w - -

Gentiobiose - + - - +

D-Turanose - - + - +

D-Tagatose - + - - +

D-Arabitol - - - - -

Potassium gluconate - - + w +

Potassium 2-ketogluconate - W - - +

-, negative; +, positive; LAB, lactic acid bacteria, Strain l, Leuconostoc mesenteroides WiKiml9; Strain 2, Pediococcus pentosaceus WiKim20; Strain 3, Lactobacillus brevis WiKim47; Strain 4. Leuconostoc lactis WiKim48; Strain 5, Lactobacillus Sakei WiKim49; w, weak reaction Q17

66 67 6B

77 7B 79 B0 B1 B2 B3 B4 BS B6 B7 BB B9

97 9B 99

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Fig. 2. Comparison of ginsenoside Rb1 bioconversion into Rg3 in cell culture supernatant of Leuconostoc mesenteroides WiKim19, Pediococcus pentosaceus WiKim20, Lactobacillus brevis WiKim47, Leuconostoc lactis WiKim48 and Lactobacillus sakei WiKim49. Concentrations of ginsenoside Rg3 (A) and Rg5 (B) after 1 d, 3 d, and 7 d incubation of each LAB strain with Rb1 at a concentration of 200цМ. Data are presented as mean ± standard deviation. *p < 0.05.

contamination. Even with varying degrees of acidity among the LABs, all culture showed the acidic property and increased accordance to the growth of bacteria.

To determine whether media acidity influences the bioconversion of ginsenoside, we measured pH and acidity of bacterial cultures (Figs. 4A and 4B). The culture supernatant of P. pentosaceus WiKim20 had a pH ~3.8, and acidity is 1.9, while

Fig. 3. Comparison of b-glucosidase activity in different LABs strains. The proteins were diluted to the concentration of ~0.5 mg/mL to assay enzyme activity and the release of p-nitrophenol was measured at 405 nm. One-tailed Student t test for unpaired samples was used to assess significance of the differences between the three LAB strains. One unit of b-glucosidase is the amount of enzyme that catalyzes the hydrolysis of 1.0 mmole substrate per min at pH 7.0. LAB, lactic acid bacteria.

L. mensenteroides WiKim19 had pH 4.4, and acidity is 1.1. The ß-glucosidase activity and ginsenoside Rg3 production was correlated with L. mensenteroides WiKim19 which have higher enzyme activity and relatively low acidity among the five LABs. The elevated Rg3 production of P. pentosaceus WiKim20 could be explained by acidic hydrolysis instead of ß-glucosidase activity. The five LABs could produce the ginsenoside Rg5 and this increase did not show the correlation with ß-glucosidase enzymatic activity, supporting the theory that the organic acid could contribute to the production of Rg5.

In the present study, we monitored the ginsenoside Rb1 bioconversion in LABs strains isolated from Kimchi with sensitive and accurate LC-MS/MS techniques. Among them, L. mesenteroides WlKim19 transformed ginsenoside Rb1 into ginsenoside Rg3 and showed a correlation with enzymatic activity, whereas, P. pentosaceus WiKim20 showed an elevated production of Rb3 in spite of lack of ß-glucosidase activity but have the highest acidity among the five LABs. This result suggests that ginsenoside bioconversion with microorganisms might review with the enzyme activities with the environmental conditions such as organic acid production and lactic acid bacteria are useful to convert minor ginsenoside by enzymatic conversion together with mild acidic conditions.

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Fig. 4. pH (A) and acidity (B) during bioconversion of ginsenoside in different LABs strains. The pH of l0 mL of each culture broth was measured using electrode pH meter and acidity was measured by titration against 0.lN NaOH to a phenolphthalein endpoint, pH 8.3. * p < 0.05. **p < 0.0l. ***p < 0.00l. ■ Lactobacillus brevis WiKim47; : Pediococcus pentosaceus WiKim20; ALeuconostoc lactis WiKim48; • Lactobacillus sakei WiKim49; □ Leuconostoc mesenteroides WiKiml9; LAB, lactic acid bacteria.

Conflicts of interest

The authors declare that there is no conflict of interest.

Uncited reference Q18 [31],[32],[33],[34].

Acknowledgments

This research was supported by grant KE1601-1 from the World Institute of Kimchi, Korea.

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77 7B 79 B0 B1 B2 B3 B4 BS B6 B7 BB B9

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