Glutathione Utilization in Lactobacillus fermentum CECT 5716

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Agricultural and Environmental Chemistry

Glutathione Utilization in Lactobacillus fermentum CECT 5716 Agustian Surya, Xiaoji Liu, and Michael J. Miller J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06136 • Publication Date (Web): 12 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018

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Journal of Agricultural and Food Chemistry

Glutathione Utilization in Lactobacillus fermentum CECT 5716

Agustian Surya1, Xiaoji Liu1, Michael J. Miller1* 1Department

of Food Science and Human Nutrition, University of Illinois at Urbana-

Champaign, 1302 W. Pennsylvania Ave., Urbana, IL 61801

*CORRESPONDENCE: Michael J. Miller, Telephone: (217) 244-1973; Fax: (217) 2650925; E-mail: [email protected]

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ABSTRACT

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Glutathione, a tripeptide antioxidant, has recently been shown to be either utilized or

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synthesized by gram-positive bacteria, such as Lactic Acid Bacteria. Glutathione plays an

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important role in countering environmental stress, such as oxidative stress. In this study,

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cellular activity regarding glutathione in Lactobacillus fermentum CECT 5716 is

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characterized. We demonstrate that L. fermentum CECT 5716 has a better survival rate in the

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presence of glutathione under both oxidative and metal stress. As L. fermentum CECT 5716

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does not possess the ability to synthesize glutathione under the conditions tested, it shows the

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ability to uptake both reduced and oxidized glutathione from the environment, regenerate

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reduced glutathione from oxidized glutathione, and perform secretion of glutathione to the

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environment.

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KEY WORDS: Glutathione, Lactobacillus fermentum, Oxidative stress, Metal Stress, Lactic

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Acid Bacteria

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INTRODUCTION

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Glutathione or γ-L-glutamyl-L-cysteinyl-glycine, the most abundant non-protein thiol

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found in many organisms1, is an important low molecular weight antioxidant. Glutathione is

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known to be able to neutralize reactive oxygen species, such as hydrogen peroxide in

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combination with glutathione peroxidase2. The resultant oxidized glutathione can be reduced

20

by glutathione reductase with NADPH as the electron donor3. Glutathione has also been shown

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to neutralize xenobiotics with glutathione-s-transferase4. In addition, glutathione acts as a

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protein reductant in the cytoplasm; either directly or indirectly through the glutaredoxin

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system5. Glutathione is synthesized in two steps by proteins GshA and GshB or by the more

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recently discovered bifunctional GshF which enables glutathione to be synthesized in one

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step6.

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Glutathione is known to be distributed widely in eukaryotic and Gram-negative

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bacteria, but scarcely present in Gram-positive bacteria such as Lactic Acid Bacteria (LAB) 7.

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However, glutathione supplementation was subsequently found to alleviate the impact of

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various environmental stresses in LAB, such as alleviating the effect of cold-related stresses

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in Lactobacillus sanfranciscensis8, and being involved in acid stress response9 and increasing

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survival rate under oxidative stress in Lactobacillus plantarum10. This suggests that at least

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some LAB possess the ability to synthesize and/or utilize exogenous glutathione which has

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been confirmed in Streptococcus pneumoniae11, Streptococcus mutans12,13 and Streptococcus

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thermophilus14. In addition, glutathione utilization components have been characterized in

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several LAB including Lactobacillus sanfranciscensis with glutathione reductase15 and

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Lactobacillus fermentum ME-3 with glutathione reductase and glutathione peroxidase16.

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This study aims to characterize glutathione utilization in glutathione-importing

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Lactobacillus fermentum. Previous screening performed by Pophaly et al.17 has demonstrated

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the lack of the complete glutathione synthesis system in several strains of L. fermentum. We

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have investigated the protective effect of glutathione in L. fermentum CECT 5716, a

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commercially available probiotic18,19 under several environmental stressors. Glutathione

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provided L. fermentum CECT 5716 protection from oxidative stress. We confirmed that

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reduced and oxidized glutathione can be imported by this strain. Furthermore, we determined

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that glutathione can be exported from the cells. Promoting survivability of a probiotic strain

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over stress conditions may promote their survival in gastrointestinal tract and during industrial

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culture production.

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MATERIALS AND METHODS

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Bacterial Strain and Chemical Reagents. Pure culture of Lactobacillus fermentum CECT 5716

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was provided by Biosearch Life (Granada, Spain). Fructose, magnesium sulfate heptahydrate,

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reduced (GSH) and oxidized glutathione (GSSG) were purchased from Sigma-Aldrich (MO,

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USA). MRS broth and Bacto Peptone were purchased from BD Biosciences (MD, USA);

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Tween 80, agar powder, yeast extract, and potassium phosphate dibasic anhydrous, copper (II)

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sulfate pentahydrate and ammonium citrate tribasic were purchased from Thermo Fisher

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Scientific (MA, USA); PBS tablets and manganese sulfate were purchased from MP

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Biomedicals (OH, USA); and peroxide solution (3%) was purchased from local retail stores.

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GSH-treated Cell Preparations. Overnight culture of L. fermentum CECT 5716 from -80 °C

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grown in semisynthetic medium20 was sub-cultured three times, incubated in 37 °C incubator,


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each for 24 hours in the semisynthetic growth medium containing 1% (w/v) fructose or the

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same media which was supplemented by glutathione. Glutathione supplementation was

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performed by adding 76.83 mg of reduced glutathione into 50 mL of 1% (w/v) fructose

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semisynthetic media to result in 50 μM of supplemented reduced glutathione, then filtering the

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medium aseptically with sterile 0.22 μM membrane (Fisherbrand) under sterile condition.

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Glutathione content in the base medium was measured with LC-MS/MS, which was 2 μM,

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exclusively present in oxidized form, while GSH supplementation of this medium increased

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the amount of total glutathione to 52 μM. These cell suspensions were used for the peroxide

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stress-response assay, cytoplasmic glutathione content, and extracellular and intracellular

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glutathione under sub-lethal peroxide treatment.

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Peroxide Stress-Response Assay. GSH-treated cells prepared as above were harvested by

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centrifugation at 16,000 x g for 2 minutes. Then the cell pellets were washed twice by

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resuspending the pellet in PBS buffer, centrifugation at 16,000 x g for 2 minutes, and

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supernatant removal. After washing, cells were resuspended in semisynthetic growth medium

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without glutathione supplementation. The cell suspensions were divided with half used for the

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control and half for the treatment group. Initial trials evaluated 1, 2, 3, 4 and 5 mM H2O2 and

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3 mM provided the desired 5-log reduction for our GSH protection study. In the treatment

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group, hydrogen peroxide was added to the resuspended cells to yield 3 mM in suspension,

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before incubating for 60 minutes at 37 °C. For the control group, cells were incubated in the

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same condition without addition of hydrogen peroxide.

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Viable cell number was quantified by serial dilutions in PBS and plating on de Man-

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Rogosa-Sharpe agar according to the Miles-Misra method21. The cell counts were determined 5 ACS Paragon Plus Environment


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following incubation of the plates for 24 hours at 37 °C anaerobically (90% N2, 5% CO2, 5%

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H2). The entire experiment was repeated 5 times independently.

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Copper Stress-Response Assay. An overnight culture of L. fermentum CECT 5716 from a

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glycerol stock stored at -80 °C, was sub-cultured three times, each for 24 hours in 1% (m/v)

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fructose-containing semisynthetic growth media. For the treatment group the same medium

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was used but containing 125 or 250 μM of copper sulfate, based on the study from Potter et

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al11. The media were filtered aseptically with sterile 0.22 μm membrane (Fisherbrand).

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Cell suspensions were homogenized by vortexing, subcultured by 1% (v/v) in 200 μL

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glutathione-supplemented (containing 50 μM of supplemented reduced glutathione) or non-

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supplemented 1% fructose-containing semisynthetic media. Cellular growth profile was

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determined with Bioscreen C (Growth Curves USA, NJ, USA) for 24 hours by measuring

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OD600nm every 10 minutes with 5 seconds of shaking before each measurement in an anaerobic

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chamber maintained at 37 °C. The entire procedures were performed in 4 independent

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biological replicates and measured in 3 technical replicates. The growth curve was analyzed

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using the algorithm developed by Hoeflinger et al.22,23 in MATLAB to determine the lag phase

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time, maximum cell density, and maximum growth rate.

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Cytoplasmic Glutathione Content. The intracellular content of glutathione in L. fermentum

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CECT 5716 was determined. GSH-treated cells were prepared according to GSH-treated cell

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preparation above, except the glutathione was supplemented in either reduced form (50 µM)

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or oxidized form (25 µM) to yield equal sulfur content. Cells were harvested by centrifugation

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at 16,000 x g for 2 minutes. Then the cell pellets were washed three times by resuspending the


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pellet in PBS buffer, centrifugation at 16,000 x g for 2 minutes, supernatant removal. After the

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third cell-washing, cells were resuspended in PBS buffer. Viable cell counts were determined

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by plating on MRS agar as described above.

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The resuspended cell pellets in PBS were homogenized by bead-beating for 10 minutes

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in an anaerobic environment (90% N2, 5% CO2, 5% H2). Cell lysates were stored at -20 °C

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until analysis. The entire experiment was repeated in 3 independent biological replicates. The

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samples were measured with LC/MS/MS as described below.

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Extracellular and Intracellular Glutathione under Sub-lethal Peroxide treatment. The cells of

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L. fermentum CECT 5716 were prepared according to GSH-treated cell preparation above.

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The cells were harvested by centrifugation at 3,220 x g for 5 minutes. Cell washing was

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performed 3 times, by resuspending in PBS buffer, centrifugation at 3,220 x g for 5 minutes,

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and supernatant removal. Recovered cell pellets were resuspended in PBS buffer to 1/5 of the

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original volume. The treatment group was added hydrogen peroxide to yield 0.25 mM,

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incubated for 30 minutes at 37 °C. The peroxide treatment was repeated once for a total

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treatment time of 1 hour. The control group was incubated for 1 hour in 37 °C without peroxide

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treatment.

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The cell suspension were centrifuged in 3220 x g for 5 minutes, the supernatant was

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decanted and recovered for analysis. The supernatants were placed in o-ring tubes sealed with

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Parafilm (Bernis NA, WI, USA), and kept at -20 °C until measurement.

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Cells were resuspended back into its original volume with PBS buffer. Viable cell counts were

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determined by plating on MRS agar as described above. Resuspended cell pellets were

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homogenized with bead-beater for 10 minutes under anaerobic condition (90% N2, 5% CO2,



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5% H2) and cell lysate were placed in o-ring tubes sealed with Parafilm. Cell lysates were kept

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at -20 °C until measurement. The samples were measured with LC/MS/MS as described below.

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LC-MS/MS Measurements. GSH, GSSG, and cystine were analyzed with the 5500 QTRAP

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LC/MS/MS system (Sciex, Framingham, MA) in the Metabolomics Lab of Roy J. Carver

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Biotechnology Center, University of Illinois at Urbana-Champaign. Software Analyst 1.6.2

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was used for data acquisition and analysis. The 1200 series HPLC system (Agilent

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Technologies, Santa Clara, CA) includes a degasser, an autosampler, and a binary pump. The

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LC separation was performed on an Agilent Eclipse Plus XDB-C18 column (4.6 x 150mm,

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5μm) using mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid

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in acetonitrile). The flow rate was 0.35 mL/min. The linear gradient was as follows: 0-3min,

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100% A; 10-11 min, 0% A; 12-18 min, 100% A. The autosampler was set at 10 °C. The

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injection volume was 10 μL. Mass spectra were acquired under positive electrospray ionization

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(ESI) with the ion spray voltage of +5000 V. The source temperature was 450 °C. The curtain

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gas, ion source gas 1, and ion source gas 2 were 32, 50, and 65, respectively. Multiple reaction

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monitoring (MRM) was used for quantitation: Cystine m/z 122.0 --> m/z 59.0; Cysteine m/z

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122.0 --> m/z 59.0; GSSG m/z 613.3 --> m/z 355.2; GSH m/z 308.1 --> m/z 179.2.

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Statistical Analysis. Experiment data with multiple factors were statistically analysed for the

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interference between each factors within the statistical model using JMP (SAS Institute, NC,

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USA). Each of the factors were further analysed with ANOVA to determine the significant

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difference. When multiple factors affect the parameter, Tukey Test was used to determine


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significance; when only a single factor affected the parameter, Student’s t-test was used. In all

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cases, significance was defined as p < 0.05.

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RESULTS & DISCUSSION

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Peroxide Stress-Response Assay. In this assay, stress-response of peroxide was performed to

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determine the protective effect of reduced glutathione supplementation on L. fermentum CECT

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5716 viability. Initial dose testing determined that 3 mM hydrogen peroxide was sufficient to

158

generate an approximate 5-log reduction. To prevent additional environmental stress to the

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cells, plated cells were grown in anaerobic condition. We hypothesized that resistance to

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peroxide can only occur if glutathione is used by the cells.

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Under peroxide treatment, the L. fermentum CECT 5716 population grown in

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glutathione-minimum growth medium (2 µM of oxidized glutathione) were reduced by 5-log

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compared to the control group; as shown in Figure 1. Under the same peroxide treatment, cells

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that were grown with abundant glutathione in their growth medium (52 µM total glutathione)

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had a better survival rate, only suffering 3-log reduction.

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This result confirmed that glutathione supplementation increases the survival rate of L.

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fermentum CECT 5716 under peroxide stress. A similar protective effect by glutathione against

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oxidative stress has been reported in L. plantarum10. As cell washing was performed prior to

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the stress treatment, resulting in a similar extracellular environment for both glutathione

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supplemented and non-supplemented groups. Thus, the difference in survival rate under the

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same peroxide treatment could be caused by the intracellular glutathione content suggesting

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that L. fermentum CECT 5716 may import glutathione. 9 ACS Paragon Plus Environment


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Copper Stress-Response Assay. To determine glutathione’s role in metal-induced stress, cells

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pretreated with copper (II) sulfate were evaluated for growth in the presence and absence of

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glutathione supplementation. The copper stress-response assay was designed to induce metal

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stress prior to glutathione supplementation to determine if exogenous glutathione can aid the

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recovery of copper-stressed cells. Cellular growth was measured in an anaerobic environment

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to prevent additional environmental stress. Pre-stressing the cells with 125 µM and 250 µM

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copper increased the lag time from 5.87 hrs to 8.01 hrs and 8.24 hrs, respectively (Figure 2 and

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Table 1). For both conditions of copper stressed cells, supplementation of their growth media

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with GSH reduced their lag time to be similar to the control condition. No significant

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differences were detected for growth rate and doubling time.

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The significantly shorter lag phase for the cells grown in glutathione-supplemented

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medium, compared to their respective control groups, suggests that glutathione can be utilized

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by L. fermentum CECT 5716. Since the lag phase represents adaptation to the new medium

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and environment, a shortened lag phase suggests that glutathione utilization by L. fermentum

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CECT 5716 facilitated a better adaptation before transitioning to the exponential phase. Such

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activity may be attributed to the antioxidant or conjugating property of glutathione24,25.

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Pre-treatment of copper sulfate was designed so that the cells would accumulate copper.

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Internalized copper mediated stress on the cells, as demonstrated by the longer lag phase for

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the copper-pretreatment groups. Since copper-pretreated cells were passed into fresh media

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without copper, we assume copper was exclusively available inside the cytoplasm. Despite

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copper increasing lag phase in the tested conditions, we were unable to provide statistically

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significant evidence that suggested a specific interaction between glutathione supplementation 10 ACS Paragon Plus Environment

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and the copper-induced stress. Hence, glutathione’s activity of reducing lag time was

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independent of the presence of copper.

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Cytoplasmic Glutathione content. The previous results provided evidence suggesting that

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glutathione was utilized by L. fermentum CECT 5716 and glutathione supplementation has a

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protective effect on this strain under oxidative stress. However, the presence of cytoplasmic

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glutathione was not determined and how glutathione is utilized was not characterized. To

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characterize the cytoplasmic GSH and GSSG, cell lysates, grown under supplementation with

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either GSH or GSSG, were analyzed by LC/MS/MS.

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Under supplementation of 50 µM of GSH, both oxidized and reduced glutathione were

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found in lysed cells, as shown in Table 2. Cells grown with 25 µM of supplemented GSSG

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also contained both GSH and GSSG inside their cytoplasm. Meanwhile, the groups that were

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not supplemented with glutathione (containing 2 µM of GSSG in MRS growth medium) did

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not contain detectable glutathione in the cell lysate with LC/MS/MS.

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Since GSH supplementation results in intracellular reduced and oxidized glutathione,

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we can imply that L. fermentum CECT 5716 may possess the ability to import GSH. GSSG

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supplementation with 25 µM caused L. fermentum CECT 5716 to uptake GSSG, as GSSG was

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not uptaken at lower GSSG concentrations in the non-supplemented MRS (2 µM). This

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suggests that there may be a minimum threshold of GSSG before influx can occur, and/or low

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affinity of the transporter for GSSG. While the non-supplemented growth medium contained

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2 μM of GSSG intrinsically, there was no GSH or GSSG detected from any of the cells growing

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in such medium. Another study conducted by Pophaly et al.17 has reported the inability of



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glutathione synthesis in several strains of L. fermentum. In addition, Peran et al.26 detected

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significant GSH in spent MRS from L. fermentum CECT 5716 suggesting that this strain can

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synthesize GSH. Although we were unable to locate glutathione synthesis genes, it may be

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possible that our experimental conditions were insufficient for L. fermentum CECT 5716 to

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synthesize glutathione in our study. Lastly, while the glutathione reductase activity may appear

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to be low, it is important to consider the difficulty in keeping glutathione in the reduced form

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prior to measurement.

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Intracellular and Extracellular Glutathione Under Stress. The previous data suggests the

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presence of cytoplasmic glutathione under supplementation and the ability to regenerate GSH

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from GSSG in L. fermentum CECT 5716. However, whether peroxide inactivation was

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performed either in intra- or extracellularly was unclear. As peroxide treatment can provide

230

both external and internal oxidative stress, both intracellular and extracellular glutathione

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contents of L. fermentum CECT 5716 were measured to determine compartmentalization of

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glutathione in this strain. We used a sub-lethal peroxide treatment, validated by comparing the

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viability between peroxide-treated and the control group that was not statistically significant,

234

to prevent the presence of unwanted extracellular glutathione that may be caused by cell lysis.

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To determine the ability of GSH to be secreted by L. fermentum CECT 5716, all cells

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were grown in the presence of GSH to allow the accumulation of GSH inside the cell prior to

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incubation with sublethal peroxide for 1 h. The GSH in the growth media was removed by

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washing with PBS. Half of the cells were exposed to a sublethal dose of peroxide for 1 h

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(treatment) while the other half remain in PBS without GSH supplementation for 1 h (control).

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While the GSH concentration was below the detection limit in the supernatant in both groups, 12 ACS Paragon Plus Environment

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the GSSG concentration in the supernatant was not altered by the sublethal peroxide. In

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addition, cystine was found in the supernatant, albeit in smaller amount compared to GSSG.

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Within the cells, cysteine and cysteine were both below the detection limit and there was no

244

change in the GSH or GSSG concentration.

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Oxidized glutathione was present in PBS supernatant after 1 hour of incubation. To

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ensure that the GSSG present in the media was not from the original growth media, we have

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washed the cells with PBS buffer 3 times to remove GSSG prior to final resuspension in PBS

248

for LC-MS/MS measurement, but we cannot eliminate the possibility for cell lysis. The result

249

implies that L. fermentum CECT 5716 may possess the ability to export glutathione into the

250

environment. Glutathione that is present in the supernatant is exclusively found in oxidized

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form; which was caused by either export of reduced glutathione that got oxidized during

252

incubation period or export of oxidized glutathione. This finding is consistent with those of

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Peran et al., 200626.

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Hypothetically, efflux of oxidized glutathione will not deactivate extracellular oxidant,

255

unlike efflux of reduced glutathione. Efflux of oxidized glutathione will only diminish their

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glutathione reserve without providing protection. Exporting the readily-oxidized GSH will

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enable L. fermentum CECT 5716 to recycle glutathione by uptaking extracellular non-

258

enzymatically oxidized GSH. However, we cannot provide evidence that only reduced

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glutathione was transported into the extracellular environment. We propose that the cells

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export glutathione in GSH form, without disregarding the possibility of GSSG export. Cystine

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presence in the supernatant may suggest that cysteine was also transported outside the cells.

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Cystine presence was likely caused by efflux of cysteine that was non-enzymatically oxidized

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in the presence of oxygen. 13 ACS Paragon Plus Environment


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Limiting the amount of environmental stress during this experiment was challenging.

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Presence of oxidized glutathione in the group untreated with peroxide may imply that oxidant

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was inherently present during the cell incubation. The incubation was not performed in

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anaerobic conditions, as added peroxide may be spontaneously reduced by the reducing

268

environment of an anaerobic chamber.

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Conclusion. In this study, glutathione utilization in L. fermentum CECT 5716 was

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characterized. L. fermentum CECT 5716 was shown to be capable of accumulating glutathione

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both in the reduced and oxidized form. In addition, L. fermentum CECT 5716 also possesses

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the ability to regenerate GSH by reduction of GSSG; and is able to export glutathione.

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ACKNOWLEDGEMENTS

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We are also thankful for constructive input and insight from Elizabeth Jeffery, Matthew

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Stasiewicz, and Maxwell Holle from Department of Food Science and Human Nutrition,

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University of Illinois. We thank Dr. Lucas Li from Metabolomics Lab of Roy J. Carver

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Biotechnology Center, University of Illinois for glutathione analysis.

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FUNDING SOURCES

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We are especially thankful for the Fulbright Scholarship, administered by Institute of

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International Education that enables this study.


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16. Kullisaar, T.; Songisepp, E.; Aunapuu, M.; Kilk, K.; Arend, A.; Mikelsaar, M.; Rehema, A.; Zilmer, M. Complete Glutathione System in Probiotic Lactobacillus Fermentum ME-3. Applied Biochemistry and Microbiology 2010, 46 (5), 481–486. 17. Pophaly, S. D.; Poonam, S.; Pophaly, S. D.; Kapila, S.; Nanda, D. K.; Tomar, S. K.; Singh, R. Glutathione Biosynthesis and Activity of Dependent Enzymes in Food-Grade Lactic Acid Bacteria Harbouring Multidomain Bifunctional Fusion Gene (GshF). Journal of Applied Microbiology 2017, 123 (1), 194–203. 18. Díaz-Ropero, M. P.; Martín, R.; Sierra, S.; Lara-Villoslada, F.; Rodríguez, J. M.; Xaus, J.; Olivares, M. Two Lactobacillus Strains, Isolated from Breast Milk, Differently Modulate the Immune Response. J. Appl. Microbiol. 2007, 102 (2), 337–343. 19. Olivares, M.; Díaz-Ropero, M. P.; Sierra, S.; Lara-Villoslada, F.; Fonollá, J.; Navas, M.; Rodríguez, J. M.; Xaus, J. Oral Intake of Lactobacillus Fermentum CECT5716 Enhances the Effects of Influenza Vaccination. Nutrition 2007, 23 (3), 254–260. 20. Barrangou, R.; Altermann, E.; Hutkins, R.; Cano, R.; Klaenhammer, T. R. Functional and Comparative Genomic Analyses of an Operon Involved in Fructooligosaccharide Utilization by Lactobacillus Acidophilus. PNAS 2003, 100 (15), 8957–8962. 21. Miles, A. A.; Misra, S. S.; Irwin, J. O. The Estimation of the Bactericidal Power of the Blood. J Hyg (Lond) 1938, 38 (6), 732–749. 22. Hoeflinger, J. L.; Hoeflinger, D. E.; Miller, M. J. A Dynamic Regression Analysis Tool for Quantitative Assessment of Bacterial Growth Written in Python. J. Microbiol. Methods 2017, 132, 83–85. 23. Hoeflinger, J. L.; Davis, S. R.; Chow, J.; Miller, M. J. In Vitro Impact of Human Milk Oligosaccharides on Enterobacteriaceae Growth. J. Agric. Food Chem. 2015, 63 (12), 3295–3302. 24. Forman, H. J.; Zhang, H.; Rinna, A. Glutathione: Overview of Its Protective Roles, Measurement, and Biosynthesis. Mol Aspects Med 2009, 30 (1–2), 1–12. 25. Pereira, T. C. B.; Campos, M. M.; Bogo, M. R. Copper Toxicology, Oxidative Stress and Inflammation Using Zebrafish as Experimental Model. J Appl Toxicol 2016, 36 (7), 876–885. 26. Peran, L.; Camuesco, D.; Comalada, M.; Nieto, A.; Concha, A.; Adrio, J. L.; Olivares, M.; Xaus, J.; Zarzuelo, A.; Galvez, J. Lactobacillus Fermentum, a Probiotic Capable to Release Glutathione, Prevents Colonic Inflammation in the TNBS Model of Rat Colitis. Int J Colorectal Dis 2006, 21 (8), 737–746.

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FIGURE CAPTIONS

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Figure 1. Protective effect of glutathione (GSH) supplementation from hydrogen peroxide

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(H2O2) on L. fermentum CECT 5716. Cells were subcultured three times with or without

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GSH and treated/untreated with hydrogen peroxide (H2O2) after washing. Cells were plated

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and grown in an anaerobic environment for 24 hours. Data are reported as the mean ±

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standard error. Letters represent statistical difference (p0.05).


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Figure 1.

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Figure 2.

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TABLE OF CONTENTS GRAPHICS


Glutathione Utilization in Lactobacillus fermentum CECT 5716

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