A metabolomics study reveals enhanced inhibition and metabolic

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A metabolomics study reveals enhanced inhibition and metabolic dysregulation in E. coli induced by Lactobacillus acidophilus fermented black tea extract Kundi Yang, Mattew Duley, and Jiangjiang Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04752 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Journal of Agricultural and Food Chemistry

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A

metabolomics

study

reveals

enhanced

inhibition

and

metabolic

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dysregulation in E. coli induced by Lactobacillus acidophilus fermented black

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tea extract

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Kundi Yang1, Matthew L. Duley2 and Jiangjiang Zhu1*

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1. Department of Chemistry and Biochemistry, Miami University, Oxford, OH, USA 45056

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2. Center for Advanced Microscopy and Imaging, Miami University, Oxford, OH, USA 45056

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*Corresponding author.

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Email address: [email protected];

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Tel: +1 513 529 3998;

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Fax: +1 513 529 5715

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1 ACS Paragon Plus Environment


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Abstract

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This study examined the ability of Lactobacillus acidophilus (LA) to ferment black tea extract

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(BTE), and the enhancement of Escherichia coli cellular uptake of phenolic compounds when

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this bacteria was incubated with fermented BTE. The inhibitory effects of BTE to E. coli

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bacteria, with and w/o fermentation, were compared. Several intracellular phenolic compounds,

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as well as metabolic profiles of E. coli with and w/o treatments, were also determined using a

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HPLC-MS/MS-based approach. Our results showed that out of three concentrations from the

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non-fermented BTE treatment, only the extract from the 25 mg/ml tea leaves solution could

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inhibit E. coli survival, while LA fermented BTE extract from 5, 10 and 25 mg/mL tea leaves

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solutions all inhibited E. coli growth significantly. Intracellular concentration of (+)-catechin-3-

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gallate/ (-)-epicatechin-3-gallate and (+)-catechin / (-)-epicatechin were significantly higher

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when E. coli was treated with fermented BTE in comparison to non-fermented BTE. Scanning

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electron microscopy (SEM) images indicated that the intracellular phenolic compounds inhibited

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E. coli growth by increasing endogenous oxidative stress. Metabolic profiles of E. coli were also

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investigated to understand their metabolic response when treated with BTE, and significant

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metabolic changes of E. coli were observed. Metabolic profile data were further analyzed using

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partial least square-discriminant analysis (PLS-DA) to distinguish the fermented BTE treatment

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group from the control group and the non-fermented BTE treatment group. The results indicated

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a large-scale E. coli metabolic dysregulation induced by the fermented BTE. Our findings

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showed that LA fermentation can be an efficient approach to enhance phenolic inhibition of

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bacterial cells through increased endogenous oxidative stress and dysregulated metabolic

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activities.


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Keywords: Black tea extract, phenolic compounds, E. coli, metabolic profiling, bacteria

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inhibitory effect.

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Introduction

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Functional foods such as tea drinks have been known for several decades to have beneficial

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health effects, although the overall clinical study outcomes are still debatable 1. Tea consumption

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has been associated with decreased incidence of cancer, reduced diabetic incidence, assistance to

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weight control, support of cognitive function, improved anti-hyperglycemic effect and

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antibacterial activities 2. Tea extract phenolic compounds, including extracts from green tea,

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black tea, yellow tea and others, have been long known to make primary contributions to these

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beneficial health effects. Out of these choices, black tea is one of the world’s most popular

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beverages, which is a polyphenol-rich aqueous infusion of dried leaves from the plant Camellia

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sinensis 1a. Over the past decade the polyphenolic constituents of black tea have been studied

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extensively to explore their biological properties. However, the antibacterial aspect of black tea

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extract (BTE) has not been fully understood in comparison to the well-studied green tea extract.

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Meanwhile, the gut bacteria population was known to modulate the utilization of food

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components, and some probiotic bacteria, such as the Lactobacillus species, can assist human

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intestinal food process/digestion/fermentation and increase the absorption of nutritional

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components 3. The existence of these probiotic bacteria is essential for a healthy gut

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environment, as they can also act as first line of defense when pathogenic bacteria invade their

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territory 4. Recently, Zhao and Shah examined lactic acid bacteria for the metabolization of tea

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phenolics in order to enhance their cellular uptake in colon cancer cells. They also observed that

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the increase in the cellular antioxidant activity of tea samples after fermentation, particularly in

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BTE, was in alignment with a bacterial altered and increased total phenolic composition. 3 ACS Paragon Plus Environment


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While both the bacterial inhibition by phenolic compounds and the function of Lactobacillus

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acidophilus in fermentation of black tea extract (LA BTE) have been explored separately, the

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synergistic effects between these two factors are still unclear, and their underlying mechanism in

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bacteria inhibition has not been fully studied. In this study, we combined several

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biotechnologies, high-performance liquid chromatography tandem mass spectrometry (HPLC-

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MS/MS) based targeted compound detection, and Lactobacillus acidophilus (LA) fermentation,

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to investigate the enhanced inhibitory capability of LA-fermented BTE to affect the bacteria E.

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coli. To better understand the molecular level events inside E. coli cells during the fermented

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BTE treatment, both intracellular level of phenolic compounds and metabolites were detected

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and compared to those found in E. coli with a non-fermented BTE treatment.

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Materials and method

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Bacterial strain and growth condition

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The model bacterial strain used in this study was Escherichia coli (E. coli) K12. The bacteria

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were grown on Difco™ LB Agar plate (BD Diagnostics, Franklin Lakes, New Jersey) and

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incubated for 24 h at 37 oC; a mono colony was selected and transferred to fresh liquid LB broth

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by a bio-loop. Overnight culture was made by growing bacteria in a 5 ml LB broth medium

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placed in a Thermo Scientific™ Nunc™ 50 mL conical sterile polypropylene centrifuge tube and

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was incubated at 37 oC for 24 h to reach a stationary phase of growth. Four testing cultures were

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made as replicates by transferring 50 µL of the overnight culture to 5 ml of fresh LB broth for

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subsequent incubation under the same conditions for another 24 h.

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Lactobacillus acidophilus ATCC 4356 was purchased from the American Type Culture Center

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(ATCC) and was used to ferment black tea extraction (BTE) and metabolize phenolic


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compounds. The growth method for this strain was similar to our previous work with minor

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modification 5. Briefly, the bacteria strain was grown on Lactobacilli MRS agar (BD

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Diagnostics, Franklin Lakes, New Jersey) for 24 h at 37 °C in an anaerobic chamber (Coy

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Laboratory Products Inc, Grass Lake, MI) following the manufacturer’s instruction. A mono

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colony was selected by bio-loop and inoculated in MRS broth medium before incubation for 24 h

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at 37 °C. Four testing cultures were made as replicates by transferring 50 µL of overnight culture

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to 5 mL of fresh MRS broth for subsequent incubation under the same conditions for another 24

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h to reach a stationary phase of growth.

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Preparation of black tea extract (BTE)

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Black tea was purchased from local grocery store. BTE was obtained from dried black tea leaves

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according to a protocol described previously but with some modification 6. Briefly, dried black

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tea leaves (2.5g) were crushed and then added to a 70% methanol-30% water solution (v/v) in

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the ratio 1:20 (w/v). This suspension was placed in a 60oC water bath for 30 min. The extract

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was filtered through a Buchner funnel with a 0.4 µm filter and dried. The final BTE stock was

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made by reconstitution with 50 mL ultrapure water to reach the concentration of 50 mg/mL

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(representing 50 mg black tea dry leaves originally extracted by 1 mL solvent). The final BTE

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stock was diluted to BTE values equivalent to the extract of 25 mg/mL and 10 mg/mL dry

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leaves, respectively. These BTE solutions were then sterilized by filtration with 0.2 µm filters

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and stored at 4 oC.

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Fermentation of BTE and co-incubation of fermented BTE with E. coli

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BTEs were fermented according to a previously published method with minor modification 6.

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Briefly, Lactobacillus acidophilus ATCC 4356 was inoculated at 50% (v/v) into BTEs of 50



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mg/mL, 20 mg/mL and 10 mg/mL dry tea leaves. and then incubation was allowed at 37 °C for

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48 h. The starting BTE concentrations were therefore now equivalent to 25 mg/mL, 10 mg/mL,

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and 5 mg/mL of dry tea leaves, respectively. Representing the control, non-fermented BTEs were

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also incubated with MRS medium without Lactobacillus acidophilus under the same conditions.

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Fermented BTE products were collected by filtering the fermented bacterial/BTEs medium with

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a 0.2 µm filter. The fermented BTE was applied to E. coli K12 culture in the ratio of 1:1 (v/v).

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Non-fermented BTEs were applied to E. coli K12 directly in the ratio of 1:1 (v/v), and the MRS

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broth was applied as a negative control group. All three BTE concentrations and controls were

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replicated 4 times. Plate count was applied before and after co-incubation to obtain the detailed

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concentration of bacterial culture.

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Measurement of total phenolic compound concentration

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The total phenolic compound concentration was analyzed according to the Folin–Ciocalteu

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method with a little modification7. Briefly, a 500 µL volume of BTE (50 mg/mL) was added into

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a 2-mL centrifuge tube which contained 500 µL of MRS broth and the tube then vortexed for

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30s. A100 µL volume of this diluted BTE (25 mg/mL) was transferred to 900 µL of distilled

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water in a 15ml test tube, followed by addition of 0.5 mL of the 1 M Folin–Ciocalteu reagent and

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a thorough mixing. A 3-mL volume of 7.5% Na2CO3 solution was added after a 3-min interval.

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Fermented BTE samples were measured using the same protocol except for no dilution with

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MRS broth at the beginning. A microplate reader (Biotek® ELx808) was applied to make all

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absorbance measurements at 630 nm. A gallic acid aqueous stock solution was used for

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preparing the solutions used to generate the standard curve by the same method. All

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measurements were carried out in triplicate. The total polyphenol sample content was calculated

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as the equivalent concentration of gallic acid in mg/mL. 6 ACS Paragon Plus Environment

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Sample preparation for HPLC-MS/MS work

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Intracellular metabolites and phenolic compounds from each biological replicate were extracted

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using a cold methanol extraction approach as previously reported . Briefly, a 200 µL volume of

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the co-incubated culture was transferred by pipet from a 96-well plate to a 2-mL centrifuge tube.

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This was followed by centrifugation and a phosphate buffer saline (PBS) wash; these steps were

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repeated three times. A 250 µL volume of methanol was added to the cell pellet in the tube and

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the sample mixed vigorously for 1 min on a vortex machine. A 150 µL volume of extracted

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supernatant was collected after centrifugation at 14000 rpm for 5 minutes and then dried. A 50

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µL volume of the isotope labeled amino acid mixture (spiking) solution prepared in 50%

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acetonitirile-50% ultrapure water was used to reconstitute the sample. The final samples were

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loaded into liquid chromatography vials for analysis.

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Targeted HPLC-MS/MS method for phenolic compound detection and metabolic profiling

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The targeted metabolite and phenolic compound detection approach applied in this study was

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similar to our previous work 5. Briefly, a Thermo Scientific TSQ Quantiva triple quadruple mass

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spectrometer equipped with an electrospray ionization (ESI) source, applied for both positive and

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negative mode compound detection, was coupled with a Thermo Scientific Ultimate 3000 high

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performance liquid chromatograph equipped with an amide hydrophilic interaction

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chromatography (HILIC) column having dimensions of 2.1 x 150 mm and particle size of 2.5 µm

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(Waters Corporation, Milford, MA, USA). The extracted bacterial intracellular reconstituted

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samples were injected on the column for gradient elution separation at 0.300 mL/min using

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solvents A (5 mM ammonium acetate in 90% water / 10% acetonitrile + 0.2% acetic acid) and B

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(5 mM ammonium acetate in 90% acetonitrile / 10% water + 0.2% acetic acid). The auto-

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sampler temperature was kept at 4 °C, the column compartment was set at 40 °C, and the 7 ACS Paragon Plus Environment


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separation time for each sample was 20 min. Retention time and selected reaction monitoring

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(SRM) transitions of targeted metabolites and phenolic compound (or compound pairs) were

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established by running pure standards (purchased from Sigma, Saint Louis, MO, USA and IROA

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Technology, Boston, MA) and collecting the tandem mass spectra (MS/MS), so the orthogonal

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information of retention time and two pairs of SRM transitions can be used to confidently detect

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and identify targeted compounds. The stable isotope-labeled amino acid mix (20 AA U-13C, 97-

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99%; U-15N, 97-99% Catalog # CNLM-6696-1,) was purchased from Cambridge Isotope

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Laboratories, (Tewksbury, MA) and used for a quality control purpose during MS runs. This

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method has been checked and validated monthly to ensure its performance. When running

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biological samples, pooled quality control samples were also tested in between every ten samples

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to monitor the instrument stability.

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Scanning electron microscope assay

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Testing culture of E. coli K12 was prepared as described above. A 100 µL volume of the testing

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culture was taken and mixed with LB broth at a 1:20 (v/v) ratio for incubation at 37 oC for 12 h.

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Three treatments were applied after pre-incubation; E. coli plus sterilized water was used as

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control group, while E. coli plus 25 mg/mL BTE and E. coli plus 25 mg/mL BTE plus 2 mM

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acetylcysteine (NAC) were used as testing groups. Each treatment was incubated at 37 oC for

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180 min.

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For initial fixation, each bacteria suspension was mixed 1:1 (v/v) with a 2.5% (v/v)

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glutaraldehyde, 2.0% (v/v) paraformaldehyde in 2x PBS buffer solution. The fixed bacteria

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suspension was then filtered through a 0.45 µm nylon transfer membrane (GVS North America,

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63 Community Dr, Sanford, ME), and an approximately 20-mm square was cut from the center

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of the filter paper. Ross Optical Lens Tissue was folded into a small envelope and the 20-mm 8 ACS Paragon Plus Environment

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square was inserted to protect it during processing. From this point on, all the samples remained

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in the envelopes. Envelopes were placed in 2% (w/v) osmium tetroxide in doubly distilled

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(dd)H2O overnight at room temperature. Samples were then washed with ddH2O 4 times for 45

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minutes each and then they proceeded through an ethanol serial dehydration, basically 3 washes

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in 100% EM grade ethanol before being critical point dried in a Samdri - 780A (Tousimis Corp.,

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2211 Lewis Ave, Rockville, MD.). After drying, the filter papers were removed from the paper

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envelopes. The bacteria were carefully scraped from the paper surface onto a carbon adhesive tab

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mounted on an aluminum stub with a new razor blade that had been cleaned with 100% acetone.

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Samples were then coated with approximately 20 nm of gold in a Denton Desk II sputter coater

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(Denton Vacuum, LLC, 1259 North Church St. Bldg 3, Moorestown, NJ). Samples were imaged

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in a Zeiss 35VP scanning electron microscope (Carl Zeiss AG, One Zeiss Dr. Thornwood, NY)

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at 4 KeV with an 8 mm working distance.

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Data processing and statistical analyses

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Xcalibur 4.0 was used to process the raw data and the data processing method was similar to that

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described in our previous work 5, 8. Briefly, the raw data were reprocessed and integrated

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manually, then exported as an Excel file. Data normalization was completed by using a viable

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count of testing culture, and both univariate and multivariate statistical analyses were conducted

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by using JMP Pro12 (SAS Institute, Cary, NC) and MetaboAnalyst 3.0 9.

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Results and discussion

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In this study, the black tea extract (BTE) was obtained following the extraction approach

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reported previously 6. The BTEs were then split into two groups, with one set of samples mixed

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with Lactobacillus acidophilus (LA) and another set mixed with the MRS medium used for LA



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growth. After LA fermentation, both types of BTE samples were filtered through 0.22 µm filters

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and then the filtered products were used to co-incubate with E. coli bacteria for the inhibitory

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test. A schematic of our workflow is shown in Figure 1A. A comparison between the E. coli

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culture co-incubated with BTE (with or without LA fermentation) was then conducted, and the

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results can be seen in Figure 1B. A concentration dependent inhibitory effect of E. coli can be

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clearly observed for the LA-fermented BTE treatment, with all three tested concentrations

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having statistically significant decreases in comparison to the control group. As for the BTE

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without LA fermentation, only the highest concentration of BTE used for co-incubation was

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showing a significant decrease of E. coli growth. It is known that black tea contains various salts

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which can cause an osmolarity change in the growth environment, and this may lead to a

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bacterial membrane leak when treated with BTE10. However, osmolarity measurements,

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conducted before and after fermentation of BTE by testing freezing points showed no significant

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difference between those two groups, indicating the similar osmolarity with or without

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BTE/FBTE treatment to the control group. Also, our focus was whether the fermentation of BTE

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changed the bacterial inhibitory function; whether the osmolarity of BTE played an important

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role in the inhibition process needs further study and will not be pursued here.

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We then examined the concentration of total phenolic compounds from the BTE, with and

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without LA fermentation. The LA-fermented BTE contained a significantly higher total of

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phenolic compounds compared to the ones without fermentation (Figure 2A), with ~ 30%

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increase of bioavailable phenolic compounds detected in the fermented BTE samples. After

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treatment of the E. coli with both types of BTEs, intracellular level of five major phenolic

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compounds (or compound pairs), namely caffeine, (+)-catechin/ (-)-epicatechin, gallocatechin,

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catechin-3-gallate/ epicatechin-3-gallate, gallocatechin-3-gallate/epigallocatechin-3-gallate, were


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targeted using our HPLC-MS/MS approach. These three compound pairs were considered

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because we cannot differentiate the isomers using our current chromatography conditions. The

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detailed detection parameters used for these compounds can be seen in Table 1. When comparing

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the intracellular level of these phenolic compounds from E. coli, two of them (gallocatechin-3-

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gallate/epigallocatechin-3-gallate and gallocatechin) cannot be detected due to relatively low

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signal intensity. Of the three compounds (compound pairs) we did detect, two of which, the

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catechin-3-gallate/epicatechin-3-gallate and the (+)-catechin/ (-)-epicatechin pairs, were detected

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at significantly higher levels in samples with the fermented BTE treatment rather than with the

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BTE treatment without fermentation (Figure 2B and 2C). This indicated the LA fermentation

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increased the amount of bioavailable total phenolic compounds, resulting in a higher intracellular

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phenolic compounds uptake, and therefore increased the inhibitory function on E. coli

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proliferation. However, we also observed the decreased concentration of caffeine in E. coli cells

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from the fermented BTE treatment (Figure 2D), which could possibly suggested the active

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metabolic degradation of caffeine by this group of bacteria, as discussed by Summers and co-

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workers in previous study 11.

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It is well-known that the bacterial metabolic activities could change during the co-incubation of

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inhibitory drugs or compounds 8, 12. Therefore, we also looked at the intracellular metabolic

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profile difference, where the metabolite data were obtained from either LA fermented or non-

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fermented BTE treated E. coli groups. The dramatic group difference of ninety detected

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metabolites are shown in Figure 4. In this heatmap, each column represented one biological

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replicate of E. coli sample and each row represented one targeted metabolite. The comparison of

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BTE treatment with and without LA fermentation clearly indicated dramatic metabolic

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dysregulation when additional phenolic compounds became available after LA fermentation.



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Metabolic profiles from both treatments were also compared to the control samples and their

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metabolic profile differences were also observed. More specific comparisons of several

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representative metabolites were conducted and are plotted in Figure 5. The detailed detection

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parameters used for these compounds can be seen in Table 2. As demonstrated, several amino

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acids, such as creatinine, phenylalanine, valine, and betaine displayed clear differences from the

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fermented-BTE treatment group comparing to the non-fermented BTE group. Other metabolites

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such as lactose, NADP, and inosine 5’-triphosphate, which are known as important indicators in

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energy metabolism, have shown variable production in the two BTE treatment groups. Cytidine

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5’-diphosphocholine, an intermediate in the generation of phosphatidylcholine from choline,

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which is a common biochemical process in cell membranes, significantly increased in the

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fermented BTE treated groups. This finding indicated the greater need for this group of bacteria

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to generate or repair cell membrane and fight the inhibitory phenolic compounds for their

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survival.

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Following the univariate analysis, which only compares individual metabolites from the

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metabolic profiles, we also conducted the multivariate statistical analysis using partial least

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square-discriminant analysis (PLS-DA). This approach is to generate an overview of the entire

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metabolic profile from each sample in a respective group, and to confirm whether the fermented

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and non-fermented BTE systematically dysregulated the metabolic activities of E. coli at the

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metabolome level. As can be seen in Figure 6A, three major clusters, determined by the

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metabolic profiles generated by the E. coli control group, the fermented BTE treated group, and

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the non-fermented BTE treated group, were clearly separated. The ovals with different colors

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indicated the 95% confidence region for the separation. Also, not to our surprise, the BTE

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concentration dependent variation of the inhibitory effect to E. coli, no matter in either the


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fermented treatment group or the non-fermented treatment group, is not significantly great

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enough to be separated from each other. Figure 6B also lists the top fifteen metabolites that

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contribute the most to the group separation achieved in Figure 6A, as measured by their variance

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importance projection (VIP) scores. Glucosamine, 4-guanidinobutanoate and glutaric acid were

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the top three metabolites that were significantly different in the three comparison groups. Based

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on the color bar on the right of the figure, all three metabolites were detected more abundantly in

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the non-fermented BTE treated culture than the fermented BTE treatment group or the control

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group.

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Extensive work has been carried out to study the beneficial health effects of tea extracts,

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including antibacterial function 1b, 13, anti-cancer function 14 and gut microbiome modulation 15.

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Although its bacterial inhibitory activity has been previously reported, the underlying mechanism

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for this effect is not fully understood yet. We argue that the inhibition of E. coli proliferation

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depends on the intracellular phenolic compounds concentration; systematic metabolic

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dysregulation induced by oxidative stress from phenolic compounds such as catechins can play a

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critical role. Several recent studies have explored the endogenous oxidative stress induced by

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phenolic compounds, such as using epigallocatechin gallate to inhibit E. coli bacteria13a; or

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investigating the reduced quorum sensing and biofilm formation through Rosa Rugosa tea

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phenolic extract 16. To test the hypothesis that the increasing level of endogenous oxidative stress

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can be induced by intracellular phenolic compounds, and therefore to generate an inhibitory

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effect to the E. coli cells, we first conducted E. coli growth tests to the control group (mock dose

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with sterilized water), the BTE treated group and BTE+NAC treated group. Our results indicated

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that BTE+NAC could partially rescue the BTE induced cell death via cell count measurement

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(data not shown). SEM experiments were also conducted to examine the cell morphology. As



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shown in Figure 3, it can be clearly observed that compared to the control group (E. coli treated

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with sterile water, Figure 3A), the cells from BTE treated E. Coli K12 group (Figure 3B)

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appeared like a clump, indicating possible impairment to the cell membrane and cell division

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process17. When the intracellular antioxidant NAC was added together with BTE (Figure 3C),

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the BTE impairment to cell integrity can be reduced. In our study, total bioavailable phenolic

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compounds were made increasingly accessible through LA fermentation, a process that could

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happen in a healthy human host, or in a human gut with additional probiotic supplement intake.

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Meanwhile, intracellular phenolic compounds catechin-3-gallate/ epicatechin-3-gallate and the

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(+)-catechin/ (-)-epicatechin were significantly increased after 48 hours LA fermentation, which

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increased the possibility for the catechins to reach desired intracellular targets and execute

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inhibitory function to E. coli. We also suspect that the increased phenolic compounds inside

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bacteria cells could enhance the oxidative stress, and therefore could contribute to the stronger

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inhibition of E. coli proliferation; future follow-up studies will be needed to confirm this

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working hypothesis. Another important phenolic compound, epigallocatechin-3-gallate, was also

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frequently linked to antimicrobial activities from a primarily green tea extract origin. In our BTE

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study, we were unable to detect intracellular level of epigallocatechin-3-gallate, probably due to

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the excessive loss of this compound during the black tea production process 2b.

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A recent review also discussed the antimicrobial effects by three out of four main catechins

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found in green tea, including (-)-epicatechin-3-gallate, (-)-epigallocatechin, and (-)-

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epigallocatechin-3-gallate 18. The known mechanisms behind this antimicrobial function include

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the damage of bacterial cell membrane (such as catechins binding to the bacterial lipid bilayer

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cell membrane, therefore inhibiting the ability of bacteria to bind to each other to form biofilms

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and to bind to host cells), inhibition of fatty acid synthesis (due to the inhibition of specific


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reductases in bacterial type II fatty acid synthesis and inhibition of bacterial production of toxic

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metabolites), and inhibition of functional enzymes such as tyrosine phosphatase and cysteine

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proteinases 18. Several of these reported mechanisms involved the disruption of metabolic

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enzymes, which strongly suggested that the downstream production of metabolites from these

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enzymes could possibly have been influenced as well. To further understand this possible

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influence, we conducted HPLC-MS/MS based targeted metabolic profiling to obtain systematic

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metabolic information. Our results have shown that massive metabolic profile changes can be

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observed from fermented BTE treated E. coli, and different classes of metabolites, such as amino

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acids, sugars, and nucleotides, were detected at significantly different levels than in the non-

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fermented BTE treatment group (Figure 4 and 5). The multivariate statistical analysis approach

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PLS-DA also summarized the group difference by projecting individual biological replicate into

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different cluster on its score plot, which was primarily based on the contribution of a group of

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significantly altered metabolites from different groups (Figure 6). Clear separation of different

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treatment groups via their metabolic profiles by PLS-DA strongly suggested that the fermented

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BTE treatment systematically disturbed the metabolic activities of the treated E. coli population,

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and inhibited their growth via enhanced oxidative stress. Our results highlighted the BTE

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fermentation by LA can contribute to increased intracellular phenolic compounds such as

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catechin-3-gallate/epicatechin-3-gallate and the (+)-catechin/(-)-epicatechin, and therefore

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dysregulate massive metabolic activities inside E. coli cells, inhibiting growth and proliferation.

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While the findings were exciting, we acknowledge the need for further follow-up studies to seek

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better understanding of specific metabolic pathway interruptions and metabolic enzyme

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inhibition induced by the intracellular phenolic compounds. Our study provided first hand

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evidence for fermented BTE induced metabolic dysregulation, which should pave the way for



333

future studies in exploring the interactions between intracellular phenolic compounds and

334

bacterial metabolic activities, and how these interactions influence the bacteria survival.

335

In summary, our study utilized state-of-the-art bioanalytical technology, mass spectrometry

336

targeted metabolite measurement and phenolic compound detection, to systematically investigate

337

the potential enhancement of Lactobacillus acidophilus bacteria to the accessibility of

338

bioavailable phenolic compounds from BTE, and its consequential bacterial inhibitory effect to

339

E. coli. Sensitive and specific detection of phenolic compounds and metabolites enabled

340

molecular level understanding of the bacterial inhibitory effect by fermented BTE. We will

341

continue this line of research in the future to further elucidate the enhanced antibacterial effect of

342

LA fermented BTE and explore its capability in other model bacteria system to prove the broader

343

utility of this synergistic effect.

344

Acknowledgement

345

This work was supported by Miami University (Startup fund to JZ). The authors thank Dr.

346

Richard E. Edelmann from the Center for Advanced Microscopy and Imaging at Miami

347

University for his assistance with our SEM experiments. We also thank a colleague at Miami

348

University for his suggestions to our manuscript.

349

Conflict of interest

350

No conflict of interest was declared by the authors.

351

352 353


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418

Table 1. Targeted phenolic compounds and their detection parameters. Two SRM transitions for

419

each compound were used for confident detection.

Phenolic compounds Caffeine (+)-Catechin/(-)Epicatechin Gallocatechin Catechin-3gallate/Epicatechin-3gallate Gallocatechin-3gallate/Epigallocatechin3-gallate 420

Collision Collision Energy Energy 1 (V) 2 (V) 22.89 19.30

RF Lens (V)

Retention time (min)

Polarity

Precursor (m/z)

1.33

positive

195.34

138.05

110.11

1.44 1.99

positive positive

291.06 307.06

139.05 151.05

123.05 139.05

15.51 15.51

16.88 10.25

70.54 72.02

1.28

positive

443.10

273.07

123.11

16.17

10.25

73.51

1.43

positive

459.10

288.75

139.00

21.33

10.25

79.19

Product Product 1(m/z) 2(m/z)

421


81.17

Page 21 of 30


422

Table 2. Example of targeted metabolites and their detection parameters. Two SRM transitions

423

for each metabolite were used for confident detection. Compound

Retention Polarity Precursor Product Time (m/z) 1(m/z) (min)

Product 2(m/z)

Collision Collision RF Energy Energy Lens 1(V) 2(V) (V)

N-acetylmannosamine

1.08

Positive

222.64

191.07

207.07

28.30

15.76

55

Lactose

1.32

Positive

361.03

342.97

352.03

10.25

10.86

84

Phenylalanine

1.86

Positive

166.15

103.07

120.11

27.65

13.79

54

Betaine

2.04

Positive

118.15

58.17

59.17

23.40

19

80

Valine

2.1

Positive

118.15

72.11

100.11

13.69

13.39

56

Creatine

2.36

Positive

132.12

86.11

114.11

10.25

10.25

39

Inosine 5'triphosphate

2.66

Positive

508.94

262.95

344.94

24.61

10.25

89

Cytidine 5'diphosphocholine

3.2

Positive

490.18

264.90

379

25.93

13.03

103



424

Figure captions:

425

Figure 1. A. The schematic of workflow used in this study. B. Inhibitory effect of BTE treatment

426

to E. coli with or w/o Lactobacillus acidophilus (LA) fermentation. C05, C10, and C25 stand for

427

different concentrations of BTE treatment without LA fermentation (BTE extracted from 5

428

mg/mL; 10 mg/mL and 25 mg/mL dry leaves, respectively); D05, D10 and D25 stand for

429

different concentrations of fermented BTE (extracted from 5 mg/mL; 10 mg/mL and 25 mg/mL,

430

respectively, then fermented with LA). All treatment groups were compared to an untreated

431

control group (UN). * p

A metabolomics study reveals enhanced inhibition and metabolic

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