Food Chemistry 270 (2019) 123–129

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Food Chemistry

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Short communication Antimicrobial and antioxidant activities of phenolic metabolites from flavonoid-producing yeast: Potential as natural food preservatives T ⁎ Kuan Rei Ng, Xiaomei Lyu, Rita Mark, Wei Ning Chen

School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore 637459, Singapore

ARTICLE INFO ABSTRACT

Keywords: We analysed the antimicrobial and antioxidant activities of phenolic metabolites secreted from a naringenin- producing Saccharomyces cerevisiae strain (a GRAS organism), against the pure flavonoid naringenin and its Food preservatives prenylated derivatives, to assess their potential as natural food preservatives. Agar disc diffusion assay was used Yeast to analyse the antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, Antimicrobial activity while DMPD% chemiluminescence assay was used to analyse antioxidant activity, based on DMPD%+-scavenging Antioxidant activity activity. Our results showed that the engineered yeast metabolites exhibited both strong antimicrobial and DMPD%+-scavenging activity, particularly the metabolite phenylacetaldehyde. Pure naringenin had poor anti- microbial and DMPD%+-scavenging effects. Prenylated varieties, 6-prenylnaringenin and 8-prenylnaringenin, inhibited only S. aureus, while only 8-prenylnaringenin exhibited moderate DMPD%+-scavenging activity. Our results suggested that phenolic metabolites secreted from naringenin-producing yeast would be a sustainable source of natural food preservatives.

1. Introduction downstream ancillary enzymes compartmentalized in plastids. There is a tremendous and still-growing body of literature on the significant Food preservatives are substances added to food to slow or prevent health benefits of flavonoids, spanning decades. These include anti- food spoilage caused by microbes or oxidation. Modern food pre- oxidant, anti-inflammatory, anticancer, antimicrobial and antidiabetic servatives are usually synthetic chemicals, such as sorbates, benzoates, properties (Araya-Cloutier, Vincken, van Ederen, den Besten, & nitrates and nitrites (Russell, 1991; Silva & Lidon, 2016). However, Gruppen, 2018; Falcone Ferreyra, Rius, & Casati, 2012; Sharma, Gupta, more research in recent times have begun to shed light on the non- Sarethy, Dang, & Gabrani, 2012; Sun et al., 2017; Tunon, Garcia- negligible health risks of consuming these synthetic food additives even Mediavilla, Sanchez-Campos, & Gonzalez-Gallego, 2009; Venturelli below recommended limits as defined by regulatory agencies, such as et al., 2016). the FDA. These health risks include allergic reactions, gastrointestinal The widespread occurrence of flavonoids in plants naturally extends disorders and cancer (Etemadi et al., 2017; Varraso & Camargo, 2014). to the human diet as well. Daily total consumption of flavonoids may There is therefore much ongoing interest in more “natural” sources of exceed 1 g (Zamora-Ros et al., 2016), with major contributing sources food preservatives, which can be plant extracts, essential oils or purified being fruits and vegetables, and beverages, such as tea, cocoa, wine and secondary metabolites (Carocho, Barreiro, Morales, & Ferreira, 2014; beer. Commercial applications of flavonoids include food additives, Gassara, Kouassi, Brar, & Belkacemi, 2016). nutraceuticals, pharmaceuticals, cosmetics and others. Global market Flavonoids, belonging to the phenolic class of plant secondary me- and demand for flavonoids was valued at over 840 million USD in 2015 tabolites, are distinguished from the other phenolics by their 15-carbon, and is expected to surpass 1 trillion USD beyond 2020 (https://www.

C6-C3-C6 (aryl-propyl-aryl) structural backbone. They are primarily zionmarketresearch.com/news/global-flavonoids-market). Currently, responsible for the vivid colours of flowering plants. Aside from colour options for large-scale commercial production of flavonoids for nu- pigments, flavonoids are typically produced in plants in response to traceutical, pharmaceutical and functional food applications are plant environmental changes, usually as a defence mechanism, e.g. UV ex- extraction and chemical synthesis. Plant extraction remains the default posure, pathogenic invasion (Winkel-Shirley, 2001). The flavonoid method, but carries significant downsides of high cost and significant biosynthetic pathway occurs mainly in the plant cell cytosol, with some carbon footprint, due to excessive energy and solvent requirements

⁎ Corresponding author. E-mail addresses: [email protected] (K.R. Ng), [email protected] (X. Lyu), [email protected] (R. Mark), [email protected] (W.N. Chen). https://doi.org/10.1016/j.foodchem.2018.07.077 Received 20 April 2018; Received in revised form 28 June 2018; Accepted 11 July 2018 0308-8146/ © 2018 Elsevier Ltd. All rights reserved. K.R. Ng et al. Food Chemistry 270 (2019) 123–129

(Guo-qing et al., 2005). Chemical syntheses have similar disadvantages, overnight seed culture was then inoculated into 50 ml fresh YPSG (1% with the additional hurdle of poor product stereoselectivity (De Luca, sucrose-1% glycerol) media to an initial OD600 of 0.05 and incubated Salim, Atsumi, & Yu, 2012). Microbial biofermentation of metabolically under the same conditions for another 72 h. engineered microbes has been touted as a superior alternative to both methods and has garnered increasing attention in recent times, due to 2.3. Extraction, HPLC and LC–MS analysis of samples several reasons. Firstly, the lower complexity of culture suspensions relative to solid plant matrices enables easier and less costly separation After completion of the shake flask cultures, the supernatants were techniques with lower carbon footprint (Ameer, Shahbaz, & Kwon, harvested for phenolic extraction and quantification. Both engineered 2017; Azmir et al., 2013). Secondly, plant extraction production levels (N2) and BY4741 negative control (WT) Saccharomyces cerevisiae cul- are gated by plant supply, which may be highly seasonal depending on tures were processed as follows: firstly, centrifugation was performed at the specific crop, whereas microbial production has no such seasonal 10,000 rpm to separate and remove the cell pellet. An equal volume of reliance owing to very short life cycles in the metric of days, and also ethyl acetate was then added to the supernatant (1:1 aqueous:organic only requiring the initial input of simple feedstock, such as complex solvent), vortexed vigorously for 30 s, and rotated at room temperature nutrient media with simple carbon sources, e.g. glucose and oxygen overnight. After another centrifugation at 10,000 rpm for 10 min, the and/or carbon dioxide, and will therefore be more reliable in meeting upper organic layer was collected and 500 μl set aside and microfiltered production demand. Thirdly, the established knowledge of common (0.45 µm) for HPLC analysis and quantification. The rest of the ethyl microbial workhorses, such as Escherichia coli and Saccharomyces cere- acetate extract was evaporated to dryness, leaving behind a con- visiae, plus the plethora of bioengineering tools at our disposal currently centrated extract residue. Quantification of naringenin in extracts was allows for high customizability, versatility and targetability, from in performed through HPLC (Agilent 1100) equipped with a variable silico design to production of target compounds. Plants in contrast, wavelength detector and C18 column (4.6 mm × 150 mm, RESTEK). accumulate these different secondary metabolites to highly variable Samples were analysed using a gradient method. The program started extents, usually yielding a range of flavonoids and their derivatives. with 25% of solvent A (methanol) and 75% of solvent B (water). The Moreover, high-resolution separation of a complex mixture of such concentration of A subsequently increased to 75% within 10 min, closely related compounds poses further challenges and cost. Therefore, continued up to 100% at 20 min, and then was held for 10 min. The whole plants are less “production-efficient” when compared to micro- solvent was returned to 25% A over 2 min and held for 13 min. The flow bial cell platforms. Lastly, engineered microbial cell factories allow rate was 0.5 ml/min, and the signal was detected at 280 nm. A pre- waste valorization (Ong, Kaur, Pensupa, Uisan, & Lin, 2017; Widsten, established calibration curve using naringenin standards was used for Cruz, Fletcher, Pajak, & McGhie, 2014), which further cheapens pro- quantification. After HPLC analysis and quantification, the extract re- duction while promoting a perfectly circular economy. sidue was re-dissolved in an appropriate amount of 100% ethanol to Plant extracts containing flavonoids, such as naringenin, have re- obtain the equivalent of 40 mg/ml concentration of naringenin in portedly demonstrated antimicrobial activities (Mandalari et al., 2007; ethanol, which served as a stock solution for use in further assays. The Nowak, Czyzowska, Efenberger, & Krala, 2016; Rauha et al., 2000). BY4741 wild-type culture extract (WT) – containing no flavonoids or Allied with their inherently potent nutraceutical benefits, the use of phenolics – was prepared identically to the N2 extract as a negative extracts containing flavonoids as novel food preservatives would control. therefore be highly desirable and profitable. Identification of other phenolic metabolites in extracts was per- The objective of our study was to investigate the antimicrobial and formed through LCMS. Samples were analysed with an Agilent 6550 antioxidant effects of microbial phenolic compounds from naringenin- iFunnel Q-TOF LC–MS using an injection volume of 2 µl. Samples were producing S. cerevisiae (N2) and compare that against the bioactivities separated on an Agilent Zorbax SB-C18 column (2.1 × 100 mm, 1.8 µm) of pure flavonoids naringenin (NAR) and its prenylated derivatives 6- with the guard column at 30 °C. The mobile phases consisted of (A) prenylnaringenin (6PN) and 8-prenylnaringenin (8PN), for evaluation water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid. The of their potential as natural substitutes for synthetic food preservatives. separation was performed using the following gradient: 0 min at 5% B, 8–10 min at 95% B, 10.1–13 min at 5% B at a flowrate of 0.2 ml/min. 2. Materials and methods Analysis was done using an Agilent Jet Stream dual electrospray in ionization negative mode, with drying gas temperature of 200 °C, 2.1. Reagents, media and strains drying gas flow of 14 l/min, nebulizer pressure of 35 psig, sheath gas temperature of 350 °C and sheath gas flow of 11 l/min. MS Scan was in Naringenin-producer Saccharomyces cerevisiae strain Y26 (herein the range of m/z 100–800. A reference solution containing mass renamed N2) was engineered based on our previous work (Lyu, Ng, Lee, 112.985587 was constantly infused as an accurate mass reference. Mark, & Chen, 2017). Escherichia coli ATCC 25922, Staphylococcus Chromatogram and MS results were subject to METLIN database search aureus ATCC 29213 and S. cerevisiae BY4741 (wild-type) were kindly (http://metlin.scripps.edu). Metabolites were singled out based on provided by the School of Chemical and Biomedical Engineering, Na- significant peak height, area and score of > 75. Compound hits that did nyang Technological University, Singapore. 6-prenylnaringenin and 8- not meet designated criteria were excluded. Hits that were not unique prenylnaringenin (both 99%) were purchased from BOC Sciences, USA. to N2 (present in both WT control and N2) were also excluded. The phenolics naringenin, phloretic acid, phenylacetaldehyde and homogentisic acid were procured from Sigma Aldrich, USA. N,N-Di- 2.4. Antimicrobial assay (agar disc diffusion) methyl-p-phenylenediamine dihydrochloride (DMPD), sodium acetate trihydrate and ferric chloride hexahydrate were also procured from The antimicrobial activity of the extract was surveyed by employing Sigma Aldrich, USA. Luria-Bertani (LB) broth formula, ethyl acetate the disc diffusion assay, to determine the inhibition zones of all samples (HPLC-grade) and ethyl alcohol (> 99%) were procured from Sigma against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC Aldrich, USA. Mueller-Hinton Broth formula was purchased from Fisher 29213 separately. Each strain was first streaked on fresh LB agar plates Scientific, USA. and incubated at 37 °C overnight. From the overnight plate, individual colonies were then re-suspended in 1 ml Mueller-Hinton (MH) broth

2.2. Shake-flask cultivation of yeast such that the OD600 value approximated 0.5 McFarland standard, cor- responding to approximately 1–3×108 CFU/ml. A sterile cotton swab Strains N2 and BY4741 wild-type were first separately cultured was used to streak-inoculate each fresh MH agar plate. N2 yeast extract overnight in 5 ml of YPD medium at 30 °C with shaking (200 rpm). This stock solution (40 mg/ml naringenin equivalent in ethanol), wild-type

124 K.R. Ng et al. Food Chemistry 270 (2019) 123–129

Fig. 1. LC–MS analysis for characterization of flavonoid metabolites in microbial phenolic extract. Top chart represents isolated chromatogram scan; bottom chart represents mass spectrum, negative ion mode at isolated retention period A. Naringenin (m/z 271) and phenylacetaldehyde (m/z 119). B. Phloretic acid (m/z 165).C. Homogentisic acid (m/z 167). yeast control and pure phenolic stock solutions (40 mg/ml each, in were done in triplicate. ethanol) were prepared. Sterile filter paper discs of 6 mm impregnated with varying amounts (400, 200, 100, 50 μg per disc) of N2 extract, WT 2.5. Preparation and measurement of DMPD%+ chemiluminescence assay extract and pure phenolic compounds were then placed on the in- oculated plates. Ampicillin antibiotic and 100% ethanol discs were also DMPD%+ scavenging assay was performed to evaluate the anti- placed as positive and negative controls, respectively. Thereafter, all oxidant activity of the phenolics, based on the protocol of Fogliano, inoculated plates with discs were incubated at 37 °C for 18 h. The an- Verde, Randazzo and Ritieni (Fogliano, Verde, Randazzo, & Ritieni, timicrobial activity was determined by measuring the zone of inhibition 1999) with some modifications. DMPD%+ (100 mM) was prepared by (mm) on the agar plates, less filter paper disc (6 mm). All measurements dissolving 20.9 mg of DMPD in 1 ml of deionized water; 500 µl of this

125 K.R. Ng et al. Food Chemistry 270 (2019) 123–129 solution was added to 50 ml of acetate buffer (0.1 M, pH 5.25), and the Table 1 coloured radical cation (DMPD%+) was obtained by adding 0.1 ml of a Major phenolics detected via LC–MS analysis and METLIN database search. solution of 0.05 M ferric chloride (final concentration 0.1 mM). 1 ml of Metabolites were singled out based on significant peak height, area and score this pink DMPD%+ radical solution blank was directly placed in a 1 ml of > 75. All compounds were not detected in wild-type S. cerevisiae extract. quartz cuvette and then its absorbance was measured at 514 nm in a Compound Formula [M-H]- RT (min) Score (based on METLIN spectrophotometer (NanoDrop 2000). An optical density of 0.950 ab- Database) sorbance units was initially obtained, which on dilution with ethanol Naringenin C H O 271 5.825 84.73 and stirring for 10 min decreased to 0.525 ± 0.020. Dilution and 15 12 5 Homogentisic acid C8H8O4 167 5.692 99.29 stirring was done to simulate experimental conditions for samples. Phloretic acid C9H10O3 165 4.25 87.36 Standard stock solution of 1.25 mg/ml of Trolox was prepared by Phenylacetaldehyde C8H8O 119 5.825 99.57 dissolving 125 mg of Trolox in 100 ml deionized water, then diluted down to a concentration range of 80 µg/ml to 1 µg/ml for establishing the Trolox calibration curve (% DMPD%+ signal inhibition against coloration of the concentrated extracts (Jones, 1976; Tieman et al., Trolox). From 40 mg/ml standard solutions of pure phenolic com- 2006; Whistance & Threlfall, 1970). pounds, N2 extract and wild-type yeast extract, first a hundred-fold dilution was performed to obtain 400 µg/ml, then subsequent two-fold 3.3. Agar disc diffusion results demonstrate antimicrobial activity of N2 and serial dilutions down to minimum 25 µg/ml concentration for assay use. metabolite PHA Each final assay sample consisted of 150 µl Trolox or phenolics, mixed with 850 µl DMPD%+ solution, making up total volume of 1 ml. After N2 showed broad-spectrum antimicrobial activity at amounts of up 10 min incubation with stirring, absorbances of test samples were to 400 µg, against both Gram-negative E. coli and Gram-positive S. measured at 514 nm. The DMPD%+ signal inhibition for each sample aureus, with inhibitions zones of 4 mm and 8.33 mm, respectively (all was expressed as follows in Eq. (1): results shown in Figs. 2 and 3). Naringenin produced no zones of in- hibition against either strain at these concentrations, while prenylated ⎛ As ⎞ flavonoids 6PN and 8PN only inhibited S. aureus, with inhibition zones %Signal inhibition = ⎜⎟1− × 100 0 fi ⎝ A ⎠ (1) of up to 4 mm and 7.67 mm, respectively. For the identi ed N2 meta- bolites, both phloretic acid and homogentisic acid demonstrated neg- where A0 is the absorbance of the stirred diluted blank, and As is the ligible antimicrobial activity, while pure phenylacetaldehyde exhibited absorbance of sample containing either Trolox or phenolics. The DMPD significant broad spectrum activity (9.33 mm and 10.33 mm). The an- %+ scavenging ability of phenolics were converted to Trolox equivalent timicrobial effect of N2 is speculated to be largely attributable to phe- antioxidant capacity (TEAC) (µg/ml Trolox), calculated from the Trolox nylacetaldehyde from this evidence. Negative control WT demonstrated 2 calibration curve, determined by linear regression in Eq. (2) (r : no antimicrobial activity, indicating that the antimicrobial effects of N2 0.9916): are solely attributable to the engineered biosynthetic pathway meta- (%Signal inhibition− 7.5276) bolites produced in strain N2. While positive ampicillin control pro- TEAC(g/mlTrolox) μ = fi 3.9811 (2) duced zones of inhibition dwar ng all other samples, it is generally accepted knowledge that an efficacious food preservative does not yield All measurements were performed in minimum of duplicates. antibiotic-levels of antimicrobial activity at comparable concentrations. The antimicrobial activities of naringenin, 6-prenylnaringenin, 8-pre- 3. Results and discussion nylnaringenin and phenylacetaldehyde have previously been studied (Barreca, Bellocco, Laganà, Ginestra, & Bisignano, 2014; Jay & Rivers, 3.1. Naringenin in phenolic extract of engineered S. cerevisiae 1984; Sánchez-Maldonado, Schieber, & Gänzle, 2011) Our results showed some differences: 8PN appears to demonstrate superior activity In our study, the N2 strain used is capable of producing more than over 6PN (Mizobuchi & Sato, 1984), while phenylacetaldehyde shows 20 mg/l naringenin extracellularly from a sucrose-glycerol carbon strong antimicrobial effects, the highest of all compounds tested here source (Lyu et al., 2017), detailed in our previous work. N2 and wild- (Zhu et al., 2011). type BY4741 (WT) yeast strains were cultured and extracted via ethyl fi + acetate liquid-liquid extraction (LLE), then redissolved to form a nal 3.4. DMPD% scavenging assay shows strong antioxidant activity of N2 ethanolic extract. and PHA

3.2. LC–MS analysis identified phenylacetaldehyde, phloretic acid and The DMPD%+ signal-Trolox calibration curve was first established homogentisic acid as metabolites (Fig. 4), and then the DMPD%+ signal inhibition of phenolic samples – at various concentrations – was then converted to Trolox equivalent LC–MS analysis of N2 was performed for further characterization of antioxidant capacity (TEAC) (Fig. 5) units based off the calibration its phenolic components. LC–MS results were compared against WT, curve. Only N2, 8PN, and the N2 metabolites HMA and PHA demon- and both cross-referenced with the METLIN database. Three other strated any significant dose-dependent antioxidant activity, with max- major phenolic metabolites were identified apart from naringenin: imums of 11.4, 6.3, 13.5 and 18.2 µg/ml Trolox, respectively. The an- Phloretic acid, homogentisic acid and phenylacetaldehyde (Fig. 1, tioxidant activity of HMA, a key phenolic of strawberry tree honey Table 1). Our preliminary quantification results indicated that nar- known for strong antioxidant activities, was previously reported by ingenin, phloretic acid and phenylacetaldehyde were detected at Rosa et al. (2011), while that of phenylacetaldehyde was characterised 25 mg/l, 207 mg/l and 18.5 mg/l, respectively. These phenolics are by Nam et al. (Nam, Jang, & Shibamoto, 2012). Compared with the associated with aromatic amino acid catabolism function as plant sec- (prenylated) flavonoids, the increased hydrophilicity of HMA and PHA ondary metabolites and are known food-grade compounds. Phenyla- could be one explanation for increased direct DMPD%+-scavenging cetaldehyde is a common food additive for fragrance, and forms strong activity due to the nature of the assay. Naringenin demonstrated neg- adducts with naringenin (Cheng et al., 2008). Phloretic acid is a me- ligible and relatively invariant direct DMPD%+-scavenging activity at tabolite of apple tree, while homogentisic acid is found in strawberry all concentrations used, and 6PN even demonstrated slight pro-oxidant tree honey, which is highly prized for its antioxidant content (Rosa effects at high concentrations, based on the negative slope obtained and et al., 2011). Homogentisic acid may also explain the dark brown TEAC value decreasing below 0. The varying antioxidant effects of

126 K.R. Ng et al. Food Chemistry 270 (2019) 123–129

25

20

15

ti on (mm) N2 10 PHA Amp 5 Zone of Inhibi

0 50 100 200 400

-5 Disc amount (μg)

Fig. 2. Agar disc diffusion assay results for E. coli ATCC 25922. Each data point is the triplicate mean, vertical bars represent standard deviation. Only N2 extract (N2), phenylacetaldehyde (PHA) and ampicillin (Amp) showed inhibitory activity.

25 Fig. 3. Agar disc diffusion assay results for S. aureus ATCC 29213. Each data point is the tri- plicate mean, vertical bars represent standard deviation. All samples except naringenin showed 20 activity at least at maximal amount tested (400 µg). PLA: Phloretic acid. HMA: Homogentisic acid. WT: Wild-type yeast nega- PLA 15 tive control. N2: N2 extract. PHA: HMA Phenylacetaldehyde. 6PN: 6-prenylnaringenin. 8PN: 8-prenylnaringenin. Amp: Ampicillin. WT ti on (mm) 10 N2 PHA 6PN 5 Zone of Inhibi 8PN Amp 0 50 100 200 400

-5 Disc amount (μg)

NAR, 8PN and 6PN, via an in vitro human low-density lipoprotein (LDL) 4. Conclusions assay, was previously reported (Miranda et al., 2000) and our results are largely in agreement: NAR, 6PN and 8PN actually demonstrate Our results show that the phenolic compounds from our engineered weak antioxidant effects. Many earlier studies have consistently re- Saccharomyces cerevisiae strain N2, exhibited good, broad-spectrum ported flavonoids, especially prenylated derivatives, such as 8PN and antibacterial activity surpassing all other pure flavonoids and phenolics 6PN, to be good to excellent antioxidants. However, the exact me- tested in this study, bar phenylacetaldehyde. Strong antioxidant activity chanisms of their antioxidant activity and the structure-activity re- – via a direct DMPD%+-scavenging mechanism – was also detected. lationships, remain to be clearly established. Based on our data, the Unknown synergistic effects between these phenolics may explain the differing prenylation positions of 6PN (pro-oxidant) versus 8PN (anti- enhanced bioactivities compared to the pure compounds. Therefore, oxidant), resulted in opposite effects and merits further investigation. complex bio-derived mixtures or extracts may inherently exhibit better Our results present more evidence suggesting indirect antioxidant me- broad-spectrum antimicrobial activity when compared to singular chanisms, rather than direct radical scavenging, may be the dominant compounds. Moving forward, combinatorial testing of these compounds mode of antioxidant activity, such as cell modulatory effects (Williams, is recommended to isolate and identify any such synergistic relation- Spencer, & Rice-Evans, 2004). Validation using other colorimetric assay ships, and also establish structure-activity relationships. methods (ABTS, DPPH), in our future investigation may provide new To be an effective food preservative replacement, candidate com- insights on the antioxidant or pro-oxidant activities of these phenolics pounds and extracts should demonstrate moderate to strong anti- and metabolites. microbial activity against common food pathogens (e.g. Campylobacter sp., Salmonella sp., S. aureus, E. coli, Listeria monocytogenes, C. botu- linum). Antimicrobial assays of this extract against more of these

127 K.R. Ng et al. Food Chemistry 270 (2019) 123–129

90 80 y = 3.9811x + 7.5276 R² = 0.9916 70 60 ti on 50 40 30 % signal inhibi % signal 20 10 0 0 5 10 15 20 Trolox (ȝg/ml)

Fig. 4. Trolox calibration curve, of DMPD%+ % signal inhibition against range of Trolox concentrations (1–80 µg/ml).

25 Fig. 5. DMPD%+ % scavenging ability (expressed as TEAC) of yeast extract and pure phenolics over a 20 range of concentrations. Each data point is the du- plicate mean, vertical bars represent standard de- 15 viation N2: N2 yeast extract. WT: Wild-type S. cer- evisiae extract as negative control. NAR: Naringenin. 6PN: 6-prenylnaringenin. 8PN: 8-prenylnaringenin. 10 PHA: Phenylacetaldehyde. HMA: Homogentisic

g/ml Trolox) acid. PLA: Phloretic acid.

μ 5

TEAC ( 0 0 10203040506070 -5

-10 Phenolics Concentration (μg/ml)

N2 WT NAR 6PN 8PN PHA HMA PLA pathogens should be conducted in future work. Mammalian cell assays Funding to ascertain cytotoxicity and therefore, human safety of the extract would also be necessary to prove its true viability. This work was supported by Nanyang Technological University In this work, we have demonstrated that microbial phenolic com- Singapore (iFood Research grant). We thank Nanyang Technological pounds from metabolically engineered, naringenin-producing S. cere- University, and Singapore International Graduate Award (SINGA) for visiae exhibited both antimicrobial and antioxidant activity surpassing scholarships (KR Ng and R Mark respectively). that of pure flavonoids. Key metabolites phenylacetaldehyde, homo- gentisic acid and phloretic acid were identified, likely responsible for Conflict of interest the enhanced bioactivities. These microbial phenolic compounds were derived from simple carbon sources and relatively low-cost LLE of a The authors declare no competing financial interest. generally regarded as safe (GRAS) microbial culture. Our work sug- gested a promising process, of biofermentation using metabolically Appendix A. Supplementary data engineered GRAS microbes, to replace large-scale plant extractions. This in turn could provide a cost-effective and sustainable source of Supplementary data associated with this article can be found, in the natural food preservatives in the future. online version, at https://doi.org/10.1016/j.foodchem.2018.07.077.

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