Eur Food Res Technol (2012) 234:535–542 DOI 10.1007/s00217-012-1669-z
ORIGINAL PAPER
Sinapic acid derivatives in defatted Oriental mustard (Brassica juncea L.) seed meal extracts using UHPLC-DAD-ESI-MSn and identiWcation of compounds with antibacterial activity Christina Engels · Andreas Schieber · Michael G. Gänzle
Received: 4 November 2011 / Revised: 2 January 2012 / Accepted: 3 January 2012 / Published online: 20 January 2012 © Springer-Verlag 2012
Abstract This study identiWed phenolic compounds from mustard seed meal and characterized their antibacterial activity. Phenolic compounds were extracted from defatted Oriental mustard (Brassica juncea L.) seed meal and characterized using ultra-high-performance liquid chromatography with diode array and electrospray ionization-mass spectrometric detection (UHPLC-DAD-ESI-MSn). Sinapic acid and several sinapoyl conjugates were identiWed based on retention time, UV spectra, MS fragmentation pattern, and by comparison with the authentic sinapic acid reference substance. The crude extract and a puriWed phenolic fraction exhibited selective antibacterial eVects against Gramnegative and Gram-positive spoilage bacteria including Staphylococcus aureus and Listeria monocytogenes; Lactobacillus plantarum was resistant. After alkaline hydrolysis, only sinapic acid could be detected, enabling quantiWcation with the authentic reference substance. Alkaline hydrolysis released 2.66 § 0.00 mg sinapic acid g¡1 dry matter defatted mustard seed meal. Minimum inhibitory concentrations of the hydrolyzed extract against Bacillus subtilis, Escherichia coli, L. monocytogenes, Pseudomonas Xuorescens, and S. aureus were 0.1 g L¡1 or less. Growth of L. plantarum remained unaVected. Sinapic acid and sinapoyl esters are generally found in members of the Brassicaceae family.
C. Engels · A. Schieber · M. G. Gänzle (&) Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton, AB T6G 2P5Canada e-mail:
[email protected] Present Address: A. Schieber Institut für Universität Bonn, Institut für Ernährungs- und Lebensmittelwissenschaften, Universität Bonn, Römerstrasse 16, 53117 Bonn, Germany
Methods for their fast identiWcation will be useful in chemotaxonomic studies. The release of sinapic acid after alkaline hydrolysis not only allows for the quantiWcation using the reference substances but also facilitates the standardization of the antibacterial activity of plant extracts for use as food preservative. Keywords Mustard seeds · Sinapic acid · Antibacterial phenolic compounds · Lactobacillus · Escherichia coli · UHPLC
Introduction Plant-derived natural antimicrobials are used as food preservatives. The application of plant extracts with antimicrobial activity as food preservatives is currently hampered by the limited knowledge on the contribution of individual compounds to the preservative eVect and diYculties regarding their characterization due to the lack of authentic reference substances. It thus remains challenging to develop optimized low-dose formulations that warrant product safety and storage life without undesirable sensory changes associated with the addition of high concentrations of botanical extracts [35]. Mustard includes black mustard (Brassica nigra L.), Oriental, brown or Indian mustard (B. juncea L.), and white/yellow mustard (Sinapis alba/B. hirta), all belonging to the Brassicaceae family (formerly called Cruciferae). Members of this family bear characteristic Xowers with four petals reminiscent of a cross. Other cruciferous plants include cabbage, broccoli, and cauliXower (all belonging to the species B. oleracea L.), and rapeseed (B. napus L. var. oleifera). Mustard seeds, leaves, and stems are edible. The seeds are used whole as a spice or prepared as a condiment.
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Mustard oil extracted from the seeds is high in omega-3 fatty acids. Mustard seeds contain several secondary metabolites such as glucosinolates. Glucosinolates are organic compounds that contain nitrogen and sulfur and occur in almost all plants of the Brassicaceae family [24]. Their corresponding hydrolytic products, isothiocyanates, are also known as mustard oil. Isothiocyanates have antibacterial activities against several bacterial strains including pathogens [1, 15]. They are diYcult to handle because they are volatile and odorous. The proWle of anthocyanins, Xavonol glycosides, and phenolic acid derivatives in red mustard greens was recently characterized in detail [14]. Hydroxycinnamic acids in mustard exhibit antimicrobial activity [31–33]. This may allow the utilization of mustard seed meal, a byproduct of mustard oil reWning, for food applications. The composition of phenolic compounds in mustard seeds, however, remains poorly characterized [33, 34]. It was, therefore, the aim of this study to characterize phenolic compounds in mustard seed meal extracts and to optimize their extraction and conversion to achieve a maximum yield of bioactive compounds. A fast and selective method for the analysis of phenolic compounds using ultra-high-performance liquid chromatography-diode array detection-electrospray ionization-mass spectrometry (UHPLC-DAD-ESI-MSn) was developed. Using this method, changes in phenolic composition when subjected to alkaline hydrolysis were investigated and subsequent alterations in antibacterial activity were determined.
Materials and methods Reference substances and chemicals The sinapic acid standard was purchased from Extrasynthèse (Genay, France) and dissolved in methanol. All solvents including water were of HPLC grade and obtained from Fisher ScientiWc (Fair Lawn, NJ, USA). Plant material, extraction, and puriWcation Defatted, coarsely ground Oriental mustard seed meal (Brassica juncea) was obtained from Biofume Technologies Inc. (Saskatoon, SK, Canada), and aliquots of 25 g were Wnely ground for 30 s in a coVee grinder. Past studies indicated that phenolic compounds from mustard are soluble in aqueous methanol or acidiWed aqueous methanol [14, 17, 34]; ground mustard seed meal was thus successively extracted with 250 mL of acetone/0.1% formic acid (FA) (80:20, v/v) or 250 mL of methanol/0.1% formic acid (FA) (80:20, v/v), respectively. The suspension was sonicated
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and stirred for 30 min, respectively, at ambient temperature in amber bottles Xushed with nitrogen. After Wltration (15-m Wlter, Whatman Inc., Clifton, NJ, USA), the extraction step was repeated. The organic solvent was removed in vacuo at 30 °C, and the remaining aqueous solution was made up to 100 mL with water (0.1% FA) and used for further puriWcation using polyamide columns (2 g CC6, Sigma-Aldrich, St. Louis, MO, USA). The columns were successively conditioned with 25 mL methanol containing 0.1% FA and 50 mL of 0.1% FA prior to the application of an aliquot of the crude extract. The polyphenolic fraction was recovered with methanol (100 mL, 0.1% FA) after washing with water (50 mL, 0.1% FA). The eluate was evaporated to dryness, and the residue was dissolved in 30 mL methanol (0.1% FA). Alkaline hydrolysis of esteriWed phenolic compounds Alkaline hydrolysis was performed to hydrolyze esters of phenolic compounds [12, 17, 20]. A 3 mL aliquot of the polyphenol fraction was added to 3 mL of distilled water and 1.5 mL of 10 M NaOH. The sample was then Xushed with nitrogen and stirred for 4 h at ambient temperature. The solution was adjusted to pH 2 using 6 M HCl prior to the extraction of phenolic compounds with ethyl acetate. Ethyl acetate extracts of repeated steps were combined, all solvents were evaporated, and the residues were re-dissolved in methanol for LC-MS analysis. UHPLC-DAD-ESI-MSn analysis and quantiWcation of the sinapic acid content LC-MS analysis was performed using a Prominence UFLCXR system (Shimadzu, Kyoto, Japan) equipped with a degasser, two solvent delivery modules, a thermoautosampler, a column oven, and a diode array detector. UV–vis spectra were recorded from 190 to 800 nm (frequency 40 Hz). The components were separated using a 100 £ 3.0 mm Kinetex 2.6 m, 100 Å, C18 column (Phenomenex, Torrance, CA, USA) with a Krudkatcher ultra column in-line Wlter and operated at 21 °C. The injection volume was 5 L for the mustard extracts and 10 L for the reference substance (0.1 g L¡1). The compounds were eluted with 0.1% (v/v) formic acid in water (eluent A) and 0.1% formic acid in an acetonitrile/water mixture (90:10, v/v; eluent B) at a Xow rate of 1.0 mL min¡1. The gradient program was as follows: 0–15% B (0.75 min), 15–40% B (15.25 min), 40–100% B (0.5 min), 100% B (0.5 min), and 100–0% B (1 min). The LC system was connected to a 4,000 QTrap mass spectrometer Wtted with an ESI source (Applied Biosystems, Streetsville, ON, Canada). Data acquisition and processing were performed using Analyst version 1.5. Mass spectrometric data were obtained in the
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Table 1 Strains and culture conditions Organisms
Origin (references)
Growth conditions
Bacillus subtilis FAD 110
Ropy bread [29] LB, aerobic, 37 °C
Escherichia coli AW 1.7
Beef [2]
LB, aerobic, 37 °C
Listeria innocua ATCC 33090
LB, aerobic, 30 °C
Listeria monocytogenes ATCC 7644
LB, aerobic, 30 °C
Pseudomonas Xuorescens ATCC 13525
LB, aerobic, 30 °C
Staphylococcus aureus ATCC 6538
LB, aerobic, 30 °C
Lactobacillus plantarum TMW 1.460
indicator strains to a cell count of about 107 cfu mL¡1 and incubated overnight. MICs were deWned as the lowest concentrations of the substances that inhibited the growth of microbial strains. Turbidity caused by bacterial growth was determined at 630 nm using a microplate reader (MultiSkan Ascent, Thermo Fisher ScientiWc Inc., Nepean, ON, Canada). The MICs of the crude extract and of the puriWed polyphenol fraction were calculated on the basis of the weight of mustard seed meal from which the active concentrations of the fraction was obtained. The MICs of the polyphenol fraction after hydrolysis and the reference substance sinapic acid were expressed as concentrations of sinapic acid. The experiments were performed in triplicate. Controls containing pure methanol were tested in the same manner.
Spoiled beer [11] MRS, anaerobic, 30 °C
enhanced MS (EMS), enhanced product ion (EPI), precursor ion scan modes, and MS3. Negative ion mass spectra were recorded in the range of m/z 50–1,300 at a scan speed of 4,000 Da s¡1 at a voltage of ¡4,000 V. Nitrogen was used as the gas source. The ion source temperature was set at 600 °C, the declustering potential was set at ¡70 V, and the collision energy (CE) varied from ¡10 to ¡50 V. MS3 was performed at an excitation energy (AF2) of 100 AU. Phenolic components were identiWed based on their retention times, UV–vis spectra, and fragmentation pattern. The sinapic acid content was quantiWed based on a calibration curve with the reference substance using appropriate dilutions. QuantiWcation was performed in duplicate, and results are shown as means § standard deviations. Bacterial strains, media, and culture conditions Bacterial strains used in this study as well as their origin and culture conditions are listed in Table 1. Strains were grown in Luria–Bertani (LB) broth (5 g L¡1 glucose, 5 g L¡1 yeast extract, and 5 g L¡1 NaCl) or de Man, Rogosa, Sharpe (MRS) broth (Becton) as indicated in Table 1. Determination of minimum inhibitory concentrations (MICs) The inhibitory activities of the crude extract, of the polyphenol fraction, of the solution after alkaline hydrolysis of this fraction, and of a sinapic acid solution (2 g L¡1 in methanol) were determined with indicator strains listed in Table 1. The MICs were determined using a critical dilution assay [10]. Methanol was completely evaporated in the air Xow of a sterile bench. Twofold serial dilutions of the solutions were inoculated with an overnight culture of
Results UHPLC-DAD-ESI-MSn analysis of phenolic compounds in the puriWed crude extract The phenolic composition of extracts from defatted mustard seed meal was investigated using a UHPLC method with diode array and mass spectrometric detection. Detection with diode array detection-electrospray ionizationmass spectrometry (DAD-ESI-MSn) in negative mode has been shown to be a powerful tool for the analysis of phenolics in botanical extracts including extracts from Brassicaceae species [9, 12, 14]. In this study, ESI-MS parameters were optimized for the components and the solvent system, respectively, to maximize the ionization eYciency. Phenolic compounds were separated within 18 min using ultrahigh-performance liquid chromatography. All compounds were assigned as sinapic acid or sinapic acid conjugates. Retention times, UV spectra, molecular masses, and main fragments of phenolic compounds in extracts using 80% acetone are listed in Table 2. To validate the qualitative composition of phenolic compounds with a diVerent extraction protocol, extracts obtained with aqueous methanol were additionally analyzed. The same proWle of phenolic compounds was observed after extraction with 80% methanol (data not shown). Compound 1 was the predominant substance in the polyphenolic fractions (Fig. 1) as well as in the crude extracts (data not shown). Based on the Wnding of ions at m/z 294, followed by the successive loss of two methyl groups (15 Da) resulting in m/z 279 and 264, respectively, and further fragmentation ions corresponding to sinapic acid (discussed below), compound 1 was tentatively characterized as sinapine, the choline ester of sinapic acid. While sinapine is often mentioned as the dominant phenolic compound in several brassicaceous species [18], information on
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Table 2 UV spectra and MS data of phenolic compounds in defatted mustard seed meal extracts No
1
tr (min) 2.83
2
3.27
m/z [M–H]¡
m/z MSn
DAD max (nm)
Identity
309a
MS2 [309] 294, 279, 264, 223, 208
977
238, 328
Sinapineb
2
238, 268, 329
Kaempferol-sinapoyl-trihexosideb
2
MS [977] 815, 609, 447, 285
3
5.48
223
MS [223] 208, 193, 179, 164, 149
238, 321
Sinapic acidc
4
8.38
385
MS2 [385] 225, 223 MS3 [385 ! 223] 225
240, 265, 328
Sinapoyl-hexosideb
5
10.80
753
MS2 [753] 529, 223, 208 MS3 [753 ! 529] 529, 223, 205, 208, 164
239, 329
Disinapoyl-dihexosideb
6
12.60
591
MS2 [591] 367, 223, 208, 205, 149 MS3 [591 ! 367] 367, 223, 205, 164, 149
239, 329
Disinapoyl-hexosideb
7
13.64
959
MS2 [959] 735, 529, 511, 223, 208, 205 MS3 [959 ! 735] 529
238, 325
Trisinapoyl-dihexosideb
8
13.84
–d
MS2 [223] 208, 164, 149
234, 287sh, 302sh, 321
Sinapoyl conjugate(s)b
223
MS2 [223] 208, 193, 179, 164, 149
236, 322
Sinapic acid
Std
5.47
Data of the authentic sinapic acid standard substance (std) compound are also included a b c d
Detected only in trace amounts Tentatively identiWed based on UV and mass spectrometric data Compounds identiWed by comparison with the authentic reference compound Several compounds were detected that all resulted in fragmentation ions corresponding to sinapic acid
Fig. 1 Separation of phenolic compounds (330 nm) from defatted mustard seed meal by ultra-high-performance liquid chromatography. Tentative peak assignment (see also Table 1): (1) sinapine, (2) kaempferol-sinapoyl-trihexoside, (3) sinapic acid, (4) sinapoyl-hexoside, (5) disinapoyl-dihexoside, (6) disinapoyl-hexoside, (7) trisinapoyl-dihexoside, and (8) sinapoyl conjugate(s)
the fragmentation pattern using mass spectrometry in negative mode is scarce. The fragmentation pattern detected in this study was in accordance with previous Wndings for phenolics in canola seed extracts [16]. Remarkably, the molecular [M–H]¡ ion at m/z 309 was detected only in traces, which can be explained by the fact that negative ionization was used and the choline derivative is a cation. Investigations on phenolic compounds from rapeseed demonstrate that positive ionization mode is more suitable for
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the detection of the molecular ion of sinapine (Lopez-Lutz and Schieber, unpublished observations). Compound 2 showed a [M–H]¡ at m/z 977. Ions corresponding to the loss of hexose (162 Da, m/z 815), sinapolyl-hexose (368 Da, m/z 609), and sinapoyl-dihexose (530 Da, m/z 447) moieties were detected, resulting in the aglycone at m/z 285, which is indicative of kaempferol. Therefore, compound 2 was tentatively identiWed as a kaempferol-sinapoyl-trihexoside. Due to its low concentration, no further fragmentation ions corresponding to kaempferol were detected. Mass spectrometric and UV data of compound 2 matched previous reports [9, 12, 21]. Sinapic acid (compound 3) was readily identiWed based on UV data, MS spectra, and by comparison with the corresponding reference substance. The successive loss of two methyl groups (15 Da) is characteristic for this hydroxycinnamic acid and resulted in m/z 208 and 193. Corresponding decarboxylated ions at m/z 164 and 149 were also detected. Additional compounds presenting the characteristic fragmentation pattern of sinapic acid were characterized as sinapate esters. Due to the loss of a monosaccharide moiety (162 Da) resulting in a fragment at m/z 223, compound 4 was tentatively characterized as sinapoyl-hexoside. Compound 5 with a [M–H]¡ ion at m/z 753 showed the loss of 224 Da, corresponding to sinapic acid. Further loss of a dihexoside moiety (306 Da) in the MS3 experiment results in the fragments (753 ! 529) at m/z 223 (sinapic acid) and leads to its tentative characterization as disinapoyl-dihexoside. Similarly, the loss of sinapic acid (224 Da) and the sugar moiety (144 Da) indicate that
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compound 6 is a disinapoyl-hexoside. Compound 7, trisinapoyl-dihexoside, was found to have a fragmentation pattern similar to compound 6 along with the additional loss of a sinapic acid moiety. MS data for sinapic acid and sinapoyl glycosides were well in accordance with Marles et al. [16], Harbaum et al. [12], and Olsen et al. [23], who investigated the phenolic composition of other Brassicaceae plants. Maximal absorbance at around 240 nm and 325 nm is characteristic of sinapic acid and its esters [5, 26]. The dihexoside moiety in sinapoyl esters in other Brassicaceae plants was identiWed as gentiobiose using 1H and 13C nuclear magnetic resonance (NMR) [5, 26]. Peak 8 was found to contain fragmentation ions corresponding to sinapic acid, and UV spectrometric data also indicated the presence of sinapic acid. Due to the broad peak shape, the presence of several sinapoyl conjugates can be assumed. Thiyam et al. [34] also described unidentiWed compounds eluding toward the end of the chromatographic run that presented fragmentation patterns similar to that of sinapine and hypothesized that these compounds may arise from temperature- and light-induced degradation of sinapine. Change in the phenolic proWle after alkaline hydrolysis and quantiWcation of the sinapic acid content The eVects of alkaline hydrolysis on the phenolic composition were determined using UHPLC-MS (Fig. 2). UV spectrum, retention time, and fragmentation pattern of compound 3 matched the authentic reference substance sinapic acid. The kaempferol aglycone was not detectable, probably due to the low concentration of compound 2. The sinapic acid content after alkaline hydrolysis conWrms the presence of sinapoyl esters in the crude extract (Table 2). The sinapic acid concentration in the hydrolyzed extract was determined and the sinapic acid concentration corresponded to 2.66 § 0.00 mg sinapic acid g¡1 defatted mustard seed meal. Antibacterial activity of phenolic compounds from mustard seed meal To investigate the eVects of alkaline hydrolysis on the antibacterial activity of the extracts, inhibitory spectra of the extracts before and after hydrolysis were determined. The MICs of the extracts were compared to aqueous acetone crude mustard seed extract as well as sinapic acid (Table 3). The antibacterial activity of the crude extract increased after puriWcation, suggesting that polyphenols are the major inhibitory compounds. Growth of several bacterial indicator strains, including the food-borne pathogens Staphylococcus aureus and Listeria monocytogenes, was inhibited.
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Fig. 2 Ultra-high-performance liquid chromatogram of released sinapic acid after alkaline hydrolysis of an extract from defatted mustard seed meal (330 nm). Peak assignment: (3) sinapic acid (in accordance with Fig. 1 and Table 1)
Lactobacillus plantarum was not aVected. MICs after alkaline hydrolysis of sinapic acid glycosides against Bacillus subtilis, Escherichia coli, Listeria monocytogenes, Pseudomonas Xuorescens, and Staphylococcus aureus were 0.1 g L¡1 or less. Growth of L. plantarum remained unaVected.
Discussion Mustard greens contain a large structural variety of hydroxycinnamic acid derivatives, including caVeoyl, coumaroyl, ferulolyl, hydroxyferulolyl, and sinapoyl esters [14]. Mustard seeds exhibited a much less complex proWle of phenolic compounds and hydroxycinnamic acids other than sinapic acid or sinapoyl esters were essentially absent. Sinapine was the predominant component in the crude extract and in the polyphenolic fraction. Only a small portion of detected phenolics was free sinapic acid. This is well in accordance with Thiyam et al. [33], who detected sinapine, sinapoyl glucose, and free sinapic acid in mustard meal. Free sinapic acid and its esters are also characteristic for other members of the Brassicaceae family. Sinapic acid was only a minor compound in kale leaves but accounted for over half of the total phenolic acid content in kale seeds [3]. Sinapine, sinapoyl glucose, and free sinapic acid also occur in canola solids extracts [13]; other phenolic compounds are present in smaller quantities [19]. Sinapoyl glycosides and derivatives of Xavonoids and other hydroxycinnamic acids were detected in pak choi leaves [12], kale [23], and cabbage leaves [9]. Esters of di- and trisinapic acid and gentiobiose were identiWed in broccoli Xorets [26].
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Table 3 Inhibitory activity of an aqueous acetone crude extract and of the polyphenol fraction, and minimal inhibitory concentrations (MICs) of a solution of this fraction after alkaline hydrolysis and of the reference substance sinapic acid Strains
Inhibitory activity (g mustard seed meal L¡1)a Crude extracta
Minimum inhibitory concentration (g L¡1)b Polyphenol fractiona
Polyphenol fraction after hydrolysisb
Sinapic acidb
Bacillus subtilis
83.3 § 0.0
3.5 § 0.0
0.1 § 0.0
0.3 § 0.0
Escherichia coli
69.4 § 24.1
1.7 § 0.0
0.1 § 0.0
0.7 § 0.0
Listeria innocua
41.7 § 0.0
3.5 § 0.0
0.1 § 0.0
0.3 § 0.0
Listeria monocytogenes Pseudomonas Xuorescens
55.6 § 24.1
4.6 § 2.0
0.1 § 0.0
0.2 § 0.0
41.7 § 0.0
3.5 § 0.0
Staphylococcus aureus Lactobacillus plantarum a b
83.3 § 0.0 >83.3
5.8 § 2.0 >55.6
>0.1 0.1 § 0.0 >0.1
0.6 § 0.2 0.3 § 0.0 >0.7
The inhibitory activity was calculated on the basis of the weight of mustard seed meal from which active concentrations were obtained MIC in g sinapic acid L¡1
Hydroxycinnamate conjugates are characteristic of brassicaceous plants [6, 8, 14]. Sinapic acid is produced in the shikimate/phenylpropanoid pathway and then converted into abundant O-ester conjugates employing a variety of enzymes [18]. Sinapine, the seed-speciWc choline ester, can be used as a chemotaxonomic marker to classify members of the Brassicaceae family [6]. It might function as a storage vehicle for phosphatidylcholine biosynthesis. The biological function of other sinapoyl esters within living plants might include UV protection [18]. Kaempferol-sinapoyl-glycosides are widely distributed in Brassicaceae species. Using spectroscopic methods including 1H and 13C NMR, Nielsen et al. [21] characterized a compound with a similar fragmentation pattern extracted from cabbage leaves as kaempferol-3-O--D-[2E-sinapoyl--D-glucopyranosyl(1 ! 2)glucopyranoside]7-O--D-glucopyranoside. IdentiWcation and quantiWcation of phenolics in crude extracts are hampered due to the large variety of conjugates and the limited number of authentic reference compounds. Alkaline hydrolysis overcame these problems by breakdown of sinapoyl conjugates into the major aglycones. Sinapic acid was the only aglycone that was released by alkaline hydrolysis, conWrming the sole occurrence of esters of sinapic acid in mustard seed meal. The release of sinapic acid from its esters enabled quantiWcation with a sinapic acid standard. Alkaline hydrolysis was applied for the identiWcation and quantiWcation purposes of phenolics in other botanical extracts [12, 20]. Alternatively, enzymatic hydrolysis may be employed. Enzymes allow for milder hydrolysis conditions when compared to alkaline hydrolysis [36]. The sinapic acid content in mustard seed meal has not been reported, but our results correspond to data for other species of the Brassicaceae family. Canola seeds, meal, and
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press cake contained from 9.6 to 15.3 mg sinapic acid equivalents per gram depending on variety and processing method [13]. Kale seeds contained a total concentration of 9.6 mg phenolic acids per gram dry weight [3]. Most phenolics remain in the solids during rapeseed processing, and only a small portion transfers to the crude oil [36]. A similar behavior can be assumed for the mustard oil process. Sinapic acid was found to be one of the antibacterial compounds in yellow mustard [32]. The increased activity of the puriWed polyphenol fraction compared to the crude extract indicates that acetone also extracts compounds that counteract the antibacterial activity of mustard polyphenolics. Comparison of the inhibitory activity of phenolic compounds after alkaline hydrolysis with the activity of sinapic acid conWrmed that this compound contributes substantially to the antibacterial activity of mustard seed extracts. Similarly, a phenolic extract of rapeseeds showed antibacterial activity only after alkaline hydrolysis releasing sinapic acid as the predominant compound [22]. The antibacterial activity of sinapic acid was remarkably selective. Gram-positive and Gram-negative bacteria were inhibited, while L. plantarum was resistant. The inhibitory eVect of sinapic acid against Gram-positive and Gram-negative bacteria including Escherichia coli and Salmonella enterica and also the resistance of lactic acid bacteria conforms with other studies [22, 25, 31]. The resistance of lactic acid bacteria toward phenolic compounds, however, is strainspeciWc. Hydroxycinnamic acids did not aVect the growth of L. plantarum, while cultures of Oenococcus oeni were inhibited [30]. Hydroxycinnamic acids were further found to delay malolactic fermentation of O. oeni [27] and decreased the cell culture viabilities due to increased membrane permeability [7]. Lactobacillus spp. metabolize phenolic acids by decarboxylase and reductase activities [4, 28, 31]; either metabolic pathway increases the resistance of lactobacilli
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[31]. L. plantarum TMW 1.460 is a hop-resistant beer isolate, which is able to metabolize several phenolic acids, and highly resistant to other hydroxycinnamic acids [31]. In conclusion, this study identiWed phenolic compounds with antimicrobial activity in mustard seed meal and thus provides a way of valorization of the by-products of the mustard oil processing. The selective inhibitory activity of sinapic acid allows food applications to eliminate foodborne pathogens without the inhibition of growth and metabolic activity of beneWcial lactic acid bacteria, which are used in food as starter cultures, protective cultures, or probiotics. Acknowledgments We thank Biofume Technologies Inc. (Saskatoon, SK, Canada) for providing mustard samples. The National Sciences and Engineering Research Council of Canada, NSERC, is acknowledged for funding. A.S. and M.G.G. acknowledge funding from the Canada Research Chair Program.
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