Food Chemistry 114 (2009) 791–797
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Thermal oxidation of vanillin affects its antioxidant and antimicrobial properties
Ioannis Mourtzinos a, Spyros Konteles b, Nick Kalogeropoulos a, Vaios T. Karathanos a,*
a
Laboratory of Food Chemistry, Biochemistry and Physical Chemistry, Department of Nutrition and Dietetics, Harokopio University, 70 El. Venizelou Ave.,
Kallithea, 176 71 Athens, Greece
Laboratory of Food Microbiology, Department of Food Technology, Technological Educational Institute of Athens, 12 Ag. Spyridonos St., Egaleo, 122 10 Athens, Greece
b
a r t i c l e
i n f o
Article history:
Received 13 March 2008
Received in revised form 6 August 2008
Accepted 8 October 2008
Keywords:
Vanillic acid
GC–MS
Differential scanning calorimetry
Oxidation studies
DPPH
a b s t r a c t
Accelerated oxidation of vanillin was studied by isothermal and non-isothermal differential scanning calorimetry (DSC) in model solutions. Exothermic peaks of DSC thermograms, due to the oxidation of vanillin, were observed. Vanillin oxidation to vanillic acid was confirmed by the detection of vanillic acid in
heated vanillin samples using GC–MS. The effect of temperature on vanillin oxidation was studied by
conducting DSC experiments with pure vanillin at several different final temperatures and by subsequent
determination of vanillin and vanillic acid by GC–MS.
Furthermore, the DPPH free radical assay was done on DSC-treated samples as well as on mixtures of
vanillin–vanillic acid. The radical-scavenging activity of the samples was increased along with the vanillic
acid content. Additionally, the antimicrobial activities and the minimum inhibitory concentrations (MIC)
of solutions containing vanillin and vanillic acid against Staphylococcus aureus, Staphylococcus epidermidis,
Bacillus cereus, Enterobacter aerogenes, Escherichia coli and Yersinia enterocolitica were determined by the
agar well-diffusion method. All tested samples exhibited inhibitory activity against all of the bacteria. Yet,
the higher the vanillic acid concentration, the lower was the MIC of the samples. It is concluded that the
thermal treatment of vanillin-containing food may lead to products with improved antioxidant and antimicrobial properties.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Vanilla is a tropical, climbing orchid (Vanilla planifolia, Vanilla
pompona or Vanilla tahitiensis). The cured or fermented beans or
pods of the orchid are treated with alcohol and the extract is the
natural vanilla. The main component of vanilla aroma is ‘‘vanillin”
(4-hydroxy-3-methoxybenzaldehyde), that is accompanied with
minor amounts of vanillic acid and up to 200 trace components
(Boyce, Haddad, & Sostaric, 2003; Korthou & Verpoorte, 2007;
Sinha, Sharma, & Sharma, 2007; Walton, Mayer, & Narbad, 2003).
Vanillin is considered to be one of the most widely appreciated
flavour compounds, with an odour threshold for humans equal to
11.8 1014 M, and has the unique characteristic that, even at
high doses, the flavour is still pleasant (Korthou & Verpoorte,
2007).
Vanillin is used as a flavouring agent and it is generally regarded
as safe (GRAS). Currently, it is added in a wide range of products,
e.g. pastry products, ice cream, soft drinks and baked products (biscuits, cereals), in concentrations ranging from 1 to 26 mM, depending on the nature of the product. Apart from flavouring properties,
vanillin exhibits several bioactive properties (Sinha et al., 2007),
e.g. antioxidant (Burri, Graf, Lambelet, & Loliger, 1989) and antimicrobial activities against yeasts, moulds (Cerruti & Alzamora, 1996;
* Corresponding author. Tel.: +30 210 9549 224x306; fax: +30 210 9577 050.
E-mail address: vkarath@hua.gr (V.T. Karathanos).
0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2008.10.014
Fitzgerald, Stratford, Gasson, & Narbad, 2004; López-Malo,
Alzamora, & Argaiz, 1997; Tipparaju, Ravishankar, & Slade, 2004)
and bacteria (Delaquis, Stanich, & Toivonen, 2005; Fitzgerald
et al., 2004; Moon, Delaquis, Toivonen, & Stanich, 2006; Rupasinghe,
Boulter-Bitzer, Ahn, & Odumeru, 2006; Tipparaju et al. 2004).
Vanillin has also been reported to possess anticlastogenic,
antimutagenic and antitumor properties and, therefore, it can be
considered as a nutraceutical molecule (Durant & Karran, 2003;
Gustafson et al., 2000; Keshava, Keshava, Whong, Nath, &
Ong, 1998; Kumar, Ghosh, Devasagayam, & Chauhan, 2000;
Lirdprapamongkol et al., 2005; Shyamala, Naidu, Sulochanamma,
& Srinivas, 2007; Sinigaglia, Reguly, & de Andrade, 2004). Moreover, it has found applications as a constituent in perfume and
pharmaceutical formulations.
From a chemical standpoint, vanillin has both aldehydic and
phenolic groups and it can undergo several types of reactions,
amongst them oxidation. Vanillin oxidation may be either enzymic
(Anklam, Gaglione, & Muller, 1997) or chemical with oxygen, in the
presence of alkalies (Fricko, Holocher-Ertl, & Kratzl, 1980) or potassium bromate (Samaddar & Banerjee, 1982) solutions. In dairy
products, the oxidation of vanillin to vanillic acid can take place
during the processing of milk by the action of enzymes that are
present in the milk, as well as by heat, during pasteurization. Furthermore, in milk and dairy products, vanillin can be oxidised into
divanillin in the presence of peroxidase (Anklam et al., 1997). Oxidation of vanillin to vanillic acid, in dairy products, was studied by
792
I. Mourtzinos et al. / Food Chemistry 114 (2009) 791–797
Anklam et al. (1997). Also, in model solutions of shoshu (Japanese
alcoholic beverage), it has been proved that ferulic acid, liberated
from rice flour, is converted to 4-vinylguaiacol and then to vanillin
and vanillic acid by microorganisms (Koseki, Ito, Furuse, Ito, &
Iwano, 1996).
Several methods have been developed to evaluate the oxidative
stability of various molecules (lipids, antioxidants) under accelerated conditions. Amongst them, thermal analytical methods were
included, such as thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) (Rudnik, Szczucinska, Gwardiak,
Szulc, & Winiarska, 2001), either isothermal or non-isothermal.
Isothermal DSC analysis has been used for evaluating the oxidation
stability of vegetable oils and antioxidants (Giuffrida et al., 2007;
Hu et al., 2007; Velasco, Andersen, & Skibsted, 2004) whilst nonisothermal or dynamic DSC analysis has been used for studying
the thermo-oxidative degradation of antioxidants (Giuffrida et al.,
2007), organic materials, polymers and petroleum products
(Litwinienko, Kasprzycka-Guttman, & Jamanek, 1999). Non-isothermal DSC is much faster and it can provide more information
about the oxidation processes than can isothermal DSC.
Despite several reports on the antioxidant and the antimicrobial
activities of vanillin, to our knowledge, the evaluation of these
properties in samples heated under several time/temperature combinations has not yet been undertaken. In this work, pure vanillin
was heat-treated, under either isothermal or non-isothermal conditions by DSC. In both cases the heat-treated samples were analysed by GC–MS. Moreover, the antioxidant and the antimicrobial
properties of the samples were assessed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay and by the ‘‘agarwell” dilution method, respectively.
2. Materials and methods
2.1. Materials
2.1.1. Reagents and solvents
Vanillin and vanillic acid were obtained from Sigma (St. Louis,
MO, USA) and Serva (Heidelberg, Germany), respectively. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) and bis-(trimethylsily)trifluoroacetamide (BSTFA), were obtained from Aldrich (Steinheim,
Germany).
2.1.2. Bacterial strains and culture preparations
Eleven bacterial strains (target-strains) – pathogenic and nonpathogenic – were used in this study. Salmonella dysenteriae NCTC
2966, Salmonella typhimurium NCTC 012023, Enterobacter aerogenes
NCTC 0100006, Yersinia enterocolitica NCTC 10460, Escherichia coli
O157:Y7 NCTC 009001, Staphylococcus aureus NTCT 006571, Staphylococcus epidermidis NCTC 011047, Bacillus cereus NCTC 7464 and Listeria monocytogenes NCTC 010357 were provided by Agrolab S.A.
(Agrolab S.A., Athens, Greece). Lactobacillus bulgaricus ACA-DC 101
and Lactococcus lactis ACA-DC 1 were provided by the ACA-DC collection (Laboratory of Dairy Research, Agricultural University of Athens,
Greece). L. bulgaricus and L. lactis working cultures were maintained
on Man Rogosa and Sharpe (MRS, Merck, Darmstadt, Germany) agar
slants whilst the remaining strains were maintained on Tryptone soy
agar (TSA, Merck) supplemented with 0.6% yeast extract (Sigma, St.
Louis, MO, USA), (TSYEA) slants, all stored at 4 °C.
2.2. Methods
2.2.1. Isothermal oxidation of vanillin by DSC
Vanillin samples (5 mg, 0.033 mmole each), were placed in
open aluminium pans and heated to 100, 120, 130 and 140 °C, at
times varying from 10 to 90 min, in a Perkin–Elmer DSC instrument (DSC-6, Boston, MA, USA). The samples obtained after ther-
mal treatment by DSC were diluted in 10 ml of ethanol:water
80:20 (v:v) and stored at 4 °C for subsequent analysis. By isothermal heating, the DSC instrument was operated as a heating apparatus, e.g. a reactor or an oven, kept at constant temperatures, in
order to affect the oxidation of vanillin.
2.2.2. Non-isothermal oxidation of vanillin by DSC
Non-isothermal DSC is a scanning procedure over a wide range
of temperatures. It offers valuable information on various thermal
transitions, which occur over the range of temperatures, such as
glass transition, melting, crystallisation, oxidation, decomposition,
evaporation and others and gives the range of temperature at
which these transitions take place (Clas, Dalton, & Hancock,
1999; Widman, 1987). All the non-isothermal heat treatments
were carried out in a DSC instrument. Vanillin samples, 5 mg each,
were placed in open aluminium pans and heated, in an oxygen
atmosphere, from room temperature (approximately 20 °C) to
120 °C, at a rate of 90 C° per min. The samples were left at 120 °C
for one minute, to ensure uniform temperature distribution in
them, and they were then heated at a rate of 10 C° per min. The
oxygen flow rate was 20 ml/s. This latter procedure was applied
eight times with the following final temperatures: 131, 179, 210,
230, 258, 262, 273 and 280 °C. The heat-treated samples at each
of the above temperatures were diluted in 10 ml of ethanol:water
80:20 (v:v) and stored at 4 °C for the subsequent GC–MS and DPPH
analyses.
2.2.3. Preparation of vanillin–vanillic acid solutions
Solutions containing vanillin and vanillic acid with the ratios
(w:w), 100:0, 77:23, 53:47, 25:75, 0:100, were prepared in 10 ml
of ethanol: water (80:20). Prior to antimicrobial testing, the samples were filter-sterilised through a 0.22 lm membrane filter (Gelman Sciences, Inc., Ann Arbor, Michigan, USA). In order to
determine the MIC (minimum inhibitory concentration) of the
samples over the range of target-strains used, several dilutions of
the samples were made (0, 1:2, 1:4, 1:8, 1:16) in ethanol:water
80:20 (v:v) solution.
2.2.4. Gas chromatography/mass spectrometry analysis
Aliquots of ethanol:water solutions of DSC-treated samples
(50 ll) or aliquots of mixtures of vanillin–vanillic acid solutions
were transferred to GC vials containing 50 ll of internal standard,
(3-(4-hydroxy-phenyl)-1-propanol, 19.2 lg/ml), evaporated to
dryness under a stream of nitrogen, and derivatized by adding
250 ll of BSTA, followed by incubation at 70 °C for 20 min (Soleas,
Diamandis, Karumanchiri, & Goldberg, 1997). An aliquot (1 ll) of
each sample was injected into the gas chromatograph (GC) at a
split ratio of 1:50. An Agilent GC (Wallborn, Germany) series
6890N, coupled with an HP 5973 Mass spectrometer (MS) detector
(EI, 70 eV), split–splitless injector and an HP 7683 autosampler was
used for the analysis. Sample separation was achieved using an HP5 MS capillary column (5% phenyl–95% methyl siloxane, 30 m
length, 0.25 mm inner diameter, 250 lm film thickness). Helium
was used as a carrier gas at a flow rate 0.6 ml/min. The injector
and transfer line temperatures were set at 280 and 300 °C, respectively. The oven temperature programme was: initial temperature
70 °C for 5 min, 70–130 °C at 15 °C per min, 130–160 °C at 4 °C per
min, held for 15 min, 160–300 °C at 10 °C per min, and finally held
at 300 °C for 15 min. Under these conditions, vanillin and vanillic
acid were eluted after 17.30 and 25.89 min, respectively. A selective ion monitoring (SIM) GC/MS method was applied for the
detection of vanillin and vanillic acid. Detection of compounds
was based on the ±0.05 RT presence of target and qualifier ions
of the standards at the predetermined ratios. Target and qualifier
ions (T, Q1, Q2) for the compounds were set as follows: vanillin:
194, 209, vanillic acid: 297, 267, 312.
793
I. Mourtzinos et al. / Food Chemistry 114 (2009) 791–797
% DPPH scavenging ¼ ½ðAcontrol Asample Þ=Acontrol 100
where Asample is the absorbance of sample after the time necessary
to reach the plateau (30 min) and Acontrol is the absorbance of DPPH.
2.2.6. Antibacterial assay
The inhibitory activities of vanillin:vanillic acid solutions were
qualitatively and quantitatively analysed by a modified ‘‘agar-well”
diffusion assay as described by Schillinger and Lucke (1989). Plates
were filled with 12 ml of agar–agar (Merck) and, when the medium
was solidified, overlaid with 10 ml of TSYE soft agar (8 g/l, agar)
that had been, previously inoculated with 100 ll of the proper target cell suspension. For Lactobacillus and Lactococcous strains, the
overlaid medium was MRS soft agar. The cell suspensions were
made by diluting overnight cultures of the target strain into saline
solution to, approximately, 107 cfu/ml, using McFarland turbidity
standards (bioMerieux S.A., Marcy l’Étoile, France). The plates were
dried for 1 h and then, using a sterile cylinder, wells of 7.0 mm
diameter were made and filled up with 100 ll of the sample. In order to obtain comparable results, all samples were treated under
the same conditions in the same plate. The plates were incubated
for 24 h at 37 °C and the results were recorded. Lactobacillus and
Lactococcous strains were incubated under anaerobic conditions
(BBL GasPak system, Becton Dickinson microbiology systems, Cockeysville, MD) for 48 h at 37 °C. The inhibitory activities of the
samples were detected as a clear zone around the wells and expressed in millimetres (mm). Minimum inhibitory concentration
(MIC) was defined as the lowest concentration of the sample that
caused a zone of inhibition (1–3 mm). As negative control, the ethanol:water (80:20) (v:v) solution was used.
2.2.7. Statistical analysis
DSC experiments were duplicated. DPPH analyses, as well as
antimicrobial tests, were performed in triplicate (n = 3). The results
presented are the averages of the obtained values. Simple descriptive statistics were analysed using Microsoft excel.
3. Results and discussion
3.1. Isothermal oxidation of vanillin
The heat stability of vanillin under isothermal conditions was
investigated with DSC. Vanillin samples (0.032 mmole) were
heated at 100, 120, 130 and 140 °C at time intervals varying from
3.2. Non-isothermal oxidation of vanillin
The heat stability of vanillin under non-isothermal conditions
was also studied. The temperature range studied was from 120
to 280 °C to examine possible thermal degradation, and oxidation
phenomena occurred at relatively high temperatures, e.g. in the
a
Vanillin (mmol)
2.2.5. Radical-scavenging activity (antioxidant activity)
The effect of the oxidised vanillin samples, as well as vanillin:vanillic acid mixtures, on DPPH was estimated according to
the procedure described by Brand-Williams, Cuvelier, and Berset
(1995). Briefly, each sample was diluted in ethanol prior to the
analysis (1 mg/ml). An aliquot (0.1 ml) of the solution was added
to 3.9 ml of DPPH solution (6 105 M in ethanol), thoroughly
mixed, and the absorbance of the sample at 515 nm was recorded
after the time necessary for the reaction to reach a plateau, which
was 30 min, as proposed by Brand-Williams et al. (1995). The
absorbance of DPPH solution in ethanol, without any antioxidant
(control), was also measured. The percentage of remaining DPPH
was calculated as follows:
10 to 90 min, and the vanillin remaining after the thermal treatment was determined by GC/MS. As can be seen in Fig. 1a, the longer the time of heat treatment, the less vanillin remained in the
samples. Moreover, the vanillin decrease was faster as the temperature increased from 100 °C to 140 °C, resulting in a drop of the
vanillin ‘‘half-life” from 53–55 min at 100–120°C to 47 min at
130°C and just 15 min at 140°C. On the other hand, vanillic acid,
an oxidation derivative of vanillin, was gradually formed in the
samples (Fig. 1b). The results showed that both temperature and
heating time governed the vanillic acid formation. Regarding the
effect of temperature, the general trend was that vanillic acid formation was favoured at higher temperatures; for example, the
vanillic acid concentrations formed after 30 min at 120, 130 and
140°C were, respectively, 3, 6 and 16 times higher than that formed
at 100 °C. By contrasting Fig.1a and b, it appears that the total
amounts of vanillin and vanillic acid (in mmoles) did not remain
constant but decreased along with temperature and time, most
likely due to evaporation and/or thermal degradation of both substances. The combined effect of vanillic acid formation from vanillin and the thermal decomposition of both substances resulted in
time–profile curves showing a maximum (Fig. 1b) at the points
where the production and deterioration rates of vanillic acid were
equal, especially at temperatures above 120 °C. These maxima appeared earlier as the temperature increased, being >90 min at
100 °C, around 60 min at 120–130 °C and around 30 min at
140 °C (Fig. 1b).
0.040
100°C
0.035
120°C
0.030
140°C
130°C
0.025
0.020
0.015
0.010
0.005
0.000
0
10
20
30
40
50
60
70
80
90
100
Time (min)
b
0.0006
100°C
Vanillic acid (mmol)
The identification of chromatographic peaks was achieved by
comparing the retention times and ratios of the fragment ions of
each compound with those of reference compounds whilst quantification was carried out using 3-(4-hydroxy-phenyl)-1-propanol as
an internal standard with target ion m/z 206 and qualifiers 191 and
179. Very good linearity (R2 > 0.999) was obtained for both compounds within the concentration range studied.
120°C
0.0005
130°C
140°C
0.0004
0.0003
0.0002
0.0001
0
0
10
20
30
40
50
60
70
80
90
100
Time (min)
Fig. 1. Time profile of: (a) vanillin oxidative loss and (b) vanillic acid formation
during heating of pure vanillin at different temperatures.
oven or during production of low-moisture foods, such as by extrusion cooking. The final temperature of the DSC programme (280 °C)
was selected in order not to exceed vanillin’s boiling point at
285 °C. An exothermic event was observed at temperatures starting at 131 °C, with a maximum at 210 °C, and then the thermogram
curve decreased until 258 °C. Over this temperature range (131–
258 °C) the thermal event was not, most likely, an oxidation phenomenon since the vanillic acid formation at some selected temperatures (131, 179, 210 and 230 °C) was relatively limited, 0.2%,
0.5%, 1.2% and 3.7%, respectively. The chemical analysis of vanillin
and vanillic acid (mmole) and their ratio was done by GC–MS (Table 1) after the contents of DSC pans at each temperature were dissolved in ethanol:water (80:20 v/v) solution. Non-isothermal DSC
scans were conducted by Svard, Gracin, and Rasmuson (2007)
and Widman (1987), who studied the glass transition, crystallization and fusion of vanillin. All of these phenomena occurred at
temperatures below 100 °C, with melting occurring at around
80.9 °C. Unfortunately, to our knowledge, there is a lack of DSC
data at higher temperatures. Thus we may propose (as possible
causes of the exothermic peak in this temperature range) a partial
decomposition or degradation of vanillin.
After the end-point (258 °C) of the previous exothermic peak, a
second exothermic thermal event was initiated. This event most
likely corresponded with vanillin’s oxidation to vanillic acid. This
was proved by the formation of increased amounts of vanillic acid,
as determined by GC–MS at higher temperatures. It should be
emphasised that, whilst over the temperature range 131–258 °C,
only 9.4% of the vanillin had been oxidised to vanillic acid, in the
much narrower range of temperatures 258–273 °C, the amount
of oxidised vanillin was changed drastically, since it was increased
from 9.4% to 47.0% (Fig. 2).
3.3. Radical-scavenging activity
The antioxidant activity of vanillin, in comparison to o-vanillin
as a scavenger of the DPPH radical, has been studied previously
(Santosh-Kumar, Priyadarsini, & Sainis, 2002). To our knowledge,
there are no data about the antioxidant activity of vanillin in the
presence of its oxidation product, vanillic acid. Brand-Williams
et al. (1995) reported that vanillin, and also vanillic acid, phenol,
coumaric acid and c-resorcylic acid, were amongst the substances
that reacted relatively slowly and very poorly with the DPPH reagent. For this reason, a relatively long reaction time (30 min)
was chosen and the results of the assay were used for comparison
of the radical-scavenging activities of several different mixtures of
vanillin and vanillic acid rather than for determination of their
antioxidant potential. The antioxidant activity of vanillin in the
presence of vanillic acid, in terms of DPPH-scavenging, was determined both in the DSC-treated vanillin samples and in solutions of
vanillin–vanillic acid and the results are presented in Table 1 and
Fig. 3, respectively. Table 1 shows that vanillin itself had an antioxidant activity, as expected and reported in the literature (Burri
Heat Flow (endo down,mW)
I. Mourtzinos et al. / Food Chemistry 114 (2009) 791–797
16.5
210oC
17
17.5
230oC
179oC
18
18.5
273oC
258oC
o
131 C
19
19.5
262oC
20
120
140
160
180
200
220
240
260
280
Temperature (°C)
Fig. 2. DSC thermogram of pure vanillin under oxidative conditions. The sampling
points are spotted.
45
% Radical scavenging
794
40
35
30
25
20
y = 0.2793x + 10.657
R 2 = 0.9847
15
10
5
0
0
20
40
60
80
100
%Vanillic acid
Fig. 3. Regression of DPPH radical-scavenging activity (%) on vanillic acid contents
(%) of vanillin–vanillic acid mixtures.
et al., 1989). The results in Table 1 also show that the antioxidant
activity of vanillin–vanillic acid mixtures, obtained after thermal
treatment of pure vanillin, increased as the percentage of vanillic
acid increased and was almost doubled when the vanillic acid
reached 47% w/w of the mixture. The same trend was observed
in the case of mixtures of pure vanillin and vanillic acid, the antioxidant activity of which increased as the vanillic acid content increased and there was a linear relationship between the % radicalscavenging and % vanillic acid (Fig. 3). Given that both vanillin and
vanillic acid exhibited antioxidant activity (Table 1 and Fig. 3), with
that of vanillic acid being more profound, it could be estimated
from Fig. 3 that the antioxidant activity of vanillic acid was about
3.3 times that of vanillin on an equimolar basis.
A similar effect of heat-processing on the antioxidant activity of
a natural compound has been reported by Kang, Kim, Pyo, and
Yokozawa (2006). They found that steaming of raw ginseng at
98–100 °C for 3 h resulted in increased antioxidant activity, compared to non-heated ginseng. The authors attributed the increased
antiradical potency of heated ginseng to the phenolic compounds
of ginseng, amongst them vanillic acid.
Table 1
Radical-scavenging (%) and vanillic acid (%) on the basis of GC–MS data of DSC-treated samples.
Temperature (°C)
Vanillin + Vanillic
acid (lmole)
Vanillin (lmole)
Vanillic acid (lmole)
Vanillic acid% (w/w)
Radical-scavenging (%) of
thermally treated vanillin
131
179
210
230
258
262
273
21.4 ± 0.3a
23.2 ± 0.2
22.7 ± 0.5
20.2 ± 0.3
21.8 ± 0.2
17.9 ± 0.6
15.1 ± 0.6
21.4 ± 0.3
23.1 ± 0.2
22.5 ± 0.5
19.5 ± 0.3
19.9 ± 0.4
14.1 ± 0.4
8.4 ± 0.2
0.04 ± 0.01
0.10 ± 0.02
0.19 ± 0.02
0.71 ± 0.05
1.90 ± 0.44
3.83 ± 0.32
6.71 ± 0.40
0.2 ± 0.1
0.5 ± 0.1
1.2 ± 0.1
3.7 ± 0.2
9.4 ± 2.0
23.2 ± 1.1
47.0 ± 1.8
11.4 ± 0.53
11.6 ± 0.40
13.7 ± 0.31
15.2 ± 1.14
17.0 ± 0.50
20.6 ± 1.15
22.2 ± 1.93
a
±S.D.
Table 2
Antimicrobial activity of vanillin–vanillic acid mixtures towards selected strains of Gram (+) and Gram () bacteria and yeasts.
Ratios
Vanillin:vanillic
acid (mM)
pH
Serial
dilutions
Target strains
Salmonella
enteritidis
Mean of
inhibition zone
(mm)
Salmonella
typhimurium
Yersinia
enterocolitica
Enterobacter
aerogenes
Bacillus
cereus
Staphylococcus
aureus
Staphylococcus
epidermidis
L.isteria
monocytogenes
Lactobacillus
bulgaricus
Lactococcus
lactis
+++
++
+++
++
++++
++
+
++++
++
++
+
++++
+++
++
+
+++
++
+++
++
++++
++
+
++++
++
++
+
++++
+++
++
+
+++
++
+++
++
++++
++
+
++++
++
++
+
++++
+++
++
+
++
+
++
+
+++
++
+
++++
++
+
++++
+++
++
+
++
+
++
+
++++
++
+
++++
+++
++
+
++
+
++
+
++++
++
+
++++
+++
++
++
+
++
+
+++
++
+
++++
++
+
++++
+++
++
++
+
++
+
+++
++
+
++++
++
+
++++
+++
++
++
+
++
+
+++
++
+
++++
++
+
++++
+++
++
Zone of inhibition (mm)
100:0.0
5.94
77:23
5.46
53:47
5.33
25:75
5.06
0:100
4.87
0
1:1
1:2
1:4
0
1:1
1:2
1:4
0
1:1
1:2
1:4
0
1:1
1:2
1:4
0
1:1
1:2
1:4
+++
++
+++
++
++++
++
+
++++
++
++
+
++++
+++
++
+
+++
++
+++
++
++++
++
+
++++
++
++
+
++++
+++
++
+
5.0
2.7
–
–
5.5
2.9
–
–
7.8
3.9
2.0
–
10.5
4.4
3.1
1.7
12.6
9.6
4.0
2.3
I. Mourtzinos et al. / Food Chemistry 114 (2009) 791–797
Escherichia
coli O157:H7
() No antimicrobial activity <1 mm zone of inhibition.
(+) Weak antimicrobial activity 1–3 mm zone of inhibition.
(++) Moderate antimicrobial activity 3–5 mm zone of inhibition.
(+++) Strong antimicrobial activity 5–7 mm zone of inhibition.
(++++) Very strong antimicrobial activity >7 mm zone of inhibition.
795
796
I. Mourtzinos et al. / Food Chemistry 114 (2009) 791–797
3.4. Antimicrobial activity
The antimicrobial activity of vanillin–vanillic acid solutions was
tested against a range of Gram-positive and Gram-negative bacteria. Vanillin exhibited the weakest inhibitory activity against all
the target strain bacteria that were tested (Table 2). The antibacterial activity of the samples was increased, along with vanillic acid
concentration, and the strongest inhibition was observed in the
100% vanillic acid sample. The antimicrobial activity of vanillin
against a broad variety of bacteria and yeasts is well documented
(Cerruti & Alzamora, 1996; Fitzgerald et al., 2004; Moon et al.,
2006). Yet, the inhibitory activity of vanillic acid against Listeria
spp. has been studied by Delaquis et al. (2005) and it was found
to be pH-dependent, with the highest antilisterial activity recorded
at pH 5.0. In the same work, it was reported that, at low pH (pH
5.0) the aqueous solutions of vanillic acid had stronger inhibitory
activities than had vanillin solutions.
The MIC of the samples was affected by the vanillic acid concentrations. In general, the higher the vanillic acid concentration, the
lower was the MIC observed. Regarding pure vanillin, the MIC for
Escherichia coli O157:H7, Listeria monocytogenes and Lactobacillus
bulgaricus was 50 mM. Fitzgerald et al. (2004) have reported MIC
values of 15, 15 and 75 mM for E. coli, Listeria innocua and Lactobacillus plantarum, respectively. Rupasinghe et al. (2006), also, reported a dose-dependent inhibitory activity of vanillin against E.
coli, Enterobacter aerogenes, Salmonella. enterica subsp. enterica
serovar Newport and Lactobacillus casei, with MIC ranging from
6–19 mM. Though our results are in agreement, from a qualitative
standpoint, with the findings of the latter work, there are discrepancies concerning the corresponding MIC. This could be attributed
to the different strains and different methods of testing of the antimicrobial activity.
The MIC for pure vanillic acid was 12.5 mM for E. coli, whilst the
corresponding MICs for Listeria monocytogenes and Lactobacillus
plantarum were 25 mM. It seems that Gram-negative bacteria are
more sensitive to vanillic acid than are Gram-positive ones.
Rodriguez-Vaquero, Alberto, and Manca de Nadra (2007) studied
the antimicrobial effect of phenolic compounds from different
wines and reported that vanillic acid showed inhibitory activity
against E. coli but not against S. aureus. This confirms that Gramnegative bacteria, like E. coli, are more sensitive to vanillic acid
than are Gram-positive (S. aureus). The non-inhibition of S. aureus
could be attributed to the very low vanillic acid concentration
used, that was 1 g/l, corresponding to 5.95 mM. Though the exact
mode of vanillin’s antimicrobial action is not fully understood, it
is considered as a membrane-active compound that destabilizes
the bacterial membrane integrity, resulting in the dissipation of
ion gradients, and therefore inhibiting the bacterial respiration
(Fitzgerald et al., 2004). It has been shown (Delaquis et al., 2005)
that pH is a critical factor for the inhibitory activity of both vanillin
and vanillic acid. In our set of samples, as the vanillic acid concentrations increased, the pH dropped (Table 2) and the antimicrobial
activity increased. Vanillic acid has two pKa values at 25 °C (pKa1
4.51 and pKa2 9.39). Therefore, at pH 5.0, most of the vanillic acid
molecules are in the non-dissociated form, which exhibits antimicrobial activity. Also vanillin, although it is an aldehyde, exhibits
acid–base behaviour with pKa 7.4 at 25 °C, a value close to the
pH of pure vanillin solution. Therefore, at higher pH values, vanillin
is expected to exhibit higher antibacterial activity, as was confirmed by our findings.
Vanillin is widely used as a GRAS flavouring agent in a variety of
food products and acts as an effective antioxidant in foods containing polyunsaturated fatty acids, whilst it has also been identified as
an antimicrobial agent. MIC values of vanillin, either for bacteria or
yeasts and moulds, have been reported to be around 6 mM
(Fitzgerald et al., 2004; López-Malo et al., 1997), which corre-
sponds to 913 ppm. Considering that vanillin has a very low taste
threshold (0.5 ppm in water) (Korthou & Verpoorte, 2007) its
application as an antimicrobial agent may be problematic, since
it will affect the original flavour of the product and hence its organoleptic acceptance. On the contrary, the vanillic acid is nearly
odourless with a taste threshold value at 30 ppm that corresponds
to 0.18 mM. According to our results and those of Delaquis et al.
(2005), the MIC for vanillic acid – under acidic conditions – was
about 10 mM. Consequently, vanillic acid seems to be a more
applicable antimicrobial agent than vanillin for food systems.
The results of the present study indicate that the antioxidant
properties of heat-processed foods (e.g. cereals), now attributed
only to vanillin, may also be partly due to its oxidised form, vanillic
acid.
Moreover, from a general point of view, our results suggest that
oxidation products of some phenolic compounds may exhibit
stronger antioxidant and/or antimicrobial activities than do their
parent compounds in food systems, and further investigation in
this direction should be carried out.
4. Conclusion
In this work, vanillin was subjected to non-isothermal, as well
as isothermal DSC analyses, followed by GC–MS analysis. The results confirmed the oxidation of vanillin to vanillic acid and allowed the monitoring of the oxidation process at different points
of the thermograms. Moreover, heat-treated vanillin was proven
to be more effective as a food constituent in terms of radical-scavenging and antimicrobial activities, as a result of the conversion of
vanillin to vanillic acid during heating. The combination of DSC and
GC–MS could be a useful tool for assaying stability of nutraceutical
constituents.
Acknowledgements
We wish to thank the Laboratory of Dairy Research (Department
of Food Science and Technology, Agricultural University of Athens,
Greece) and Agrolab S.A. that kindly provided the bacterial strains
used in this study. One of the authors (I. Mourtzinos) is thankful to
the Greek Foundation of Fellowships for financial support.
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