Production, stability, and antioxidative and antimicrobial activities of two l-ascorbate analogues from phycomyces blakesleeanus: D-erythroascorbate and d-erythroascorbate glucoside

Article abstract of DOI:10.1021/jf102202e

D-Erythroascorbate (D-EAA), a five-carbon analogue of L-ascorbate (L-AA), and D-erythroascorbate monoglucoside (D-EAAG) are accumulated in Phycomyces blakesleeanus grown on glucose (99.5 and 1084 μg/g mycelial dry weight, respectively) and also excreted into the culture medium. Both compounds showed UV spectral properties and ionization constants similar to those of L-AA. D-EAAG was much more stable to aerobic oxidation than D-EAA and L-AA at acidic pH. D-EAAG is synthesized from D-erythroascorbate by a mycelial glucosyltransferase activity that uses UDP-glucose as glucose substrate donor with K_m = 2.5 mM and 41.3 μM for D-EAA. This glucosyltransferase activity was maximal in the stationary growth phase in parallel with maximal production of D-EAAG. The presence of D-arabinose or D-arabinono-1,4-lactone in the culture medium produces the maximal accumulation of D-EAA and D-EAAG (about 30- and 4-fold with respect to that obtained in glucose culture). Both compounds showed greater antioxidant activity than L-AA and other standard antioxidants, with a capacity similar to that of L-AA to inhibit the growth of Escherichia coli.

Full text of DOI:10.1021/jf102202e

J. Agric. Food Chem. 2010, 58, 10631–10638 10631
DOI:10.1021/jf102202e
Production, Stability, and Antioxidative and Antimicrobial
Activities of Two -Ascorbate Analogues from
Phycomyces blakesleeanus: -Erythroascorbate
and -Erythroascorbate Glucoside
L
D
D
†
,
M
ARTA
G
UTIERREZ-LARRAINZAR,† CRISTINA DE
C
ASTRO,† PILAR DEL
V
ALLE
†
J
AVIER
R
UA,† MARIA
R
OSARIO
G
ARCIA-ARMESTO,‡ FELIX
,†
B
USTO
,
AND
D
OLORES DE
A
RRIAGA
*
†Departamento de Biologıa Molecular and ‡Departamento de Ciencia y Tecnologıa de los Alimentos,
Universidad de Leon, E-24071, Leon, Spain
D
-Erythroascorbate (
monoglucoside ( -EAAG) are accumulated in Phycomyces blakesleeanus grown on glucose (99.5 and
1084 μg/g mycelial dry weight, respectively) and also excreted into the culture medium. Both compounds
showed UV spectral properties and ionization constants similar to those of -AA. -EAAG was much
more stable to aerobic oxidation than -EAA and -AA at acidic pH. -EAAG is synthesized from
-erythroascorbate by a mycelial glucosyltransferase activity that uses UDP-glucose as glucose substrate
donor with Km = 2.5 mM and 41.3 μM for -EAA. This glucosyltransferase activity was maximal in the
stationary growth phase in parallel with maximal production of -EAAG. The presence of -arabinose
or -arabinono-1,4-lactone in the culture medium produces the maximal accumulation of -EAA and
-EAAG (about 30- and 4-fold with respect to that obtained in glucose culture). Both compounds
showed greater antioxidant activity than -AA and other standard antioxidants, with a capacity similar to
that of -AA to inhibit the growth of Escherichia coli.
D-EAA), a five-carbon analogue of L-ascorbate (L-AA), and D-erythroascorbate
D
L
D
D
L
D
D
D
D
D
D
D
D
L
L
KEYWORDS: D-Erythroascorbate; D-erythroascorbate monoglucoside; physicochemical properties; stability;
UDP-glucosyltransferase activity; antioxidant activity; Phycomyces blakesleeanus; filamentous fungus
INTRODUCTION
-Erythroascorbate (
in structure and physicochemical properties to ascorbate (1). We
have previously reported that -EAA and a -erythroascorbate
glucoside ( -EAAG) are naturally molecules occurring in
Phycomyces blakesleeanus, a Zygomycete fungus (Figure 1),
providing evidence that both compounds protect the glutathione
pool and β-carotene from hydrogen peroxide-induced depletion,
suggesting that they can act as antioxidants in vivo (2). The
derivatives of ascorbic acid show effective antioxidative activity
with enhanced oxidative stability (8).
In the present study, the molecular mass, UV spectral properties,
D
D-EAA) is a C5 ascorbate analogue similar
and oxidative stability of
D-EAAG synthetized by P. blakesleeanus
D
D
are described, and the antioxidative activity of
D
-EAA and -EAAG
D
D
against commercial antioxidants is also evaluated. Glycosyl-
ascorbic acids have been obtained by transglycosylation with
R-glucosidases and maltogenic amylase (8,9). Here we report the
existence of a glucosyltransferase activity responsible for the
glycosylation reaction of
have analyzed the capacity of P. blakesleeanus to synthetize
and -EAAG under different growth conditions that demon-
strate the ability of this fungus to produce -EAA and -EAAG
D-EAA to form
D-EAAG. Finally, we
presence of
reported in two other fungal groups, the Ascomycetes and
Basidiomycetes (3, 4), suggesting that -EAA is characteristic
D-EAA and the absence of L-ascorbate have been
D-EAA
D
D
D
D
of the true fungi and that the ability to synthetize it must have
evolved in the common ancestor of the three fungal groups.
in amounts larger than those described in other microorganisms.
D-EAA can be replaced by ascorbate in several industrial pro-
MATERIALS AND METHODS
cesses (5), but does not have antiscorbutic activity in humans (6).
Ascorbic acid can be used as a common antioxidant in many food
systems and also as a cosmetic ingredient for skin care (7). However,
the instability of ascorbic acid against oxidative environments
with the consequent losses in antioxidative activity is disadvanta-
geous for its applications. It has been described that glycosylated
Materials, Strains, and Growth Conditions. All carbohydrates
used, D-aldoses, D-cetose (D-fructose), D- and L-pentoses, and dissacharides
(sucrose and maltose); Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-
2-carboxylic acid), 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS), and 2,20,diphenyl-1-picrylhydrazyl (DPPH) were purchased from
Sigma Chemical Co. (St. Louis, MO). Lactones and 2,4,6-tris(2,4,6-
tripyridyl-2-triazine) (TPTZ) were obtained from Fluka Chemie A.G.
(Buchs, Switzerland).
P. blakesleeanus (strain NRRL 1555, genotype (-), wild type) was
grown routinely in liquid minimal medium (SIV) as described by
*Author to whom correspondence should be addressed (phone þ34
987 291 229; fax þ34 987 291 226; e-mail dolores.arriaga@unileon.es).
© 2010 American Chemical Society
Published on Web 09/03/2010
pubs.acs.org/JAFC

10632 J. Agric. Food Chem., Vol. 58, No. 19, 2010
Gutierrez-Larrainzar et al.
and
D
-EAAG ([M - H]- m/z 307, collision energy, 8 and 44 eV). Data
were acquired with MassLynx 4.0 software (Waters Corp.) in con-
tinuous mode, from m/z 50 to 400 to obtain complete fragmentation
patterns.
Spectrophometric Analysis and Stabilities of D-Erythroascorbic
Acid and D-Erythroascorbic Glucoside in Aqueous Solution. Ultra-
violet (UV) absorption spectra were measured in solutions at different
pH values on a Shimadzu UV-260 double-beam spectrophotometer, and
extinction coefficients were determined. Ionization constants were deter-
mined by the changes in inflections of curves of λ max versus pH (1). To
determine the aerobic stabilities of
(final concentrations of each at 0.23 and 0.7 mM in 3 mL of buffer) of
EAA and -EAAG were prepared in 0.1 M sodium citrate buffer, pH 4,
D-EAA and D-EAAG, the solutions
Figure 1. Chemical structures of L-ascorbate (1), D-erythroascorbate (2),
and D-erythroascorbate monoglucoside (3).
D-
D
and 0.1 M sodium phosphate buffer, pH 6 and 8, respectively, in open-
topped glass tubes (15 ꢀ 85 mm, 15 mL). The tubes were placed in a
constant-temperature bath at 25, 35, or 45 °C. The decline in UV absor-
Baroja-Mazo et al. (2). For experiments of production of
D-EAA and
D-EAAG, (1) several carbohydrates were used as carbon source or (2) the
precursor was added to the culture medium with glucose 2% (w/v) at a
concentration of 10 mM at the onset of growth or at the indicated time of
growth on glucose. Mycelia were obtained by filtration at different stages
of growth. Growth of P. blakesleeanus was determined as a function of
mycelial dry weight. Aliquots of culture were taken and passed through a
glass wool filter. They were kept at 50 °C until their weights were constant.
Ten strains of Staphylococcus aureus, 3 collection strains (ATCC 25923,
CCTM La 2812, and ATCC 27664) and 7 foodborne isolates (6 from
bulk tank ewe’s milk and 1 from rabbit carcasses), and 10 strains of
Escherichia coli foodborne isolates from bulk tank ewe’s milk provided by
Dr. M. R. Garcia Armesto, Dr. J. M. Rodriguez-Calleja, and Dr. I. Caro
Canales, respectively, from the Department of Food Hygiene and Food
Technology (University of Leon, Leon, Spain) were used in this study.
Stock cultures were maintained in eppendorfs with brain-heart infusion
broth (BHI; Oxoid Ltd., Basingstoke, Hampshire, U.K.) in the presence of
30% (v/v) glycerol at -40 °C. A microdilution test assay was used to
bance at 251 nm (pH 4), 263 nm (pH 6), and 261 nm (pH 8) for
D-EAA and
at 255 nm (pH 4) and 265 nm (pH 6 and 8) for
follow losses of both compounds.
Glucosyl Transferase Activity Assay and Analysis of Reaction
D-EAAG was determined to
Product. The glucosyltransferase assay contained 9 mM UDP-glucose,
0.2 mM
D-EAA, and P. blakesleeanus mycelial extract (after passing
through a PD-10 column) in 50 mM sodium phosphate buffer, pH 7,
containing 14 mM 2-mercaptoethanol (MSH) and 2 mM dithiothreitol
(DTT) in a final reaction volume of 600 μL. The reaction mixture was
incubated at 30 °C for 30 min, and then 500 μL of the reaction mixture was
loaded onto an anion exchange column (Amprep-trimethylaminopropyl
SAX, 1 mL bed volume), washing this with 1 mL of H2O and eluting the
reaction product with 500 μL of 60 mM formic acid. The reaction product
was analyzed by HPLC (as described above) and identified as
Antioxidant Activity Determination. The antioxidant activity of
-EAA, -EAAG, and the standard antioxidants ascorbic acid, gallic acid,
D-EAAG.
D
D
determine the MICs of D-EAA, D-EAAG, and other selected antioxidants
resveratrol, butylated hydroxyanisole (BHA), and butylated hydroxy-
toluene (BHT) was determined according to three procedures: (1) The
FRAP method was conducted according to the procedure of Benzie and
Strain (11). A solution of 10 mM TPTZ and 20 mM ferric chloride was
diluted in 300 mM sodium acetate buffer, pH 3.6, at a ratio of 1:1:10. One
according to the method of Barry (10). MIC values were determined as the
lowest concentrations that inhibited growth of the strain tested. At least
two trials on different days were carried out in duplicate for each strain and
compound.
Extraction, Quantification, and Identification of
-EAAG. P. blakesleeanus mycelium grown in liquid medium under the
indicated conditions in each experiment was used to purify -EAA and
-EAAG as described by Baroja-Mazo et al. (2). The routine identification
of -EAA and -EAAG was carried out by high-performance liquid
D-EAA and
hundred microliters of standards or
D
-EAA (25 μM) or -EAAG (25 μM)
D
D
was added to 3 mL of the TPTZ solution, and absorbance at 593 nm was
determinedafter 15 min of reaction. A Trolox standard curve was obtained
in a concentration range of 0-30 μM. (2) The ABTS assay was carried out
as described by Ozgen et al. (12), with minor modifications. ABTS stock
solution (7 mM) with 2.45 mM potassium persulfate was prepared in
20 mM sodium acetate buffer, pH 4.5, and was allowed to stand for 12-16 h
at room temperature in the dark, remaining stable for several weeks under
these conditions. On the day of analysis, the ABTS solution was diluted
with the same buffer to an absorbance of 0.7 at 734 nm. For the spectro-
photometric assay, 1 mL of the ABTS solution and 10 μL of antioxidant
(100 μM) were mixed, and the absorbance at 734 nm was registered for
6 min at 30 °C. A Trolox standard curve was obtained in a concentration
range of 0-30 μM. (3) The ability to scavenge the DPPH radical was
measured as described by Brand-Williams et al. (13) with modifications.
The assay mixture contained 1 mL of 200 μM DPPH• (dissolved in 100%
ethanol) and 1 mL of antioxidant (2 μM) in 100 mM Tris-HCl buffer, pH
7.4. The mixture was mixed and left to stand in the dark at room tempe-
rature for 20 min, after which the absorbance at 517 nm was determined.
A Trolox standard curve was obtained in a concentration range of
0-100 μM. All data were expressed as Trolox equivalents from the three
assay methods.
D
D
D
D
chromatography (HPLC) using an Alliance Waters 2690 liquid chromato-
graph equipped with an Array 996 photodiode detector (Waters) and an
anionic exclusion column Supelcogel C-610H (300 ꢀ 7.8 mm) with a
Supelcogel C-610 coupled precolumn (50ꢀ4.6 mm) (Sigma Chemical Co.).
Separation and identification of D-EAA and D-EAAG were achieved by
isocratic elution with 0.75 mM H2SO4 at a flow rate of 1 mL/min.
-EAA and -EAAG were detected by their absorbance at 254 nm.
The peaks of -EAA and -EAAG were identified by their retention times,
D
D
D
D
8.30 ( 0.15 and 6.15 ( 0.20 min, respectively, as described by Baroja-
Mazo et al. (2). The areas of the peaks were determined by using the
Empower software and the quantification of
an -ascorbate standard curve (0.025-5 mM).
Mass Spectrometry Analysis. A Q-Tof micro (Waters Corp.) tandem
mass spectrometer with an ESI LockSpray source in negative ion mode
was used for the analysis of -EAA and -EAAG. Samples were intro-
D-EAA and D-EAAG from
L
D
D
duced in the mass spectrometer by direct infusion using as mobile phase
50% methanol and 1 mM ammonium acetate in high purity grade water.
The mobile phase flow rate was between 5 and 20 μL/min. Ionization
parameters were optimized by direct infusion of standard solutions and
were as follows: capillary voltage, 2.2 kV; sample cone voltage, 25 V; extrac-
tion cone voltage, 1.0 V; multiplier voltage, 2500 V; source temperature,
80 °C; and desolvation temperature, 120 °C. Nitrogen was used as cone
(50 L/h), nebulizing, and desolvation gas (200 L/h). Argon was used as
collision gas (vacuum pressure = 2.6 ꢀ 10-7 mbar) with a low collision
energy of 2 eV, which did not cause fragmentation of the analytes for MS
spectra. MS/MS analyses were carried out using the same settings for ioni-
zation, and the following deprotonated molecular ions were used for the
fragmentation experiments: glucose ([M - H]- m/z 179, collision energy, 4
and 30 eV); chlorine adduct of glucose ([M þ Cl]-,m/z 215, collision energy,
RESULTS AND DISCUSSION
Molecular Mass, UV Spectral Properties and Stability of D-
Erythroascorbate Glucoside from Phycomyces blakesleeanus. We
have previously described the characterization and biosynthesis
of
well as some properties of the glucosylated form (2). Here, we
have analyzed a sample of the -EAAG purified (as described
D-EAA and the presence of a D-EAAG in P. blakesleeanus, as
D
under Materials and Methods) by ESI mass spectrometry using a
Q-Tof micro mass spectrometer (Water Corp.) equipped with an
ESI LockSpray source. The ESI mass spectra acquired in the
4 and 12 eV);
-EAA ([M - H]- m/z 145, energy collision, 8 and 32 eV);
D

Article
J. Agric. Food Chem., Vol. 58, No. 19, 2010 10633
Table 1. UV Spectral Properties and Ionization Constants of D-Erythroascorbate Glucoside (D-EAAG) Obtained from Phycomyces blakesleeanus
pH 2.0
pH 4.0
pH 6.0
pH 7.0
pH 8.0
λ
max (nm) ε (mM-1 cm-1
)
λ
max(nm) ε (mM-1 cm-1
)
λ
max(nm) ε (mM-1 cm-1
)
λ
max(nm) ε (mM-1 cm-1
)
λ
max(nm) (mM-1 cm-1
)
pKa1 pKa2
D-EAAG
D-EAA
L-AA
243a
243b
243b
255
251
254
11.2
17.2
13.7
265
263
258
19.8
16.5
11.3
262
264
258
18.6
23.2
12.0
265
261
260
19.0
24.1
15.1
3.70 12.40
3.96 12.10
4.03 11.92
10b
10b
a From Baroja-Mazo et al. (2). b From Shao et al. (1).
negative ion mode showed an intense molecular ion [M - H]-
signal at m/z 307, which is in line with the molecular formula of
C11O10H16 (M = 308) corresponding to the molecular mass of
Table 2. Stability of D-Erythroascorbate (D-EAA) and D-Erythroascorbate
Glucoside (D-EAAG) under Aerobic Conditionsa
half-life (h)
D
-EAAG in which the glucosyl moiety is composed by a single
D-EAA
D-EAAG
glucose unit. This observed molecular ion is in agreement with the
observed ion at m/z 331.1 by ESI-MS analysis in the positive ion
mode from the fungus Sclerotinia sclerotiorum corresponding to
M þ H of the Na salt of the galactopyranosyl-
bate (14) and is also similar to that obtained with the glucopyr-
anosyl- -erythroascorbate from the basidiomycete Hypsizigus
temperature
(°C)
pH
4
0.23 mM
0.70 mM
0.23 mM
0.70 mM
D-erythroascor-
25
35
45
40.9 ( 3.90
16.2 ( 2.70
15.2 ( 0.06
stable for
156 h
stable
for 156 h
stable for
156 h
D
mamoreus (4). The fragmentation pattern of the ion with m/z
307 obtained by the MS/MS procedure involves formation of an
intense fragment ion at m/z 127 that could correspond to loss of
180 mass units to glucose moiety from the [M - H]- ion. It has
been reported that in flavonoid glycosides the cleavage at the
glycosidic O linkage with a concomitant H arrangement leads to
the elimination of dehydrated monosaccharide residues (15). On
6
8
25
35
45
64.3 ( 7.72
28.5 ( 0.06
15.6 ( 2.34
89.0 ( 11.40 182.4 ( 1.75 257.2 ( 37.54
31.6 ( 2.30
11.7 ( 1.20
91.1 ( 21.94 229.7 ( 56.81
9.7 ( 0.83
28.1 ( 7.7
25
35
45
69.1 ( 10.37 164.0 ( 23.00
35.0 ( 0.11
31.7 ( 7.00
6.2 ( 0.83
3.7 ( 0.01
1.5 ( 0.11
16.3 ( 0.45
13.5 ( 0.91
2.5 ( 0.17
38.9 ( 1.55
17.0 ( 0.60
the other hand, when a solution of D-EAAG was hydrolyzed with
a The half-lives were determined in 0.1 M sodium acetate buffer, pH 4, and 0.1 M
sodium phosphate buffer, pH 6 and 8, as described in the text. Values are the mean
of at least two experiments in duplicate ( standard deviation of the mean.
3 M HCl for 15 min at 98 °C, the MS spectra of the hydrolysate
showed [M - H]- and [M þ Cl]- signals at m/z 145 and 215 asso-
ciated with the molecular mass of D-EAA and the chlorine adduct
of glucose, respectively, as compared with the ESI mass spectra
obtained for the purified -EAA and glucose under the same
conditions. The MS/MS fragmentation patterns of the ion peaks
at m/z 145 and 215 obtained in the negative ion mode were similar
in both cases. A signal at m/z 145 was also obtained by negative
ion electrospray mass spectrometry by Spickett et al. (16) in the
D
-EAA display two acidic protons of pKa values similar to pKa
values for the 3- and 2-hydroxyls of -AA (Table 1). It has been
described that the substitution of the 2-OH of -AA increased the
D
L
L
acidity of the 3-OH, resulting in a decrease in the pKa value from
4.2 to 3.11-3.40, whereas substitution at 3-OH produces a drop
in the pKa value of the 2-OH from 11.8 to 6.22-7.9 (18). The
spectral properties of
as the pKa values that we obtained are similar to those of
characterization of
D-EAA from S. cerevisiae.
D
-EAAG from P. blakesleeanus as well
-AA,
-EAA
The glycosidic residue is sometimes crucial for the activity of
the biological compounds, and in other cases glycosylation pro-
vided an increase in its hydrophilicity (mainly affecting mem-
brane transport) and stability against UV light, heat, and oxida-
tion (17). Glycosylated ascorbic acids have been synthesized by
using the transglycosylation activity of Bacillus stearothermophilus
maltogenic amylase, with maltotriose showing effective anti-
oxidative activity with enhanced oxidative stability (8). Thus,
L
indicating that the glucosyl unit is attached to C-5 of
(Figure 1).
D
We evaluated the stability of D-EAAG and D-EAA for com-
parison in aqueous solution under aerobic conditions at three
different pH values, 4, 6, and 8 and three different temperatures,
25, 35, and 45 °C, respectively. Under all of the tested conditions,
oxidative degradation of both compounds follows first-order
kinetics. On the basis of analysis by nonlinear regression of plots
of percent residual concentration versus incubation time we
we have analyzed the effect of the glucosylation of D-EAA testing
the UV spectral properties, ionization constants, and stability
against chemical oxidation in aqueous solution under aerobic
calculated the half-lives of
aerobic conditions described in Table 2. As can be seen,
was much more stable at acidic pH values than at basic pH.
-EAAG maintained its spectrophotometric activity completely
for almost 7 days at pH 4 for all of the temperatures studied in the
concentration range between 0.033 and 0.11 mg/mL. -EAA was
more stable at pH 8 than -EAAG, whereas the opposite occurs
at acidic pH values. Shao et al. (1) stated that -AA and -EAA
are more stable at pH 4 and 8 than at pH 6, as was observed for
-EAA at the high concentration used in this study. On the other
D-EAA and
D-EAAG under the
conditions of the
Table 1 illustrates the pH effect on UV spectral properties and
ionization constants of the -EAAG from P. blakesleeanus.
-EAAG showed a bathochromic shift and hyperchromic effect
D-EAAG.
D-EAAG
D
D
D
when the pH was increased from acidic to basic values. λmax
shifted toward a longer wavelength, about 20 nm, when the pH
was increased from 2 to 8, and ε (molar extinction coefficient) was
increased from 11.2 to 19.0 mM-1 cm-1 when the pH rose from
4 to 8. As can be seen in Table 1, these properties resembled those
D
D
L
D
D
of
of
D
-EAA and
-AA, 6-O-R-
L
-ascorbate(
L
-AA). On the other hand, a glucoside
-ascorbic acid (AA-6G) (9),
-AA, -EAA,
-EAAG. However, 2-O-substituted AA derivatives showed
hand, the increase in initial concentration significantly decreased
the oxidative degradation rate of both compounds, which means
that D-EAA and D-EAAG stability can be substantially increased
by increasing their concentrations. This behavior can be expla-
ined because the concentration of both compounds is varied while
the oxygen concentration remains constant. It is known that ascor-
bateoxidationisdependentupontheoxygenconcentration(19,20).
L
D-glucopyranosyl-L
shows spectral data similar to those observed with
and
a characteristic bathochromic shift of about 5 nm to the shorter
wavelength at both pH 2 and 7, whereas the hypochromic effect
L
D
D
was observed only in 3-O-substituted AA (8, 18). D-EAAG and

10634 J. Agric. Food Chem., Vol. 58, No. 19, 2010
Gutierrez-Larrainzar et al.
Figure 2. Glucosyltransferaseactivity fromP. blakesleeanus mycelium: (A) HPLCchromatogram ofpurified
glucosyltransferase reaction product with D-EAA and UDP-glucose as susbtrates [(inset) D-EAAG formation at different reaction times]; (C, D) incubation time
D-EAA and
D-EAAG;(B) HPLC chromatogram of
andmycelial dependence of glucosyltransferaseactivity. Glucosyltransferase activity andHPLC chromatograms werecarried out asdescribed under Materials
and Methods.
The effect of temperature on the aerobic oxidation of
D
-EAA and
have a stabilizing effect. Thus, we analyzed the oxidative degra-
dation of -EAA (0.23 mM) in 0.1 M sodium phosphate buffer,
D-EAAG is expressed by the activation energy value (Ea) of the
D
oxidative degradation process, obtained from linear plots of log k
versus 1/T, k being the oxidative degradation constant. At pH 6,
we obtained Ea values of 14.7 and 24.1 kcal/mol, whereas at pH 8
pH 8, in the presence of an equimolar amount of glucose, at 25 °C.
The presence of glucose diminished about 5-fold the half-life of
D-EAA (from 69.2 to 14.8 h). It is possible that at alkaline pH
these values were 25.3 and 15.4 kcal/mol for
D
-EAA and
values, glucose facilitates the formation of a metal-
D
-EAAG
D-EAAG, respectively. An Ea value of 14.8 kcal/mol has been
monoanion-molecular oxygen complex, opposing thus the che-
described for aerobic oxidation of ascorbic acid in commercial
water (20), whereas Ea values of 3.3, 9.0, and 13.3 kcal/mol have
been reported for ascorbic acid degradation in tomato juice (21),
in pasteurized packed table olives (22), and in pasteurized orange
juice (23). Thus, because the pH values of many food systems,
such as fruit jams and jellies, fruit and vegetable juices, and
cheeses, are in the acidic pH range, we think that D-EAAG can be
the focus of attention due to its possible antioxidant role in many
food systems.
lating effect of phosphate ions and favoring the oxidative degra-
dation of
D-EAAG under these conditions.
Enzymatic Production of
D
-Erythroascorbate Glucoside. In
general, glycoside formation involves the presence of glycosyl-
transferase activity (GTs; EC 2.4.x.y), which in the vast majority
uses sugar donors containing diphosphate leaving groups, with
UDP/TDP leaving groups as the most common (for a review see
ref 25). We have found glucosyltransferase activity in mycelia
of P. blakesleeanus grown in a liquid minimal medium with 2%
(w/v) glucose ascarbon source in darkness and 13 mM asparagine
as nitrogen source. The glucosyltransferase activity was deter-
mined in the forward direction using UDP-glucose as sugar
As can be see in Table 2,
D-EAAG was much more unstable
that -EAA at pH 8. In the presence of molecular oxygen and
D
trace levels of transition metals (Cu2þ, Fe 3þ), it has been
proposed that the oxidation of ascorbic acid involves the forma-
tion of a metal-ascorbate monoanion-molecular oxygen com-
plex (19). Shao et al. (1) proposed that the metallic ions present
are probably more highly chelated by the phosphate ions in the
buffer at pH 8 than at pH 4 or 6, and this effect could explain the
substrate donor for
Figure 2A, incubation of
mycelial extract in the presence of 9 mM UDP-glucose resulted in
the time-dependent formation of -EAAG as determined by HPLC.
The retention time of the reaction product (6.15 ( 0.2 min)
corresponds to -EAAG by comparison with the retention times
obtained for authentic -EAAG and -EAA (Figure 2B) purified
as described by Baroja-Mazo et al. (2). In addition, UV spectra of
the reaction product from -EAA were indistinguishable from
the corresponding authentic -EAAG. As the incubation time
increased, the peak area corresponding to -EAAG increased
with a concomitant decrease in the peak area corresponding to
D-EAAG formation. As can be seen in
D-EAA with 60-h-old P. blakesleeanus
D
greater stability of
D-EAA and ascorbic acid at alkaline pH value,
D
because the oxidation of ascorbic acid by molecular oxygen
with various metal chelate compounds of Cu(II) and Fe(III) as
catalysts occurs at much slower rates than the corresponding
metal ion catalyzed reactions (24). However, this effect is not
D
D
D
D
observable in
D-EAAG oxidation at pH 8, suggesting that the
D
presence of glucose in the molecule at alkaline pH value does not

Article
J. Agric. Food Chem., Vol. 58, No. 19, 2010 10635
Table 3. Contents of D-EAA and D-EAAG in P. blakesleeanus Growing on Different Carbon Sources in the Dark with 13 mM Asparagine as the Nitrogen Source
μg/g of dry weighta
D-EAA
D-EAAG
carbon source
24 h
48 h
24 h
48 h
D-glucose
D-sorbitol
D-xylose
78 ( 10.3
157 ( 13.2
100 ( 12.6
47 ( 5.1
32 ( 1.4
0.55 ( 0.05
47 ( 5.6
74.8 ( 7.3
42.9 ( 1.5
88 ( 5.2
119 ( 8.7
94.7 ( 6.6
20.4 ( 1.8
56 ( 5.0
4.7 ( 0.3
nd
35 ( 3.4
186.5 ( 18
55 ( 3.7
173.4 ( 10.4
184.4 ( 5.7
111 ( 14.4
78 ( 7.6
470 ( 37.3
337 ( 29
nd
538 ( 56.5
458 ( 44.9
145 ( 9.5
287 ( 31.4
346 ( 38.1
427 ( 41.2
151.5 ( 14.5
nd
D-fructose
L-arabinose
D-galactose
D-mannose
sucrose
48.8 ( 4.3
nd
140 ( 12.4
2 ( 0.13
nd
131 ( 18.1
87 ( 5.1
maltose
D-mannitol
4.7 ( 0.2
nd
37.8 ( 3.5
69.4 ( 4.4
60.5 ( 5.1
30.5 ( 1.8
53 ( 6.1
D-glucose þ D-arabinoseb
63 ( 7.4
1.76 ( 0.15
4.2 ( 0.4
5.5 ( 0.38
1442 ( 129
1011 ( 149
746 ( 9.7
566 ( 55.1
D-glucose þ L-fucoseb
D-glucose þ L-gulono-1,4-lactoneb
D-glucose þ L-galactone-1,4-lactoneb
a Values are the mean of at least two experiments in duplicate ( standard deviation of the mean. nd, not detected. b D-Arabinose (10 mM), L-fucose (10 mM), L-gulono-1,4-
lactone (10 mM), or L-galactono-1,4-lactone (10 mM) was added at the onset of growth on D-glucose 2% (w/v).
D
-EAA substrate. Assays in the absence of the UDP-glucose
respectively, when P. blakesleeanus was grown on 2% (w/v)
glucose as carbon source (2). We have determined the accumula-
donor substrate or in the absence of mycelial extract yielded no
product (data not shown). On the other hand, in the enzymatic
mycelial extract used, prepared as described under Material and
Methods, no
HPLC analysis.
The glucosyltransferase activity shows a linear dependence on
the quantity of mycelial extract present in the incubation mixture
(Figure 2D) and also a linear dependence on the incubation
reaction time up to 60 min (Figure 2C). Thus, the enzymatic
activity measured under these experimental conditions corres-
ponding to an initial velocity of 78.2 pmol of
protein. The addition of Mg2þ (13.5 mM) to the reaction mixture
has no effect upon the extent of -EAA glucosylation, similar to
tion of
when it was grown in liquid minimal medium with the following
carbon sources: 2% (w/v) -mannose, 2% (w/v) -galactose, 2%
(w/v) -fructose, 1.8% (w/v) -arabinose, 2% (w/v) sucrose, 2%
(w/v) maltose, 2% (w/v) -xylose, 2% (w/v) -mannitol, and 2%
(w/v) -sorbitol. On the other hand, P. blakesleeanus did not grow
with 2% (w/v) -xylose, 1.8% (w/v) -arabinose, 2% (w/v)
-gulono-1,4-lactone, 2% (w/v) -galactono-1,4-lactone, or 1.8%
-fucose, as the carbon source. Thus, the effect of these
-EAA and -EAAG was deter-
mined from P. blakesleeanus cultures obtained with 2% (w/v)
-glucose plus 10 mM of each of them individually added at the
D-EAA and D-EAAG in mycelium of P. blakesleeanus
D-EAA or
D-EAAG presence was detected by
D
D
D
L
D
D
D
L
D
L
L
(w/v)
L
D-EAAG/min/mg of
sugars on the accumulation of
D
D
D
D
what has been described for UDP-glucose:flavonoid 3-O-gluco-
syltransferase from Vitis vinifera (26) and for UDP-glucose:
anthocyanin 30,50-O- glucosyltransferase from Clitoria ternatea
(27). We have not detected either the reverse reaction, that is, the
onset ofgrowth. Inall cases, asparagine (13 mM) was the nitrogen
source. The effect of all these carbon sources on the accumulation
of D-EAA and D-EAAG was determined in 24- and 48-h-old
mycelia of P. blakesleeanus corresponding to the second half of
generation of
glucosylation reaction using maltose as sugar donor substrate
for -EAAG formation under our experimental conditions. The
formation of glucosyl derivatives of -ascorbic acid with mam-
malian R-glucosidase by enzymatic tranglucosylation from mal-
tose to -ascorbic acid (9) has been described.
No significant glucosyltransferase activity was present at the
onset and during the exponential growth phase of P. blakesleea-
nus, increasing at the onset of the stationary growth phase and
reaching maximal activity at 72 h of growth and then decreasing
from this time. This maximal glucosyltransferase activity was in
D
-EAA from UDP and
D
-EAAG, or trans-
the exponential growth phase and the onset of the stationary
growth phase, respectively, when 2% (w/v)
carbon source.
D-glucose was the
D
L
Table 3 shows the contents of
blakesleeanus growing on the different carbon sources used in
this study under the conditions indicated above. -EAA and
-EAAG were detected in 24-h-old mycelia under all conditions.
D-EAA and D-EAAG in P.
L
D
D
With respect to 48-h-old mycelia,
D
-EAA was not detected in
-sorbitol cul-
-mannitol cultures. It is
-EAA accumulates 10- 13-, and 30-fold, respec-
tively, in comparison with -glucose cultures in 48-h-old mycelia
grown with -fructose, -arabinose, and -galactose. The cul-
tures on -galactose and -fructose showed a larger lag in com-
parison with the -glucose cultures. Thus, at 48 h, the -galactose
L
-arabinose,
tures, and -EAAG was not detected in
noticeable that
D-xylose, D-mannitol, sucrose, and D
D
D
D
parallel with the production of
levels also in the stationary growth phase (data not shown).
The kinetics of -EAA glucosyltransferase were studied for
D-EAAG, which shows the higher
D
D
D
D
D
D
D
both donor and acceptor substrates. Hyperbolic saturation curves
were obtained for both substrates, from which Lineweaver-Burk
transformation gave an apparent Km values of 2.5 mM for UDP-
D
D
cultures are at a stage of growth that could be equivalent to
accumulation on glucose cultures at 24 h of growth. The produc-
glucose and 41.3 μM for
D
-EAA, with apparent Vmax values of
tion of
L
D
-EAA and
-arabinose as carbon source may be due to the fact that
-arabinose catabolism is closely linked to the -xylose pathway
D-EAAG when P. blakesleeanus grew on
0.11 and 0.07 nmol -EAAG/min/mg of protein, respectively.
D
The apparent Km value obtained for UDP-glucose is similar to
those reported for other glucosyltransferase from V. vinifera (26)
and C. ternatea (27).
L
D
in filamentous fungi (28), involving a series of redox transforma-
tions from which -arabinose is converted into -xylulose, which
is reduced to xylitol and which can be oxidized to -xylose by a
xylose reductase. This hypothesis is supported by the higher
accumulation of -EAA in P. blakesleeanus grown with -xylose.
L
L
D
-Erythroascorbate and
tion from P. blakesleeanus.
during the exponential and stationary phases of mycelial growth,
D
-Erythroascorbate Glucoside Produc-
D
D
-EAA and -EAAG accumulated
D
D
D

10636 J. Agric. Food Chem., Vol. 58, No. 19, 2010
Gutierrez-Larrainzar et al.
Table 4. Effect of D-Arabinose and Light on the Maximal Mycelial Content of
D-EAA and D-EAAG
Table 5. Maximal Accumulation of D-EAA and D-EAAG in the Culture Medium
When P. blakesleeanus Grows under the Described Conditionsa
μg/g of mycelial dry weighta
culture medium content
(μg/mL)
mycelial content
(μg/mL)
growth conditions
D-EAA
D-EAAG
growth conditions
D-EAA
D-EAAG
D-EAA
D-EAAG
D-glucoseb
99.5 ( 5.8
125.4 ( 1.4
2664.5 ( 15.7
3180.0 ( 90.0
1084.2 ( 40.3
3755.0 ( 221
3997.0 ( 758
4683.0 ( 586
D-glucose in lightc
þD-arabinosed
þD-arabinosee
D-glucoseb
0.45
43.10
102.1
1.80
13.04
15.25
0.52
6.12
57.0
52.22
281.50
272.61
þD-arabinosec
þD-arabino-1,4-lactonec
a Values are the mean of at least two experiments in duplicate ( standard
deviation of the mean. b Growth on D-glucose 2% (w/v) in the dark as the control.
c Growth on D-glucose 2% (w/v) in white light (0.5 W/m2). d After 60 h of growth on
D-glucose 2% (w/v) in the dark, 10 mM D-arabinose was added to the culture follow-
ing growth under the same conditions for almost 36 h. e After 60 h of growth on
D-glucose 2% (w/v) in light, 10 mM D-arabinose was added to the culture following
growth under the same conditions for almost 36 h.
D-EAA and D-EAAG were determined by HPLC as described under Materials
and Methods. For comparison, the mycelial content was expressed in μg/mL by
assuming a water content of 90% for Phycomyces sporangiophore (32). b 48 h of
growth on D-glucose 2% (w/v) in the dark. c After 12 h of growth on D-glucose 2%
(w/v) in light, 10 mM D-arabinose or 10 mM D-arabino-1,4-lactone was added, and
the cultures were kept under these conditions for another 36 h.
a
The maximal amount of
D
-EAA present in glucose-grown
growth phase (18 h) with all nitrogen sources studied, except
glycine and leucine. In both cases P. blakesleeanus cultures
showed a large lag in the growth curve (2 days for glycine), and
mycelia at 30 h of growth of P. blakesleeanus in darkness (100 (
5.8 μg/g of mycelial dry weight) is much higher than that reported
for S. cerevisiae (1.67 ( 0.26 μg/g of dry weight) (5) and for
mycelium of S. sclerotiorium (<40 μg/g of dry weight) (29). The
this could be the reason of no detection of
D-EAA in these con-
ditions. The higher accumulation of -EAA was obtained with
D
presence of 10 mM
produced by P. blakesleeanus (about 120 μg/g of mycelial dry
weight). This value is close to the amount of -EAA produced by
the functional expression of S. cerevisiae -arabinono-1,4-lactone
oxidase in E. coli (178 μg/g of wet cells) (30) and lower than
S. cerevisiae grown in the presence of -arabinose (200.8 (
21.2 μg/g of dry weight) (5). However, P. blakesleeanus also pro-
duced -EAAG, and thus the total amount of both compounds
exceeded the content reported for S. cerevisiae by about 8-fold.
The presence of -arabinose (10 mM) at the onset of growth
increases the amounts of -EAA and -EAAG present in the
cultures (Table 3), and because only -EAA and -EAAG are
accumulated in these conditions (2), we analyzed the -arabinose
effect when it was added at 12, 24, 48, and 60 h of growth on 2%
(w/v) -glucose after following the incubation for almost 36 h. In
general, the addition of -arabinose at the different times of
glucose culture appears to have a greater effect on the level of
-EAA, the maximum being reached when -arabinose is added
at the stationarygrowth phase. Table 4 shows the maximum levels
of -EAA and -EAAG obtained in the presence of 10 mM
-arabinose. The maximum content of -EAA increases 27-
32-fold in comparison with that obtained in -glucose culture as
the control, whereas the maximum -EAAG level was only about
4-fold that of the control culture. It appears to be evident that the
synthesis of -EAAG is more dependent on the glucosyltrans-
ferase activity responsible for the glucosylation of -EAA, whereas
the synthesis of the latter is naturally more influenced by the
presence of -arabinose, which is the substrate for -arabinose
dehydrogenase that produces the immediate precursor of -EAA,
-arabino-1,4-lactone. The addition of -arabino-1,4-lactone
D
-arabinose increases the amount of
D
-EAA
urea plus zinc ions with values 8- and 13-fold higher than those
obtained from control cultures. It has been reported that
P. blakesleeanus growth is much better on glucose-urea media
when cultures are supplied with 10-4 M zinc ions than glucose-
D
D
asparagine (for revision see ref 32). In all cases D-EAAG accu-
D
mulates in the highest amounts at 96 h of growth with values
similar to those shown by control cultures with asparagine. These
D
results seem to indicate that
D-EAA production was more depen-
dent on the nitrogen source than
D-EAAG formation and
D
reaffirm the association between mycelial growth and D-EAA
as we have previously reported (2).
D
D
D
D
Finally, we had already reported that P. blakesleeanus excretes
into -EAA and -EAAG growth medium (2). Table 5 shows the
maximal accumulation of -EAA and -EAAG in the culture
D
D
D
D
D
D
medium. Assuming a water content of 90% for Phycomyces spor-
angiophore (32), we calculated the mycelial content (expressed in
μg/mL) of both compounds for comparison. As can be seen,
D
D
D
P. blakesleeanus excreted
medium, although it is clear that the mycelial content of
is much greater than that excreted into the culture medium,
whereas the content of -EAA is higher in the culture medium
than inside the mycelium, except in the control on -glucose. The
addition of -arabinose or -arabino-1,4-lactone to the culture
medium increased mycelial content of -EAA with a parallel
increase in its content in the culture medium, but the increase in
mycelial -EAAG observed under the same conditions is not
accompanied by a parallel increase in -EAAG into the culture
D-EAA and
D-EAAG into the culture
D-EAAG
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
medium. Specific transport systems for ascorbate and dehydroas-
corbate in animal, human, and plant cells has been described
(33, 34), indicating that simple diffusion plays only a very minor
role. Therefore, assuming the existence of a similar transport
D
D
D
(10 mM) to the culture medium did not have a significant effect
on the maximum levels of either compound. P. blakesleeanus
shows responses to the light that involve changes in metabolism,
growth, and positive phototropism (31). Thus, we tested the effect
system in P. blakesleeanus, it seems clear that it prefers
D-EAA
over -EAAG. High-affinity transporters have been described for
D
both ascorbate and dehydroascorbate (33, 34), suggesting that
they potentially regulate the level and redox status in the cell.
Obviously, more studies are needed to establish the existence of a
similar transport system in P. blakesleeanus, which was respon-
of light on
presence of
light, in the absence or presence of
increase in the maximum levels of
D
-EAA and
-arabinose in the culture medium. The presence of
-arabinose, resulted in a slight
-EAA and -EAAG (Table 4).
We also tested the effect of the nitrogen source on -EAA and
-EAAG production when asparagine was replaced by the follow-
D-EAAG production in the absence or
D
D
D
D
sible for the excretion of both
culture medium.
D-EAA and D-EAAG into the
D
D
Evaluation of the Antioxidative and Antimicrobial Activities of
-Erythroascorbate and -Erythroascorbate Glucoside. We deter-
mined the antioxidant activities of -EAA and -EAAG in order
ing compounds in P. blakesleeanus cultures: glycine, alanine,
glutamic acid, aspartic acid, leucine, and ammonium chloride, all
at 26 mM, arginine (9 mM), and urea (13 mM) plus Zn2þ (10-4 M).
D
D
D
D
to discuss the possibility of their being used as antioxidants. As it
is well-known that the numerous methods available for assessing
D-EAA accumulation was maximal at the onset ofthe exponential

Article
J. Agric. Food Chem., Vol. 58, No. 19, 2010 10637
Table 6. Antioxidant Activity of D-EAA, D-EAAG, and Standard Antioxidants
Measured by DPPH, ABTS, and FRAP Assays
BHA against S. aureus (14-32 times), whereas they are virtually
unchanged for E. coli, and could therefore be used as substitutes
for BHA and gallic acid in inhibiting the growth of Gram-
μM Trolox equivalent
negative bacteria. The MIC50 values for
L-AA, D-EAA, and
antioxidant
DPPH
ABTS
FRAP
D-EAAG were identical to those of MIC90 for E. coli strains
against these compounds. However, they are less effective against
S. aureus than BHA and gallic acid. Obviously, further extensive
work should be performed to establish precisely the antimicrobial
D-EAA
D-EAAG
L-AA
2.80
4.91
1.00
4.80
1.05
1.15
1.48
1.80
0.93
1.17
3.73
3.25
0.69
1.12
2.50
2.50
1.12
2.09
0.30
0.30
1.11
potency of D-EAA and D-EAAG.
gallic acid
resveratrol
BHT
ABBREVIATIONS USED
-EAA, -erythroascorbate;
coside; -AA, -ascorbate; Trolox, 6-hydroxy-2,5,7,8-tetramethyl-
BHA
D
D
D-EAAG, D-erythroascorbate glu-
Table 7. MICa Values of D-EAA, D-EAAG, and Other Selected Antioxidants
MIC (μg/mL)
L
L
chroman-2-carboxylic acid; ABTS, 2,20-azinobis(3-ethylbenzothia-
zoline-6-sulfonic acid); DPPH, 2,20,diphenyl-1-picrylhydrazyl;
TPTZ, 2,4,6-tris-2,4,6-tripyridyl-2-trizine; BHA, butylated hydro-
compound
BHA
bacterium
range
MIC50
MIC90
xyanisole; BHT, butylated hydroxytoluene; AA-6G, 6-O-R-
D-gluco-
S. aureus
E. coli
200-300
1600-6400
87.5-1600
3200-6400
1600-2400
6400
200
3200
125
280
6400
pyranosyl- -ascorbic acid; MIC, minimal inhibitory concentration.
L
gallic acid
ascorbic acid
D-EAA
S. aureus
E. coli
1600
6400
ACKNOWLEDGMENT
4800
1600
6400
6400
6400
1600
>6,400
S. aureus
E. coli
2,400
6400
We thank Dr. M. Paniagua (Molecular Biology, Genomic and
Proteomic Center of University of Leon) for the mass spectro-
metry analysis of D-erythroascorbate glucoside.
S. aureus
E. coli
6400
6400
6400
6,400
>6400
>6,400
D-EAAG
S. aureus
E. coli
1600->6400
>6400
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Received for review March 2, 2010. Revised manuscript received August
4, 2010. Accepted August 6, 2010. C. de Castro was the recipient of
fellowships from the Spanish Ministry of Education and Science (FPI).
This work was supported by the Spanish Ministry of Education and
Science Research Grant AGL2005-01793. D.d.A. and P.d.V. contributed
with equal responsibility to conducting this study.