NMR study of histidine metabolism during alcoholic and malolactic fermentations of wine and their influence on histamine production

Article abstract of DOI:10.1021/jf402489g

The metabolic pathways of amino acids play a crucial role in the organoleptic and hygienic quality in wines. In particular, histidine is one of the most studied amino acids of wines due to histamine toxicity in humans, a biogenic amine derived from histidine by enzymatic decarboxylation. The development of new tools to increase knowledge on metabolism that produces histamine in wine is critical. This study investigated by using nuclear magnetic resonance (NMR) spectroscopy the transformation of histidine into histaminol and histamine during alcoholic and malolactic fermentations. The transformations of histidine into histaminol during alcoholic fermentation and into histamine during malolactic fermentation were observed. This paper highlights the importance of selecting lactic acid bacteria for malolactic fermentation to avoid the production of biogenic amines such as histamine.

Full text of DOI:10.1021/jf402489g

Article
pubs.acs.org/JAFC
NMR Study of Histidine Metabolism during Alcoholic and Malolactic
Fermentations of Wine and Their Influence on Histamine Production
Eva Lopez-Rituerto, Alberto Avenoza, Jesus H. Busto,* and Jesus M. Peregrina*
́ ́ ́
́
̃
Departamento de Quımica, Centro de Investigacion en Sıntesis Quımica (CISQ), Universidad de La Rioja, E-26006 Logrono, Spain
́ ́ ́
ABSTRACT: The metabolic pathways of amino acids play a crucial role in the organoleptic and hygienic quality in wines. In
particular, histidine is one of the most studied amino acids of wines due to histamine toxicity in humans, a biogenic amine derived
from histidine by enzymatic decarboxylation. The development of new tools to increase knowledge on metabolism that produces
histamine in wine is critical. This study investigated by using nuclear magnetic resonance (NMR) spectroscopy the
transformation of histidine into histaminol and histamine during alcoholic and malolactic fermentations. The transformations of
histidine into histaminol during alcoholic fermentation and into histamine during malolactic fermentation were observed. This
paper highlights the importance of selecting lactic acid bacteria for malolactic fermentation to avoid the production of biogenic
amines such as histamine.
KEYWORDS: wine, nuclear magnetic resonance, histidine, histamine, histaminol
INTRODUCTION
decarboxylation of the corresponding amino acid precursors
Figure 1). These higher alcohols and biogenic amines are
■
(
Amino acid content in wine depends on many factors, such as
grape berry variety, viticultural techniques, optimal maturation,
ammonium content, enological techniques, yeast, bacteria,
storage, and aging. Amino acids together with proteins and
peptides play an important role as nitrogen sources for yeast
and lactic acid bacteria during alcoholic and malolactic
formed during fermentative processes, aging, and storage of
7
foods, and they may produce adverse effects in humans in high
concentrations, especially when ethanol is present. Biogenic
amines may induce headaches, respiratory distress, heart
palpitations, hyper- or hypotension, and several allergic
8
1
,2
disorders. Therefore, studies on the evolution of amino acids
fermentations, respectively. The term YAN (yeast assimilable
nitrogen) represents the optimal nitrogen level for a good
performance of fermentation. The amount of nitrogen
influences the wine quality. A low level of YAN in grape
must leads to low yeast populations and also to the reduction of
yeast growth during wine fermentation, thus increasing the risk
of “stuck” fermentation and the production of undesirable
during alcoholic and malolactic fermentations should be taken
into account to evaluate the quality of wines. However, only
studies applying some analytical techniques, mainly HPLC,
9
have been performed to date.
In this regard, NMR spectroscopy is being applied as an
alternative technique for evaluating wine quality. In metab-
olomics, NMR has emerged as an effective method to
3
,4
compounds.
1
0,11
investigate metabolic changes
and then to compare,
Amino acids are precursors of low molecular weight
compounds such as fusel alcohols and biogenic amines, which
have high impacts on wine quality. Fusel or higher alcohols,
containing more than two carbon atoms in their structure,
together with their ester derivatives play a critical role in the
aromatic complexity of wines.
into higher alcohols through the well-known Ehrlich pathway
Figure 1). Biogenic amines are produced by microbial
1
2−19
distinguish, and classify different wines
or grape
20−22
1
berries.
Consequently, Hong and co-workers used H
NMR to evaluate the behavior of different yeast and lactic acid
23−25
bacteria strains
and, by combining NMR spectroscopy
1
,5,6
Yeasts convert amino acids
with multivariate statistics, they could classify wines and grape
2
6,27
berries according to variety and geographical origin.
1
(
Recently, we have reported the use of quantitative H NMR
for monitoring and controlling biological processes such as the
alcoholic and malolactic fermentations, as well as for wine
classification according to vintage, region of production, and
28−30
winery.
The combination of 13C NMR spectroscopy with the use of
isotopically C-labeled molecules as tracers is a well-established
13
protocol for the identification of metabolic pathways in
31−33
microbiology.
Although this protocol is very valuable for
the identification of metabolites, and hence the metabolic
Received: June 7, 2013
Revised: September 6, 2013
Accepted: September 9, 2013
Figure 1. Transformation of amino acids into alcohols by the Ehrlich
pathway and into biogenic amines (top) as well as transformation of L-
histidine into its higher alcohol and into histamine (bottom).
©
XXXX American Chemical Society
A
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Journal of Agricultural and Food Chemistry
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1
pathways, it has not been commonly used for quantification.
alcoholic and malolactic fermentations were monitored by H NMR
28,29
1
3
using a quantitative method described previously.
On this basis, we have recently applied C NMR to study the
1
3
Alcoholic Fermentation. To follow the metabolic transformation
of L-histidine during alcoholic fermentation, we added 40.9 mg of
labeled L-histidine into an Erlenmeyer flask with 500 mL of grape must
to obtain a final concentration of 81.0 mg/L of L-[2- C ring]-histidine
99% C, sample A), and 41.0 mg of nonlabeled histidine was added
into an Erlenmeyer flask with 500 mL of grape must to obtain a final
concentration of 81.2 mg/L (sample B). Moreover, we carried out
another experiment in an Erlenmeyer flask with 500 mL of grape must
without addition of histidine (sample C). Alcoholic fermentation was
carried out under optimal conditions of temperature (25 °C), and
samples were collected at different time intervals (0, 9, 21.5, 26, 30, 34,
metabolic transformations of some C-labeled amino acids into
their corresponding higher alcohols during the alcoholic
3
4
fermentation. Because histidine is present in grape juice and
wine, its concentration could be a parameter for quality
assesment. Therefore, herein, we have investigated the
transformation or metabolism of histidine during alcoholic
13
13
(
1
13
and malolactic fermentations through H and C NMR
1
3
experiments of samples containing C-labeled histidine.
According to the Ehrlich pathway, histidine is transformed by
yeast into a higher alcohol (Figure 1). However, this alcohol
has not been extensively studied even though its presence in
4
6, 50, 54, 58, 71.5, 78, 84.5, 98, and 105.5 h). The alcoholic
fermentations were monitored by H NMR, and the ethanol signal at
1
35
wine has been detected by HPLC methods, by Bordiga et al.
The same authors indicated the need to study the origin of this
1
.17 ppm was initially observed at 30 h for all samples.
Spontaneous Malolactic Fermentation. To follow the meta-
3
6,37
alcohol on the basis of previous papers
showing that yeast
bolic transformation of L-histidine during malolactic fermentation, we
added 20.0 mg of labeled L-histidine into 250 mL of wine (activated
with bacterial biomass from the winery) to obtain a final concentration
of 79.2 mg/L of L-[2- C ring]-histidine (99% C, sample A) and 19.0
mg of nonlabeled histidine to obtain a final concentration of 75.2 mg/
L (sample B). Furthermore, we carried out another fermentation
without addition of histidine (sample C). Spontaneous malolactic
fermentation was induced under optimal conditions of temperature
25 °C), and samples were collected at different time intervals (1, 4, 7,
0, 15, and 60 days). Malolactic fermentations were monitored by H
NMR, and the transformation of malic acid (2.83 ppm) into acid lactic
(1.40 ppm) was observed for all samples.
Activated Malolactic Fermentation. To follow the metabolic
transformation of L-histidine during the malolactic fermentation with
commercial lactic acid bacteria O. oeni, we added 39.4 mg of labeled L-
histidine into 500 mL of wine (activated with Uvaferm Alpha,
Lallemand Bio S.L.) to obtain a final concentration of 78.0 mg/L of L-
and mycobacteria are able to transform histamine into the same
higher alcohol. In this regard, a previous study showed that
higher alcohols such as 2-phenylethanol, 3-methyl-1-butanol,
and (S)-2-methyl-1-butanol were produced through the Ehrlich
13
13
34
transformation of the corresponding amino acids.
On the other hand, lactic acid bacteria such as Oenococcus
oeni transform histidine into histamine during the malolactic
fermentation. Such transformation has been widely studied
(
1
1
38
39,40
41
from microbiological, biochemical,
and chemical points
of view. Histidine decarboxylase enzyme (HDC) is responsible
for the transformation of histidine into histamine (Figure 1).
The production of histamine has been recently reviewed by
42
̃
Garcia-Moruno and Munoz. Histamine is one of the biogenic
amines in wine that may cause negative effects on human
health. The presence of histamine in wine has a great impact,
and there are countries that recommend upper limits for this
13
13
[
2- C ring]-histidine (99% C, sample A) and 41.0 mg of nonlabeled
histidine to obtain a final concentration of 81.0 mg/L (sample B). In
addition, we carried out another experiment without addition of
histidine (sample C). Activated malolactic fermentation was carried
out under optimal conditions of temperature (25 °C), and samples
were collected at different time intervals (1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9,
43
biogenic amine ranging between 2 and 10 mg/L. Several
methods have been developed for the analysis of biogenic
amines, including histamine, in foods: thin-layer chromatog-
raphy (TLC), gas chromatography (GC), a capillary electro-
phoretic method (CE), and high-performance liquid chroma-
1
1, 13, 16, 19, 21, 23, 27, 30, 33, and 37 days). Malolactic
1
44
fermentations were monitored by H NMR, and the transformation of
malic acid (2.83 ppm) into acid lactic (1.40 ppm) was observed for all
samples.
tography (HPLC).
In the current study, we introduce a simple and easy 13C
NMR analysis to study the metabolism or transformation of
histidine into histamine and histaminol during wine fermenta-
tion processes.
1
NMR Spectroscopy. H NMR spectra were recorded on a Bruker
Avance 400 spectrometer equipped with a 5 mm inverse probe (BBI
H-BB Z-GRD). Acquisition of spectra was carried out with TOPSPIN
software (version 1.2). Processing was performed with MestReNova
4
5
(
version 7.0.2). The spectrometer was locked onto D O in a mixture
2
MATERIALS AND METHODS
■
H O/D O (9:1), and all spectra were acquired at 298 K.
2
2
1
Samples. Alcoholic Fermentation. Red grapes (Tempranillo grape
variety, Vitis vinifera) were manually collected from Urun
Spain. Must chemical composition was as follows: sugar (density),
H NMR spectra were recorded with the standard pulse sequence
̃
uela, La Rioja,
for presaturation of the water signal at 1875 Hz; zgpr (1D sequence
with f1 presaturation) with pl9 (power level for presaturation) at 60
dB and a flip angle of 90°. Experiments were carried out with
automatic tuning and matching (ATM) and with GRADSHIM tools,
using the NMR CASE as a NMR sample changer allowing automatic
1
.110 g/mL; total acidity, 3.33 g/L (H SO ); pH, 3.35; malic acid,
2 4
3.23 g/L. Destemmed and crushed grapes were homogenized, and a
prefermentation cold maceration was carried out. Color extraction
enzymes were used (750 g/420 hL), and the concentration of free
sulfur dioxide was 39 mg/L. Alcoholic fermentations were carried out
in sterile Erlenmeyer flasks kept in an incubator regulated at 25 °C
with activated Uvaferm BC (Lallemand Bio S.L.).
1
analysis of several samples. Quantitative H NMR was carried out
2
8,29
using the methodology previously reported.
1
3
C NMR spectra were recorded on a Bruker Avance 400
spectrometer equipped with a 5 mm inverse probe (PABBO BB-
1H/D Z-GRD). Inverse gated decoupled methodology was used.
Proton decoupling was applied only during the acquisition period. In
this case, no polarization transfer from H to C via NOE takes place;
therefore, the resulting H-coupled C spectrum should be used for
quantitative measurements.
Phase-sensitive gradient-enhanced 2D heteronuclear single-quan-
tum correlation (HSQC) spectra were recorded by using hsqcedgpph
pulse program (z filter and selection before t removing decoupling
during acquisition) with CNST2 J( C, H) = 145 Hz [heteronuclear
46
Malolactic Fermentation. Alcoholic fermentation to produce red
wine was carried out at winery and chemical composition as follows:
alcoholic degree, 12.4 g/L; total acidity, 4.42 g/L (H SO ); acetic acid,
1
13
2
4
1
13
0.45 g/L; reducing sugar, 2.62 g/L; pH, 3.75; malic acid, 3.41 g/L.
Malolactic fermentations were carried out in sterile Erlenmeyer flasks
kept in an incubator regulated at 25 °C, activated with commercial
preparations of O. oeni lactic acid bacteria, Uvaferm Alpha (Lallemand
Bio S.L.), which was considered as the activated malolactic
fermentation and also with bacterial biomass from the winery, which
was considered as the spontaneous malolactic fermentation. Both
1
13
1
13
1
scalar J( C, H) coupling].
B
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Journal of Agricultural and Food Chemistry
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Processing of Spectra. Free induction decays (FIDs) from one-
dimensional files were exported into the MestReNova software and,
prior to carrying out Fourier transformation, an exponential window
4
7
function was applied to obtain an optimal signal-to-noise ratio. The
number of data points in the real part of spectra was set to 64K.
Spectra phases were manually corrected by selecting the submenu
“
Phase Correction”, and baselines were adjusted by the “Polynomial
46
Fit” function in accordance with the literature. Signal integration, in
1
13
both H NMR and C NMR spectra, was manually carried out, and
processing data were achieved twice. Metabolites were assigned by
two-dimensional NMR experiment, spiking experiments, and/or
2
8,29
information published elsewhere.
RESULTS AND DISCUSSION
■
Histidine is an essential amino acid for humans, and the
4
concentration range in grape juice is 5−197 mg/L; in all
experiments of the current study, the quantity of histidine
added was therefore within this range. In NMR experiments,
13
13
the C NMR chemical shift of the L-[2- C ring]-histidine was
observed at 137.0 ppm and the proton attached to this carbon
1
at 8.66 ppm in the H NMR spectrum. Notably, chemical shifts
of histidine protons substantially depend on pH. Thus, the
imidazole ring of the histidine is protonated at the pH of wine,
3
4
0
8
modifying the proton chemical shifts around 1 ppm
compared to the chemical shifts of histidine at a neutral pH.
Alcoholic Fermentation. Three different processes
1
3
(
samples A, with C-labeled histidine; B, with histidine; C,
1
without histidine) were monitored using H NMR to evaluate
the consumption of histidine through alcoholic fermentation
process and the influence of the labeled amino acid in these
processes. Histidine was quantified by the methodology
2
8,29
developed in our group,
which allows a simultaneous
1
quantification of several compounds in wine by H NMR
spectroscopy. The histidine transformation was similar for
samples A and B, with the added amino acid disappearing at
1
05.5 h. Moreover, we could conclude that yeast does not
discriminate between labeled and nonlabeled amino acids
Figure 2A).
Although we visualized by H NMR the disappearance of
(
1
histidine during alcoholic fermentation, we did not know the
nature of the metabolites generated. For this purpose, in
subsequent experiments, we used isotopically labeled histidine.
13
The C NMR spectrum at initial time (0 h) showed a peak at
37.0 ppm, corresponding to labeled C2 carbon of histidine
1
1
Figure 2. (A) Time course evolution of L-histidine (by H NMR, g/L)
in samples A, with 13C-labeled histidine; B, with histidine; and C,
without histidine. (B) C NMR spectra for monitoring trans-
formations of L-histidine into histaminol: (a) sample A at 0 h; (b)
sample A at 71.5 h; (c) sample A at 105.5 h; (d) sample C (control) at
05.5 h. (C) Time course evolution of L-histidine, histaminol (by C
NMR, absolute integrals), and alcoholic degree (by H NMR, v/v).
aromatic ring (Figure 2B, spectrum a). When alcoholic
fermentation began, this peak decreased quickly, disappearing
at 84.5 h after initiation of alcoholic fermentation. At 71.5 h, the
spectrum showed two different peaks (Figure 2B, spectrum b),
the histidine signal and a new one located at 136.0 ppm,
13
1
3
1
1
1
3
corresponding to a novel C-labeled compound derived from
labeled histidine. This new compound was assigned to the
higher alcohol 2-(1H-imidazol-4-yl)ethanol (histaminol). The
assignment of histaminol was confirmed by spiking with
commercial histaminol and by two-dimensional (2D) hetero-
nuclear multi bond correlation (HMBC) spectroscopy,
main parameter in wine, such as ethanol, is shown. According
to our previous works, histidine was completely transformed
into histaminol in the beginning of the alcoholic fermentation,
when the alcoholic degree is nearly 5 v/v (Figure 2C). No
other metabolites associated with the Ehrlich pathway (α-keto
acids and/or aldehydes derived from histidine or histamine)
were detected in any spectra. Considering the data reported, it
is reasonable to think that histamine in wine does not originate
from the metabolism of histidine in the alcoholic fermentation
process but in malolactic fermentation, depending on bacteria
used. Therefore, we concluded that the production of
histaminol in wine was likely a consequence of the metabolism
of histidine by yeast.
1
13
between two bonded H− C atoms, as shown in Figure 3.
In the spectrum at 105.5 h (Figure 2B, spectrum c), we were
able to observe the total disappearance of the representative L-
1
3
[
2- C ring]-histidine signal and its complete transformation to
13
labeled [2- C ring]-histaminol, likely following an Ehrlich
process (Figure 1). All of these spectra were compared with
those obtained from the control sample C, in which we did not
observe any 13C signals (Figure 2B, spectrum d). Time course
evolution of labeled metabolites together with progress of the
C
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Journal of Agricultural and Food Chemistry
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Figure 3. HMBC for sample A at 71.5 h.
Spontaneous Malolactic Fermentation. The other
essential metabolic process in wine elaboration is malolactic
fermentation by lactic acid bacteria. In most cases, these
bacteria are present in the tanks in which fermentation takes
place, and this process can occur spontaneously without the
addition of more bacteria by indigenous strains. Taking into
account the different metabolic processes that occur in
malolactic fermentation, it is necessary to study the behavior
1
3
of histidine in these processes. The C NMR spectrum at
initial time (day 1) showed a peak at 137.0 ppm corresponding
to labeled histidine (Figure 4A, spectrum a). When malolactic
fermentation began, this peak decreased quickly, and at day 4
two adjacent peaks (Figure 4A, spectrum b) were observed in
spectrum, consisting of the labeled histidine at 137.0 ppm and
the labeled histamine at 136.9 ppm. To corroborate this
assignment, we compared this value with that obtained in the
1
3
C NMR experiment carried out on commercially available
histamine. In the spectrum at day 15 (Figure 4A, spectrum c),
we were able to observe the presence of histamine, but not
histaminol or other metabolites associated with histidine
(
Figure 4A). Time course evolution of labeled metabolites
13
was checked by C NMR during malolactic fermentation, and
transformation of malic acid into lactic acid was monitored by
1
13
H NMR. Both progressions are described in Figure 4B ( C
1
NMR) and 4C ( H NMR), showing that the transformation of
histidine by lactic acid bacteria took place simultaneously with
transformation of malic acid. The complete transformations of
histidine and malic acid were observed around 15 days. At the
same time, the histamine and lactic acid were appearing. In view
of these results, the presence of large quantities of histidine at
the beginning of malolactic fermentation could likely be the
main source of production of histamine in wine.
Figure 4. (A) 13C NMR spectra for monitoring transformations of L-
13
histidine into histamine for samples A, with C-labeled histidine, and
C, without histidine: (a) sample A at 1 day; (b) sample A at 4 days; (c)
sample A at 15 days; (d) sample A at 60 days; (e) sample C at 60 days.
(B) Time course evolution of histidine and histamine in sample A by
Activated Malolactic Fermentation. In this section, we
attempt to determine if the use of an appropriate selection of
lactic acid bacteria could control the production of histamine.
We used O. oeni lactic acid bacteria commercialized as Uvaferm
1
3
C NMR (absolute integrals). (C) Time course evolution of malic
1
and lactic acids in sample A by H NMR (mol/L).
1
3
Alpha. Transformation of L-[2- C ring]-histidine was inves-
tigated by 13C NMR analysis to evaluate its metabolic process
during the malolactic fermentation induced by commercial O.
completed, the histidine level was stable. Therefore, we could
establish that histidine was not metabolized by lactic
commercial bacteria Uvaferm Alpha. In addition, during
malolactic fermentation the appearance of histamine was not
observed; neither were other 13C-labeled metabolites such as
histaminol. The metabolism of histidine during malolactic
fermentation with various strains of lactic acid bacteria remains
for further study. This protocol could be very useful to analyze
1
3
oeni. Unlike the previous case, in C NMR spectra, the
histidine signal was the only one observed in all cases; no
histamine or histaminol signals were detected (Figure 5A). To
confirm that malolactic fermentation had taken place correctly,
1
transformation of malic acid into lactic acid was followed by H
NMR (Figure 5B). Once malolactic fermentation was
D
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AUTHOR INFORMATION
■
*
Corresponding Authors
(J.H.B.) Fax: +34 941 299621. E-mail: hector.busto@unirioja.
es.
*
(J.M.P.) Fax: +34 941 299621. E-mail: jesusmanuel.
peregrina@unirioja.es.
Funding
E.L.-R. thanks the Universidad de La Rioja (predoctoral grant)
and Gobierno de La Rioja (Instituto de Estudios Riojanos
́
grant). We are grateful to the Ministerio de Economıa y
Competitividad/FEDER (CTQ2012/36365).
Notes
The authors declare no competing financial interest.
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histidine during alcoholic and malolactic fermentations in wine.
Using this methodology, transformation of histidine was
monitored during these processes. In the beginning of the
alcoholic fermentation, transformation of histidine into
histaminol was observed. Moreover, the transformation of
histidine into histamine during malolactic fermentation may
depend on strains of lactic acid bacteria, highlighting the
importance of lactic acid bacteria selection for malolactic
fermentation to avoid the production of biogenic amines such
as histamine. The use of certain bacteria as Uvaferm Alpha
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