Stability of monosodium glutamate in green table olives and pickled
cucumbers as a function of packing conditions and storage time
Antonio de Castro, Antonio Higinio Sánchez, Víctor Manuel Beato, Francisco Javier
Casado, Alfredo Montaño*
Antonio de Castro (amillan@cica.es), Food Biotechnology Department, Instituto de la
Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012 Seville, Spain
Antonio Higinio Sánchez (ahiginio@cica.es), Food Biotechnology Department,
Instituto de la Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012 Seville, Spain
Víctor Manuel Beato (vmbeagal@ig.csic.es), Food Biotechnology Department, Instituto
de la Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012 Seville, Spain
Francisco Javier Casado (FJavier.Casado@uni-hohenheim.de), Institute of Food
Science and Biotechnology, Chair Plant Foodstuff Technology, Hohenheim University,
Garbenstrasse 25, D-70593 Stuttgart, Germany
* Corresponding autor. Alfredo Montaño (amontano@cica.es), Food Biotechnology
Department, Instituto de la Grasa (C.S.I.C.), Avenida Padre García Tejero 4, 41012
Seville, Spain. Tel: +34 95 4691054, fax: +34 95 4691262
1
Acknowledgements
This work was supported by the Spanish government through Project AGL 2007-62686
(partially financed by the European Union, FEDER funds) and the Junta de Andalucía
through financial support to group AGR-208.
2
Abstract
The effects of different packing conditions and storage time on the stability of
monosodium glutamate (MSG) added to two different fermented vegetables (Spanishtype green table olives and pickled cucumbers) were studied. Factors such as packaging
material (glass bottle vs. plastic pouch), heat treatment (pasteurization vs. nonpasteurization), and the presence or not of a preservative compound (potassium sorbate)
were considered. The MSG content in pickled cucumbers was stable for up to one year
of storage in all packing conditions studied. The MSG content also remained stable in
pasteurized green table olives. On the contrary, MSG was extensively degraded (>75%
degradation) after 54 weeks of storage in unpasteurized green olives with a higher
degradation rate in glass bottles compared to plastic pouches. In the presence of
potassium sorbate, MSG was also considerably degraded in olives packed in plastic
pouches (>50% degradation) but hardly degraded in glass bottles. The results indicate
that MSG degradation in olives is due to the action of both lactic acid bacteria and
yeasts, with the formation of γ-aminobutyric acid as the major end-product.
Keywords: γ-aminobutyric acid; cucumbers; degradation; glutamate; pyroglutamic
acid; table olives
3
Introduction
Glutamic acid and its salts (especially the sodium salt) are important food additives for
enhancing the flavour of many foods. In pickled vegetables, mainly green table olives,
monosodium glutamate (MSG) is widely used to impart a typical flavour known as
“anchovy flavour”, which is highly appreciated by consumers. Despite its extended use,
the effect of MSG on the chemical, microbiological, and sensory characteristics of
pickled vegetables has hardly been studied. The fate of MSG in pickled vegetables at
prolonged storage has not been investigated either. Rejano & Sánchez (1996)
investigated the effect of MSG on the physicochemical characteristics and flavour of
green table olives. Since the addition of MSG provoked an increase in pH, a
pasteurization treatment was recommended by these authors in order to guarantee
product stability. The degradation of MSG could result in a decrease in the typical taste
of these products, or even in the formation of off-flavours if MSG is largely converted
to pyroglutamic acid (pGlu) (Shallenberger et al. 1959; Mahdi et al. 1961). The
chemical degradation of MSG to pGlu has been reported in model aqueous solutions
during storage at room temperature or during heat treatments (boiling, autoclaving at
135 ºC) (Gayte-Sorbier et al. 1985). Aside from this chemical degradation of MSG,
microorganisms from the fermentation step, lactic acid bacteria (LAB) and yeasts could
metabolize MSG during the storage period as the needed enzymes (glutamate
dehydrogenase or glutamate decarboxylase) have been reported to be present in many
species of LAB or yeasts (Christensen et al. 1999; Albers et al. 1998; Williams et al.
2006). A strain of Lactobacillus pentosus isolated from fermenting green olives was
demonstrated to utilize free glutamic acid in the olive brine (Montaño et al. 2000),
although the end-products of glutamic acid catabolism were not identified.
4
The main objective of the present work was to study the stability of MSG added
to Spanish-type green olives and pickled cucumbers during long-term storage under
different packing conditions. In addition to pGlu, possible metabolites from MSG
catabolism by LAB or yeasts were also analysed in order to know the mechanism of
MSG degradation. The above-mentioned vegetables are the most important fermented
vegetables in Western countries and their processing methods and corresponding
fermentations have been extensively studied (Rejano et al. 2010; Breidt et al. 2013).
Materials and methods
Materials
Pitted Spanish- style green olives (Manzanilla cultivar) and pickled cucumbers were
supplied in brine by a local processor. Physico-chemical characteristics of the
corresponding brines were the following: Olives: pH, 2.74; titratable acidity, 0.9% (as
lactic acid); and salt, 8.2% NaCl; Cucumbers: pH, 3.43; titratable acidity, 5%; and salt,
3.5% NaCl. Cylindrical glass bottles (type “B250”, 125 g fruits plus 120 ml brine
capacity) were obtained from Juvasa Co. (Dos Hermanas, Spain). Flexible plastic
pouches (type “XSARAN/PLTN”, made of three different materials: polyester,
polyethylene, and saran; 75 g fruits plus 105 ml brine capacity, oxygen permeability of
7.6 ml/m2/day; Plastienvase Co., Córdoba, Spain) were a gift from the Jolca Co.
(Seville, Spain). Olives and cucumbers were directly packed without any conditioning
(washing) step.
Chemicals
Monosodium glutamate (MSG), potassium sorbate, L-pGlu, γ-aminobutyric acid
(GABA), 2-oxoglutaric acid, and succinic acid, all of analytical grade, were purchased
5
from Sigma-Aldrich (St Louis, MO). Deionized water was obtained from a Milli-Q
system (Millipore, Bedford, MA). De Man, Rogosa, Sharpe (MRS) agar and
oxytetracycline-glucose-yeast extract (OGYE) agar were from Oxoid (Basingstoke,
England). All other chemicals and solvents were of analytical or chromatographic grade
from various suppliers.
Packing of olives and cucumbers
Olives and cucumbers were divided into three lots which were packed using an acidified
brine as cover liquor with the following additives: (1) control, no additive was added
(packing C); (2) MSG was added to adjust the MSG level to 10 g kg-1 net weight
(packing G); and (3) as lot 2 plus potassium sorbate adjusted to a level of 1 g kg-1 net
weight (olives) or 2 g kg-1 (cucumbers) (packing G+S). The mentioned concentrations
are the maximum permitted concentrations of MSG and sorbic acid for each product in
the European Union (EU 2008; European Commission 2011). Each lot was further
divided into two sub-lots, one of which was packed in glass bottles and the other in
plastic pouches. For olives, acidified cover brine consisted of lactic acid and NaCl to
give equilibrium values of 0.5% titratable acidity (as lactic acid) and 5.0 % salt. The
moisture content of pitted olives was assumed to be 75% (w/w). For cucumbers, a cover
brine containing 3% (w/v) NaCl was used in all cases. When packing in glass bottles,
cover brine was added hot (≈ 70 ºC) to achieve and maintain a vacuum inside the
bottles. After packing, glass bottles were divided into two lots: one lot was subjected to
pasteurization (80 ºC for 7.5 min) and then stored at room temperature (20-24 ºC), and
the remaining lot was directly stored at room temperature. Plastic pouches were stored
similarly to glass bottles, except that a pasteurized lot was not included. All samples
were kept in the dark.
6
Chemical analyses
Duplicate containers (glass bottle or plastic pouch) for each sample were analysed for
glutamic acid and its degradation products. The whole content of a container was
blended with the same weight of distilled water. A portion (40 g) of the slurry was
diluted with water in a 100 ml volumetric flask and then filtered through Whatman No.
41 filter paper. The filtrate was kept at –30 ºC until analysis.
Monosodium glutamate (MSG) and GABA were analysed by HPLC after
derivatization with 9-fluorenylmethylchloroformate (FMOC-Cl). An aliquot of the
filtratewas derivatized after proper dilution with water according to Montaño et al.
(2000) and then analysed using an Ultrabase C18 (2.5 μm, 100 x 4.6 mm i.d.; Análisis
Vínicos, Tomelloso, Spain) column, held at 40 ºC. Mobile phase was composed of 90%
eluent A and 10% eluent B. The eluent A was prepared as follows: 3 ml acetic acid and
7 ml 1M triethylammonium acetate were added to 900 ml water plus 100 ml
acetonitrile, and the pH was adjusted to 6.8 with 20 M NaOH. The eluent B was
acetonitrile-water (90:10). The flow rate was constant at 1.0 ml min-1. To purge the
column, after the elution of FMOC-glutamic acid, the eluent B percentage was
increased to 80% within 5 min and maintained for 5 min. The HPLC system consisted
of a Waters 2695 separations module (Waters Assoc., Milford, MA, USA) connected to
a Jasco FP-920 fluorescence detector (excitation wavelength 263 nm, emission
wavelength 313 nm, flow cell 5 μl) (Jasco Corp., Tokyo, Japan).
Pyroglutamic acid (pGlu), 2-oxoglutaric acid, and succinic acid were analysed
by HPLC using an Aminex HPX 87H column (300 x 7.8 mm i.d., Bio Rad Labs.) and
UV detection at 210 nm. The system consisted of a Waters 2695 connected to a Waters
996 photodiode array detector.The column was kept at 60 ºC and 0.005 M H2SO4 was
7
used as mobile phase at a flow rate of 0.7 ml min-1. In the case of samples from green
olives, before injection, a clean-up treatment of the filtrate was necessary to eliminate
interfering peaks due to polyphenols. For this, after adjusting the pH to 3 units with 6 N
HCl, an aliquot (0.5 ml) of filtrate was applied to an SPE cartridge (C18, 500 mg;
Waters) that had been conditioned with methanol (1 ml) and water (5 ml). The SPE
eluate was collected into a test tube, then 4.5 ml of 0.005 M H2SO4 were applied to the
cartridge, and the eluate was collected into the same test tube. An aliquot of the solution
was filtered through a 0.45 μm membrane filter, and 50 μl of the filtrate were injected
into the chromatograph. In order to confirm the peak identities, the filtrates were
analysed by HPLC interfaced with an electrospray ionization mass spectrometer (ESIMS). The LC flow was directed to the ESI-MS using a flow splitter. Typical settings of
the main tuning parameters were as follows: capillary voltage, 3 kV; cone voltage, 15
V; source temperature, 100 ºC; and desolvation temperature, 350 ºC. Ions were formed
using ESI in positive mode. The expected ions were obtained in all cases confirming the
peak identities. The HPLC/ESI-MS system consisted of a Waters 2695 separation
module connected to a Waters ZMD mass detector and controlled by MassLynx
software (Micromass, Wythenshawe, U.K.)
Sorbic acid and trans-4-hexenoic acid were analysed by GC-FID using the
method described by Montaño et al. (2013). The physico-chemical characteristics of the
brines, such as pH, titratable acidity, and salt content, were determined by the routine
methods used in our laboratory (Fernández-Díez et al. 1985).
Microbiological analyses
The microbial population during storage was determined by plating the brines on the
appropriate solid media, both spreading 0.1 ml onto the surface and plating their
8
decimal dilutions (in 0.1% peptone water) with a Spiral Plater (Don Whitley Sci. Ltd.,
Shipley, England). De Man, Rogosa, Sharpe (MRS) agar with and without 0.02%
sodium azide was used for lactic acid bacteria determination, and oxytetracyclineglucose-yeast extract (OGYE) agar was used for yeasts. The plates were incubated at 32
ºC (MRS) or 26 ºC (OGYE) for up to 5 days, and colonies were enumerated using an
automatic counter (Countermat, IUL Instruments, Barcelona, Spain).
Statistical data analysis
The data were subjected to analysis of variance using the STATISTICA software,
version 7.0 (Statsoft, Inc., Tulsa, OK). The Scheffé test was used for the comparison of
means. Significant differences were determined at p < 0.05.
Results and discussion
Microbiological and physicochemical characteristics of olives and cucumbers
containing MSG
As expected, neither LAB nor yeasts were detected (< 1.3 log cfu ml-1) in the
pasteurized samples from each vegetable. However, clear differences were found
between the olives and cucumbers with regards to the microbiological characteristics of
unpasteurized samples during storage (Tables 1 and 2). For olives, no LAB population
was detected in the control (without MSG) but high populations of LAB (>106 cfu ml-1)
were detected in packing G (with MSG) both in plastic and glass packages stored at
room temperature. Also, LAB were found in packing G+S (with MSG + potassium
sorbate), although in glass bottles LAB were not detected until the sampling performed
after 25 months of storage. Yeasts were detected in all packings, except for packing
G+S due to the addition of potassium sorbate in this case. Regarding physico-chemical
9
characteristic, the olive samples from packing G had initial pH values of 3.7-3.8 units,
about 1 unit higher than the control samples (Table 1). An additional increase in the
initial pH of about 0.1 units was found in packing G+S, compared to packing G, due to
the presence of potassium sorbate together with MSG. The values of pH and titratable
acidity of brines remained stable in all samples of packed olives for at least up to 25
weeks of storage, but a significant increase in pH with a concomitant decrease in
titratable acidity was apparent from 25 to 54 weeks for samples from packing G packed
in both plastic and glass containers. Values of pH as high as 4.88 and 5.28 are not
acceptable for a proper preservation of green olives, and their packing with MSG added
without further pasteurization should be discouraged. To a lesser extent, these changes
in pH and titratable acidity were also observed in the samples packed in glass bottles
from packing G+S.
For cucumbers, no LAB populations were detected (<1.3 log cfu ml-1)
throughout storage in all the different storage conditions studied (Table 2). However,
relatively high yeast populations (>103 cfu ml-1) were found in the control and packing
G, mainly in plastic pouches. Apparently, the highly acid environment where the
cucumbers were supplied and the relatively high acidity level in the different packings
(1.7-2.3% as lactic acid) resulted in LAB inhibition; but yeasts were more resistant to
this acid environment. The addition of MSG provoked an increase in the initial pH in
packing G compared to control of about 0.5 units, and a higher increase of initial pH
was observed in packing G+S (about 0.6 units). During storage, pH and titratable
acidity remained stable in all cases.
MSG stability in olives and cucumbers
10
In case of olives, ANOVA showed that both the storage time and the packing method
significantly affected the MSG content (expressed as g kg-1 net weight of glutamic acid)
(Table 3). The mean content of MSG significantly decreased during storage but only at
prolonged times (> 23 weeks) and, among the packing methods, the lowest MSG
content was found in the unpasteurized samples from packing G. From Table 3, it
appears that MSG was not significantly degraded in packing G+S. However, when the
interaction storage time x packing method was taken into account, some MSG
degradation was apparent in packing G+S, particularly in plastic pouches, although to a
lesser extent than in packing G (Figure 1).
The following MSG degradation percentages with respect to the initial level were found
after 54 weeks storage: packing G glass bottles, 96%; packing G plastic pouches, 76%;
packing G+S glass bottles, 12%; and packing G+S plastic pouches, 53%. The higher
MSG degradations in packing G compared to packing G+S could indicate that both
LAB and yeasts metabolized MSG whereas only LAB were active in packing G+S, as
yeasts were inhibited by the presence of sorbate. In addition, it appears that the presence
of oxygen could be an important factor for the metabolism of MSG by LAB, explaining
the higher degradation in plastic compared to glass bottles in packing G+S. Although
panel tests were not carried out, the MSG degradation should be taken into account
when “best before” dates are given. The shelf-life of packed green table olives
established by manufacturers is usually 3 years (Sánchez-Gómez et al., 2013). However,
our results have shown that the initial characteristics of the unpasteurized products are
lost between 23 and 54 weeks when MSG is added. As stated above in relation to pH
and acidity, only pasteurized products maintained MSG concentration during the study
period.
11
Pyroglutamic acid (pGlu), the product of chemical degradation of glutamic acid
or MSG, was detected in all samples from green table olives, but its concentration was
relatively low ranging from 60 to 265 mg kg-1 net weight at the end of storage (0.5-2.0
mM, which corresponded to < 4% of the initial MSG content) (Table 4).
It is known that the identified pathways of glutamate catabolism in LAB are
initiated by an aminotransferase, a dehydrogenase, or a decarboxylase (Christensen et
al. 1999). The first two enzymes result in the formation of 2-oxoglutarate from
glutamate, while GABA is the product of decarboxylation, and it appears unlikely that
these products are catabolized further. When samples were analysed for 2-oxoglutarate
and GABA after 54 weeks, it was found that 2-oxoglutarate was detected in trace
amounts (< 1 mM, data not shown) in all cases while relatively high yields of GABA
were found in the unpasteurized samples from both packings, particularly from packing
G. In general, the amount of GABA formed between 23 and 54 weeks was similar to the
amount of MSG degraded (Table 4) indicating that the mechanism of MSG degradation
in olives at prolonged storage was mainly a decarboxylation reaction. Yeasts could also
metabolize glutamate forming GABA, explaining the higher concentration of this
compound in samples from packing G compared to packing G+S. In fact, yeast is the
most popular microorganism used for the production of foods containing GABA
(Kanbara et al. 2009). The species of LAB and yeasts responsible for MSG degradation
were not identified in the present study. Previously, we identified different species of
LAB (Lactobacillus parafarraginis, L. pentosus, L. paracollinoides) which were
responsible of the sorbate degradation in packed green table olives with the formation of
trans-4-hexenoic acid as the sole end-product (Montaño et al. 2013). In the present
study, this sorbate degradation occurred at prolonged storage (> 5 months, data not
shown). Therefore, it is reasonable to assume that the above-mentioned LAB species
12
might be involved in the MSG degradation as well. In fact, L. pentosus was
demonstrated to degrade glutamic acid in green olive brine (Montaño et al. 2000).
Further research is needed to confirm this assumption.
For cucumbers, the level of MSG remained constant during the storage time (at
least up to 54 weeks storage) in all samples. Contrary to olives, the presence of
microorganisms (yeasts in samples without added sorbate according to the previous
section) appeared not to affect the MSG content in pickled cucumbers. Factorial
analysis of variance (ANOVA) showed that the effects of storage time and packing
method as well as their interaction on MSG content were not significant (data not
shown). The measured content of MSG was 9.4 g kg-1 net weight as glutamic acid, on
average of whole data, with a relative standard deviation of 7.9%. As occurred in the
case of olives, the formation of pGlu in pickled cucumbers under the different packing
conditions was slight (1.1-2.2 mM after 54 weeks, corresponding to <3.5% of initial
MSG content). Therefore, similarly to olives, the conversion of MSG to pyroglutamic in
cucumbers was almost non-existent. In comparison, Gayte-Sorbier et al. (1985) found
higher conversion percentages (8-11%) in model solutions at room temperature and pH
values ranging from 2 to 8 after 50 days of storage, with conversion percentages as high
as 43-53% in the presence of oxygen. Therefore, the vegetable matrix appears to
significantly affect the rate of conversion of MSG to pGlu.
Conclusions
Monosodium glutamate (MSG) added to packed pickled cucumbers was stable for up to
one year of storage in all packing conditions studied. The same conclusion was found in
pasteurized green table olives. MSG was highly degraded at prolonged storage (> 5
months) in unpasteurized green table olives packed in both glass bottles and plastic
13
pouches. This was mainly due to microbial action, the chemical degradation to pGlu
being negligible. LAB and yeasts were responsible for the MSG disappearance in
olives, with MSG being almost completely converted into GABA. The addition of
potassium sorbate, which resulted in yeast inhibition, decreased the MSG degradation
rate, but this preservative has the drawback of also being degraded by LAB at storage
times greater than 5 months. A pasteurization treatment appeared to be the most
appropriate method to stabilize packed olives, including the maintenance of the physicchemical characteristics and MSG content.
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Figure captions
Figure 1. Changes in monosodium glutamate (expressed as g of glutamic acid /kg net
weight) during storage at room temperature of green table olives, packed in glass and
plastic containers, in the absence (packing G) and presence of sorbate (packing G+S).
Values are means of duplicate containers. Error bars show the range of the data (n = 2).
16
Table 1. Changes in lactic acid bacteria (LAB), yeasts, pH, and titratable acidity (TA) in brine during storage of
unpasteurized green table olives packed under different conditions a
Storage time
(weeks)
2
Packing b
C, glass
C, plastic
G, glass
G, plastic
G+S, glass
G+S, plastic
LAB
(log cfu ml-1)
<1.3
< 1.3
6.0
>6.0
<1.3
2.7
Yeasts
(log cfu ml-1)
4.1
4.6
4.8
4.6
2.3
<1.3
2.90
2.82
3.81
3.78
3.97
3.97
TAc
(%)
0.51
0.50
0.54
0.54
0.54
0.53
7
C, glass
C, plastic
G, glass
G, plastic
G+S, glass
G+S, plastic
<1.3
<1.3
6.0
6.7
<1.3
5.3
4.0
4.9
3.9
4.9
<1.3
<1.3
2.94
2.86
4.02
3.96
3.93
4.04
0.49
0.48
0.51
0.52
0.55
0.51
12
C, glass
C, plastic
<1.3
3.4
2.88
0.49
<1.3
4.9
2.83
0.49
G, glass
5.5
6.0
<1.3
2.8
4.3
<1.3
3.98
3.96
3.97
0.51
0.49
0.56
5.7
<1.3
4.01
0.55
<1.3
<1.3
3.2
3.6
4.7
3.1
2.92
2.91
4.20
0.51
0.51
0.50
5.5
4.4
4.03
0.52
G+S, plastic
4.7
4.8
<1.3
<1.3
4.09
4.13
0.54
0.52
C, glass
C, plastic
<1.3
<1.3
2.9
2.96
0.51
4.7
2.95
0.52
G, glass
3.6
1.9
4.88
0.34
G, plastic
6.1
3.9
5.28
0.16
G+S, glass
4.7
<1.3
4.48
0.46
G, plastic
G+S, glass
G+S, plastic
23
C, glass
C, plastic
G, glass
G, plastic
G+S, glass
54
G+S, plastic
pH
4.14
0.53
4.3
<1.3
-1
Neither LAB nor yeasts were detected (<1.3 log cfu ml ) in the pasteurized samples from
each packing. b C, no additive added to the acidified cover brine (control ); G, packing with
MSG added to the acidified cover brine; G+S, packing with MSG plus potassium sorbate added
to the acidified cover brine. c % as lactic acid. Values are means of two containers. Coefficients
of variation, on average, were 5.9% (LAB), 3.7% (yeasts), 1.1% (pH), and 2.4% (TA).
a
17
Table 2. Changes in lactic acid bacteria (LAB), yeasts, pH, and titratable acidity (TA) in brine during
storage of unpasteurized pickled cucumbers packed under different conditions a
Storage time
(weeks)
2
Packing b
C, glass
C, plastic
G, glass
G, plastic
G+S, glass
G+S, plastic
LAB
(log cfu ml-1)
< 1.3
< 1.3
< 1.3
< 1.3
< 1.3
< 1.3
Yeasts
(log cfu ml-1)
1.90
3.19
1.60
3.63
< 1.3
< 1.3
3.43
3.42
3.86
3.89
3.97
3.99
TAc
(%)
2.02
1.78
2.04
1.84
2.18
1.94
7
C, glass
C, plastic
G, glass
G, plastic
G+S, glass
G+S, plastic
< 1.3
< 1.3
< 1.3
< 1.3
< 1.3
< 1.3
< 1.3
3.57
1.90
3.91
< 1.3
< 1.3
3.36
3.36
3.87
3.89
3.91
3.92
2.16
1.96
2.24
2.04
2.36
2.18
12
C, glass
C, plastic
< 1.3
< 1.3
3.41
2.46
< 1.3
3.66
3.41
2.18
G, glass
< 1.3
< 1.3
< 1.3
< 1.3
3.75
< 1.3
3.85
3.88
3.94
2.54
2.26
2.26
< 1.3
< 1.3
3.98
2.04
< 1.3
< 1.3
< 1.3
< 1.3
3.6
1.7
3.41
3.42
3.86
2.08
1.82
2.12
< 1.3
3.7
3.90
1.86
< 1.3
< 1.3
< 1.3
< 1.3
3.97
4.00
2.32
2.06
C, glass
C, plastic
< 1.3
< 1.3
3.42
2.04
< 1.3
3.2
3.44
1.86
G, glass
< 1.3
< 1.3
3.84
2.22
G, plastic
< 1.3
2.7
3.90
1.82
G+S, glass
< 1.3
< 1.3
3.90
2.24
G, plastic
G+S, glass
G+S, plastic
23
C, glass
C, plastic
G, glass
G, plastic
G+S, glass
G+S, plastic
54
G+S, plastic
pH
< 1.3
< 1.3
3.93
2.12
-1
Neither LAB nor yeasts were detected (<1.3 log cfu ml ) in the pasteurized samples from
each packing. b C, no additive added to the acidified cover brine (control ); G, packing with
MSG added to the acidified cover brine; G+S, packing with MSG plus potassium sorbate added
to the acidified cover brine. c % as lactic acid. Values are means of two containers. Coefficients
of variation, on average, were 5.4% (yeasts), 0.2% (pH), and 2.5% (TA).
a
18
Table 3. Main effects of storage time and packing method on MSG content in
green table olives
Main effects
MSG content
(g kg-1 of glutamic
acid)a
nb
F valuec
Storage time (weeks)
6.69***
2
7.1 ± 0.3b
12
7
7.3 ± 0.3b
12
12
6.7 ± 0.2b
12
23
6.9 ± 0.3b
12
54
4.6 ± 0.9a
12
Packing method
4.62**
G, glass
5.0 ± 0.8a
10
G, plastic
5.7 ± 0.7a
10
G, glass, pasteurized
7.2 ± 0.2b
10
G+S, glass
7.0 ± 0.4b
10
G+S, plastic
6.6 ± 0.6b
10
G+S, glass, pasteurized 7.5 ± 0.2b
10
a
Means with different letters for each effect are significantly different (p<0.05). b Number of
experimental data. c F value, assessment of overall differences obtained from analysis de
variance; ***, p<0.001; **, p<0.01
19
Table 4. Changes in MSG and its degradation products (pGlu and GABA) in packed green olives between 23 and 54 weeks of storagea
MSG (mM)
pGlu (mM)
GABA (mM)
Packingb
G, glass
23 w
33.6 ± 1.8
54 w
1.6 ± 0.2
Consumed
32
23 w
0.47 ± 0.00
54 w
0.56 ± 0.03
Formed
0.1
23 w
12.2 ± 0.1
54 w
42.5 ± 1.6
Formed
30
G, plastic
41.6± 2.7
10.7 ± 5.5
31
0.52 ± 0.09
1.39 ± 0.1
0.9
7.9 ± 0.5
33.1 ± 4.8
25
G, glass,
pasteurized
49.1 ± 3.2
48.0 ± 0.6
NS
0.53 ± 0.07
2.02 ± 0.04
1.5
ND
ND
ND
G+S, glass
50.4 ± 1.6
40.9 ± 11
10
0.53 ± 0.01
1.66 ± 0.1
1.1
ND
9.0 ± 9.1
9
G+S, plastic
53.2 ± 4.2
24.7 ± 7.9
29
0.49 ± 0.04
1.29 ± 0.07
0.8
ND
23.9 ±7.3
24
G+S, glass,
46.3 ± 1.3 47.9 ± 8.3 NS
0.53 ± 0.03 1.9 ± 0.01
1.4
ND
ND
ND
pasteurized
a
Values are means ± SD of two bottles. b G, packing with MSG added to the acidified cover brine; G+S, packing with MSG plus potassium
sorbate added to the acidified cover brine. ND = not detected. NS = non-significant
20
Figure 1
Packing G
Glutamic acid (g kg-1 net weight)
10
8
6
4
2
0
2
7
12
23
Storage time (weeks)
Packing G+S
10
Glutamic acid (g kg-1 net weight)
Glass bottle
Plastic pouch
Glass bottle, pasteurized
54
Glass bottle
Plastic pouch
Glass bottle, pasteurized
8
6
4
2
0
2
7
12
23
Storage time (weeks)
54
21