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Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20

Stability of monosodium glutamate in green table olives and pickled cucumbers as a function of packing conditions and storage time a

a

a

Antonio de Castro , Antonio Higinio Sánchez , Víctor Manuel Beato , Francisco Javier b

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Casado & Alfredo Montaño a

Food Biotechnology Department, Instituto de la Grasa (C.S.I.C.), Seville, Spain

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Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology, Hohenheim University, Stuttgart, Germany Accepted author version posted online: 10 Apr 2014.Published online: 12 May 2014.

To cite this article: Antonio de Castro, Antonio Higinio Sánchez, Víctor Manuel Beato, Francisco Javier Casado & Alfredo Montaño (2014) Stability of monosodium glutamate in green table olives and pickled cucumbers as a function of packing conditions and storage time, Food Additives & Contaminants: Part A, 31:7, 1158-1164, DOI: 10.1080/19440049.2014.913320 To link to this article: http://dx.doi.org/10.1080/19440049.2014.913320

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Food Additives & Contaminants: Part A, 2014 Vol. 31, No. 7, 1158–1164, http://dx.doi.org/10.1080/19440049.2014.913320

Stability of monosodium glutamate in green table olives and pickled cucumbers as a function of packing conditions and storage time Antonio de Castroa, Antonio Higinio Sáncheza, Víctor Manuel Beatoa, Francisco Javier Casadob and Alfredo Montañoa* a

Food Biotechnology Department, Instituto de la Grasa (C.S.I.C.), Seville, Spain; bInstitute of Food Science and Biotechnology, Chair Plant Foodstuff Technology, Hohenheim University, Stuttgart, Germany

Downloaded by [Universite Laval] at 13:33 07 July 2014

(Received 13 January 2014; accepted 4 April 2014) The effects of different packing conditions and storage times on the stability of monosodium glutamate (MSG) added to two different fermented vegetables (Spanish-type green table olives and pickled cucumbers) were studied. Factors such as packaging material (glass bottle versus plastic pouch), heat treatment (pasteurisation versus non-pasteurisation), and the presence or not of a preservative compound (potassium sorbate) were considered. The MSG content of pickled cucumbers was stable for up to 1 year of storage in all packing conditions studied. The MSG content also remained stable in pasteurised green table olives. On the contrary, MSG was extensively degraded (>75% degradation) after 54 weeks of storage in unpasteurised green olives with a higher degradation rate in glass bottles compared with 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

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 during prolonged storage has also not been investigated. 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 pasteurisation 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 RT or during heat treatment (boiling, autoclaving at 135ºC) (Gayte-Sorbier et al. 1985). Aside from this chemical degradation of MSG, microorganisms from the fermentation step, lactic acid bacteria (LAB) and yeasts could metabolise MSG during the storage period as the needed enzymes (glutamate dehydrogenase or glutamate *Corresponding author. Email: [email protected] © 2014 Taylor & Francis

decarboxylase) have been reported to be present in many species of LAB or yeasts (Albers et al. 1998; Christensen et al. 1999; Williams et al. 2006). A strain of Lactobacillus pentosus isolated from fermenting green olives was demonstrated to utilize free glutamic acid in olive brine (Montaño et al. 2000), although the end-products of glutamic acid catabolism were not identified. 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

Food Additives & Contaminants: Part A 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 m–2 day–1; Plastienvase Co., Cordoba, Spain) were a gift from Jolca Co. (Seville, Spain). Olives and cucumbers were directly packed without any conditioning (washing) step.

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Chemicals Monosodium glutamate (MSG), potassium sorbate, L-pGlu, γ-aminobutyric acid (GABA), 2-oxoglutaric acid and succinic acid, all of analytical grade, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Deionised water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). De Man, Rogosa, Sharpe (MRS) agar and oxytetracycline–glucose–yeast extract (OGYE) agar were from Oxoid (Basingstoke, UK). 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 (≈70ºC) to achieve and maintain a vacuum inside the bottles. After packing, glass bottles were divided into two lots. One lot was subjected to pasteurisation (80ºC for 7.5 min) and then stored at RT (20–24ºC); the remaining lot was directly stored at RT. Plastic pouches were stored similarly to glass bottles, except that a pasteurised lot was not included. All samples were kept in the dark.

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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 –30ºC until analysis. MSG and GABA were analysed by HPLC after derivatisation with 9-fluorenylmethylchloroformate (FMOC-Cl). An aliquot of the filtrate was derivatised after proper dilution with water according to Montaño et al. (2000) and then analysed using an Ultrabase C18 (2.5 μm, 100 × 4.6 mm i.d.; Análisis Vínicos, Tomelloso, Spain) column, held at 40ºC. 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 1 M 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 Associates, 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 × 7.8 mm i.d., Bio Rad Labs, Hercules, CA, USA) 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 60ºC and 0.005 M H2SO4 was 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 ionisation mass spectrometer (ESI-MS). 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, 100ºC; and desolvation temperature, 350ºC. Ions were formed using ESI in positive mode. The

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Table 1. Changes in lactic acid bacteria (LAB), yeasts, pH and titratable acidity (TA) in brine during storage of unpasteurised green table olives packed under different conditions.a Storage time (weeks) 2

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23

54

Packingb

LAB (log cfu ml−1)

Yeasts (log cfu ml−1)

pH

TA (%)c

C, glass C, plastic G, glass G, plastic G + S, glass G + S, plastic C, glass C, plastic G, glass G, plastic G + S, glass G + S, plastic C, glass C, plastic G, glass G, plastic G + S, glass G + S, plastic C, glass C, plastic G, glass G, plastic G + S, glass G + S, plastic C, glass C, plastic G, glass G, plastic G + S, glass G + S, plastic

Stability of monosodium glutamate in green table olives and pickled cucumbers as a function of packing conditions and storage time.

The effects of different packing conditions and storage times on the stability of monosodium glutamate (MSG) added to two different fermented vegetabl...
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