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EFFECT OF SULFITES AND SORBATES ON THE PRESERVATION AND
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COLOR OF PICKLED BLANCHED GARLIC UNDER DIFFERENT STORAGE
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CONDITIONS
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Francisco Javier Casado, Antonio Higinio Sánchez, Víctor Manuel Beato, Antonio de
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Castro, Alfredo Montaño*
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Food Biotechnology Department, Instituto de la Grasa IG-CSIC, Avenida Padre García
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Tejero 4, 41012 Seville, Spain
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*Tel: +34 95 4691054, fax: +34 95 4691262, e-mail: amontano@cica.es
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Short title: additives in pickled garlic
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ABSTRACT
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The influence of sulfites and sorbates on the microbial stability and color of pickled
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blanched garlic stored under different conditions was studied. Two storage temperatures
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(ambient, 20–24 ºC ; refrigeration, 6–9 ºC) and two types of packaging (glass bottle,
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plastic pouch consisting of polyester/polyethylene/ saran) were considered. Both
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sulfites and sorbates gave satisfactory results as antimicrobial agents, even at room
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temperature. Sulfite was also effective as an anti-browning agent, but sorbate produced
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the opposite effect on color. The samples packed in plastic pouches stored at room
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temperature had the highest values of the parameters b* (yellowness), C (color purity),
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and BI (browning index). The sorbate level hardly changed during storage, regardless
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of the storage conditions used, but the sulfite level decreased, especially in samples
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packed in plastic pouches stored at room temperature, in which oxidation to sulfate was
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demonstrated to be the sole mechanism of sulfite loss.
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PRACTICAL APPLICATIONS
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Long-term preservation of pickled blanched garlic without the need for expensive
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preservation treatments such as pasteurization or refrigeration is possible with the help
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of sulfites and/or sorbates as preservatives. However, these preservatives yield opposite
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effects on garlic color. The data collected in this study demonstrated that sulfite at
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approximately 100 mg/L of SO2 at equilibrium was an effective antimicrobial and
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antibrowning agent. For practical purposes, we recommend the use of sulfites alone and
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glass packages to maintain the quality of pickled blanched garlic as the presence of
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sorbates and/or the use of plastic packages enhance browning reaction.
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Keywords: color, pickled garlic, preservation, sorbate, storage, sulfite
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INTRODUCTION
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Pickled blanched garlic is an interesting vegetable product since, in addition to its
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appreciated organoleptic properties, it may present beneficial health effects due to its
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considerable contents of organosulfur compounds (Beato et al. 2012). For its long-term
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storage, blanched garlic is normally packed in small containers using acidified brine as
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cover liquid with the optional addition of spices and/or olive oil. Although microbial
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stability during storage can be guaranteed by pasteurization or the addition of
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sorbates/benzoates plus refrigeration (Rejano et al. 1997), garlic browning could be an
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inconvenience which could limit the product’s shelf life. To prevent this problem, the
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use of additives such as sulfites is a practice widely used by the industry, but their effect
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on garlic color both in the absence and presence of other additives such as sorbates has
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not been evaluated in an objective manner. In addition, studies on the evolution during
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storage of added preservatives have not been carried out in the case of pickled blanched
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garlic. Since combined sulfites in foods appear to be less toxic than free sulfites (Taylor
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et al. 1986), knowledge on the levels of both free and bound sulfites in pickled blanched
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garlic would be interesting from a toxicological point of view. The term “sulfite”
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includes sulfur dioxide, sodium sulfite, sodium and potassium bisulfite, and sodium and
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potassium metabisulfite, among others. In the EU, the maximum permitted
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concentration of sulfites in vegetables in brine is 100 mg/kg, expressed as SO2, with the
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exception of olives, in which sulfites are not permitted (EU 2011).
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Sorbates (sodium or potassium sorbate, sorbic acid) are frequently added to
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pickled vegetables as adjuncts to preservation. In the EU, the maximum permitted
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concentration of sorbates in vegetable products in brine is 2000 mg/kg as sorbic acid
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(with the exception of olives and olive-based products, 1000 mg/kg) (EU 2011).
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Sorbates are generally used to inhibit yeasts and moulds, although they are also active
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against many bacteria (Stopforth et al. 2005). Sorbates have been demonstrated to be
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rapidly degraded during the storage of pickled green olives (Casado et al. 2010), which
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is attributed to the action of lactic acid bacteria (LAB) accompanying the olives. In
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other pickled vegetables such as cucumbers and caperberries, where LAB were
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undetected during storage, sorbate levels remained practically unchanged (unpublished
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results). In all the above-mentioned pickled vegetables, the addition of sorbates was
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demonstrated to negatively affect the vegetable color. Sorbates could take part in
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nonenzymatic browning reactions deteriorating the color (Wedzicha et al. 1991).
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The main objective of the present work was to study the effect of both sulfites
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and sorbates on the microbial stability and color of pickled garlic stored under different
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packing conditions. Another aim of the work was to monitor the levels of both additives
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during storage, with special emphasis on analyzing both free and combined forms of
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sulfites.
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MATERIALS AND METHODS
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Materials and chemicals
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Pickled garlic cloves in acidified brine were supplied in bulk by a local industry.
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The physico-chemical characteristics of the corresponding brine were: pH, 4.02;
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titratable acidity, 1.11% (as lactic acid); and salt, 13.2% NaCl. Prior to packing, the
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garlic cloves were partially desalted by immersion in water for 2 days.
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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
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“XSARAN/PLTN”, 75 g vegetable plus 105 mL brine capacity, oxygen permeability of
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7.6 mL/m2/day; Plastienvase Co., Córdoba, Spain) were a gift from Jolca Co. (Sevilla,
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Spain).
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Sodium bisulfite and potassium sorbate were purchased from Sigma-Aldrich (St
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Louis, MO). Deionized water was obtained from a Milli-Q system (Millipore, Bedford,
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MA). All other chemicals and solvents were of analytical or chromatographic grade
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from various suppliers.
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Packing
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Garlic cloves were divided into four lots which were packed using an acidified
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brine as cover liquid with the following additives: (1) control, no additive was added
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(packing C); (2) sodium bisulfite was added to adjust the SO2 level to 100 mg/kg net
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weight (packing Su); (3) potassium sorbate was added to adjust the sorbic acid level to
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2000 mg/ kg net weight (packing So); and (4) sodium bisulfite + potassium sorbate was
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added at the same levels as packing Su and packing So, respectively (packing Su + So).
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Each lot was further divided into two sub-lots, one of which was packed in a glass bottle
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and the other in a plastic pouch. The amounts of garlic and cover brine actually added in
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each type of packaging were: glass bottle, 150 g garlic plus 110 mL brine; and plastic
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pouch, 75 g garlic plus 105 mL brine. Acidified cover brine consisted of lactic acid and
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NaCl to give equilibrium values of 1.5% titratable acidity (as lactic acid) and 5.0% salt,
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respectively. For the calculations, the moisture content of pickled garlic was assumed to
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be 87% (w/w) (Casado et al. 2004). In the case of packing in glass bottles, hot cover
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brine was added (≈ 70 ºC) in order to achieve and maintain a vacuum inside the bottles.
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In order to minimize SO2 losses in the packing step, bisulfite from a stock solution (80
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g/L of sodium bisulfite) was added after the addition of the cover brine and the
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containers were immediately sealed. Finally, the containers were divided into two lots;
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one of them was stored at room temperature (20–24 ºC) and the other one under
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refrigeration (6–9 ºC). All samples were kept in the dark for up to 46 weeks.
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Chemical and microbiological analyses
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Determinations of sulfite and sorbic acid in brine were performed in duplicate
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containers (glass bottle or plastic pouch) for each sample. Sulfites, both free and
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combined, were determined by iodometric titration according to the Ripper method
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(Vahl and Converse 1980) with slight modifications. Briefly, for the determination of
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free sulfite, a 50 mL aliquot of brine sample was placed in a 250 mL Erlenmeyer flask
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and 2 g of KI, 1 mL of 50 mM EDTA, 0.5 mL of 2% starch solution, and 3 mL of 10%
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(v/v) H2SO4 were added; the mixture was immediately and rapidly titrated with 0.02 N
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I2 to the lightest blue end point. Then, for the combined sulfite analysis, 10 mL of 4 N
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NaOH were added and, after 5 min of incubation at room temperature, 15 mL of 10%
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(v/v) H2SO4 were added and the mixture was titrated again as mentioned above. Free
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sulfite values were corrected for non-SO2 iodine-reducing substances by performing
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titration in another 50 mL aliquot of sample, prior to the addition of 5 mL of 0.7% (v/v)
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acetaldehyde and incubation for 30 min. Sorbic acid was analyzed by
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spectrophotometry after separation by steam distillation, according to the method
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described by Montaño et al. (1995). Sulfate was determined by the turbidimetric
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method proposed by Kolmert et al. (2000). The brine sample was diluted 1:5 with water
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and 1 mL of the diluted brine was mixed with 1 mL of conditioning reagent (150 g
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NaCl, 100 mL glycerol, 60 mL concentrated HCl, 200 mL 95% ethanol, and deionized
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water to make up 1 l). Then, approximately 60 mg of crushed BaCl2 were added and the
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mixture was vortex mixed for 30 s, and the absorbance at 420 nm was immediately
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measured against a blank. The concentration of sulfate in the sulfite-containing samples
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(packing Su and Su + So) was corrected by the average concentration of sulfate in
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samples in which sulfite was not added (packing C and So). Analytical procedures for
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pH, titratable acidity, and sodium chloride were the same as described by Rejano et al.
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(1997).
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The microbiological analysis comprised aerobic plate count, lactic acid bacteria
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(LAB), and molds and yeasts. Media (plate count agar, PCA; de Man, Rogosa and
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Sharpe agar MRS; and oxytetracycline glucose yeast extract agar, OGYE, respectively)
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were from Oxoid Ltd (Basingstoke, England). PCA and MRS plates were incubated at
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30ºC for 48 h, and OGYE agar at 25ºC for up to 5 days.
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Color measurements
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The surface color of the garlic cloves was measured by a Color-View Model
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9000 spectrophotometer (BYK-Gardner, Inc., Silver Spring, MD) with a measurement
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area of 11 mm diameter, 45º circumferential illumination, and observation angle of 0º.
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All measurements were done on the CIE 1976 L*a*b* scale using illuminating
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conditions CIE type C, 10º observer. The results were expressed as the mean of 10
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replicate measurements, each made on one garlic clove. Hue angle (hº) was calculated
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from the equation: hº = 180 + tan-1 (b*/a*) (20). Chroma (C) was calculated as (a*2 +
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b*2)1/2. The browning index (BI) was calculated as follows (Palou et al. 1999): BI =
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[100(x – 0.31)]/0.172, where x = (a* +1.75L*)/(5.645L* + a* – 3.012b*).
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Statistical analysis
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Analysis of variance (ANOVA) was performed with the Statistica software,
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version 7.0 (Statsoft Inc., Tulsa, OK). The Duncan test was used for the comparison of
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means. Significant differences were determined at p < 0.05.
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RESULTS AND DISCUSSION
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Changes in chemical and microbiological characteristics during storage
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The titratable acidity and pH during storage for the four treatments studied
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(packing C, Su, So, and Su + So) under the different storage conditions are shown in
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Figure 1. The presence of the additives, both of them added as salts, was reflected in the
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slightly lower values of titratable acidity in comparison with the control samples.
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Significant differences in pH compared to the control were only noted in the presence of
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sorbate (packing So and Su + So). In all cases, the pH values were always lower than
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3.3 units and pH changes during storage were negligible. However, a slight increase in
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titratable acidity during storage was observed in the control samples stored at room
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temperature. Aerobic mesophilic bacteria, LAB, and yeasts were not detected (< 1 log
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cfu/mL) in any sample, with the exception of an appreciable number of atypical or
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damaged yeasts in control samples at room temperature (3-5 log cfu/mL after 2 months
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storage) (data not shown). At longer storage, this growth, which was not observed in
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the refrigerated control samples, resulted in the appearance of visual defects related to
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gas formation inside the containers such as swollen pouches or swollen lids on the glass
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bottles with eventual breakage of the pouches or brine leaks, respectively. On the other
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hand, these undesirable effects were not found in the presence of additives (packing Su,
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So, and Su + So) under the different conditions at any time during storage. It must be
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stressed that sulfite alone was an effective antimicrobial agent despite the relatively low
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amounts of free sulfite (as discussed below), which is the active form of the sulfites in
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terms of their antimicrobial actions (Taylor et al. 1986).
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Influence of additives and packing conditions on color parameters
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The ANOVA of the color parameters of garlic cloves and brine browning after
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46 weeks of storage (Table 1) showed that the sulfite only-containing samples (packing
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Su) had lower values of parameters b* (yellowness), C (color purity), and BI (browning
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index) but higher hº (hue angle) than the control samples or those containing sorbate
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(packing So and Su + So). The browning index (BI) represents the purity of the brown
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color and is considered as an important parameter associated with browning (Palou et
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al. 1999). All values of hº (hue angle) were in the very slightly green-predominantly
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yellow region (hue angle between 90° and 180°) of the color solid dimensions. The
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parameters L* (lightness), -a* (greenness), and A440-A700 (brine browning) of the
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additive-containing samples were not significantly different from the control samples.
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The positive effect of sulfites on garlic color could be attributed to the inhibition of non-
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enzymatic browning (Maillard reactions) (Wedzicha and Vakalis 1988). Enzymatic
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browning in pickled blanched garlic is unlikely, as enzymes there would have been
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deactivated during the blanching treatment. Sorbic acid could act as a catalyst in the
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browning reaction mechanism. It has been demonstrated that Maillard browning in
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model solutions of glucose and glycine is accelerated by the presence of sorbic acid
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(Wedzicha et al. 1991). The influence of the packing conditions on garlic color was
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significant for all color parameters, except A440-A700. It can be noted (Table 1) that
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samples packed in plastic pouches stored at room temperature had the worst color (i.e.
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they showed the highest browning, as reflected in the highest values of parameters b*,
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C, and BI).
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Evolution of sulfites and sorbates during storage
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In the present work, it was considered important to measure both free and bound
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forms of sulfite in the garlic samples. Since sample preparation for analysis from
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pickled garlic cloves could cause important losses in SO2 in the homogenization step, all
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analyses of sulfites were performed on the packing brines. The Ripper direct iodometric
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titration is a simple and rapid method for determining both free and total SO2 widely
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used in wines (Vahl and Converse 1980). When this method (see Materials and
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Methods) was applied to a brine garlic sample with added sulfites at a concentration
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level of 100 mg/L , expressed as SO2, a recovery of 86% for total SO2 was found, which
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is close to the minimum value established by the AOAC (AOAC 1993) of 90% for an
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analyte concentration of 100 mg/L . The coefficient of variation for duplicate analyses
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of all samples analyzed (n=50) in the present work following this method was, on
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average, 5.1% for total sulfites, which is slightly lower than the maximum value
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established by the AOAC (AOAC 1993) of 5.3% for an analyte concentration of 100
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mg/L . However, in the case of the determination of free sulfites, the coefficient of
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variation was too high (34%, on average). Therefore, the Ripper method was considered
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adequate for the analysis of total sulfites, but it provided only a semi-quantitative
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determination of free sulfites in pickled blanched garlic.
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The analysis of variance (ANOVA) showed that the mean value of total sulfites
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in pickled garlic packed according to packing Su was not significantly (p=0.278)
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different from that of packing Su + So. The same conclusion was obtained for the free
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sulfites (p=0.367). The presence of sorbic acid slightly increased the combined sulfite
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proportion, presumably due to the formation of a 1:1 addition product (Khandelwal and
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Wedzicha 1990), but the increase was not statistically significant (p=0.058). Therefore,
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samples from both packings were used as a whole for evaluating the effects of packing
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conditions and storage time on the values of free, combined, and total sulfites. In all
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three variables, the ANOVA revealed highly significant (p<0.001) effects for the two
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factors and their interaction (data not shown). Evolution of free, combined, and total
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sulfites during storage of pickled blanched garlic under the different storage conditions
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is shown in Figure 2. Initial mean values of total sulfites ranged between 85 and 97
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mg/L, expressed as SO2, without taking into account the analytical method of recovery.
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These values agree well with the intended value (100 mg/L of SO2), indicating that the
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amount of sulfites lost by oxidation or irreversibly bound to garlic compounds was
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negligible at the beginning of storage. It is clear that most added sulfites were reversibly
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bound to garlic components forming the combined sulfites, which at initial sampling
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ranged from 59% in samples packed in plastic pouches stored under refrigeration to
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89% in the samples packed in bottles stored at room temperature. It can be noted that,
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regardless of storage temperature, the initial level of free SO2 was lower in the samples
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packed in glass bottles than in pouches, which could be related to fact that the cover
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brine was hot when added in the former. With storage time the amount of free sulfites
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diminished and eventually disappeared (in most cases before 27 weeks of storage).
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Therefore, in all cases, at longer storage (>27 weeks) all retained sulfite was in the form
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of combined sulfite. This fact is interesting from a toxicological point of view, as
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previous findings in the literature suggest that some of the combined sulfites are less
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toxic than the inorganic sulfites. However, further studies, both in garlic and foods in
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general, are needed to confirm this hypothesis. Total sulfites diminished during storage
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but apparently the major change occurred between 9 and 27 weeks of storage, with
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greater losses in plastic (air-permeable) pouches than in glass (air-impermeable) bottles.
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Also, as expected, the sulfite losses were significantly lower in the case of refrigerated
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storage. At the end (46 weeks storage), in the samples stored at room temperature,
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average sulfite retention was 24% (pouches) and 67% (bottles) (Table 2). It can be
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noted that the amount of retained sulfite was slightly higher (% retention was exactly
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8% greater) in the presence than in the absence of sorbate, although this effect was only
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significant (p<0.05) in the samples packed in plastic pouches. In addition, in these
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samples the sulfite loss corresponded exactly to the amount of sulfate formed, indicating
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that oxidation to sulfate was the sole mechanism of sulfite loss. However, another
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mechanism, alone or in addition to the mentioned oxidation mechanism, appeared to be
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present in the samples packed in glass bottles. The oxidation of sulfite to sulfate during
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storage, especially in oxygen-permeable packages, has been found in other foods
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(Taylor et al. 1986).
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Contrary to sulfites, initial values of sorbate (measured after 2 weeks storage)
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were considerably lower than the intended value (≈600 mg/L in brine, on average,
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versus 2000 mg/L), and in general this level of sorbate did not decrease significantly
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during storage (data not shown). The unexpectedly low levels of sorbic acid found in
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brine can be partially attributed to the precipitation of sorbic acid (its solubility in water
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at pH 3.1 and room temperature is 1500 mg/L ; Stopforth et al. 2005), and also to a
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possible autoxidation mechanism and/or irreversible reaction of sorbic acid with food
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components (Thakur et al. 1994; Scotter and Castle 2004). However, the degradation of
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sorbic acid due to LAB action can be ruled out, as LAB were not detected during
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storage.
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CONCLUSION
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The use of sulfites and sorbates, both separately and as a mixture, gave
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satisfactory results in relation to the long-term preservation of pickled garlic, even at
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ambient temperature. In addition, sulfite addition had a positive impact on garlic color
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through browning reduction. On the contrary, sorbate addition tended to enhance garlic
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browning. Consequently, the positive effect on color was not apparent when sulfites
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were added together with sorbates. Storage conditions also had a significant effect on
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garlic color, with plastic package plus ambient temperature being the worst conditions
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to preserve the original “white” color of blanched garlic. The stabilities of additives
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during storage were also quite different. Whereas the sorbate level hardly changed,
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regardless of the storage conditions used, losses in added sulfite during storage were
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apparent, particularly (>70%) in plastic pouches due to oxidation to sulfate. In all the
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conditions assayed, after 6 months of storage, all the retained sulfite was in the form of
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combined sulfite. Packers or processors should be aware of all these results if they use
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sulfites and/or sorbates as additives in pickled blanched garlic.
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STOPFORTH, J.D., SOFOS, J.N. and BUSTA, F.F. 2005. Sorbic acid and sorbates. In
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ACKNOWLEDGEMENTS
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This work was supported by the Spanish government through grant AGL-
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2007-62686 (partially financed by EU FEDER funds), and by the Junta de Andalucía
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financial support to group AGR-208.
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FIGURE CAPTIONS
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Figure 1. Values of pH (open symbols) and titratable acidity (filled symbols) during
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storage of pickled blanched garlic under different storage conditions. ○/●, packing C;
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□/■, packing Su;
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pouches, analyses after 27 and 46 weeks were not performed in packing C stored at
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room temperature. Values are means of two samples (n=2).
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Figure 2. Free (□) and combined (■) form of sulfites during storage of pickled blanched
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garlic under different storage conditions. Values are means of two samples,
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corresponding to packing Su and Su + So, each analysed in duplicate. Error bars show
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standard deviation of total sulfite (n=4). Total sulfite = free + combined sulfite.
/▼, packing So;
/▲, packing Su+So. Due to breakage of the
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TABLE 1.
INFLUENCE OF ADDITIVES AND STORAGE CONDITIONS ON COLOR
PARAMETERS OF PACKED PICKLED GARLIC AFTER 46 WEEKS STORAGEa
Color parameter
Factor
Additivesb
C
Su
So
Su + So
Storage
conditionsc
Amb, G
Amb, P
R, G
R, P
L*
a*
b*
hº
C
BI
Brine color
A440-A700
81.3a
80.9a
80.8a
82.0a
-2.1ab
-2.4a
-1.8b
-2.3ab
10.8b
9.3a
12.0b
10.8b
101.4ab
105.0c
99.4a
102.3b
11.0b
9.6a
12.1b
11.0b
12.0bc
9.6a
14.0c
11.7ab
0.038ab
0.021a
0.045b
0.036ab
80.2a
80.7ab
81.8ab
82.2b
-2.0ab
-1.8b
-2.5a
-2.4a
10.8b
13.1c
9.2ab
8.8a
101.0a
98.4a
104.6b
105.7b
11.0b
13.3c
10.0ab
9.3a
12.2b
15.6c
10.0ab
8.9a
0.043a
0.041a
0.028a
0.027a
a
Means within columns followed by the same letter are not significantly different.
C, no additive added to the cover brine (control); Su, sodium bisulfite added to the cover brine; So, potassium
sorbate added to the cover brine; Su + So, sodium bisulfite plus potassium sorbate added to the cover brine.
c
Amb, ambient temperature (20– 24 ºC); R, refrigeration (6–9 ºC); G, glass bottle; P, plastic pouch.
b
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TABLE 2.
FATE OF SULFITE ADDED TO PICKLED GARLIC STORED UNDER DIFFERENT
CONDITIONS AFTER 46 WEEKS AT ROOM TEMPERATURE
Packinga
Sulfite retainedb
Sulfite oxidized to sulfateb
17.3 ± 1.2a (20)
68.6 ± 9.4b (81)
Su, P
Su + So, P
23.6 ± 0.6b (28)
60.6 ± 0.6b (72)
Su, G
55.9 ± 6.3c (63)
28.3 ± 7.1a (32)
Su + So, G
64.5 ± 4.6c (71)
0 (0)
a
Su, sodium bisulfite added to the cover brine; Su + So, sodium bisulfite plus
potassium sorbate added to the cover brine; P, plastic pouch; G, glass bottle.
b
Mean ± SE, expressed as mg/L of SO2 in brine. Percentage of sulfite in
comparison with its initial concentration in parenthesis. Sulfite retained was 100%
in combined form. Values with the same letter indicate that there was no
significant differences between them at 95% level.
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