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Effects of salt mixtures on Spanish green table olive fermentation performance
LWT - Food Science and Technology 46 (2012) 56e63
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Effects of salt mixtures on Spanish green table olive fermentation performance F. Rodríguez-Gómez, J. Bautista-Gallego*, V. Romero-Gil, F.N. Arroyo-López, A. Garrido-Fernández, P. García-García Departamento de Biotecnología de Alimentos, Instituto de la Grasa (CSIC), Avda, Padre García Tejero 4, 41012 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2011 Received in revised form 16 September 2011 Accepted 3 November 2011
This work investigates the effect of sodium, potassium, and calcium chloride salts on the performance of Spanish green table olive fermentation using a simple centroid mixture design. The presence of calcium chloride hindered the diffusion of all sugars and delayed the period required to reach their respective maximum concentrations in brines. Such effects can prevent tumultuous processes and gas pocket spoilage. Acetic acid was present at low concentrations in all treatments but was not generated during fermentation. The production rate of lactic acid was either decreased or delayed by the presence of calcium chloride but adequate final conditions were always reached. The chemometric analysis classified treatments into groups according to the presence of calcium chloride and disclosed a stimulating effect of potassium on lactic acid production. Therefore, these techniques can be a useful tool to investigate olive fermentation performance. According to the results, acceptable Spanish green table olives can be produced using salt mixtures, with the subsequent reduced sodium content in the final products. The results obtained in this study could also be of interest for other fermented vegetables. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Chloride salts Green table olives Sugars Organic acids
1. Introduction Storage and fermentation in brines is a traditional system for food preservation which is still useful because of its low energy consumption and cost. In this method of preservation, salt decreases the water activity, which helps to control the growth of undesirable microorganisms and improve the taste (Ross, Morgan, & Hill, 2002). However, authorities are increasingly concerned about the use of NaCl because of the relationship between high dietary sodium (Na) intake and cardiovascular diseases (British Scientific Advisory Committee, 2003). The recommended daily intake of Na in the US is currently 2500 mg (Code of Federal Regulations, 2003), but a survey has shown that the average sodium urinary excretion during the period 1957e2003 was 3526 mg Na/day (Berstein & Willet, 2010). In Europe the situation is similar and, particularly in Spain, the NaCl intake was about 9800 mg/day (Ortega et al., 2011). Recently, the European Union (WHO, 2010) has adopted a strategy to reduce salt levels in those foods most contributing to salt intake. The Spanish “Agencia Española de Seguridad Alimentaria y Nutrición” (AESAN) has passed the question to the food industry (AESAN, 2010). Table olives are potential candidates for action, particularly Spanish style green * Corresponding author. Tel.: þ34 954 690850; fax: þ34 954 691262. E-mail addresses: [email protected], [email protected] (J. BautistaGallego). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.11.002
olives. In this style olives are treated with a diluted sodium hydroxide solution (lye) until the alkali reaches 2/3 of the flesh; then the fruits are washed to remove the excess alkali and placed in a 100e110 (g/L) NaCl solution (brine) where they undergo a lactic fermentation. In spring, the salt content is raised to about 85 g/L to preserve the fermented product and, finally, fruits are packed in fresh brine (Garrido Fernández, Fernández Díaz, & Adams, 1997). Therefore, sodium is generously used in all processing steps of these olive products. Combinations of sodium, calcium and magnesium chloride salts have been widely used in cucumber fermentation (Guillou & Floros, 1993; Yoo, Hwang, Eog, & Moon, 2006). Solutions containing KCl and CaCl2 produced green directly brined olives with good commercial characteristics (Di Silva, 2000). The substitution of common salt in cracked green olives also led to normal processing (Bautista Gallego, Arroyo López, Durán Quintana, & Garrido Fernández, 2010). A study of the mass transfer and solute diffusion in brined cucumbers was carried out by Fasina, Fleming, and Thompson (2002), who found that the sugar exchange between cucumber and cover brine was slower than the exchange of malic acid or NaCl. In cucumber juice, glucose and fructose were degraded simultaneously but at different rates (Lu, Fleming, & McFeeters, 2001). However, no information on the changes of individual sugars and acetic or lactic acid production in the presence of sodium, potassium and calcium chloride salt mixtures is available. Thus, the aim of this work was to investigate healthier alternatives to the Spanish style green table olive brines by substituting
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them with different sodium, potassium and calcium chloride salt mixtures, focusing the attention on their effects on the fermentation performance (individual sugar release into brines and acetic and lactic acid production). The results were studied using the General Linear Model (GLM), mixture design and chemometric analyses. 2. Material and methods 2.1. Samples and experimental design Fresh Gordal cv. olives were obtained from a local producer (JOLCA SA, Seville, Spain). The fruits were treated with a 20 g/L sodium hydroxide solution up to 2/3 flesh (ca. 10 h). Then the olives were washed with tap water for 18 h and after removing the exhausted water, immersed in diverse brines with selected concentrations of sodium, potassium and calcium chloride salt mixtures. The experimental design, generated with Design Expert v 6.06 software package (State Easy, INC., Minneapolis, USA), consisted of a simplex centroid mixture design with NaCl, KCl and CaCl2 ranging from 40 to 100 g/L, from 0 to 40 g/L, and from 0 to 60 g/L, respectively (Table 1). After 24 h of immersion, CO2 was bubbled to saturation through the mixture of olives and brine to neutralize the excess alkali. One day later, the containers were inoculated with a starter culture of Lactobacillus pentosus TOMCLAB1 (previously isolated from table olives) and from this moment, fermentation was left to evolve naturally at ambient temperature (15 Ce25 C) at the pilot plant.
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(BioRad Labs, Hercules, Ca. U.S.A.) held at 85 C. Deionized water was used as eluent at 0.6 mL/min. For the organic acids, a Spherisorb ODS-2 (5 mm, 250 4.6 mm, Waters Inc.) column with deionized water (pH adjusted to 2.3 with phosphoric acid) as mobile phase was used. The flow rate was 1.2 mL/min. Samples (0.5 mL) were diluted (1:1) with deionized water, centrifuged at 11,600 g for 5 min and an aliquot of 20 mL was injected into the chromatograph. The system was composed of a Water 2690 Alliance (which includes a pump, column, heater and auto-sampler modules). The detections were achieved using a Water 410 Differential Refractometer. Quantification was achieved by comparison of the peak areas with the corresponding standards. Then, the production of lactic acid was modeled, as an empirical approach for estimating the corresponding pseudo parameters, using a re-parameterized Gompertz equation (Zwietering, Jongengburger, Rombous, & van’t Riet, 1990) which took the form:
1 m l ½ *e*ð tÞ C B A @ Aþ1 e Acidity ¼ Ae 0
(1) 1
where A is the upper asymptote, m a pseudo production rate (h ), and l (h) a pseudo lag phase for lactic acid production. The fit was accomplished using the non-linear module of the Statistica 6.0 package and Table Curve 2D v5.1 (Systat Software Inc., 2002). Parameters were considered significant when their probabilities to be null, according to the F of Fisher, were p 0.05. 2.3. Effect of mixture composition on the individual sugar concentrations in brine and lactic acid production
2.2. Physicochemical analysis Individual reducing sugars (glucose, fructose, sucrose and mannitol) and organic acids (lactic and acetic) were determined by HPLC according to the methods developed by Montaño, Sánchez, and Castro (1993). For the analysis of sugars, 0.5 mL of liquid and 1.5 mL of sorbitol internal standard (1 bg/L, w/v) were put into contact with 1 g IRA 120 (Hþ-form, 16e45 mesh, Fluka) and 1 g IRA 96 (free base, 20e50 mesh, Fluka) resins. After 1 h of contact, 0.5e1.0 mL was centrifuged. The clarified liquid was used for analysis using an Aminex HPX-87C carbohydrate analysis column
Table 1 Simplex centroid mixture design used to study the effect of diverse mixtures of NaCl, KCl and CaCl2 (g/L) on the fermentation of Gordal cultivar processed according to Spanish style. Lactic acid production rate (standard error in parenthesis) deduced from the fit of the reparameterized Gompertz model to lactic acid changes over time. Run number
NaCl
KCl
CaCl2
m (h1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
100 50 40 70 80 60 50 40 100 40 40 40 70 40 60
0 10 30 30 10 20 40 0* 0 0 30 0 0 0* 20
0 0* 30 0 10 20 10 0 0 60 30 60 30 0 20
0.105 0.126 0.064 0.079 0.042 0.043 0.085 0.059 0.059 0.057 0.057 0.046 0.049 0.086 0.045
0.019d 0.003d 0.006ab 0.012abc 0.004a 0.004a 0.016bc 0.007ab 0.008ab 0.006ab 0.007ab 0.005a 0.004ab 0.011bc 0.004a
Notes: Ranges of individual salts were: NaCl, 40e100 g/L; KCl, 0e40 g/L; and CaCl2, 0e60 g/L. *Experiments not subjected to the general constrain (NaCl þ KCl þ CaCl2 ¼ 100 g/L); m (pseudo specific production (g L1) rate); pooled standard error, 0.009; t0.05, 15 ¼ 2.13: Rows with different superscripts are significantly different at p 0.05.
Data were first studied using the General Linear Model (GLM) analysis and, later, introduced as responses of the mixture experimental design to deduce their respective Response Surface (RSM) as a function of the initial chloride salts (Myers & Montgomery, 2002). To achieve this, the maximum concentration or area under the curves of individual sugars or lactic acid vs. time were used as responses and subjected to quadratic regression, which can be represented by the following equation, expressed in the canonical (Sheffé) form:
R ¼
4 X 1
bi xi þ
4 XX
bij xi xj þ
i
4 X X X
bijk xi xj xk þ ε
(2)
i
where x1, x2, and x3 stand for NaCl, CaCl2, and KCl, respectively, R is the response (any physicochemical parameter or transformed value), and bi are the coefficients to be estimated. Successive sequential sums of squares to suggest the model, study of this by ANOVA analysis and selection of the coefficients (backward option) were achieved to deduce the final equation. Only significant (p 0.05) models (assessed by fit, lack of fit and precision) were considered. The coefficients retained were those significant (p 0.05) or re-introduced in application of the hierarchical principle. The models were then transformed so that they could be used with percentages. A more detailed discussion of the application of these models can be found elsewhere (Bautista Gallego et al., 2010). 2.4. Chemometric analysis Before chemometric analysis, the data were standardized according to Kowalski and Bender (1972). Standardized data were then subjected to hierarchical clustering and Principal Component Analysis (PCA), using a varimax rotation. For the selection of the
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number of PCS, the Kaiser criterion (Jolliffe, 1986) was followed and only factors with eigenvalues higher than 1.00 were retained. Statistica software version 7.0 (Statsoft Inc, Tulsa, USA) was used for data processing. 3. Results and discussion 3.1. Changes in the sugar composition of brines In olive fermentation, sugars diffuse from the flesh into the surrounding solution just after brining. In this work, the individual changes in the main olive sugar components (sucrose, glucose, fructose and mannitol) in brine vs. time were followed; such changes include diffusion from the flesh into the brine and use by microorganisms. Data were first subjected to GLM analysis, considering the successive contents as repeated measurements, and then to mixture design analysis, using individual parameters as responses. Sucrose sugar was detected in low proportions but differences among runs were found. Fig. 1a shows only changes in runs 1, 2, 9, 10 and 11 as examples to simplify the figure, but two main behaviors, with similar trends, could be observed. The first group includes all treatments without calcium chloride in their brines. In this group,
run 1 showed the highest sucrose level during most of the fermentation period, with sharp changes and a rapid decrease after 188 h. Other runs in the first group, which also had about 0.4e0.8 g/L sucrose, were 2, 4, 8, 9, and 14. In the second group (runs with diverse proportions of calcium chloride in the initial brine), the sucrose concentrations increased slowly and reached their maximum (about 0.2e0.4 g/L) after longer periods of time (>236 h) with levels below those in the first group for most of the time. However, at the end of the fermentation process, sucrose concentrations were low and fairly close in all runs. At industrial scale this sugar practically disappears from brines at the end of fermentation (Montaño, Sánchez, Casado, de Castro, & Rejano, 2003). Sucrose may be used instead of glucose for supplementing brines to prevent acetic acid production by LAB at the end of the fermentation (Chorianopoulus, Boziaris, Stamatiou, & Nychas, 2005). Using maximum sucrose concentration as a response, the model suggested by the sequential sums of squares was linear and the transformed equation took the form: Maximum sucrose (g/L) ¼ 0.0094)NaCl 0.0022)KCl þ 8.41) 104)CaCl2
(3)
Interpretation of the equation through the two dimension contour lines (Fig. 1b) showed that sucrose in brines (always low) markedly decreases as one moves from the NaCl vertex toward the base of the triangle. The contour lines are inclined toward the CaCl2 vertex, indicating that the decreasing effect of substituting NaCl with CaCl2 is stronger than that of exchanging NaCl by KCl. The analysis of the area under the sucrose curve (which reflects the overall changes with time) led to identical conclusions. In cucumbers, sugars were diffused more slowly than acids and the effect was assigned to their higher molecular weight (Fasina et al., 2002). Glucose is the most abundant sugar in olives (Garrido Fernández et al., 1997). Its changes with time were similar to those described for sucrose but with more relevant effects (Fig. 2a). Glucose concentrations in the brines of treatments without calcium (1, 2, 4, 8, 9 and 14) increased rapidly and reached their maximum (8e10 g/L) at about 48 h after brining while those with calcium rose more progressively and required about 90e180 h to reach a marked lower maximum (w4 g/L) (Fig. 2a). After the maximum, there was always a further progressive reduction in the glucose level because in this period the consumption rate was higher (due to fermentation) than diffusion. In this case, only the area under glucose was fit properly by a special cubic model. The transformed equation was: Area for glucose ¼ 17.08)NaCl 13.93)KCl þ 5.58)CaCl2 þ 0.351) NaCl)KCl 0.097)NaCl)CaCl2 þ 2.674)KCl)CaCl2 0.0664) (4) NaCl)KCl)CaCl2
Fig. 1. a) Changes in sucrose concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations in the mixtures. Graph was obtained by GLM considering values at the diverse sampling times as repeated measurements. Symbols in the graph are: , Run 1; , Run 2; , , Run 10; Run, 11. Vertical bars denote 0.95 confidence intervals. b) Run 9; Contour lines of maximum sucrose concentration in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The interactions NaCl*KCl and NaCl*CaCl2 in the equation were reintroduced after the coefficient selection to maintain the hierarchical principle. The graph of the two dimension countor lines (Fig. 2b) shows that the minimum area can be found in a region between 60 g/ L NaCl (center of the design) and the base of the triangle. Possibly, as one approaches the CaCl2 vertex, the predominant process might be the slow diffusion whereas as one approaches the KCl vertex, the dominant process could be consumption because there is no evidence that potassium causes any delay in diffusion. Glucose is usually the sugar of choice to supplement brines in the case of stuck fermentations (Chorianopoulus et al., 2005; Panagou & Katsaboxakis, 2006). Residues of glucose at the end of fermentation were found in industrially fermented olives (Montaño et al., 2003). Fructose changes in brine were similar to those described for sucrose and glucose. Runs without calcium chloride (1, 2, 4, 8, 9, and 14, although run 4 was removed from the graph because some
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Fig. 2. a) Changes in glucose concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. The graph was obtained by GLM considering values at the diverse sampling , Run 1; , Run 2; , times as repeated measurements. Symbols in the graph are: , Run 4; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; , Run 3; , Run 12; , Run 13; , Run 14; , Run 15. Vertical bars denote 0.95 Run 11; confidence intervals. b) Contour lines of the area under glucose in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
analyses were lost) showed a more rapid increase in fructose and an intermediate value for their maximum (3e4 g/L) but in those containing calcium, concentrations of sugar rose progressively and reached their maximum below 2.5 g/L (with marked differences among treatments) (Fig. 3a). Changes could be modeled properly using the area under fructose as a response. The analysis suggested a special cubic equation, which took the form: Area for fructose (g/L) ¼ 7.07)NaCl þ 35.21)KCl 2.13) CaCl2 0.717)NaCl)KCl þ 0.080)NaCl)CaCl2 0.805)KCl) (5) CaCl2 þ 0.0174)NaCl)KCl)CaCl2 The graph of this curve in two dimensional contour lines (Fig. 3b) showed that the region with a lower area was quite similar to that for glucose, with the only exception that, in this case, there was also a minimum in the center of the line connecting the NaCl and KCl vertexes, possibly due to its rapid consumption in this specific condition. The similarities of the contour lines of the models for the area under glucose and fructose indicate a fairly
59
Fig. 3. a) Changes in fructose concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations in the mixtures. The graph was obtained by GLM considering values at the diverse sampling , Run 1; , Run 2; , times as repeated measurement. Symbols in the graph are: Run 3; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; , Run 11; , Run 12; , Run 13; , Run 14; , Run 15. Vertical bars denote 0.95 confidence intervals. b) Contour lines of the area under fructose in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
similar behavior of both sugars during the fermentation process; however, the use of sucrose might be more directly dependent on the potassium concentration in brine and, possibly, LAB growth (Panagou & Katsaboxakis, 2006). Fructose was found to be completely exhausted during cucumber fermentation but not glucose (Lu, Fleming, & McFeeters, 2001). Mannitol is a sugar alcohol relatively abundant in olives (Garrido Fernández et al., 1997). The initial changes in this sugar were similar to those observed in sucrose, glucose or fructose (Fig. 4) with the same runs included in the first (without calcium) and second group (with calcium). Later, however, the trend was quite different because in most of the runs its consumption was limited and no appreciable decrease was observed (Fig. 4). As a result, in most runs, concentrations at the end of the process were fairly close to their maximum. The model suggested for maximum concentration was special cubic and the transformed equation was: Maximum mannitol (g/L) ¼ 0.060)NaCl þ 0.148)KCl 0.017) CaCl2 0.0019)NaCl)KCl þ 0.0012)NaCl)CaCl2 þ 0.0015) (6) KCl)CaCl2 0.8 þ 104)NaCl)KCl)CaCl2
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Fig. 4. Changes in mannitol concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. The graph was obtained by GLM considering the analysis at each , Run 1; , sampling time as repeated measurements. Symbols in the graph are: Run 2; , Run 3; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; Run 11; , Run 12; , Run 13; , Run 14; , Run 15. Vertical bars denote 0.95 confidence intervals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Its presentation as a two dimensional contour line (Fig. 5a) showed that, apparently, the substitution of sodium with potassium (one moves from the NaCl vertex toward the KCl vertex) does not have an influence on the maximum concentration; both cause the same effect on diffusion. However, when sodium was substituted with calcium the maximum concentration in mannitol decreased, indicating an interference in diffusion due to the presence of calcium. The highest overall effect (diffusion þ use) was observed in a region limited by the contour line corresponding to 4.06 g/L, inside of which the mannitol in brine was at its minimum. The model suggested to fit the area under mannitol was quadratic and the transformed equation took the form: Area for mannitol ¼ 17.35)NaCl þ 63.64)KCl 21.13) CaCl2 1.432)NaCl)KCl þ 0.752)NaCl)CaCl2
(7)
A different behavior of this parameter with respect to maximum concentrations was reflected in the contour lines of this equation (Fig. 5b). The higher proportions were expected in the line connecting the NaCl and CaCl2 vertexes, where its consumption was the lowest. The presence of calcium not only causes a maximum due to the interference in the diffusion but does not promote its consumption. On the contrary, the minimum area (maximum utilization during fermentation) was observed on the opposite side for a mixture of approximately 70 g/L NaCl þ 30 g/L KCl. The presence of a certain proportion of potassium does not cause any interference with mannitol diffusion but also produces a possible stimulating effect at specific combinations. Therefore, the graphs corresponding to maximum concentration and area under the mannitol curve (Fig. 5a and b) were complementary and contributed to the clarification of the different behaviors of potassium and calcium chloride salts in the mannitol changes in brine. Residual mannitol was also found by Montaño et al. (2003) in industrially fermented olives. 3.2. Production of organic acids Panagou, Schillinger, Franz, and Nychas (2008) found several organic acids during the Conservolea natural black olive
Fig. 5. a) Contour lines of maximum mannitol concentration in brine as a function of NaCl, KCl and CaCl2 concentrations in the mixtures. The graph was obtained by GLM considering time as repeated measurements. b) Contour lines of the area under mannitol in brine as a function of the NaCl, KCl and CaCl2 concentrations in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them.
fermentation, but in this work only acetic and lactic acids were found. Low proportions of acetic acid were present in the brines immediately after brining. It should have been produced during the lye treatment (Garrido Fernández et al., 1997). However, acetic acid concentration did not change during the process and therefore it was not produced during fermentation. Its average content ranged from 1.27 (0.39) g/L to 2.08 (0.46) g/L. These concentrations were of the same order as those found in Conservolea cv processed by the Spanish method (Chorianopoulus et al., 2005). However, in naturally fermented black olives, the presence of acetic acid was attributed to a shift from the homo to the heterofermentative
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metabolism of LAB (Panagou et al., 2008). A similar behavior could have occurred when brines from the Spanish style were supplemented with sucrose (Chorianopoulus et al., 2005). In a survey at industrial scale, markedly higher proportions of acetic acid, together with propionic acid were found, which were related to a possible development of the propionibacteria species (Montaño et al., 2003). The overall changes in lactic acid vs. time showed that lactic acid was absent before L. pentosus inoculation (w24 h) and was progressively produced during fermentation to reach a maximum between 13 and 16 g/L (Fig. 6a). Thus, in general, all runs achieved a proper fermentation without relating the final lactic acid contents to the initial chloride salts in brines. Montaño et al. (2003) found similar levels in the brines of industrially fermented green olives of Gordal, Manzanilla and Hojiblanca cultivars. In this work, lactic formation was very fast in treatments containing only NaCl (runs 1, 8, 9 and 14) or NaCl with small proportions of KCl (run 2) but concentrations showed a sharp decrease after 188 h due to a shift in
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production with respect to diffusion into the flesh. In each treatment this decrease was proportional to the maximum acid concentration (e.g. run 1 vs. run 8). However, in runs containing calcium chloride, the production was slower, diffusion into the flesh comparatively faster (with respect to production) and only a progressive increase was observed. Recently, fermentation of Conservolea natural olives using the following brines: i) 80 g/L NaCl, ii) 40 g/L NaCl þ 40 g/L KCl, iii) 40 g/L NaCl þ 40 g/L CaCl2, iv) 40 g/L KCl þ 40 g/L CaCl2, and v) 26 g/L NaCl þ 26 g/L KCl þ 26 g/L CaCl2, developed vigorous lactic process which led to fairly low pH (3.9e4.2) and lactic acid (w7.0e8.6 g/L). However, the most critical factor for olives acceptability was the sensory characteristics (Panagou, Hondrodimou, Mallouchos, & Nychas, 2011). To study acid formation and delay among runs after inoculation (Fig. 6a), the lactic acid changes vs. time curves were fitted using the reparameterized Gompertz equation (eq. (1)) and the corresponding pseudo-parameters obtained. The fit was always significant (p 0.05) and led to significant coefficients (p 0.05). The estimated delay in acid production as a function of salt concentrations was linear and the transformed equation was: Delay in lactic production (h) ¼ 0.634)NaCl þ 0.327)KCl þ 1.298) (8) CaCl2 The delay contour lines in the graph (Fig. 6b) were almost perpendicular to the base so that delay does not change as one moves from the NaCl vertex toward the base, provided the proportion of KCl to CaCl2 remains constant. However, the estimated lag phase decreases as one moves to the left (KCl vertex) or increases as ones moves toward the right (CaCl2 vertex). In summary, the initiation of lactic acid production is faster as the KCl content raises while progressive concentrations of CaCl2 delay its formation. Estimated production rates were not related to initial salt concentration but there were significant differences among runs (Table 1). In agreement with Fig. 6a, the highest production rate (Table 1) was found in run 2 followed by run 1, whose brines did not contain added calcium. High rates were also obtained in runs 14 and 7, which were characterized by the absence of, or low calcium contents. The lowest rates were found in runs 5, 6, 15, and 12, all of which with moderate or high calcium chloride contents. Complete information on homogeneous groups can be found in Table 1. The literature reports total acidity and production rate increases when brines were supplemented with sucrose, glucose or both (Chorianopoulus et al., 2005; Panagou & Katsaboxakis, 2006). The estimated maximum lactic acid production and area under the lactic acid curves vs. time were not related to the initial concentration in the diverse salts. In part, this behavior can be due to L. pentosus inoculation because spontaneous processes usually led to lower acidification (Panagou & Katsaboxakis, 2006; Panagou et al., 2008). 3.3. Multivariate analysis
Fig. 6. a) Changes in lactic acid concentrations (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations in the mixtures. The graph was obtained by GLM considering the analysis at sampling , Run 1; , Run 2; , times as repeated measurements. Symbols in the graph are: , Run 4; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; Run 3; , Run 11; , Run 12; , Run 13; , Run 14; Run 15. Vertical bars denote 0.95 confidence intervals. b) Contour lines of pseudo lag phase lactic acid production in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Using the maximum concentrations in sucrose, glucose, fructose, and mannitol as well as lactic and acetic acids in brines as variables, clustering showed that all replicate runs were perfectly recognized and grouped (Fig. 7); the separation between runs 1 and 9 was more apparent than real because of its short distance. In addition, Fig. 7 shows the presence of two well differentiated groups. The first, on the left, was characterized by including all treatments with CaCl2 while the second, on the right, was composed of runs without CaCl2. Therefore, the presence of CaCl2 was the most decisive factor for clustering. Treatments containing KCl, on the contrary, were distributed into both groups, supporting
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Fig. 7. Results of the hierarchical cluster analysis, using maximum sucrose, glucose, fructose, mannitol, acetic acid and lactic acid concentrations in brine variables and runs as clustering factors.
the hypothesis that the substitution of NaCl by KCl does not cause fermentation disturbances. Descending in the distances, treatment 13 also shows a clearly differentiated position in the first group because Enterobacteriaceae growth was practically prevented in this run and the lag phase of yeasts was particularly long (data not shown). The PC analysis extracted two eigenvalues higher than 1, indicating that the total number of variables could be grouped into only two Factors explaining 80.5% of the variability (i.e., the first principal component explained 58.1% and the second 22.4%, respectively). The relationship between the Factors and the original variables can be deduced from the projection of the original variables onto the plane of the first two Factors (Fig. 8a). The variables maximum sucrose, glucose, fructose and mannitol contents were strongly related to Factor 1 (their cosines or projections on it were high) which can then be re-named as “sugars” and may be used in substitution of the four individual sugars. However, acetic and lactic maximum concentrations have their higher projections (higher cosines) on Factor 2, so this must be associated to acids. Fig. 8a also shows that while sugars and acids were close to each other, the two groups were quite different (overall angle between them was close to 90 , with zero cosine and no relation at all). The diverse runs could be characterized with respect to Factors (and variables) when their scores are represented in the plane of the two Factors (Fig. 8b). Run 1 and its duplicate run 9, placed in the fourth quadrant, were characterized by an above average sugar concentration and below average acidity (negative Factor 2 values). Run 3 was characterized by the production of high acidity and slightly low sugar in brine; its position can be linked to an eventual stimulating effect of potassium. On the contrary, runs 10 and 12 share low sugar concentration but run 10 has the second lowest proportion of acid because of the previously shown delaying effect of CaCl2 on acid production. Treatments 2, 3, 4, 5, 7, 11, and 14, containing KCl in diverse proportions, were situated on the positive side of Factor 2 (acidity), indicating an eventual stimulating effect of potassium on the production of lactic acid (because no acetic acid was produced in any run), as also found in a previous work (Arroyo López, Durán Quintana, Romero, Rodríguez Gómez, & Garrido Fernández, 2007). On the contrary, most of the treatments related to the negative values of Factor 2
Fig. 8. a) Projection of variables (“sugars” and “acids”) onto the plane formed by the first two Factors. b) Projection of cases onto the plane formed by the first two Factors.
did not have potassium in their brines, particularly those with fairly low acidity values (runs 9 and 10). 3.4. Optimization Mixture design analysis may also disclose (based on the models previously deduced) the optimum combination of chloride salts which lead to specific targets (minimum concentrations and areas under sucrose, glucose, fructose, mannitol and acetic acid as well as maximum lactic acid content and area under its curve). The selected concentrations were 51.3 g/L NaCl, 36.2 g/L KCl and 12.5 g/L CaCl2. Therefore, the composition of this brine would be recommended for optimum Spanish style green table olive fermentations at industrial scale. In Conservolea natural black olives, consideration of sensory characteristics led to recommend a brine composed of 40 g/L NaCl þ 40 g/L KCl. This brine was then recommended to the Greek table olive industry to process olives with less sodium without affecting the traditional taste of the product (Panagou et al., 2011). 4. Conclusions The initial concentrations of sodium, potassium and calcium chloride can markedly influence the diffusion (and use) of glucose, sucrose, fructose and mannitol into the brine as well as the
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production of lactic acid. The major effect was due to the presence of calcium which slowed down the diffusion of all types of sugars and caused a delay in the production of lactic acid. Multivariate analysis was efficient in clustering the runs into two mains groups which corresponded to runs with and without CaCl2 in the brine. The application of PCA grouped the variables into only two Factors which included sugars and acids, respectively. The graph of run scores in the plane of the two Factors was useful to observe the situation of certain treatments containing potassium on the positive side of Factor 2 (acidity) and deduce the eventual stimulating effect of this cation on L. pentosus growth and lactic acid production. Data also allowed for the optimization of the process, according to specific targets, information that can be of great interest for the design of the new processes at industrial scale. Results obtained in this work can also be useful for other fermented vegetables. Acknowledgments This work was supported by the European Union (Probiolives, contract 243471), Spanish Government (projects AGL-2006-03540/ ALI, AGL2009-07436/ALI and AGL2010-15494/ALI), partially financed by European regional development funds (ERDF), CSIC (project 201070E058), and Junta de Andalucía (through financial support to group AGR-125). J. Bautista-Gallego and F.N. ArroyoLópez want to thank CSIC for their JAE-PREDOC fellowship and JAEDOC postdoctoral research contract, respectively. References AESAN (Agencia Española de Seguridad Alimentaria y Nutrición). (2010). Plan to reduce the salt intake, within the Estrategia NAOS. http://www.naos.aesan.msps. es/naos/observatorio/observatorio00102.html. Arroyo López, F. N., Durán Quintana, M. C., Romero, C., Rodríguez Gómez, F., & Garrido Fernández, A. (2007). Effect of storage process on the sugars, polyphenols, color, and microbiological changes in cracked Manzanilla-Aloreña table olives. Journal of Agricultural and Food Chemistry, 55, 7434e7444. Bautista Gallego, J., Arroyo López, F. N., Durán Quintana, M. C., & Garrido Fernández, A. (2010). Fermentation profiles of Manzanilla-Aloreña cracked green table olives in different chlorides salt mixtures. Food Microbiology, 27, 403e412. Berstein, A. M., & Willet, W. C. (2010). Trends in 24-h urinary sodium excretion in the United States, 1957e2003: a systematic review. American Journal of Clinical Nutrition, 92, 1172e1180. British Scientific Advisory Committee. (2003). Salt and health. The Stationary Office. www.tso.co.uk/bookshop Last access March 2011.
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Chorianopoulus, N. G., Boziaris, I. S., Stamatiou, A., & Nychas, G. J. E. (2005). Microbial association and acidity development of unheated and pasteurized green table olives fermented using glucose or sucrose supplements at various levels. Food Microbiology, 22, 117e124. Code of Federal Regulations. (2003). Title 21, Part 101.9. Nutrition labeling of foods. Washington: Federal Register Printing Office. Di Silva, A. (2000). Preliminary results of a new processing in order to obtain green table olives with a low sodium content. Industrie Alimentari, 39, 844e847. Fasina, O., Fleming, H., & Thompson, R. (2002). Mass transfer and solute diffusion in brined cucumbers. Journal of Food Science, 67, 181e187. Garrido Fernández, A., Fernández Díaz, M. J., & Adams, R. M. (1997). Table olives. Production and processing. London: Chapman & Hall. Guillou, A., & Floros, J. D. (1993). Multiresponse optimization minimizes salt in natural cucumber fermentation and storage. Journal of Food Science, 58, 1381e1389. Jolliffe, I. T. (1986). Principal component analysis. New York: Springer-Verlag. Kowalski, B. R., & Bender, C. F. (1972). Pattern recognition. A powerful approach to interpreting chemical data. Journal of the American Chemical Society, 94, 5632e5639. Lu, Z., Fleming, H. P., & McFeeters, R. F. (2001). Differential glucose and fructose utilization during cucumber juice fermentation. Journal of Food Science, 66, 162e166. Montaño, A., Sánchez, A. H., Casado, F. J., de Castro, A., & Rejano, L. (2003). Chemical profile of industrially fermented green olives of different varieties. Food Chemistry, 82, 297e302. Montaño, A., Sánchez, A. H., & Castro, A. (1993). Controlled fermentation of green table olives. Journal of Food Science, 58, 842e852. Myers, R. H., & Montgomery, D. C. (2002). Response surface methodology (2nd ed.). New York: John Wiley & Sons, Inc. Ortega, R. M., López Sobaler, A. M., Ballesteros, J. M., Pérez Farinós, N., Rodríguez Rodríguez, E., Aparicio, A., et al. (2011). Estimation of salt intake by 24 h urinary sodium excretion in a representative sample of Spanish adults. British Journal of Nutrition, 105, 787e794. Panagou, E. Z., Hondrodimou, O., Mallouchos, A., & Nychas, G.-J. E. (2011). A study on the implications of NaCl reduction in the fermentation profile of Conservolea natural black olives. Food Microbiology, 28, 1301e1307. Panagou, E. Z., & Katsaboxakis, C. Z. (2006). Effect of different brining treatments on the fermentation of cv. Conservolea green olives processed by the Spanish method. Food Microbiology, 23, 199e204. Panagou, E. Z., Schillinger, U., Franz, C. M. A. P., & Nychas, G. J. E. (2008). Microbiological and biochemical profile of cv. Conservolea naturally black olives during controlled fermentation with selected strains of lactic acid bacteria. Food Microbiology, 25, 348e358. Ross, R. P., Morgan, S., & Hill, C. (2002). Preservation and fermentation: past, present and future. International Journal of Food Microbiology, 79, 3e16. WHO. (2010). Geneve (Switze Council, Council conclusions of 8 June on “Action to reduce population salt intake for better health” Adoption of the conclusion. Official Journal of the European Union 11.11.2010 C305/3-C305-5.Irland). Yoo, K. M., Hwang, I. K., Eog, G., & Moon, B. (2006). Effects of salts and preheating temperatures of brine on the texture of pickled cucumbers. Journal of Food Science, 71, C97eC101. Zwietering, M. H., Jongengburger, I., Rombous, F. M., & van’t Riet, K. (1990). Modelling of the bacterial growth curve. Applied and Environmental Microbiology, 56, 1875e1881.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Effects of salt mixtures on Spanish green table olive fermentation performance F. Rodríguez-Gómez, J. Bautista-Gallego*, V. Romero-Gil, F.N. Arroyo-López, A. Garrido-Fernández, P. García-García Departamento de Biotecnología de Alimentos, Instituto de la Grasa (CSIC), Avda, Padre García Tejero 4, 41012 Sevilla, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 June 2011 Received in revised form 16 September 2011 Accepted 3 November 2011
This work investigates the effect of sodium, potassium, and calcium chloride salts on the performance of Spanish green table olive fermentation using a simple centroid mixture design. The presence of calcium chloride hindered the diffusion of all sugars and delayed the period required to reach their respective maximum concentrations in brines. Such effects can prevent tumultuous processes and gas pocket spoilage. Acetic acid was present at low concentrations in all treatments but was not generated during fermentation. The production rate of lactic acid was either decreased or delayed by the presence of calcium chloride but adequate final conditions were always reached. The chemometric analysis classified treatments into groups according to the presence of calcium chloride and disclosed a stimulating effect of potassium on lactic acid production. Therefore, these techniques can be a useful tool to investigate olive fermentation performance. According to the results, acceptable Spanish green table olives can be produced using salt mixtures, with the subsequent reduced sodium content in the final products. The results obtained in this study could also be of interest for other fermented vegetables. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Chloride salts Green table olives Sugars Organic acids
1. Introduction Storage and fermentation in brines is a traditional system for food preservation which is still useful because of its low energy consumption and cost. In this method of preservation, salt decreases the water activity, which helps to control the growth of undesirable microorganisms and improve the taste (Ross, Morgan, & Hill, 2002). However, authorities are increasingly concerned about the use of NaCl because of the relationship between high dietary sodium (Na) intake and cardiovascular diseases (British Scientific Advisory Committee, 2003). The recommended daily intake of Na in the US is currently 2500 mg (Code of Federal Regulations, 2003), but a survey has shown that the average sodium urinary excretion during the period 1957e2003 was 3526 mg Na/day (Berstein & Willet, 2010). In Europe the situation is similar and, particularly in Spain, the NaCl intake was about 9800 mg/day (Ortega et al., 2011). Recently, the European Union (WHO, 2010) has adopted a strategy to reduce salt levels in those foods most contributing to salt intake. The Spanish “Agencia Española de Seguridad Alimentaria y Nutrición” (AESAN) has passed the question to the food industry (AESAN, 2010). Table olives are potential candidates for action, particularly Spanish style green * Corresponding author. Tel.: þ34 954 690850; fax: þ34 954 691262. E-mail addresses: [email protected], [email protected] (J. BautistaGallego). 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.11.002
olives. In this style olives are treated with a diluted sodium hydroxide solution (lye) until the alkali reaches 2/3 of the flesh; then the fruits are washed to remove the excess alkali and placed in a 100e110 (g/L) NaCl solution (brine) where they undergo a lactic fermentation. In spring, the salt content is raised to about 85 g/L to preserve the fermented product and, finally, fruits are packed in fresh brine (Garrido Fernández, Fernández Díaz, & Adams, 1997). Therefore, sodium is generously used in all processing steps of these olive products. Combinations of sodium, calcium and magnesium chloride salts have been widely used in cucumber fermentation (Guillou & Floros, 1993; Yoo, Hwang, Eog, & Moon, 2006). Solutions containing KCl and CaCl2 produced green directly brined olives with good commercial characteristics (Di Silva, 2000). The substitution of common salt in cracked green olives also led to normal processing (Bautista Gallego, Arroyo López, Durán Quintana, & Garrido Fernández, 2010). A study of the mass transfer and solute diffusion in brined cucumbers was carried out by Fasina, Fleming, and Thompson (2002), who found that the sugar exchange between cucumber and cover brine was slower than the exchange of malic acid or NaCl. In cucumber juice, glucose and fructose were degraded simultaneously but at different rates (Lu, Fleming, & McFeeters, 2001). However, no information on the changes of individual sugars and acetic or lactic acid production in the presence of sodium, potassium and calcium chloride salt mixtures is available. Thus, the aim of this work was to investigate healthier alternatives to the Spanish style green table olive brines by substituting
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them with different sodium, potassium and calcium chloride salt mixtures, focusing the attention on their effects on the fermentation performance (individual sugar release into brines and acetic and lactic acid production). The results were studied using the General Linear Model (GLM), mixture design and chemometric analyses. 2. Material and methods 2.1. Samples and experimental design Fresh Gordal cv. olives were obtained from a local producer (JOLCA SA, Seville, Spain). The fruits were treated with a 20 g/L sodium hydroxide solution up to 2/3 flesh (ca. 10 h). Then the olives were washed with tap water for 18 h and after removing the exhausted water, immersed in diverse brines with selected concentrations of sodium, potassium and calcium chloride salt mixtures. The experimental design, generated with Design Expert v 6.06 software package (State Easy, INC., Minneapolis, USA), consisted of a simplex centroid mixture design with NaCl, KCl and CaCl2 ranging from 40 to 100 g/L, from 0 to 40 g/L, and from 0 to 60 g/L, respectively (Table 1). After 24 h of immersion, CO2 was bubbled to saturation through the mixture of olives and brine to neutralize the excess alkali. One day later, the containers were inoculated with a starter culture of Lactobacillus pentosus TOMCLAB1 (previously isolated from table olives) and from this moment, fermentation was left to evolve naturally at ambient temperature (15 Ce25 C) at the pilot plant.
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(BioRad Labs, Hercules, Ca. U.S.A.) held at 85 C. Deionized water was used as eluent at 0.6 mL/min. For the organic acids, a Spherisorb ODS-2 (5 mm, 250 4.6 mm, Waters Inc.) column with deionized water (pH adjusted to 2.3 with phosphoric acid) as mobile phase was used. The flow rate was 1.2 mL/min. Samples (0.5 mL) were diluted (1:1) with deionized water, centrifuged at 11,600 g for 5 min and an aliquot of 20 mL was injected into the chromatograph. The system was composed of a Water 2690 Alliance (which includes a pump, column, heater and auto-sampler modules). The detections were achieved using a Water 410 Differential Refractometer. Quantification was achieved by comparison of the peak areas with the corresponding standards. Then, the production of lactic acid was modeled, as an empirical approach for estimating the corresponding pseudo parameters, using a re-parameterized Gompertz equation (Zwietering, Jongengburger, Rombous, & van’t Riet, 1990) which took the form:
1 m l ½ *e*ð tÞ C B A @ Aþ1 e Acidity ¼ Ae 0
(1) 1
where A is the upper asymptote, m a pseudo production rate (h ), and l (h) a pseudo lag phase for lactic acid production. The fit was accomplished using the non-linear module of the Statistica 6.0 package and Table Curve 2D v5.1 (Systat Software Inc., 2002). Parameters were considered significant when their probabilities to be null, according to the F of Fisher, were p 0.05. 2.3. Effect of mixture composition on the individual sugar concentrations in brine and lactic acid production
2.2. Physicochemical analysis Individual reducing sugars (glucose, fructose, sucrose and mannitol) and organic acids (lactic and acetic) were determined by HPLC according to the methods developed by Montaño, Sánchez, and Castro (1993). For the analysis of sugars, 0.5 mL of liquid and 1.5 mL of sorbitol internal standard (1 bg/L, w/v) were put into contact with 1 g IRA 120 (Hþ-form, 16e45 mesh, Fluka) and 1 g IRA 96 (free base, 20e50 mesh, Fluka) resins. After 1 h of contact, 0.5e1.0 mL was centrifuged. The clarified liquid was used for analysis using an Aminex HPX-87C carbohydrate analysis column
Table 1 Simplex centroid mixture design used to study the effect of diverse mixtures of NaCl, KCl and CaCl2 (g/L) on the fermentation of Gordal cultivar processed according to Spanish style. Lactic acid production rate (standard error in parenthesis) deduced from the fit of the reparameterized Gompertz model to lactic acid changes over time. Run number
NaCl
KCl
CaCl2
m (h1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
100 50 40 70 80 60 50 40 100 40 40 40 70 40 60
0 10 30 30 10 20 40 0* 0 0 30 0 0 0* 20
0 0* 30 0 10 20 10 0 0 60 30 60 30 0 20
0.105 0.126 0.064 0.079 0.042 0.043 0.085 0.059 0.059 0.057 0.057 0.046 0.049 0.086 0.045
0.019d 0.003d 0.006ab 0.012abc 0.004a 0.004a 0.016bc 0.007ab 0.008ab 0.006ab 0.007ab 0.005a 0.004ab 0.011bc 0.004a
Notes: Ranges of individual salts were: NaCl, 40e100 g/L; KCl, 0e40 g/L; and CaCl2, 0e60 g/L. *Experiments not subjected to the general constrain (NaCl þ KCl þ CaCl2 ¼ 100 g/L); m (pseudo specific production (g L1) rate); pooled standard error, 0.009; t0.05, 15 ¼ 2.13: Rows with different superscripts are significantly different at p 0.05.
Data were first studied using the General Linear Model (GLM) analysis and, later, introduced as responses of the mixture experimental design to deduce their respective Response Surface (RSM) as a function of the initial chloride salts (Myers & Montgomery, 2002). To achieve this, the maximum concentration or area under the curves of individual sugars or lactic acid vs. time were used as responses and subjected to quadratic regression, which can be represented by the following equation, expressed in the canonical (Sheffé) form:
R ¼
4 X 1
bi xi þ
4 XX
bij xi xj þ
i
4 X X X
bijk xi xj xk þ ε
(2)
i
where x1, x2, and x3 stand for NaCl, CaCl2, and KCl, respectively, R is the response (any physicochemical parameter or transformed value), and bi are the coefficients to be estimated. Successive sequential sums of squares to suggest the model, study of this by ANOVA analysis and selection of the coefficients (backward option) were achieved to deduce the final equation. Only significant (p 0.05) models (assessed by fit, lack of fit and precision) were considered. The coefficients retained were those significant (p 0.05) or re-introduced in application of the hierarchical principle. The models were then transformed so that they could be used with percentages. A more detailed discussion of the application of these models can be found elsewhere (Bautista Gallego et al., 2010). 2.4. Chemometric analysis Before chemometric analysis, the data were standardized according to Kowalski and Bender (1972). Standardized data were then subjected to hierarchical clustering and Principal Component Analysis (PCA), using a varimax rotation. For the selection of the
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number of PCS, the Kaiser criterion (Jolliffe, 1986) was followed and only factors with eigenvalues higher than 1.00 were retained. Statistica software version 7.0 (Statsoft Inc, Tulsa, USA) was used for data processing. 3. Results and discussion 3.1. Changes in the sugar composition of brines In olive fermentation, sugars diffuse from the flesh into the surrounding solution just after brining. In this work, the individual changes in the main olive sugar components (sucrose, glucose, fructose and mannitol) in brine vs. time were followed; such changes include diffusion from the flesh into the brine and use by microorganisms. Data were first subjected to GLM analysis, considering the successive contents as repeated measurements, and then to mixture design analysis, using individual parameters as responses. Sucrose sugar was detected in low proportions but differences among runs were found. Fig. 1a shows only changes in runs 1, 2, 9, 10 and 11 as examples to simplify the figure, but two main behaviors, with similar trends, could be observed. The first group includes all treatments without calcium chloride in their brines. In this group,
run 1 showed the highest sucrose level during most of the fermentation period, with sharp changes and a rapid decrease after 188 h. Other runs in the first group, which also had about 0.4e0.8 g/L sucrose, were 2, 4, 8, 9, and 14. In the second group (runs with diverse proportions of calcium chloride in the initial brine), the sucrose concentrations increased slowly and reached their maximum (about 0.2e0.4 g/L) after longer periods of time (>236 h) with levels below those in the first group for most of the time. However, at the end of the fermentation process, sucrose concentrations were low and fairly close in all runs. At industrial scale this sugar practically disappears from brines at the end of fermentation (Montaño, Sánchez, Casado, de Castro, & Rejano, 2003). Sucrose may be used instead of glucose for supplementing brines to prevent acetic acid production by LAB at the end of the fermentation (Chorianopoulus, Boziaris, Stamatiou, & Nychas, 2005). Using maximum sucrose concentration as a response, the model suggested by the sequential sums of squares was linear and the transformed equation took the form: Maximum sucrose (g/L) ¼ 0.0094)NaCl 0.0022)KCl þ 8.41) 104)CaCl2
(3)
Interpretation of the equation through the two dimension contour lines (Fig. 1b) showed that sucrose in brines (always low) markedly decreases as one moves from the NaCl vertex toward the base of the triangle. The contour lines are inclined toward the CaCl2 vertex, indicating that the decreasing effect of substituting NaCl with CaCl2 is stronger than that of exchanging NaCl by KCl. The analysis of the area under the sucrose curve (which reflects the overall changes with time) led to identical conclusions. In cucumbers, sugars were diffused more slowly than acids and the effect was assigned to their higher molecular weight (Fasina et al., 2002). Glucose is the most abundant sugar in olives (Garrido Fernández et al., 1997). Its changes with time were similar to those described for sucrose but with more relevant effects (Fig. 2a). Glucose concentrations in the brines of treatments without calcium (1, 2, 4, 8, 9 and 14) increased rapidly and reached their maximum (8e10 g/L) at about 48 h after brining while those with calcium rose more progressively and required about 90e180 h to reach a marked lower maximum (w4 g/L) (Fig. 2a). After the maximum, there was always a further progressive reduction in the glucose level because in this period the consumption rate was higher (due to fermentation) than diffusion. In this case, only the area under glucose was fit properly by a special cubic model. The transformed equation was: Area for glucose ¼ 17.08)NaCl 13.93)KCl þ 5.58)CaCl2 þ 0.351) NaCl)KCl 0.097)NaCl)CaCl2 þ 2.674)KCl)CaCl2 0.0664) (4) NaCl)KCl)CaCl2
Fig. 1. a) Changes in sucrose concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations in the mixtures. Graph was obtained by GLM considering values at the diverse sampling times as repeated measurements. Symbols in the graph are: , Run 1; , Run 2; , , Run 10; Run, 11. Vertical bars denote 0.95 confidence intervals. b) Run 9; Contour lines of maximum sucrose concentration in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
The interactions NaCl*KCl and NaCl*CaCl2 in the equation were reintroduced after the coefficient selection to maintain the hierarchical principle. The graph of the two dimension countor lines (Fig. 2b) shows that the minimum area can be found in a region between 60 g/ L NaCl (center of the design) and the base of the triangle. Possibly, as one approaches the CaCl2 vertex, the predominant process might be the slow diffusion whereas as one approaches the KCl vertex, the dominant process could be consumption because there is no evidence that potassium causes any delay in diffusion. Glucose is usually the sugar of choice to supplement brines in the case of stuck fermentations (Chorianopoulus et al., 2005; Panagou & Katsaboxakis, 2006). Residues of glucose at the end of fermentation were found in industrially fermented olives (Montaño et al., 2003). Fructose changes in brine were similar to those described for sucrose and glucose. Runs without calcium chloride (1, 2, 4, 8, 9, and 14, although run 4 was removed from the graph because some
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Fig. 2. a) Changes in glucose concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. The graph was obtained by GLM considering values at the diverse sampling , Run 1; , Run 2; , times as repeated measurements. Symbols in the graph are: , Run 4; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; , Run 3; , Run 12; , Run 13; , Run 14; , Run 15. Vertical bars denote 0.95 Run 11; confidence intervals. b) Contour lines of the area under glucose in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
analyses were lost) showed a more rapid increase in fructose and an intermediate value for their maximum (3e4 g/L) but in those containing calcium, concentrations of sugar rose progressively and reached their maximum below 2.5 g/L (with marked differences among treatments) (Fig. 3a). Changes could be modeled properly using the area under fructose as a response. The analysis suggested a special cubic equation, which took the form: Area for fructose (g/L) ¼ 7.07)NaCl þ 35.21)KCl 2.13) CaCl2 0.717)NaCl)KCl þ 0.080)NaCl)CaCl2 0.805)KCl) (5) CaCl2 þ 0.0174)NaCl)KCl)CaCl2 The graph of this curve in two dimensional contour lines (Fig. 3b) showed that the region with a lower area was quite similar to that for glucose, with the only exception that, in this case, there was also a minimum in the center of the line connecting the NaCl and KCl vertexes, possibly due to its rapid consumption in this specific condition. The similarities of the contour lines of the models for the area under glucose and fructose indicate a fairly
59
Fig. 3. a) Changes in fructose concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations in the mixtures. The graph was obtained by GLM considering values at the diverse sampling , Run 1; , Run 2; , times as repeated measurement. Symbols in the graph are: Run 3; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; , Run 11; , Run 12; , Run 13; , Run 14; , Run 15. Vertical bars denote 0.95 confidence intervals. b) Contour lines of the area under fructose in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
similar behavior of both sugars during the fermentation process; however, the use of sucrose might be more directly dependent on the potassium concentration in brine and, possibly, LAB growth (Panagou & Katsaboxakis, 2006). Fructose was found to be completely exhausted during cucumber fermentation but not glucose (Lu, Fleming, & McFeeters, 2001). Mannitol is a sugar alcohol relatively abundant in olives (Garrido Fernández et al., 1997). The initial changes in this sugar were similar to those observed in sucrose, glucose or fructose (Fig. 4) with the same runs included in the first (without calcium) and second group (with calcium). Later, however, the trend was quite different because in most of the runs its consumption was limited and no appreciable decrease was observed (Fig. 4). As a result, in most runs, concentrations at the end of the process were fairly close to their maximum. The model suggested for maximum concentration was special cubic and the transformed equation was: Maximum mannitol (g/L) ¼ 0.060)NaCl þ 0.148)KCl 0.017) CaCl2 0.0019)NaCl)KCl þ 0.0012)NaCl)CaCl2 þ 0.0015) (6) KCl)CaCl2 0.8 þ 104)NaCl)KCl)CaCl2
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Fig. 4. Changes in mannitol concentration (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. The graph was obtained by GLM considering the analysis at each , Run 1; , sampling time as repeated measurements. Symbols in the graph are: Run 2; , Run 3; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; Run 11; , Run 12; , Run 13; , Run 14; , Run 15. Vertical bars denote 0.95 confidence intervals. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Its presentation as a two dimensional contour line (Fig. 5a) showed that, apparently, the substitution of sodium with potassium (one moves from the NaCl vertex toward the KCl vertex) does not have an influence on the maximum concentration; both cause the same effect on diffusion. However, when sodium was substituted with calcium the maximum concentration in mannitol decreased, indicating an interference in diffusion due to the presence of calcium. The highest overall effect (diffusion þ use) was observed in a region limited by the contour line corresponding to 4.06 g/L, inside of which the mannitol in brine was at its minimum. The model suggested to fit the area under mannitol was quadratic and the transformed equation took the form: Area for mannitol ¼ 17.35)NaCl þ 63.64)KCl 21.13) CaCl2 1.432)NaCl)KCl þ 0.752)NaCl)CaCl2
(7)
A different behavior of this parameter with respect to maximum concentrations was reflected in the contour lines of this equation (Fig. 5b). The higher proportions were expected in the line connecting the NaCl and CaCl2 vertexes, where its consumption was the lowest. The presence of calcium not only causes a maximum due to the interference in the diffusion but does not promote its consumption. On the contrary, the minimum area (maximum utilization during fermentation) was observed on the opposite side for a mixture of approximately 70 g/L NaCl þ 30 g/L KCl. The presence of a certain proportion of potassium does not cause any interference with mannitol diffusion but also produces a possible stimulating effect at specific combinations. Therefore, the graphs corresponding to maximum concentration and area under the mannitol curve (Fig. 5a and b) were complementary and contributed to the clarification of the different behaviors of potassium and calcium chloride salts in the mannitol changes in brine. Residual mannitol was also found by Montaño et al. (2003) in industrially fermented olives. 3.2. Production of organic acids Panagou, Schillinger, Franz, and Nychas (2008) found several organic acids during the Conservolea natural black olive
Fig. 5. a) Contour lines of maximum mannitol concentration in brine as a function of NaCl, KCl and CaCl2 concentrations in the mixtures. The graph was obtained by GLM considering time as repeated measurements. b) Contour lines of the area under mannitol in brine as a function of the NaCl, KCl and CaCl2 concentrations in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them.
fermentation, but in this work only acetic and lactic acids were found. Low proportions of acetic acid were present in the brines immediately after brining. It should have been produced during the lye treatment (Garrido Fernández et al., 1997). However, acetic acid concentration did not change during the process and therefore it was not produced during fermentation. Its average content ranged from 1.27 (0.39) g/L to 2.08 (0.46) g/L. These concentrations were of the same order as those found in Conservolea cv processed by the Spanish method (Chorianopoulus et al., 2005). However, in naturally fermented black olives, the presence of acetic acid was attributed to a shift from the homo to the heterofermentative
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metabolism of LAB (Panagou et al., 2008). A similar behavior could have occurred when brines from the Spanish style were supplemented with sucrose (Chorianopoulus et al., 2005). In a survey at industrial scale, markedly higher proportions of acetic acid, together with propionic acid were found, which were related to a possible development of the propionibacteria species (Montaño et al., 2003). The overall changes in lactic acid vs. time showed that lactic acid was absent before L. pentosus inoculation (w24 h) and was progressively produced during fermentation to reach a maximum between 13 and 16 g/L (Fig. 6a). Thus, in general, all runs achieved a proper fermentation without relating the final lactic acid contents to the initial chloride salts in brines. Montaño et al. (2003) found similar levels in the brines of industrially fermented green olives of Gordal, Manzanilla and Hojiblanca cultivars. In this work, lactic formation was very fast in treatments containing only NaCl (runs 1, 8, 9 and 14) or NaCl with small proportions of KCl (run 2) but concentrations showed a sharp decrease after 188 h due to a shift in
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production with respect to diffusion into the flesh. In each treatment this decrease was proportional to the maximum acid concentration (e.g. run 1 vs. run 8). However, in runs containing calcium chloride, the production was slower, diffusion into the flesh comparatively faster (with respect to production) and only a progressive increase was observed. Recently, fermentation of Conservolea natural olives using the following brines: i) 80 g/L NaCl, ii) 40 g/L NaCl þ 40 g/L KCl, iii) 40 g/L NaCl þ 40 g/L CaCl2, iv) 40 g/L KCl þ 40 g/L CaCl2, and v) 26 g/L NaCl þ 26 g/L KCl þ 26 g/L CaCl2, developed vigorous lactic process which led to fairly low pH (3.9e4.2) and lactic acid (w7.0e8.6 g/L). However, the most critical factor for olives acceptability was the sensory characteristics (Panagou, Hondrodimou, Mallouchos, & Nychas, 2011). To study acid formation and delay among runs after inoculation (Fig. 6a), the lactic acid changes vs. time curves were fitted using the reparameterized Gompertz equation (eq. (1)) and the corresponding pseudo-parameters obtained. The fit was always significant (p 0.05) and led to significant coefficients (p 0.05). The estimated delay in acid production as a function of salt concentrations was linear and the transformed equation was: Delay in lactic production (h) ¼ 0.634)NaCl þ 0.327)KCl þ 1.298) (8) CaCl2 The delay contour lines in the graph (Fig. 6b) were almost perpendicular to the base so that delay does not change as one moves from the NaCl vertex toward the base, provided the proportion of KCl to CaCl2 remains constant. However, the estimated lag phase decreases as one moves to the left (KCl vertex) or increases as ones moves toward the right (CaCl2 vertex). In summary, the initiation of lactic acid production is faster as the KCl content raises while progressive concentrations of CaCl2 delay its formation. Estimated production rates were not related to initial salt concentration but there were significant differences among runs (Table 1). In agreement with Fig. 6a, the highest production rate (Table 1) was found in run 2 followed by run 1, whose brines did not contain added calcium. High rates were also obtained in runs 14 and 7, which were characterized by the absence of, or low calcium contents. The lowest rates were found in runs 5, 6, 15, and 12, all of which with moderate or high calcium chloride contents. Complete information on homogeneous groups can be found in Table 1. The literature reports total acidity and production rate increases when brines were supplemented with sucrose, glucose or both (Chorianopoulus et al., 2005; Panagou & Katsaboxakis, 2006). The estimated maximum lactic acid production and area under the lactic acid curves vs. time were not related to the initial concentration in the diverse salts. In part, this behavior can be due to L. pentosus inoculation because spontaneous processes usually led to lower acidification (Panagou & Katsaboxakis, 2006; Panagou et al., 2008). 3.3. Multivariate analysis
Fig. 6. a) Changes in lactic acid concentrations (unweighted means) in Spanish style green olive brines vs. sampling times, according to NaCl, KCl and CaCl2 concentrations in the mixtures. The graph was obtained by GLM considering the analysis at sampling , Run 1; , Run 2; , times as repeated measurements. Symbols in the graph are: , Run 4; , Run 5; , Run 6; , Run 7; , Run 8; , Run 9; , Run 10; Run 3; , Run 11; , Run 12; , Run 13; , Run 14; Run 15. Vertical bars denote 0.95 confidence intervals. b) Contour lines of pseudo lag phase lactic acid production in brine as a function of the NaCl, KCl and CaCl2 concentrations (g/L) in the mixtures. Design points. Duplicate design points are indicated by a 2 close to them. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Using the maximum concentrations in sucrose, glucose, fructose, and mannitol as well as lactic and acetic acids in brines as variables, clustering showed that all replicate runs were perfectly recognized and grouped (Fig. 7); the separation between runs 1 and 9 was more apparent than real because of its short distance. In addition, Fig. 7 shows the presence of two well differentiated groups. The first, on the left, was characterized by including all treatments with CaCl2 while the second, on the right, was composed of runs without CaCl2. Therefore, the presence of CaCl2 was the most decisive factor for clustering. Treatments containing KCl, on the contrary, were distributed into both groups, supporting
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Fig. 7. Results of the hierarchical cluster analysis, using maximum sucrose, glucose, fructose, mannitol, acetic acid and lactic acid concentrations in brine variables and runs as clustering factors.
the hypothesis that the substitution of NaCl by KCl does not cause fermentation disturbances. Descending in the distances, treatment 13 also shows a clearly differentiated position in the first group because Enterobacteriaceae growth was practically prevented in this run and the lag phase of yeasts was particularly long (data not shown). The PC analysis extracted two eigenvalues higher than 1, indicating that the total number of variables could be grouped into only two Factors explaining 80.5% of the variability (i.e., the first principal component explained 58.1% and the second 22.4%, respectively). The relationship between the Factors and the original variables can be deduced from the projection of the original variables onto the plane of the first two Factors (Fig. 8a). The variables maximum sucrose, glucose, fructose and mannitol contents were strongly related to Factor 1 (their cosines or projections on it were high) which can then be re-named as “sugars” and may be used in substitution of the four individual sugars. However, acetic and lactic maximum concentrations have their higher projections (higher cosines) on Factor 2, so this must be associated to acids. Fig. 8a also shows that while sugars and acids were close to each other, the two groups were quite different (overall angle between them was close to 90 , with zero cosine and no relation at all). The diverse runs could be characterized with respect to Factors (and variables) when their scores are represented in the plane of the two Factors (Fig. 8b). Run 1 and its duplicate run 9, placed in the fourth quadrant, were characterized by an above average sugar concentration and below average acidity (negative Factor 2 values). Run 3 was characterized by the production of high acidity and slightly low sugar in brine; its position can be linked to an eventual stimulating effect of potassium. On the contrary, runs 10 and 12 share low sugar concentration but run 10 has the second lowest proportion of acid because of the previously shown delaying effect of CaCl2 on acid production. Treatments 2, 3, 4, 5, 7, 11, and 14, containing KCl in diverse proportions, were situated on the positive side of Factor 2 (acidity), indicating an eventual stimulating effect of potassium on the production of lactic acid (because no acetic acid was produced in any run), as also found in a previous work (Arroyo López, Durán Quintana, Romero, Rodríguez Gómez, & Garrido Fernández, 2007). On the contrary, most of the treatments related to the negative values of Factor 2
Fig. 8. a) Projection of variables (“sugars” and “acids”) onto the plane formed by the first two Factors. b) Projection of cases onto the plane formed by the first two Factors.
did not have potassium in their brines, particularly those with fairly low acidity values (runs 9 and 10). 3.4. Optimization Mixture design analysis may also disclose (based on the models previously deduced) the optimum combination of chloride salts which lead to specific targets (minimum concentrations and areas under sucrose, glucose, fructose, mannitol and acetic acid as well as maximum lactic acid content and area under its curve). The selected concentrations were 51.3 g/L NaCl, 36.2 g/L KCl and 12.5 g/L CaCl2. Therefore, the composition of this brine would be recommended for optimum Spanish style green table olive fermentations at industrial scale. In Conservolea natural black olives, consideration of sensory characteristics led to recommend a brine composed of 40 g/L NaCl þ 40 g/L KCl. This brine was then recommended to the Greek table olive industry to process olives with less sodium without affecting the traditional taste of the product (Panagou et al., 2011). 4. Conclusions The initial concentrations of sodium, potassium and calcium chloride can markedly influence the diffusion (and use) of glucose, sucrose, fructose and mannitol into the brine as well as the
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production of lactic acid. The major effect was due to the presence of calcium which slowed down the diffusion of all types of sugars and caused a delay in the production of lactic acid. Multivariate analysis was efficient in clustering the runs into two mains groups which corresponded to runs with and without CaCl2 in the brine. The application of PCA grouped the variables into only two Factors which included sugars and acids, respectively. The graph of run scores in the plane of the two Factors was useful to observe the situation of certain treatments containing potassium on the positive side of Factor 2 (acidity) and deduce the eventual stimulating effect of this cation on L. pentosus growth and lactic acid production. Data also allowed for the optimization of the process, according to specific targets, information that can be of great interest for the design of the new processes at industrial scale. Results obtained in this work can also be useful for other fermented vegetables. Acknowledgments This work was supported by the European Union (Probiolives, contract 243471), Spanish Government (projects AGL-2006-03540/ ALI, AGL2009-07436/ALI and AGL2010-15494/ALI), partially financed by European regional development funds (ERDF), CSIC (project 201070E058), and Junta de Andalucía (through financial support to group AGR-125). J. Bautista-Gallego and F.N. ArroyoLópez want to thank CSIC for their JAE-PREDOC fellowship and JAEDOC postdoctoral research contract, respectively. References AESAN (Agencia Española de Seguridad Alimentaria y Nutrición). (2010). Plan to reduce the salt intake, within the Estrategia NAOS. http://www.naos.aesan.msps. es/naos/observatorio/observatorio00102.html. Arroyo López, F. N., Durán Quintana, M. C., Romero, C., Rodríguez Gómez, F., & Garrido Fernández, A. (2007). Effect of storage process on the sugars, polyphenols, color, and microbiological changes in cracked Manzanilla-Aloreña table olives. Journal of Agricultural and Food Chemistry, 55, 7434e7444. Bautista Gallego, J., Arroyo López, F. N., Durán Quintana, M. C., & Garrido Fernández, A. (2010). Fermentation profiles of Manzanilla-Aloreña cracked green table olives in different chlorides salt mixtures. Food Microbiology, 27, 403e412. Berstein, A. M., & Willet, W. C. (2010). Trends in 24-h urinary sodium excretion in the United States, 1957e2003: a systematic review. American Journal of Clinical Nutrition, 92, 1172e1180. British Scientific Advisory Committee. (2003). Salt and health. The Stationary Office. www.tso.co.uk/bookshop Last access March 2011.
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