Journal of Food Engineering 89 (2008) 479–487

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Journal of Food Engineering

journal homepage: www.elsevier .com/locate / j foodeng

Improvement of the storage process for cracked table olives

F.N. Arroyo-López *, J. Bautista-Gallego, M.C. Durán-Quintana, F. Rodríguez-Gómez,C. Romero-Barranco, A. Garrido-FernándezDepartamento de Biotecnologia de Alimentos, Instituto de la Grasa (C.S.I.C), Avda Padre García Tejero No. 4, 41012 Seville, Spain

a r t i c l e i n f o

Article history:Received 4 December 2007Received in revised form 27 May 2008Accepted 1 June 2008Available online 7 June 2008

Keywords:Cracked table olivesCarbon dioxideCalcium chlorideMagnesium chlorideOzonePolyphenolsSodium metabisulphiteStorage

0260-8774/$ - see front matter � 2008 Elsevier Ltd. Adoi:10.1016/j.jfoodeng.2008.06.001

* Corresponding author. Tel.: +34 954 692516; fax:E-mail address: fnarroyo@cica.es (F.N. Arroyo-Lóp

a b s t r a c t

The aim of this work was to improve the storage method for cracked table olives. First, a study wascarried out of the washing phase in 5% (wt/vol) NaCl brine with ascorbic acid and sodium metabisulphiteadded to it. Its use removed half the proportion of the initial sugar content in the flesh and markedlydecreased the content of polyphenols in the fruits, confirming the effectiveness of this treatment.Washing with 5% NaCl and sodium metabisulphite was selected and applied in storage phase whichfollows. Storage of the washed cracked olives produced an additional decrease in sugars and polyphenolsin the flesh, although the product still retained a fairly high concentration of residual sugars at the end ofthe storage period. The growth of lactic acid bacteria in this phase was prevented by the high NaCl level(10–13% at the equilibrium) but yeast growth, followed by a decline phase, was found in all storage sys-tems studied. The use of a CO2 atmosphere led to a partial inactivation of yeasts for about 300 h andshowed a protective effect on polyphenols. Application of ozone had a favourable effect on initial solu-bilisation of sugars and polyphenols but caused a negative effect on fresh appearance, colour retentionand polyphenol degradation. CaCl2 + MgCl2 treatment led to a significantly faster decline phase of yeasts.A tentative improved process for cracked olive storage may consist of a previous washing of olives for24 h with a 5% NaCl solution (optionally added with 0.1% sodium metabisulphite), and a subsequent brin-ing in a 15% NaCl solution with 0.25% CaCl2 + 0.20% MgCl2 or in a 15% NaCl brine under CO2 atmosphere.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction bination with pH, temperature, and NaCl concentration) (Fernán-

Green ‘‘seasoned” Manzanilla-Aloreña is a table olive specialtythat is gaining the favour of consumers due to a progressive aware-ness for traditional and natural foodstuffs. When these fruits arestored in brine (�10% NaCl), they become progressively brownishin colour and loose commercial value. The cold (�7 �C) storage ofcracked olives could delay this phenomenon but the process in-creases the risk of spoilage and fruits packed as ‘‘seasoned” olivesare still prone to browning (Garrido Fernández et al., 2002). A pre-vious screening of alternative treatments has shown some promis-ing perspectives (Arroyo-Lopez et al., 2007) but further studies arestill necessary to establish a satisfactory procedure. The improve-ment of the storage process should preserve the freshness of the ol-ives (green surface colour) for further packing. In addition, a deeperstudy of the microbiological changes throughout the process is apriority for the industry.

Carbon dioxide (CO2) is effective for extending the shelf life ofperishable foods by increasing both the lag phase and the genera-tion time of spoilage organisms. Its effect increases with concen-tration. CO2 negatively influenced the growth rate of Lactobacillussake (Devlieghere et al., 1998) and Listeria monocytogenes (in com-

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+34 954 691262.ez).

dez et al., 1997). Panagou et al. (2002) found a CO2 atmospheremost effective at keeping the number of yeasts low in dry-saltedolives. The use of CO2 during the storage process of Manzanilla-Aloreña olives may displace oxygen and, thus, prevent browningreactions and retard microbial growth.

Ozone has been declared Generally Recognized As Safe (GRAS)and in 2001 it was admitted as a direct food additive for the treat-ment, storage and processing of food (Khadre et al., 2001a). It isrecommended as a sanitizer to eliminate contaminating sporeson packing materials and food contact surfaces (Khadre et al.,2001b). Its use during the storage process of these cracked fruitscould help to control the microbial population.

During green Spanish-style fermentation, chlorophylls aretransformed into pheophytins and pheophorbides while chloro-phyllides are detected as intermediate products. The exchange ofmagnesium ions for those of hydrogen in all the porphyrincompounds is favoured by the acid environment developed inthe process (Mínguez-Mosquera et al., 1994). The use of Mg2+ inthe storage solution may then delay the green colour degradation.Calcium ions inhibited the softening rate of fresh and Spanish stylegreen olives (Brenes et al., 1994). The presence of Ca2+ during thestorage of cracked Mananilla-Aloreña olives could prevent soften-ing and contribute to the maintenance of the fresh appearance ofthe fruits.

480 F.N. Arroyo-López et al. / Journal of Food Engineering 89 (2008) 479–487

Sulphite, together with calcium to improve texture, has beenused by Papageorge et al. (2003) as a microbial preservative for asalt-free storage of red bell peppers and for preventing the soften-ing caused by the oxygen in acidified peppers (McFeeters et al.,2004). Sulphite can inhibit the polyphenoloxidase action (Sayave-dra and Montgomery, 1986). Ascorbic acid has also shown anti-browning activity in bruised olive fruits. So sulphites and ascorbicacid may help to maintain the fresh appearance of cracked olives.

The aim of this study was to improve the current storage systemof cracked Manzanilla-Aloreña table olives by (i) studying theeffects of several washing systems with respect to their sugar,polyphenol removal, and microbial growth, (ii) investigating alter-native storage systems incorporating the previously selectedwashing procedure.

2. Materials and methods

2.1. Samples and experimental design

Fresh cracked Manzanilla-Aloreña olives were obtained directlyfrom the industry (Aceitunas Bravo S.A., Málaga, Spain). The studyconsisted of two phases, which are shown in Fig. 1. First, a previousexperiment to select the most appropriate washing procedure wascarried out. Six kilogram of cracked olives were placed in 10 l con-tainers and washed for six days (�144 h) with the following solu-tions: (a) 5% NaCl (wt/vol) brine (control), (b) 5% NaCl brine addedwith 1% ascorbic acid, and (c) 5% NaCl brine added with 0.1% so-dium metabisulphite. In a second step, a complete storage process(washing and storage phases) was carried out, using similar con-tainers. In this case, olives were first subjected to a washing stepwith a 5% NaCl + 0.1% sodium metabisulphite solution for 24 hand then stored under diverse treatments, in which the initial NaClconcentration was always 15%; they consisted of storage: (1) under

Fig. 1. Scheme of the diverse treatments used in this work.

a CO2 atmosphere, (2) subjected to a periodical application of ozo-nated air (7.27 � 10�4 g O3/h) at 200 l/h flow rate for 30 min eachweek, (3) in a solution containing CaCl2 (0.25%) + MgCl2 (0.20%),and (4) in 15% NaCl brine alone (control). The ozonate air treat-ment was also applied to unwashed fruits. The volume of liquidsused for sampling was replaced with brine of the same initial com-position and the fruits removed for analysis substituted with ster-ile glass spheres of similar size. All experiments were carried out induplicate and analyzed for microbial and physicochemical charac-teristics for three months. The ozone was produced by a TODOZ-ONO�, Mod TD ZN (Colmenar Viejo, Madrid, Spain) equipmentand applied by injecting the ozonated air through a diffusion glassdevice introduced at the bottom of the container.

2.2. Physicochemical analysis

The analyses of olive brines for pH, titratable acidity (expressedas grams of lactic acid per 100 ml brine), combined acidity (as mil-liequivalents of HCl acid added to 1 l brine to reach pH 2.6, mEq/l),and salt (g NaCl per 100 ml brine) were carried out using the meth-ods described by Garrido Fernández et al. (1997).

Surface colour analyses on olives were performed using a BYK-Gardner Model 9000 Colour-view spectrophotometer, equippedwith computer software to calculate the CIE coordinates: L* (light-ness), a* (red–green), and b* (yellow–blue) values. Hue is the angu-lar component of the polar representation, while chroma is theradial component. Interference by stray light was minimized bycovering samples with a box, which had a matt black interior.The data of each measurement are the mean of 20 olives. Thechanges in the green colour of vegetables were expressed as �a*

(greenness) parameter, the ratio �a*/b*, and the Hue angle (Kocaet al., 2006). Tijskens et al. (2001), proved that the ratio �a*/b*

considerably reduced the observed variance within measuringsamples and could thus be considered as a kind of internalstandardization. Hue values (hab) were estimated from theequation:

hab ¼ arctanb�

a�ð1Þ

C* (chroma) values were obtained from the equation:

C� ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffia�2 þ b�2

qð2Þ

Changes in the L*, �a*/b*, hab and C* parameters were modelledaccording to a pseudo second order decay kinetic model that tookthe following equation:

p� ¼ P0 þc

1þ ckxð3Þ

where P0 was the constant (parameter value t1), c was a specific va-lue for each fit (parameter estimated change), and k was the con-stant rate, units per days (Taoukis et al., 1997). The time requiredto reach a 50% decrease (t50) in a particular parameter, i.e. loss of50% green (or 50% red formation) colour was estimated as (Taoukiset al., 1997):

t50 ¼1ck

ð4Þ

Polyphenols in fruits and brines were analyzed by high perfor-mance liquid chromatography (HPLC) as described Romero et al.(2004), using a Waters 2695 Alliance chromatograph and a25 cm � 46 mm i.d. 5 lm Lichrospher 100 (Merck, Darmstard, Ger-many) column. Sucrose, glucose, fructose and mannitol in fruitsand brines were also analyzed by HPLC as described by Sánchezet al. (2000), using an Aminex HPX-87C carbohydrate analysiscolumn (Bio Rad Labs) held at 85 �C.

F.N. Arroyo-López et al. / Journal of Food Engineering 89 (2008) 479–487 481

2.3. Microbiological analyses

Brine samples and their decimal dilutions were plated using aSpiral System model dwScientific (Dow Whitley Scientific Limited,England) on the appropriate medium. Subsequently, plates werecounted using a CounterMat v.3.10 (IUL, Barcelona, Spain) imageanalysis system. Enterobacteriaceae were counted on VRBD (Crys-tal-violet Neutral-Red bile glucose) agar (Merck, Darmstadt, Ger-many), lactic acid bacteria (LAB) on MRS (de Man, Rogosa andSharpe) agar (Oxoid) with 0.02% (wt/vol) sodium azide (Sigma,St. Luis, USA), and yeasts on YM (yeast–malt–peptone–glucosemedium) agar (DifcoTM, Becton and Dickinson Company, Sparks,MD, USA) with oxytetracycline and gentamicin sulphate as selec-tive agents for yeasts. Plates were incubated at 30 �C for 48–72 h.

The partial inhibition and later growth observed in some treat-ments was modelled according to the following equation, proposedby Pruitt and Kamau (1993):

N ¼ ðNmax=ð1þ expð�lðt � sÞÞÞ þ Ndying expð�atÞ ð5Þ

where N is the population size at time t, Nmax is the maximum pop-ulation reached after growth, l is the growth rate at which growthwould be observed with excess growth factor, s is the time at whichgrowth reaches Nmax/2, Ndying is the part of the subpopulationfatally damaged by the effect of the applied treatment, and a isthe death rate constant in the inhibition period. Nmax was consid-ered a parameter, making a total of 5 estimates. To facilitate thefit at the normal plot of log10 CFU/ml vs. time used in microbiology,the log10 transformation at both sides of the equation was achieved.A transformation of Nmax ¼ 10log Nmax and Ndying ¼ 10log Ndying was alsoaccomplished.

The microbial growth and decay were described by the modeldeveloped by Peleg (1996) based on the continuous logistic equa-tion on which a Fermi’s term was superimposed. The first compo-nent of the combined model accounts for growth and the secondfor decay. It has the form:

NðtÞ ¼N0 þ Ns�N0

ð1þexp½kgðtcg�tÞ�Þ

1þ exp½k1ðt � tclÞ�ð6Þ

where N(t) is the number of microorganisms at time t, N0 the initialnumber of microorganisms, Ns the maximum number that the envi-ronment can support, kg a growth rate constant, tcg a characteristictime indicating the time required to reach half the environmentalcapacity (i.e. N(tcg)/Ns = 1/2), kl a lethality or decline rate constantand tcl the time to reach 50% survival. Since N0 is usually known,the equation may be reduced to one with only five adjustableparameters. A transformation similar to that mentioned for theapplication of the equation of Pruitt and Kamau (1993) was also ap-plied for N0 and Ns.

2.4. Statistical data analysis

Statistica software version 6.0 was used for data processing.Microbial and pseudo second order decay kinetic models were fit-ted using the non-linear module of the package. Parameters wereconsidered significant when p < 0.05 and are expressed as esti-mates ± standard errors. The least significant difference betweenvariables or parameters was estimated as

LSD ¼ t0:025

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiMSW

1naþ 1

nb

� �sð7Þ

where the t0.025 is the t(Student)-value corresponding to the degreeof freedom of the pooled standard, MSW stands for the pooled meansquares (within groups), and na and nb are the number of samplesfrom which each error came from or the degree of freedom (df) ofeach parameter’s error. A simplified formula was applied in the case

of comparing averages or parameters with the same df (http://cas-tleton.edu/math/statistics).

3. Results

3.1. Physicochemical and microbiological evolution during washings

During the experimental washing waters (six days), the NaClconcentration decrease in brine was of �1.0% (solutions withascorbic acid and sodium metabisulphite) and �2.2% (control).The solutions containing ascorbic acid had the lowest initial pH(3.0), which went up to 3.9 at the end of the washing. In solutionswith sodium metabisulphite, the pH (�4.5) was not modified dur-ing this phase; and, in 5% NaCl brine alone the pH decreased from5.5 to 4.6.

No differences in polyphenol removal among washing systemswere observed. So, the effect of washings was evaluated combiningthe data from the three treatments. Results are then expressed asthe average ± pooled standard deviation. Oleuropein (190 ±40 mg/l) and hydrotyrosol glucoside (170 ± 20 mg/l) were thepolyphenols found in highest proportions in these solutions.Hydroxytyrosol (74 ± 7 mg/l), rutin (35 ± 7 mg/l), tyrosol (25 ±3 mg/l), luteolin-7-glucoside (22 ± 5 mg/l), tyrosol glucoside(14 ± 3 mg/l), and verbascoside (�1 mg/l) were removed in pro-gressively lower concentrations. Polyphenol concentrations in theolive flesh after washing were still high, especially for hydrotyrosolglucoside (400 ± 20 mg/kg). Residual concentrations of other poly-phenols were: tyrosol glucoside, 120 ± 3 mg/kg; hydroxytyrosol,69 ± 7 mg/kg; luteolin-7-glucoside, 62 ± 5 mg/kg; and rutin, 55 ±7 mg/kg.

The most abundant sugar in the raw material was glucose(25,700 ± 300 mg/kg), followed by fructose (7140 ± 50 mg/kg),mannitol (5960 ± 80 mg/kg) and sucrose (2100 ± 200 mg/kg). Theexperimental washings (six days) dramatically reduced sugar con-centrations in the flesh but no effect among the diverse solutionswas observed. The remaining sugars in the flesh after the washingswere 13,300 ± 200 mg/kg, 3080 ± 20 mg/kg, 2400 ± 50 mg/kg, and1000 ± 40 mg/kg, for glucose, fructose, mannitol and sucrose,respectively.

Yeasts, always present in the washing waters, had an initial per-iod of inactivation followed by one of growth (Fig. 2), which wasmodelled by the Pruitt and Kamau (1993) equation. Their decayand growth parameters are shown in Table 1. The population frac-tion fatally damaged was very high in all treatments (0.80–0.99).The highest death rate was observed in the control (0.271 h�1)and the lowest in ascorbic acid (0.114 h�1); the highest growth ratewas found in the washing water containing sodium metabisulphite(0.337 h�1) and the lowest in the control (0.258 h�1) but their con-fidence limits also overlapped. The time to reach half the finalaffordable population was significantly lower (46 h) in sodiummetabisulphite and the longest delay was observed in ascorbic acid(60 h), which, in turn, was not significantly different from the con-trol (56 h). Final yeast populations were not significantly different.LAB were observed after 50 h washing in the presence of sodiummetabisulphite, where they reached a final population of 5 log10

CFU/ml. Enterobacteriaceae were detected at 40 h in the presenceof sodium metabisulphite and at 70 h in the control. Their popula-tions at the end of washing were 5 log10 CFU/ml and 7 log10 CFU/ml, respectively.

3.2. Physicochemical and microbiological evolution during storage

The pH values stabilized at �4.2 at the end of storage in alltreatments. Titratable acidity (g lactic acid (l.a.)/100 ml brine) inbrine increased from 0.12 ± 0.02 (CaCl2 + MgCl2 and control) and

Fig. 2. Yeast growth during the application of the different washing systems. Valuesare the average of two replicates. Confidence limits of fits were intentionallyomitted to improve readability and their statistics are shown in Table 1.

Table 2Average (two replicates) residual sugar content (±standard error) in the brines at theend of the storage period, according to treatment

Treatment Glucose (mg/l) Fructose (mg/l) Mannitol (mg/l) Total (mg/l)

CO2 3033 ± 52b 235 ± 3a 1232 ± 16b 4500 ± 36b

Ozone 3415 ± 11c 225 ± 5a 1311 ± 11c 4951 ± 26c

CaCl2 + MgCl2 2718 ± 29a 224 ± 7a 1118 ± 9a 4060 ± 24a

Control 3302 ± 11c 264 ± 9b 1363 ± 6d 4929 ± 4c

Rows followed by different super indexes, within parameter, means significantdifferences based on their pooled standard error and its degree of freedom (4).

482 F.N. Arroyo-López et al. / Journal of Food Engineering 89 (2008) 479–487

0.17 ± 0.03 (CO2 and ozone) up to 0.32 ± 0.40 (CO2 atmosphere,CaCl2 + MgCl2) and 0.28 ± 0.06 (ozone and the control). Combinedacidity values were always slightly higher in CO2 atmosphereand ozone treated brines. No marked differences in NaCl contentamong treatments were observed in the equilibrium (final valuesranged from �10% to �13%).

CO2 atmosphere and ozone treatments had the highest initialsugar concentrations in brine which were between 400 and500 mg/l, 6500 and 7500 mg/l, 1200 and 1300 mg/l, and 1200and 1300 mg/l for sucrose, glucose, fructose and mannitol, respec-tively, whereas treatment using CaCl2 + MgCl2 and the control hadlower initial concentrations (below half the levels above men-tioned). Then, concentrations of glucose and fructose in brine de-creased, sucrose was practically exhausted, and mannitol, whosecontent initially increased in CaCl2 + MgCl2 and the control treat-ments, stabilized after 10–20 days. Changes after this moment ofstorage were reduced but glucose, fructose and mannitol (in se-lected treatments) showed a slight decreasing tendency. At theend of the storage period the average concentration of sugars inbrines are shown in Table 2. The lowest significant content of glu-cose, mannitol and total sugars was found in the CaCl2 + MgCl2

treatment, followed by that under CO2 atmosphere, the controland that with ozone, except for mannitol which content in the con-trol was higher. The level of fructose was always higher in the con-trol than in the alternative storage treatments.

The total sugar concentration in the flesh at the end of the stor-age period was fairly high (1457–1880 mg/kg). Differences amongtreatments were not significant. The composition of sugars at thismoment was: sucrose, not detected; fructose, 354–426 mg/kg: glu-cose, 622–680 mg/kg; and mannitol, 332–713 mg/kg.

The trend and relative concentrations of the diverse polyphe-nols during storage are shown in Fig. 3. Hydroxytyrosol glucoside

Table 1Growth/decay biological kinetic parameters for yeast evolution during the washing of crackto curves shown in Fig. 2

Treatment l (h�1) s (h)

Ascorbic acid 0.271 ± 0.025a 60 ± 3b

Sodium metabisulphite 0.337 ± 0.052a 46 ± 3a

Control 0.258 ± 0.019a 56 ± 2b

Notes: l, growth rate; s, time to reach Nmax/2; Ndying fatally damaged population fraction;standard error. The adj. R2 for the ascorbic acid, sodium metabisulphite and control treatthe fit was always <0.0001. Rows followed by different super indexes, within parameter,freedom (33).

and oleuropein were the main polyphenols that passed progres-sively into the brine and reached their maximum concentrationsbetween the 10th and 20th day (Fig. 3). Then contents decreaseddue to their progressive hydrolysis with time. The highest initialconcentrations in brine were always found in the presence ofCO2 atmosphere and in those treated with ozone, althoughhydroxytyrosol glucoside and oleuropein rapidly decreased in thepresence of ozone. Oleuropein also showed high contents and a ra-pid decrease in the control (Fig. 3).

Storage significantly affected the evolution of colour. The valuesof L*, chroma (C*), Hue angle (hab), and �a*/b* are shown in Fig. 4.All the parameters showed a rapid decrease with time for aboutthe first 20 days and then continued at a slower rate. The changeswere more clearly observed in L* and C* than in Hue angle and –a*/b*. Fruits treated with ozone had the lowest measurements forlightness, Hue angle, chroma and �a*/b* value. The comparisonamong treatments was made by the corresponding kinetic param-eters (Table 3). Changes in L* were 10.76 units for the ozone treat-ment, which was statistically higher than the values obtained inCaCl2 + MgCl2, the control and CO2 atmosphere treatments butwithout statistical differences among these. Similar trends weredetected when changes were assessed by the �a*/b*, and Hue an-gle. The t50 parameter was markedly higher for the treatment inCO2 atmosphere when colour was assessed by C* which indicatesa lower contribution of black and gray to colour in this case.

Microbial evolution was also affected by the different storagesystems (Fig. 5). Yeasts were the only microorganisms detected.The strongest initial inactivation was observed in treatmentsusing CO2 atmosphere. This phase lasted for about 150 h andkept the population below the rest of the treatments for about300 h. A shorter inactivation period (<50 h) was observed in therest of the treatments. After this period, vigorous growth wasalways observed. The maximum population was reached at�300 h. From then on, a slow progressive decrease was ob-served. The growth and decay phases were modelled accordingto the equation developed by Peleg (1996) always obtaining aninsignificant lack of fit (the pure error for this comparison wasobtained from the variance between replicates). The parametersof the model and some other characteristics of the fits areshown in Table 4. No significant differences were found amongexpected populations (5.7–5.9 log10 CFU/ml) and the growthrates (0.070–0.092 h�1) but the time to reach half the final

ed Manzanilla-Aloreña olives, obtained by fitting the Pruitt and Kamau model (1993)

Ndying a (h�1) Nmax (log10 CFU/ml)

0.80 ± 0.20a 0.114 ± 0.049a 6.40 ± 2.8a

0.99 ± 0.30a 0.153 ± 0.029a 5.84 ± 1.63a

0.99 ± 0.26a 0.271 ± 0.086a 6.18 ± 2.79a

a, death rate; Nmax maximum population reached. Each parameter is followed by itsments was 0.981, 0.962, and 0.984, respectively. Probability of the ANOVA F test formeans significant differences based on their pooled standard error and its degree of

Fig. 3. Changes in polyphenols (hydroxytyrosol glucoside, hydroxytyrosol, tyrosol glucoside, tyrosol, luteolin-7-glucoside, rutin, oleuropein and verbascoside) in brine duringthe storage period of cracked Manzanilla-Aloreña olives, according to the different storage treatments.

F.N. Arroyo-López et al. / Journal of Food Engineering 89 (2008) 479–487 483

population, tcg, was significantly higher in CO2 atmosphere thanin CaCl2 + MgCl2 and the control. The inactivation phase wasdifferent according to treatments and the death rate observedin olives treated with ozone was similar to CO2 and the controlbut significantly lower than in CaCl2 + MgCl2 treatment. In ozo-nated unwashed olives the yeast population was markedlyhigher, although followed a similar trend. There was also a ten-dency to show the lowest tcl values (shorter inactivation) forCaCl2 + MgCl2 and the control.

4. Discussion

4.1. Physicochemical and microbiological evolution during washingwaters

The characteristics of fruits (cracked), which offered an easy andrapid osmotic exchange between the flesh and the surroundingsolution, were essential to removing a significant proportion ofpolyphenols from the fresh of the olives during washing. The

Fig. 4. Changes in the surface colour of fruits (expressed as L*, hab, �a*/b*, and C*) during the storage period of cracked Manzanilla-Aloreña olives, according to the differentstorage treatments. Values are the average of two replicates. Confidence limits of fits were intentionally omitted to improve readability and their statistics are shown in Table 3.

Table 3Kinetic parameters of surface colour changes for L*, �a*/b*, hab and C* in fruitssubjected to the different storage systems studied

Parameter P0 c k (days�1) t50 (days)

L*

CO2 54.9(0.6)b 6.02(0.66)a 0.029(0.005)a 24Ozone 50.6(0.7)a 10.76(1.06)b 0.025(0.004)a 28CaCl2 + MgCl2 56.6(0.5)b 4.96(0.75)a 0.072(0.018)a 10Control 56.5(0.8)b 4.82(1.37)a 0.070(0.035)a 10

�a*/b*

CO2 �0.133(0.006)a 0.332(0.011)a 1.33(0.15)a 0.5Ozone �0.184(0.009)b 0.382(0.016)b 1.12(0.17)a 0.6CaCl2 + MgCl2 �0.147(0.010)a 0.347(0.018)ab 1.30(0.23)a 0.5Control �0.143(0.008)a 0.340(0.015)ab 1.39(0.20)a 0.5

hab

CO2 82.37(0.33)b 18.87(0.62)a 0.023(0.003)a 30Ozone 79.55(0.49)a 21.68(0.91)b 0.020(0.003)a 35CaCl2 + MgCl2 81.62(0.58)b 19.75(1.01)ab 0.023(0.004)a 30Control 81.81(0.43)b 19.32(0.82)ab 0.024(0.003)a 29

C*

CO2 35.2(1.2)b 8.7(1.1)b 0.0072(0.0009)a 99Ozone 30.3(1.1)a 14.2(1.2)c 0.0123(0.0015)a 58CaCl2 + MgCl2 37.6(1.1)bc 7.0(1.3)ab 0.0326(0.0097)b 22Control 38.2(0.5)c 5.5(0.6)a 0.0360(0.0061)c 19

Notes: P0, constant; c, specific value for each fit; k, constant rate; t50, time for 50%colour change in each parameter. Each parameter is followed by its standard error.Adj. R2 for the fits ranged from 0.875 to 0.998. Probability of the ANOVA F test forthe fit was always <0.0001. Rows followed by different super indexes, within kineticparameter and colour parameter, means significant differences based on theirpooled standard error and its degree of freedom (33).

Fig. 5. Yeast growth in the different storage brines during the storage period ofcracked Manzanilla-Aloreña olives. Inside main graph is a detail of the first 400 h ofstorage. Values are average of two replicates. Confidence limits of fits wereintentionally omitted to improve readability and their statistics are shown in Table 4.

484 F.N. Arroyo-López et al. / Journal of Food Engineering 89 (2008) 479–487

presence of hydroxytyrosol and tyrosol (in a lower proportion) inthese solutions may indicate that the hydrolysis of their corre-

sponding precursors was initiated from the very beginning of thisphase. Washing was also very effective in removing sugars fromthe olive flesh, which were decreased to approximately half of theirinitial concentration. Such a rapid solubilisation in cracked fruits isin contrast with the very slow rate in directly brined whole green(Garrido Fernández et al., 1997) or naturally black olives (Romeroet al., 2004).

Table 4Values of the Peleg model (1996) parameters obtained by fitting it to yeast growth and decay during cracked Manzanilla-Aloreña olive storage

Treatment Ns kg (h�1) tcg (h) kl (h�1) tcl (h�1)

CO2 5.9 ± 1.7a 0.092 ± 0.027a 261 ± 34b 0.0021 ± 0.0007ab 545 ± 210a

Ozone 5.9 ± 0.6a 0.070 ± 0.018a 211 ± 24ab 0.0002 ± 0.0001a 301 ± 123a

CaCl2 + MgCl2 5.8 ± 0.6a 0.071 ± 0.007a 166 ± 14a 0.0033 ± 0.0007b 250 ± 60a

Control 5.7 ± 0.6a 0.077 ± 0.022a 193 ± 23a 0.0020 ± 0.0009ab 250 ± 80a

Notes: Ns, maximum population reached (log10 CFU/ml); kg, growth rate constant; tcg, time to reach Ns/2; kl, decline rate constant; tcl, time to reach 50% survival. Eachparameter is followed by its standard error. The adj. R2 for treatments with CO2, ozone, CaCl2 + MgCl2, and the control were 0.943, 0.920, 0.967, and 0.962, respectively.Probability of the ANOVA F test for the fit was always <0.0001. Rows followed by different super indexes, within kinetic parameter, means significant differences based ontheir pooled standard error and its degree of freedom (CO2, 13; ozone, CaCl2 + MgCl2 and control, 17).

F.N. Arroyo-López et al. / Journal of Food Engineering 89 (2008) 479–487 485

Apparently, washing waters were a good media for supportingmicrobial activity, which was always detected from the very begin-ning of their application. The initial population consisted only ofyeasts but, after 40 h of immersion, LAB (solution containing so-dium metabisulphite) and Enterobacteriaceae (metabisulphiteand control) were also detected. The growth of these may be re-lated to the low NaCl concentration and with the pH values ofthe solutions. The period after which Enterobacteriaceae appeared(�40 h) was longer than that observed in the water or brine solu-tions used for washing untreated naturally black olives (GarridoFernández et al., 1997). The initial inactivation period for yeastslasted 10–20 h. The greatest growth rate was found in solutionsusing sodium metabisulphite. Furthermore, the intermediate deathrate in sodium metabisulphite is in contrast with its successful usefor preserving salt-free red bell peppers (McFeeters et al., 2004;Papageorge et al., 2003). The mechanism through which metab-isulphite did not prevent but stimulated the growth of yeasts, atthe concentrations used in this work, requires additional research.

As a result, the use of a dilute solution of NaCl for washingcracked Manzanilla-Aloreña olives removed a marked proportionof sugars, polyphenols and initiated salt absorption (�1% to 2%).The rate of leaching decreased as time progressed but most ofthese compounds passed into the brine during the first 24 h, whichcoincided with the phase of inactivation of the yeast population.Then, a period of 24 h was considered appropriate for the washingstep of the olives subjected to storage. The use of ascorbic acid ledto an excessively low pH (3.0–3.9) which may have a negative ef-fect on the green surface colour due to the degradation of chloro-phylls at these pH levels (Mínguez-Mosquera et al., 1994), andthe washing with fresh brine always had a pH higher than 4.5 units(from 5.5 to 4.6), which can be considered the limit for the inacti-vation of some Enterobacteriaceae. On the contrary, washing solu-tions containing sodium metabisulphite always had pH valuesslightly below the limit for the Enterobacteriaceae inhibition anddid not show any growth of LAB and Enterobacteriaceae for a per-iod 624 h. So the use of sodium metabisulphite brine solution maybe useful for controlling colour degradation in the washing step,because of its pH value around 4.5, and Enterobacteriaceae growth,provided the washing period were limited to 24 h, although brinealone may also be acceptable. The application of only one washingis more advisable than the use of two (Arroyo-Lopez et al., 2007),because of a lower contribution to pollution, a carry over of micro-bial contamination on the olives and residual metabisulphite.

4.2. Physicochemical and microbiological evolution during storage

The use of brine containing sodium metabilsuphite has beenpreviously recommended for Manzanilla-Aloreña storage (Ar-royo-Lopez et al., 2007). The US-EPA (2007) has maintained theinorganic sulphites as GRASS under certain limitations and label-ling requirements (US-EPA, 2007). However, the use of this chem-ical must be considered with caution, especially in long storage

periods, because it may cause health problems in some sensibleconsumers (Lester, 1995; WHO, 1999). Therefore, the study ofalternative systems, as intended in this work, is advisable.

There was an apparent contradiction between the increase inacidity and pH values because, in olive fermentation, an incrementin the first usually leads to a decrease in the second (Garrido Ferná-ndez et al., 1997). In this case, such an effect could have been coun-teracted by a simultaneous increase in combined acidity (RejanoNavarro, 1985), due to the progressive solubilisation of compoundslike polyphenols and organic acids from the flesh. This buffercapacity was similar to that found in directly brined olives andlower than those in green, in which the lye treatments cause agreater formation of organic salts (Rodríguez de la Borbolla y Alcaláand Rejano Navarro, 1978).

Cracking the olives had a similar effect to lye treatment in theelaboration of green olives (Garrido Fernández et al., 1997) infavouring a rapid solubilisation of sugars and their initial accumu-lation in the brine. Apparently, the use of sucrose, glucose, andfructose by the microorganisms was very rapid during the phaseof exponential growth (data not shown) but the rate of consump-tion varied among sugars. Sucrose was the only sugar completelyused up during the process (disappeared after 10–20 days).Glucose and fructose were always found in marked residualconcentrations after the maximum microbial (yeast) population,although with a slight tendency to decrease with time. The sugar(alcohol) which was least affected by storage was mannitol, possi-bly due to the low rate of its use by the spontaneous microbial pop-ulation, except in treatments containing CaCl2 + MgCl2 and, in alower proportion, CO2, in which a decreasing trend and the lowestconcentration at the end of the storage period were seen. Thisbehaviour may be an indication that the presence of these cationsmay contribute to improving stability and to inducing the use ofsugars and, especially mannitol.

The presence of residual sugars in brines also corresponded to aproportional level in the olive flesh. Consequently, sugars canhardly be completely eliminated (from brine or flesh), under theconditions used in these treatments in spite of their easy solubili-sation (cracked fruits). Packing these olives may re-start the micro-bial activity, produce gas and swell the containers (GarridoFernández et al., 2002). To prevent final product spoilage, the fruitsshould then be subjected to new washings before their elaborationas ‘‘seasoned” olives.

The initial NaCl concentration (15%) was very efficient for pre-venting LAB growth. This level is fairly common in home-madeor in dry-salted table olives (Panagou et al., 2002) but not in Man-zanilla-Aloreña in which the production of titratable acidity is nor-mal (Garrido Fernández et al., 2002). The fermentation (by yeasts)process of cracked olives was always more rapid than any of theother untreated (natural) fruits, a circumstance that may acceler-ate the degradation of chlorophylls and the browning of theolive surface and brine (Garrido Fernández et al., 2002). However,CO2 bubbling was effective in improving the solubilisation of

486 F.N. Arroyo-López et al. / Journal of Food Engineering 89 (2008) 479–487

polyphenols, its atmosphere had a protective effect on them andled to the highest concentrations of these compounds. The bub-bling of ozonated air also favoured solubilisation but it had a par-allel oxidative effect and caused a more rapid degradation ofcompounds like hydroxytyrosol glucoside, luteolin-7-glucoside,oleuropein, and rutin (Fig. 3). In treatments using CaCl2 + MgCl2,polyphenols followed a generally similar trend to that describedin the presence of CO2 atmosphere but their concentrations inbrines were always lower. A progressive hydrolysis of tyrosol glu-coside, luteolin-7-glucoside, and oleuropein was less evident dur-ing the first part (up to 10–20 days) of the storage periodbecause their solubilisation rates were higher than the hydrolysisrate but, after this period, the hydrolysis degradation predomi-nated and the products resulting were increasing progressively,except tyrosol glucoside which always showed an increasing trendthroughout the storage period (possibly due to its reduced hydro-lysis rate). Treatments had a limited effect on rutin and verbasco-side which were in lower concentrations. Overall, the sweeteningof fruits required only a short storage period since in just 10–20days a marked proportion of polyphenols passed into the brine.This time was shorter than that required by naturally black olives,which may be of 6 months (Balatsouras, 1995), or (whole) directlybrined olives subjected to an aerobic process, which also need alonger period (�3 months) (Durán-Quintana et al., 1985).

The mechanisms of green colour changes in fruits may followfirst order kinetics (Koca et al., 2006) or more complex processes,e.g. two consecutives reactions: one that increases colour andone that decreases it (Tijskens et al., 2001). In cracked Manzanil-la-Aloreña olives, appearance changes affected all the CIE L*a*b*

(Clydesdale, 1978) colour space parameters, especially L* (light-ness) and C* (chroma) and data were fit better by a second orderkinetic decay. Fruits became progressively darker (lightness movedcloser to the centre of the colour space); the Hue angle decreased,indicating that the surface colour changed from green–yellow(negative values of a* and positive of b*) to yellow–red, althoughthe colour remained closer to the first than to the second (�90�);changes in the values of C* were related to colour displacement to-wards the centre of the colour diagram and were slower in olivesstored under CO2 because they showed the highest value of t50.Application of ozone produced the greatest colour degradation,regardless of the procedure used to measure this attribute, and thiseffect was in contrast with the protective effect in maintaining thefreshness of some fruits (Bai and Zhang, 2003). The treatment withCaCl2 + MgCl2 showed a similar trend to the control regardless ofthe method used to measure colour; however, the general favour-able effect of calcium for improving texture is well established(Brenes et al., 1994). The tendency of the olive fruits to loose fresh-ness with storage time may be not only due to the progressivepolymerization of phenols but also to the degradation of chloro-phylls and the subsequent decrease in the green surface colour(Mínguez-Mosquera et al., 1994).

Panagou et al. (2002) showed an initial decline in yeast popula-tion followed by a steady increase until the end of the storage per-iod of dry-salted olives. In directly brined olives, an initialmicrobial inactivation was related to a lack of nutrients becauseof their slow solubilisation from whole fruits (García et al., 1992).In cracked olives, this behaviour must be due to the high NaCl con-centrations (10–13%) which prevented the growth of LAB andmade yeast the main microorganisms responsible for fermentationduring storage. The longest inactivation period was observed in ol-ives subjected to CO2 atmosphere which maintained the yeast pop-ulation below the levels observed in other treatments for a periodof about 300 h (possibly, after the nutrient content had reached acritical level); however, the final yeast populations were alwaysfairly similar. This behaviour was in agreement with the effect ex-pected from CO2: an extension of the lag phase and a decrease in

the growth rate (Daniels et al., 1985), as found previously withsimilar products (Arroyo-Lopez et al., 2007). The effectiveness ofthe previous washing water was confirmed by following microbialevolution in ozonated unwashed cracked olives (see Fig. 5) whichshowed maximum population and markedly higher growth ratethan in any of the other treatments.

5. Conclusions

This work has shown that the application of only one washingwater previous to the storage process of cracked table olives re-moved about 50% of the original sugars in the flesh and a markedproportion of the polyphenols thus contributing to lower availabil-ity of fermentable substrates in the storage and packing phases andto an earlier sweetening of the fruits. Storage under CO2 atmo-sphere led to a better preservation of polyphenols. The lowestresidual concentrations of sugars were found in CaCl2 + MgCl2, fol-lowed by the CO2 treatment. Ozone had a significantly negative ef-fect on the preservation of the fresh appearance during storage.The storage under CO2 atmosphere produced a complete inactiva-tion of yeast for a period of about 150 h and maintained the yeastpopulation below that in the rest of the treatments for an addi-tional period of about 300 h. As a result, a tentative process forcracked olive storage may consist of (1) one washing water with5% NaCl brine (optionally added with 0.1% sodium metabisulphite,which provides a suitable pH level for both colour stabilization andmicrobial control), (2) storage in: a 15% NaCl brine (to prevent LABgrowth) with CaCl2 (0.25%) + MgCl2 (0.20%) added; or 15% NaClwhile maintaining the container under a CO2 protective atmo-sphere. Possibly, the combination of both storage treatments willlead to better results but checking their additive effect requiresfurther research. The procedure may have some advantages overother previously recommended systems: limiting the use of so-dium metabisulphite (if added) to only the washing step, less con-tamination (only one washing water), preserving the oxidation ofpolyphenols, delaying and controlling microbial growth, reducingthe residual sugars in the storage brine, contributing to the reten-tion of the freshness appearance of the fruits, improving the nutri-tional value of the product (higher Ca2+ and Mg2+ concentrations),and opening new possible uses of CaCl2, MgCl2 and CO2 in tableolives.

Acknowledgments

This work was supported by the Spanish Government (AGL-2003-00779 and AGL-2006-03540/ALI), Junta de Andalucía(through financial support to group AGR-125), and EU (TDC-Olive,FOOD CP 2004 505524). F.N. Arroyo López also thank an I3P fellow-ship from CSIC.

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