The effect of ZnCl2 on green Spanish-style table olive packaging, a
presentation style dependant behaviour
J. Bautista-Gallego*, F.N. Arroyo-López, V. Romero-Gil, F. Rodríguez-Gómez & A.
Garrido-Fernández.
Department of Food Biotechnology. Instituto de la Grasa (CSIC). Avda\ Padre García Tejero,
nº 4. 41012, Seville (Spain).
*Corresponding author: Joaquín Bautista-Gallego, PhD.
Tel: +34 954 692 516. Fax: +34 954 691 262.
E-mail address: joaquinbg@ig.csic.es; joaquin.bautistagallego@unito.it;
Short title: Effect of ZnCl2 addition on green table olive packaging
1
1
Abstract
2
BACKGROUND: Zinc chloride has been used previously as preservative in directly brined
3
olives with promising results. However, this is the first time that the effects of ZnCl2 addition
4
(0-1 g L-1) on green Spanish-style table olives (Manzanilla cv.) packaging has been studied.
5
RESULTS: The presence of ZnCl2 affected the physicochemical characteristics of the
6
products; presence of the Zn led lower pH values (particularly just after packaging) and
7
titratable and combined acidity values than the control but did not produced clear trends in the
8
colour parameters. No Enterobacteriaceae were found in any of the treatments evaluated. At
9
the highest ZnCl2 concentrations, the lactic acid bacteria were inhibited while, unexpectedly,
10
its presence showed lower effect than potassium sorbate against yeast population. Regardless
11
of the use of potassium sorbate or ZnCl2, the packages had a reduced microbial biodiversity
12
because only Lactobacillus pentosus and Pichia galeiformis were found at the end of the shelf
13
life. With respect to organoleptic characteristics, the presentations containing ZnCl2 were not
14
differentiated from the traditional product.
15
CONCLUSION: Zn chloride was less efficient than potassium sorbate as yeast inhibitor in
16
green Spanish-style olives, showing clear presentation style dependant behaviour for this
17
property. Its presence produced significant changes on chemical parameters but scarcely
18
affected colour or sensory characteristics.
19
20
21
Keywords: Zinc; preservation; shelf life; green Spanish-style olive; table olive packaging.
2
22
1. Introduction
23
The use of olives for human consumption is a well documented tradition in the area
24
around the Mediterranean Basin1 but, currently, table olive processing is found all around the
25
world. In the 2010/2011 season, its production reached 2,440,000 tons.
26
presentation is the so-called green Spanish-style, which accounts for, approximately, 60% of
27
total consumption. These olives are included in the category of treated olives, as defined in
28
the Trade Standard Applying to Table Olives issued by the IOC. 3 Briefly, the process consists
29
of debittering the olives with a diluted sodium hydroxide solution (18-25 g L-1 NaOH) until
30
the alkali reaches 2/3 of the flesh, followed by one or several washing waters to remove the
31
excess alkali. Then, the fruits are covered with brine (100-110 g L-1 NaCl solution) where
32
they typically undergo spontaneous lactic acid fermentation1, although starter cultures can
33
also be used. 4 Finally, the olives are packed according to various commercial presentations.
2
The most popular
34
No zinc (Zn) fortified/preserved green olives are currently available in the market,
35
although this microelement is allowed to be added to foods5,6 and has 18 authorized possible
36
claims in the current European Union legislation. 7 Zn is used in food technology because it
37
forms green colour complexes with chlorophyll derivatives. Thereby, Zn has been applied
38
(followed by heat treatment) to preserve the green colour of pears8 and olives. 9 Moreover, its
39
presence in the continuous alcoholic fermentation reduced the size of flocks, increased the
40
tolerance to alcohol and temperature, decreased the production of glycerol, and accumulated
41
in the yeast dry matter10. Zinc oxide has also shown antimicrobial activity against Listeria
42
monocytogenes, Salmonella enteriditis and Escherichia coli O157:H711, while ZnCl2 has
43
shown a strong inhibitory effect on table olive related yeasts.
44
Aloreña olives, a type of directly brined table olive elaboration, the presence of ZnCl2 led to a
45
marked reduction in Enterobacteriaceae and yeast populations14 and improvement of the
46
nutritional value of the product.
15,16
12,13
In cracked Manzanilla-
Furthermore, zinc sulphate and zinc perchlorate
3
47
completely inhibited fungal growth and their ability to produce mycotoxins from toxigenetic
48
strains of Fusarium graminearum, Penicillium citrinum and Aspergillus flavus; the conidia
49
production of all fungi was reduced and hyphae damage was also noticed. 17 Therefore, all
50
these findings suggest the use of Zn as preservative in food industry to control fungal growth.
51
The aim of the present work was to investigate the effect of ZnCl2 as preservative on
52
green Spanish-style table olives packaging. Particularly, the relationships between its
53
presence and the changes in physicochemical, microbiological, and sensory characteristics
54
during shelf life were studied.
55
2. Experimental
56
2.1. Samples and experimental design
57
The experiment was carried out with fully fermented Spanish-style Manzanilla green
58
olives supplied by JOLCA S.A. (Huévar del Aljarafe, Seville, Spain). The experiment
59
consisted of packaging olives in plastic pouches (polyester-polyethylene, 105 mL brine and
60
75 g olives) using the habitual industrial packaging conditions (0.12 g L-1 potassium sorbate,
61
50 g L-1 NaCl, 3.33 g L-1 citric acid, 0.66 g L-1 ascorbic acid and 1.66 g L-1 lactic acid) plus
62
additional treatments in which the presence of potassium sorbate was replaced with 0.25,
63
0.50, 0.75, and 1.00 g L-1 concentrations of ZnCl2; therefore, there were five different
64
treatments in total. The plastic pouches were manually filled with the appropriate brine and
65
closed twice with a thermal sealing at 177ºC for 3 s at the industry. Then, the pouches (a total
66
of 120) were transported and stored at room temperature (20±3 ºC) at the pilot plant of
67
Instituto de la Grasa (CSIC, Seville, Spain). Periodically, two replicate pouches per treatment
68
were removed and analyzed. The packed olives were also subjected to sensory tests at the end
69
of the study (3 months, to mimic the commercial shelf life).
70
2.2. Microbiological analysis
4
71
Brine samples and their decimal dilutions were plated using a Spiral System model
72
dwScientific (Don Whitley Scientific Limited, England) on the media described below.
73
Subsequently, plates were counted using a CounterMat v.3.10 (IUL, Barcelona, Spain) image
74
analysis system, and the results expressed as log10 CFU/mL. Enterobacteriaceae were
75
counted on VRBD (Crystal-violet Neutral-Red bile glucose)-agar (Merck, Darmstadt,
76
Germany), lactic acid bacteria (LAB) on MRS (de Man, Rogosa and Sharpe)-agar (Oxoid
77
LTD., Basingstoke, Hampshire, England) with 0.2 g L-1 sodium azide (Sigma, St. Luis, USA),
78
and yeasts on YM (yeast-malt-peptone-glucose medium)-agar (DifcoTM, Becton and
79
Dickinson Company, Sparks, MD, USA) supplemented with oxytetracycline and gentamicin
80
sulphate as selective agents for yeasts. The plates were incubated at 30 ºC for 48-72 h.
81
Changes in the microbial populations versus time were also assessed by estimating the area
82
under the corresponding growth/decline curves. The areas were calculated by integration
83
using OriginPro 7.5 software (OriginLab Corporation, Northampton, USA).
84
At the end of the study (~3 months), brine samples from the packaging (100 mL) were
85
collected in sterile conditions and plated on the yeast and LAB selective media. Then, a total
86
of 100 isolates, 50 LAB and 50 yeasts (10 for each treatment) were randomly selected and
87
purified by subsequent re-streaking on YM or MRS agar, respectively, and subjected to
88
molecular identification. The different LAB isolates were identified at species level using a
89
multiplex PCR analysis of the recA gene with species-specific primers for Lactobacillus
90
pentosus, Lactobacillus plantarum and Lactobacillus paraplantarum, following the protocol
91
described by Torriani et al.18. Yeasts were identified by an RFLP analysis of the 5.8S-ITS
92
rDNA region according to the procedure described by Esteve-Zarzoso et al.19
93
2.3. Physicochemical analyses
94
95
The analyses of brines for pH, NaCl concentration, titratable acidity, and combined
acidity were carried out using the standard methods described for table olives. 1
5
96
Olive surface colour analyses were performed using a BYK-Gardner Model 9000
97
Color-view spectrophotometer, equipped with computer software to calculate the CIE
98
coordinates: L* (lightness), a* (negative values indicate green while positive values indicate
99
magenta), and b* (negative values indicate blue and positive values indicate yellow).
100
Interference by stray light was minimized by covering the samples with a box which had a
101
matt black interior. The value for each treatment was the mean of the determinations from 20
102
olives.
103
The colour index (Ci) was calculated according to Sanchez et al.20
Ci 
104
2 R560  R590  2 R630  2 R640
3
105
where Rs are for the reflectance values at 560, 590, 630 and 640 nm, respectively.
106
2.5. Sensory analysis
107
Tests were conducted by a panel of 12 members (5 men and 7 women) of experienced
108
judges from the staff of Instituto de la Grasa (CSIC, Seville, Spain). Their age ranged from 25
109
to 55 years and their experience in participating on sensory panels for testing table olives
110
ranged from 4 to 15 years. All judges were familiar with ranking and the A-Not A tests. They
111
were subjected to two sessions during which the objectives and characteristics of the
112
experiment were explained. In addition, they were trained in the specific characteristics of the
113
new products and tests as described below. Two protocols were used: ranking and a modified
114
version of the A-Not A test.
115
In the first case, the control was presented to the judges as the standard and the judges
116
were able to taste the control as much as desired until they felt they had become familiar with
117
its sensory characteristics. Then, the judges were given the control (no ZnCl2) and the
118
products containing from 0.25 to 1.00 g L-1 ZnCl2. The products were presented, in random
119
order, in cups similar to those used in the olive oil tests, and numbered with a combination of
120
letters and numbers. Judges were asked to rank them in order of dissimilarity to the standard
6
121
(1 more similar; 5 less similar). In essence, the test was similar to the first protocol described
122
by Lee et al.21
123
For the A-Not A analysis, the version used by Lee et al.21 of this protocol was used.
124
As in the first protocol, the control was presented to the judges as the standard and the judges
125
were able to taste the control as much as desired until they felt they had become familiar with
126
its sensory characteristics. Then, the control and the four Zn treatments (0.25 - 1.00 g L-1)
127
were given to the judges, using the same methodology described for protocol 1, and were
128
required to report whether the product tasted the same or different from the standard.
129
Responses were given in terms of five categories “same sure”, “same not sure”, “same”,
130
“different not sure”, “different sure”. For the analysis of results, data from all the judges were
131
pooled onto a single response matrix and single R-index was computed. For the calculus, the
132
procedure described in Lee and van Hout22 was used. Significance of the obtained R values
133
was compared with the critical values for two side tests, expressed in percentages of the R-
134
index-50%.23
135
2.6. Statistical data analyses
136
Sigma Plot 12.0 (Systat Software, Inc., Chicago, IL, USA) and Statistica software
137
version 7.0 (Statsoft Inc, Tulsa, USA) were used for data presentation and analysis.
138
3. Results and discussion
139
3.1. Effects of ZnCl2 presence on the physicochemical characteristics
140
Initially, pH values were significantly lower in the Zn containing brines than in the
141
control. This was because the ZnCl2 added behaved as a Lewis acid and, although is order of
142
strength is lower than other usual strong acids like phosphoric, nitric or sulphuric acids24, its
143
presence produces a pH decrease in the fresh brines which is approximately correlated with its
144
concentration. During storage, there was a general tendency for pH to increase, possibly due
145
to equilibrium with the flesh, which lasted for about 10 days, followed by a new decrease
7
146
(Figure 1, upper panel). The treatments containing Zn retained the initial significant lower (as
147
assessed by the no overlapping of their respective confidence intervals) pH values than the
148
control (with sorbate) during about the first 500 h (~20 d) of packaging. Then, the presence of
149
ZnCl2 improves the product safety in such period. This contrasts with the reduced effect that
150
the addition of ZnCl2 in directly brined cracked Aloreña de Málaga olives had on the initial
151
pH of brines. 14
152
There was later an increase in pH that raised the level to a maximum of 3.55-3.60 at
153
approximately 1800 h (75d) (Figure 1, upper panel), followed by a new decreasing trend
154
which lowered this parameter to 3.50-3.55 at the end of the study (~2500 h or ~100 d). Some
155
of the Zn treatments (particularly those with the highest concentrations) showed also the
156
lowest pH values during this time but the differences were rather reduced. On the contrary, in
157
directly brined cracked Aloreña de Málaga olives changes with time were reduced. 14
158
At the end of the shelf life, the range of the average pH values among treatments was
159
of only 0.05 units, which probably would be of limited influence on the product stability.
160
Hence, the results have shown that pH did not remain stable during shelf life but
161
experimented some cycles, although with a general tendency to increase with time and a
162
slightly final lowering, leading the presence of Zn to lower pH values than the control en
163
many cases due to it acidic character of this compound.
164
brined cracked Aloreña de Málaga olives were markedly higher than in this style (4.03 vs.
165
3.5), although they were also similar among treatments. 14
24
The final pH values in directly
166
The concentration of NaCl in the samples had a marked variability during the first
167
1000 h (~42 days) period of packaging (Figure 1, lower panel).These oscillations could be
168
due to the equilibrium phenomenon between brine and flesh because, during the rest of the
169
time, the concentrations were more stable. In directly brined cracked Aloreña de Málaga
8
170
olives there was a marked NaCl initial decrease due to its absorption by the unsalted fresh
171
fruits used. 14 The levels of NaCl in the control treatment were higher than in the products
172
packed with ZnCl2 in most of the sampling points and, particularly, as time progressed; the
173
NaCl concentrations oscillated between 4.8 and 5.2, which is not an excessive range for this
174
product. The final concentrations in NaCl of the Zn added olive samples were significantly
175
lower than in the control. A similar effect of the ZnCl2 presence was observed in directly
176
brined cracked Aloreña de Málaga olives which final NaCl level was even lower (42 g L-1)14.
177
With respect to titratable acidity (Figure 2, upper panel), the presence of ZnCl2 clearly
178
affected the titratable acidity changes.; however, the acidic character 24 of ZnCl2 was observed
179
only initially and for the highest concentrations but, later, the contents were mostly below that
180
of the control, particularly after 1500 h and, approximately, correlated with concentrations in
181
the fresh brine. In general, many punctual significant differences during the study were
182
observed; although, due to the variability, commenting the overall trend is more convenient.
183
The trends observed in the control, which titratable acidity clearly increased, and that of
184
treatments added with ZnCl2, in which it decreased, particularly during the last part of the
185
study, were quite different. In some cases, the lowest titratable acidity was observed in the
186
treatments containing 0.50 g L-1 ZnCl2, which also had the lowest final titratable acidity
187
(Figure 2, upper panel) while at the end of the study the highest titratable acidity was
188
observed for the control (>6.0 g L-1). This trend in the evolution of the titratable acidity
189
indicates a production of acid by the LAB in the control (with potassium sorbate) and a clear
190
inhibition in the ZnCl2 added treatments. At the end of the study, the average titratable acidity
191
values of the Zn treated olives ranged from 4.8 to 5.2 g L-1, with the lowest value being
192
observed in the olives treated with 10.00 g L-1 ZnCl2; thus, in general, presence of ZnCl2
193
prevented lactic acid production and resulted in a greater stability. On the contrary, the
194
traditional preservative did not prevent LAB activity and led to a titratable acidity in control
9
195
around 10.00 g L-1. The high titratable acidity level is not a safety drawback1 but it is an
196
indication of a deficient stabilization of the product by just sorbate. In this aspect, ZnCl2 had a
197
more convenient behaviour. The trend observed for titratable acidity in this experiment
198
contrasts with that observed in directly brined cracked Aloreña de Málaga olives in which all
199
treatments increased acidity levels, being observed the highest values in treatments containing
200
0.5 g L-1 and 0.75 g L-1 ZnCl2 (and not in the control); furthermore, the levels in directly
201
brined cracked Aloreña de Málaga olives were markedly higher and only comparable to the
202
proportion reached in the green Spanish-style control. 14
203
The changes in combined acidity showed a fairly similar trend and had less variability
204
than those observed in pH but with outstanding differences among treatments (Figure 2, lower
205
panel). Initially, this parameter ranged from 0.020 to 0.025 Eq L-1, which is the recommended
206
level for proper packaging1. The acidic character24 of ZnCl2 also affected the levels of
207
combined acidity which was reduced in all treatments with respect to the control. In directly
208
brined cracked Aloreña de Málaga olives (non lye treated) the initial values were higher
209
(0.045-0.052 Eq L-1)14. However, as time progressed, there was a tendency for combined
210
acidity to increase, especially above 1500 h (~ 63 d). This increase was more relevant for the
211
control and can be related to the production of acid by the LAB. Possibly, part of the lactic
212
acid formed remained as lactate and contributed to increasing the combined acidity, with a
213
more evident effect in the control treatment. On the contrary, all treatments with ZnCl2 added
214
were efficient for mitigating combined acidity changes and led to quite similar values at the
215
end of the study. This increase in combined acidity could possibly be, at least in part, the
216
cause of the pH increase observed in parallel during the same period (Figure 1, upper panel).
217
On the contrary, in directly brined cracked Aloreña de Málaga olives the combined acidity
218
increase with time was reduced and oscillated between narrow limits (0.060-0.066 EqL-1)
219
without substantial effect of the ZnCl2 addition. 14
10
220
Colour is also an important quality attribute, particularly in the case of transparent
221
packaging materials. Higher values of Ci, specifically developed for green Spanish-style
222
olives20, are associated with better colour appreciations. The variability observed in this
223
parameter during the first period of the study was high and led to only a few significant
224
differences (Figure 3, panel A) while the Ci values were more homogeneous as the study
225
progressed. In any case, in the long run, Zn presence had a reduced effect on Ci. A similar
226
behaviour was also detected by Gallardo-Guerrero et al.9 However, the use of ZnCl2 in fresh
227
directly brined Aloreña de Málaga olives gave rise to a significantly lower Ci than in the
228
control15, indicating a clear different effect of ZnCl2 in both products.
229
High values of L* are related to clearer and more attractive olives. The changes
230
observed in L* (Figure 3, panel B) had smaller variability than most of the previously
231
described parameters. Overall, L* was fairly stable throughout the study although, at the end
232
of the study, two groups were noticed: the first one, with a higher value of L*, consisted of the
233
treatments containing from 0.50 to 1.00 g L-1 ZnCl2, while the second group had a slightly
234
lower L* value and consisted of the control and the treatment with 0.25 g L-1 ZnCl2. Hence,
235
the presence of Zn above a certain proportion may lead to higher final luminance and better,
236
more characteristic, green Spanish-style colour. The trend above described contrast with that
237
observed in directly brined cracked Aloreña de Málaga olives in which L* values markedly
238
decreased with time and only showed a slight increase at the end of the shelf life. 15
239
The values of a* and b* (Figure 3, panels C and D, respectively) showed high
240
variability and had marked changes from one sampling point to another. Both parameters had
241
at the end of the study slightly higher values in the control than in ZnCl2 added treatments but,
242
in general, the trend with time observed did not show clearly significant changes. In any case,
243
the trends of both parameters were quite different from those observed in directly brined
244
cracked Aloreña de Málaga olives in which a* increased progressively during shelf life while
11
245
b* had an initial increased followed by a decrease fairly similar to that experimented by L* in
246
the same experiment. 15
247
3.2. Effect of ZnCl2 addition on the microbial population
248
No Enterobacteriaceae were found in the plastic pouches of the packed green
249
Spanish-style Manzanilla olives. This is in agreement with the observation reported in studies
250
on the packaging of stored Aloreña cultivar where Enterobacteriaceae were never isolated25
251
and the main cause of its instability was attributed to the growth of yeasts. 26 On the contrary,
252
Enterobacteriaceae were present during approximately half shelf life of fresh directly brined
253
cracked Aloreña de Málaga olives, in spite of the favourable effect of ZnCl2 addition for
254
reducing their populations. 14
255
Regardless of the packaging conditions, in the green Spanish-style Manzanilla olive
256
packages of this study, only LAB and yeast populations were observed in the cover brines
257
(Figure 4). Level of ZnCl2 affected LAB growth/survival, which counts followed a very
258
similar trend in all treatments; however, the curve for 1.00 g L-1 ZnCl2 was below those
259
corresponding to the other treatments in most of the samples (Figure 4, upper panel). Initially,
260
the populations were around 4.5-5.5 log10 CFU mL-1 but a tendency to increase with time was
261
observed, reaching a maximum of 5.5-6.5 log10 CFU mL-1 at ~800 h (~33 d). Later, a new
262
slow general decrease was noticed which led to population levels close to the original ones or
263
below; particularly, the average final LAB population in samples containing 1.00 g L-1 ZnCl2
264
was fairly low, although its differences with respect to some others also containing this salt
265
were not significant due to its high variability. The LAB changes observed in this work are
266
quite different from those described in directly brined cracked Aloreña de Málaga olives,
267
because in this product, the initial counts were very low (absence of previous lactic acid
268
fermentation) but the evolution resembled more a classical fermentation, in spite of the
12
269
inhibitory effect of the 1.00 g L-1 ZnCl214, than to changes in a stabilized packaging. The
270
different behaviour was possibly caused by the abundance of nutrients in the case of (fresh)
271
Aloreña olives.
272
A convenient approach to studying the overall population changes is the estimation of
273
the area below the growth curve, directly related to the counts of microorganisms throughout
274
the period studied provided this is the same for all curves. Concentrations of 0.25 and 0.75 g
275
L-1 ZnCl2 showed an overall similar behaviour to the control because the confidence limits of
276
their respective areas overlap (Figure 5. upper panel). However, the addition of 1.00 g L-1
277
ZnCl2 had a marked inhibitory effect on LAB population because, in spite of the high
278
variability, the area of this curve was the lowest and significantly different with respect to the
279
other treatments (Figure 5, upper panel). The LAB inhibition by 1.00 g L-1 ZnCl2 in brines
280
had also been detected in fresh directly brined cracked Aloreña de Málaga olives14, a quite
281
different presentation (non lye treated, cracked, and seasoned) from the green Spanish-style
282
olives studied in this work. The trend observed in the curve of areas in directly brined cracked
283
Aloreña de Málaga olives had a more pronounced peak because the stronger stimulating
284
effects of the intermediate ZnCl2 concentrations. 14
285
The changes in yeast populations were particularly monitored (Figure 4, lower panel).
286
Initially, they were between 4.5-5.0 log10 CFU mL-1 and, with only a couple of sampling
287
exceptions, a clear decrease with time was noticed until about 2000 h (83 d) of packaging.
288
These populations are higher than those initially found in directly brined cracked Aloreña de
289
Málaga olives because the absence of any previous fermentative process in this
290
presentation14. Overall, data from this work show that the inhibitory effect of ZnCl2 on yeasts
291
has been slightly less effective than that of potassium sorbate (control) during a large part of
292
the study. These results contrast with the strong inhibitory effect observed in synthetic
293
medium for ZnCl2.
13
In any case, complete inhibition of the yeast population was not
13
294
achieved in the control either, although its curve was below those of treatments containing
295
ZnCl2 during a marked period of time. In directly brined cracked Aloreña de Málaga olives,
296
yeasts curves for ZnCl2 treatments were, on the contrary, below the control. 15
297
As in the case of LAB growth, a more general comparison among the yeast
298
populations in the different treatments was made through the representation of the areas under
299
the respective curves (Figure 5, lower panel). There was observed significant effects of ZnCl2
300
levels. Treatments with 0.25 and 1.00 g L-1 showed a great variability, which has led to their
301
confidence limits overlapping. The highest areas were obtained in brines containing 0.25 and
302
0.50 g L-1 ZnCl2 (without significant difference among them), while the presence of 0.75 and
303
1.00 g L-1 ZnCl2 led to lowest areas (higher inhibition) among Zn treatments. On the contrary,
304
the area under the yeast curve corresponding to the control (with potassium sorbate) was
305
significantly lower than any treatment containing ZnCl2. This behaviour indicates a relatively
306
low inhibitory effect of yeast by ZnCl2 with respect to that observed in synthetic culture
307
media where the inhibition was clearly achieved. 13 Furthermore, the present trend contrasts
308
with that observed during the shelf life of directly brined cracked Aloreña de Málaga olives in
309
which yeasts clearly decreased (the curve of areas was concave) in the presence of ZnCl2, and
310
particularly when the concentration was 0.75 g L-1 ZnCl2.
311
packaging conditions (previous microbial load, salt concentration, type of acid or pH) and
312
olive composition (polyphenols, nutrients, cell wall components or minerals) can also
313
markedly modify the inhibitory action of ZnCl2.
14
Apparently, differences in
314
The identification of the survival microorganisms was considered of interest in order
315
to investigate those LAB and yeasts with the highest resistance to the presence of ZnCl2 or
316
potassium sorbate as preservatives. With this objective, 50 isolates of LAB and 50 isolated of
317
yeasts were subjected to molecular identification. All the isolates from LAB were assigned to
318
the species Lactobacillus pentosus while all yeast strains were identified as Pichia galeiformis
14
319
(data not shown). Therefore, the inter-specific biodiversity in these packages at the end of the
320
shelf life was very limited. These species were also found by Rodríguez Gómez et al.27 during
321
the storage of Hojiblanca and Manzanilla olives intended for the elaboration of ripe olives in
322
an acidic medium. Apparently, the survival of P. galeiformis in these packed olives cannot
323
only be related to the presence of sorbate or ZnCl2, although Bautista-Gallego et al.13
324
described P. galeiformis as well-resistant yeast to ZnCl2 in synthetic medium, with MIC
325
values around 0.15 g L-1, but also to its good adaptation to the table olive environment. 27
326
3.3. Effect of the ZnCl2 addition on the sensory characteristics
327
The ranking test and the modified version of the A-Not A method have the advantage
328
of comparing a series of samples at the same time. 21,28 In the ranking test, the sum of all ranks
329
for the control, 0.25, 0.50, 0.75, and 1.00 g L-1 ZnCl2 treatments were 36, 31, 40, 34, and 39,
330
respectively. The critical value for 12 judges and 5 treatments, for p0.05, were 25 (the lowest
331
insignificant rank sum, any treatment) to 47 (the highest insignificant rank sum, any
332
treatment). 28 As the sums of all ranks were within these critical values, it can be deduced that
333
according to this test, there was no significant organoleptic differences among the treatments.
334
The results from the version of the A-Not A ranking test are shown in Table 1. The R-
335
indices for 0.25, 0.50, 0.75 and 1.00 g L-1 with respect to the control were 43.75%, 52.43%,
336
49.56% and 54.17%.22 The critical values for two side tests were 29.07% and 70.93% (12
337
judges and p=0.025). Therefore, using the R-index, all the treatments can be considered
338
similar to the control (the traditional product) from the sensory point of view.
339
It is apparent that the presence of ZnCl2 has in this study a limited effect on the
340
sensory characteristics. However, in directly brined cracked Aloreña de Málaga olives, the
341
addition of this salt affected the preferences of the panellists, particularly in the case of 0.75 g
342
L-1 ZnCl2 (78% preferred this treatment against the control)14and caused products with
15
343
marked different profiles15. The absence of lye treatment of fruits, maturation degree,
344
bitterness levels of both products may be among the causes of the different responses.
345
4. Conclusions
346
The addition of ZnCl2 to the cover brines used for the packaging of fermented green
347
Spanish-style Manzanilla table olives affected the physicochemical characteristics of the
348
traditional product and led to others with specific profiles. The treatment with the highest
349
ZnCl2 in the brine showed a clear inhibitory effect on LAB growth, with L. pentosus as the
350
only LAB species detected at the end of the study. The yeast population decreased
351
progressively along the shelf life in the presence of both potassium sorbate (mainly) and
352
ZnCl2 (less efficiently), with only P. galeiformis survival. This behaviour of Zn chloride was
353
unexpected and specific-dependent for the product; apparently, certain compounds or
354
conditions prevailing in green Spanish style olives may prevent/mitigate the demonstrated Zn
355
inhibitory effect on yeasts. On the contrary, the presence of Zn scarcely affected the sensory
356
profile of the traditional product while the same salt markedly affected non lye, fresh fruits,
357
packed Aloreña olives. Apparently, the effect of ZnCl2 on the organoleptic characteristics of
358
the packed olives is also quite dependant on the presentation style.
359
Acknowledgements
360
This work was supported by the Spanish Government (projects AGL2009-07436/ALI
361
and AGL2010-15494/ALI, partially financed by European regional development funds,
362
ERDF), and Junta de Andalucía (through financial support to group AGR-125). J. Bautista-
363
Gallego and F.N. Arroyo-López wish to thank the CSIC and the Spanish government for their
364
JAE predoctoral fellowship and Ramón y Cajal postdoctoral research contract, respectively.
365
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366
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Lipolytic
activity
of
the
19
yeast
species
associated
with
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438
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28 Jellinek G, Ranking tests, in Sensory evaluation of foods. VCH, Weinheim, Federal
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440
20
441
442
Figure 1. Effect of ZnCl2 on pH (upper panel) and chloride concentration (lower panel) in
443
brines of packed “Manzanilla” during shelf life. For each sample point, confidence limits that
444
do not overlap indicate significant differences at p <0.05. Symbols and treatments
445
correspondence:
446
, Control;
, 0.25 g L-1;
, 1.00 g L-1 of ZnCl2.
447
448
449
450
451
452
453
21
, 0.50 g L-1;
, 0.75 g L-1;
454
455
456
457
458
459
Figure 2. Effect of ZnCl2 on the titratable acidity (upper panel) and combined acidity (lower
460
panel) in brines of packed “Manzanilla” during shelf life. For each sample point, confidence
461
limits that do not overlap indicate significant differences at p <0.05. Symbols and treatments
462
correspondence:
463
, Control;
, 0.25 g L-1;
, 1.00 g L-1 of ZnCl2.
464
465
466
22
, 0.50 g L-1;
, 0.75 g L-1;
467
468
469
470
471
472
Figure 3. Effect of ZnCl2 on colour index (panel A), L* (panel B), a* (panel C) and b* (panel
473
D) according to treatments in packed “Manzanilla” olives. For each sample point, confidence
474
limits that do not overlap indicate significant differences at p <0.05. Symbols and treatments
475
correspondence:
476
,
Control;
, 0.25 g L-1;
, 1.00 g L-1 of ZnCl2.
477
478
479
480
23
, 0.50 g L-1;
, 0.75 g L-1;
481
482
483
484
485
Figure 4. Effect of ZnCl2 on the microbial populations in the brine of packed "Manzanilla”
486
olives during shelf life. Changes in lactic acid bacteria (upper panel) and yeasts (lower panel)
487
vs. time, according to treatments. For each sample point, confidence limits that do not overlap
488
indicate significant differences at p <0.05. Symbols and treatments correspondence:
489
Control;
490
ZnCl2.
, 0.25 g L-1;
, 0.50 g L-1;
491
492
493
494
495
496
24
, 0.75 g L-1;
,
, 1.00 g L-1 of
497
498
499
500
501
Figure 5. Effect of ZnCl2 on the areas under lactic acid bacteria (upper panel) and yeasts
502
(lower panel) curves vs. time, according to treatments (concentrations of ZnCl2 expressed as g
503
L-1). For each sample point, confidence limits that do not overlap indicate significant
504
differences at p <0.05.
25
Table 1. Results of the response matrix for the computation of the R-index when the five
products (including the control as reference) were tested using a similarity ranking to a
reference product.
Different Different Different Same Same Total
sure
unsure
unsure sure
*
12
1
2
2
5
2
0.25
12
1
4
6
0
1
0.50*
12
4
0
1
4
3
0.75*
12
3
3
2
2
2
1.00*
12
3
3
1
1
4
Control
-1
*Note: ZnCl2 concentrations in g L .
Treatments
26