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Use of central composite design in food microbiology: A case study on the

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DOI: 10.3109/09637486.2015.1064866

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Use of central composite design in food microbiology:

a case study on the effects of secondary phenols on
lactic acid bacteria from olives
a a a a
Barbara Speranza , Angela Racioppo , Milena Sinigaglia , Maria Rosaria Corbo & Antonio
a
Bevilacqua
a
Department of the Science of Agriculture, Food and Environment (SAFE), University of
Foggia, Foggia, Italy
Published online: 15 Jul 2015.

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To cite this article: Barbara Speranza, Angela Racioppo, Milena Sinigaglia, Maria Rosaria Corbo & Antonio Bevilacqua (2015)
Use of central composite design in food microbiology: a case study on the effects of secondary phenols on lactic acid bacteria
from olives, International Journal of Food Sciences and Nutrition, 66:5, 520-525

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ISSN: 0963-7486 (print), 1465-3478 (electronic)

Int J Food Sci Nutr, 2015; 66(5): 520–525

! 2015 Informa UK Ltd. DOI: 10.3109/09637486.2015.1064866

FOOD COMPOSITION AND ANALYSIS

Use of central composite design in food microbiology: a case study on

the effects of secondary phenols on lactic acid bacteria from olives
Barbara Speranza, Angela Racioppo, Milena Sinigaglia, Maria Rosaria Corbo, and Antonio Bevilacqua

Department of the Science of Agriculture, Food and Environment (SAFE), University of Foggia, Foggia, Italy

Abstract Keywords
This paper focuses on the use of statistical Design of Experiments (DoE) to investigate the Central composite design, growth, lactic acid
effects of two anti-lactic acid bacteria compounds on growth and metabolism of lactobacilli
Downloaded by [Antonio Bevilacqua] at 10:22 09 August 2015

bacteria, phenolic compounds, pH

isolated from Italian table olives. p-Coumaric and vanillic acids (0.0–0.4%) were used as phenolic
compounds, which were combined with salt (0.0–6.0%) and glucose (0.0–4.0%) through a History
Central Composite Design. Three strains of Lactobacillus plantarum (5 log cfu/ml) were used as
test organisms, samples were stored at 37  C, and cell counts and pH were evaluated Received 1 December 2014
periodically. The growth of lactobacilli was affected in a significant way by salt, p-coumaric and Revised 11 February 2015
vanillic acids, being the salt the most significant factor after 24 h (short storage time), then Accepted 3 June 2015
replaced by p-coumaric acid. p-Coumaric acid also played a significant role on the acidifying Published online 14 July 2015
ability, expressed as decrease of pH of the medium: microbial metabolism, in fact, appeared as
completely inhibited at 0.2% of p-coumaric acid.

Introduction In vegetable systems, biophenols play an important role, as

defence mechanism against pathogens and insects attacks (Bitonti
Table olives are the most popular fermented vegetable in Europe
et al., 2000); their polident functionality is due to the ‘‘aromatic
and Mediterranean area, with a worldwide production of
core’’, having a hydroxyl group. Oleuropein and oleisides,
approximately 2.5 millions of tons (International Olive Oil
instead, are esterified structures with terpene moieties (Uccella,
Council, 2013). They can be prepared by different methods
2001).
(Spanish and Greek types, Californian method) (Brenes & de
Simple biophenols can be divided in two groups: hydro-
Castro, 1998; Garrido-Ferandez et al., 1997), but lactic acid
xycinnamic (p-coumaric, caffeic, and ferulic acids) and hydro-
bacteria (LAB) are traditionally recognized as responsible of the
xybenzoic families (p-hydroxybenzoic, protocatechuic, gallic,
correct course of fermentation in green table olives (Perricone
vanillic, and syringic acids) but, despite the similarity between
et al., 2010). Although many variables can affect the growth of
the two classes, their effect on the growth and survival of lactic
LAB in brines (temperature, nutrients, salt concentration, and pH)
acid bacteria can be very different (Campos et al., 2003). Stead
(Bevilacqua et al.,, 2010a), the presence of anti-LAB compounds
(1993) reported that hydroxycinnamic acids inhibited the growth
have been recognized since 1960s (Fleming & Etchells, 1967;
of Lactobacillus brevis, Escherichia coli, Staphylococcus aureus,
Juven & Henis, 1970). For many years, oleuropein and its
Bacillus cereus, and several yeasts. Campos et al. (2003) reported
hydrolysis products (oleuropein aglycon, elenolic acid, and
that hydroxycinnamic acids were more inhibitory to the growth of
hydroxytyrosol) have been pointed out as responsible for the
Oenococcus oeni than hydroxybenzoic acids. On the contrary,
inhibition of LAB growth in olive brines (Federici & Bongi, 1983;
some acids, like ferulic, vanillic and caffeic acids, showed a
Fleming et al., 1973; Medina et al., 2009; Ruiz-Barba et al.,
beneficial effect on the growth of Lactobacillus hilgardii. Only
1993). Recently, Medina et al. (2009) found that the main
p-coumaric acid had a strong inhibitory effect on both bacteria.
antimicrobial compound of Manzanilla olives was the dialdehydic
Despite the importance of biophenols in table olives, to our
form of decarboxymethyl elenolic acid either free (EDA) or
knowledge, no data are available in the literature regarding the
linked to hydroxytyrosol (HyEDA). Apart from oleuropein and
influence of phenolic compounds on growth and metabolism of
fatty acids (free or linked to glycerol), olive oil and table olives
lactic acid bacteria with this kind of fermented food; in addition,
are characterized by non-nutrient minor ingredients, such as
this topic was never approached using the statistical Design of
phenolic compounds, also labelled biophenols (Uccella, 2001).
Experiments (DoE) which is an efficient procedure able to
combine two or more variables and point out the correlation
Correspondence: Antonio Bevilacqua, Department of the Science of
amongst them.
Agriculture, Food and Environment (SAFE), University of Foggia, Via As stated by Romano et al. (2004), ‘‘A variety of real-world
Napoli 25, 71122, Foggia, Italy. Tel: +39 0881 589232. E-mail: problems calls for a multi-response approach in robust design
antonio.bevilacqua@unifg.it because industrial applications routinely present more than one
 DOI: 10.3109/09637486.2015.1064866 Growth and acidification of Lactobacillus plantarum 521

quality characteristic to be targeted’’. This problem could find
one possible solution into the use of the Response Surface
Methodology (RSM), i.e. a collection of mathematical and
statistical techniques useful for developing, improving, and
optimizing process and for searching optimum conditions of
factors for desirable responses, and for evaluating the relative
significance of several affecting factors even in the presence of
complex interactions (Yin et al., 2010; Zhang & Mu, 2011).
The Box–Wilson Central Composite Design, usually called the
Central Composite Design, is one of the most powerful and
efficient experimental design among other response surface
designs (central composite, Doehlert matrix, and three-level full
factorial designs), because its ability to estimate the parameters of
the quadratic model, building of sequential designs, detection of
lack of fit of the model and process optimization (Bevilacqua
et al., 2010b).
Therefore, the main aim of this research was the evaluation of
the effects of two phenols (p-coumaric and vanillic acids) on the
growth/survival of a cocktail of lactobacilli strains isolated from
table olives using of methodology of DoE; these compounds were
combined with salt and glucose, as they are, respectively, the
Downloaded by [Antonio Bevilacqua] at 10:22 09 August 2015
incubated at 37  C for 48–72 h under anaerobic conditions); the
analyses were performed twice over two different samples.
For each time of analysis, data were modeled as increase of
cell count referred to ‘‘time zero’’ (beginning of the experiment-
just after inoculation). Statistical differences were revealed
through one-way analysis of variance (ANOVA) and Tukey’s
test (p50.05).
pH
pH was evaluated through a pH-meter Crison (Crison
Instruments, Barcelona, Spain); for each time of analysis two
different samples were analyzed.
Data were modeled through a modified Gompertz equation
(Zwietering et al., 1990), cast in the negative form as follows:
pH ¼ pH0  DpH  expfexp½ðdmax  e=AÞ  ð  tÞ þ 1g
where pH0 is the initial pH; DpH, the maximal decrease of pH;
dmax, the maximal rate of acidification (DpH/h) over time; a, the
time (h) before the beginning of acidification and t the time
(h, independent variable).

main compound in brine and a coadjutant of fermentation of

table olives. Second-level model
The increase of cell number after 24, 48, and 168 h, as well as the
Materials and methods extent of acidification over time (DpH) were used as input values
Strains and cocktail preparation for a black-box model analysis; the statistical analysis was
performed through the option DOE of the software Statistica for
A number of three strains of Lactobacillus plantarum, labeled as Windows (Statsoft, Tulsa, OK).
c9, c10, and c15 and isolated from Italian table olives Bella di The polynomial equation was in the following form:
Cerignola (Bevilacqua et al., 2010a), were used as test organisms.
The strains were stored at 20  C in MRS broth (Oxoid, Milan, X
n n X
X n X
n
y ¼ 0 þ j  Xj þ jk  Xj  Xk þ jj  Xj2
Italy) added with 33% of sterile glycerol (J.T. Baker, Milan, Italy) j¼1 j¼1 k¼jþ1 j¼1
and grown before each assay in MRS broth incubated at 37  C Pn
for 24 h. where: j¼1 j  Xj is the individual effect of each factor
Then, each strain was centrifuged at 3000 rpm for 10 min; the (independent
Pn Pn variable; salt, glucose, and phenolic compounds);
supernatant was discarded and the pellet suspended in sterile j¼1 k¼jþ1 jk  Xj  Xk indicates the interactions among the

distilled water; each suspension was diluted in saline solution
(0.9% NaCl) in order to attain a cell concentration of 5 log cfu/ml
and the microbial cocktail was prepared by mixing 10 ml of cell Table 1. Combinations of the design.
suspension of each strain.
p-Coumaric Vanillic
Glucose (%) NaCl (%) acids (%) acids (%)
Medium
1 1.0 1.5 0.1 0.1
The assays were performed in the following medium: yeast extract 2 1.0 1.5 0.1 0.3
(Oxoid), 2 g per liter of tap water. The pH of the medium was 3 1.0 1.5 0.3 0.1
adjusted to 5.3 through HCl 0.1 N. 4 1.0 1.5 0.3 0.3
5 1.0 4.5 0.1 0.1
6 1.0 4.5 0.1 0.3
Design 7 1.0 4.5 0.3 0.1
8 1.0 4.5 0.3 0.3
The medium was added with different amounts of NaCl, glucose, 9 3.0 1.5 0.1 0.1
p-coumaric acid (trans-p-hydroxycinnamic acid; C9H8O3) 10 3.0 1.5 0.1 0.3
(Sigma-Aldrich, Milan, Italy) and vanillic acid (4-hydroxy-3- 11 3.0 1.5 0.3 0.1
12 3.0 1.5 0.3 0.3
metoxybenzoic acid; C8H8O4) (Sigma-Aldrich, Milan, Italy), 13 3.0 4.5 0.1 0.1
following a four variables – Central Composite Design. Table 1 14 3.0 4.5 0.1 0.3
shows the 27 combinations of the design. 15 3.0 4.5 0.3 0.1
Then, the different combinations of the design were inoculated 16 2.0 4.5 0.3 0.3
with the microbial cocktail, in order to attain a cell concentration 17 2.0 3.0 0.2 0.0
of lactobacilli of ca. 3 log cfu/ml and stored at 37  C for 7 d; pH 18 2.0 3.0 0.2 0.4
19 2.0 3.0 0.0 0.2
and cell concentration of LAB were evaluated periodically (just
20 2.0 3.0 0.4 0.2
after inoculation and after 24, 48, 72, and 168 h). For each 21 2.0 0.0 0.2 0.2
combination, 10 different samples were prepared. 22 2.0 6.0 0.2 0.2
23 0.0 3.0 0.2 0.2
24 4.0 3.0 0.2 0.2
Cell number 25 2.0 3.0 0.2 0.2
26 2.0 3.0 0.2 0.2
Samples were serially diluted in saline solution and cell number
27 2.0 3.0 0.2 0.2
was evaluated through the pour plate method (MRS agar,
 522 B. Speranza et al.
Pn
variables; the term j¼1 jj  Xj2 takes into account a possible
non-linear/quadratic effect of some factors; y is the dependent
variable (acidification or increase of cell count over time).
The significance of the polynomial equation was evaluated
through the adjusted regression coefficient, as well as with the
Fisher test and standard error of the model. In addition, the
statistical weight of each term was pointed out through the Pareto
chart of the standardized effects, reporting as bars the standar-
dized effects associated with each individual, quadratic and
interactive factors of the equations. These standardized effects
were evaluated as the ratio of the mathematical coefficient of each
term of the equation versus the respective standard error.
Finally, the quantitative effect of the variables of CCD on the
decrease of pH and cell counts was studied through the surface
response plots.
Results
Cell count
The trend of Lb. plantarum within the running time relied on the
factors of CCD (NaCl, glucose, p-coumaric, and vanillic acid);
Downloaded by [Antonio Bevilacqua] at 10:22 09 August 2015
Int J Food Sci Nutr, 2015; 66(5): 520–525
the increase of cell count was related to NaCl, glucose, and
phenols through the following polynomial equation:
DC ðlog cfu=mlÞ ¼ 2:69  0:75  glucose  1:52  NaCl
 0:53  NaCl2  1:88  p-coumaric acid
 1:38  vanillic acid  0:62  NaCl
 p-coumaric acid
being 0.949 the adjusted regression coefficient. Thus, the increase
of cell count was affected negatively by all the factors of CCD;
moreover the interaction [NaCl]*[p-coumaric acid] influenced the
process in a significant way, too. The standardized effects of these
factors are reported in Table 2: the most significant term was
p-coumaric acid, followed by NaCl, vanillic acid, glucose and
finally by the interaction [NaCl]*[p-coumaric acid].
A better description of the effects of NaCl, glucose and
phenols could be achieved through the surface response plots; the
graphs show the effects of two factors of the design when the third
one was set to the coded level ‘‘0’’ (Figure 2A–C).
Concerning the interaction [NaCl]*[glucose (Figure 2A), the

despite the significant differences amongst the different runs,
there were some common details related to LAB evolution.
Figure 1 shows cell counts of Lb. plantarum in some selected
combinations of the design; namely, some sampling points could
be related to the growth/death kinetic of a bacterial population in
a batch system.
After 24 h, LAB population was at the end of the exponential
phase (like in the combinations 19 and 23) or at its beginning
(runs 20 and 22), while after 48 h cell counts attained the
maximum level (stationary phase). As expected, death occurred
for prolonged storage time, thus a decrease of cell count was
found after 96 h (start of the death phase). At the end of the
running time (168 h) in some combinations (e.g. in the samples
20 and 22), Lb. plantarum was at the undetectable level, whereas
in other runs, a variable number of survivors was recovered
(late death phase).
This trend, with different cell levels, was found for all the
combinations of the design, thus three time intervals were chosen
as representative of LAB trend: 24 h, (early or late exponential
phase), 48 h (stationary phase), and 168 h (late death phase).
The increase of cell counts at these intervals was used as an
input value for a DoE analysis to pinpoint the significant effects of
the factors of CCD and build a polynomial equation. After 24 h,
model predicted the maximum increase of cell count after 24 h
(ca. 3.5 log cfu/ml) when neither NaCl nor glucose was added to
the medium; otherwise, growth did not occur (no increase or
negative increase) at the highest concentrations of these two
compounds. It is important to underline that this prediction was
performed by setting phenols at 0.2%, thus the extent of cell
increase was not the maximum one, but the maximum achievable
with phenols at the coded level ‘‘0’’.
A similar effect was found for phenols (Figure 2B), i.e. the
increase of population was the highest one (ca. 6 log cfu/ml) with
the lowest concentrations of p-coumaric and vanillic acids added
(0.0%). Finally, Figure 2(C) shows the effects of the interaction
[NaCl]*[p-coumaric acid]; both NaCl and p-coumaric acid alone
played a slight effect on the increase of cell count, while the
combination showed an additive/synergistic effect and Lb.
plantarum decreased by 3 log cfu/ml.
Table 2 shows also the significance of the factors of CCD in
the stationary (48 h) and in the late death phase (168 h) and
highlights that p-coumaric acid was the only significant term
within the storage.
Table 2. Effects of salt, glucose, vanillic, and p-coumaric acids on the
increase of cell count after 24 h, 48 h, and 168 h.

24 h 48 h 168 h
Linear terms
-Coumaric acid 14.03 5.73 5.94
NaCl 11.31 – –
Vanillic acid 10.25 – –
Glucose 5.61 – –
Interactive terms
NaCl  -coumaric acid 3.77 – –
-Coumaric acid  vanillic acid – – –
Glucose  -coumaric acid – – –
NaCl  vanillic acid – – –
Glucose  NaCl – – –
Glucose  vanillic acid – – –
Quadratic terms
-Coumaric acid2 – – 3.00
NaCl2 3.75 – –
Vanillic acid2 – – –
Glucose2 – – –
R2ad * 0.949 0.798 0.820
MSE** 0.109 1.271 1.094

Figure 1. Evolution of lactic acid bacteria (LAB) count in some selected *R2ad , adjusted regression coefficient.
combinations of the design. Bars represent standard deviation. **MSE, mean squared error.
 DOI: 10.3109/09637486.2015.1064866 Growth and acidification of Lactobacillus plantarum 523
Table 3. Gompertz parameters ( ± standard error) for the runs where
acidification occurred.

DpH* dmax a R2
Run 1 1.81 ± 0.02f** 0.18 ± 0.03c 25.87 ± 0.72a 0.996
Run 2 1.32 ± 0.01c,d 0.07 ± 0.01a 35.11 ± 1.43b 0.998
Run 5 1.41 ± 0.01e 0.07 ± 0.01a 34.95 ± 1.30b 0.998
Run 6 1.11 ± 0.01a 0.06 ± 0.01a 52.90 ± 2.06c 0.998
Run 9 1.80 ± 0.01f 0.12 ± 0.01b 28.18 ± 0.34a,b 0.998
Run 10 1.39 ± 0.02d,e 0.06 ± 0.00a 34.58 ± 1.25b 0.998
Run 13 1.27 ± 0.01b,c 0.04 ± 0.00a 36.67 ± 0.53b 0.998
Run 14 1.22 ± 0.02b 0.05 ± 0.01a 52.29 ± 2.32c 0.998
Run 19 1.80 ± 0.04f 0.11 ± 0.02b 24.19 ± 1.14a,b 0.996

*DpH, maximal decrease of pH; dmax, maximal rate of acidification

(DpH/h) over time; a, time (h) before the beginning of acidification; R2,
regression coefficient.
**For each parameter, letters indicate significant differences (one-way
ANOVA and Tukey’s test, p50.05); the alphabetical order of the letters
follows the increase of each parameter

of Gompertz equation produced some mathematical parameters,

Downloaded by [Antonio Bevilacqua] at 10:22 09 August 2015

related to the three steps of the acidification kinetics, i.e. a (the

time before the beginning of the acidification), dmax (rate of pH
decrease), and DpH (maximal decrease of pH attained in the tail
phase). These parameters were submitted to one-way analysis of
variance, to point out the combinations of the design where
Lb. plantarum performed the best acidification (the highest DpH
and dmax and the lowest a parameters): the minimal decrease of
pH and the lowest value of the acidification rate, as well as the
highest value of the parameter a (respectively, 1.11, 0.06 DpH/h
and 52.90 h) were recovered in the combination 6 (glucose, 1%;
NaCl, 4.5%; p-coumaric acid, 0.1%; vanillic acid, 0.3%). The
highest value of the parameter a, combined with the lowest
acidification rate, was found also in the run 14 (glucose, 3%;
NaCl, 4.5%; p-coumaric acid, 0.1%; vanillic acid, 0.3%). Using
DpH values as input data for a black-box model, Pareto chart
highlights that acidification was affected only by p-coumaric acid
(Figure 3); 3-D plots revealed the existence of a break-point at ca.
0.2% of p-coumaric acid, able to completely inhibit LAB
metabolism (Figure 4).

Figure 2. Three-dimensional plot for the interactions glucose/NaCl
(A); p-coumaric acid/vanillic acid (B); NaCl/p-coumaric acid (C) on
the increase of cell count after 24 h.
Acidification
The primary tool of LAB in olive brine is acidification; the effects
of glucose, NaCl, and phenols also were studied on the kinetics of
acidification.
Among the 27 combinations of the design, we found a
significant decrease of pH only in nine combinations out of 27, as
reported in Table 3. Following the approach proposed elsewhere
(Bevilacqua et al., 2008a), data fitting through a modified version
Discussion
Evolution of olive microbiota throughout the fermentation relies
on some intrinsic and environmental variables, like the concen-
tration of NaCl in brines, storage temperature, olive variety,
addition of starter cultures, and/or fermentation coadjutants, like
glucose (Garrido-Fernandèz et al., 1997; Nychas et al., 2002;
Perricone et al., 2010). The role of NaCl on LAB is well known
and documented (Bevilacqua et al., 2010a); moreover, in the past,
it was found that the addition of glucose in brine could increase
the performance of a starter culture (Perricone et al., 2010).
In addition, some minor phenolic components could play a
significant role on growth/survival of Lb. plantarum, as reported
elsewhere (Bevilacqua et al., 2006).
Thus, the combined effects of these variables were investigated
through the theory of DoE; a Central Composite Design (CCD)
was chosen as a convenient approach, as it is a robust design,
useful to describe the effect of the different factors and for
predictive purposes (Box et al., 2005).
A CCD is useful to build a second-order model, e.g. an
equation showing the effects of some variables or factors of the
design (independent variables) on some physiological and math-
ematical parameters related to microbial growth and/or death
survival (death or growth rate, lag phase); in the present paper, a
different kind of approach was proposed, as cell counts at selected
 524 B. Speranza et al.
Figure 3. Pareto chart of standardized effects
of salt, glucose, vanillic, and p-coumaric acid
on the extent of acidification (DpH).
Downloaded by [Antonio Bevilacqua] at 10:22 09 August 2015
Figure 4. Three-dimensional plot for the interaction NaCl/p-coumaric
acid on the extent of acidification (DpH).
time intervals were used as dependent variables. The result is the
building of static models, offering a picture of lactic microflora at
selected time intervals and useful to describe, if any, a different
weight of the factors of CCD throughout the time.
After 24 h (exponential growth phase), the increase of cell
count was affected significantly by all the factors of CCD, being
p-coumaric acid the most significant variable. The results for
phenolic components and NaCl were in line with those reported
by other authors; in particular, the bioactivity of p-coumaric acid,
higher than vanillic acid, confirmed the results of Campos et al.
(2003): hydroxycinnamic acids, in fact, behaves in a more
hydrophobic way than hydroxybenzoic compounds. This property
would make easier their entrance throughout membrane, thus in
the cell they could dissociate and cause enzyme inactivation and
Int J Food Sci Nutr, 2015; 66(5): 520–525
metabolic exhaustion (Campos et al., 2003; McDonnel &
Russell, 1999; McManus et al., 1985).
A result quite different from the expected one was found for
the glucose: in the past, the effect of glucose was studied on the
production of biomass from a strain of Lb. plantarum isolated
from table olives (Bevilacqua et al., 2008a), finding that biomass
increased by increasing the amount of glucose up to a critical
break-point (20 g/l), after which a further increase exerted a
negative effect on lactobacilli. In the present paper, the effect of
glucose was negative; however, there are no details in the
literature useful to find a possible reason for this behavior. In
addition, this effect was found only for the cocktail, but not when
the strains were used alone (data not published) or when the
assays were performed in a complex medium (Bevilacqua et al.,
2008b).
DoE approach underlined that both in the stationary and death
phases, the significance of factors changed and p-coumaric acid
was the only factor playing a significant role for the entire running
time. These results were in line with those found for other
microorganisms (Enterobacter cloacae, yeasts) in brines and
showing a shift or a change of the effects of the different variables
within the time, being the most important event the decrease of
the weight of salt (Bevilacqua et al., 2013).
The strong effect of p-coumaric acid on cell counts for the
entire running time could explain its role in the extent of
acidification expressed by lactobacilli, as we can suppose that the
increase in cell count and primary metabolism are strictly related.
Conclusions
The results of this paper showed that the growth of a cocktail of
lactobacilli, isolated from table olives, could be affected in a
strong way by some variables, like the concentrations of salt,
p-coumaric and vanillic acid into the medium. Thanks to the
approach used, it was possible to pinpoint that the statistical
weight of these variables could change within the time: salt, in
fact, exerted a negative effect only after 24 h; the same effect was
recovered also for vanillic acid. On the contrary, the significance
of p-coumaric acid was clearly recovered for the entire running
time and its strong effect was evidenced also for the acidifying
 DOI: 10.3109/09637486.2015.1064866
ability: the microbial metabolism, in fact, was completely
inhibited at 0.2%.
Declaration of interest
This paper was supported by the Italian Ministry of Education, University
and Research through the Grant PROINNOBIT (Sviluppo di prodotti
alimentari innovativi mediante soluzioni biotecnologiche, impiantistiche
e tecnologiche – PON02_00186_3417037 – Development of innovative
food products through biotechnological, plant design and technological
solutions) – PON Ricerca e Competitività 2007–2013 – Programma
Cofinanziato dal Fondo Europeo di Sviluppo Regionale (FESR).
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