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Production and characterization of emulsion


filled gels based on inulin and extra virgin olive
oil
Article in Food Hydrocolloids November 2014
Impact Factor: 4.09 DOI: 10.1016/j.foodhyd.2014.10.027

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Food Hydrocolloids 45 (2015) 30e40

Contents lists available at ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Production and characterization of emulsion lled gels based on inulin


and extra virgin olive oil
Vito M. Paradiso*, Mariagrazia Giarnetti, Carmine Summo, Antonella Pasqualone,
Fabio Minervini, Francesco Caponio
Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Via Amendola 165/A, 70126 Bari, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 9 April 2014
Accepted 28 October 2014
Available online 11 November 2014

Emulsion lled gels (EFG) can help food producers to reduce fat content in foods. The present study
evaluated the physical, chemical, sensorial and microbiological properties of emulsion lled gels based
on inulin, a gel-forming prebiotic carbohydrate, and extra virgin olive oil (EVOO), well-known for its high
nutritional value and phenolic antioxidant content. EFG based on inulin and EVOO were produced by
means of both mechanical shearing and ultrasound homogenization. Three different ingredient ratios
lead to high, medium and low oil content EFG (H, M, L respectively). H EFG had also lower inulin/water
ratio. The resulting gels could be considered as a healthy alternative to spreads, rich in ber, unsaturated
fatty acids and phenolic antioxidants. Compared to mechanical shearing, ultrasound homogenization
gave more consistent EFG, characterized by lower lightness respect to mechanical processing. Lower
coarseness and fusion-like behavior and higher greasiness, perceived by panelists, conrmed the
structural and textural differences conferred by ultrasound. Higher inulin content and inulin/water ratios
determined consistency increase. The EFG with the best sensory prole (melting, less coarse texture,
higher consistency, greasy mouthfeel) was submitted to consumer test and liked by over 70% consumers
(n 80). The volatile pattern was characterized by compounds found in fresh oils, mainly 2-hexenal.
Oxidized triacylglycerols showed a slight increase in the EFG oil fraction respect to the fresh oil,
particularly when using ultrasound homogenization. The residual phenolic compounds were in the range
50e76%, with losses minimized in mechanically sheared EFG. Ultrasound improved the microbiological
stability of EFG.
2014 Elsevier Ltd. All rights reserved.

Chemical compounds studied in this article:


Inulin (PubChem CID: 24763)
(E)-2-hexenal (PubChem CID: 5281168)
Keywords:
Emulsion lled gel
Inulin
Extra virgin olive oil
Ultrasound

1. Introduction
Reducing the intake of fats, especially saturated ones, is a
mandatory target for consumers of developed countries
(Utzschneider et al., 2013). Recent recommendations from food
safety authorities invite to minimize the assumption of saturated
fats (European Food Safety Authority, 2011). This reects in a
challenging task for food producers: reducing the fat content of
foods, with special attention to saturated ones, without giving up
the fat-related desirable textural properties of food (Abhyankar,
Mulvihill, & Auty, 2014). The combined use of gelling agents and

* Corresponding author. University of Bari Aldo Moro, Department of Soil, Plant


and Food Sciences, Food Science and Technology Unit, Via Amendola 165/A, I-70126
Bari, Italy. Tel.: 39 (0)80 5442272.
E-mail address: vito.paradiso@uniba.it (V.M. Paradiso).
http://dx.doi.org/10.1016/j.foodhyd.2014.10.027
0268-005X/ 2014 Elsevier Ltd. All rights reserved.

oils, in order to obtain emulsion lled gels (EFG) is considered by


both researchers and food producers as an effective approach to
reach this aim. Both proteins (e.g. soybean or whey protein isolate,
gelatin) and polysaccharides (e.g. alginate, agar, k-carrageenan)
have been used as gelling agents in studies regarding EFG. Mainly
structural, rheological and sensorial properties have been studied
as a function of several variables (oil volume fraction, gelling agent
type and concentration, droplet size, homogeneity of fat distribution) (Chojnicka, Sala, de Kruif, & van de Velde, 2009; Kim,
Renkema, & van Vliet, 2001; Mosca, Rocha, Sala, van de Velde, &
Stieger, 2012; Sala, de Wijk, van de Velde, & van Aken, 2008; Sala,
van Vliet, Cohen Stuart, van de Velde, & van Aken, 2009).
Inulin is recognized as food ingredient in most countries, and
often labeled as dietary ber (Franck, 2002). A wide literature
proves its properties as prebiotic and functional ingredient (Kolida,
Tuohy, & Gibson, 2007; Roberfroid, 1999). In fact, due to its
chemical structure, inulin is neither hydrolyzed nor adsorbed in the

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

small intestine but is extensively fermented by the colon bacteria


(Bot, Erle, Vreeker, & Agterof, 2004). Recently Adebola, Corcoran,
and Morgan (2013) found an anticytotoxic and antigenotoxic effect of inulin beyond its prebiotic role. Inulin gels are formed by a
network of small crystallites of about 100 nm diameter that
aggregate to form larger clusters of 1e5 mm, which trap a large
amount of water in the network. The properties of this network
resemble that of a network of fat crystals in oil. Because of this
similarity, inulin has been identied as an interesting ingredient for
structuring in low- or zero-fat food products (Barclay, GinicMarkovic, Cooper, & Petrovsky, 2010; Bot et al., 2004). Recently,
inulin has begun to be considered as gelling agent in EFG. Glibowski
(2010), considered the behavior of a model EFG obtained homogenizing an inulin solution with hot canola oil, in presence of an
emulsier. Rheometry, hardness, spreadability and structure (by
scanning electron microscopy) were the parameters measured. An
EFG having similar composition was successively submitted to
storage at 8  C in order to evaluate its microbiological and rheological stability (Glibowski, Kordowska-Wiater, & Glibowska, 2011).
Mantzouridou and coworkers evaluated an inulin-based dressing
emulsion as a carrier for probiotics (Mantzouridou, Spanou, &
Kiosseoglou, 2012). Alimi et al., instead, used inulin together with
modied starch in order to obtain a low-fat mayonnaise, that was
characterized for microstructure and viscoelastic properties (Alimi,
Mizani, Naderi, & Shokoohi, 2013). No other studies seem to be
available about the physicechemical properties of inulin-based
EFG.
Moreover, scarce attention has been paid until now to the
quality of the oil fraction of these products. Still, potential health
benets could be achieved also by means of the correct choice of
the oil to be emulsied, with regard to both the fatty acid composition and minor compounds (e.g. antioxidants, phytosterols) content. To this regard, extra virgin olive oil presents a well-known
potential (Aparicio & Harwood, 2013). EFG based on inulin and
extra virgin olive oil could constitute potential healthy alternatives
to commercial spreadable fatty products. According to European
regulations, products containing more than 3 g or 6 g/100 g of ber
can be labeled as source of bre and high bre (Ofcial Journal
of the European Communities, 2006).
The present study was aimed to the production of EFG based on
inulin and extra virgin olive oil, by means of both mechanical
shearing and ultrasound homogenization, and their textural,
chemical and microbiological characterization.
2. Materials and methods
2.1. Materials
Inulin (Orafti HPX, Beneo-Orafti SA, Oreye e Belgium), with
degree of polymerization 5 accounting for not less than 99.5% of
total, and soy lecithin were kindly supplied by Eigenmann & Veronelli SpA (MilaneItaly). Extra virgin olive oil was purchased from a
local retailer. All reagents were of analytical grade, from Sigma
Aldrich (Steinheim, Germany). Nile Red was supplied by SigmaeAldrich Chemicals, MO, USA.
2.2. EFG production
Three formulations of EFG were selected after preliminary trials
based on the evaluation of consistency and visual homogeneity
(data not shown). The ingredients ratios in weight (extra virgin
olive oil:inulin:water) were the following: 38:19:43 in high oil
content EFG (H); 27:27:46 in medium oil content EFG (M); 21:29:50
in low oil content EFG (L). Lecithin, added as emulsier, accounted

31

for 2% on the whole in all of three EFG. The inulin/water (i/w) ratio
for the three formulations was 0.44, 0.59 and 0.58 respectively.
The EFG were prepared in 200 g batches using three different
homogenization technologies:
- Me: mechanical homogenization with Ultraturrax T25 (IKA,
Staufen, Germany) for 10 min at 24,000 rpm (nal temperature
of the emulsion 30  C);
- US: ultrasonic homogenization with Sonopuls HD 3200 (Bandelin Electronic, Berlin, Germany) with tapered tip KE 76 (6 mm
diameter) for 10 min for M and L formulation and 5 min for H
formulation (nal temperature of the emulsion 55  C);
- CUS: cold (ice bath) ultrasonic homogenization with the same
apparatus and times of US (nal temperature of the emulsion
45  C).
The duration of homogenization corresponded to the time
necessary to obtain a homogenous system. No pre-hydration was
carried out. After production, the EFG were cooled down at ambient
temperature and then kept at 5  C.
As a whole, nine EFG typologies were produced and compared,
as a combination of formulation and homogenization system: LMe, M-Me, H-Me, L-US, M-US, H-US, L-CUS, M-CUS, H-CUS.
A portion of the formulations was used for sensory, color, volatile compounds and texture analyses; the remaining part was
placed at 18  C for about 4 h to facilitate water crystallization and
subsequently lyophilized prior to extract the oil fraction.
Six more spreads were produced, according to a hexagon design,
by varying of 4% the proportions of inulin, olive oil and water
respect to the H formulation and using ultrasound (US) homogenization, to be submitted to texture analysis, in order to better
assess the relations existing between formulation and textural
properties.
2.3. Fluorescence microscopy
Fluorescence microscopy was adopted to obtain information
about the morphology of the emulsion formed during homogenization of EFG. To label the oil phase in the EFG, a solution of the fat
specic dye Nile Red (0.15%, w/w, in 1,2-propanediol) was added to
the extra virgin olive oil at a level of 20 mL/ml. The labeled oil was
homogenized with inulin, water and lecithin to produce EFG. The
microscopy observations were carried out using a uorescent microscope (DMLS, Leica) with an excitation lter of 450e490 nm and
a barrier lter of 515 nm. Images were acquired at 40
magnication.
2.4. Texture analysis
The texture analysis of the EFG obtained was performed by back
extrusion with a 3340 Series Single Column Systems (Instron,
Milan, Italy). The test consisted in the penetration of a piston of
40 mm diameter into a sampling tube of 45 mm diameter containing a sample layer of 30 mm of the sample. The penetration
occurred for a stroke of 25 mm at a speed of 0.7 mm/s. The area
under the load/time curve was calculated and converted in N$mm
lez-Martnez et al.,
as a measure of the sample consistency (Gonza
2002).
The analysis was carried out on two samples obtained in independent trials for all of fteen spread formulations.
2.5. Color analysis
Color measurement was carried out by a Minolta Chromameter
2 reectance colorimeter (Minolta, Tokyo, Japan) equipped with the

32

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

measurement head CR 300 and CIE Standard Illuminant D65. The


results were expressed as L* (lightness), a* (redness) and b* (yellowness). Two samples obtained in independent trials were
analyzed. The mean of three measurements was considered for
each sample.
2.6. Oil extraction
The extraction on lyophilized samples (50 g) was conducted
using 200 mL of hexane added in two aliquots of 100 mL; after each
addition, a magnetic stirring was performed for 30 min. Subsequently, the mixture was centrifuged for 20 min (10,000 rpm) at
10  C. The hexane solution was ltered on anhydrous sodium sulfate and evaporated under reduced pressure at 40  C.
The extraction and subsequent analyses of lipid degradation
were carried out on two samples obtained in independent trials.
Each analysis was carried out in triplicate.
2.7. Lipid degradation
The chemical analyses were carried out on both the starting
extra virgin olive oil and the oils extracted from the EFG. The determinations of the free fatty acids (FFA), peroxide value (PV) and
spectrophotometric constants were carried out according to the
Ofcial Journal of the European Communities (1991).
Polar compounds (PC) were separated by silica gel column
chromatography according to the AOAC method no. 982.27 (2003).
After being recovered in THF, they were analyzed by means of high
performance size-exclusion chromatography (HPSEC) using THF as
eluant at a ow rate of 1 mL/min. The HPSEC system consisted of a
series 200 pump (PerkineElmer, Norwalk, CT, USA) with Rheodyne
injector, a 50 mL loop, a PL-gel guard column (PerkineElmer, Beaconseld, UK), 5 cm length  7.5 mm i.d., and a series of two PL-gel
columns (PerkineElmer, Beaconseld, UK) 30 cm length  7.5 mm
i.d. each. The columns were packed with highly cross-linked styrene-divinylbenzene copolymer with particle of 5 mm and a pore
diameter of 500 . The refractive index detector was a series 200A
(PerkineElmer, Norwalk, CT, USA). Peak identication and quantication were carried out as described elsewhere (Gomes &
Caponio, 1999).
2.8. Total phenolic compounds (TPC) content
TPC were recovered from the EFG by liquid extraction and
determined according to Gambacorta et al. (2010) with some
modications. In particular, 5 mL of methanol/water (70:30, v/v)
and 1 mL of hexane were added to 1 g of EFG and mixed with vortex
for 10 min. The hydroalcoholic phase containing phenolic compounds was separated from the oily phase by centrifugation
(6000 rpm, 4  C, 10 min). The hydroalcoholic phase was submitted
to another centrifugation (9000 rpm at room temperature, 5 min).
Finally, hydroalcoholic extracts were recovered with a syringe and
then ltered through a 0.45 mm nylon lter before analysis.
The determination of the TPC included the use of the FolinCiocalteu reagent. In a test tube, 100 mL of phenolic extract were
mixed with the Folin-Ciocalteu reagent (100 mL, 2 M) and, after
4 min, with an aqueous solution of Na2CO3 (800 mL, 5%). The
mixture was heated in a water bath at 40  C for 20 min and the total
phenol content was determined colorimetrically at 750 nm. The
standard curve was prepared using diluted solutions of gallic acid in
a methanol:water (70:30, v/v). The TPC content was expressed as
mg of gallic acid equivalents per kg of oil.
The analysis was carried out on two samples obtained in independent trials and the mean value of three analysis replicates was
considered.

2.9. Volatile compounds analysis


Samples of EFG (0.5 0.005 g) were weighed in 20-mL vials.
Next, the vials were sealed with a screw top aluminum cap and
pierceable butyl rubber septa. Volatiles were extracted by solidphase microextraction (SPME). The extraction was performed by
exposing a 75 mm Carboxen/polydimethylsiloxane (CAR/PDMS) ber (Supelco, Bellefonte, PA, USA) in the headspace of the sample at
40  C for 50 min. When the extraction process was completed, the
ber was inserted into the injector port of the gas chromatograph
for thermal desorption of volatiles in splitless mode. The GC/MS
instrumentation included an Agilent model 6850 gas chromatograph coupled to a mass spectrometer Agilent 5975. The volatile
compounds were separated on an HP-Innowax (60 m  0.25 mm,
0.25 mm lm thickness) polar capillary column under the following
conditions: ow 1.5 mL/min; injector temperature, 250  C; pressure of the carrier (helium), 30 kPa. The oven temperature was held
for 5 min at 35  C, then increased by 5  C/min to 50  C and held
constant for 5 min, then raised to 230  C at 5.5  C/min and nally
held at 230  C for 5 min.
The mass spectrometer was operated in the electron impact
mode (electron energy 70 eV), and the ion source temperature
was 250  C. A continuous scan mode was employed with a scan
time of 7.7 scans/s over a mass range of 33e200 amu. The Linear
Retention Index (LRI) of each volatile compound was calculated by
comparing the retention times with those of a series of n-alkanes
(Alkane standard solution C8eC20, Sigma Aldrich, Milan, Italy). The
volatile compounds were identied by their LRI and by comparison
with the mass spectra present in the NIST and Wiley libraries.
Semiquantitative data were obtained, expressed as peak area
counts.
The analysis was carried out on two samples obtained in independent trials and the mean value of three analysis replicates was
considered.
2.10. Sensory analysis
2.10.1. Sensory proling
A panel of 8 assessors evaluated the sensory characteristics and
structural properties of gels.
The training of the panelists was carried out by several sessions
including the following steps: familiarization with the product;
identication of the sensory descriptors qualifying the EFG; standardization of the ranges and of the references for the descriptors.
These tasks were addressed by means of: consensus discussions, in
which the panel evaluated reference samples and tested samples,
developing terminology and scores; individual sessions followed by
statistical evaluation of the performances of the panel and of each
panelist, and subsequent discussion. The descriptors adopted were:
Gloss (glossy appearance of the surface of the sample); Homogeneity
(uniform appearance of the sample); Oil odor (orthonasal perception of a fresh olive oil odor); Oil avor (retronasal perception of a
fresh olive oil avor); Lecithin odor (orthonasal perception of lecithin odor); Lecithin avor (retronasal perception of lecithin avor);
Rancid odor (orthonasal perception of an oxidized oil odor); Rancid
avor (retronasal perception of an oxidized oil avor); Greasiness
(greasy sensation in mouth after tasting the sample); Consistency
(degree in which a teaspoonful of sample keeps its shape when
manipulating in mouth to swallow); Coarseness (perception of a
coarse/grainy/sandy texture detected by moving the tongue parallel to palate); Melting (ease of disrupting in mouth the product
structure); Spreadability (ease with which the sample can be spread
on a slice of bread). The panel group was selected among researchers and technicians of the research laboratory and Ph.D
students. Besides the EFG, the olive oil and the soy lecithin were

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

used as odor and avor references, as well as commercial soft foods


(mayonnaise, spreadable cheeses, butter, margarine, fermented
milks) as references for the textural descriptors consistency, melting,
greasiness, spreadability, in order to establish consensus on the
descriptors and their ranges.
Panelists received a tray containing samples, some apple slices,
some bread slices and an evaluation form with a pencil. Samples
(10 g of gel) were served in containers coded with three digit
random numbers. Each descriptor was assessed using an unstructured linear scale of 10 cm with the ends of the scales corresponding to the intensity of the attribute: minimum value of
perception (lower limit: 0) and maximum value (upper limit: 10).
Attributes were evaluated according to the following protocol:
- Visual evaluation of the appearance (gloss, homogeneity);
- Snifng to evaluate odors;
- Introduction in mouth of the sample to evaluate textural attributes (consistency, coarseness, melting, greasiness) and avors;
- Evaluation of the spreadability of the sample on a bread slice;
The analysis was carried out on all of the nine EFG typologies.
Three samples obtained in independent trials were analyzed by
each panelist. The median value of the scores assigned by panelists
was assumed.
2.10.2. Consumer test
One EFG typology (H-US) was chosen to carry out the consumer
test, being the formulation with the best sensory prole (see Section 3.4 in Results and discussion). A total of 80 consumers (age
range 19e56 years, mean 24.5, 42 males and 38 females) were
recruited to participate in a consumer test. A four digit random
code was assigned to the emulsion lled gels tested that were
served at room temperature spread on a bread slice. Tapwater and
1 min break between samples were chosen as palate cleansers. For
each product, subjects were asked to indicate their level of liking
giving a score from 1 (dislike) to 5 (like very much).

33

2.12. Statistical analysis


Analysis of variance (either two-way ANOVA with Formulation
and Homogenization as independent variables or one-way ANOVA,
as stated in the text) and Fisher's LSD test were performed using
XLStat software (Addinsoft SARL, New York, NY, USA). Surface
response regression was carried out using Minitab 16 (Minitab Inc.,
State College, Pennsylvania).

3. Results and discussion


3.1. Fluorescence microscopy
Fig. 1 represents the emulsion morphology as detected by
means of the uorescence microscopy. Mechanically homogenized
systems presented clearly distinguishable droplets, larger than in
sonicated systems. Ultrasound homogenization gave distinguishable smaller droplets and a ne, cloudy dispersion, probably due to
the smallest droplets. This agrees with Beherend, Ax, & Schubert
(2000), who reported that, when applied in emulsication, ultrasound allowed to obtain smaller particle size than other mechanical techniques. The ne dispersion of smallest droplets was
more evident in low-oil US EFG and in CUS EFG. This could mean
that, in M-US and H-US, ultrasound, besides disrupting droplets,
induced also re-coalescence. In fact the nal emulsion droplet size
results as the equilibrium between droplet disruption and their
subsequent re-coalescence due to droplet collisions. This phenomenon is called over-processing and can appear when
increasing the energy input during emulsication (Jafari,
Assadpoor, He, & Bhandari, 2008). Such re-coalescence would be
enhanced by both temperature (in US EFG) and oil volume fraction
(in M and H EFG), and lead to a certain decrease of the amount of
the smallest droplets.

2.11. Microbiological stability


Ten grams of each gel were homogenized with 90 mL of sterile
saline (sodium chloride 0.9% w/v) for 3 min in a 400P Bag Mixer
(Interscience, St. Nom, France). After decimal dilution, homogenates were plated on Plate Count Agar (PCA), Wort Agar (WA), and
Violet Red Bile Glucose Agar (VRBGA), for enumeration of total
mesophilic aerobic microorganisms, of molds and yeasts, and of
Enterobacteriaceae, respectively. Plates were incubated at 30  C
for 48 h (PCA and WA) and 37  C for 24 h (VRBGA). For enumeration of Listeria monocytogenes, the ISO 11290-2:2005 method was
used. In details, ten grams of each gel were homogenized with
Buffered Peptone Water (1% [wt/vol] peptone, 0.5% [wt/vol] NaCl,
0.35% [wt/vol] disodium phosphate, and 0.15% [wt/vol] potassium
dihydrogen phosphate), pH 7.2 0.2. After homogenization,
samples were kept at 20  C for 1 h. One hundred microliters of
serially diluted homogenates were plated on Listeria Selective
Agar plates supplemented with modied Listeria Selective Supplement. Plates were incubated at 35  C for 48 h. Only colonies
surrounded by a black halo were counted and ascribed to
L. monocytogenes after biochemical and serological analyses
(Microbact Listeria 12L, Oxoid). The absence of L. monocytogenes
in the samples showing no colony ascribed to this species was
ascertained through an enrichment procedure. The analyses were
carried out in triplicate on duplicate samples of each gel at time
0 and after 10, 20 and 30 days (t10, t20, t30, respectively) of
storage at 5  C.

Fig. 1. Images of the EFG obtained by uorescence microscopy. Oil droplets in yellow
(H, high oil content; M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound homogenization). (For interpretation of the references to color in this gure legend, the reader is
referred to the web version of this article.)

34

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

3.2. Textural characterization


Fig. 2 reports the consistency of the EFG, expressed as the area
under the curve of the back extrusion test (Gonz
alez-Martnez et al.
2002). A wide range of variability was observed for this parameter.
A signicant decrease of the EFG consistency was observed when
proceeding from L to M to H formulations. According to this effect,
emulsion droplets in inulin gels could be considered inactive llers
and unbound to the matrix (Sala et al., 2008). As regards the processing technology, a signicant increase of consistency was obtained when using ultrasound homogenization. Similar effects
were probably achieved in the EFG production, with a ner
dispersion of the components and an improvement of the gel
network formation (Bot et al., 2004), due to an optimized distribution of inulin and oil droplets and consequently improved hydration and gelling interaction of inulin.
A favorable effect of heating on the gel consistency could be
argued from the differences observed between US and CUS trials
(p < 0.05), being the former able to give more consistent EFG than
the latter. This effect was more marked in the EFG with higher
inulin contents and lower i/w ratio, indicating that the effect of
temperature on gel consistency directly involved the gelation
process, probably enhancing the hydration of inulin granules. A
previous study by Glibowski (2010), regarding inulin gels, reported
that the preparation temperature did not considerably affect the
rheological parameters, but signicant differences were observed
for different heating rates. Though it was hard to nd unambiguous
tendencies, the highest heating rate caused signicant hardness

decrease. Kim, Faqih, and Wang, 2001, instead, found


that
thermally-induced gelation gave gels with higher strength than
shear-induced ones. Our ndings seem to agree with the latter
authors.
A wide variability of rheological properties of EFG could be
therefore obtained by modulating homogenization temperatures
and techniques, even using the same amounts of EFG components.
Nevertheless, in order to better assess the role of the EFG
components in determining gel consistency, further trials were
performed on EFG obtained by varying of 4% each ingredient
respect to the H-US spread. The ternary plot with the response

Fig. 3. Ternary plot with surface response regression of the spread consistency (area
under the load/time curve of texture analysis was calculated and converted in N$mm)
as a function of inulin, water and oil contents. Data points indicate the tested
formulations.

contour of the area under the curve (Fig. 3) showed a substantial


dependence of the spread consistency on the inulin content,
though a signicant interaction (p 0.022 from the analysis of the
variance of the quadratic regression) between water and oil pointed
out that a certain balance between the two liquid phases should be
kept in order to avoid drops in the consistency. In particular, consistency showed a positive relation with the inulin/water ratio, as
pointed out by the linear regression of area as a function of i/w ratio
(p < 0.001), conrming the central role of inulin hydration.
3.3. Color analysis
The obtained EFG are shown in Fig. 4. Their appearance was that
of homogenous, white-yellowish creams, with varying consistency.

Fig. 2. Results of the texture analysis carried out on the EFG. Different letters mean
signicant difference at p < 0.05 according to the Fishers test performed after the
analysis of variance (H, high oil content; M, medium oil content; L, low oil content; Me,
mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound
homogenization).

Fig. 4. Appearance of the EFG under investigation (H, high oil content; M, medium oil
content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound homogenization).

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

35

Fig. 5. Results of the color analysis carried out on the EFG. Different letters in the legend mean signicant difference at p < 0.05 according to the Fisher's test performed after the
analysis of variance (H, high oil content; M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound
homogenization).

Fig. 5 reports the results of the color analysis. All the EFG were
characterized by high lightness (L*), due to the white color of inulin
gels (Chiavaro, Vittadini, & Corradini, 2007; Kim, Faqih, et al., 2001)
and a dominant yellow color (b*), attributable to the olive oil, with
very low values of the red index (a*). The analysis of variance
showed no signicant effect of the formulation on the values of L*
and a*, while it gave a p-value equal to 0.08, slightly above the
chosen signicance threshold, for the yellow index, that became
higher as the oil content increased. On the contrary, the homogenization system signicantly affected all of the three color indices.
The legend of Fig. 5 reports also the results of the Fisher's test
performed to compare the homogenization systems. Ultrasound
treatment determined a slight but signicant decrease of the L*
index and an increase of both red and yellow indices. The use of the
ice bath signicantly reduced these effects.
The color of such complex systems is affected by several factors.
As regards concentrated emulsions, that have been widely studied
(McClements, 2002a, 2002b), their color is determined not only by
the concentration and absorbance of the existing chromophores,
but mainly by their physical and microstructural characteristics,
such as reectance properties, which depend on droplet characteristics (radius, concentration) and scattering parameters on one
hand, and on chromophore characteristics (concentration and
absorbance) on the other hand. Decreasing the droplet size and
their concentration leads to higher lightness and lower chromaticness (a* and b* indices). The scattering properties of such systems depend, besides the droplet characteristics, also on the
refractive index of both oil droplets and continuous phase: the
lightness decreases and the chromaticness increases as the
refractive index of the droplets tended towards that of the
continuous phase. This could be achieved by adding a watersoluble solute to the aqueous phase of an oil-in-water emulsion
(McClements, 2002b). Moreover, the magnitudes of a* and b* are
approximately proportional to the reciprocal of the lightness so
that an increase in lightness tends to cause a decrease in chromaticness (Billmayer & Saltzman, 1981). As regards the EFG under
investigation, due to their opacity conferred by inulin gel, probably
absorbance of chromophores from olive oil (carotenoids and
chlorophylls) was responsible of color, more than scattering of oil
droplets. Therefore, smaller oil droplets increased the surface-tovolume ratio of the oily phase and its light absorbance. Possible
incipient browning due to localized temperature peaks could have
contributed to color and have led to lower lightness and higher
chromaticness in US EFG.

3.4. Sensory analysis


3.4.1. Sensory proling
The results of the sensory analysis are reported in Fig. 6, while
Table 1 reports the results of the statistical analysis. The EFG
appeared quite opaque, but their gloss increased as the oil content
increased and was signicantly higher in mechanically homogenized EFG, due to higher amounts of oil and higher droplets dimensions, respectively. They were characterized by low perception
of intensity of odor and avor, of both oil and lecithin. Only a signicant increase of oil odor was assessed in H EFG. No perception of
rancid odors and avors was reported by panelists, pointing out
that no signicant oxidative stress was determined by neither ultrasound nor mechanical homogenization. Yet, an optimization of
the avor properties of these EFG is needed, since the lecithin avor
was perceived as unpleasant by the panelists. No other statistical
signicance was observed for the inuence of both formulation and
homogenization variables on avor attributes. As regards the
textural properties of the EFG, interesting results were obtained.
Quite a high consistency was perceived by the panelists, with great
variability among the samples. The scores ranged from 4.9 (H-Me)
to 8.2 (L-US), with a signicant inuence of both formulation and
homogenization technique. The increase of the oil content and the
decrease of inulin and i/w ratio determined a signicant decrease of
consistency. A signicant improvement of the EFG consistency,
instead, was obtained by the use of US homogenization, in accordance with the texture analysis results. On the other hand, when
formulation had high oil content and low i/w ratio, and when
mechanical homogenization was used, EFG showed more marked
fusion-like behavior in mouth, as a consequence of a soft gel
structure with melting perception. The EFG coarseness, perceived
as a sandy mouthfeel, was dramatically reduced by ultrasound,
being the US and CUS EFG particularly smooth: the sandy perception could be a consequence of a less ne dispersion of components
and worse inulin hydration with mechanical homogenization. High
oil contents signicantly decreased the coarseness of EFG, that
could be attributed, therefore, to the gel fraction, As regards the
greasiness perception, it was greater as the oil content increased.
This agrees with Sala et al. (Sala, Vandevelde, Cohen Stuart, &
Vanaken, 2007), who found positive correlations for fatty and
creamy perception with the oil content of EFG. The EFG spreadability showed the same trend, as the incorporated emulsion
intermingled with the gel network, reducing its strength. The effects of US homogenization in the EFG production, i.e. a ner

36

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

Fig. 6. Results of the sensory analysis carried out on the EFG. Different letters mean signicant difference at p < 0.05 according to the Fishers test performed after the analysis of
variance (H, high oil content; M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound homogenization).

dispersion of the components and an improvement of the gel


network formation, observed by instrumental texture analysis,
were therefore conrmed by the sensory evaluation of consistency
and coarseness. The increase in temperature seemed to enhance
this effect, since US EFG showed higher (though not signicantly)
scores than CUS ones. Kim, Faqih, et al. (2001) also found sandy
texture in inulin gels, when gel formation lead to worse dispersion
of inulin particles and aggregation of large particles.
3.4.2. Consumer test
The results of the consumer test are reported in Fig. 7. H-US EFG
was liked (scores equal or higher than 3) by 72.5% of consumers.
The quite high acceptability of H-US EFG suggests that, though
being model systems, such gels could provide functional food
products as potential alternatives to traditional spreadable with
high fat/saturated fat contents.

amounts of oxidized triacylglycerols and total phenolic compounds


were 2.6 g/kg and 513 mg/kg respectively. These parameters point
out a low oxidative degradation level. The peroxide value complied
with the standards provided by Regulations (Ofcial Journal of
European Communities, 1991) for extra virgin olive oils, while the
phenolic compounds content was quite appreciable, in consideration of their well assessed antioxidant activity.
The data of oxidative degradation indices of the oil fraction of
the EFG are reported in Fig. 8. Quite high variability characterized
the data obtained. Nevertheless, the increase of peroxides as a
consequence of the processing was limited and not signicant. On
the contrary the increase of oxidized triacylglycerols (including
peroxides and other oxidized forms of triacylglycerols) was signicant (p < 0.05) when comparing the oil phase of the EFG

3.5. Oxidative degradation of the oil phase


The extra virgin olive oil used for the production of the EFG
presented a peroxide value equal to 9.5 meq O2/kg of oil, while the
Table 1
Results of the statistical analysis on the sensory data of the EFG.a
Fisher's LSD testb

p-values

Gloss
Homogeneity
Oil odor
Oil avor
Lecithin odor
Lecithin avor
Rancid odor
Rancid avor
Coarseness
Greasiness
Consistency
Melting
Spreadability

Formulation

Homogenization

Me

US

CUS

<0.001
0.955
0.003
0.205
0.527
0.998
0.111
0.373
0.006
0.023
0.041
<0.001
0.041

0.005
0.193
0.904
0.855
0.665
0.837
0.226
0.554
<0.001
0.477
0.004
<0.001
0.112

A
B
A
B
B

A
B
A
B
AB

B
A
B
A
A

B
A

A
B

A
B

H, high oil content; M, medium oil content; L, low oil content; Me, mechanical
homogenization; US, ultrasound homogenization; CUS, cooled ultrasound
homogenization.
a
p-values of the ANOVA for the signicance of each factor and results of the
Fisher's LSD test. Values lower than 0.05 are highlighted in bold.
b
Different letters mean a signicant difference at p < 0.05.

Fig. 7. Results of the consumer test carried out on two EFG (n 80). Frequency of
attribution of liking scores from 1 e dislike to 5 e like very much (H, high oil
content; US, ultrasound homogenization).

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

37

Fig. 8. Results of the analysis of oxidation carried out on the EFG (H, high oil content; M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound
homogenization; CUS, cooled ultrasound homogenization).

(irrespective of formulation and processing technology) with the


starting oil. This could mean that the EFG production lead to a
certain oxidation, with both peroxide formation and degradation,
resulting in apparently unvaried peroxides and slightly increased
oxidized triacyglycerols. Both mechanical aeration and US are
suitable causes of lipid oxidation. Chemat and coworkers (Chemat,
Grondin, Sum Cheong Sing, & Smadja, 2004) reported that US determines oxidative deterioration in edible oils.
3.6. Total phenolic compounds
Signicant differences were observed among the systems as
regards the residual total phenols content. Fig. 9 shows TPC of the
systems. The concentrations have been reported respect both to the
EFG and to the oil fraction allowing a correct evaluation of the residual antioxidants in the systems. The TPC content in the EFG was
obviously affected by the oil content, resulting higher in the highoil EFG. This trend was almost inverted when comparing the TPC
reported to the actual oil content of the EFG. The high oil EFG
showed, in fact, a more marked decrease (mean value of 312 mg/kg)

respect to medium- and low-oil EFG (mean values 364 and 363 mg/
kg respectively, different at p < 0.05).
As regards the processing technology, mechanically homogenized EFG presented the highest contents of TPC; US samples were
not signicantly different, while, unexpectedly, CUS systems presented signicantly lower amounts of TPS respect to M, in spite of
the lower temperatures reached than in US processing. The
decrease of TPC in mechanically homogenized EFG respect to the
fresh oil should be attributed to the intense aeration induced to the
system. Ultrasound, instead, is known to induce oxidation of
phenolic compounds in edible oils, with an enhancing effect of
temperature (Luque de Castro & Priego-Capote, 2007), and lead,
therefore, to a more pronounced decrease of TPC than that due to
mechanical homogenization. The reason why CUS systems showed
the maximum decrease of TPC should be studied. An hypothesis
could be made. US are commonly used to degas liquid media
(Luque de Castro & Delgado-Povedano, 2014): decreasing the
temperature could have increased the oxygen solubility and
therefore reduced the degassing effect of US, leaving higher
amounts of oxygen in the EFG.

Fig. 9. Results of the analysis of total phenolic compounds (TPC) carried out on the EFG. The box plot reports the TPC concentration in the EFG, while the corresponding scatter plot
reports the TPC respect to the oil content. Different letters mean signicant difference at p < 0.05 according to the Fishers test performed after the analysis of variance (H, high oil
content; M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound homogenization).

38

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

The residual amount of TPC, however, remained appreciable for


all systems, varying from 50 to 76 % of the starting oil content, with
a concentration in the EFG in the range of about 70e130 mg/kg.
3.7. Headspace volatile compounds
The analysis of volatile compounds allowed to compare the data
relative to 31 compounds, as reported in Table 2. Most of them
derived from both enzymatic and non-enzymatic lipid oxidation
and could be therefore attributed to olive oil. Also compounds
whose formation involves amino acids were detected (Strecker
aldehydes, pyrazines, furans). They probably derived in part from
the oil, where Strecker aldehydes are commonly found and derive
from enzymatic deamination of amino acids (Kalua et al., 2007). As
regards pyrazines and furans, their appearance could be due to
carbohydrate caramelization or a minimal development of Maillard
reaction products from residual peptides in inulin or in lecithin
(Sabik, Fortin, & Martin, 2012). Both formulation and processing
technology affected the semiquantitative levels of several compounds. Obviously, compounds deriving from olive oil resulted
more abundant as the oil content increased. In particular, all the
interested compounds derived from the lipoxygenase pathway and
characterize the volatile pattern of a fresh olive oil (Kalua et al.
2007). Among them, 2-hexenal was the most abundant volatile
compound, accounting for the 56% of the sum of the peak areas
(data not shown), remaining within the range 46e66% in all the
samples analyzed.
As regards the effect of the homogenization system, the EFG
obtained by sonication presented richer volatile patterns. The effect
of sonication respect to mechanical homogenization was double.

On one hand, higher levels of lipoxygenase pathway-derived


compounds point out that the original avor compounds were
better preserved, probably due to the minor aeration induced by
ultrasound compared to mechanical homogenization. On the other
hand, compounds from autoxidation of oleic acid (i.e. nonanal) and
from carbohydrate thermal degradation/Maillard reaction (acrolein/2-propenal, methyl-pyrazine) are markers of a certain thermal
stress induced to the system.
Fig. 10 reports the sums of the compounds ascribable mainly to
the lipoxygenase pathway (lipoxygenase-pathway-compounds,
LPC, including selected C5 and C6 aldehydes, alcohols and esters)
and of the C7eC9 aldehydes, deriving from autoxidation (autoxidation compounds, AOC), as well as an oxidative index given by
their ratio. These values are reported also for the starting oil.
As can be expected, LPC were much lower in the EFG than in the
oil, due to the dilution effect of the other ingredients of the recipe.
Nevertheless, the LPC still remained abundant in EFG, much higher
than AOC. A signicant increase of LPC was observed as the oil
content increased and when US were used to homogenize. The use
of cooled US unexpectedly attenuated this effect. The sum of AOC
showed high variability, pointing out that the thermal stress requires further effort to be controlled respect to other effects of the
processing technologies under comparison. Yet, it seems that a
slight increase in US and CUS systems should be assumed. The
amount of AOC did not seem to increase along the oil content. As a
consequence, the LPC/AOC ratio, that could be assumed as a synthetic index of the impact of the EFG production on the volatile
prole of the oil, resulted higher in the EFG with high oil contents,
especially when mechanically homogenized. The adopted cooling
system was not able to improve the properties of sonicated EFG.

Table 2
Volatile compounds analyzed and results of the comparisons for Formulation and Homogenization variables.a
Fishers LSD Testb

p-values

Propanal
Methyl acetate
2-Propenal
Ethyl acetate
2-Methyl-butanal
3-Methyl-butanal
Pentanal
Hexanal
2-Pentenal
Penten-3-ol
2-Hexenal
Pentanol
Methylpyrazine
Octanal
3-Hexenol acetate
2-Pentenol
2-Heptenal
Hexanol
3-Hexenol
Nonanal
2,4-Hexadienal
2-Hexenol
2-Octenal
Acetic acid
Furfural
Copaene
2,4-Heptadienal
2-Nonenal
5-Methyl-furfural
2-Furanmethanol
Hexanoic acid

LRI

Formulation

Homogenization

657
809
828
874
905
909
974
1081
1135
1168
1227
1259
1277
1294
1324
1331
1334
1363
1374
1401
1414
1417
1442
1473
1485
1498
1512
1551
1599
1681
1870

0.879
0.020
0.830
0.305
0.192
0.842
0.276
0.425
0.047
0.100
0.026
0.039
0.253
0.559
0.097
<0.001
0.696
0.004
0.002
0.780
0.320
0.007
0.361
0.620
0.552
0.172
0.262
0.217
0.061
0.279
0.167

0.268
0.065
<0.001
0.490
0.005
<0.001
0.931
0.242
0.171
0.341
0.024
0.714
0.039
0.189
0.036
0.030
0.403
0.583
0.541
0.044
0.066
0.042
0.172
0.159
0.218
0.978
0.125
0.476
0.473
0.133
0.717

LPC

LPC

AOC
LPC
AOC
LPC
LPC
AOC
LPC
AOC

AOC
AOC

AB

AB

B
AB

AB
B

A
A

B
B

A
A

A
A

AB

Me

US

CUS

a
a

b
b

b
b

ab

ab

b
a

a
b

ab
ab

ab

ab

H, high oil content; M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound homogenization;
AOC, selected autoxidation deriving compounds; LPC, selected lipoxygenase pathway deriving compounds.
a
p-values of the ANOVA for the signicance of each factor and results of the Fishers LSD test.
b
Different letters mean a signicant difference at p < 0.05. Values lower than 0.05 are highlighted in bold.

V.M. Paradiso et al. / Food Hydrocolloids 45 (2015) 30e40

39

Fig. 10. Results of the analysis of volatile compounds carried out on the EFG. Different letters mean signicant difference at p < 0.05 according to the Fisher test performed after the
analysis of variance (H, high oil content; M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound
homogenization).

Table 3
Mean values (standard deviation) of cell density (log cfu/g) of total mesophilic aerobic microorganisms, molds and yeasts found in the EFG after 0 (T0), 10 (T10), 20 (T20) e 30
(T30) days of storage at 5  C.a
Total mesophilic aerobic microorganisms

L-Me
L-US
L-CUS
M-Me
M-US
M-CUS
H-Me
H-US
H-CUS

T0

T10

2.4 0.1j
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

4.5
0.0
2.9
3.2
0.0
0.0
1.4
0.0
3.8

0.5ef
0.9hi
0.7ghi

2.0k
1.0g

T20
4.6
0.0
4.4
4.7
0.0
1.3
3.8
0.0
5.5

1.1ef
0.1f
1.0ef
1.9k
0.4g
0.3 ab

Molds
T30
5.4
2.6
3.7
5.0
5.1
1.0
3.4
0.0
5.8

0.1bc
0.2ij
0.2g
0.2de
0.3cd
1.4k
1.3gh

0.1a

Yeasts

T0

T10

T20

T30

T0

T10

T20

T30

0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

0.0
0.0
2.7 0.3ef
0.0
0.0
0.0
0.0
0.0
0.0

0.0
0.0
3.0
2.4
0.0
1.3
3.9
0.0
3.6

0.0
0.0
3.6
4.2
3.1
1.3
3.1
0.0
4.6

0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0

0.0
0.0
0.0
3.1 0.5d
0.0
0.0
0.0
0.0
3.7 1.0d

4.6 1.1c
0.0
0.0
4.6 1.0c
0.0
0.0
0.0
0.0
5.4 0.6 ab

5.2
0.0
0.0
5.1
5.0
0.0
0.0
0.0
6.0

0.2e
0.6f
1.8g
0.2bc
1.1cd

0.1cd
0.1 ab
0.8de
1.8g
1.2de

0.4a

0.2b

0.2bc
0.1bc

0.2a

H, high oil content, M, medium oil content; L, low oil content; Me, mechanical homogenization; US, ultrasound homogenization; CUS, cooled ultrasound homogenization.
a
For a given microbial group, different letters mean a signicant difference at p < 0.05.

Probably, better results could be achieved under modied


atmosphere.
3.8. Microbiological stability
Table 3 shows the results of microbiological stability. Overall,
cell density increased for all the microbial groups during refrigerated storage. It is well known that refrigerated storage either slows
down or inhibits the growth of most microorganisms. Anyway, it
may be hypothesized that some of the microorganisms contaminating the formulations were psycrotrophic (Mossel & Zwart,
1960).
The H-US formulation showed the highest microbiological
quality, given the absence of all the microbial groups over the
whole storage period (Table 3). This may be due the combination of
two hurdles: (i) ultrasonic homogenization causing the inactivation
n
~ ez & Burgos, 1976); (ii)
of contaminating microorganisms (Ordo
higher oil content (corresponding to lower value of water activity)
of the H-US with respect to L-US and M-US formulations, inhibiting
the growth of eventually surviving microorganisms. Enterobacteriaceae (a common food quality indicator) (Tortorello, 2003)
and the pathogenic bacterium L. monocytogenes were absent in all
the formulations, regardless of time of storage (results not shown).
4. Conclusions
The production of functional EFG based on inulin and extra
virgin olive oil was set up with the present study. The characteristics of the obtained EFG depended on both the ingredients' proportions and the homogenization technique: the textural

properties were improved using ultrasound homogenization, while


both consistency and coarseness decreased as the oil content and
melting, greasy perceptions increased. Also the ratio inulin/water
affected signicantly the gel textural properties. The avor was not
intense and slightly affected by the presence of lecithin. Homogenization caused a moderate oxidative stress, in terms of both volatile and non-volatile products, with differences depending on the
technique adopted. Nevertheless, the nal content of phenolic antioxidants remained appreciable. The EFG showed a good microbiological stability, measured over 30 days at 5  C, that was
improved by ultrasound.
Ultrasound homogenization appeared to be, therefore, a suitable technique to obtain EFG with enhanced textural and microbiological properties, though an optimization of processing
temperature would be desirable in order to balance textural and
microbiological benets with drawbacks regarding the slight
oxidative stress and color changes.
The obtained systems showed good consumers acceptability
and could constitute the basis for functional spreadable products.
Acknowledgments
The Authors wish to thank Dr. Annalisa Paradiso for precious
contribute in uorescence microscopy analysis.
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