CETvol87
DOI: 10.3303/CET2187091
Paper Received: 9 October 2020; Revised: 10 February 2021; Accepted: 6 April 2021
Please cite this article as: Cejudo C., Gallardo M., Fernandez-Ponce M.T., Casas L., Mantell C., Martinez De La Ossa E.J., 2021, Preservation of
Food with High Oil Content by Supercritical Impregnation Techniques, Chemical Engineering Transactions, 87, 541-546
DOI:10.3303/CET2187091
CHEMICAL ENGINEERING TRANSACTIONS
VOL. 87, 2021
A publication of
The Italian Association
of Chemical Engineering
Online at www.cetjournal.it
Guest Editors: Laura Piazza, Mauro Moresi, Francesco Donsì
Copyright © 2021, AIDIC Servizi S.r.l.
ISBN 978-88-95608-85-3; ISSN 2283-9216
Preservation of Food With High Oil Content by Supercritical
Impregnation Techniques
Cristina Cejudo*, Miriam Gallardo, María Teresa Fernández-Ponce, Lourdes
Casas, Casimiro Mantell, Enrique J. Martínez de la Ossa
Chemical Engineering and Food Technology Department Wine and Agrifood Research Institute (IVAGRO)
University of Cadiz. Avda. República Saharaui, s/n, 11510, Puerto Real, Cádiz, SPAIN
cristina.cejudo@gm.uca.es
Nuts are very important foods for human nutrition due to their high content of mono- and polyunsaturated fatty
acids. However, they are substrates very sensitive to oxidation, appearing rancidity that affects both the taste
and the smell, that led to the consumer’s rejection. To avoid this, the food industry uses some strategies that
sometimes substantially alter the composition and nutritional value of nuts. A promising possibility for the
conservation and stability against its oxidation is to impregnate a natural antioxidant such as methyl gallate
using CO2 in supercritical conditions. This technology is being used recently for the impregnation of different
natural substances on polymers for its use in the food and biomedicine field. However, the direct supercritical
impregnation of compounds into food has been scarcely investigated.
In the present work, it is studied the effect of pressure and temperature in the impregnation process of methyl
gallate on peeled peanuts. Additionally, the effect of the depressurization rate and impregnation time was
studied at the most convenient conditions. The results obtained indicate the best conditions for impregnation
were reached at 150 bar and 45 ºC. These conditions improved the antioxidant capacity of the nuts while
preserving the quality by avoiding breakages during the process.
1. Introduction
Lipid oxidation and microbial growth are the main causes of most foodstuff spoilage, being particularly
important for fresh food, fourth range products, or peeled nuts, causing losses of both sensory and nutritional
qualities. Lipid oxidation is of primary importance on food rich in mono- and polyunsaturated fatty acids since
the rancidity are sensorial perceived yet in early states (B. Weinreich, 2010). Peanuts and other shelled nuts
have a longer shelf life since they are protected from oxygen. However, those marketed in the peeled or
ground format are especially sensitive to oxidation reactions (Grüner-Richter et al., 2012), so one of the
challenges in nut production is to avoid oxidation during the manufacture and storage. Apart from the use of
modified atmosphere packaging, there are some commercial strategies to fight the oxidation of nuts, such as
adding sugar or chocolate coatings, drying, or toasting. However, these processes severely affect the sensory
properties and the nutritional composition by increasing calories or reducing the nutritional value (Weidner,
2018).
New technological treatments as coating with encapsulated antioxidant agents are being explored to stabilized
the lipid fraction of nuts (Dodorni et al., 2017). Another suitable technology is direct-food impregnation with
active compounds, such as enzymes, minerals, vitamins, and antioxidants, among others, into the edible parts
of the food product. Some of the techniques traditionally used are osmosis impregnation, vacuum
impregnation, ultrasound, or edible coating (Radziejewska-Kubzdela et al., 2014). However, these processes
possess certain limitations. On one side, the mass transfer mechanisms are restricted by diffusion and
capillary and are dependent on some food properties such as humidity, geometry, or food composition. On the
other hand, generally, subsequent drying stages are needed to eliminate remained liquids on the food’s
surface, which produce Maillard Reactions and causes changes both in taste and texture (Yang et al., 2019).
The use of supercritical fluids has arisen great interest in recent decades for improving these industrial
processes as it solves the problems presented by traditional impregnation methods. Active packaging is one
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of the most innovative applications aimed at increasing the shelf life of these products with minimal
modification of its formulation. These packages contain compounds with antioxidant and antimicrobial activity
that aim to extend the shelf life of the food while maintaining its composition intact. For instance, Cejudo
Bastante et al. (2018) demonstrated that a PET/PP food package impregnated with bioactive compounds from
olive leaf extracts significantly reduced the peroxide index of peeled, packaged sunflower seeds compared to
not impregnated packaging. Another alternative is the direct supercritical impregnation of products, which is
based on the facility of CO2 in solving active compounds and then incorporate them into different matrices
thanks to its high diffusivity, while providing a solvent-free product once the process ends. The supercritical
impregnation of rosemary extract and carnosic acid into different peeled nuts has been previously reported in
the literature with promising results attending to its preservation, but previous steps of decoloration and
deodorization of the extract were required to reduce the sensorial impact of the aromatic terpenes (Grüner-
Richter et al., 2012). Considering this, the present study aims to incorporate a lower sensorial impact
compound as methyl gallate (MG) and evaluate the structural modification and the antioxidant capacity of the
peanuts after impregnation treatment. MG is a phenolic compound already present in nuts (Fuente-Maqueda
et al., 2020) with a strong antioxidant capacity that intervene in hinders lipid oxidation (Mansouri et al., 2020).
In this sense, their incorporation into peanuts would help in maintaining longer their functional compounds and
sensory properties by adding a compound naturally contained among their constituents.
2. Material and methods
2.1 Raw materials and reagents
Carbon dioxide (99.99%) was purchased from Abello-Linde S.A. (Barcelona, Spain). Peeled penuts were
obtained from a local market (Cadiz, Spain). 2,2-Diphenyl-1-pricrylhydrazyl reagent (DPPH) and methyl galate
were obtained in Sigma-Aldrich (Steinheim, Germany). Organic solvents (hexane, ethyl acetate, acetonitrile,
methane and formic acid) were purchased in Panreac (Barcelona, Spain).
2.2 Supercritical impregnation procedure
Experiments were developed in a lab-scale high-pressure equipment provided by Thar Technologies
(Pittsburgh, PA, USA), which includes a condenser, a P50 high-pressure pump, a pre-heater, a 104 mL vessel
with a thermostatic jacket, and a back-pressure regulator (BPR) (Figure 1). All units are monitored by a
controller.
The impregnation process was developed in batch mode. A stir bar was placed at the bottom of the vessel to
ensure the homogenization of the headspace with constant stirring. Then, 86 mg of methyl gallate (MG) and
~30 g of raw entire peeled peanuts were introduced into the vessel, separated by a metallic mesh to avoid
physical contact. CO2 was pumped at a flow rate of 10 g/min until reaching the pressure value set in each
experiment. Once passed 5 h of impregnation time, the system was depressurized at 2 bar/min. Samples
were collected and stored at 4 ºC in darkness until analysis. The impregnation flowchart is depicted in Figure
1.
Figure 1. Impregnation process flowchart
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A 2
3
screening experimental design was analysed varying pressure (100, 150, and 200 bar), and temperature
(35, 45, and 55 ⁰C). Then, the depressurization rate and impregnation time were also studied at the most
convenient pressure-temperature conditions selected in the experimental design.
2.3 Quantification of MG impregnated
Impregnated nuts were extracted aid with ultrasounds for 60 min with hexane in order to obtain the peanut oil
that contains the MG deposited. Then, 300 µL of the oil were pre-concentrated by solid-phase extraction
(SPE) using a C18 column (40 µm of particle size) previously pre-conditioned using water, methanol, and
hexane. 1 mL of methanol was used as the elution solvent. The fraction was recovered and analysed in an
HPLC Agilent 1100 (Agilent, Germany) coupled with a UV/Vis detector and a column SynergiHydro-RP C18 (4
μm, 150 mm x 3 mm i.d.) (Phenomenex, US.). The mobile phase consists of a solvent A of 0.1% formic acid in
water, and a solvent B of 0.1 %formic acid in acetonitrile. The amount of MG was quantified at 278 nm using
the following calibration curve
Area = 101962 CMG + 4.0023; R
2
= 0.999 (1)
Then, the impregnation yield (I%) was calculated as the amount of MG quantified respecting the amount of the
oil recovered in the extraction. To evaluate possible interference of the measured, the initial amount of MG on
the samples was analysed and quantified in trace levels, so it was not considered a variation factor.
Quantification of the MG impregnated was done in duplicate.
2.4 Antioxidant capacity of impregnated nuts
The antioxidant activity of the impregnated nuts was determined by analysing the reaction of the extracted oil
in the presence of the 2,2-Diphenyl-1-pricrylhydrazyl reagent (DPPH). To do so, 100 µL of extracted oil were
introduced in 3.9 mL of 6·10
-5
M DPPH solution prepared in ethyl acetate. The reaction was monitored at 515
nm until the steady state was achieved. The efficient concentration (EC50) of extracted oil was determined in
each impregnation condition in order to define the oil concentration required to reduce the DPPH reagent by
50%. Analyses were done in duplicate.
2.5 Scanning Electron Microscopy SEM
Nuts impregnated under the most convenient conditions were analysed by SEM (Quanta 200) in order to
observe the deposition of MG on the nuts’ surface and evaluate possible structural damages.
3. Results and discussion
3.1 Study of the impregnation conditions
The impregnation parameters were optimized according to the I% of MG. The CO2 density at each condition
has to be taking into consideration since it would determine both the solubility of MG and the diffusivity of the
compound in the matrix to be impregnated (Champeau et al., 2015).
The density of supercritical CO2 increases with an isothermal increase of pressure, with the consequent
increase in solubility of the MG in the CO2 (Murga et al., 2002). According to the results of Murga and
researchers, the solubility of MG at 40 ºC increase with pressure, reaching values from 1.2∙107 to 5.56∙107 M
in the range of 100 to 200 bar. When increasing temperature until 50 ºC, the solubility drastically increments
until ca. 10∙107 M when pressure is above 200 bar. These values are in concordance with the I% obtained
(Figure 2A), since pressures over 150 bar turn into a decrease in the impregnation efficiency. At those
conditions, the partition coefficient of the MG deviates to the supercritical phase instead of to the peanut
surface, favouring the dragging of the compound during the depressurization. Besides, the decrease in the
diffusivity hinders yet the penetration of the MG into the peanut matrix. When analysing the experimental
design, the pressure was pointed as the only significant parameter, being a positive variable of the
impregnation process (Figure 2B). However, it should be mention that pressures of 200 bar cause a high
impact on the nuts integrity so that conditions were discarded. Attending to the I% values (Figure 1a), a
Gauss-Bell behaviour seems to be observed in the data obtained, in which low and very high levels of
solubility of MG lead to low impregnation yields, being the most optimal levels those obtained at 150 bar,
where the solubility of MG in the supercritical phase is ca. 5 to 6∙107 M. At 150 bar, the increase from 45 to 55
ºC did not cause a significant change in the impregnation yield, maintaining adequate yields above 2
mg MG/g oil . In this sense, 45 ºC was selected as the most convenient temperature as this would entail energy
savings in the process.
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Figure 2. A) Impregnation yield (%I, mg MG/g peanut oil) and CO2 density at each impregnation conditions; B)
Pareto diagram of the impregnation yield
Both pressure and temperature are the determining factors in the impregnation processes, since they
influence the solute’s solubility in the supercritical solvent. Moreover, the impregnation time and the
depressurization step are also critical variables. On one side, impregnation is a mass transfer process so
knowing the kinetics of the reaction would aid to develop the process more efficiently. Moreover, extreme
pressure changes during depressurization could improve the deposition of the compound but also affect the
integrity of the product (Ameri et al., 2020). Therefore, both parameters have to be also defined to optimize
impregnation conditions. As far as we know, it is the first time that both parameters have been studied in nut
impregnation.
Figure 3. Effect of A) depressurization rate and B) impregnation time on the impregnation yield (I%) on
samples impregnated at 150 bar and 45 ºC.
The impregnation yields obtained at a low depressurization rate of 2 bar/min were compared with a fast one of
10 bar/min. The operating conditions of pressure and temperature are those selected previously, i.e. 150 bar
and 45 ºC. The impregnation time was kept constant at 5 hours in those experiments.
As can be seen in the Figure 3A, the I% is significantly higher when depressurization is fast. As the
supercritical conditions are modified in a short period, the CO2 rapidly reduced its density, decreasing the
solubility of MG and favouring its deposition on the peanut’s surface. However, that extreme pressure change
in the vessels may cause the breakage of the nuts and therefore those conditions were not advisable for the
impregnation of entire peanuts. The depressurization step is crucial in the impregnation of fragile products,
where lower impregnation yield has to be sometimes assumed to obtain a product without mechanical
damages still accepted by the consumer (Ongkasin et al., 2020).
Grüner-Richter et al. (2012) developed the impregnation of rosemary extract during 1 h on different nuts
obtaining good impregnation efficiency and significant delay in its peroxidation. In these experiments, longer
periods were studied to favour the solubilisation and subsequent impregnation into a complex matrix as nuts.
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Time increments positively influenced the impregnation yield (Figure 3B), since 3 h were insufficient for
achieving higher impregnation yields, obtaining better results at 5 h of treatment. Although longer periods
should be explored, preliminary experiments with 24 h of impregnation time were discarded due to a co-
extraction of peanut oil was observed (data not shown), which led to an early deterioration of the product and
an adequate impregnation process.
3.2 Antioxidant capacity of the impregnated peanuts
The antioxidant capacity gained by the peanuts after the treatment is showed in Table 1 in comparison to the
untreated peanuts. As can be seen, all conditions favoured the increment of the antioxidant capacity of the
peanut oil extracted, which eventually intervene in delaying the lipid oxidation. The lower the EC50 value is,
the higher is the antioxidant activity reached, since lower amounts of oil efficiently react with the 50% of the
DPPH reagent. Nevertheless, there was no direct correlation between I% and EC50 values since the peanut
oil contains other reactive compounds that could cause a synergistic effect in the reaction with the DPPH.
Samples impregnated at 55 ºC seemed to achieve slightly higher antioxidant activity than the rest of
conditions.
Table 1. Antioxidant capacity of impregnated peanuts.
Pressure (bar) Temperature (⁰C) EC50 (μg antioxidant/mL)
No impregnated peanuts 21.90 ± 1.99
100 35 12.20 ± 0.99
45 12.50 ± 3.96
55 9.95 ± 0.49
150 35 10.35 ± 3.18
45 12.10 ± 0.99
55 7.80 ± 2.55
4. Scanning electron microscopy
Impregnated and not impregnated peanuts were analysed by SEM. A comparison of both samples can be
seen in Figure 4. The superficial homogeneity of the not impregnated peanut can be observed. Besides, there
is no apparent damage of the impregnated peanut surface since any microscopic cracks were observed.
However, certain superficial modifications can be perceived after the impregnation treatment (Figure 4B, 4C,
and 4D) since some spherical particles, possibly attributed to the MG, were deposited in the peanut surface.
The spherical morphology of this compound after supercritical impregnation has been previously described on
impregnated food-grade films (Belizón et al., 2018). Although a partial internal diffusion of MG into the peanut
matrix could not be discarded, the superficial deposition of the antioxidant is a good approach, since the outer
surface of the peanut would be in contact with the oxygen that causes the lipid peroxidation in a greater
extent, so the MG particles will be available for their rapid action.
Figure 4. A) No impregnated peanut; B, C and D) Different images of the peanuts impregnated at 150 bar and
55 ºC.
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5. Conclusions
Supercritical impregnation of nuts is presented as an alternative to prolong its shelf life. All conditions provide
statistically higher antioxidant capacity respecting to the untreated samples, which will undoubtedly intervene
in delaying lipid oxidation. Pressures above 150 bar and depressurization rates over 2 bar/min, although
showing higher impregnation yields, were not recommended for the impregnation of entire nuts, since the
breakage of the sample was observed. Nevertheless, those results have to be positively considered, since
those conditions could be employed in crushed nuts, which in turn needs higher impregnation yields since the
higher superficial area make them even more sensitive to oxidation.
All in all, the impregnation conditions of 150 bar 45 ºC and 5 h of impregnation, with a depressurization rate of
2 bar/min provide the lower physical modification of the product under study with the highest impregnation
yield. Independently of the impregnation conditions, the antioxidant activity of the product improves
significantly comparing with the untreated product. Even though further sensory and peroxide index analysis
should be done in order to assess the suitability of the process, these results offer a good perspective of the
use of this alternative treatment for nut’s preservation.
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