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Quality by design approach to optimize cladodes soluble fiber processing

extraction in Opuntia ficus indica (L.) Miller

Article in Journal of Food Science and Technology -Mysore- · June 2019

DOI: 10.1007/s13197-019-03794-7

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J Food Sci Technol
https://doi.org/10.1007/s13197-019-03794-7

ORIGINAL ARTICLE

Quality by design approach to optimize cladodes soluble fiber

processing extraction in Opuntia ficus indica (L.) Miller
Bacchetta Loretta1 • Maccioni Oliviero1 • Martina Vittorio1 •
Emanuel Bojórquez-Quintal2 • Persia Franca1 • Procacci Silvia1 •

Zaza Fabio1

Revised: 22 March 2019 / Accepted: 25 April 2019

Ó Association of Food Scientists & Technologists (India) 2019

Abstract Opuntia ficus indica by-products can be exploi- of soluble polymers from cladodes. Further studies on
ted as sources of high-value components for applications in green assisted extraction tools and their effects in terms of
food and other industries. The aim of the present work is to quality of extracts are required in order to obtain high
elucidate and optimize the mucilage extraction process added value bio-products.
from cladodes. The effect of five water-to-biomass ratios
(1:1, 1:3, 1:5, 1:7, 1:9 w/v), pH range (2.0, 4.5, 7.0, 9.5, Keywords Mucilage  Prickly pear  Cladodes  Green
12.0) and ionic strength (water supplemented with NaCl or extraction processes  Response surface methodology
CaCl2 at the concentration of 0.1, 1.0, 10.0 and 100.0 mM)
were evaluated on mucilage yield. The analysis of the
critical factors was done by the response surface method- Introduction
ology. Ultrasound and microwave assisted extractions were
evaluated to improve the mucilage recovery and quality. In New economic models based on efficient use of the
this work: (1) the development of a multivariate model to resources in agriculture are strongly promoted by European
predict mucilage recovery on the basis of biomass/water Union policies with the twofold objectives of creating new
ratio and time of extraction; (2) pH, ionic strength and productive chains recycling by-products and to reduce the
temperature were found critical process variables by the cost of waste along the agro industrial processes. Opuntia
application of Plackett–Burman design; (3) the optimal ficus-indica is a species native to Mexico, commonly
operating conditions obtained were found to be: 1:9 bio- known as prickly pear, belonging to the family Cactaceae,
mass/water ratio, pH 12.0, ionic strength 1.0 mM NaCl; (4) well adapted to arid lands and to diversity of climates all
ultrasonic or microwave treatments are efficient tools to over the world, including the Mediterranean basin, Middle
enhance the recovery of mucilage depending on its final East, South Africa, Australia and India (Griffith 2004;
uses. Within a multi-disciplinary approach, this work pro- Stintzing and Carle 2005). In the Mediterranean areas,
vides achievements for a more efficient extraction process South Africa and South America this species is cultivated
for its edible fruits (prickly pear), although in some
countries different parts of the plant are utilized in food and
Electronic supplementary material The online version of this cosmetic industry (Retamal et al. 1987; Saenz 2000; Sáenz
article (https://doi.org/10.1007/s13197-019-03794-7) contains sup-
plementary material, which is available to authorized users.
2002; Stintzing and Carle 2005; Cushman et al. 2015). The
cactus pads (or cladodes) commonly known as nopales,
& Bacchetta Loretta when whole or nopalitos when cut into small pieces, are
loretta.bacchetta@enea.it regularly consumed as vegetable or forage (16% or 22% of
1 productive lands, respectively) in Mexico, since cactus
Department of Sustainability of Production and Territorial
Systems, ENEA, Via Anguillarese, 301 Rome, Italy pear is deeply embedded in local culture. Nevertheless, the
2 last scientific studies demonstrated cladodes nutritional and
CONACYT-El Colegio de Michoacán Laboratorio de
Análisis y Diagnóstico del Patrimonio, Cerro de Nahuatzen functional value, hence there is a growing demand for O.
85, La Piedad, Michoacán, Mexico ficus indica consumption as vegetable in US and Canada,

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 J Food Sci Technol

where the product reaches sales equivalent to 31 and 2.86 acids content in the extracts, in order to ameliorate the
million dollars, respectively (Ventura-Aguilar et al. 2017). efficiency of the process.
The mucilage is a hetero-polysaccharide of high molecular
weight (2.3 9 104 to 3 9 106 g mol-1) with ecological,
biological, chemical and physical properties (Medina- Materials and methods
Torres et al. 2000; Sáenz et al. 2004; León-Martı́nez et al.
2010); it constitutes approximately 14% of the dry weight Plant materials
of the cladodes and consists mainly of six sugars: arabi-
nose, galactose, rhamnose, xylose, uronic acid and galac- The cladodes were obtained from O. ficus-indica cv. San-
turonic acid (Trachtenberg and Mayer 1981). The guigna plants grown in the region of Maccarese, Rome.
economic importance of mucilage lies in its gelling prop- The cladodes were collected early in the morning, in the
erties with a multifunctional use in food, cosmetics and summer–autumn months, from the beginning of June
pharmaceutical industry (Del-Valle et al. 2005; Allegra through October. The 2–3 years old cladodes were imme-
et al. 2017). Medina-Torres et al. (2000) suggested vis- diately used for the experimental trials; these were washed
cosity values of mucilage solution at 10% comparable with with tap water in order to remove impurities and spines,
a xanthan solution at 3%, with interesting implications for peeled from epidermis in both sides and then chopped into
the economic and technical importance of xanthan gum. 1–2 cm pieces.
Furthermore, many studies based on clinical trials docu-
mented its antihyperlipidemic, antiobesity and hypoc- Extraction conditions
holesterolemic effects (Frati-Munari et al. 1983). Different
authors discussed methods of extraction and purification of Biomass-to-water ratio (w/v), pH, ionic strength,
mucilage (Sáenz et al. 2004; Sepúlveda et al. 2007; Con- temperature
treras-Padilla et al. 2016) or pectin (Bayar et al. 2017a, b)
from Opuntia spp. However, in the last year, efforts to We defined five biomass-to-water ratios: 1:1, 1:3, 1:5, 1:7,
exploit extraction methods are carried out on industrial 1:9 (w/v); 200 g of fresh chopped cladodes were put in
scale, in order to evaluate possible productive chains uti- flasks with distilled water (200 mL in 1:1, 600 mL in 1:3,
lizing cladodes as by-products or as main commodity in 1000 mL in 1:5, 1400 mL in 1:7 and 1800 mL in 1:9), then
dedicated crops. The use of water as extraction buffer is a we left them soaking for 24 h at 25 °C, in dark condition
traditional method in Mexico, which is effective for without stirring. We filtered all the samples with a fine
homemade applications, ease to apply. In the aqueous cloth (0.5 mm) until there was no mucilage dropping
method, different factors such as temperature, mechanical down. The mucilage was maintained at - 5 °C until the
processes (blending), extraction period and liquid/solid analysis. Each experiment was replicated three times.
ratio can induce changes in the chemical and nutritional Since the cladodes as experimental plant material have a
qualities of Opuntia cladodes, affecting the yield and buffering capacity (Corrales-Garcı́a et al. 2004), we
quality of bio-product (Bayar et al. 2017a, b; De Santiago investigated a range of pH from 2 to 12 with and without
et al. 2018). The aim of the present research is to optimize buffer as a control. Fifty grams of fresh chopped cladodes
the efficiency of mucilage extraction by water, reducing its in 250 mL of distilled water at pH 2.0, 4.5, 7.0, 9.5, 12.0
cost and ensuring safe and high quality extract/product. with and without a buffer (biomass-to-water ratio was 1:5)
The analysis of the critical factors affecting the extraction were replicated three times each. We used either
process was done by the response surface methodology hydrochloric acid or sodium hydroxide to adjust the pH in
which implicates the application of multivariate techniques the samples without buffer and the McIlvaine’s
on factors and responses relating to the process. Therefore, tables (Sigma-Aldrich) to prepare the samples with buffer.
the specific objectives aimed to depict the most appropriate The pH value was determined before and after 24 h of
conditions for mucilage extraction from O. ficus-indica soaking.
cladodes are the following: (1) to investigate the effects of In order to evaluate the effect of ionic strength, 50 g of
dilution/ratio/time/biomass, pH and ionic strength on fresh chopped cladodes were added to 250 mL of distilled
mucilage yield from cladode parenchyma, expressed as water (biomass/water 1:5) supplemented with NaCl or
total sugar content; (2) to apply response surface method- CaCl2 at the concentration of 0.1, 1.0, 10.0 and 100.0 mM.
ology and Plackett–Burman design (Analytical Methods A total of eight samples were compared to the control
Committee 2013) to optimize the extraction procedure; (3) sample (50 g of plant material in 250 mL distilled water)
to compare the effects of ultrasonic and microwave assisted after 24 h of soaking. Each experiment was carried out in
extraction (UAE and MAE, respectively) with respect to triplicate. Mucilage was extracted from 50 g of fresh
conventional method, also evaluating protein and uronic chopped cladodes in 250 mL of distilled water for 24 h at

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J Food Sci Technol

20 or 80 °C. All solvents were purchased from Sigma– solution of known concentration of glucose or xylose ?
Aldrich (Milan, Italy). 250 lL 80% phenol ? 5 mL 8% H2SO4 were added. After
stirring for 1 min and waiting for 30 min to complete the
Ultrasonic and microwave assisted extraction of mucilage reaction, the absorbance value was read at 490 nm (Varian
Scary 50 Scan spectrophotometer). The results were
In order to increase the mucilage recovery, two sets of obtained by interpolation of a calibration curve built from a
three times replicated samples (50 g of fresh chopped series of points representing Abs vs known concentration
cladodes in 250 mL of distilled water, 1:5 w/v), have been of glucose or xylose. Total neutral sugar content was
subject to: (1) ultrasonic bathing (EMAG ZCC009, United expressed as microgram equivalent of xylose per milligram
States) at 40 kHz for 30 min; (2) microwaves (SMEG ME/ of extracted soluble polymer x 100. All solvents were
203FX, Italy) at 500 W for 5 min. The percentage muci- purchased from Sigma–Aldrich (Milan, Italy).
lage yield extracted from each samples was compared to
the extraction obtained by conventional method (50 g of Determination of proteins and uronic acids content
fresh chopped cladodes in 250 mL of distilled water at
room temperature in the dark). Data were recorded a t0 The protein content was assessed using the method of
(immediately after treatment); at t1 (after 16 h of macer- Lowry et al. (1951), which combines two redox reactions:
ation); at t2 (after 24 h of treatment). Cu2? ions with the peptide bonds under alkaline conditions
and the oxidation of aromatic protein residues (tryptophan
Chemical analysis and tyrosine) by Folin–Ciocalteau reagent. The Cu?2 ions
produced in the first reaction react with the reduced Folin
Determination of total polysaccharides content reagent producing an intense blue molecule known as
heteropolymolybdenum blue. The concentration of the
Forty mL of 96% ethanol was added to 10 mL of each reduced Folin reagent (heteropolymolybdenum blue) was
water extract. Each sample (3 replicates per sample) was detected at 750 nm absorbance. The total concentration of
vortexed and left to form a precipitate for about 20 min. protein in the sample can be deduced from the concentra-
All the samples were then centrifuged at 9000 rpm for tion of tryptophan and tyrosine residues that reduce the
5 min to let the mucilage precipitate on the bottom of the Folin–Ciocalteu reagent. Uronic acid contents were deter-
test tube. The supernatant was then eliminated and 5 mL of mined according to the method of Blumenkrantz and
sulfuric acid 2 M was added in order to start the hydrolysis Asboe-Hansen (1973), 1.2 mL of sodium tetraborate
process. The tubes were left in a hot bath at 80 °C for 2 h. 0.0125 M in 98% H2SO4 was added to 200 lL of sample at
Then 1.4 g of CaCO3 was added in order to neutralize the 4 °C. After cooling, the samples were shaken in a Vortex
excess of acid. The sample was centrifuged at 2.500 g for mixer and heated in a water bath at 100 °C for 5 min. After
15 min and the supernatant was separated and centrifuged cooling in a water–ice bath, 20 lL of the 0.15% m-hy-
again at the same conditions. Due to the reduced quantity droxydiphenyl reagent in 0.5% NaOH was added. The
of mucilage precipitate samples and the uncertainty of the tubes were shaken again and, within 5 min, absorbance
error in the weighted quantity, we decided to proceed with measurements made at 520 nm (Varian Scary 50 Scan
total polysaccharides determination considered in propor- spectrophotometer). A blank sample was prepared adding
tion to 100 g of fresh biomass as a benchmark for deter- 200 lL of distilled water ? 1.2 mL of sodium tetraborate
mination of the mucilage yield (% yield). Similarly, Lefsih 0.0125 M in 98% H2SO4 ? 0,5% NaOH. A calibration
et al. (2016) report the % yield of the mucilage extraction curve with this method was built using galacturonic acid as
according to the total polysaccharides content. standard at six known concentrations from 0.4 to 0.05 mg/
mL. The amount of uronic acids was expressed as % yield:
Determination of neutral sugar content grams of galacturonic acid equivalents/100 g of FW or DW
of cladode.
Total neutral sugar content was determined with the
method of DuBois et al. (1956), which is based on the Chemiometric statistical analysis
reaction among 98% sulphuric acid and the carbohydrate
contained in the liquid samples as described above. All The analysis of variance was used in order to compare the
carbohydrates are transformed in furfurals (pentoses) and means of mucilage yield (%) obtained at different condi-
hydroxymethylfurfurals (hexose) which, in turn, react with tions of dilution, time of process, pH or ionic strength of
phenol originating a golden yellow complex with a con- the buffer. Differences among the means were statistically
centration, directly proportional to the amount of sugars in significant depending on the random errors or the real
the sample. In a test tube 200 mL of sample or standard effect of the operating changes in conditions. In addition,

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the process optimization by the quality by design (QbD) are coefficients describing the importance of the direct
approach was applied, including the variation of two effect of extraction time and dilution; bt;H2 O=BM is the
selected factors at one time in agreement with a defined coefficient for the indirect effect and the synergy of time
design of experiment (DoE) and the multi-regression and dilution; finally bt2 and bH2 O=BM2 are the coefficients
analysis of the experimental data. In detail, a full-factorial describing the importance of the quadratic effect of the
experimental design with two factors and three levels was factors. The bar charts in Fig. 1 show the values of every b-
performed in order to study the dependence of mucilage coefficient obtained from the regression, highlighting both
yield on the experimental process conditions, such as the standard deviation and the level of significance. The
dilution and time. Nine experiments displayed geometri- results indicate that both bt and bH2 O=BM are statistically
cally as a face-edge centered square in agreement with the significant with positive values to indicate that both factors
model matrix and the correlated experimental matrix were have a direct positive effect on the extraction process.
carried out. A nonlinear multiple regression to investigate According to these data, significant differences were
the direct and indirect effects of each factor was applied to detected between the water/biomass ratios, with the % of
define the most suitable working conditions and develop mucilage yield at different dilutions largely dependent on
predictive mathematical models. the solid–liquid proportion. The efficiency of extraction
Other independent experimental variables, such as pH, process was very low at 1:1 ratio (0.95%) due to the rapid
temperature and ionic strength can affect the mucilage saturation of the solvent. By increasing progressively the
extraction. In order to define which factors have the most ratio solid–liquid from 1:1 to 1:9, the increment of % yield
significant effect, the experimental Plackett–Burmann was quadruplicate (3.8%). No plateau effect was detected,
design, according to the experimental conditions, was displaying additional potential mucilage to be extracted.
applied. Since this method with 4n experiments is suit- This achievements agree with Sepúlveda et al. (2007) who
able for studying 4n - 1 factors, the authors have per- reported the best mucilage yield was obtained with 1:7 pad/
formed a Plackett–Burmann design with eight experiments water ratio (at 40 °C), suggesting future research on greater
and seven factors, two of which are dummy factors without quantity of water as solvent. Therefore the diffusivity of
real meaning, only required for the mathematical the solvent into mucilaginous cells increases in these
elaboration. conditions, enhancing the desorption of soluble polysac-
charides from the tissue (Bayar et al. 2017a, b). The pre-
cision of the predicted response is very high, with the
Results and discussion experimental error decreasing towards the inner zone due
to the interpolation which allows the minimization of the
Full-factorial experimental design leverage. Our results indicate the proposed model fits well
the experimental data being appropriate for describing the
Full-factorial experimental design was applied to evaluate relationship between independent variables and the
the relationship between the predicted response and the
independent variables. The following polynomial function
(Eq. 1), was proposed to evaluate the mathematical
regression:

y ¼ b0 þ bt Xt þ bH2 O XH2 O þ bt;H2 O Xt XH2 O þ bt2 Xt2 þ bH2 O2 XH22 O

BM BM BM BM BM BM

ð1Þ
where y is the response related to the mucilage percentage
yield expressed as the grams of total sugars extracted from
100 grams of cladode; Xt and XH2 O=BM are dimensionless
and normalized surrogate variables, respectively, defined
on a coded scale from ? 1 to - 1; they represent the true
experimental factors from their lowest to highest level. Xt
is related to the extraction time expressed in hours and
XH2 O=BM refers to the dilution factor, expressed as the ratio
between milliliters of water and the grams of cladode used Fig. 1 Bar charts related to the values of the b-coefficients of the
in the experiment. Apart from b0, that is a constant with mathematical motel describing the direct and indirect effects of both
dilution and time on mucilage yield. In addition, it is showed the
only a mathematical meaning, the b-coefficients represent standard deviation and the statistical significance for every coeffi-
the magnitude of direct and indirect effects: bt and bH2 O=BM cient. The bars are not statistically significant

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response. Consequently, this model can be used to navigate coefficient bt2 is statistically significant, showing that the
the design space. In Fig. 2 the three-dimensional response dependence between extraction time and yield is linear
surface (A) and the corresponding contour plot (B) indicate only at the first step of the extraction process, later the
the trend of mucilage % yield as a function of both dilution aqueous solution becomes progressively saturated by
and extraction-time conditions. In the level curves of the mucilage solute and, consequently, the yield does not
contour plot, the effect of ratio water/biomass on mucilage increase any more.
extraction, results more understandable. As expected from
the full-factorial experimental design, the effect of dilution Effect of pH, ionic strength and temperature
is more effective than maceration time. The coefficient on extraction efficiency
bt;H2 O=BM is significant, showing an interaction between
dilution and time factors. Our results showed a higher Among the external factors able to affect the extraction
water/mass ratio promotes not only the efficiency of pro- process, pH, ionic strength and temperature can be crucial
cess by enhancing the dissolved mucilage threshold limit, in terms of mucilage removal and quality (Trachtenberg
but also the process rate by keeping high mucilage con- and Mayer 1981; Medina-Torres et al. 2000). As showed in
centration gradients close to the water-cladode interface. Fig. 3, the pH showed a positive effect on mucilage
For this reason, the time required for extraction decreases extraction with statistically significant values. The opti-
by increasing the water/mass ratio. Furthermore, the mum extraction condition was found at pH 12 with buffer
(5.2%) followed by the treatment at pH 2 without buffer
(4.3%). In the absence of buffer the percentage of mucilage
extracted showed a reversed bell trend, due to pH-buffering
capacity of Opuntia ficus-indica tissues rich in numerous
small organic molecules typical of CAM plants, such as
organic acids, which bear multiple acid–base groups able to
act as buffer components in the physiological pH range
(Corrales-Garcı́a et al. 2004). In this regard, data on the
effects of chemical factors such as pH or ionic strength on
mucilage recovery are required in order to optimize the
process extraction. High methoxyl pectin can form a gel
under acidic conditions in the presence of high sugar
concentrations at pH \ 3.5 (Ngouémazong et al. 2012).
From our results both acid and alkaline pH showed positive
influence on mucilage yield (%) probably contributing to

Fig. 3 Effect of pH without or with buffer on mucilage yield at

Fig. 2 Multiple regression analysis related to the full-factorial cladode–water ratio 1:5. The open squares and open circles represent
experimental design where it is showed the dependence of mucilage the effect of pH without buffer (F = 8.91; P = 3.1E-05) or with
yield % on both dilution H2O/BM (g/g) and time t(h): a 3-D plot with buffer (F = 12.74; P = 1.9E-06) respectively. The points represent
yield surface, b contour plot with yield line levels the mean of the yield % at the indicated pH, mean ± SD (n = 9)

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 J Food Sci Technol

break mucilage cells structure letting them to free a major cations, particularly Ca2? (Ngouémazong et al. 2012);
quantity of mucilage. Trachtenberg and Mayer (1981) therefore, the addition of high concentrations of Ca2?
demonstrated mucilage molecules shapes are clearly promotes (Cárdenas et al. 2008; Lira-Ortiz et al. 2014) the
affected by pH, with viscosity loss at acidic conditions. We formation of Ca-bridges with carboxyl groups (Han et al.
observed the same effect in the acid region, indeed the 2017) in the wall cells of parenchyma tissue sample,
viscosity of mucilage varied according to the dissociation decreasing the extraction and yield of mucilage. Different
of acidic groups in polysaccharides and the repulsion authors (Sepúlveda et al. 2007; Yoshida et al. 2010;
between negative-charged, being more evident at increas- Rodriguez-Felix and Cantwell 1988) showed an efficient
ing alkaline range, as reported by Medina-Torres et al. Opuntia ficus-indica mucilage extraction from raw clado-
(2000). In this condition the polysaccharide solubility in des directly dependent on temperature; we included these
water and viscosity was enhanced allowing a best yield parameter in our study, however our preliminary trials at
extraction (Felkai-Haddache et al. 2015). Figure 4 presents 80 °C demonstrated that this factor could negatively
the effect of ionic strength on mucilage yield (%) at cla- influence the viscosity of extract by contributing to the
dode water ratio 1:5 using 0, 0.1, 1,10, and 100 mM NaCl hydrolysis of polysaccharides (Glibowski and Wasko
or CaCl2. It is represented as box charts where box is 2008).
determined by the 25th and 75th percentiles and the
whiskers are determined by the 1st and 99th percentiles. Plackett–Burmann design
The mean values are represented with white squares; while
the medians are horizontal lines inside every box. The A multi-regression analysis following the Plackett–Bur-
results showed significant differences among the treatments mann design was performed by using a polynomial func-
confirming a positive effect of cations concentration on tion with the linear terms only, Eq. 2.
mucilage recovery as expected for a polyelectrolyte. The y ¼ b0 þ bt Xt þ bH2 O XH2 O þ bpH XpH þ bT XT þ bl Xl
presence of either NaCl or CaCL2 enhance the recovery of BM BM

mucilage from Opuntia ficus indica cladodes; the most þ bd1 Xd1 þ bd2 Xd2 ð2Þ
efficient extraction was observed at the concentrations of where y is the mucilage yield, while Xt, XH2 O=BM , XpH, XT,
1 mM NaCl or CaCl2, where the percentage yield values Xl are surrogate variables related to time, dilution, pH,
were ranging from 5.2% to 4.6%, respectively with an temperature and ionic strength, while Xd1 and Xd2 are
increment of ? 35% or ? 22% compared to the control dummy factors. The b-coefficients related to the real fac-
(0 mM). However, as ionic strength increases, mucilage tors are in the bar chart in Fig. 5, where both the standard
yield decreases. This behavior is more pronounced when deviation and the level of significance are showed. By
using divalent ions, which are able to influence the physical comparing the b-coefficient values, it is possible to claim
parameters of mucilage such as the viscosity. In fact, the that biomass/water, temperature and ionic strength are the
low-methoxyl pectin forms gel by interacting with divalent

Fig. 4 Effect of ionic strength on mucilage yield (%) at cladode

water ratio 1:5 using different concentrations of NaCl (F = 11.43; Fig. 5 Bar chart related to the values of the b-coefficients of the
P = 2.8E-6) or CaCl2 (F = 29.14; P = 2.2E-11). It is represented mathematical motel obtained by following the Plackett–Burmann
box charts where box is determined by the 25th and 75th percentiles method, that describes the direct effects of dilution, time, pH,
and the whiskers are determined by the 1st and 99th percentiles. The temperature and ionic strength on the extracted total sugar amounts. It
mean values are represented with the squares; while the medians are is showed the standard deviation and the statistical significance for
horizontal lines inside every box every coefficient. The bars are not statistically significant

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J Food Sci Technol

factors that have the most significant effect on mucilage showed UAE treatment to be more effective allowing the
yield. best of mucilage recovery in a shorter processing time,
MAE provided more proteins and uronic acids than con-
Assisted mucilage extraction ventional and UAE extractions. These data are in agree-
ment with Felkai-Haddache et al. (2015), who also reported
Furthermore Felkai-Haddache et al. (2015) and Bayar et al. the highest molecular weight in the polysaccharides
(2017a, b) reported an increment in mucilage yield from extracted from MAE. In our experiments, we observed a
Opuntia ficus-indica peeled cladodes using microwave or loss of viscosity of mucilage extracted with microwave, as
ultrasonic assisted extractions. Studing Arabidopsis as reported by Zheng et al. (2011), an excessive time-power
plant model for mucilage extraction from seeds, Zhao et al. exposure under the microwave fields leads to possible
(2017) showed the ultrasonic treatment as an effective degradation of polysaccharide molecules. Thus, additional
method to remove, in a very short time (20 s), the adherent studies on extraction pre-treatments should be undertaken
hydrophobic mucilage compacted in seed coat and resistant in order to depict the most appropriate conditions for the
to water extraction. Figure 6 presents the mean mucilage highest yield as well as for enhancing rheological mucilage
yield (%) after 0, 16 and 24 h (t0, t1, t2, respectively) from properties depending on its final use.
cladode–water ratio 1:5 comparing conventional, ultra-
sound or microwave assisted extraction methods. Signifi-
cant differences were observed among the treatments and
Conclusion
time of extraction. At t0 the microwave treatment resulted
to be more efficient than the ultrasonic one, showing an
Within a multi-disciplinary approach, the present work
increment in percentage yield of ?83% compared to the
elucidated and optimized the water technological process
control (2.2% and 1.2, respectively). However, after 16 h
to extract mucilage from cladodes. We propose a multi-
of extraction the mucilage yield (%), quadrupled in ultra-
variate model to predict mucilage recovery at different
sound-treated material (7.5%), remained at lower values in
levels of biomass/water ratio/extraction time, and we dis-
the other cases (about 5% after 24 h of extraction).
cussed a statistical methodology to identify the critical
Regarding the quality of extraction, we observed a loss of
factors affecting the extraction procedure. Further studies
viscosity in extract from microwave instead of ultrasonic
on green assisted extraction tools and their effects in terms
assisted extraction process. According to our results, MAE
of quality of extracts are required in order to obtain high
provided more proteins (0.19 ± 0.01 mg/mL) and uronic
added value co-products. In particular research activities
acids (0.19 ± 0.05 mg/mL) contents compared to UAE
concerning the nutritional, functional and health benefits
(0.01 ± 0.01 mg/mL; and 0.01 ± 0.01 mg/mL) or con-
are recommended to make this by-product a potential
ventional methods (0.006 ± 0.01 mg/mL and
thickening and stabilizing agent for food industry. Finally
0.06 ± 0.01 mg/mL). Although our preliminary results
mucilage content could differ as a result of cultivar/geno-
types, stage of development, geographical growth area;
these data should be thorough as a part of a green strategy
for a large scale utilization of Opuntia ficus indica agro-
products that yields a range of target products from a given
biomass.

Acknowledgements With the contribution of the Ministry of Foreign

Affairs and International Cooperation, Directorate-General for the
promotion of the Country System—Scientific and Technological
Bilateral Cooperation Project Italy—Mexico. This scientific work is
dedicated to our colleague and friend Tiziana Coccioletti, who died
prematurely. Her enthusiasm and technical skills have enhanced and
enriched the team work.

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Fig. 6 Mucilage yield (%) after 0, 16 and 24 h from cladode–water (2017) The effectiveness of Opuntia ficus-indica mucilage edible
ratio 1:5 comparing conventional extraction and ultrasound or coating on post-harvest maintenance of Dottato fig (Ficus carica
microwave pretreatment at t0. The figure displays mean values and L.) fruits. Food Packag Shelf Life 12:135–141. https://doi.org/
standard errors from three replications (F = 132,288.8; P = 4.06E19) 10.1016/j.fpsl.2017.04.010

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