Accepted Manuscript

Optimization of high pressure processing parameters to preserve

quality attributes of a mixed fruit and vegetable smoothie

M.V. Fernandez, G.I. Denoya, M.V. Agüero, R.J. Jagus, S.R.

Vaudagna

PII: S1466-8564(17)30583-0
DOI: doi:10.1016/j.ifset.2018.02.011
Reference: INNFOO 1931
To appear in: Innovative Food Science and Emerging Technologies
Received date: 24 May 2017
Revised date: 6 February 2018
Accepted date: 11 February 2018

Please cite this article as: M.V. Fernandez, G.I. Denoya, M.V. Agüero, R.J. Jagus, S.R.
Vaudagna , Optimization of high pressure processing parameters to preserve quality
attributes of a mixed fruit and vegetable smoothie. The address for the corresponding
author was captured as affiliation for all authors. Please check if appropriate.
Innfoo(2017), doi:10.1016/j.ifset.2018.02.011

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
 ACCEPTED MANUSCRIPT

Optimization of high pressure processing parameters to preserve quality

attributes of a mixed fruit and vegetable smoothie

1,4* 2,3 1,3,5 1,5 2,3,5

Fernandez, M.V. ; Denoya, G.I. ; Agüero, M.V. ; Jagus, R.J. ; Vaudagna, S.R.

T
1
Universidad de Buenos Aires (UBA), Consejo Nacional de Investigaciones Científica y

IP
Técnicas (CONICET), Instituto de Tecnologías y Ciencias de la Ingeniería (INTECIN),

CR
Facultad de Ingeniería. Departamento de Ingeniería Química. Laboratorio de

Microbiología Industrial: Tecnología de alimentos, Av. Int. Guiraldes 2620, (C1428EGA),

US
Ciudad Autónoma de Buenos Aires, Argentina.
2
Instituto Tecnología de Alimentos, Centro de Investigación de Agroindustria, Instituto
AN
Nacional de Tecnología Agropecuaria (INTA), CC 77 (B1708WAB) Morón, Buenos Aires,

Argentina
M

3
Consejo Nacional de Investigaciones Científicas y Técnicas, Av. Rivadavia 1917,
ED

(C1033AAJ) Ciudad Autónoma de Buenos Aires, Argentina

4
Peruilh Foundation, Facultad de Ingeniería, Universidad de Buenos Aires.
PT

5
These authors contributed equally to the manuscript
CE

*Corresponding Author:
AC

María Verónica Fernández

Departamento de Ingeniería Química, Facultad de Ingeniería, Universidad de Buenos

Aires, Ciudad Universitaria, Intendente Güiraldes 2620, (C1428EGA), Buenos Aires,

Argentina

(+54) 11-4576-3240/41

[email protected]

1
 ACCEPTED MANUSCRIPT

ABSTRACT

Fruit & Vegetable (F&V) smoothies are rich in nutrients and other health related

compounds. However, they have a short shelf-life and the traditional methods applied to

preserve them generate losses in their natural flavor and nutrients. The aim of this study

was to optimize the pressure level (350-650MPa) and holding time (1-9min) of High

T
IP
Pressure Processing (HPP), performed at an initial temperature of 20°C and only modified

by adiabatic heating of a F&V smoothie in order to achieve microbial and enzymatic

CR
inactivation while maintaining its natural attributes. Response surface methodology with a

US
Doehlert design and Desirability function were employed to simultaneously optimize these

quality attributes. Results showed that HPP enhances microbial quality and does not affect
AN
pH, total soluble solids, texture and total phenolic content. Moreover, the optimal HPP

treatment (627.5MPa/6.4min) leads to reductions of 85%, 45% and 10% on PME, POD
M

and PPO, increases antioxidant capacity by 75% and maintains or slightly improves the
ED

color of the smoothie.

PT

Industrial relevance text:

F&V smoothies are tasty, healthy, convenient and ready to drink, fulfilling all the
CE

current demands of consumers. This has led to an accelerated increase in their popularity.
AC

However, they have a short shelf life mainly attributed to microbial and enzymatic

spoilages. HPP is proposed as a non-thermal method able to prolong shelf-life of the

products by means of microbial and enzymatic inactivation, while preserving bioactive

compounds and quality characteristics. An optimization assay was carried out in order to

find optimal process conditions for the F&V smoothie´s preservation. The promising results

obtained can help to promote the use of HPP as an alternative technology for the

preservation of this kind of products.

2
 ACCEPTED MANUSCRIPT

1. Introduction

In recent years consumers have become aware of the impact that their diet has on

their health. This is why the demand for healthy, nutritious, free of additives products has

noticeably increased. Additionally, the current lifestyle has led consumers to look for more

convenient, ready-to-eat products (Hendrickx & Knorr, 2002). In this sense, consumption

T
IP
of smoothies is an excellent way to increase the intake of nutrients and bioactive

CR
compounds, present in fruits and vegetables (F&V). Their sensory properties, mainly

appearance and taste, and the fact that they are ready-to-drink are all decisive factors for

US
the consumption success of smoothies (Andrés, Villanueva, & Tenorio, 2016). Moreover,

blending is a good way to incorporate non-traditional and underutilized, yet highly nutritive,
AN
vegetables (Jayachandran, Chakraborty, & Rao, 2015) such as beet leaves and stems

(Fernandez, Jagus, & Agüero, 2014) into value-added products.

M

However, untreated F&V beverages have a short shelf-life that generally can be
ED

attributed to both microbial and enzymatic spoilage. Although they are usually highly acidic

products (pH lower than 4) and this condition inhibits the growth of most of bacterial
PT

spores, some acid tolerant microorganisms such as yeast, lactic acid bacteria
CE

(Lactobacillus, Leuconostoc, Pediococcus and Streptococcus), Alicyclobacillus

acideoterrestris, Listeria monocytogenes, some species of Salmonella and some strains of

AC

E. coli, among others, could survive and grow (Gram et al. 2002; Jayachandran et al.,

2015). Moreover, the natural presence of enzymes such as peroxidase (POD), polyphenol

oxidase (PPO) and pectinmethylesterase (PME), among others, causes loss of phenolic

compounds, induces browning and cloud loss in beverages, resulting in a reduced

nutritional and sensory quality (Chakraborty, Kaushik, Rao, & Mishra, 2014).

Traditionally, fluid foods have been preserved by thermal treatments such as

pasteurization and sterilization. These processes are capable of ensure safety and

3
 ACCEPTED MANUSCRIPT

preventing spoilage; however, they can also result in a loss of heat-labile nutrients such as

certain vitamins or bioactive phytochemicals (Rickman, Bruhn, & Barrett, 2007), among

other compounds responsible for nutritional and organoleptic attributes, during the

preservation/processing treatment (Barba, Esteve, & Frigola, 2012). Retaining the

nutritional value and fresh-like quality of F&V beverages is a major challenge for the food

industry. Therefore, research in non-thermal preservation processes is rapidly increasing

T
IP
(Duong & Balaban, 2014). In this sense, High Pressure Processing (HPP) is an alternative

CR
technology that involves applying very high hydrostatic pressures (100-1000 MPa) at

refrigeration or room temperature for a short time (a few seconds to some minutes) on

US
packed food in order to eliminate vegetative microorganisms (pathogens and spoilage

microbiota), and to inactivate enzymes, with minimal modifications in nutritional and

AN
sensory quality (Oey et al., 2008). Certainly, one of the main advantages of this technology

is its ability to maintain different compounds, such as pigments, volatiles, vitamins and
M

other health-promoting compounds, rather unaffected (Denoya et al., 2016; Varela-Santos

ED

et al., 2012; Patras, Brunton, Da Pieve, & Butler, 2009b). This has been attributed to the

stability of covalent bonds under high pressure (Knorr, 1993). Regarding microbial
PT

inactivation, Lado & Yousef (2002) reported that HPP alters cell structure and
CE

physiological functions by breaking DNA strands, disrupting cell membrane integrity,

inactivating key enzymes, irreversibly denaturing proteins and disabling membrane

AC

selectivity. Enzyme inactivation is also associated with conformational changes induced in

the protein structure (Ludikhuyze, Van Loey, Indrawati, Smout, & Hendrickx, 2003;

Rastogi, Raghavarao, Balasubramaniam, Niranjan, & Knorr, 2007). However, depending

on the intensity of the treatment applied, HPP can either enhance or inhibit the enzymatic

activity (Oey et al., 2008). This also depends on the enzyme type, its origin, its

microenvironmental condition and process conditions (Duong & Balaban, 2014).

4
 ACCEPTED MANUSCRIPT

Therefore, the present study aims to investigate the effect of HPP on quality

attributes of a mixed F&V smoothie and to optimize the main processing parameters

(pressure level and holding time) in order to achieve microbial and enzymatic inactivation

while maintaining nutritional quality attributes, texture and color of the product.

2. Materials and methods

T
IP
2.1. Smoothie preparation

CR
Smoothie formulation was selected based on previous studies of our group

(Denoya et al., 2017) in which a sensory acceptability test was conducted where

US
properties like color, appearance, taste, phase separation, among others, were considered

and characteristics such as the intense red color, fresh fruits taste and cloud stability, were
AN
positively valued. The composition by weight of the selected smoothie was: orange juice

(59%), apples (15%), carrots (15%), beet leaves (6%) and beet stems (5%). Oranges (cv.
M

Salustiana, Argentina) were harvested from an experimental orchard in Concordia, Entre

ED

Ríos, Argentina, their juice was extracted using a home squeezer (Oster, USA). Apples

(cv. Granny Smith, Argentina), carrots (cv. Flakee, Argentina) and beets (cv. Detroyt,
PT

Argentina) were obtained from a local retailer. Before processing, all fruits and vegetables
CE

were washed and disinfected by dipping in chlorinated water for 5 min and dried. The

carrots and apples were then manually peeled and chopped into small pieces. Finally, all
AC

the ingredients were blended in a homogenizer (JTC OmniBlend, Guangdong, China) for

60 s. The smoothie was packed into polyethylene terephthalate (PET) bottles (100 mL).

Bottles containing fresh control smoothies were immediately stored at 4 ± 1 °C, while the

other samples were kept under refrigeration (4 ± 1 °C) until HHP treatments were applied.

Four bottles were prepared for each treatment.

5
 ACCEPTED MANUSCRIPT

Since the developed product aims to meet the needs of consumers who are aware

of the importance of food on health and consequently look for nutritious and fresh tasting

products, thermal treatment was not considered as an option in this study.

2.2. High-pressure processing

Samples were subjected to different pressure levels and holding times, selected

T
IP
according to the experimental design. HPP was performed in a high hydrostatic pressure

CR
equipment with a 2-L capacity (Stansted Fluid Power Ltd. High Pressure Iso-Lab System

Model: FPG9400:922, UK) and a maximum working pressure of 900 MPa. A mix of

US
distilled water and propylene glycol (70/30) was used as the compression fluid. The

proportion of water and propylene glycol was selected considering the working
AN
temperature applied, in order to avoid changes in the physical state of the water. Pressure

was increased at 5 MPa s−1. Conditioning temperature of vessel and initial temperature of
M

compression fluid were between 21°C and 24°C. After processing, samples were kept at 4
ED

± 1 °C until further analysis.

PT

2.3. Experimental design

CE

Response surface methodology (RSM) was used to estimate the main effects and

interactions of HPP parameters on quality attributes of the smoothie. A total of 9

AC

experiments were conducted according to a Doehlert uniform shell design with two factors:

pressure level (P) and holding time at working pressure (t). This method has been

successfully applied to optimize different process parameters (Denoya et al., 2016; Bup,

Abi, Tenin, Kapseu, & Tchiegang, 2012; Mayor, Moreira, & Sereno, 2011). In this

research, pressure was studied at five levels (350, 425, 500, 575, and 650 MPa) and

holding time at three levels (1, 5, and 9 min). Furthermore, the central point of the design

was triplicated in order to validate the model by means of an estimate of experimental

6
 ACCEPTED MANUSCRIPT

variance. Table 1 shows the coded and actual values of the factors of the experimental

shell design and their levels.

For each response variable the linear, quadratic, and simple interaction effects of

the factors were compared with each other. Each response variable (Y) was analyzed as a

function of the two independent factors (P, t) and the significance of the equation

coefficients for each response variable was obtained by multiple regression analysis using

T
IP
the F test with a p<0.05:

Y= b0+ b1 P + b2 t + b11 P2+ b22 t2 + b12 Pt

CR
where b0 is the regression coefficient for the mean effect; b1 and b2 for the linear effect of

US
pressure level and holding time, respectively; b11 and b22 for the quadratic effect of

pressure level and holding time, respectively, and b12 for the interaction effect of these
AN
variables. Four bottles of each treatment were analyzed to determine quality parameters.

It is important to highlight that the initial effects of HPP on the product is

M

determinant for its quality and after treatment, during the storage time, the losses will be
ED

due to coexisting chemical reactions, such as oxidation, and biochemical reactions when

endogenous enzymes or microorganisms are incompletely inactivated (Oey et al., 2008).

PT

That is why numerous researchers optimized the process with the same criterion (Denoya
CE

et al., 2016; Duong & Balaban, 2014; Kaushik et al., 2016; Swami Hulle, Chakraborty, &

Rao, 2017).
AC

2.4. Sample analysis

2.4.1. Microbiological analyses

Smoothie samples (10 g) were taken aseptically from the bottles and homogenized

with 90 mL of sterile 0.1% peptone water (Biokar Diagnostics, France) in a stomacher

(Interscience Laboratories Inc. BagMixer ® 400P, France) for 60 s. Decimal dilutions were

prepared with sterile 0.1% peptone water and plated in the appropriate media for microbial

7
 ACCEPTED MANUSCRIPT

counts. The mesophilic aerobic bacteria (MAB) count was determined in plate count

agar (PCA, Biokar Diagnostics, France) incubated at 37 °C during 24-48 h;

Enterobacteriaceae (EB) were determined in Mac Conkey agar (Biokar Dignostics,

France) incubated at 37 °C during 24-48 h and molds and yeast (M&Y) counts were

determined in yeast extract glucose chloramphenicol agar (YGC, Biokar Diagnostics,

France) incubated at 28 °C during 48-72 h. The results were expressed as the logarithm of

T
IP
colony forming units per gram of smoothie (log CFU g-1). The detection limit of the

methods was 2.00 log CFU g-1.

CR
US
2.4.2. pH and total soluble solids (TSS)

The pH was measured in smoothies at room temperature (20 ± 1 °C) using a digital
AN
pH meter (Hanna, HI99163, Rumania) with a pH electrode (FC232D, Italy). The TSS was

determined as °Brix at room temperature (20 ± 1 °C) using a Milwaukee MA871

M

Refractometer (Milwaukee Instrument, Rocky Mount, USA). Three measurements were

ED

performed for each sample and the results were averaged.

PT

2.4.3. Chromatic parameters

CE

Chromatic parameters of smoothies were determined with a Minolta CR-400

chromameter provided with a sample holder CR A505 and a Glass Cell 20 mm CM-A99 for
AC

measuring liquids (Konica Minolta, Japan), using the CIE scale L*a*b*. These values were

then used to calculate Hue degree (h0 =arctangent [b*/a*]) and Chroma [Ch=(a*2 +b*2)1

/2], which is the color intensity or saturation. The instrument was set up for illuminant D65

and 2° observer angle. Three measurements were performed for each sample i.e. glass

cell was filled three times, and the results were averaged.

2.4.4. Back Extrusion analysis

8
 ACCEPTED MANUSCRIPT

Back-extrusion (pseudo-compression) test was performed using a Texture

Analyzer model XT plus (Stable Micro Systems LTD, Surrey, England) equipped with a 5

kg load cell and a 50-mm diameter back extrusion cell (A/BE Rig). The samples were

tested immediately after removal from storage (4 ± 1°C), using an extrusion disc (Ø=45

mm) operating at a fixed test speed of 0.5 mm s-1 to a depth of 30 mm. The force-time

curves were analyzed using Exponent Software version 6.1.10.0 (Stable Micro Systems

T
IP
Ltd., U.K.) and the textural parameters derived were: maximum force in compression

CR
(firmness (g)) and positive area of the curve (consistency (g s)), which indicates the

thickness of the sample.

US
2.4.5. Antioxidant capacity and total phenolic content
AN
The extraction phenolic compounds and antioxidants was conducted following the
M

methodology proposed by Viacava, Roura, & Agüero (2015) with some modifications.

Briefly, 5 g of smoothie was mixed with 20 mL of extracting solvent (ethanol acidified with
ED

2% citric acid) in a 150 mL Erlenmeyer flask. Extraction was carried out during 1 h, under

agitation at 28 ºC. Once extraction finished, homogenates were centrifuged (11,000 rpm)
PT

for 15 min at 4 ºC. The supernatant was the source of phenolic compounds and
CE

antioxidants. Antioxidant capacity was determined using the DPPH and FRAP assays,

according to Viacava et al. (2015) and Chen et al. (2015), calculated by using 6-hydroxy-2,
AC

5,7,8-tetramethylchroman-2-carboxilic acid (trolox, Sigma- Aldrich, St. Luis, USA) as

standard and expressed as μmol of trolox equivalents antioxidant capacity (TEAC) per 100

g of smoothie. Total phenolic content was determined by Folin-Ciocalteau methodology

and calculated by using gallic acid (Merk, Germany) as standard and expressed as mg of

Gallic acid equivalent per 100 g of smoothie (GAE100 g-1). These determinations were

9
 ACCEPTED MANUSCRIPT

carried out by duplicated for each sample. The detail of each technique can be found in

complementary material section (S.2.4.5.a, S.2.4.5.b, S.2.4.5.c, respectively).

2.4.7. Betaxanthins and betacyanins

Betaxanthins (Bx) and betacyanins (Bc) were determined by an adaptation of the

T
methodology proposed by Moßhammer, Stintzing, & Carle (2006). Briefly, 5 g of smoothie

IP
was weighed and diluted in 10 mL of McIlvaine buffer (pH = 6.3). Absorbances at 600, 536

CR
and 476 nm were determined and the contents of Bx and Bc in the extracts were

calculated as:

US
𝐴 ∙ 𝐷𝐹 ∙ 𝑀𝑊 ∙ 1000
𝐵𝑥 𝑜𝑟 𝐵𝑐 (𝑚𝑔⁄𝐿) =
𝑒∙𝑙
AN
where A is the absorbance at 536 or 476 nm for Bc or Bx corrected by reading at 600 nm

(baseline), respectively; DF is the dilution factor; l is the path length (1 cm) of the cuvette;
M

MW is the molecular weight of Bc (550 g mol-1) or Bx (308 g mol-1); and e is the molar
ED

extinction coefficient (60,000 or 48,000 L mol-1 cm in H2O for Bc and Bx, respectively).
PT

2.4.8. Enzyme activities analyses

The enzyme activity was determined as described by Vicente, Costa, Martínez,

CE

Chaves, & Civello (2005) for pectinmethylesterase (PME) and as described by Augustin,
AC

Ghazali, & Hashim (1985) for poliphenoloxidase (PPO) and peroxidase (POD) with

some modifications. One unit of enzyme activity was defined as the change of 0.001 of

absorbance at the corresponding wavelength. The detail of each technique can be found

in complementary material section (S.2.4.8.a and S.2.4.8.b).

2.5. Simultaneous optimization and model validation

10
 ACCEPTED MANUSCRIPT

A simultaneous optimization was carried out using the Desirability function. This

function is commonly used for multi-response simultaneous optimization and was applied

in similar studies (Denoya et al., 2016; Duong & Balaban, 2014; Kaushik, Rao, & Mishra,

2016). For this purpose, predicted values obtained from each model (Yn) were

transformed to a dimensionless desirability scale (dn). The desirability scale ranges from 0

to 1, where d=0 for an unacceptable response value, and d=1 for a completely desirable

T
IP
one. The individual desirability functions from the considered responses are then

CR
combined to obtain the overall desirability D, defined as the geometric average of the

individual desirability. An algorithm is then applied to this function in order to determine the

US
set of values of design factors that maximize it (Bezerra, Santelli, Oliveira, Villar, &

Escaleira, 2008).
AN
In order to test the reliability of the simultaneous optimization, a new set of

experiments using optimal values of design factors obtained with the Desirability function
M

were performed. The relative deviation between predicted and experimental value of the
ED

response variables (in both cases related to control values) were compared in order to

determine the validity of the model.

PT

2.6. Statistical Analysis

CE

All the procedures were carried out using the statistical software STATISTICA (trial
AC

version 12, Stat Soft, OK, USA). The Lack of Fit test was performed for each model with a

95% confidence level. The significant factors affecting each response variable were

selected according to the Student t-test establishing a 95% confidence level (Kuehl, 2000).

Moreover, in order to analyze the presence of significant differences between treatments

for the responses that do not fit the quadratic model, a one-way ANOVA was performed,

also with a 95% confidence level.

11
 ACCEPTED MANUSCRIPT

3. Results and discussion

Table 2 shows experimental mean values of quality attributes of untreated mixed

fruit & vegetable (F&V) smoothie (control sample). Among all the evaluated responses,

some were not affected by treatment (pH, TSS, total phenolic, betaxanthin content and

some chromatic parameters), others were affected but do not fit the quadratic model

T
IP
(microbiologic and textural parameters) while others were affected by treatment and fit the

CR
model (betacyanins content, antioxidant capacity, PME, POD and PPO activities and

Chroma value). The mean values for the experimental responses that fit the model can be

US
found in supplementary material section (Table S1), while Table 3 shows the regression

coefficients of adjusted models for each response variable. Coefficients of determination

AN
(R2) and lack of fit for each equation are also presented. R2 in all cases was higher than

0.8, indicating that the equations obtained for each response variable explained the
M

variation adequately. Following, HPP effects on the different parameters evaluated are
ED

presented and analyzed.

PT

3.1. Microbial quality

CE

Counts observed on untreated samples (Table 2) were in the range of those

usually found in F&V beverages (Andrés et al., 2016; Barba, Esteve, & Frigola, 2013;
AC

Chen et al., 2013). As expected, the treatments were effective and samples showed

reductions in all microbial counts. For MAB, reductions between 1-2 log cycles were

observed, without significant differences among HPP treatments. Only the strongest

treatments (650 MPa/5min and 575 MPa/9 min) allowed to achieve reductions of 2 log

cycles or more. Both EB and M&Y were more sensitive to HPP than MAB, presenting

counts below the detection limit (DL < 2.00 log CFU g-1) in all treated samples.

12
 ACCEPTED MANUSCRIPT

According to Mújica-Paz, Valdez-Fragoso, Samson, Welti-Chanes, & Torres (2011)

differences in the sensitivities of microorganisms to HPP processing arise from their

dissimilar cell wall morphology, as well as from the environment in which they are found.

Similar results to those presented in this research were obtained by Chen et al. (2015) who

worked with papaya beverage with initial counts of 5.54 and 3.73 log CFU g -1 of MAB and

M&Y. They observed that HPP treatments at 450 MPa or higher reduced M&Y under DL,

T
IP
whereas for MAB, pressure level at least 550 MPa were needed to obtain counts under

CR
DL, regardless of treatment time. Moreover, Chen et al. (2013) working with pomegranate

juice treated at 400 MPa, found that applying a holding time of 2.5 min, M&Y were

US
eliminated, although 20 min were needed in order to eliminate MAB counts. It is important

to mention that the different pressure levels and holding times observed for microorganism
AN
inactivation in different studies may be due to the different food matrices (TSS, pH, sugar

concentration), type of microorganisms (species and strains) and pressurization and

M

depressurization rates (Chen et al., 2015; Koseki & Yamamoto, 2014).

ED

3.2. Physicochemical characteristics

PT

In the treated samples pH varied between 3.76 to 3.81 and total soluble solids
CE

(TSS) between 9.03 to 9.80 ºBrix. In both cases the observed differences found to be not

statistically significant (p> 0.05). Moreover, these values were not different than those of
AC

the control (Table 2). These results are in agreement with numerous studies on the effect

of HPP on different fruits and/or vegetable based beverages (Barba et al., 2013; Chen et

al., 2013; Chen et al., 2015; Swami Hulle et al., 2017; Jayachandran et al., 2015; Varela-

Santos et al., 2012) that show that pH and TSS are generally unaffected by the treatment.

3.3. Chromatic parameters

13
 ACCEPTED MANUSCRIPT

The untreated smoothies presented a reddish color with chromatic values as

showed in Table 2. The treated smoothies presented the same reddish color. The

variations induced by HPP treatment in the chromatic parameters were proven to be

statistically insignificant (p>0.05), except for Ch which had a significant (p< 0.05) negative

correlation coefficient with the quadratic term of the holding time (Table 3). According to

the developed second order equation, in low to medium holding times an increase in the

T
IP
value of Ch was observed until reaching a critical point from which the values begin to

CR
descend. A high Chroma is associated with high intensity or saturation of color. In this

sense, in this study domain, treatments of intermediate pressures and times seem to be

US
the most suitable.

HPP was applied largely to preserve fresh color in many F&V products (Oey et al.,
AN
2008; Andrés et al., 2016; Patras, Brunton, Da Pieve, Butler, & Downey, 2009a).

Nevertheless, like in this study, many authors have informed changes in chromatic
M

parameters. Particularly related with Ch changes, Barba, Esteve, & Frigola (2010) found
ED

that HPP treated (100-400 MPa / 2-9 min) vegetable beverage had higher color saturation

(Chroma) than the unprocessed beverage. Similar results were found by Patras et al.
PT

(2009a) in tomato purees. Moreover, González-Cebrino, Durán, Delgado-Adámez,

CE

Contador, & Ramírez ( 2013), working with plum purée found similar or higher values of

the parameters Ch or h° in HPP treated samples (400-600MPa / 1-300 s), indicating that
AC

HPP maintained or even improved the color of the plum puree. In the case of this smoothie

the higher Chroma value, may be related to a greater extractability of the red pigments

(betacyanins). According to Oey et al. (2008) the cell disruption caused during HPP can

result in the leakage of pigments into the intercellular space, which could yield a more

intense bright color.

3.4. Back Extrusion analysis

14
 ACCEPTED MANUSCRIPT

All treated samples showed significant reductions (p <0.05) in the values of

consistency and firmness in relation to the untreated samples (Table 2). In the case of

firmness, reductions between 23 and 36% were observed, without significant differences

among HPP treatments. A similar result was observed for consistency with reductions

between 17 and 37%.

Similar results were found by Ahmed, Ramaswamy, & Hiremath (2005) who

T
IP
observed significant reductions in the viscosity of mango pulp after HPP (300-400 MPa/15-

CR
30 min). Furthermore, Verlent, Hendrickx, Rovere, Moldenaers, & Loey (2006) who worked

with high pressure treated tomato pulp (0.1-500MPa/15 min/ 30-70ºC) observed drastic

US
losses in consistency of the pulp pressurized at 300-400 MPa, regardless of the

temperature applied. They associated these losses with PME and polygalacturonase (PG)
AN
activities which were higher in this range. Indeed, due to cell disruption, HPP facilitates the

occurrence of enzymatic and non-enzymatic reactions that cause transformations in the

M

cell wall polymers (Oey et al., 2008). Particularly on F&V puree, smoothie or juice texture,
ED

activities of PME and PG are highly relevant, since they act synergistically leading to a

decrease in textural integrity (Chakraborty et al., 2014). It is important to mention that PG

PT

is considerably more baro-sensitive than PME (Chakraborty et al., 2014), hence, achieving
CE

PME inactivation will probably prevent texture losses from being greater during the storage

of the product.
AC

3.5. Total phenols content

The total phenols content (TPC) of the high pressure treated samples ranged

between 62.07 ± 0.91 and 68.33 ± 1.35 mg GAE 100 mL-1. Thus, TPC was preserved or

increased around 10% after treatment, however this increase was proven to be not

statistically significant (p > 0.05) in relation to untreated samples (Table 2).

15
 ACCEPTED MANUSCRIPT

This result is in agreement with other studies reporting similar behavior of total

polyphenol content after high-pressure treatment. For instance, Andrés et al. (2016)

obtained increases of 6.6% and 4.2% in the TPC of mixed F&V smoothies high pressure-

treated at 450 MPa/3 min and 600 MPa/3 min, respectively. Varela-Santos et al. (2012)

also reported TPC increments between 3.3% and 11.9% in pomegranate juices treated at

350 MPa/30-150 s and 550 MPa/30-150 s, respectively.

T
IP
According to Chen et al. (2013), TPC increase could be related with the fact that

CR
during the compression stage, the volume of system tends to be reduced, the extracting

solvent comes into cells to interact with bioactive components and the pressurized cells

US
show increased permeability, hence, an increased extractability of some components.
AN
3.6. Betaxanthins (Bx) and betacyanins (Bc)

The betalains family represents the principal pigment in red beet, present in their
M

root, stems and in the leaves veins. In particular, two classes of betalains are well-known:
ED

the red violet betacyanins, and the yellow orange betaxanthins (Ninfali & Angelino, 2013).

For betaxanthin, all high pressure-treated samples presented higher values (2-
PT

15%) than control (Table 2). Nevertheless these increases induced by the treatment, were
CE

proven to be statistically non-significant (p>0.05).

AC

On the other hand, betacyanin content of the treated samples were affected by

treatments and ranged between 92.1 and 107.1% in relation to control (Table 2).

Differences between the behavior of betalains could be associated to the molecular

structure of these compounds which differ by the residues attached to the main structure:

betacyanins exhibit a closed structure of cyclo-DOPA (cyclo-3,4-dihydroxy-phenylalanina)

and can be substituted with sugars and acyl groups, whereas betaxanthines are

conjugated with amines and amino acids. In line with these results Celli & Brooks (2016),

16
 ACCEPTED MANUSCRIPT

who reviewed the effect of different processing conditions and technologies on stability of

betalains, also associated differences between betacyanins and betaxanthines behavior to

the molecular structure of pigments.

Betacyanin content in high pressure-treated samples had significant (p<0.05)

positive correlation coefficients with the quadratic terms of pressure and holding time

T
(Table 3). As in all cases when the quadratic term is significant, there is a critical value (in

IP
this case for both, pressure and holding time) that must be considered. The highest

CR
contents of betacyanins were found in the most intense treatments: 575MPa/ 9min,

425MPa/ 9min and 650MPa/ 5min (Table S1).

US
There are few previous studies in which the effect of HPP on these particular
AN
pigments was evaluated. Interestingly, Paciulli, Medina-Meza, Chiavaro, & Barbosa-

Cánovas (2016) working with beetroot HPP-treated (650MPa/ 3-30min) found significant
M

increases in betanin (betacyanin) content of treated samples, with a time-dependent

ED

behavior. They observed that up to 7 min, more than 6-fold increase in betanin contents

was achieved, because of a better extraction from the broken cells, whereas holding times
PT

of 15 min onwards decreased the yields. According to the authors, due to the high

sensitivity of betanin to oxidation, the baro-induced increase of oxygen partial pressure in

CE

HPP treated samples could be the reason for the observed reduction at more extended

holding times. Even though during smoothies’ homogenization significant amounts of

AC

oxygen are introduced into the system this effect was not observed in our work, probably

because we worked with relatively short holding times (1-9 min).

3.7. Antioxidant capacity

After HPP, antiradical antioxidant capacity (DPPH) of the treated samples ranged

between 96.3 and 114.3% in relation to the untreated smoothie (Table 2). Table 3 shows
17
 ACCEPTED MANUSCRIPT

the regression coefficients of antioxidant capacity as estimated by the application of

multiple linear regression analysis. DPPH had a significant (p<0.05) positive correlation

coefficient with the quadratic term of holding time. The surface plot of DPPH

corresponding to pressure and holding time (Figure 1a) has a concave shape and provides

evidence that holding time had a stronger impact on DPPH values than pressure, meaning

that the best results could be obtained working with lower pressures and longer times.

T
IP
Indeed, with longer holding times (9 min) the highest DPPH values were achieved.

CR
Ferric-reducing capacity (FRAP) was increased after HPP with values between

16.4 and 82.9% higher than the control (Table 2). The effects of the pressure level and the

US
holding time were not strictly linear since the equation contains both negative lineal

coefficients (first order term) and positive quadratic coefficients for both factors. Therefore,
AN
an increase in P and t determines a reduction in FRAP capacity, however, the negative

quadratic terms indicate that there are critical values for these parameters from which the
M

tendency is reversed. Furthermore, as it can be observed in Figure 1b, within the domain,
ED

the highest values for FRAP are achieved for the highest pressure level and holding time

(650 MPa and 9 min).

PT

Regarding antioxidant capacity, literature data is very variable. While some authors
CE

found that is not affected by HPP (Andrés et al., 2016; Chen et al., 2015) others found

decreases (Barba et al., 2013; González-Cebrino et al., 2013) and others increases
AC

(Swami Hulle et al., 2017; Varela-Santos et al., 2012). Certainly, the effect of HPP on

antioxidant activity depends not only on the pressurization conditions but also on the type

of food matrix under study and also on the method used for its evaluation. It is therefore

fundamental to study the HPP effect for each particular product

Similar results to those obtained in this study were found by Patras et al. (2009b)

who also reported higher antiradical activities at higher level of pressure compared to

lower one in strawberry purees (600 vs. 400 MPa). Also, an increase of 37 and 27% in

18
 ACCEPTED MANUSCRIPT

antioxidant potential was reported in carrot and tomato purees, respectively, at 600

MPa/15 min (Patras et al., 2009b). Again, the most accepted reason for this increase is the

better extractability of antioxidant components due to changes in the tissue matrix, induced

by HPP, resulting in the release of compounds with antioxidant actions into the

extracellular environment (Jayachandran et al., 2015).

T
IP
3.8. Enzyme activities

CR
The PME activity of treated samples ranged between 6.9 and 36.8% in relation to

the one of the untreated samples (Table 2). Regardless of the holding time, which did not

US
affected PME activity, the higher the pressure level, the more effective was the inactivation

of PME, which was reflected by a significant (p<0.05) negative regression coefficient

AN
(Table 3). Thus, for the pressure range studied (350- 650 Mpa), an increase in the

pressure level will result in a decrease in the PME activity (Figure 2a). Accordingly, the
M

treatment at 650MPa and 5min achieved the highest PME inactivation (81.5%).
ED

Similar results were found by Nienaber & Shellhammer (2001) working with orange

juice treated with HPP (400-600MPa/ 3min). They found that PME inactivation increased
PT

with the level of pressure, achieving almost complete inactivation at 600 MPa/3 min.
CE

Moreover, Timmermans et al. (2011) found a similar level of PME inactivation (92%) in

orange juice treated at 600 MPa /1 min. A higher baroresistence was observed by Rao et
AC

al. (2014) in HPP-treated (400–600 MPa/ 5–25 min) peach juice. They observed that PME

was inactivated significantly with increasing P and t, although the maximum inactivation

achieved was only 50% (600MPa/ 25min).

There have been many studies reporting pressure induced changes in PME, and

the extent of changes varies with different commodities, indicating that the source of the

enzyme and substrate can also affect the barosensitivity of enzymes (Swami Hulle et al.,

19
 ACCEPTED MANUSCRIPT

2017). Since our smoothie had 60% of orange juice, it was not surprising that our results

were closer to those observed in orange juice.

The POD activity of treated samples ranged between 63.1 and 93.1% in relation to

the one of the untreated smoothie (Table 2). POD activity was affected by both, pressure

level and holding time. The effect of P was not strictly linear since the equation contains

both a significant (p< 0.05) positive lineal coefficient and a significant (p< 0.05) negative

T
IP
quadratic coefficient. The effect of t in this case was strictly linear, with a significant (p<

CR
0.05) positive regression coefficient. Accordingly, an increase in P and t would determine

an increase in POD activity. However, the negative quadratic term indicate that there is a

US
critical pressure from which the situation is reversed, thus the maximum inactivation is

finally achieved with the highest pressure level and holding time, as shown in Figure 2b.
AN
Interestingly, Duong & Balaban ( 2014) reported a similar behavior in feijoa puree,

since they observed that at low pressure level (200–400 MPa) and/or with short holding
M

time (1–7 min), POD activities tended to increase. On the other hand, when holding time or
ED

pressure level were increased, the enzyme activity decreased. Applying treatments

between 400 and 600 MPa, for more than 8 min, they achieved a maximum reduction, with
PT

a residual activity below 70%. Similarly, Liu, Zhao, Zou, & Hu (2013), studyingthe effect of
CE

HPP (200- 600 MPa and 5- 60 min) in watermelon juice, reported a maximum POD

inactivation of about 42% at 600 MPa/60 min. Moreover, they observed a stronger impact
AC

of pressure level on the POD activity values.

The PPO activity of treated samples ranged between 78.8 and 106.6% in relation

to the one of untreated smoothie (Table 2), resulting in the most baroresistant enzyme

among the studied ones. Additionally, under certain conditions, some activation was

observed. The PPO activity had a significant (p<0.05) positive correlation coefficient with

the quadratic term of holding time (Table 3). In the surface plot of PPO as a function of

pressure level and holding time (Figure 2c) can be observed the concave shape and the

20
 ACCEPTED MANUSCRIPT

stronger impact of holding time on the PPO activity values. In low to medium times a

reduction of activity values is observed until a critical point is reached from which the

values begin to increase. Within the study domain, the lowest activity values were

observed in the treatments with an intermediate (5 min) holding time.

Very variable responses of PPO activity have been observed on different food

matrices subjected to HHP treatments. For instance, Duong & Balaban (2014) in feijoa

T
IP
puree observed that at low pressure level (200–400 MPa) and/or with short treatment time

CR
(1–7 min), PPO activities tended to increase up to around 120% residual enzyme activity

(REA), but as the holding time increased, the enzyme activity tended to decrease.

US
According to Gonzalez Cerebino (2013), who worked with plum purée, the higher activity

of the PPO after processing could be attributed to the release of membrane-bound

AN
enzymes, which could increase the extractability of PPO, counteracting the inactivating

effect of the HPP. Another factor which may contribute to this behavior is the activation of
M

latent PPO by the interaction with other constituents in the extract (Terefe, Yang,
ED

Knoerzer, Buckow, & Versteeg, 2010). In other studies different degrees of inactivation

have been observed. For instance, Keenan, Rößle, Gormley, Butler, & Brunton (2012),
PT

working with a mixed fruit smoothie found that a treatment at 450 MPa/5min resulted in
CE

35% reduction of PPO activity, while a treatment at 600 MPa/10 min resulted in a

considerable reduction (83%) of PPO activity.

AC

In this study, low degrees of inactivation of POD and PPO were achieved.

Nevertheless, characteristics such as polyphenol content and color remained unchanged

or improved. However, future studies are necessary to evaluate how quality evolves with

storage time.

3.9. Optimization of HPP conditions and validation

21
 ACCEPTED MANUSCRIPT

The response variables having at least one coefficient statistically significant in the

effects considered in the regression models (betacyanin content, DPPH capacity, FRAP

capacity, PME, POD and PPO activities, Chroma) were selected for simultaneous

optimization of process condition. As detailed above, HPP affected dissimilarly each

response; hence, this tool is fundamental to achieve a compromise solution that allows

obtaining good results for all the variables under study. The Figure 3 shows the predicted

T
IP
profiles at the different levels assayed for each independent variable (pressure level and

CR
holding time), while maintaining constant the level of the other independent variable at the

estimated optimal value. Figure 3 also shows each individual desirability function and the

US
global desirability function profiles.

The criteria selected for the optimization of process parameters were: maximization
AN
of bioactive component concentration and antioxidant capacity (betacyanins, DPPH and

FRAP); maximization of color saturation (Chroma) and minimization of enzyme activity

M

(PME, POD, PPO). Based on the above criteria, the predicted optimal process condition
ED

leading to the maximum value of global desirability function for the process under study

was a combination of a pressure level at 627.5 MPa and a holding time at 6.4 min (which
PT

would correspond to practical operating values at 630 MPa and 6 min).

CE

Further, the smoothie was processed under these conditions and its quality was

compared with the predicted response values. The relative deviation was found to be <5%
AC

(Table 4), verifying that with the optimized HPP parameters a high quality smoothie is

obtained.

4. Conclusions

The results of the present study show that HPP is a promising technology for the

preservation of the mixed fruit & vegetable smoothie, reducing spoilage microorganisms

counts and enzymes activity. Moreover, HPP does not affect pH, total soluble solids,

22
 ACCEPTED MANUSCRIPT

texture and total phenolic content, increases antioxidant capacity largely and maintains or

slightly improves color of the smoothie. The optimization analysis suggests that HPP

applied at 627.5 MPa and 6.4 min would lead to a product with high quality and maximum

reduction of spoilage causing factors.

Moreover, the developed quadratic models might be useful to predict the quality

characteristics of smoothie during the HPP within the studied domain of process

T
IP
conditions. Future studies will be oriented to evaluate the stability of the different quality

CR
attributes during storage, moreover, shelf-life estimation and scale-up studies may be

explored in order to transfer HPP to the smoothies industry.

Acknowledgements
US
AN
This research was financially supported by Universidad de Buenos Aires

(20020130100176BA; 20020120300008BA), Instituto Nacional de Tecnología

M

Agropecuaria, INTA (PNAIyAV 1130033) and Agencia Nacional de Promoción Científica y

ED

Tecnológica (PICT 2013-0636) projects. The authors also gratefully acknowledge the

Peruilh Foundation (FIUBA) for Fernandez MV scholarship.

PT

References:
CE

Ahmed, J., Ramaswamy, H.S., & Hiremath, N. (2005). The effect of high pressure treatment on
AC

rheological characteristics and colour of mango pulp. International journal of food

science & technology, 40(8), 885-895.
Andrés, V., Villanueva, M.J., & Tenorio, M.D. (2016). The effect of high-pressure processing on
colour, bioactive compounds, and antioxidant activity in smoothies during refrigerated
storage. Food chemistry, 192, 328-335.
Augustin, M.A., Ghazali, H.M., & Hashim, H. (1985). Polyphenoloxidase from guava (Psidium
guajava L.). Journal of the Science of Food and Agriculture, 36(12), 1259-1265.

23
 ACCEPTED MANUSCRIPT

Barba, F.J., Esteve, M.J., & Frigola, A. (2010). Ascorbic acid is the only bioactive that is better
preserved by high hydrostatic pressure than by thermal treatment of a vegetable
beverage. Journal of Agricultural and Food Chemistry, 58(18), 10070-10075.
Barba, F. J., Esteve, M. J., & Frígola, A. (2012). High Pressure Treatment Effect on
Physicochemical and Nutritional Properties of Fluid Foods During Storage: A Review.
Comprehensive Reviews in Food Science and Food Safety, 11(3), 307–322.
Barba, F.J., Esteve, M.J., & Frigola, A. (2013). Physicochemical and nutritional characteristics of

T
blueberry juice after high pressure processing. Food Research International, 50(2), 545-

IP
549.

CR
Bezerra, M.A., Santelli, R.E., Oliveira, E.P., Villar, L.S., & Escaleira, L.A. (2008). Response surface
methodology (RSM) as a tool for optimization in analytical chemistry. Talanta,
76(5), 965-977.

US
Bup, D. N., Abi, C. F., Tenin, D., Kapseu, C., & Tchiegang, C. (2012). Optimisation of the
cooking process of sheanut kernels (Vitellaria paradoxa Gaertn.) using the Doehlert
AN
experimental design. Food and bioprocess technology, 5(1), 108 –117.
Celli, G.B., & Brooks, M.S. (2016). Impact of extraction and processing conditions on betalains
M

and comparison of properties with anthocyanins—A current review. Food Research

International. 100, 501-509.
ED

Chakraborty, S., Kaushik, N., Rao, P.S., & Mishra, H.N. (2014). High‐pressure inactivation of
enzymes: a review on its recent applications on fruit purees and juices. Comprehensive
PT

Reviews in Food Science and Food Safety, 13(4), 578-596.

Chen, D., Pang, X., Zhao, J., Gao, L., Liao, X., Wu, J., & Li, Q. (2015). Comparing the effects of high
CE

hydrostatic pressure and high temperature short time on papaya beverage. Innovative
Food Science & Emerging Technologies, 32, 16-28.
AC

Chen, D., Xi, H., Guo, X., Qin, Z., Pang, X., Hu, X.,Liao,X., Wu, J. (2013). Comparative study of
quality of cloudy pomegranate juice treated by high hydrostatic pressure and high
temperature short time. Innovative Food Science & Emerging Technologies, 19, 85-94.
Denoya, G.I., Polenta, G.A., Apóstolo, N.M., Budde, C.O., Sancho, A.M., & Vaudagna, S.R. (2016).
Optimization of high hydrostatic pressure processing for the preservation of minimally
processed peach pieces. Innovative Food Science & Emerging Technologies, 33, 84-93.
Denoya,G.I., Fernandez, M.V., Sanow, L.C., Bello, F., Jagus, R.J., Vaudagna, S.R., Agüero, M.V.
(2017). Revalorización de subproductos hortícolas: desarrollo de un batido mezcla de

24
 ACCEPTED MANUSCRIPT

frutas y verduras estabilizado con altas presiones hidrostáticas. I Congreso Argentino de

Biología y Tecnología Poscosecha. Concordia, Argentina (October 25-27).
Duong, T., & Balaban, M. (2014). Optimisation of the process parameters of combined high
hydrostatic pressure and dense phase carbon dioxide on enzyme inactivation in feijoa
(Acca sellowiana) puree using response surface methodology. Innovative Food Science &
Emerging Technologies, 26, 93-101.
Fernandez M.V., Jagus R.J., Agüero M.V. (2014). Revalorización de hortalizas subutilizadas:

T
caracterización y evaluación de hojas de remolacha y su potencial como fuente de

IP
compuestos beneficiosos para la salud. In Fito P. and Gerard J. (ed). Book of abstracts

CR
“International Conference on Food Innovation". Concordia, Argentina. ISBN OC 978-950-
698-376-5. ISBN 978-950-698-380-2, pp. 64-68.
González-Cebrino, F., Durán, R., Delgado-Adámez, J., Contador, R., & Ramírez, R. (2013). Changes

US
after high-pressure processing on physicochemical parameters, bioactive compounds,
and polyphenol oxidase activity of red flesh and peel plum purée. Innovative Food
AN
Science & Emerging Technologies, 20, 34-41.
Gram, L., Ravn, L., Rasch, M., Bruhn, J. B., Christensen, A. B., & Givskov, M. (2002). Food
M

spoilage–interactions between food spoilage bacteria. International Journal of Food

Microbiology, 78(1–2), 79–97.
ED

Hendrickx, M.E.G., & Knorr, D. (2002). Ultra high pressure treatments of foods. food
engineering series. US: Springer, 0306472783, 9780306472787.
PT

Jayachandran, L.E., Chakraborty, S., & Rao, P.S. (2015). Effect of high pressure processing on
physicochemical properties and bioactive compounds in litchi based mixed fruit
CE

beverage. Innovative Food Science & Emerging Technologies, 28, 1-9.

Kaushik, N., Rao, P. S., & Mishra, H. N. (2016). Process optimization for thermal-assisted high
AC

pressure processing of mango (Mangifera indica L.) pulp using response surface
methodology. LWT - Food Science and Technology, 69, 372–381.
Keenan, D.F., Rößle, C., Gormley, R., Butler, F., & Brunton, N.P. (2012). Effect of high hydrostatic
pressure and thermal processing on the nutritional quality and enzyme activity of fruit
smoothies. LWT-Food Science and Technology, 45(1), 50-57.
Knorr, D. (1993). Effects of high-hydrostatic-pressure processes on food safety and quality. Food
technology (USA).
Koseki, S., & Yamamoto, K. (2014). High Pressure Microbial Inactivation : Modelling and its

25
 ACCEPTED MANUSCRIPT

Features. In Novel Food Preservation and Microbial Assessment Techniques (pp. 3–24).
Taylor & Francis Group.
Kuehl, R.O.O. (2000). Designs of experiments: statistical principles of research design and
analysis: Duxbury Press.
Lado, B.H., & Yousef, A.E. (2002). Alternative food-preservation technologies: efficacy and
mechanisms. Microbes and Infection, 4(4), 433-440.
Liu, Y., Zhao, X.Y., Zou, L., & Hu, X.S. (2013). Effect of high hydrostatic pressure on overall quality

T
parameters of watermelon juice. Revista de Agaroquimica y Tecnologia de Alimentos,

IP
19(3), 197-207.

CR
Ludikhuyze, L., Van Loey, A., Indrawati, S.C., & Hendrickx, M. (2003). Effects of combined
pressure and temperature on enzymes related to quality of fruits and vegetables: from
kinetic information to process engineering aspects. Critical reviews in food science and

US
nutrition, 43(5), 527-586.
Mayor, L., Moreira, R., & Sereno, A. (2011). Shrinkage, density, porosity and shape changes
AN
during dehydration of pumpkin (Cucurbita pepo L.) fruits. Journal of Food Engineering,
103(1), 29 –37.
M

Moßhammer, M.R., Stintzing, F.C., & Carle, R. (2006). Cactus pear fruits (Opuntia spp.): a review
of processing technologies and current uses. Journal of the Professional Association for
ED

Cactus Development, 8, 1-25.

Mújica-Paz, H., Valdez-Fragoso, A., Samson, C.T., Welti-Chanes, J., & Torres, J.A. (2011). High-
PT

pressure processing technologies for the pasteurization and sterilization of foods. Food
and Bioprocess Technology, 4(6), 969-985.
CE

Nienaber, U., & Shellhammer, T.H. (2001). High‐pressure processing of orange juice: Kinetics of
pectinmethylesterase inactivation. Journal of Food Science, 66(2), 328-331.
AC

Ninfali, P., & Angelino, D. (2013). Nutritional and functional potential of Beta vulgaris cicla and
rubra. Fitoterapia, 89, 188-199.
Oey, I., Lille, M., Van Loey, A., & Hendrickx, M. (2008). Effect of high-pressure processing on
colour, texture and flavour of fruit-and vegetable-based food products: a review. Trends
in Food Science & Technology, 19(6), 320-328.
Paciulli, M., Medina-Meza, I.G., Chiavaro, E., & Barbosa-Cánovas, G.V. (2016). Impact of thermal
and high pressure processing on quality parameters of beetroot (Beta vulgaris L.). LWT-
Food Science and Technology, 68, 98-104.

26
 ACCEPTED MANUSCRIPT

Patras, A., Brunton, N., Da Pieve, S., Butler, F., & Downey, G. (2009a). Effect of thermal and high
pressure processing on antioxidant activity and instrumental colour of tomato and
carrot purées. Innovative Food Science & Emerging Technologies, 10(1), 16-22.
Patras, A., Brunton, N.P., Da Pieve, S., & Butler, F. (2009b). Impact of high pressure processing
on total antioxidant activity, phenolic, ascorbic acid, anthocyanin content and colour of
strawberry and blackberry purées. Innovative Food Science & Emerging Technologies,
10(3), 308-313.

T
Rao, L., Guo, X., Pang, X., Tan, X., Liao, X., & Wu, J. (2014). Enzyme activity and nutritional

IP
quality of peach (Prunus persica) juice: effect of high hydrostatic pressure. International

CR
Journal of Food Properties, 17(6), 1406-1417.
Rastogi, N. K., Raghavarao, K. S. M. S., Balasubramaniam, V. M., Niranjan, K., & Knorr, D.
(2007). Opportunities and challenges in high pressure processing of foods. Critical

US
Reviews in Food Science and Nutrition, 47(1), 69 −112.
Rickman, J. C., Bruhn, C. M., & Barrett, D. M. (2007). Review: Nutritional comparison of fresh,
AN
frozen, and canned fruits and vegetables. Part I. Vitamins C and B and phenolic
compounds. Journal of the Science of Food and Agriculture, 87, 930–944.
M

Swami Hulle, N.R., Chakraborty, S., & Rao, P.S. (2017). Effect of high pressure thermal processing
on the quality attributes of Aloe vera-litchi mixed beverage. Innovative Food Science &
ED

Emerging Technologies,40,68-77.
Terefe, N.S., Yang, Y.H., Knoerzer, K., Buckow, R., & Versteeg, C. (2010). High pressure and
PT

thermal inactivation kinetics of polyphenol oxidase and peroxidase in strawberry puree.

Innovative Food Science & Emerging Technologies, 11(1), 52-60.
CE

Timmermans, R.A.H., Mastwijk, H.C., Knol, J.J., Quataert, M.C.J., Vervoort, L., Van der Plancken,
I.,Hendrickx,M.E., Matser, A.M. (2011). Comparing equivalent thermal, high pressure
AC

and pulsed electric field processes for mild pasteurization of orange juice. Part I: Impact
on overall quality attributes. Innovative Food Science & Emerging Technologies, 12(3),
235-243.
Varela-Santos, E., Ochoa-Martinez, A., Tabilo-Munizaga, G., Reyes, J.E., Pérez-Won, M., Briones-
Labarca, V., & Morales-Castro, J. (2012). Effect of high hydrostatic pressure (HHP)
processing on physicochemical properties, bioactive compounds and shelf-life of
pomegranate juice. Innovative Food Science & Emerging Technologies, 13, 13-22.

27
 ACCEPTED MANUSCRIPT

Verlent, I., Hendrickx, M., Rovere, P., Moldenaers, P., & Loey, A.V. (2006). Rheological Properties
of Tomato‐based Products after Thermal and High‐pressure Treatment. Journal of food
science, 71 (3) 243-248.
Viacava, G.E., Roura, S.I., & Agüero, M.V. (2015). Optimization of critical parameters during
antioxidants extraction from butterhead lettuce to simultaneously enhance polyphenols
and antioxidant activity. Chemometrics and Intelligent Laboratory Systems, 146, 47-54.
Vicente, A.R., Costa, M.L., Martínez, G.A., Chaves, A.R., & Civello, P.M. (2005). Effect of heat

T
treatments on cell wall degradation and softening in strawberry fruit. Postharvest

IP
Biology and Technology, 38(3), 213-222.

CR
US
AN
M
ED
PT
CE
AC

28
 ACCEPTED MANUSCRIPT

Table 1- Coded and actual values of independent variables in the Doehlert design.

Coded values Actual values

Exp. No.

T
X1 X2 Pressure (MPa) Holding time (min)

IP
1 0 0 500 5

CR
2 0 0 500 5
3 0 0 500 5
4 -1 0 350 5

US
5 1 0 650 5
6 -0.5 -0.866 425 1
AN
7 -0.5 0.866 425 9
8 0.5 -0.866 575 1
9 0.5 0.866 575 9
M
ED
PT
CE
AC

29
 ACCEPTED MANUSCRIPT

Table 2- Experimental values for untreated fruit & vegetable smoothies quality
attributes

Microbiological quality
MAB (log CFU mL-1) 3.9 ± 0.1
EB (log CFU mL-1) 3.7 ± 0.2
M&Y (log CFU mL-1) 2.5 ± 0.3
Physicochemical parameters
pH 3.79 ± 0.03

T
Total Soluble Solids (ºBrix) 9.6 ± 0.2
Chromatic parameters

IP
L* 40.2 ± 0.2
a* 9.4 ± 0.3

CR
b* 14.6 ± 0.1
hº 57 ± 1
Ch 17.3 ± 0.1

US
Textural parameters
Firmness (g) 624 ± 13
Consistency (g s) 18430 ± 2381
AN
Nutritional indicators
Betacyanin (mg L-1) 13.1 ± 0.5
Betaxanthin (mg L-1)
M

4.9 ± 0.1
DPPH (TEAC 100 g-1) 350 ± 3
FRAP (TEAC 100 g-1) 1040 ± 92
ED

Total phenols content

(GAE 100 g-1) 62.6 ± 0.6
Enzymatic activity
PT

PME 37.8 ± 4.0

POD 84.9 ± 2.3
PPO 30.4 ± 2.5
CE

MAB: mesophilic aerobic bacteria, EB: Enterobacteriaceae, M&Y: Mold and yeast, DPPH: radical
AC

scavenging capacity, FRAP: Ferric reducing capacity, PME: pectinmethylesterase, POD:

Peroxidase, PPO:Poliphenoloxidase.

30
 ACCEPTED MANUSCRIPT

Table 3- Regression coefficients, R2 values and lack of fit test results for the response variables of the fruits &
vegetable mixed smoothie subjected to HPP.

PME POD PPO

Regression
coefficient
Betacyanin DPPH FRAP Activity Activity Activity

P T Chroma

Constant 26.113 *
747.389*
6741.105*

*
86.946 *

*
7.808*
*
I
74.613

R
*
-0.499*

P (linear)

t (linear)
-0.055

-0.254
-1.290

-28.247
-20.281

-297.726*
-0.133

-1.738
S C
0.357

0.257*
-0.139

-4.357
0.066

0.868

P2 (Quadratic) 0.00006* 0.0012 0.020*

N
0.00004U -0.0004* 0.00011 -0.00007

t2 (Quadratic) 0.060* 2.461* 20.587*

A
0.1656 -0.211 0.2543* -0.102*

P*t -0.00059 0.0109 0.245

M -0.0021 0.0014 0.0036 0.00025

R2 0.938 0.937

E D 0.968 0.963 0.982 0.879 0.895

Lack of fit 0.2578

P T
0.7634 0.1072 0.1173 0.2606 0.8631 0.6586

E
Reduced equations for process parameters:
Betacyanin (mg L-1)= 0.00006 P2+ 0.060 t2+ 26.113

C
DPPH (TEAC 100 g-1)= 2.461 t2+ 747.389

A C
FRAP (TEAC 100 g-1)= -20.281 P- 297.726 t +0.020 P2 +20.587 t2+6741.105
PME Activity= -0.133P+ 86.946
POD Activity= 0.357 P+ 0.257 t -0.0004 P2+ 7.808
PPO Activity= 0.2543 t2+ 74.613
Chroma= -0.102 t2 -0.499

* Significant at 0.05 level.

31
 ACCEPTED MANUSCRIPT

DPPH: radical scavenging capacity, FRAP: Ferric reducing capacity, PME: pectinmethylesterase, POD: Peroxidase, PPO:
Poliphenoloxidase.
Table 4- Relative error between predicted and actual values for fruits & vegetable smoothie processed under optimized HPP
conditions

P T
Values
Optimized process
parameters
Pressure Holding
R I
Response Variables

DPPH FRAP
Level
(MPa)
Time
(min)
PME
ACTIVITY
POD
ACTIVITY
PPO
ACTIVITY
S C
Bc
(mg/L)
(μmol
TEAC/100g)
(μmol
TEAC/100g)
Chroma

Predicted
Actual
627.5
630.0
6.4
6.0
5.5
4.8
54.9
81.2
27.4

N
15.0U 13.5
14.9
368
363
1812
1797
16.0
17.5
% error** 0.33 -4.76 3.1 -3.1
A 0.5 1.2 3.0 0.4 0.2

M
PME: pectinmethylesterase, POD: Peroxidase. PPO: Poliphenoloxidase, Bc: Betacyanin, DPPH: radical scavenging capacity, FRAP: Ferric
reducing capacity.

E D
T
**For the calculation of the error, the values of the response variables were relativized to the value of the corresponding control, since in each
new elaboration there may be differences associated to the variability of the raw material. The low %error means that the response was similar

E P
regardless of the initial values of the product. The %error was calculated as follows:

%actual from control−%predicted from control

%error =
C
%predicted from control

C
A

32
 ACCEPTED MANUSCRIPT

Highlights:

- HPP is a promising technology for the preservation of a F&V smoothie

- HPP does not affect physicochemical attributes and enhances microbiological quality
- The process condition that maximizes global desirability was 627.5 MPa for 6.4 min
- Optimal treatment leads to reductions of 85%, 45% and 10% on PME, POD and PPO
- Optimal treatment increases antioxidant capacity (75%) and slightly improves color

T
Industrial relevance text:

IP
F&V smoothies are tasty, healthy, convenient and ready to drink, fulfilling all the current

CR
demands of consumers. This has led to an accelerated increase in their popularity. However, they

have a short shelf life mainly attributed to microbial and enzymatic spoilages. HPP is proposed as a

US
non-thermal method able to prolong shelf-life of the products by means of microbial and enzymatic

inactivation, while preserving bioactive compounds and quality characteristics. An optimization

AN
assay was carried out in order to find optimal process conditions for the F&V smoothie´s

preservation. The promising results obtained can help to promote the use of HPP as an alternative
M

technology for the preservation of this kind of products.

ED
PT
CE
AC

33
Figure 1
Figure 2
Figure 3