Journal of Food Measurement and Characterization (2023) 17:2519–2536

https://doi.org/10.1007/s11694-022-01719-1

ORIGINAL PAPER

Effect of the multi-stage block freeze concentration process on the

physicochemical and biological properties of noni tea (Morinda
citrifolia L.): a case study in Brazil to obtain a promising functional
food
Édipo da Silva Almeida1 · Giordana Demaman Arend1 · Mateus Antônio Knapp1 · Kátia Rezzadori2 · Silvani Verruck2 ·
Dachamir Hotza1 · Débora de Oliveira1

Received: 10 March 2022 / Accepted: 19 November 2022 / Published online: 11 January 2023
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022

Abstract
Morinda citrifolia, widely known as “noni”, provides fruits of which it is a raw material not only for the preparation of
foods, such as beverages, but is also used, for example, in the preparation of cosmetics. However, processing the fruit
results in the generation of waste, in which the leaves represent the majority. Taking into account that noni leaves are
proven to hold a high amount of bioactive compounds with phytotherapeutic properties, this study proposes the applica-
tion of multi-stage block freeze concentration performed through the passive thaw method with the aid of the microwave-
assisted technique to promote a greater speed and efficiency in the process of concentration of the bioactive components
present in Morinda citrifolia L. leaves. The effect of the cryoconcentration steps on the physical, chemical and biological
characteristics of the concentrated fractions and residual ice was evaluated in order to quantitatively verify presence and
content of phenolics and total solids, viscosity, antioxidant activity, and bioavailability gastrointestinal in vitro. An increase
in the phenolic and total solids values was observed at the end of the process, resulting in efficiency in the retention of
phenolics above 90%. High-performance liquid chromatography assay detected catechin as bioactive compound of the
largest amount in the final product. The use of the microwave-assisted concentration system allowed concentrated fractions
with high biological, nutritional and phytotherapic value; indicating that the technique can contribute as a reference in the
use of a sustainable technology in the generation of a promising product of high biological value, since the execution and
elaboration of the microwave-assisted method allows for a lower energy consumption and waste production, in addition
to providing less expensive and easy-to-operate tests.

Keywords Cryoconcentration · Noni · Microwave-assisted · Phenolic compounds · Antioxidant activity · Bioavailability

Débora de Oliveira Silvani Verruck

debora.oliveira@ufsc.br silvani.verruck@ufsc.br
Édipo da Silva Almeida Dachamir Hotza
edypobrito@gmail.com d.hotza@ufsc.br
Giordana Demaman Arend 1
Department of Chemical Engineering and Food Engineering
giordana.darend@gmail.com
(EQA), Federal University of Santa Catarina (UFSC),
Mateus Antônio Knapp 88040-970 Florianópolis, SC, Brazil
mateus_knapp@hotmail.com 2
Department of Food Science and Technology (CAL), Federal
Kátia Rezzadori University of Santa Catarina (UFSC),
katia.rezzadori@gmail.com 88034-001 Florianópolis, SC, Brazil

13
2520
Main abbreviations
ABTS 2,2’-azino-bis (3-ethylbenzo-
thiazoline-6-sulfonic acid)
ANOVA Analysis of variance
BFC Block freeze concentration
CC1, CC2, CC3, and CC4 Cryoconcentrated 1, Cryo-
concentrated 2, Cryoconcen-
trated 3 and Cryoconcentrated
4 In fact, due to the complexity of biochemical components
Cf Concentration factor
DPPH 2,2-Diphenyl-1-picrylhydrazyl
HPLC-DAD-MS High-Performance Liquid
Chromatography with a
coupled mass spectrometer
GAE Equivalent to gallic acid
ORAC Oxygen Radical Absorbance
Capacity
TPC Total Phenolic Content
TSC Total Solids Content
Introduction
According to the Food and Agriculture Organization of the
United Nations (FAO), about 1.3 billion tons of food are
likely to be wasted every year, where around 15% of the
waste from the agro-food industries comes from the pro-
duction and processing of fruit and vegetables [1, 2]. One
strategy to reduce such alarming amounts of food waste
involves the use of all parts of plants for human consump-
tion. A challenge is to combine an emerging and sustainable
technology that promotes the reduction of the waste previ-
ously mentioned.
Morinda citrifolia is the scientific name of the vegeta-
ble widely known by the commercial name of “noni”. M.
citrifolia belongs to the Rubiaceae Juss. family, constituted
by approximately 6500 genera and 13,000 species [3]. It
has a cosmopolitan distribution, predominantly in tropical
regions, with about half of its species being neotropical and
most found in South America. In Brazil, the attempt to cul-
tivate noni is quite recent, empirically carried out by people
who brought some seeds from the Caribbean or Polyne-
sia. The fruit was introduced as a raw material with strong
commercial appeal due to all the beneficial characteristics
attributed to it and the benefits related to its consumption.
However, there are few research works carried out with this
species in the country [4].
The processing of the noni fruit results in a remarkable
generation of residues from which the leaves make up a large
part [5]. Besides the use in tea preparation, noni leaves are
also characterized by bioactive components with phytother-
apeutics properties of which the presence of acids, alcohols,
13
É. d. S. Almeida et al.
phenols, saccharides, anthraquinones, carotenoids, esters,
triterpenoids, flavonoids, glycosides, lactones, iridoids,
ketones, lignans, nucleosides, triterpenoids, sterols, among
other minor bioactives have already been reported [6–8]. It
is proven that the solution elaborated with the leaf extract
of noni presents a phytotherapeutic effect against various
diseases such as cancer and obesity disorders, besides pre-
senting antibacterial properties and antioxidant activity [3].
present in noni tea, technologies that promote the increase of
solids content are valid for the aqueous leaf extract of noni
[9]. Also, the production of aqueous extracts from industrial
waste has been the subject of several studies, since these can
be excellent sources of bioactive compounds [10].
The concentration of liquid foods had led to innovation
in the food industry [11], considering that the removal of
water will increase the solids content of food, prolonging
shelf-life and consequently promoting microbial stability.
Seeking to reuse the nutritional value of the structural com-
ponents of vegetables, which are generally rejected, the goal
of this study was to evaluate the potential of the microwave-
assisted cryoconcentration technique on the behavior of the
physical, chemical and biological properties of bioactive
compounds present in extracts of Morinda citrifolia leaves
(noni), quantitatively checking the presence and content of
phenolics and solids total, viscosity, antioxidant activity and
gastrointestinal bioavailability in vitro.
This method presents the advantage of being a one-step
procedure, characterized by simple construction and opera-
tion of the equipment, where all liquid food is frozen, then
thawed and the concentrated fraction is separated from the
ice fraction by the gravitational method. The use of micro-
wave will have an auxiliary role, acting as an external
force to promote a solid/liquid separation more quickly and
efficiently [12, 13]. Besides, considering that M. citrifolia
leaves have large amounts of bioactive compounds with
phytotherapeutic properties, this work presents to the best
of our knowledge the first application of cryoconcentration
for separation and accumulation of bioactive substances of
this plant species. It is expected that the results of this study
provide useful information on the application of cryocon-
centration in the development of the nutritional and phyto-
therapeutic properties of M. citrifolia leaves.
Materials and methods
Raw material
The raw material used in this study was kindly provided by
rural landowners in the city Pombal, in the state of Paraíba,
Brazil. The leaves were taken randomly in different trees of
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties…
Morinda citrifolia. The species was identified by the local
owners and later confirmed by a local botanist. Morinda
citrifolia L. is an authenticated plant as per the International
Plant Names Index (IPNI Life Sciences Identifier (LSID):
urn:lsid:ipni.org:names:756359-1 – www.theplantlist.com/
tpl1.1/record/kew-129789).
After the pruning, the leaves were washed in running
water aiming at the removal of any soil residues or insects.
Afterward, the leaves of M. citrifolia were immersed in
chlorinated water solution (200 ppm) for 15 min for final
sanitization, followed by a new wash with running water.
Finally, the leaves were subjected to a bleaching process as
described by Xavier et al. [14]. The leaves of M. citrifolia
were immersed in hot water with controlled temperature at
70 ºC ± 5 ºC, for a period of 5 min, immediately after cooling
in order to stop the cooking process of the vegetable. After
bleaching, the material was oven-dried and maintained at
45 °C for not more than 2 days as described by Shalan et
al. [15]. The noni leaves were then milled in a Wiley mill
(Tecnal, TE-605/1, Piracicaba, SP, Brazil), packed in poly-
ethylene bags, and stored at -24 °C for up to 48 h when the
preparation of the extracts started.
Extract preparation
The preparation of the extract consisted of the infusion of
dried and crushed Morinda citrifolia leaves into ultrapure
water. The preparation of the aqueous extract followed the
methodology described by Shalan et al. [15, 16] with few
modifications. The experimental apparatus for preparing the
extract consisted of a closed system with continuous agi-
tation and controlled temperature maintained at 40 ± 2 °C,
composed of 40 g of suspended leaves for each 1 L (4%
w/v) of ultrapure water for 20 min. After preparation of the
extract, the solution was subjected to vacuum filtration with
qualitative filter paper (125 mm - Unifil, Zilquimica Prod-
ucts Ltd., São Paulo, Brazil) to remove suspended solids.
Subsequently, the material was cooled (~ 4 °C) for 30 min,
so that the cryoconcentration process was started. An infu-
sion of commercial matte tea (Matte Leão®, The Coca-
Cola® Co., Linhares, Brazil), purchased at local retail,
containing leaves and toasted mate stalks (Ilex paraguarien-
sis St. Hil.) prepared according to the manufacturer’s speci-
fications, were used as the positive control for comparison.
In this study, we will call “control” all the samples obtained
in this extraction assay and which were not subjected to the
cryoconcentration test.
Cryoconcentration assay
Block freeze concentration (BFC) process of Morinda citri-
folia leaves followed the methodology described by Adorno
2521
et al., Aider et al. and Khajehei et al. [17–19] with few
modifications. Samples of M. citrifolia leaves extract were
frozen by indirect cooling in a chilled air chamber (Bosch
Space REBS37, SP, Brazil) until the samples reached a
temperature of -20 ± 2 °C. After freezing the extract in 200
mL portions, the thawing method was performed to sepa-
rate each frozen portion into two phases: the concentrated
fraction and the fraction corresponding to the residual ice.
The BFC was performed through the passive thaw method
in blocks, where each block consisted of one step of the pro-
cess. In this study, the passive thawing followed with the aid
of the microwave-assisted technique to promote a greater
speed and efficiency in the process of concentration of the
bioactive components present in the noni leaves.
In the microwave-assisted defrosting of portions of the
frozen extract, the frozen initial solution was kept under
a controlled temperature of 20 ± 2 °C. The melting for the
formation of the concentrated samples was divided into 4
steps or cycles. In each cycle, 50% of the volume of the
frozen samples was thawed. The volume thawed during the
first BFC cycle was again frozen and used as a feed solu-
tion for the second BFC cycle. This procedure was repeated
in the third and fourth BFC cycles, resulting in 4 cryocon-
centrated volumes: cryoconcentrated 1 to cryoconcentrated
4 (CC1, CC2, CC3, and CC4); and 4 volumes of residual
ice. After each BFC cycle, 50 mL of the cryconcentrated
sample was collected. Each collected cryoconcentrated
sample was again frozen at -24 °C for further analysis. For
this test, defrosting was performed in a microwave oven
(Panasonic GT68LBRU 900 W, São Paulo, SP, Brazil) with
heating power set at 70% of the appliance’s total capacity.
The defrosting time was not measured, since the defrost
occurred until the acquisition of the initial representative
volumes for each stage of the cryoconcentration. Figure 1
show, respectively, the experimental apparatus and the gen-
eral flowchart of the microwave-assisted BFC process per-
formed in this study.
Physical and chemical properties
Total phenolic content (TPC)
The determination of the content of total phenolic com-
pounds in the cryoconcentrated fractions and the ice frac-
tions for each stage of the BFC process was carried out
following the methodology presented by Singleton and
Rossi [20], described by Martin-Diana et al. [21], via colori-
metric scanning of Folin-Ciocalteu. The method consists of
the reaction of 100 µL sample, 7 mL ultrapure water, 0.5 mL
Folin-Ciocalteu (Sigma-Aldrich, Inc., St. Louis, MO, US),
and 1.5 mL of 20% w/v sodium carbonate solution (Merck
Milipore Brasil, Barueri, SP, Brazil). The mixture was kept
13
2522
Fig. 1 Illustrative representation of the BFC by microwave-assisted process (a) and stages of BFC process (b): for each cryoconcentrated fraction
(CC1 to CC4), 50 mL aliquots were removed for analysis
in the dark at room temperature (24 ºC) for 120 min. The
absorbance was determined with wavelength adjustment at
765 nm in a spectrophotometer (UV-Vis mini-1240 Tokyo,
Japan) using ultrapure water as white. The same procedure
was used to construct the analytical curve, elaborated from
solutions of gallic acid (Metaquímica, Ltd., SC, Brazil) in
the range of 200–800 mg·mL− 1. The results expressed as
milligram equivalent to gallic acid per milliliters of extract
(mgGAE·mL− 1).
Total solids content (TSC)
All samples concerning the cryoconcentrated fractions and
the ice fractions of the extract of leaves of M. citrifolia
were analyzed for the content of total solids. The method
followed the description of AOAC [22], to determine the
mass loss after drying the samples at 105 °C for 24 h. The
results are expressed as dry matter per total mass content
(g.100.g− 1).
Viscosity of cryoconcentrated samples
The rheological behavior of the concentrated extracts in
each BFC step of the samples of the M. citrifolia leaves
extract was measured in a rotational viscometer (VT 550,
Thermo Haake, Karlsruhe, Germany) with concentric cylin-
ders (NV ST 807 − 0713 CE and NV 807 − 0702). Data were
collected through software (Pro Rheowin, version 2.93).
The rheological analyzes were obtained with a variation of
the deformation rate of 200 to 1800 s− 1 (ascending curve)
and 1800 to 200 s− 1 (descending curve), with 3 min duration
for each curve. The measurements were made at 25 ± 0.1 °C
13
É. d. S. Almeida et al.
by water circulation through a thermostatic bath coupled
to the equipment (Phoenix P1, Thermo Haake, Karlsruhe,
Germany). The analyzes were performed in triplicate, and
in each measurement, a new sample was used. The rheologi-
cal behavior was described by Newton’s model (Eq. 1) and
Power law (Eq. 2) as follows:
τ = µγ (1)
τ = Kγ n (2)
where τ is the shear stress (Pa); µ, the absolute or dynamic
viscosity (Pa·s); γ , the shear rate (s− 1); K, the consistency
index (Pa·s); and n , the behavior index (dimensionless).
Major phenolic compounds by high-performance
liquid chromatography assays
The detection analyses of the major phenolic compounds
present in Morinda citrifolia leaves cryoconcentrated
extracts were performed using high-performance liquid
chromatography (HPLC-DAD-MS, Shimadzu, Kyoto,
Japan). The equipment consisted of a binary pump, an auto-
matic injector, and a diode array detector with a coupled
mass spectrometer containing an electrospray ionization
source and a quadrupole analyzer (LCMS-2020, Shimadzu,
Kyoto, Japan).
The separation process was performed by using a C18
column, 25 mm long, 4.6 mm internal diameter, and 5 μm
particle size (NST, Santos, Brazil), with the oven at 40 °C.
The composition of the mobile phase had the components:
A (acidified water with 0.1% formic acid) and B (acidified
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties…
methanol with 0.1% formic acid) in a gradient elution sys-
tem. The mobile phase was initially composed of 14% B,
with a linear increase of up to 55% at 16 min. From 16
to 17 min the composition was 100% B for cleaning the
column and from 17 to 20 min the reconditioning was car-
ried out with 14% B. Column cleaning required 100% B
between min 16 and 17, between min 17 and 20 the col-
umn was reconditioned with 14% B. The flow of the mobile
phase was 1.2 mL.min− 1 and the injection volume was 5 µL.
The identification of the phenolic compounds was carried
out by comparing the retention times and absorption spectra
of the peaks of the Morinda citrifolia leaves cryoconcen-
trated extracts samples with those of standard compounds.
The electrospray interface temperature was 300 °C, the neb-
ulizing gas flow was 1.5 L.min− 1; the heat block, 200 °C;
and the drying gas flow, 15 L.min− 1. The interface voltage
was 4 kV and, the array radio frequency (RF) voltage was
60 V. Detection and quantification of phenolic compounds
of M. citrifolia, based on the study by Koop et al. [23],
which promoted the concentration of bioactive compounds
from extracts of Jambolan (Syzygium cumini (L.)) by Nano
and Ultrafiltration. The quantification of the compounds
was performed by calculating the peak chromatographic
area obtained from the signals from the diode array detec-
tor (SPD-20 A/20AV, Shimadzu, Kyoto, Japan) present in
the spectrometer, where the standards made available had
their respective detection wavelengths adjusted according
to the biochemical identity of the components: gallic acid;
vanillic acid; catechin; epicatechin; caffeic acid; p-coumaric
acid; quercetin; ferulic acid; trans-resveratrol and myric-
etin. The structural identity of the individual components
was adjusted with high molecular specificity and detection
sensitivity in accordance with the chemical nature of the
described patterns.
The method was validated for the parameters of the lin-
ear band (analytical curve of 7 points, in random triplicates),
limits of quantification and detection (with 3 and 6 times
the height of the signal noise, respectively), and precision in
three levels (limit of intermediate point, quantification, and
maximum point of the analytical curve), with n = 3 for each
level. Analysis of variance (ANOVA) was used to validate
the data presented by the analytical curve models.
Evaluation of process parameters
Concentration factor and efficiency of cryoconcentration
process
The concentration factor for each cryoconcentrated fraction
and ice at different stages of BFC was calculated according
to Arend et al. [24]. The concentration factor (Cf) was calcu-
lated as a function of the increase of the total solids content
2523
in each step of the process (TSi) concerning the total solids
content in the initial extract (TS0). The concentration factor
is calculated by the following Eq. 3:
T Si
Cf = (3)
T S0
where TSi is the total solids content for each cryoconcentra-
tion stage (i = 1, 2, 3 and 4th stage) (mg.L− 1); and TS0 is the
total solids content in the initial extract (mg.L− 1).
The efficiency of the BFC process was defined as the
increase in phenolic compounds content for each process
step in relation to the residual phenolic content present in
each ice fraction for each process step. Theoretically, the
lower the content of phenolic compounds present in each
fraction of ice, the higher the concentration of the concen-
trated solution in its respective cryopreservation step. The
efficiency of the process η (%) was calculated as the follow-
ing Eq. 4:
Pi − Pf
η (%) = .100 (4)
Pi
where Pi is the amount of total phenolic compounds pres-
ent in the cryoconcentration solutions for each stage of
the process; and Pf is the content of phenolic compounds
present in the corresponding fraction of ice. Pi and Pf were
expressed in milligrams equivalent to gallic acid per mil-
liliter of extract (mg.GAE.mL− 1).
Biological properties of the cryoconcentrated
samples
Antioxidant activity via ABTS method
The ABTS free radical scavenger activity was determined
according to the ABTS· + (2,2’-azino-bis (3-ethylbenzothia-
zoline-6-sulfonic acid)) method described by Martin-Diana
et al. [21]. In summary, absorbances were measured 6 min
after the addition of the sample and the spectrophotom-
eter reading (UV-Vis mini-1240 spectrometer, Shimadzu,
Tokyo, Japan) was performed at the wavelength of 30 µL
diluted sample. Each sample was reacted as 3 mL of ABTS
radical solution. The same assay was used to construct
the analytical curve, using solutions of gallic acid in the
range of 200–800 mg·mL− 1. The results were expressed in
mg.GAE.mL− 1 of extract. All readings were performed in
duplicate.
13
2524
Antioxidant activity via DPPH method
Also known as 2,2-Diphenyl-1-picrylhydrazyl, the method
of evaluating the antioxidant potential via DPPH assay was
first established by Brand-Williams et al. [25], and per-
formed as described by Martin-Diana et al. and Adorno et
al. [17, 21] with few modifications. The method consists
of reacting 100 µL of each sample with 3.9 mL of DPPH
(2,2-Diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, Inc., St.
Louis, MO, US) methanolic solution (60 µM). After 30 min
of reaction, absorbances were measured at 515 nm wave-
length spectrophotometer (UV-Vis mini-1240 spectrometer,
Shimadzu, Tokyo, Japan) using methanol as white. The
same procedure was used to construct the analytical curve,
using gallic acid solutions in the range of 200–800 mg.
mL− 1. The results were expressed in mg.GAE.mL− 1 of
extract. All readings were performed in duplicate.
Antioxidant activity via ORAC method
Also known as Oxygen Radical Absorbance Capacity, the
ORAC method performed in this study is based on the
exposition by Mazzucotelli et al. [26]. The ORAC method
is performed by the addition of 25 µL of the sample, or Tro-
lox solution ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylic acid - Sigma-Aldrich, Inc., CAS Number:
53188-07-1), in potassium phosphate buffer solution 75
mmol.L− 1 (pH 7.4), incubated at 37 °C for 10 min. Posteri-
orly, 150 µL of disodium fluorescein solution (81 nmol.L− 1)
was added as an indicator, and 25 µL of AAPH dihydro-
chloride (2,2′-Azobis(2-methylpropionamidine - Sigma-
Aldrich, Inc., CAS Number: 2997-92-4) (152 mmol.L− 1)
added as peroxide radical generator. The fluorescence of the
samples was measured every minute (emission and excita-
tion wavelengths of 530 ± 25 and 485 ± 20 nm, respectively)
at 37 °C, for 90 min in a microplate reader (GloMax®
Discover, Promega, Madison, WI, US). The ORAC values
were obtained in comparison with the Trolox curve with a
concentration between 0 and 96 µmol.L− 1 and with the cal-
culation of the area under the curve (AUC) of fluorescein
reduction. The results were expressed as µmol equivalent
of Trolox per mL of sample and the AUC obtained by fol-
lowing Eq. 5:
f1 f2 f3 fn
AU C = 1 + + + +? + (5)
f0 f0 f0 f0
where fn is the fluorescein at one reading cycle (1 min) and
f0 is the fluorescein at the initial time.
13
É. d. S. Almeida et al.
In vitro gastrointestinal digestion simulation
The gastrointestinal digestion simulation assays were per-
formed following the methodology developed by Prestes
et al. and Yuan et al. [27, 28] with few modifications. The
test simulates the conditions of digestion of the cryoconcen-
trated product in the mouth, stomach, duodenum, and ileum
in sequence. Twenty-five grams of each sample of the last
stage of BFC and of in natura sample were prepared in test
tubes. Peristaltic movements and temperature (37 ± 1 °C)
were simulated in a thermostated bath. The enzyme solu-
tions were filtered-sterilized using a 0.22 μm membrane
filter (MF-Millipore™, Sigma-Aldrich, San Luis, Missouri,
USA). Before and during all the analysis, the enzyme solu-
tions were kept cooled and gradually added according to the
digestion steps. The effect of in vitro digestion was evalu-
ated on TPC and the antioxidant activity via ABTS.
Statistical analysis
All data presented in this study were presented as
mean ± standard deviation (SD) in which all tests were per-
formed in triplicate. The differences between means were
compared by Tukey’s least significant difference test (LSD),
with a significance level of 5%. The Pearson correlation
analysis (r) was applied to verify the strength of the cor-
relation between the responses evaluated at p < 0.05. All
analyzes were performed using software (Statistica, v. 8.0,
TIBCO, Palo Alto, CA, USA).
Results and discussion
Total solids content and concentration factor
The increase in the total solids content in the cryocon-
centrated fractions and residual ice beyond the concentra-
tion factor is presented in Fig. 2, and the analytical data in
Table 1. Statistical analysis of the data showed that the BFC
stage had a significant effect on the evolution of total dry
matter (p < 0.05). The significant increase in TSC at each
stage of the process makes the microwave-assisted cryo-
concentration method satisfactory (Fig. 2a) The increase in
TSC ranged from 15.40 mg.L− 1 to 35.29 mg.L− 1 for samples
CC1 and CC4, respectively (Table 1). Such TSC value at
the end of the fourth stage was presented a similar behavior
with the studies carried out by Adorno et al. and Boaventura
et al. [17, 29], who applied the cryoconcentration process to
retain solids in samples of the strawberry juice and extract
of mate (Ilex paraguariensis A. St. Hil.) respectively. Fig-
ure 3 shows the samples of Morinda citrifolia extracts after
each BFC stage.
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties… 2525

Fig. 2 Total solids content in cryoconcentrated fractions and residual samples and ice in each cryoconcentration stage, different superscript
ice (a); and the concentration factor for the presence of solids (b) for lowercase letters indicate a significant difference (p < 0.05) between
each stage of BFC of Morinda citrifolia leaf extract: data are expressed the samples of each BFC stage
as mean ± SD (n = 3) of the total solids content in the concentrated

Table 1 Total solids content in concentrated fractions and residual ice

in each BFC step concentration, and Orellana-Palma et al. [32] on the concen-
Stage of BFC Samples Total solids con- tration of orange juice.
tent (mg·L− 1) Figure 2b illustrates the behavior of the TSC concentra-
-- Control 11.12e ± 0.04 tion factor for samples of M. citrifolia leaf extracts cryocon-
1st CC1 15.40d ± 0.20 centrated. As previously described, Cf indicates how many
Ice 1 3.78hi ± 0.31 times the sample was concentrated compared to the control
2nd CC2 19.12c ± 0.20 sample. The statistical analysis of the data obtained showed
Ice 2 2.70i ± 0.09 that the cryoconcentration steps had a significant effect on
3rd CC3 30.37b ± 0.23
the TSC concentration factor, as expected (p < 0.05) with
Ice 3 4.02 g ± 0.10
values of 1.39 ± 0.04; 1.72 ± 0.02; 2.73 ± 0.02 and 3.18 ± 0.18
4th CC4 35.29a ± 0.32
corresponding to the first, second, third and fourth stage
Ice 4 5.59f ± 0.19
of BFC respectively. According to Aider et al. [30, 33],
Data are expressed as mean ± SD (n = 3) of the total solids content
in the cryoconcentrated samples and ice in each cryoconcentration the effectiveness of the retention of solids via microwave-
stage assisted method occurs due to the low dielectric constant of
Different superscript lowercase letters indicate a significant differ- the ice, compared to that of pure water or aqueous solution
ence (p < 0.05) between the samples of each BFC stage present in the extracts of the leaf of M. citrifolia. Accord-
ing to the authors, the dielectric of pure water is approxi-
An important data to be considered during the BFC mately 78.13. It is well known that microwaves in the range
process refers to the TSC present in the residual ice. It is of 300 MHz up to 300 GHz can effectively heat solid and
noted that the representative TSC values for samples Ice 1, liquid foods. However, some particularities regarding the
Ice 2, and Ice 3 remained statistically similar, with a small use of the microwave in the defrosting process should be
increase in TSC in Ice 4 (p < 0,05), a result that was already discussed.
expected due to the accumulation of solids present in CC4 Unlike laser lights, whose photon energy is several eV,
for this method of cryoconcentration. The TSC present in the microwave photon energy is as small as 5 eV, and its
the ice residue ranged from 3.78 to 5.59 mg.L− 1 between period is in orders of magnitude greater than the electronic
Ice 1 and Ice 4, respectively. According to Orellana-Palma processes that occur in molecules. However, microwaves
et al. [11], the increase in the dry matter content in the ice can control the heating of solids and liquids with high energy
can be explained due to the high content of solids retained efficiency. Thus, water in the ice state is hardly heated by
in the ice fractions with the advance of the process steps. low-frequency microwaves, accrediting the technique as
The same behavior was reported by Aider et al. [30] in the favorable for the application in the retention of solids in
milk whey concentration, Benedetti et al. [31] for whey tofu food solutions [18, 30, 33, 34].

13
2526
Fig. 3 Samples of M. citrifolia leaf extracts after each BFC stage
Total phenolics content
Figure 4 illustrates the behavior of the TPC for samples of
aqueous extract of M. citrifolia leaves applied in the BFC
method. The analytical data together with the statistical
analysis of the data are shown in Table 2. The simultane-
ous behavior of the process efficiency percentage as well
as the concentration factor for phenolic compounds is pre-
sented in Fig. 5. The effect of the freezing concentration
cycle was highly significant (p < 0.05) on the TPC, in the
efficiency percentage and concentration factor in all cryo-
concentrated samples. The TPC increased significantly
during the BFC stages (p < 0.05), thus favoring the reten-
tion of phenolic compounds associated with the increase
in the total solids content already mentioned. The values
varied from 0.70 ± 0.02 mg.GAE.mL− 1 for the control
sample, 0.82 ± 0.02 mg.GAE.mL− 1 for CC1 finished with
4.01 ± 0.08 mg.GAE.mL− 1 in the last stage of the process
(Fig. 4). The accumulation of TPC in the last stage of BFC
represented a concentration factor of 5.74 ± 0.05 times
higher than the control sample and reaching an efficiency of
up to 94.86% ± 0.02 retention of phenolic compounds in the
last stage of cryopreservation (Fig. 5), a similar result in the
percentage recovery of bioactive components in an apple
juice cryoconcentration process was reported by Qin et al.
[35]. According to Adorno et al. [17], the maintenance of
phenolic compounds during the process is enhanced at low
temperatures. The intermediate freezing time of the extract
13
É. d. S. Almeida et al.
is also, according to Safiei and Alaudin [36], an important
variable in the most efficient condition to simultaneously
produce a percentage increase and preservation of the TPC
contents. The behavior described in Fig. 4, as previously
discussed, is a result of an increase in dry matter, conse-
quently causing an increase in TPC, thus suggesting the
preservation of these compounds.
Regarding the residual ice fractions resulting from cryo-
concentration, it is noted that there was no significant differ-
ence (p < 0.05) in the TPC. This fact shows the effectiveness
of BFC process, as it is clear that almost all of the TPC in the
studied samples was directed to the concentrated samples
(CC1 to CC4), resulting in greater purity in the ice samples.
As described by Orellana-Palma et al. and Boaventura et
al. [13, 37], due to hydrogen bonds, phenolic compounds
can bind to a large number of water molecules. As the con-
centration of phenolics is increased in the solution, intersti-
tial water becomes less available for freezing. As a result,
during the procedure of separating the ice and the concen-
trated fluid, the frozen fractions remain with small portions
retained of phenolic compounds, which explains the per-
manence of these chemical species in the ice. According to
Zielinski et al. [38], this is also reflected in the increase in
viscosity at the interface (solute-ice), consequently resulting
in greater difficulty of mass transference.
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties… 2527

Fig. 4 Total phenolic content (TPC) in the cryoconcentrated fluid and in the ice fractions at each BFC stage

Table 2 Total phenolics content for the cryoconcentrated and ice frac-
tions of the aqueous extract of M. citrifolia leaves Viscosity tests of cryoconcentrated samples
Stage of BFC Samples Total pheno-
lics content Figure 6 illustrates the graph of viscosity versus shear rate
(mgGAE·ml− 1) for control samples and cryoconcentrate (CC1 to CC4) of
-- Control 0.70d ± 0.02 the aqueous extract of M. citrifolia leaves. The viscosities
1st CC1 0.82d ± 0.02
are presented in the range of shear rate between 200 and
Ice 1 0.11e ± 0.00
1800 s− 1 due to the limitations of the equipment. It is known
2nd CC2 2.33c ± 0.03
that the increase in viscosity occurs due to a greater deposi-
Ice 2 0.15e ± 0.01
tion of solids in the concentrated fractions, which is a reflec-
3rd CC3 3.10b ± 0.08
Ice 3 0.17e ± 0.02
tion of the increase in mass transfer at the ice-concentrate
4th CC4 4.01a ± 0.08 interface, as described by Zielinski et al. [38]. However,
Ice 4 0.21e ± 0.01 although it is an expected phenomenon that corroborates
Data are expressed as mean ± SD (n = 3) of the total phenolics content the effectiveness of the separation of the solute from the ice
in the cryoconcentrated samples and ice in each cryoconcentration fraction, the increase in viscosity is according to Sánchez
stage et al. [39] one of the factors that limit the efficiency of the
Different superscript lowercase letters indicate a significant differ- process. Few studies experimentally address the rheological
ence (p < 0.05) between the samples of each BFC stage
behavior of fluids concentrated, such as those developed by
Belén et al. [40], analyzing the behavior of functional com-
pounds during frozen tofu whey concentration; and Sánchez

13
2528
Fig. 5 Percentage efficiency in TPC retention and concentration factor for TPC for each stage of BFC of M. citrifolia leaf extracts: different super-
script lowercase letters indicate a significant difference (p < 0.05) between the samples of each BFC stage for each analyzed variable
et al. [41] analyzing the frozen serum concentration in a
pilot plant based on a descending film.
Although the increase in viscosity interferes with the effi-
ciency of the process, it is noted that here, this interference
was not abrupt, since significant differences (p < 0.05) are
noted in the TSC of the ice fractions in steps 3 and 4 of the
process (Table 1). Moreover, the percentage of efficiency in
the retention of TPC did not decrease significantly (Fig. 5).
This suggests that the addition of new BFC stage may result
in an inverse relationship between efficiency and viscosity,
as demonstrated by Orellana-Palma et al. [32]. In Fig. 6, it
can be seen that the viscosity remains at constant values,
without great variations for higher shear rate in all stages of
BFC. This viscosity behavior is considered characteristic of
Newtonian fluids, whose viscosity is constant. Examples of
typical Newtonian fluids are water, coffee, beer, soft drinks,
more honey, sugar syrup, milk, filtered juices, orange juice,
and wines [42, 43]. However, at higher concentrations (CC4,
for example), viscosity values are higher, as expected. In
13
É. d. S. Almeida et al.
our study, samples referring to the last stage of cryocon-
centration reached viscosity values ranging from 3.8 to 4.9
mPa·s (Fig. 6).
Table 3 presents the adjusted models (p < 0.05) for the
cryoconcentrated samples (CC1 to CC4) of the aqueous
extracts of M. citrifolia leaves. The Power Law and Newton
models were applied to describe the rheological behavior
of the samples at each stage of the process. Both rheologi-
cal models fitted the experimental data satisfactorily and
consistently. However, based on the statistical treatment of
these data, it is concluded that the Power Law model was the
most appropriate, as it presents the best adjustments (high-
est values of R2). Also, the Power Law model presented val-
ues for the fluid behavior index (n) close to 1, an aspect that
is consistent with a characteristic behavior of a fluid with
Newtonian behavior for the analyzed samples.
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties… 2529

Fig. 6 Apparent viscosity versus shear rate for each stage of BFC in the concentrated extracts of M. citrifolia leaves

Table 3 Rheological parameters obtained by adjusting the data to the Power Law and Newton model, for the aqueous extract of cryoconcentrated
M. citrifolia
Sample Power law Newton model
K (Pa·sn) n R² µ (Pa·s) R²
Control 0.0018 0.96 0.98 1.48e ± 0.20 0.95
CC1 0.0021 0.99 0.99 2.01 cd ± 0.48 0.91
CC2 0.0014 1.00 0.95 3.10bc ± 0.28 0.95
CC3 0.0006 1.12 0.96 3.50b ± 0.14 0.95
CC4 0.0003 1.17 0.96 4.00a ± 0.07 0.95
Data are expressed as mean ± SD (n = 3)
Values followed by the same letter do not differ (ANOVA, followed by Tukey’s test; p˂0.05)
Different symbols indicate significant differences (p < 0.05) for each rheological model separately analyzed
µ is the absolute or dynamic viscosity (Pa·s); K the consistency index (Pa·sn); the behavior index (dimensionless) and R 2 is the correlation
n
coefficient

13
2530 É. d. S. Almeida et al.

Data are expressed as mean ± SD (n = 3). Different superscript letters indicate a significant difference (p < 0.05) between the control extract and the cryoconcentrated samples of each BFC stage
HPLC-DAD-MS assays

0.287 ± 0.017ab

0.002 ± 0.001ab
0.144 ± 0.009a
13.66 ± 0.469a
2.774 ± 0.252a
0.300 ± 0.022a
0.679 ± 0.004a
4.856 ± 0.154a
0.920 ± 0.012a

4.311 ± 0.123a
M. citrifolia is widely known due to its nutritional charac-
ter, where about 200 biochemical compounds with bioactive
properties have already been identified and isolated from

CC4
different parts of this plant [44]. It is also important to note
that the biochemical composition of these compounds dif-

0.234 ± 0.044bc

bc

0.002 ± 0.003bc
b
b

b
b

4.034 ± 0.410b
0.601 ± 0.009b

3.841 ± 0.232b
0.094 ± 0.008
12.09 ± 0.982
1.883 ± 0.356
0.246 ± 0.029
0.574 ± 0.006
fers not only in terms of quantity according to the structure
of the plant, but also concerning the place of origin and har-
vest time of these structures [45]. The phenolic compounds
identified and their respective amounts isolated from the

CC3
control sample and the cryoconcentrated samples are shown
in Table 4. Figure 7 presents an HPLC-DAD-MS chromato-

bcd
gram representative of the phenolic compounds identified

0.193 ± 0.032c
c
c

3.340 ± 0.173c
0.119 ± 0.005c
0.002 ± 0.001c
2.779 ± 0.376c
c
0.076 ± 0.002
8.332 ± 0.291
1.536 ± 0.228

0.410 ± 0.003
0.200 ± 0.011
and quantified in the extracts of M. citrifolia leaves.
The detection of phenolic compounds made use of a
validated method in which it presented low detection limits
(0.0104 mg.g− 1; 0.0269 mg.g− 1; 0.0345 mg.g− 1; 0.01395 mg

CC2
.g− 1; 0.0105 mg.g− 1; 0.0090 mg.g− 1; 0.0082 mg.g− 1;
0.0042 mg.g− 1; 0.0075 mg.g− 1; 0.0028 mg.g− 1; according

2.710 ± 0.823 cd
0.136 ± 0.005d
d
d
d
d
d

0.031 ± 0.010d
0.001 ± 0.000d
1.981 ± 0.044d
0.068 ± 0.004
5.788 ± 0.387
1.246 ± 0.238
0.162 ± 0.027
0.311 ± 0.001
to the listing of compounds in Table 4 identified from 1 to

Table 4 Major phenolic compounds identified via HPLC-DAD-MS assays for control and cryoconcentrated samples
10 respectively). The analytical curves showed linear behav-
ior from the limit of quantification up to 26 mg.g− 1, with
models without lack of adjustment (p < 0.05) and distribu-
tion of random residues. A standard deviation ranging from CC1
0.29 to 4.95% was calculated, considering the three levels

0.001 ± 0.000d
0.065 ± 0.006e
e
e
e
e
e

1.291 ± 0.248e
0.007 ± 0.002e

0.099 ± 0.047e
0.054 ± 0.002
2.178 ± 0.950
0.063 ± 0.018
0.008 ± 0.002
0.013 ± 0.001
(limit of quantification, intermediate point, and maximum
curve). Satisfactory values were obtained ​​as recommended
by the International Union of Pure and Applied Chemistry
Phenolic compounds (mg/g) ID Wavelength (nm) Retention time (min) Control

(IUPAC), as well as demonstrated by Thompson et al. [46].

As seen in Table 4, the levels of phenolic compounds
increased as the stages of BFC progressed. The total phe-
nolic content representative of the last stage of BFC was
10.62

12.44
10.11

11.09
27.93 mg.g− 1 for all species detected and quantified. This
4.42
5.94
7.34
8.08
8.28
9.92

value represents an increase of 7.4 times the total content

present in the control sample or 640.8% ± 0,03 concerning
the initial content. Lin et al. [47] presented phenolic acids
as the main polyphenolic components in noni juice. In our
study, the main components were gallic acid, vanillic acid,
272
279
279
324
262
310
323
370
306
372

caffeic acid, p-coumaric acid, and feluric acid, with respec-

tive quantitative increases of 4.48; 2.70; 39.86; 56.58, and
3.75 times regarding the initial quantities detected in the
control fractions (Table 4). The increase in the quantities
of these species was expected and corroborated with the
1
2
3
4
5
6
7
8
9
10

premise that the BFC process effectively separated the sol-

ids in greater quantity in the CC4 fractions. Lin et al. [48]
detected the presence of chlorogenic acid in samples of noni
juice, which allowed the reduction of the risk of cardiovas-
trans-Resveratrol

cular diseases by reducing the oxidation and inflammation

p-Coumaric acid

of LDL-C. Bittová et al. [49] also promoted the identifica-

Vanillic acid

Caffeic acid

Ferulic acid
Epicatechin
Gallic acid

Quercetin

Myricetin

tion among other organic acids, caffeic acid, and p-cumaric

Catechin

acid in several noni products (Morinda citrifolia L.). At last,

13
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties…
Fig. 7 Chromatogram containing the identification of the main phe-
nolic species present in the control sample and the cryoconcentrated
samples of the aqueous extracts of the leaves of M. citrifolia: the wave-
Nowak et al. [50] detected the presence of caffeic acid in
noni berries. The aforementioned studies highlight the pres-
ence of organic acids in M. citrifolia and its relationship,
among other phytotherapeutic properties, to antioxidant
activity.
Epicatechin is a flavonoid present especially in woody
plants in the forms (+)-catechin and (-)-epicatechin (cis) and
stands out for having antioxidant properties [51]. In the pres-
ent study, a significant increase (p < 0.05) was achieved in
the amounts of epicatechin of ~ 44.67 times in the last stage
of cryoconcentration in comparison to the content present in
the control samples (Table 4). The occurrence of epicatechin
in samples of M. citrifolia leaves corroborates the studies
developed by Shalan et al., Osman et al. and Ahmadi et al.
[52–54], which promoted the isolation of epicatechin via
HPLC, in addition to the analysis of the phytotherapeutic
efficacy of this bioactive in different biological tests. This
2531
lengths, the retention time, and the identification of each compound are
detailed in Table 4
proves that this compound in the leaves of M. citrifolia pre-
sented satisfactory experimental results concerning the anti-
inflammatory activity and even in bone regeneration tests.
Catechin was the most detected phenolic compound after
the four stages of BFC. Its increase in relation to the sample
in natura was approximately 6.27 times (Table 4) and its
presence in M. citrifolia is corroborated by Nowak et al.
and Shalan et al. [50, 52]. In the latter study, the amounts of
catechin were detected in samples obtained from the leaves
of M. citrifolia. Thus, the high amount obtained after the
end of the BFC process is fundamentally due to the greater
deposition of bioactive compounds in the CC4 samples.
Moreover, as BFC advanced, the content of total solids
and, consequently, bioactive components also increased
(Table 1; Fig. 2).
According to Ay et al. [55], quercetin is a natural fla-
vonoid found abundantly in vegetables and fruits. As
13
2532 É. d. S. Almeida et al.

Table 5 Summary of the results of the analysis of antioxidant activity via ABTS, DPPH and ORAC methods for the cryoconcentrated and ice
fractions of the aqueous extract of M. citrifolia leaves
Sample/ ABTS method DPPH method ORAC method (µMOL
Antioxidant activity method (mgGAE·ml− 1) (mgGAE·ml− 1) TE.mL− 1)
de e
Control 0.13 ± 0.02 0.87 ± 0.01 2.49 g ± 0.83
de d
CC1 0.18 ± 0.01 1.28 ± 0.01 17.61c ± 0.81
Ice 1 0.05 g ± 0.01 0.11f ± 0.00 7.45f ± 0.61
CC2 0.25c ± 0.06 1.72c ± 0.00 20.91b ± 0.53
Ice 2 0.08f ± 0.01 0.10f ± 0.00 9.03e ± 0.39
CC3 0.46b ± 0.03 1.80b ± 0.00 71.66a ± 1.87
Ice 3 0.13de ± 0.00 0.11f ± 0.00 10.45d ± 0.24
CC4 0.60a ± 0.04 1.90a ± 0.01 76.91a ± 3.79
Ice 4 0.15de ± 0.00 0.12f ± 0.00 10.61d ± 0.21
Data are expressed as mean ± SD (n = 3)
Values followed by the same superscript letters do not differ (ANOVA, followed by Tukey’s test; p˂0.05)
Different superscript letters indicate significant differences (p < 0.05) for each stage of BFC analyzed in each method separately

mentioned by Deng et al. and Nitteranon et al. [56, 57],
the potential anti-inflammatory activity of this bioactive
through samples of noni fruits includes an effective activ-
ity. Also, Masuda et al. [58] described the antioxidant activ-
ity of quercetin present in noni. In our study, the content of
quercetin increased significantly (p < 0.05) compared to the
initial content after the four stages of BFC, representing an
increase of approximately 128 times its content (Table 4).
The presence of quercetin in leaves of M. citrifolia is also
confirmed by Deng et al. and Sevallo & Rocha [59, 60], cor-
roborating our study.
The other phenolic compounds detected in this study can
be seen in Table 4. In short, there was a linear behavior in the
face of the increase in the content of phenolic compounds,
where no reductions in the amounts of the compounds were
observed after 4 BFC stages.
Antioxidant activity assays
The ABTS, DPPH, and ORAC methods were evaluated to
verify whether the BFC caused an increase in the antioxi-
dant activity values of the studied extracts. Table 5 shows a
comparison between the effect of radical scavenging activ-
ity using the ABTS, DPPH, and ORAC assays, for the aque-
ous extracts of M. citrifolia leaves.
Significant differences (p < 0.05) were achieved in the
different concentrated fractions (CC1 to CC4) accord-
ing to both methods of antioxidant activity, ABTS and
DPPH (Table 5). In both cases, the radical scavenging
activity increased as the BFC steps advanced. Based on
the analytical results regarding the antioxidant activity
via the ABTS test, the BFC steps had a significant effect
(p < 0.05) on the activity of free radical capture. Values ​​of
0.13 ± 0.02 mg.GAE.mL− 1 were observed for the sample
in natura, increasing to 0.18 ± 0.01 mg.GAE.mL− 1 in CC1,
comprising 0.60 ± 0.04 mg.GAE.mL− 1 in the last step of
the process, a value greater than 4.5 times higher in the
antioxidant activity via ABTS for CC4 compared to the
in natura sample (Table 5). Concerning the DPPH tests,
a significant effect (p < 0.05) of the process steps on the
antioxidant activity was also obtained. The values ranged
from 0.87 ± 0.01 mg.GAE.mL− 1 for the sample in natura,
increasing to 1.28 ± 0.01 mg.GAE.mL− 1 in CC1, ending
with 1.90 ± 0.01 mg.GAE.mL− 1 in the last step of the pro-
cess. Therefore, the antioxidant activity exposed by the
DPPH method showed higher average values than in the
analysis via ABTS.
In similar studies involving the cryoconcentration of
apple juice and cabernet sauvignon wine by Wu et al. and
Zielinsky et al. [9, 38] respectively, the authors report that
there is a strong indication that the increase in antioxidant
activity is closely related to the increase in the TPC of cryo-
concentrate extracts. Besides, according to Krishnaiah et al.
[61], most phenolic compounds belong to flavonoids. These
are considered as part of the antioxidant members. Thus, the
phenolic content is closely related to the radical scavenging
activity. The antioxidant potential is also attributed to the
content of biochemically active compounds with the ability
to interact in the elimination of free radicals [62].
In summary, the antioxidant activity increased with the
advance of the BFC stages for ABTS and DPPH methods,
thus showing the efficiency of the methods to increase the
free-radical-scavenging capacity of the aqueous extracts of
M. citrifolia leaves. However, according to Arend et al. [24],
it is difficult to compare the results obtained by different
methods of determining antioxidant activity. Nascimento et
al. [63] reported that the TPC assays with Folin-Ciocalteu
reagents determined both free phenolics and bound pheno-
lics in M. citrifolia products. Moreover, the bound antioxi-
dants in noni may not contribute to free-radical-scavenging
activity in the DPPH assay. Another important aspect is
that the reactions of antioxidants to the Folin-Ciocalteu

13
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties…
Fig. 8 Behavior of TPC and antioxidant activity via ABTS method in the in vitro simulation of gastrointestinal digestion
reagent were different from DPPH free radicals’ reactions
in the TPC assays. The Folin-Ciocalteu reagent is sensitive
to a broad range of substrates that are easily oxidized, but
the DPPH free radicals exhibit different sensitivity to vari-
ous antioxidants, which present fast, intermediate, or slow
kinetic reactions to the DPPH free radicals [64].
Finally, ORAC is based mainly on a radical initiator to
generate the peroxyl radical, and its mechanism of action is
the transfer of hydrogen atoms [65]. Table 5 illustrates the
effect of antioxidant activity using the ORAC method for
cryoconcentrated extracts. Similar to what happened with
the use of ABTS and DPPH methods, the stages of the BFC
process had a significant effect (p < 0.05) on the antioxidant
activity by the ORAC method. In this case, the values of
this parameter increased as the stages of BFC progressed.
However, it is noted that significant differences (p < 0.05)
were obtained for the cryoconcentrated fractions except for
samples CC3 and CC4. ORAC assay values for concen-
trated fractions ranged from 2.49 ± 0.83 µMOL TE.g− 1 for
the control sample, 17.61 ± 0.81; 20.91 ± 0.53; 71.66 ± 1.87
and 76.91 ± 0.81 µMOL TE.g− 1 for fractions CC1, CC2,
CC3 and CC4 respectively. Although analytically, the value
of antioxidant activity at the end of CC4 was about 31 times
greater compared to the value obtained in the fresh sample,
we saw that there was no significant difference (p < 0.05)
between CC3 and CC4 (Table 5), suggesting that the ORAC
assay could be completed in step 3. Such response is similar
to the results presented by Adorno et al. and Arend et al.
[17, 24]. Both works suggested that in an industrial process,
BFC could be interrupted in the third stage, thus reducing
the processing time and the consumption of energy.
2533
In vitro assays of simulation of gastrointestinal
digestion
Figure 8 illustrates the behavior of TPC and antioxidant
activity via ABTS, of samples in natura and CC4 samples
submitted to the in vitro simulation of gastrointestinal diges-
tion by the BFC by microwave-assisted. One of the most
important considerations to be carried out in the analysis of
the behavior of such variables submitted to the simulated
digestion process is that the evaluation of the parameters
in each cryoconcentrated and ice sample will be centered
mainly on how much the TPC and the antioxidant activity it
is susceptible to sudden changes in pH [28].
Considering the behavior of TPC during in vitro diges-
tion in different gastrointestinal digestion sites in the thaw-
ing method applied, it is noted that statistically the TPC
values were not sharply affected by the enzymatic attack
promoted by simulated digestion. In this case, there were no
intense reductions in TPC values for CC4 samples in each
digestion site (Fig. 8a). Moreover, the applied BFC method
had a significant effect on the behavior of TPC for CC4 sam-
ples during simulated digestion when compared to samples
in natura. In summary, there were significant differences
(p < 0.05) regarding the enzymatic attack that simulated
digestion in different portions of the gastrointestinal tract,
mainly those related to digestion in the stomach, either for
the CC4 sample or for the sample in natura without passing
through the digestive process (Table 6). However, the TPC
values for cryoconcentrated samples remained much higher
when compared to samples in natura even submitted to the
digestive process. For CC4 samples, TPC showed values
of 1.10 ± 0.14 mg.GAE.mL− 1 before simulated digestion;
1.30 ± 0.07 mg.GAE.mL− 1 for digestion referring to the
mouth; 1.15 ± 0.02 mg.GAE.mL− 1 for stomach digestion;
13
2534 É. d. S. Almeida et al.

Table 6 Behavior of TPC and antioxidant activity via ABTS method during in vitro simulation of gastrointestinal digestion
Representative samples Gastrointestinal region Behavior of TPC Behavior of antioxidant activity via ABTS
(mgGAE·ml− 1) (mgGAE·ml− 1)
f
Control samples Initial 0.49 ± 0.01 0.04gh ± 0.00
f
Mouth 0.53 ± 0.00 0.05 g ± 0.00
Stomach 0.42f ± 0.00 0.01i ± 0.00
Duodenum 0.50f ± 0.00 0.01i ± 0.00
Ileus 0.51f ± 0.00 0.21 f
± 0.00
CC4 samples CC4 Initial 1.10e ± 0.14 0.15 cd ± 0.00
CC4 Mouth 1.30bc ± 0.07 0.17b ± 0.01
CC4 Stomach 1.15d ± 0.02 0.07e ± 0.01
CC4 Duodenum 1.31bc ± 0.03 0.13d ± 0.01
CC4 Ileus 1.41a ± 0.01 0.47a ± 0.02
Data are expressed as mean ± SD (n = 3)
Values followed by the same superscript letters do not differ (ANOVA, followed by Tukey’s test; p˂0.05)
Different superscript letters indicate significant differences (p < 0.05) for each sample evaluating the methods separately

1.31 ± 0.03 mg.GAE.mL− 1 for digestion referring to the
duodenum region, and 1.41 ± 0.01 mg.GAE.mL− 1 for diges-
tion referring to the illeus region (Table 6). Theoretically,
the TPC should remain intact at the end of the digestion
of the CC4 samples. The significant difference (p < 0.05)
verified in the CC4 samples suggests that the BFC method
affects the significant variation of the TPC for this region
of the gastrointestinal tract. Ryu & Koh [66], evaluating in
vitro digestion process to verify the stability of the anthocy-
anin content Rubus occidentalis L., found that during simu-
lated digestion, gastric digestion had no significant effect on
anthocyanins.
It is important to note that variations in TPC and anti-
oxidant activity prescribe an expected behavior during gas-
trointestinal digestion in vitro. Wu et al. [67], verified that
the different pH conditions and digestive enzymes present
in in vitro digestion assays directly interfere in the chemical
composition and in the dynamics of the molecular weights
of bioactive compounds present in loquat leaves (Eriobotrya
japonica L.). In fact, as described in Table 6, the lowest val-
ues ​​of TPC and antioxidant activity for CC4 samples sub-
mitted to in vitro digestion occurred in the digestion stage
in the stomach, whereas in the intestinal portion, duodenum
and ileum, these values ​​increased significantly. According to
Liu et al. [68], in plants, phenolic compounds as secondary
metabolites generally combine with some macromolecules,
such as proteins and carbohydrates, to form copolymers,
thus keeping the bioaccessibility of phenolic substances
low. During digestion of the gastrointestinal tract, variations
between the acid and alkali content, as well as the action
of some digestive enzymes, can destroy the plant cell wall
and hydrolyze the binding bonds, thus allowing the release
of more phenolic substances at the end of the gastro intes-
tinal tract. Moreover, De Santiago et al. [69] reported that
the bioaccessibility of phenolic compounds increased with
microwaving and griddling processes. Likewise, Dalmau et
al. [70] lets us know his study involving the influence of
freezing on the bioaccessibility of bioactive compounds of
beet (Beta vulgaris), that the morphology of ice crystals and
the structure of the porous layers formed during the freezing
stages at a very low temperature they can result in an easier
release of phenolic compounds during the in vitro biodiges-
tion process.
Considering the effect of antioxidant activity via ABTS
for CC4 samples submitted to the in vitro digestion process,
it is notable that the digestion process significantly affected
free-radical-scavenging activity in the BFC method used to
the different in vitro digestion sites (p < 0.05). The highest
value of antioxidant activity via ABTS for the CC4 sample
was found in the ileum region: 0.47 ± 0.02 mg.GAE.mL− 1
(Table 6). It is also noted that the region corresponding to
digestion in the stomach was the one that most affected radi-
cal capture activity via ABTS when compared to other in
vitro digestion sites (Fig. 8b). Such reduction in antioxidant
activity by ABTS in the digestion region corresponding to
the stomach may be related to how the pH influences the
principle of the antioxidant reaction, where the compound
can supply electrons or hydrogen. According to Lee et al.
[71], this is related to the principle of the antioxidant reac-
tion. However, the antioxidant mechanism of the bioactive
compounds present in the different structures of M. citrifo-
lia is not yet fully understood due to their high molecular
weight, complex constituents, and its respective structures
[72]. Yuan et al. [28] describe that the low pH value exerted
by simulated in vitro digestion in the stomach region can
contribute precisely to the ability to supply electrons to cap-
ture free radicals that such bioactive compounds have, thus
compromising the reduction of antioxidant activity in this
region.

13
Effect of the multi-stage block freeze concentration process on the physicochemical and biological properties… 2535

laborated in supervision and Writing - Review & Editing.

Conclusion
Funding This work was funded by CNPq (National Council of Sci-
BFC by microwave-assisted process provided satisfactory entific and Technological Development, Brazil), FAPESC (Santa Ca-
results concerning the accumulation of bioactive com- tarina State Research and Innovation Support, Brazil), which provide
financial support to this study.
pounds as well as promoting the maintenance of the nutri-
tional properties of M. citrifolia leaves extracts. It could be
noted that the progression of the freeze and thaw cycles had Declarations
a significant effect on the increase of the total solids content, Conflict of interest Statement The authors declare that they have no
with high dry matter content at the end of the process, being known competing financial interests or personal relationships that
3 times higher than the initial value. Moreover, as the stages could have appeared to influence the work reported in this paper.
of the BFC progressed, higher efficiency in the retention
of total phenolic content and higher amounts of phenolic
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