foods
Article
Concentrating Model Solutions and Fruit Juices Using CO2
Hydrate Technology and Its Quantitative Effect on Phenols,
Carotenoids, Vitamin C and Betanin
Alexander Rudolph *,†, Amna El-Mohamad , Christopher McHardy †and Cornelia Rauh †
Citation: Rudolph, A.; El-Mohamad,
A.; McHardy, C.; Rauh, C.
Concentrating Model Solutions and
Fruit Juices Using CO2Hydrate
Technology and Its Quantitative
Effect on Phenols, Carotenoids,
Vitamin C and Betanin. Foods 2021,10,
626. https://doi.org/10.3390/
foods10030626
Academic Editor: M. Azad Emin
Received: 17 February 2021
Accepted: 11 March 2021
Published: 16 March 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
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nal affiliations.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Department of Food Biotechnology and Food Process Engineering, Technische Universität Berlin, Straße des 17.
*Correspondence: a.r[email protected]
† Current address: Königin-Luise-Straße 22, D-14195 Berlin, Germany.
Abstract:
Fruits have an important economic impact in the context of plant-based food production.
The consumption of fruit juices, mostly produced from concentrates, is particularly noteworthy.
Conventional concentration methods do not always enable a sustainable and gentle concentration.
The innovative gas hydrate technology addresses this point with its energy-saving, gentle character,
and high concentration potential. In this study, the concentration of fruit juices and model solutions
using CO
2
hydrate technology was investigated. To find a suitable operating point for hydrate
formation in the used bubble column, the hydrate formation in a water–sucrose model solution was
evaluated at different pressure and temperature combinations (1, 3, 5
°
C and 32.5, 37.5, 40 bar). The
degrees of concentration indicate that the bubble column reactor operates best at 37.5 bar and 3
°
C.
To investigate the gentle processing character of the hydrate technology, its quantitative effects on
vitamin C, betanin, polyphenols, and carotenoids were analyzed in the produced concentrates and
hydrates via HPLC and UV/VIS spectrophotometry. The results for fruit juices and model solutions
imply that all examined substances are accumulated in the concentrate, while only small amounts
remain in the hydrate. These amounts can be related to an inefficient separation process.
Keywords: food processing; fruit juice concentration; gas hydrate; food quality; sustainability
1. Introduction
Consumers are increasingly aware of the importance of healthy and plant-based
foods. Among these foods, fruit juices enjoy great popularity. They are considered to be
both natural and healthy. Health benefits are directly related to phytochemicals, such as
polyphenols or carotenoids, which are present in fruits [
1
]. For this reason, juices experience
high demand in the market. Across geographic regions in 2010, 0.66 to 0.013 servings/day
were consumed worldwide while the global market revenue was around $111 billion in
2014 with an annual growth of 3% between 2010 and 2014 [
2
,
3
]. In 2018, 35.9 billion L of fruit
juice and nectar were consumed worldwide, with 9.1 billion L being consumed in the EU [
4
]
and 2.3 billion L consumed in Germany [
5
]. Large quantities of fruit juices are concentrated
during production. Thereby, further processing and storage properties are improved,
while, at the same time, logistical advantages open up, as costs for packaging, storage,
and transport can be saved [
6
,
7
]. 30.35 billion L of the global fruit juice consumption were
related to powdered and concentrated juices in 2018 [
8
]. In the EU, 3.8 billion L of the
consumed juices were produced from concentrates in 2018 [
9
], while the corresponding
value for Germany was 1.39 billion L in 2014 [10].
Removing water using conventional methods involves a high level of equipment and
energy consumption. In many cases, evaporation is used to concentrate juices, since a de-
gree of concentration up to 85% can be reached [
7
]. Because of the prevailing temperatures
Foods 2021,10, 626. https://doi.org/10.3390/foods10030626 https://www.mdpi.com/journal/foods
Foods 2021,10, 626 2 of 17
of at least 70
°
C [
6
], 180 to 2160 kJ/kg water are required to remove water from
juices [11]
.
Besides, the process impairs product quality, because heat-sensitive substances, like vi-
tamins or polyphenols, are destroyed or altered [
6
,
11
,
12
]. Moreover, recovery systems
are used to maintain product quality concerning volatile compounds, such as aroma [
13
].
Freeze concentration is available as an alternative to avoid the loss or damage of heat-
sensitive or volatile substances [
6
,
7
]. It is based on a multistage crystallization of water
to ice and its separation (e.g., by centrifuges). Thereby, a degree of concentration of up to
55% can be reached [
7
,
11
]. The typical energy requirement of 936–1800 kJ/kg water in this
process is less than the amount of energy that is required for evaporation [
11
]. Nevertheless,
there is still a need to improve conventional concentration methods for sustainability and
energy efficiency reasons.
The need for a resource- and product-saving process has brought gas hydrate technol-
ogy into the focus of industrial and scientific research. Gas hydrates were discovered in
the early 19th century by Sir Humphry Davy, but their first industrial relevance came in
1934 when Hammerschmidt discovered that hydrates were responsible for blocking gas
pipelines [
14
]. More recent scientific studies concern carbon capture and storage [
15
–
19
],
water withdrawal from ionic liquids [
20
], and seawater desalination [
21
–
24
]. Moreover,
hydrate technology is used in the food sector, as it provides an innovative approach to con-
centrate juices with low energy requirements of 252–360 kJ/kg water [
11
]. The conditions
for hydrate formation are in the range of low temperatures (below 27
°
C) and moderate
pressures (above 6 bar), depending on the process gas [
25
–
27
]. CO
2
hydrate forms between
30 bar and 80 bar and from 1
°
C to 8
°
C [
11
]. This combination of temperature and pressure
enables gentle and sustainable processing. So far, the concentration of liquid foodstuffs
using gas hydrate technology has been carried out on a laboratory scale with fruit and
vegetable juices or coffee [
11
,
13
,
28
–
33
]. The main focus of the studies concerning juices was
either on hydrate formation kinetics and equilibrium conditions or achievable degrees of
concentration, indicating the need for new data, especially on the preservation of valuable
fruit juice components. It was possible to concentrate apple juice and orange juice in a
bubble column reactor to values between 20
°
Brix and 27
°
Brix at 37 bar and
2.5 °C
with a
yield of 40% [
11
]. Furthermore, the influence of pressures between 30 bar and
45 bar
was
analyzed in more detail. From 30 bar to 37 bar and 42.5 bar to 45 bar produced concentrates
had sugar contents from 12
°
Brix to 15.5
°
Brix, whereas, at 40 bar, concentrates showed
values of 20
°
Brix [
32
]. Besides temperature and pressure, the ratio of sample volume to
reactor volume directly influences the hydrate formation. For a bubble column configura-
tion, the optimum ratio was found between 33% and 40% [
11
,
32
]. For stirred tanks, it has
been reported that the ratio is between 33% and 35% [
13
,
28
,
29
]. Concentrates with over
40 °Brix could be produced with this ratio in stirred tanks at 1 °C to 2 °C and 35 bar [13].
The results reported in the literature highlight the importance of choosing the optimal
temperatures and pressures. However, hydrate formation conditions for juices are shifted
towards lower temperatures and higher pressures as compared to CO
2
-water systems
due to inhibitory effects of food ingredients [
29
,
31
–
33
]. Especially, sugars, such as sucrose,
fructose, and glucose, are known for inducing this shift [
34
], because they are the major
components in juices. Nevertheless, the presented studies imply that concentrating juices
using CO
2
hydrate technology is promising, even though further process optimization is
required to reach the required degree of concentration for industrial applications. More-
over, there is a need for studies evaluating the concentrate quality, since no parameters
concerning the preservation of valuable fruit juice compounds have been evaluated so far.
Data that were collected on this topic for the first time would contribute to underlining
the advantage of the innovative gas hydrate technology over conventional concentration
methods. Therefore, several valuable juice components have to be investigated to show
that product quality is not impaired during fruit juice concentration using gas hydrate
technology. To estimate the preservation of fruit juice compounds, such as polyphenols,
carotenoids, or vitamins, knowledge of hydrate formation and structure is indispensable.
The basic principle of gas hydrate technology is based on water molecules (host molecules)
Foods 2021,10, 626 3 of 17
forming an ice-like cage structure in whose cavities guest molecules with low molecular
weight are trapped [
14
]. Water builds up the cage structure, while the guest molecules
stabilize the gas hydrate by van der Waals forces [
35
]. The thermodynamically preferred
crystal structure will form, depending on the combination of temperature, pressure, and
the availability of hydrate-forming guest components [
14
]. To this day, three crystal struc-
tures sI, sII, and sH have been identified [
14
,
26
,
27
,
36
]. CO
2
as a food-safe gas forms sI
hydrates [
35
]. Water-soluble acid gases, hydrophobic components, water-soluble polar
components, and water-soluble alkylammonium salts are other known types of guest
molecules in hydrate structures [
37
]. The main aspects defining whether a substance can
participate in hydrate formation are its shape, size, and chemical nature [35,38].
Hydrate formation in technical systems is governed by the reaction and transport
kinetics as well as the equilibrium thermodynamics. The driving force is proportional to
the distance of the prevailing temperature and pressure conditions from the equilibrium
state [39]
. The main principle to induce hydrate formation is to inject gas into an aqueous
phase or water into a gaseous phase. CO
2
could be even liquid, as described for the
formation of thin CO
2
hydrate films referring to oceans [
40
]. For several applications,
reactors must ensure the efficient removal of heat during the hydrate formation [
41
]. This
is achieved by improving heat transfer by mechanical mixing in stirred tanks or bubble
towers [
42
]. At the laboratory scale, stirred tanks are the most used reactor types [
43
]. A
disadvantage of stirred tanks is that the higher the volume fraction of hydrate becomes
more energy is needed to reach the required degree of turbulence due to the increased
viscosity of the hydrate slurry [
42
]. Therefore, bubble columns have been introduced as
one alternative. In bubble columns, gas hydrate directly forms around the bubble at the
gas–liquid interface [
44
]. Any further formation is hindered by additional resistance for
interphase mass transport that was caused by the hydrate shell [
42
,
45
]. Larger interface
areas have to be created by either reducing the bubble sizes or increasing the gas velocity to
overcome this issue or shockwaves could be used to crack the shells to renew the reaction
sites [
42
,
44
–
46
]. For this reason, the gas hydrate process cannot only be based on the
equilibrium conditions, but also on the conditions in the reactor.
The preservation of quality is considered to be a great advantage of gas hydrate tech-
nology. Therefore, this research mainly aims to analyze whether polyphenols, carotenoids,
vitamin C, and betanin are participating in hydrate formation within fruit juices and a
water-sucrose-betanin model solution for the first time. Consistent with the knowledge
regarding gas hydrate structures, no incorporation of valuable substances into the hydrate
is expected. According to the literature, Brix values for concentrates produced by CO
2
gas
hydrate in bubble columns vary depending on pressure and temperature. Therefore, in
this study, a water-sucrose model solution representing juices is used to define a specific
working point in a bubble column reactor because mass and energy transport will influence
the hydrate formation and thus the concentration process. Thereby, possible interactions in
the complex juice matrix are eliminated, and the hydrate formation is expected to be more
reproducible. Although the used solution is a simplified model matrix, sugars are the main
components of juices. Thus, the model solution is stated to be an adequate replacement
for juices.
2. Materials and Methods
2.1. Materials
A water-sucrose model solution (10
°
Brix) was used to identify a suitable temperature
and pressure combination for the hydrate formation within the bubble column reactor.
After the operating point had been identified, a water-sucrose-betanin model solution
with 10
°
Brix and 1 g/L betanin was used for further experiments. Its purpose was to
investigate whether betanin is going to be part of the hydrate structure. Furthermore,
the colored solution made it possible to examine optically if the hydrate contains the
dye. This shows how efficient the separation of hydrate and concentrate is working. In
a third series of experiments, clear apple juice, cloudy apple juice, and orange juice were
Foods 2021,10, 626 4 of 17
used to test whether polyphenols, carotenoids, or vitamin C are incorporated into the
hydrate structure. The juices were purchased from local supermarkets. Table 1provides
an overview of all used media, the analyzed substances, the investigation aim, and the
number of experimental repetitions. In all experiments, CO
2
hydrate was formed with
high purity CO2(99.95%, Air Liquide, Paris, France).
Table 1.
An overview of the media used, indicating the substances considered, the experimental
target, and the experimental repetition.
Medium Analyzed
Substances Aim of Experiment Hydrate Formation
Repetitions
Model
solution
(water,
sucrose)
Sucrose
Identification of working
point. 2
Model
solution
(water,
sucrose,
betanin)
Sucrose
Betanin
Evaluation of concentrate
quality and preservation dur-
ing hydrate formation.
Visual assessment of separa-
tion quality by red/pink color
of betanin.
3
Apple
juice
(clear)
Total phenolics
Vitamin C
Evaluation of concentrate
quality and preservation dur-
ing hydrate formation.
3 for total phenolics
2 for vitamin C
Apple
juice
(cloudy) Vitamin C
Evaluation of concentrate
quality and preservation dur-
ing hydrate formation. 2
Orange
juice
Total phenolics
Total carotenoids
Vitamin C
Evaluation of concentrate
quality and preservation dur-
ing hydrate formation.
3 for total phenolics
and total carotenoids
2 for vitamin C
2.2. Reactor System
The high-pressure reactor that is shown in Figure 1is designed to withstand pressures
of up to 5000 bar. It has a volume of 1.5 L and was operated as a bubble column.
Figure 1. Schematic of the used bubble column reactor.
Foods 2021,10, 626 5 of 17
To control and measure the pressure, a pressure controller (SLA5810, Brooks Instru-
ment, Dresden, Germany) and a pressure sensor (DRTR-AL-10V-R100B, B+B Thermo-
Technik GmbH, Donaueschingen, Germany) were used. A cooling jacket with a circulating
chiller (L002326 Proline RP 855, Lauda, Lauda-Königshofen, Germany) realized the cooling
of the system, while the temperature within the reactor was measured with a thermocouple.
Thus, the conditions that are needed for gas hydrate formation were measured, controlled,
and recorded (OMB-DAQ-2408, OMEGA Engineering, Deckenpfronn, Germany).
2.3. Experimental Procedure
To find a suitable operating point, experiments at different levels of temperature
(
1 °C
, 3
°
C and 5
°
C) and pressure (32.5 bar, 37.5 bar and 40 bar) were performed and
the formation of gas hydrate in the water–sucrose model solution was evaluated for each
temperature–pressure combination. Similar conditions are reported in the literature for
concentrating juices using gas hydrate technology [
11
,
13
,
32
]. The hydrate formation in the
water–sucrose–betanin model solution and the three juices for the quality analysis was
investigated at 37.5 bar and 3 °C.
For all experiments, the reactor was first cleaned with hot water at a temperature of
100
°
C. After cleaning, the interior of the reactor was dried with a cloth to remove any
foreign particles. 550 mL juice or model solution was then filled into the reactor. This
corresponds to a sample to reactor volume ratio of about 36%, which is in the suitable
range of 33% to 40% for bubble columns that were reported in literature [
11
,
32
]. Shortly
before cooling to the desired temperature for a specific experiment, the system was flushed
with CO
2
. Thereby, air was removed and pressure built up in the reactor. Subsequently,
the pressure was set to the desired value by an automatic adjustment of the inlet gas flow.
Figure 2shows the typical pressure and temperature curves during an experiment with the
water–sucrose model solution at 32.5 bar and 3
°
C. To start the process, the outlet valve
was opened. The start of hydrate formation was indicated by a rising temperature within
the reactor, which is related to the exothermal character of hydrate formation (see Figure 2).
The experiments were stopped two hours after the start of hydrate formation to guarantee
a solid hydrate structure.
Figure 2.
Temperature and pressure curves are exemplarily shown for hydrate formation in the
water-sucrose model solution at 32.5 bar and 3
°
C: the system is cooled to the desired temperature (
A
).
Subsequently, pressure is build up (
B
) and set to the desired value (
C
). The start of hydrate formation
is indicated by a rising temperature (
D
to
E
). This temperature rise is followed by another cooling
step (F). The experiments were stopped two hours after the start of hydrate formation (G).
After the hydrate had formed, it was removed from the reactor as a solid block.
Claßen et al. (2020)
[
13
] describe that the use of a pellet press and an additional washing
Foods 2021,10, 626 6 of 17
step can improve the separation of the hydrate and concentrate. They advise that the
separation should take place under hydrate stable conditions for sugar contents above
30
°
Brix. This separation concept was adapted from seawater desalination [
21
]. Because
around 20% to 40% of the hydrate was adhering concentrate in the present study, the gas
hydrate was pressed by a wire press for 60 s applying a pressure of 450 bar to ensure a
separation of the phases. The hydrate is metastable under the pressing conditions and,
consequently, a dissociation of the hydrate is induced. However, the time scale of the
pressing procedure was short enough to prevent the hydrate from excessive dissociation.
After the separation, the hydrate was carefully dissociated in a microwave in a maximum
of two to three time steps of 10 to 15 s, depending on the amount of hydrate. Finally,
the concentrate, the dissociated hydrate, and the drained liquid phase obtained from the
pressed hydrate were analyzed for total phenolics, total carotenoids, vitamin C, or betanin,
depending on the initial solution according to Table 1.
2.4. Sugar
The sugar content in
°
Brix of the samples was determined in triplicate without any
further preparation in an oscillating U-tube (DMA 4500M, Anton Paar, Graz, Austria)
and a digital refractometer (RFM 80, Bellingham + Stanley, Xylem Analytics, Weilheim,
Germany). For calculating a yield, the mass of the concentrate, the hydrate, and the drained
liquid pressed from the hydrate were measured once. Equation (3) in Section 3.2 presents
the corresponding calculation of the yield.
2.5. Betanin
The betanin content in the samples was photometrically evaluated at 538 nm (Lambda
25 UV/VIS Spectrometer, Perkin Elmer, Rodgau, Germany), and the betanin concentration
was then calculated from a calibration line. The measurements were conducted in duplicate.
For the determination of the calibration line 0.375 g, 0.75 g, 1 g, 1.5 g, 2.25 g, and 3 g of
betanin (AB137484, abcr GmbH, Karlsruhe, Germany) were dissolved in 1 L distilled water.
These solutions were measured at 538 nm against a blank of pure distilled water. The
calibration line was determined twice with a triplicate determination on each data point.
Furthermore, a calibration line with a 10
°
Brix solution and the betanin concentrations
given above was produced. The added sucrose had no effects on the calibration line.
2.6. Total Phenolics
One of the most used methods for determining the total phenolics is the Folin–
Ciocalteu assay [
47
]. The procedure used in this research is based on the publication
of Singleton, Orthofer, and Lamuela-Raventós (1994) [
48
], with some modifications. 200
µ
L of diluted samples was obtained from the concentration process of clear apple juice
and orange juice (1:100 with distilled water) or distilled water as a blank was mixed with
1 mL of diluted Folin–Ciocalteau reagent (Sigma-Aldrich, Merck, Darmstadt, Germany,
diluted 1:10 with distilled water) in triplicate. After 30 s, 800
µ
L of 7.5% Na
2
CO
3
solution
were added. All of the samples were then incubated at 40
°
C for 30 min., shaken, and
subsequently measured in the spectrophotometer at
765 nm
. Finally, the total phenolics
were calculated from a gallic acid calibration line.
2.7. Total Carotenoids
Oranges tend to contain more carotenoids than apples, and the carotenoids are also
present in orange juice [
49
]. Therefore, only orange juice was considered in the analysis
of total carotenoids. The method was taken from a DIN EN standard [
50
]. 50 mL of each
orange juice sample were analyzed for total carotenoids in triplicate. The carotenoids were
precipitated with 1 mL Carrez-I and Carrez-II solution, respectively. Subsequently, the
carotenoids were extracted from the precipitate with acetone and transferred to petroleum
ether. The total carotenoid content was spectrophotometrically determined against a blank
Foods 2021,10, 626 7 of 17
consisting of pure petroleum ether at a wavelength of 450 nm. The total carotenoids are
expressed as β-carotene equivalents ρ(C40H56), according to the DIN EN standard [50]:
ρ(C40H56) = A·4.00 ·
V2
V1. (1)
In Equation
(1)
,
A
is the extinction of the petroleum ether extract, 4.00 represents a
mean conversion coefficient,
V2
is the volume of the petroleum ether extract, and
V1
is the
initial volume of the analyzed sample.
2.8. Vitamin C
The vitamin C content was measured by HPLC (Ultimate 3000, Thermo Fisher Sci-
entific, Waltham, MA, USA) with a hyperchrome HPLC column. The temperature of
the HPLC column oven (TCC-3000SD) was 25
°
C. The mobile phase was isocratic with
10 mM sulfuric acid (pH-value of 2.2). The method used was developed following the
publications of Rückemann (1980) [
51
] and Sood et al. (1976) [
52
]. 25 mg of L-ascorbic
acid (purity 99%, Sigma–Aldrich, Merck, Darmstadt, Germany) were dissolved in 25 mL
of 3% meta-phosphoric acid and further diluted in 3% meta-phosphoric acid to produce
a standard solution. All the phases from the concentration process of orange juice, clear
apple juice, cloudy apple juice, and the initial solutions of the respective juices were frozen
until the analysis. The samples were then filtered and the filtrate was used for HPLC
analysis. Therefore, all of the samples were mixed 1:10 with 3% meta-phosphoric acid
to stabilize the vitamin C. The samples and standard solution were pressed through an
HPLC syringe filter to remove coarser particles and prevent membrane clogging. A defined
volume of 20
µ
L of the sample was injected into the dosing loop. In the separation column,
the samples were analyzed by the detector (diode array UV detector, DAD-3000) at 254 nm.
The measurements of the samples were carried out in duplicate.
2.9. Statistical Data Evaluation
For all experiments, the mean values
¯
x
and the standard errors of the mean
σ¯
x
were
calculated. The standard error of the mean is defined as:
σ¯
x=s
√n. (2)
In Equation
(2)
,
s
represents the standard deviation and
n
is the number of samples.
The mean of the Brix values of the hydrate formation repetitions was determined for
identifying the working point. Within the sugar content measurements, the standard error
of the mean is approximately zero. Consequently, the standard error of the mean between
the repetitions of hydrate formation (
n=
3) is considered to be the main error occurring
during this experiment and, thus, it is presented in the figures of Section 3.2. Because the
masses needed for the calculation of the yield were measured once, the calculated mean is
the mean of the hydrate formation repetitions. The standard error of the mean represents
the standard error between these repetitions (n=3).
The mean of the data that were obtained from the quality analyses was calculated
from all determinations of one analysis (e.g., total phenolics) and all respective repetitions
of hydrate formation. For example, for total phenolics, this would take the three repetitions
of the hydrate formation and the triple determination of the Folin–Ciocalteu assay into
account (
n=
9). The standard error of the mean of these values is finally calculated. For
the determination of total carotenoids the number of samples is nine (
n=
9), for vitamin C
four (n=4), and for betanin six (n=6).
3. Results
3.1. Hydrate Structure
The hydrate produced in the bubble column reactor has a different consistency, de-
pending on the temperature and pressure conditions used.
Foods 2021,10, 626 8 of 17
Figure 3a depicts that at 40 bar and 1
°
C a hydrate consisting of a solid block and
hydrate slurry is formed in the water-sucrose-betanin model solution. At 37.5 bar and 3
°
C,
a solid and porous block forms from the same initial solution as shown in Figure 3b. This
block fills almost the entire reactor volume of 1.5 L. The structure of the hydrate formed
at 37.5 bar and 3
°
C is porous, and Figure 3b, as well as Figure 3c, show that concentrate
either adheres to the surface of the hydrate block or the porous structure. This underlines
the need for a suitable separation of hydrate and concentrate.
(a) (b) (c)
Figure 3.
CO
2
hydrate formed in the water-sucrose-betanin model solution at different temperature
and pressure conditions using a bubble column reactor: (
a
) hydrate block with hydrate slurry formed
at 40 bar and 1
°
C, (
b
) solid hydrate block formed at 37.5 bar and 3
°
C, and (
c
) porous hydrate
structure formed at 37.5 bar and 3 °C.
3.2. Identification of the Operating Point
In bubble columns, CO
2
hydrate is formed in fruit juices between 30 bar and 42 bar at
temperatures between 1
°
C and 10
°
C [
11
,
32
]. Because of the shift in equilibrium conditions
towards higher pressures and lower temperatures, corresponding values can be expected
for the water–sucrose model solution [34].
The collected results in Figure 4show that, for the tested pressure and temperature
combinations, concentrates with Brix values from around 10
°
Brix to nearly 18
°
Brix can be
produced from an initial solution with 10
°
Brix. In the literature, values between 12
°
Brix
and
27 °Brix
were reported for fruit juices [
11
,
32
]. Within all experiments of the present
study, the Brix value of the hydrate phases was between 2
°
Brix and 6
°
Brix. At a pressure
of
37.5 bar
and a temperature of 3
°
C, the best possible concentration in the used reactor
system was achieved. Values of nearly 18
°
Brix are reached, which corresponds to a
concentration factor of about 1.8. At the same temperature, pressures of 32.5 bar and
40 bar
lead to a slightly less satisfactory concentration by a factor of 1.6. If a temperature of 1
°
C
is used in the process, 14
°
Brix to 16.6
°
Brix are achieved at the same pressure levels. At
32.5 bar
, the lowest value is obtained in the resolved range, but the achievable concentration
increases at higher pressure. However, the difference between 37.5 bar and
40 bar
is no
longer significant and, thus, the influence of pressure decreases. The concentration process
is least efficient at 5
°
C. While, at this temperature and 37.5 bar, about 13
°
Brix are still
achieved and the Brix value for 32.5 bar is slightly above 12
°
Brix. At a combination of 5
°
C
and 40 bar, nearly no concentration took place, since the sucrose content of the concentrate
does not differ significantly from the starting solution in its sucrose content of 10
°
Brix. The
calculated standard errors of the mean are in the range of 0.05 and 0.7
°
Brix for all samples.
This indicates a high reproducibility of the hydrate formation.
Foods 2021,10, 626 9 of 17
Figure 4.
Reached sucrose content in
°
Brix in the concentrate produced from the water-sucrose
model solution (10
°
Brix) using CO
2
hydrate technology at pressures of 32.5 bar, 37.5 bar, and 40 bar
combined with temperatures of 1
°
C, 3
°
C, and 5
°
C (error bars: standard error of the mean according
to Section 2.9, the standard errors of the mean are given in Table S2).
The amount of the produced concentrate was in the further focus of the performed
experiments. In order to determine the concentration yield for the used pressure and
temperature conditions, the quotient of the achieved concentrate mass
mc
, and the mass of
the initial solution m0was determined:
Yield[%] = mc
m0·100%. (3)
Figure 5visualizes the yields for every analyzed temperature and pressure combina-
tion. The calculated standard errors of the mean are between 0.3 and 6.9% for all yields.
Figure 5.
Reached yield in % expressed as a quotient of the reached concentrate mass
mc
after the
concentration using CO
2
hydrate technology and the mass
m0
of the initial water-sucrose model
solution (10
°
Brix) at pressures of 32.5 bar, 37.5 bar, and 40 bar combined with temperatures of 1
°
C,
3 °C
, and 5
°
C (error bars: standard error of the mean according to Section 2.9, the standard errors of
the mean are given in Table S2).
At 5
°
C from 32.5 bar to 37.5 bar, the yield is nearly at 65%, whereas, at 5
°
C and
40 bar
, nearly 75% are reached. The yields at 1
°
C and 3
°
C are in the same range. At
32.5 bar, between 37% (3
°
C) and 42% (1
°
C) of the initial mass are present as concentrate
Foods 2021,10, 626 10 of 17
after the process. If a pressure of 37.5 bar is used to produce a concentrate from the
solution, the yield is between 18% to 22%, while at 40 bar yields of 30% to 32% were
achieved. In comparison, the literature reports values of 40% for the concentration in a
bubble column configuration [
11
]. From the data in the present research, it appears that
a higher concentration results in a lower yield, since the values approximately directly
correspond to the sucrose concentrations of the concentrate. This can be attributed to the
fact that a lower amount of water is present in a higher concentrated concentrate.
3.3. Effect of the Concentration Using Gas Hydrate Technology on Valuable Ingredients
Because CO
2
hydrate formation requires gentle processing conditions, valuable juice
ingredients should not be negatively affected during the concentration of fruit juices and
the water–sucrose–betanin model solution. Therefore, the quality experiments aimed to
assess the preservation of betanin, polyphenols, carotenoids, and vitamin C.
Quotients of the achieved concentration
c
and the initial concentration within the
water–sucrose–betanin model solution
c0
of sucrose and betanin, in each phase (concentrate,
drained liquid pressed from the hydrate, hydrate), respectively, are presented in
Figure 6
.
Thus, every value can be considered as a concentration factor and quotients above 1 indicate
that a concentration took place. The absolute values of the betanin contents can be found
in Table S1 of the Supplementary Material. For the water–sucrose–betanin model solution,
the data show that the calculated quotients for sucrose and betanin in the concentrate are
1.65 and around 1.5 for the drained liquid from the pressed hydrate. Consequently, both
sucrose and betanin are concentrated. Because the values for the drained liquid pressed
from the hydrate are in the same range, it is assumed that the drained liquid is adhering
concentrate. Nevertheless, sucrose and betanin are both found in the hydrate phase, but
the calculated quotient for betanin is 0.5, whereas, for sucrose, it is 0.4. This means that
more betanin than sugar is present in the hydrate phase. The standard errors of the mean
are between 0.03 and 0.11 for all data presented in Figure 6.
Figure 6.
Quotients of the reached concentrations
c
after hydrate formation in the water-sucrose-
betanin model solution and the initial concentrations
c0
, respectively, for sucrose and betanin; C:
concentrate, L: drained liquid from the hydrate, H: hydrate (error bars: standard error of the mean
according to Section 2.9, the standard errors of the mean are given in Table S3).
Figure 7a shows the results of the vitamin C measurements. Being analogous to the be-
tanin determinations, the quotient
c
/
c0
was calculated to obtain a measure of concentration.
The standard errors of the mean are between 0.001 and 0.1 for all vitamin C data. The results
indicate that the highest amounts of vitamin C are found in the concentrates of orange
juice and both apple juices. During juice concentration using gas hydrate technology, the
vitamin is concentrated by a factor of about 1.7 for orange juice. In both apple juices, the
Foods 2021,10, 626 11 of 17
concentration factors for vitamin C are around 1.2. For all samples, the concentration ratios
in the drained liquids pressed from the hydrate are between 0.5 to 1. It is noticeable that
the gas hydrate contains only small amounts of vitamin C (maximum quotients of 0.3).
The values for the hydrate phase produced in both apple juices differ from the values for
orange juice. The absolute amounts of vitamin C in orange juice are much higher than the
absolute amounts of vitamin C in apple juice (both clear and cloudy). In the hydrate phase,
these values are even smaller (see Table S1). Because the data presented in
Figure 7
are
relative, even small differences in the absolute values of vitamin C content in the apple
juices have a great impact on the presented data. The results on vitamin C imply that
separating the gas hydrate and adhering concentrate has to be further improved.
(a) (b)
(c)
Figure 7.
Quotients of the reached concentrations c produced and the corresponding initial concentrations
c0
for all phases
from the concentration process: (
a
) vitamin C for orange juice, apple juice (clear) and apple juice (cloudy), (
b
) total phenolics
for orange juice and apple juice (clear), and (
c
) total carotenoids for orange juice. C: concentrate, L: drained liquid from the
hydrate, H: hydrate (error bars: standard error of the mean according to Section 2.9, the standard errors of the mean are
given in Table S3).
The results regarding total phenolics for the concentration of clear apple juice and
orange juice using gas hydrate technology that are shown in Figure 7b underline the
inefficient separation. The quotient
c
/
c0
was determined, as before. The standard errors
Foods 2021,10, 626 12 of 17
of the mean for total phenolics are between 0.03 and 0.3. Polyphenols are concentrated
in the concentrate by more than factor 2 via CO
2
hydrate formation. Again, quotients
around 1 are found regarding the liquids from the pressed hydrate. The hydrate phases
contain the smallest amount of polyphenols. For clear apple juice, this value is at 0.3 and
for orange juice at 0.4. Figure 7c visualizes the results for the quotient
c
/
c0
concerning
total carotenoids in all the phases gained during the concentration of orange juice. The
standard errors of the mean for total carotenoids are between 0.003 and 0.06. Most of the
carotenoids are part of the concentrate, which is indicated by the concentration factor of
1.4. A value below 1 is found regarding the drained liquid from the pressed hydrate. The
hydrate phases contain just slightly fewer carotenoids with a quotient of 0.7.
All of the results indicate that polyphenols, carotenoids and vitamin C are concentrated
by a factor of 1.2 to more than 2 within the juice during the gas hydrate process. The
factors for the hydrate phase are 0.7 and below. Although only small amounts remain in
the hydrate, the separation of the hydrate and the adhering concentrate is not efficient.
Especially, carotenoids are not separated sufficiently by pressing. For the support of the
presented data, the absolute values corresponding to the relative data that are presented in
Figure 7can be found in Table S1 of the Supplementary Material.
4. Discussion
4.1. Identification of the Operating Point
Overall, the results regarding the operation point highlight the effect of pressure on
the concentration process. From 32.5 bar to 37.5 bar, the concentration of the water–sucrose
model solution at temperatures of 1
°
C, 3
°
C and 5
°
C improves. Beyond these pressures,
the concentration process does not get more efficient or even deteriorates. This effect is
described for fruit juices in literature as pressures below 37 bar or 40 bar lead to a decreased
concentration efficiency as well as pressures beyond these values [
32
]. For the effect of
different temperatures, the results indicate that temperatures of 1
°
C and 3
°
C should be
used instead of 5
°
C. According to the literature, a temperature of 2.5
°
C is suitable for
concentrating juices in a bubble column reactor [
11
]. In the present study, between 1
°
C
and 3
°
C, the reached sugar contents in the hydrate are close, which implies that lowering
the temperature is just applicable to a certain degree. Because hydrate technology is stated
to consume less energy than conventional concentration methods [
11
] cooling to 3
°
C saves
energy as compared to the processing at 1
°
C. Thus, pressure and temperature are both
limiting factors for the concentration process. This limiting influence is the reason that
nearly no concentration took place at 5
°
C and 40 bar. The thermodynamic driving force
is proportional to the distance of the working point to the equilibrium
conditions [39]
.
All of the results indicate that high pressures can reduce concentration efficiency. At 40
bar, the concentration process is no longer efficient and in combination with the reduced
driving force due to the temperature of 5
°
C the hydrate formation is hindered. Thus, at
this temperature and pressure combination, the concentration process is not taking place,
since nearly no hydrate is formed. Furthermore, the state of aggregation of CO
2
influences
the concentration process in the bubble column at pressure levels above 37.5 bar and
temperatures of 1
°
C or 3
°
C. At these conditions, CO
2
is liquid, and
Shindo et al. (1993) [40]
assume that a formation of thin hydrate films takes place in liquid CO
2
. However, the
results that are presented in this research imply a decrease in concentration efficiency if
liquid CO
2
is present. Liquid CO
2
changes the reaction and transport kinetics in the bubble
column, which might directly affect the hydrate formation. Consequently, for a better
understanding of every detail, kinetic and thermodynamic studies should be conducted
and connected to this research.
Concerning the maximum achievable sugar concentration in the concentrate produced
in bubble columns by hydrate formation, the values for fruit juices in the literature are
higher with 20
°
Brix to 27
°
Brix [
11
,
32
], as compared to nearly 18
°
Brix reached in this
study for the water–sucrose model solution. This can be related to the complexity of the
fruit juice matrix. Inhibitory ingredients in fruit juices and sugar solutions affect hydrate
Foods 2021,10, 626 13 of 17
formation by shifting the hydrate formation conditions towards higher pressures and lower
temperatures [29,31–34]
. The best formation conditions in the present study for the water–
sucrose model solution and juices reported in the literature [
11
,
32
] are in the same range.
Therefore, it can be assumed that hydrate formation in juices is strongly influenced by
the effect of sucrose. Because the formation conditions are very similar, the concentration
process is further affected by either process kinetics or other components of the juices that
might promote hydrate formation. Besides the need for additional data on kinetics and
thermodynamics concerning the concentration process, further studies on the effect of fruit
juice components or components of liquid foodstuff, in general, should be conducted.
For an industrial application, high sugar contents within the concentrate are needed.
The reached sugar contents in the bubble column reactor during the concentration of the
water-sucrose model solution of nearly 18
°
Brix are much lower than over 40
°
Brix, which
can be achieved in stirred tanks for fruit juices in literature [
13
]. The results imply that
further hydrate formation is hindered in the bubble column reactor used in the present
study. According to the literature, the hydrate shell can negatively influence the hydrate
formation [
42
,
45
]. Additional to the hydrate shell, inhomogeneities of the mass and energy
distributions can be expected in the bubble column reactor. Thus, a better mixing, as
achieved in a stirred tank, might help to improve the concentration process. Because the
reactor contains more hydrate with increasing hydrate formation, solid block structures
form almost throughout the entire bubble column reactor (see Figure 3b). In a stirred tank,
shear forces break up hydrate blocks, leading to a slurry-like consistency. Consequently,
the energy requirements of stirred tanks are higher. Nevertheless, the break-up of large
hydrate structures improves further hydrate formation, which makes higher degrees of
concentration possible.
4.2. Effect of the Concentration Using Gas Hydrate Technology on Valuable Ingredients
The results of this study indicate that polyphenols, carotenoids, vitamin C, and betanin
are concentrated during the concentration of fruit juices or a water-sucrose-betanin model
solution using gas hydrate technology. For all of the examined substances, the concentration
factors are between 1.2 to 2.2 in one concentration step. Only small amounts remain in the
hydrate phase. Based on the present results, no conclusions can be made regarding the
mechanisms behind the incorporation of polyphenols, carotenoids, vitamin C or betanin,
as further research is needed. However, the dissociation enthalpies of fruit juices and pure
CO
2
-water systems that are calculated in the literature are in the same range. CO
2
-water
systems have a dissociation enthalpy of 85.19 kJ/mol, and the dissociation enthalpies of
orange and apple juice are 85.32 kJ/mol and 86.64 kJ/mol, respectively [
32
]. Thus, the
produced gas hydrate only should consist of water and CO
2
. A reason for the preservation
of valuable substances is the hydrate structure itself. Referring to the literature, CO
2
hydrate forms a sI structure consisting of cavities that only allow for molecules of a certain
size and a low molecular weight to be part of the hydrate [
14
]. The maximum guest
molecule size is about 9 Å in sH structure, whereas molecules with a size of around 4 Å
to 7 Å form sI or sII hydrates [
35
]. Typical substances that are part of sI hydrates are CO
2
,
ethane, and
methane [25]
. Furthermore, the guest molecule must not contain either a single
strong hydrogen-bond group or several moderately strong hydrogen-bonding groups [
38
].
Because vitamin C, polyphenols, and carotenoids are either large molecules or molecules
with hydroxyl groups, their size and chemical nature do not allow them to be part of the
hydrate structure. Consequently, the reason for the remaining amount of these substances
in the hydrate phase is probably not related to hydrate formation. Much more likely is an
influence of processing, especially concerning the separation technique.
Figure 8depicts the hydrate pellet obtained via pressing from experiments conducted
with the water-sucrose-betanin model solution. This figure visualizes the separation
quality within the scope of this research, which clarifies the need for further optimization.
Figure 3b,c present the concentrated water–sucrose–betanin model solution adhered to
the hydrate structure. After pressing, parts of this concentrate are still visible on the
Foods 2021,10, 626 14 of 17
hydrate pellet. In other studies pressing was used for the separation of hydrate and
concentrate with promising results [
13
,
21
]. However, Claßen et al. (2020) [
13
] highlight that
pressing should be performed under hydrate stable conditions for sugar contents above
30
°
Brix. As this requires a high technical effort, the separation was not performed under
hydrate stable conditions within the present research. Pressing outside of the stability
range induced hydrate dissociation of the metastable hydrate. From the results of the
water–sucrose–betanin model solution, in this research it has been shown that the drained
liquid from the pressed hydrate is adhering concentrate with high sucrose and betanin
content. During the investigation of fruit juices, it appeared that polyphenols, carotenoids,
and vitamin C are not as much present in the drained liquid from the pressed hydrate as
in the concentrate. The dilution that is induced by the pressing step contributes to this
result. For this reason, in this research, the additional washing step that was suggested by
Claßen et al. (2020) [
13
] was not realized, since no further dilution of adhering concentrate
should take place. The inefficient separation is an issue that needs to be addressed to
produce high-quality concentrates while using hydrate technology. Besides the separation
technique, the water solubility of the analyzed compounds could influence the separation
efficiency. Especially, carotenoids that are insoluble in water remain in the hydrate phase
during pressing. For this reason, the continuous removal of hydrate crystals should take
place before valuable fruit juice components could adhere to the hydrate and its porous
structure. As an alternative, hydrate slurries could be produced. This approach is an
advantage over bulky hydrate blocks, especially for continuous processes. The produced
slurries could be separated by centrifugation or filtration.
Figure 8.
Hydrate pellet obtained from pressing after hydrate formation (at 37.5 bar and 3
°
C) in the
water–sucrose–betanin model solution.
5. Conclusions
This research had the main aim to investigate whether valuable ingredients of juices
are accumulated in the concentrate or the hydrate during the concentration of fruit juices
and model solutions while using gas hydrate technology. The presented results show, for
the first time, that all examined substances can be mainly found in the concentrate. Only
small amounts remain in the hydrate phase due to an inefficient separation technique.
Therefore, improved processing could lead to concentrates of even higher quality. For an
industrial application, the continuous removal of hydrate particles could enable a better
separation. In future studies, either the mechanisms behind the possible incorporation of
valuable juice compounds or quantitative effects on more specific phenols or carotenoids
should be analyzed instead of total phenolics and total carotenoids. This would take dif-
ferent molecular structures and sizes into account. Besides, it might become important
to characterize the inhibitory or promoting effects of certain compounds of liquid food-
stuffs. Furthermore, the present study showed that a suitable operating point for hydrate
Foods 2021,10, 626 15 of 17
formation is at 37.5 bar and 3
°
C for concentrating a water–sucrose model solution from
10
°
Brix to nearly 18
°
Brix. Because reactor types, like stirred tanks, provide higher sugar
concentrations than the used bubble column reactor, in the future it will be necessary to
clarify what the most suitable reactor concept is.
Supplementary Materials:
The following are available online at https://www.mdpi.com/2304-815
8/10/3/626/s1, Table S1: Absolute values for the contents of betanin [g/L], vitamin C [mg/L], total
phenolics [mg/L] and total carotenoids [mg/L] for the concentrate (C), the drained liquid pressed
from the hydrate (L) and the hydrate phase (H) within all analyzed media and the corresponding
standard errors of the mean (SEM), Table S2: Standard errors of the mean (SEM) of sucrose measure-
ments [
°
Brix] and the concentration yields [%] for the concentrate produced from the water-sucrose
model solution by gas hydrate technology presented in Figures 4and 5, Table S3: Standard errors of
the mean (SEM) of c/c
0
for sucrose, betanin, vitamin C, total phenolics and total carotenoids for the
concentrate (C), the drained liquid pressed from the hydrate (L) and the hydrate phase (H) within all
analyzed media presented in Figures 6and 7.
Author Contributions:
Conceptualization, C.R. and C.M.; software, A.R.; validation, A.R. and
A.E.-M.; formal analysis, A.R. and A.E.-M.; investigation, A.R. and A.E.-M.; writing—original
draft preparation, A.R. and C.M.; writing—review and editing, A.R. and C.M.; visualization, A.R.;
supervision, C.R. and C.M.; project administration, C.R. and C.M.; funding acquisition, C.R. and C.M.
All authors have read and agreed to the published version of the manuscript.
Funding:
This IGF Project of the FEI (Grant number: AiF EWN 11) is/was supported via AiF within
the programme for promoting the Industrial Collective Research (IGF) of the German Ministry
of Economic Affairs and Energy (BMWi), based on a resolution of the German Parliament. We
acknowledge support by the German Research Foundation and the Open Access Publication Fund of
TU Berlin.
Conflicts of Interest: The authors declare no conflict of interest.
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