6480 J. Agric. Food Chem.

2008, 56, 6480–6487

Impact of High-Pressure Carbon Dioxide Combined

with Thermal Treatment on Degradation of Red Beet
(Beta vulgaris L.) Pigments
XUAN LIU, YANXIANG GAO,* HONGGAO XU, QI WANG, AND BIN YANG
College of Food Science and Nutritional Engineering, China Agricultural University,
Beijing 100083, China

A combined high-pressure carbon dioxide (HP-CO2) and thermal degradation reaction of betanin
and isobetanin in aqueous solution was investigated and can be described by a first-order decay. At
45 °C, the degradation rate constant (k) for each pigment component significantly increased (the
half-life (t1/2) decreased, p < 0.05) with elevated pressure. Furthermore, HP-CO2 treatment led to
lower k values (higher t1/2 values) than thermal treatment. However, k and t1/2 values approached
those of thermal treatment when the pressure was >30 MPa combined with temperatures exceeding
55 °C. Moreover, betanin was more stable than isobetanin under HP-CO2. Ea values ranged from
94.01 kJ/mol for betanin and 97.16 kJ/mol for isobetanin at atmospheric pressure to 170.83 and
142.69 kJ/mol at 50 MPa, respectively. A higher pressure and temperature as well as longer exposure
time resulted in higher values of L*, b*, C*, and h°. HP-CO2 induced more degradation products from
betanin and isobetanin than thermal treatment with an identical temperature and exposure time.

KEYWORDS: Betanin; isobetanin; high-pressure carbon dioxide; HP-CO2; degradation kinetics; color
difference; degradation products

INTRODUCTION systematic and quantitative studies are rather scarce concerning

the effect of HP-CO2 and increasing temperature on nutrients
To produce safe and shelf stable products is a primary
in food. Recently, there have been a few reports on HP-CO2 in
objective of food manufactures and was achieved by multifarious
traditional thermal methods in the past decades; food quality commercial use for fruit and vegetable products, but the
was considered of secondary importance (1). It is commonly technology is still under investigation in the laboratory. One of
known that thermal treatment, widely used in commercial food the biggest challenges for the application of HP-CO2 in fruit
production, impacts product quality, including flavor, texture, juices or nonthermal food products is to evaluate the availability
and nutrients such as vitamins, phenolics, etc. However, with and safety of this processing, including changes of main
the increasing demand by consumers for fresher, higher quality, nutritional components.
minimally processed, and safer food, there is a strong interest Red beet (Beta Vulgaris L.) is a traditional vegetable
in the food industry to develop nonthermal processing techniques distributed in many parts of the world and has been used
to replace traditional thermal methods for food preservation (2, 3). commercially to produce red beet juice and natural pigment.
High-pressure carbon dioxide (HP-CO2) treatment is one The essential pigment in red beet is a group of bioactive
nonthermal method that has been the subject of intense scrutiny compounds called betalains (10, 11). Ca. 80-90% of the total
for potential applications in a number of food processing fields pigments of red beet is ascribed to betacyanins (violet), mainly
including inactivation of enzymes that can adversely affect the betanin (betanidin 5-O-β-glucoside), and its C15 isomer isobea-
quality of food products (4). HP-CO2 at lower temperatures can nin, whereas vulgaxanthin I represents the predominant beta-
inactivate microorganisms and food deteriorative enzymes in xanthin (yellow) (12–14). The red-violet color is regarded as
fruit juices while only slightly affect nutritional and sensorial a major quality attribute in determining red beet product
food quality aspects (e.g., ascorbic acid content, total acidity, acceptability. Like many other natural pigments, betacyanins
color, flavor, and cloud), most of which are sensitive to are very sensitive to heat, light, and oxidation, which especially
heat (5–9). With the consideration of extreme pressure stability result from peroxidase (POD) and these are major reasons for
of some microbial species in low-acid food/vegetable juice (such discoloration of the pigment (15–18). Moreover, enzymatic
as carrot, red beet, spinach juice, etc.), HP-CO2 is required to browning induced by polyphenol oxidase (PPO) in red beet is
be combined with proper heat treatment. However, experimental another frequently encountered problem in the production of
red beet juice. HP-CO2 combined with thermal treatment has
* Author to whom correspondence should be addressed. Tel.: + 86- been shown to be effective in inactivating POD and PPO in
10-6273-7034; fax: + 86-10-6273-7986; e-mail: gyxcau@126.com. red beet extracts (19).
10.1021/jf800727q CCC: $40.75  2008 American Chemical Society
Published on Web 07/12/2008
Combined HP-CO2 and Thermal Degradation of Betalains J. Agric. Food Chem., Vol. 56, No. 15, 2008 6481

Table 1. Concentrations (mg/L) of Betanin and Isobetanin before and after Different Treatments

atmospheric pressure 10 MPa

temp (°C) treatment time (h) betanin isobetanin betanin isobetanin
control 0 18.34 ( 0.14 17.04 ( 0.20 18.34 ( 0.14 17.04 ( 0.20
0.5 17.62 ( 0.04 16.73 ( 0.03 18.18 ( 0.12 16.71 ( 0.15
45 1.5 16.92 ( 0.15 15.90 ( 0.12 17.44 ( 0.08 15.93 ( 0.07
2.5 15.39 ( 1.56 14.20 ( 1.73 17.40 ( 0.08 15.77 ( 0.08
0.5 17.14 ( 0.14 15.75 ( 0.12 17.40 ( 0.09 16.00 ( 0.13
55 1.5 14.81 ( 0.05 13.20 ( 0.04 17.39 ( 0.09 15.49 ( 0.07
2.5 13.49 ( 0.05 11.78 ( 0.04 16.40 ( 0.07 14.39 ( 0.06
0.25 16.46 ( 0.04 15.14 ( 0.07 17.08 ( 0.06 16.19 ( 0.05
65 0.75 13.01 ( 0.04 11.45 ( 0.04 15.77 ( 0.10 13.61 ( 0.12
1.3 9.92 ( 0.13 8.36 ( 0.10 12.35 ( 0.09 10.57 ( 0.11

30 MPa 50 MPa
temp (°C) treatment time (h) betanin isobetanin betanin isobetanin
control 0 18.34 ( 0.14 17.04 ( 0.20 18.34 ( 0.14 17.04 ( 0.20
0.5 17.54 ( 0.15 15.95 ( 0.18 17.51 ( 0.08 16.66 ( 0.08
45 1.5 17.16 ( 0.06 15.32 ( 0.04 17.13 ( 0.08 15.57 ( 0.06
2.5 17.00 ( 0.09 14.96 ( 0.11 16.92 ( 0.06 15.00 ( 0.13
0.5 16.47 ( 0.10 14.75 ( 0.15 17.24 ( 0.05 15.55 ( 0.11
55 1.5 14.22 ( 0.11 12.53 ( 0.10 15.10 ( 0.12 12.97 ( 0.07
2.5 13.11 ( 0.03 11.44 ( 0.04 11.88 ( 0.03 10.30 ( 0.03
0.25 17.02 ( 0.40 15.36 ( 0.36 16.26 ( 0.06 14.81 ( 0.02
65 0.75 10.96 ( 0.08 9.68 ( 0.06 7.76 ( 0.16 6.71 ( 0.16
1.3 4.09 ( 0.02 3.34 ( 0.06 3.90 ( 0.02 3.31 ( 0.04

However, there is little information on the degradation and 65 °C. For each pressure, the samples were treated separately for
characteristics of betalains under HP-CO2. The objective of this 0, 0.5, 1, 1.5, 2, and 2.5 h (45 and 55 °C) and 0, 0.25, 0.5, 0.75, 1, and
work was to verify as to whether HP-CO2 combined with 1.3 h (65 °C). At the end of each treatment, the pressure was slowly
thermal processing can result in improved betalain retention as released over 15 min, and the samples were removed from the treatment
compared to thermal treatments, to investigate the degradation vessel and cooled to room temperature in an ice-water bath. The
concentrations of betanin and isobetanin were determined by HPLC at
kinetics of betanin and isobetanin for predicting pigment
25 °C. CO2 (g99.9%) was purchased from Beijing Analytical Apparatus
retention under HP-CO2, and to describe color changes by CIE Co. (Beijing, China).
parameters and characterize degradation products by spectro- Thermal Treatment of RBPS. To compare HP-CO2 treatments with
photometry and HPLC-MS. Furthermore, a degradation mech- conventional thermal ones, aliquots of 15 mL of RBPS in the treatment
anism for betanin and isobetanin under HP-CO2 also is proposed vessel (the same volume as before) were placed in a water bath set at
and discussed. 45, 55, and 65 °C for the same times as stated previously. After that,
the containers were moved to an ice-water bath to the room
MATERIALS AND METHODS temperature, and the concentrations of betanin and isobetanin were
Preparation of Red Beet Pigment (RBP) Solution. RBP powder determined by HPLC at 25 °C.
extracted from red beets, purified, and spray-dried with dextrin (the Color Measurements of RBPS. The spectrum of RBPS was scanned
content of betanin and isobetanin was 98% of total pigments, determined using a UV-vis spectrophotometer (UV757CRT, Lengguang, Shanghai,
by HPLC) was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, China) at ambient temperature; the scanning wavelength ranged from
Japan). The major colored components of the powder were betanin 400 to 700 nm in steps of 1 nm. RBPS was also analyzed with a color
and isobetanin, which were 917 ( 5 and 852 ( 7 µg/g, respectively difference meter (Xingguang Instrument Co., Beijing, China) using the
(determined by HPLC, see Determination of Betanin and Isobetanin). transmission mode. Samples were poured in 5 cm × 3 cm × 1 cm
A 2% (w/w) red beet pigment solution (RBPS) was prepared with glass cell and measured. CIE L*, a*, and b* parameters were recorded
doubly distilled water. as L* (lightness), a* (redness), and b* (yellowness). The hue angle
HP-CO2 Treatment System. The HP-CO2 system was manufactured (h°) and chroma (C*) were calculated as h° ) arctan(b*/a*) and C* )
by Huali Pump Co., Ltd. (Hangzhou, China). It consisted of a three- (a*2 + b*2)0.5.
plunger type pump, a 1 L stainless steel treatment vessel, a temperature Determination of Betanin and Isobetanin. The concentrations of
controller and monitor, two pressure gauges (with an accuracy of (0.01 betanin and isobetanin in RBPS were analyzed by HPLC following the
MPa), a water bath, and a back pressure regulator. The treatment vessel method defined by Schwartz and Von Elbe (10), Pourrat et al. (20), and
was enveloped by an electrical water bath heating jacket with a Stintzing et al. (21) with minor modifications. HPLC analysis was
thermocouple attached into the top to monitor the temperature inside performed on an Agilent 1100 HPLC system with a diode array detector
the vessel. Another thermocouple, connected to the temperature monitor, (DAD). Samples were separated on an Agilent Zorbax SB C18 column
was placed in the water bath to control the vessel temperature. The (250 mm × 4.6 mm i.d., 5 µm) with a gradient elution system. Solvent A
temperature inside the treatment vessel was controlled to an accuracy was a mixture of 100% methanol and 0.2% (v/v) formic acid (88%) in
of (0.5 °C. The maximum operating pressure of the system was 55 water with a ratio of 18: 82 (v/v). Solvent B was 100% methanol. At a
MPa. flow rate of 1.0 mL/min, the first 6 min was performed isocratically with
HP-CO2 Treatment of RBPS. For each treatment, aliquots of 15 100% solvent A, followed by a linear gradient from 0 to 7% B in 6 min,
mL of RBPS in a 200 mL stainless steel container (60 mm i.d. × 80 then from 7 to 12% in 5 min, then from 12 to 20% B in 4 min, and finally
mm) were placed in the treatment vessel, which was preheated to a with isocratic 100% B over 9 min. The injection volume was 20 µL, and
given temperature and then pressurized by CO2. A series of trials was peaks were detected at 538 nm. Concentrations of betanin and isobetanin
carried out with varying values of pressure and temperature (based on were obtained by comparisons with standard lines. HPLC grade methanol
the experimental conditions in ref 19with an extension of pressure up was purchased from Merck Chemical Inc. (Darmstadt, Germany). Other
to 50 MPa and temperature to 65 °C): 10, 30, and 50 MPa and 45, 55, chemicals used were of analytical grade.
6482 J. Agric. Food Chem., Vol. 56, No. 15, 2008 Liu et al.

daughter ions with those published in the literature (20, 22–25). All
samples were kept at 4 °C between treatments and analyses to eliminate
the degradation effect of room temperature and light.
Data Analysis. The degradation data of betanin and isobetanin were
subjected to regression analysis using the following first-order models:

ln []
Ct
C0
) -kt (1)

where Ct and C0 are the concentrations of betanin/isobetanin at time t

and time 0, and k is the reaction rate constant (h-1).
The Arrhenius equation was applied to estimate the activation energy
Ea (kJ/mol) of the degradation reaction

ln [] [
k1
k2
)
Ea 1
-
R T2 T1
1
] (2)

where k1 and k2 are the reaction rate constants at temperatures T1 and

T2, respectively, R is the gas constant (8.3144 J/K mol), and T is the
absolute temperature (K).
Statistical Analysis. All treatments were conducted in duplicate,
and all measurements were performed in triplicate. Data were subjected
to analysis of variance (ANOVA) using the software package SPSS
12.0 for Windows (SPSS Inc., Chicago, IL). Means of degradation data
were separated at the 5% significance level using the LSD method.

RESULTS AND DISCUSSION

Thermal/Combined HP-CO2 and Thermal Degradation
Kinetics of Betanin and Isobetanin. Concentrations of betanin
and isobetanin in RBPS before and after treatments at different
parameters are presented in Table 1. As the treatment time
extended, the residual contents of betanin and isobetanin in
RBPS were remarkably decreased (p < 0.05) apart from the
treatment at 10 MPa, 45 °C, and 0.5 h where the concentrations
of betanin and isobetanin remained largely unchanged (p >
0.05). Treatment at 50 MPa, 65 °C, and 1.3 h led to the greatest
pigment loss from initial concentrations of 18.34 ( 0.14 and
17.04 ( 0.20 mg/L to only 3.90 ( 0.02 and 3.31 ( 0.04 mg/L
for betanin and isobetanin, respectively. As expected, the
degradation reaction of the two compounds was obviously
accelerated with an elevation of temperature.
From a log linear plot (Figure 1) of the relative residual
response value [ln(Ct/C0)] versus treatment time at constant
temperature and pressure, it was verified as to whether combined
HP-CO2 and thermal degradation of betanin and isobetanin could
be adequately described by a first-order reaction. The kinetic
data tabulated in Table 2 indicated that the degradation reaction
of betanin and isobetanin under HP-CO2 combined with
temperature could be described by first-order decays on the basis
of regression coefficients >0.81 (p < 0.05). As shown in Figure
1 and Table 2, with an elevated temperature, k values for the
degradation of betanin and isobetanin increased significantly
(p < 0.05) at constant pressure. Moreover, the degradation
kinetic parameters listed in Table 2 revealed that the degradation
Figure 1. Effect of HP-CO2 on degradation kinetics of betanin and rate of betanin was slightly slower than that of isobetanin at
isobetanin at different pressures and temperatures as compared to each identical pressure and temperature. This implied that
traditional thermal treatment. betanin was more stable than isobetanin under HP-CO2. As
compared to treatments at atmospheric pressure and 45 °C, HP-
HPLC-MS Analysis of Pigment Patterns of RBP Treated by HP- CO2 treatments led to significant decreases in k values and
CO2 Combined with Thermal Conditions. Untreated and RBPS increases in half-life t1/2 values (p < 0.05) at the same
treated by combined HP-CO2 (10-50 MPa) and thermal (55 °C for temperaturesthe t1/2 value was 12.160 and 11.198 h for betanin
2.5 h and 65 °C for 1.3 h) treatments were analyzed by HPLC-MS
and isobetanin, respectively, at atmospheric pressure. In contrast,
performed on an Agilent 1100 HPLC-MS system with the same method
for HPLC-DAD analysis. The mass spectrometer was equipped with the t1/2 value was >16 and 13 h for betanin and isobetanin,
an ESI source operating in the positive ionization mode. Nitrogen was respectively, with pressures of 10-50 MPa, indicating that
used as the dry gas at a flow rate of 8 L/min and a pressure of 35 psi. betanin and isobetanin were even more stable under HP-CO2
The nebulizer temperature was set at 350 °C. Compounds were than atmospheric pressure at 45 °C. Furthermore, with the
identified by comparing their protonated molecular ions and derived elevation of pressure, k values for both pigments dramatically
Combined HP-CO2 and Thermal Degradation of Betalains J. Agric. Food Chem., Vol. 56, No. 15, 2008 6483

Table 2. Degradation Kinetic Parameters of Betanin and Isobetanin

betanin isobetanin
-1 2 -1
temp (°C) pressure (MPa) k (h ) t1/2 (h) R k (h ) t1/2 (h) R2
45 atmospheric pressure 0.057 12.160 0.817 0.062 11.198 0.892
10 0.022 31.364 0.811 0.034 20.447 0.927
30 0.025 28.292 0.811 0.045 15.576 0.851
50 0.027 25.297 0.925 0.053 13.078 0.986
55 atmospheric pressure 0.127 5.475 0.987 0.152 4.554 0.989
10 0.048 14.321 0.983 0.075 9.267 0.988
30 0.136 5.108 0.939 0.156 4.435 0.960
50 0.173 4.018 0.989 0.196 3.536 0.992
65 atmospheric pressure 0.470 1.476 0.997 0.547 1.267 0.997
10 0.472 1.469 0.939 0.507 1.367 0.949
30 1.110 0.624 0.939 1.201 0.577 0.943
50 1.212 0.572 0.986 1.299 0.534 0.988

increased, and t1/2 values decreased (p < 0.05). However, when < 0.05). In other words, the influence of HP-CO2 on the stability
the temperature was 55 °C, the degradation parameter values of betanin and isobetanin is attributed to both pressure and
for betanin and isobetanin at 30 and 50 MPa approached those temperature. This suggests that betanin and isobetanin become
at atmospheric pressure (p > 0.05). Meanwhile, when the less stable at pressures >30 MPa combined with temperatures
temperature increased to 65 °C at 10 MPa, the parameters for exceeding 55 °C than atmospheric pressure with accordant
both pigments were almost the same as those at atmospheric temperatures. In conclusion, with HP-CO2 application in
pressure, respectively (p > 0.05). Nevertheless, k values for microorganism sterilization or enzyme inactivation for red beet
betanin and isobetanin at 30 and 50 MPa were much larger than products, the combination of pressure below 30 MPa (if the
those at atmospheric pressure, and t1/2 values were smaller (p temperature is >55 °C) or temperature below 55 °C (if the

Figure 2. Spectrum of RBPS after HP-CO2 treatments at different pressures (a, atmospheric pressure; b, 10 MPa; c, 30 MPa; d, 50 MPa) and temperatures
(treatment time: 2.5 h at 45 and 55 °C and 1.3 h at 65 °C).
6484 J. Agric. Food Chem., Vol. 56, No. 15, 2008 Liu et al.

Figure 3. Change in L*, h°, and C* values of RBP exposed to HP-CO2 at various conditions (a, 45 °C; b, 55 °C; c, 65 °C).

pressure is >30 MPa) is considered to be effective for a higher HP-CO2. This phenomenon demonstrated that the thermal
retention of betacyanins. sensitivities of betanin and isobetanin were changed when
The activation energy (Ea) (energy required for the degrada- exposed to HP-CO2.
tion reaction) is another important parameter for evaluating Spectra of Betanin and Isobetanin with Thermal/Com-
pigment stability under different treatments. The Ea values for bined HP-CO2 and Thermal Treatment. The spectra from
betanin and isobetanin under atmospheric pressure and HP-CO2 400 to 700 nm of RBPS after HP-CO2 treatments at different
were obtained from the Arrhenius equation (eq 2). At atmo- temperatures are shown in Figure 2. The absorbance at 538
spheric pressure, Ea values were 94.01 and 97.16 kJ/mol for nm was reported to be the characteristic peak of betanin and
betanin and isobetanin, respectively, where as compared to the isobetanin (23). As the treatment temperature and pressure
thermal treatment, the degradation reaction under HP-CO2 had increased, the absorbance at 538 nm declined. However, the
considerably higher Ea values: 136.12, 170.25, and 170.83 kJ/ shape of the spectra at different pressures is similar to those
mol for betanin and 120.38, 146.89, and 142.69 kJ/mol for under atmospheric pressure except for HP-CO2 treatment at
isobetanin at pressures of 10, 30, and 50 MPa, respectively. 30-50 MPa and 65 °C for 1.3 h where the maximum
Therefore, this seems to suggest that betanin and isobetanin are absorbance wavelength (λmax) moved to ∼445 nm. This might
more sensitive to heat under atmospheric pressure than HP- be induced by extra degradation of betanin and isobetanin to
CO2, and it also was found that Ea values increased significantly some yellow pigments at 50 MPa and 65 °C for 1.3 h.
from 10 to 30 MPa (p < 0.05) and then remained stagnant Color Developments. Color developments of different treat-
between 30 and 50 MPa for betanin and isobetanin (p > 0.05). ment samples were monitored, and the values are shown in
Additionally, the Ea value for isobetanin was higher than that Figure 3. With the rise in temperature and pressure and
for betanin at atmospheric pressure. In contrast, the Ea value extension of treatment time, C* and h° values increased
for isobetanin was relatively lower than that for betanin under significantly (p < 0.05), which possibly is attributed to
Combined HP-CO2 and Thermal Degradation of Betalains J. Agric. Food Chem., Vol. 56, No. 15, 2008 6485

Figure 4. HPLC profiles of RBPS. (a, control (untreated sample); b, heated for 2.5 h at 55 °C; c, heated for 1.3 h at 65 °C; d, treated for 2.5 h by
HP-CO2 at 50 MPa and 55 °C; e, treated for 1.3 h by HP-CO2 at 50 MPa and 65 °C).
Table 3. HPLC-DAD and HPLC-MS Data of Betanin and Isobetanin and Their Degradation Products in RBPS

peak retention HPLC-DAD HPLC-ESI(+)-MS2 name/proposal

numbera time (min) λmax (nm) m/z (M + H)+ experiment m/z name
1 7.3 538 551 MS2 [551]:389 betanin
2 7.9 538 551 MS2 [551]:389 betaninb
3 9.1 505 507 MS2 [507]:345 17-decarboxy-betanin
4 10.4 538 551 MS2 [551]:389 isobetanin
5 11.8 538 551 MS2 [551]:389 isobetaninb
6 13.3 505 507 MS2 [507]:345 17-decarboxy-isobetanin
7 14.1 533 507 MS2 [507]:345 12-decarboxy-betanin
8 16.4 505 507 MS2 [507]:345 decarboxy-betaninb
9 16.8 538 507 MS2 [507]:345 15-decarboxy-betanin

a
Peak numbers correspond to Figure 4. b Proposal name and structures were not determined.
betacyanin loss and creation of orange-red/yellow pigments. compounds, which resulted in an increase in the b* value and
As shown in Figure 2, a higher temperature and pressure as a decrease in the a* value, and then the h° and C* values
well as longer exposure time under HP-CO2 caused a hypso- increased. Besides, higher C* values reflected an increasing
chromic shift of the absorption maxima, resulting in the color color purity through HP-CO2 combined with thermal
changing from purple to orange-red. Moreover, it is known treatment.
that with an increasing betacyanin concentration, the hue angle Pigment Pattern of Betanin and Isobetanin with Thermal/
shifts from yellow-red to purple (26). On the contrary, with Combined HP-CO2 and Thermal Treatment (Peak and
decreasing betacyanin content, the hue angle shifts from purple Compound Numbers Correspond to Figure 4). As shown in
to yellow-red for betanin and isobetanin when exposed to Figure 4a-e, two main peaks were identified to be betanin (1)
combined HP-CO2 and thermal treatments. Therefore, the and isobetanin (4) according to retention times, absorption
increment of the h° value probably was induced by the larger maxima, protonated molecular ions (m/z 551), and fragments
b* value, which was due to the orange-red degradation [m/z 389 ) 551 - 162 (Glc)] yielded by MS2 experiments (22).
products. These consistent results could be disclosed by HPLC- As compared to untreated RBPS, thermal treatments at 55 °C
MS analysis. Betanin and isobetanin were degraded to yellow-red (for 2.5 h) and 65 °C (for 1.3 h) could only result in the
6486 J. Agric. Food Chem., Vol. 56, No. 15, 2008 Liu et al.

Table 4. Stability of Pigments with Different Treatmentsa

control APc, 55 °C, AP, 65 °C, 10 MPa, 55 °C, 10 MPa, 65 °C, 30 MPa, 55 °C, 30 MPa, 65 °C, 50 MPa, 55 °C, 50 MPa, 65 °C,
b
compound (untreated) 2.5 h 1.3 h 2.5 h 1.3 h 2.5 h 1.3 h 2.5 h 1.3 h
1 51.16 51.22 52.37 47.21 42.53 47.13 35.41 46.40 34.05
2 0.00 0.00 0.00 1.43 5.54 3.85 6.03 4.15 6.64
3 0.64 1.58 1.40 1.30 1.35 1.06 2.86 1.19 3.11
4 46.18 43.52 42.39 44.78 37.52 39.83 26.55 38.71 25.11
5 0.00 0.00 0.00 1.82 4.56 3.13 5.87 3.64 6.05
6 0.83 1.39 1.24 0.90 1.12 1.03 2.26 1.12 2.83
7 0.49 1.27 0.73 0.53 1.07 0.66 2.06 0.75 2.40
8 0.70 0.54 0.94 0.96 2.97 1.44 9.12 1.97 9.67
9 0.00 0.49 0.92 1.07 3.34 1.87 9.84 2.08 10.15

a
Nine compounds were quantified by HPLC with peak area (%). b Compound numbers correspond to Figure 4. c AP: atmospheric pressure.

generation of 9, while 2, 5, and 9 were induced by HP-CO2 at evaluation of degradation products as well as the degradation
50 MPa (at 65 °C for 1.3 h), and each compound was primarily mechanism of betanin and isobetanin under HP-CO2 combined
identified by HPLC-MS analysis. As represented in Table 3, with thermal treatment also need further investigation and should
1, 2, 4, and 5 had the same m/z, and they might be isomeric be discussed in a future study.
compounds since there are two chiral carbon atoms in the
molecular structure of beanin; however, 2 and 5 were identified
as undescribed structures. LITERATURE CITED
In accordance with thermal treatment in previous findings, (1) Loey, A. V.; Ooms, V.; Weemaes, C.; Van de Broeck, I.;
HP-CO2 combined with thermal treatment led to a decar- Ludikhuyze, L.; Indrawati; Deny, S.; Hendrickx, M. Thermal and
boxylated reaction. Peaks 3 and 6-9 with typical fragmenta- pressure-temperature degradation of chlorophyll in broccoli
tion data in the MS2 analyses [(m/z 345 ) 507 - 162 (Glc)] (Brassica oleracea L. italica) juice: A kinetic study. J. Agric.
were regarded as decarboxy-betanin with different decar- Food Chem. 1998, 46, 5289–5294.
boxylation sites except for 8 (not determined). Moreover, (2) Farr, D. High pressure technology in the food industry. Trends
the loss of a carboxy moiety was reported to reduce polarity, Food Sci. Technol. 1990, 1, 14–16.
leading to longer retention relative to the respective nonde- (3) Riahi, R.; Ramaswamy, H. S. High pressure inactivation kinetics
carboxylated structure (21, 23–25). On the basis of retention of amylase in apple juice. J. Food Eng. 2004, 64, 151–160.
(4) Damar, S.; Balaban, M. O. Review of dense phase CO2 technol-
time, structure, and absorption maxima (505 nm, hypsochro-
ogy: Microbial and enzyme inactivation and effects on food
mic shift of 33 nm relative to betanin and isobetanin), peaks
quality. J. Food Sci. 2006, 71, 1–11.
3 and 6 were identified as 17-decarboxy-betanin and 17- (5) Arreola, A. G.; Balaban, M. O.; Marshall, M. R.; Peplow, A. J.;
decarboxy-isobetanin, respectively. Similarly, peak 9 was Wei, C. I.; Cornell, J. A. Supercritical carbon dioxide effects on
identified as 15-decarboxy-betanin because of the identical some quality attributes of single strength orange juice. J. Food
absorption maxima with betanin, loss of the chiral center, Sci. 1991, 56, 1030–1033.
and absence of a second compound (22). By analogy, peak (6) Arreola, A. G.; Balaban, M. O.; Wei, C. I.; Peplow, A. J.;
7 could be assumed to be 2-decarboxy-betanin with a slight Marshall, M. R. Effect of supercritical carbon dioxide on microbial
hypsochromic shift of 5nm relative to betanin (25). population in single strength orange juice. J. Food Sci. 1991, 14,
From the chromatograms shown in Figure 4, the peak area 275–284.
(%) of each compound is listed and illustrated in Table 4. As (7) Haas, G. J.; Prescott, H. E.; Dudly, E.; Dick, R.; Hintlian, C.;
compared to thermal treatments, HP-CO2 treatments induced Keane, L. Inactivation of microorganisms by carbon dioxide under
high levels of 3 and 6-9 and new isomeric compounds of pressure. J. Food Safety 1989, 9, 253–265.
betanin. In addition, a higher pressure and temperature induced (8) Kincal, D.; Hill, S.; Balaban, M. O.; Marshall, M. R.; Wei, C. A
continuous high pressure carbon dioxide system for microbial
higher levels of 2, 3, 5-7, and 9. HP-CO2 treatments with higher
reduction in orange juice. J. Food Sci. 2005, 70, 249–254.
pressure and temperature accelerate the isomerization and
(9) Sims, M.; Estigarribia, E. Continuous sterilization of aqueous
decarboxy reactions of betanin and isobetanin. Therefore, the pumpable food using high pressure. In Proceedings of the 4th
major degradation products of betanin and isobetanin treated International Symposium on High Pressure Process Technology
by HP-CO2 and thermal treatments were decarboxy-betanin and Chemical Engineeing; Bertucco, A., Ed.; Chemical Engineer-
isomers with different decarboxylation sites (C-15, -17, -2, etc.) ing Transactions: Venice, Italy, 2002; pp 2921-2926.
and two new isomeric compounds of betanin that were (10) Schwartz, S. J.; von Elbe, J. H. Quantitative determination of
particularly caused by HP-CO2 treatments. individual betacyanin pigments by high-performance liquid chro-
In conclusion, the stability of betanin and isobetanin under matography. J. Agric. Food Chem. 1980, 28, 540–543.
HP-CO2 was affected by both pressure and temperature, and (11) Vincent, K. R.; Scholz, R. G. Separation and quantification of
betanin was even more stable than isobetanin. Treatments with red beet betacyanins and betaxanthins by high-performance liquid
a pressure above 30 MPa and temperature >55 °C led to a more chromatography. J. Agric. Food Chem. 1978, 26, 812–816.
(12) Piattelli, M.; Minale, L. Pigments of centrospermae. II. Distribution
rapid loss of betanin and isobetanin and a color change from
of betacyanins. Phytochemistry 1964, 3, 547–557.
violet to orange-red. At the same treatment temperature and (13) Piattelli, M.; Minale, L.; Prota, G. Pigments of centrospermae.
time, more products were generated, and decarboxylation was III. Betaxanthins from Beta Vulgaris L. Phytochemistry 1965, 4,
accelerated by a higher pressure than thermal treatment alone. 121–125.
Therefore, it is necessary to explore proper pressure and (14) Mégard, D. Stability of red beet pigments for use as food colorant:
temperature ranges for HP-CO2 treatments to achieve not only A review. Foods Food Ingred. J. 1993, 158, 130–150.
better pigment retention but also effective microorganism (15) Lashley, D.; Wiley, R. C. A betacyanine decolorizing enzyme
sterilization and enzyme inactivation. However, the safety found in red beet tissue. J. Food Sci. 1979, 44, 1568–1569.
Combined HP-CO2 and Thermal Degradation of Betalains J. Agric. Food Chem., Vol. 56, No. 15, 2008 6487

(16) Martinez-Parra, J.; Munoz, R. Characterization of betacyanin (22) Herbach, K. M.; Stintzing, F. C.; Carle, R. Impact of thermal
oxidation catalyzed by a peroxidase from Beta Vulgaris L. roots. treatment on color and pigment pattern of red beet (Beta Vulgaris
J. Agric. Food Chem. 2001, 49, 4064–4068. L.) preparations. J. Food Sci. 2004, 69, 491–498.
(17) Shi, C. C.; Wiley, R. C. Betacyanin and betaxanthine decolorizing (23) Strack, D.; Vogt, T.; Schliemann, W. Recent advances in betalain
enzymes in the red beet (Beta Vulgaris L.) root. J. Food Sci. 1981, research. Phytochemistry 2003, 62, 247–269.
47, 164–166, 172. (24) Hilpert, H.; Siegfried, M. A.; Dreiding, A. S. Totalsynthese von
(18) Wasserman, B. P.; Guilfoy, M. Peroxidative properties of betanin decarboxybetalainen durch photochemische ringöffnung von 3-(4-
decolorization by cell walls of red beet. Phytochemistry 1983, pyridyl)alanin. HelV. Chim. Acta 1985, 68, 1670–1678.
22, 2653–2656. (25) Stintzing, F. C.; Carle, R. Functional properties of anthocyanins
(19) Liu, X.; Gao, Y.; Peng, X.; Yang, B.; Xu, H.; Zhao, J. Inactivation and betalains in plants, food, and human nutrition. Trends Food
Sci. Technol. 2004, 19–38.
of peroxidase and polyphenol oxidase in red beet (Beta Vulgaris
(26) Stintzing, F. C.; Schieber, A.; Carle, R. Evaluation of color
L.) extract with high pressure carbon dioxide. InnoV. Food Sci.
properties and chemical quality parameters of cactus juices. Eur.
Emerg. Technol. 2008, 9, 24–31.
Food Res. Technol. 2003, 216, 303–311.
(20) Pourrat, A.; Lejeune, B.; Grand, A.; Pourrat, H. Betalains assay
of fermented red beet root extract by high performance liquid
Received for review March 10, 2008. Revised manuscript received May
chromatography. J. Food Sci. 1988, 53, 294–295.
15, 2008. Accepted May 23, 2008. This research was supported by the
(21) Stintzing, F. C.; Kammerer, D.; Schieber, A.; Adama, H.;
National Natural Science Foundation of the People’s Republic of China
Nacoulma, O. G.; Carle, R. Betacyanin and phenolic compounds
(Project 20376084).
from Amaranthus spinosus L. and BoerhaVia erecta L. Z.
Naturforsch, C: J. Biosci. 2004, 59, 1–8. JF800727Q