THE EFFECT OF PRE-TREATMENT, TEMPERATURE AND LENGTH OF FROZEN STORAGE ON THE RETENTION OF CHLOROPHYLLS IN FROZEN BRASSICAS

TIARUM POLONO
N
R
IE
UM
S
CACTA Acta Sci. Pol., Technol. Aliment. 7(2) 2008, 21-34

THE EFFECT OF PRE-TREATMENT, TEMPERATURE
AND LENGTH OF FROZEN STORAGE
ON THE RETENTION OF CHLOROPHYLLS
IN FROZEN BRASSICAS*
Waldemar Kmiecik, Zofia Lisiewska, Jacek S?upski,
Piotr G?bczy?ski
University of Agriculture in Krakow
Abstract. The investigation covered broccoli, green cauliflower and Brussels sprouts. The
evaluation concerned the raw material; the material after blanching; the material after
cooking; and frozen products from blanched (traditional method) and cooked (modified
method) material, stored at –20°C and –30°C then prepared for consumption using water-
cooking in the traditional method and thawing by microwave in the modified method after
0, 4, 8 and 12 months of frozen storage. Depending on the investigated sample, the vege-
tables prepared for consumption after 12 months of frozen storage retained total chloro-
phylls as follows: broccoli, 45-66% of the content in the raw material; green cauliflower,
30-45%; and Brussels sprouts, 66-78%. In comparison with the traditional method, the
mean content of chlorophylls in Brussels sprouts obtained using the modified method was
16% higher; however, in broccoli the content was lower by 23% and in cauliflower by
21% on average. Lower storage temperature resulted in higher content of chlorophylls in
all investigated vegetables.
Key words: brassicas, chlorophylls content, blanching, cooking, frozen storage, preparing
for consumption
INTRODUCTION
Green brassicas such as broccoli, green cauliflower and Brussels sprouts are widely
grown and consumed. In temperate climates their supply is limited to a few months of
the year. In order to provide year-round availability, various storage conditions improv-

* This work was supported from means on science in years 2006-2008 as a scientific project
N31203831/2450.

Corresponding author – Adres do korespondencji: Prof. dr hab. Zofia Lisiewska, Department of
Raw Materials and Processing of Fruit and Vegetables of University of Agriculture in Krakow,
Balicka 122, 30-149 Cracow, Poland, e-mail: rrlisiew@cyf-kr.edu.pl

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22
W. Kmiecik ...
ing shelf-life are used [Pogson and Morris 1997, Yamauchi and Watada 1998]. They are
also processed, the best available method being freezing [Dong et al. 2004, G?bczy?ski
2003, G?bczy?ski and Kmiecik 2006, G?bczy?ski and Lisiewska 2006]. The low tem-
peratures commonly prescribed for frozen foods (–18ºC) can maintain initial quality and
nutritive value practically unchanged, so that frozen and fresh vegetable products differ
only in texture [Canet 1989]. The freezing of vegetables immediately post-harvest guar-
antees consumers higher vitamin C content than could be attained by any other form of
preservation and distribution. Furthermore, if properly handled before freezing and
during distribution, there is no possibility of growth of microbial contaminants between
freezing and thawing [Kennedy 2000].
The colour of green vegetables depends on the level of chlorophyll a and chloro-
phyll b and on their ratio. The content of chlorophylls depends not only on the species
and cultivar of the vegetable and its edible part [Murcia et al. 2000], but also on the
developmental stage; possible signs of senescence [Suda et al. 1994]; and conditions of
storage after harvest [Yamauchi and Watada 1998]. In the product prepared for con-
sumption the level of chlorophyll is also affected by pre-treatment before processing.
The significant parameters are the temperature, the length of thermal processing and the
pH of the medium in which the pre-treatment is carried out [Canet et al. 2005, Murcia et
al. 2000, Tijskens et al. 2001]. Freezing and frozen storage also are important factors in
retaining the attractive bright green colour [G?bczy?ski 1999, Lisiewska et al. 2004].
In recent years the consumption of frozen vegetables has rapidly increased, prompt-
ing efforts to improve their quality and shelf-life. Appearance is taken as an indication
of freshness, palatability and nutritional value [López et al. 1998]. Particular attention is
paid to food products of the “do-it-for-me” or “ready-to-eat” type. Frozen vegetable
products of this type are obtained when, in the pre-treatment stage, blanching is re-
placed by cooking to consumption consistency. After frozen storage, such products only
require thawing and heating to consumption temperature. Thus the product is prepared
for consumption using a microwave oven instead of cooking in water, which always
increases the leaching of nutritional compounds.
Modifications to processes should be the outcome of basic research into the physi-
cal, chemical, and biological features of phytosystems at low temperatures aimed at
improving our understanding of the behavioural processes that take place during freez-
ing. The development of new generations of frozen vegetables products with higher
added values and competitive pricing will depend on the creative efforts and techno-
logical development arising out of cooperation between scientists and manufacturers.
The first aim of the present work was to study and compare the effect of two pre-
treatments before freezing (traditional and new modified technology) on the chlorophyll
content of frozen broccoli, cauliflower and Brussels sprouts. The second was to ascer-
tain, in each studied frozen vegetable, the effect on the cited content of chlorophylls of
the temperature and length of frozen storage prior to both the final culinary methods
(boiling and microwaving). This paper follows on from a series of papers describing the
content of minerals, heavy metals and antioxidative compounds in brassicas [G?bczy?-
ski and Lisiewska 2006, Kmiecik et al. 2007, Lisiewska et al. 2007].
Acta Sci. Pol.

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The effect of pre-treatment, temperature and length of frozen storage ...
23
MATERIAL AND METHODS
Plant materials
The investigated material consisted of three species of brassicas recommended for
freezing: broccoli cultivar Lord F1, green cauliflower cultivar Trevi F1 and Brussels
sprouts cultivar Lunet F1. Analytical procedures were carried out by analysing the raw,
blanched and cooked vegetables and frozen vegetables obtained using the two different
pre-treatments (blanching and cooking) and prepared for consumption using two domes-
tic culinary treatments (cooking and microwaving). The products were evaluated di-
rectly after freezing (0 months) and after 4, 8, and 12 months of frozen storage at –20°C
and –30°C.
The raw material was harvested in the experimental field of the Agricultural Univer-
sity of Krakow in 2005. The field lies in southern Poland, on the western outskirts of
Krakow. The soil was of good horticultural structure, with neutral pH and high levels of
potassium, phosphorus and calcium. The application of mineral fertilizers was deter-
mined by the fertility of soil and the nutritional requirements of the investigated species.
The doses of mineral fertilizers were of 120 kg N ha-1, 80 kg P2O5 ha-1 and 150 kg K2O
ha-1 for the Brussels sprouts, and 150 kg N ha-1, 100 kg P2O5 ha-1 and 150 kg K2O ha-1
for the broccoli and the cauliflower. The cultivation included mechanical weed control,
sprinkler irrigation, and protection against diseases and pests as necessary. The harvest
of broccoli and cauliflower was carried out in the first 10 days of October and of Brus-
sels sprouts in mid-October.
A sample representing the whole batch of a given cultivars was taken for the analy-
sis of the raw material and the preparation of frozen products. Heads of broccoli and
cauliflower were trimmed of leaves and divided into florets about 5 cm in diameter, the
stalks being cut to a depth of 2 cm below the lowest ramification. Heads of Brussels
sprouts 25-30 mm in diameter were cleaned of stipules and their stalks were shortened.
Pre-treatment before freezing
Two different pre-treatments were used in preparing the fresh vegetables for freez-
ing. Using the traditional technology (pre-treatment I) the fresh vegetables were
blanched and, after freezing and frozen storage, the frozen product required traditional
water cooking. With the modified technology (pre-treatment II) the fresh vegetables
were cooked before freezing to an approximately consumption consistency; giving a
ready-to-eat products which merely requires defrosting and heating in a microwave
oven after freezing and frozen storage.
In pre-treatment I the fresh material was blanched in a stainless steel vessel in water,
the proportion of water to the fresh material being 5:1 and the blanching temperature
95-98°C. The blanching time (Table 1) used for the different vegetables was determined
in preliminary experiments. These blanching conditions permitted a decrease in the
activity of catalase and peroxidase to a level below 5% of the initial value. The total
absence of peroxidase activity indicates overblanching and there is a substantial body of
evidence suggesting that the quality of products frozen after blanching is superior if a
certain level of peroxidase activity remains at the end of the blanching process. As
Canet (1989) showed, the residual activity of peroxidase in blanched material of:
Technologia Alimentaria 7(2) 2008

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24
W. Kmiecik ...
Table 1. Time of blanching and cooking before freezing and time of preparing for consumption
after frozen storage
Tabela 1. Czas blanszowania i gotowania przed mro eniem oraz czas przygotowania do spo ycia
po sk?adowaniu zamra alniczym
Before freezing
After frozen storage
Przed mro eniem
Po sk?adowaniu zamra alniczym
thawing and heating in microwave
Vegetable
oven, goods stored at:
Warzywo
rozmra anie i podgrzewanie
blanching
cooking
cooking*
w kuchence mikrofalowej
blanszowanie
gotowanie
gotowanie*
produktów przechowywanych w:
–20°C
–30°C
Broccoli
3 min
6 min
5 min
7 min 45 s
8 min 15 s
Broku?
Green cauliflower
3 min 15 s
6 min
5 min
7 min 45 s
8 min 15 s
Kalafior zielony
Brussels sprouts
5 min
15 min
9 min
7 min 45 s
8 min 15 s
Kapusta brukselska
* Time was the same for both storage temperature (–20°C and –30°C).
* Czas gotowania by? taki sam dla obydwu temperatur sk?adowania (–20°C i –30°C).
2.0-6.3% in peas; 0.7-3.3% in green beans; 2.9-8.2% in cauliflower; and 7.5-11.5% in
Brussels sprouts did not reduce the quality of frozen products. During frozen storage no
regeneration of these enzymes was recorded. After blanching the material was immedi-
ately cooled in cold water and left for 30 min on sieves to drain the water remaining on
the surface.
In the pre-treatment II the vegetables were cooked in a stainless steel vessel in water
with 2% added salt (NaCl), the proportion of the weight of the raw material to brine being
1:1. The vegetables were placed in boiling water. The cooking time measured from the
moment when the medium began boiling again is given in Table 1. After cooking to con-
sumption consistency, the material was left on sieves and cooled in a stream of cold air.
Freezing process and frozen storage
The materials from blanched and cooked samples were placed on trays and frozen at
t = –40°C in a Feutron blast freezer type 3626-51. The time required for the inside of
the product to reach the storage temperature of –20°C was 90 min and of –30°C was
120 min. The frozen vegetables were then packed in 500 g polyethylene bags and stored
at –20°C and –30°C respectively for 0, 4, 8, and up to 12 months.
Preparation of the frozen product for evaluation
Frozen samples of the vegetables blanched before freezing (subjected to the pre-
treatment I) were cooked in 2% brine, the proportion in weight of brine to processed
products being 1:1. As was the case when cooking fresh vegetables, the frozen product
Acta Sci. Pol.

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The effect of pre-treatment, temperature and length of frozen storage ...
25
was placed in boiling water. The cooking time measured from the moment when the
medium began boiling again is given in Table 1. After cooking, the water was immedi-
ately drained and the products were cooled to 20°C for analyses. Frozen samples of the
vegetables cooked before freezing (subjected to the pre-treatment II) were thawed and
heated in a Panasonic NN-F621 microwave oven. In this case a 500 g portion was
placed in a covered heatproof vessel. The time required for thawing and heating to con-
sumption temperature [Codex Alimentarius 1993] is given in Table 1. The samples were
then cooled to 20°C and analysed. The changes in the weight of vegetables resulting
from all the technological processing and culinary treatments applied are shown in Ta-
ble 2. Because these changes were relatively small and similar for both types of prod-
ucts, their effect on the content of chlorophylls was not taken into consideration in the
discussion section.
Table 2. Weight* of vegetables after freezing and preparing for consumption (weight of raw
material = 100%)
Tabela 2. Masa warzyw* po procesie technologicznym zamra ania i przygotowaniu do spo ycia
(masa surowca = 100%)
Material
Broccoli
Green cauliflower
Brussels sprouts
Materia?
Broku?
Kalafior zielony
Kapusta brukselska
Blanched before freezing and cooked
95-98
95-98
98-100
Blanszowany przed mro eniem i ugotowany
Cooked before freezing and prepared
95-97
94-96
95-98
in microwave oven
Gotowany przed mro eniem i przygotowany
w kuchence mikrofalowej
* Range of values for all stages and temperatures.
* Zakres warto?ci dla wszystkich terminów i temperatur.
The table salt used in the experiment was always taken from the same batch; the pH
of the tap water varied within 7.3-7.7.
Chemical analyses
Dry matter content was determined by gravimetric as the mass loss of the sample at
96-98°C [AOAC 1984]. The content of chlorophylls a and b was determined using the
Lichtenthaler and Buschmann [2001 a, b] method. Chlorophylls were extracted with
pure acetone until all colour was removed from the sample. After suitable dilutions were
obtained, colorimetric measurements were carried out. The measurement of absorbance
was conducted in a Shimadzu UV-160A spectrophotometer at 661.6 and 644.8 nm
wavelengths, i.e. at the absorbance maxima for chlorophylls a and b. The pigment con-
centrations were calculated on the basis of the absorbance coefficients at the above
wavelengths.
The content of chlorophylls in the raw material, in the partly processed material and
in frozen products prepared for consumption was calculated per 100 g fresh matter. The
level of dry matter given in Tables 3-5 allows the reader to apply the presented results to
calculate the content of dry matter.
Technologia Alimentaria 7(2) 2008

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26
W. Kmiecik ...
Table 3. Content of chlorophyll in broccoli prepared for consumption depending on pre-
treatment before freezing and temperature of frozen storage, in fresh matter
Tabela 3. Zawarto?? chlorofilu w broku?ach przygotowanych do konsumpcji w zale no?ci od
obróbki przed mro eniem i temperatury sk?adowania, w ?wie ej masie
Storage
Chlorophyll, mg·100 g-1*
temperature
Dry matter
Chlorofile, mg·100 g-1*
Stage of
Material
°C
g·100 g-1*
estimation
Materia?
Temperatura
Sucha masa
Etap bada?
sk?adowania
g·100 g-1*
a
b
a + b
°C
Before
raw – surowy
–
10.34 ±0.129 10.3 ±0.26
5.1 ±0.22
15.4 ±0.50
freezing
after blanching – blanszowany
–
8.37 ±0.108
8.5 ±0.25
4.2 ±0.05
12.7 ±0.30
Przed
mro eniem after cooking – gotowany
–
10.74 ±0.120
7.3 ±0.28
3.6 ±0.13
10.9 ±0.35
0
blanched before freezing and cooked
–20
9.19 ±0.075
8.1 ±0.13
4.0 ±0.26
12.1 ±0.39
blanszowany przed mro eniem
i ugotowany
–30
9.23 ±0.079
7.7 ±0.24
4.2 ±0.12
11.9 ±0.34
cooked before freezing and prepared
–20
11.02 ±0.091
6.8 ±0.14
3.4 ±0.19
10.2 ±0.32
in microwave oven
gotowany przed mro eniem
–30
11.13 ±0.103
6.9 ±0.13
3.5 ±0.15
10.4 ±0.27
i przygotowany w kuchence
mikrofalowej


c
i
a
n
y
o 4
blanched before freezing and cooked
–20
9.17 ±0.080
7.2 ±0.29
3.9 ±0.06
11.1 ±0.46
t
i
o
p

s
p
blanszowany przed mro eniem
m
o
s
u

d
i ugotowany
–30
9.22 ±0.076
7.6 ±0.24
4.2 ±0.14
11.8 ±0.33
n
i
u
n
a
r

c
o
cooked before freezing and prepared
–20
11.18 ±0.071
6.1 ±0.17
3.0 ±0.13
9.1 ±0.29
w

f
o
t
o
in microwave oven
g
o
g
gotowany przed mro eniem
–30
11.22 ±0.074
6.2 ±0.24
3.2 ±0.21
9.4 ±0.34
r
i
n
y
a
r
z
i przygotowany w kuchence
p
r
e
mikrofalowej

p
d
e
)

i


p
s
)

a
n
s
i
?
c
i
e 8
blanched before freezing and cooked
–20
9.05 ±0.081
6.7 ±0.28
3.4 ±0.10
10.1 ±0.33
t
h
n
blanszowany przed mro eniem
o

(
m
i
u
i ugotowany
–30
9.09 ±0.069
7.4 ±0.10
3.6 ±0.15
11.0 ±0.21
n
e

(
m
a
g
w
y
cooked before freezing and prepared
–20
11.22 ±0.102
5.5 ±0.14
2.8 ±0.08
8.3 ±0.25
r
a
w
in microwave oven
o
r

s
t
o
h
gotowany przed mro eniem
–30
11.24 ±0.084
5.9 ±0.10
3.0 ±0.10
8.9 ±0.29
f
t
e
e
c
r
z
i przygotowany w kuchence
A

p
mikrofalowej
o
P
12 blanched before freezing and cooked
–20
9.09 ±0.075
6.2 ±0.19
3.1 ±0.10
9.3 ±0.29
blanszowany przed mro eniem
i ugotowany
–30
9.02 ±0.083
6.9 ±0.20
3.3 ±0.15
10.2 ±0.33
cooked before freezing and prepared
–20
11.28 ±0.092
4.6 ±0.19
2.4 ±0.08
7.0 ±0.26
in microwave oven
gotowany przed mro eniem
–30
11.21 ±0.099
5.4 ±0.08
2.7 ±0.10
8.1 ±0.17
i przygotowany w kuchence
mikrofalowej


LSD, ? < 0.01

0.142
0.38
0.28
0.61
NIR, ? < 0,01
*Mean value of four samples and standard deviation.
*?rednia dla czterech oznacze? i odchylenie standardowe.
Acta Sci. Pol.

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The effect of pre-treatment, temperature and length of frozen storage ...
27
Table 4. Content of chlorophyll in green cauliflower prepared for consumption depending on
pre-treatment before freezing and temperature of frozen storage, in fresh matter
Tabela 4. Zawarto?? chlorofilu w kalafiorze zielonym przygotowanym do konsumpcji w zale no?ci
od obróbki przed mro eniem i temperatury sk?adowania, w ?wie ej masie
Storage
Chlorophyll, mg·100 g-1*
temperature
Dry matter
Chlorofile, mg·100 g-1*
Stage of
Material
°C
g·100 g-1*
estimation
Materia?
Temperatura
Sucha masa
Etap bada?
sk?adowania
g·100 g-1*
a
b
a + b
°C
Before
raw – surowy
–
9.30 ±0.057
8.6 ±0.36
3.8 ±0.13
12.4 ±0.45
freezing
after blanching – blanszowany
–
8.08 ±0.059
6.4 ±0.10
3.2 ±0.14
9.6 ±0.21
Przed
mro eniem after cooking – gotowany
–
10.46 ±0.059
5.6 ±0.15
2.9 ±0.17
8.5 ±0.22
0
blanched before freezing and cooked
–20
9.22 ±0.052
5.5 ±0.05
2.7 ±0.10
8.2 ±0.14
blanszowany przed mro eniem
i ugotowany
–30
9.24 ±0.053
5.6 ±0.05
2.7 ±0.13
8.3 ±0.16
cooked before freezing and prepared
–20
11.19 ±0.065
5.0 ±0.06
2.4 ±0.13
7.4 ±0.24
in microwave oven
gotowany przed mro eniem
–30
11.28 ±0.064
5.0 ±0.06
2.4 ±0.13
7.4 ±0.18
i przygotowany w kuchence
mikrofalowej


c
i
a
n
y
o 4
blanched before freezing and cooked
–20
9.22 ±0.059
4.6 ±0.10
2.3 ±0.10
6.9 ±0.16
t
i
o
p

s
p
blanszowany przed mro eniem
m
o
s
u

d
i ugotowany
–30
9.17 ±0.057
5.0 ±0.14
2.4 ±0.10
7.4 ±0.24
n
i
u
n
a
r

c
o
cooked before freezing and prepared
–20
11.17 ±0.073
3.8 ±0.06
2.0 ±0.10
5.8 ±0.14
w

f
o
t
o
in microwave oven
g
o
g
gotowany przed mro eniem
–30
11.33 ±0.070
4.0 ±0.10
2.0 ±0.13
6.0 ±0.22
r
i
n
y
a
r
z
i przygotowany w kuchence
p
r
e
mikrofalowej

p
d
e
)

i


p
s
)

a
n
s
i
?
c
i
e 8
blanched before freezing and cooked
–20
9.24 ±0.056
3.9 ±0.13
1.9 ±0.19
5.8 ±0.19
t
h
n
blanszowany przed mro eniem
o

(
m
i
u
i ugotowany
–30
9.22 ±0.050
4.4 ±0.10
2.2 ±0.15
6.6 ±0.23
n
e

(
m
a
g
w
y
cooked before freezing and prepared
–20
11.15 ±0.067
3.1 ±0.08
1.5 ±0.13
4.6 ±0.17
r
a
w
in microwave oven
o
r

s
t
o
h
gotowany przed mro eniem
–30
11.26 ±0.074
3.5 ±0.13
1.6 ±0.13
5.1 ±0.24
f
t
e
e
c
r
z
i przygotowany w kuchence
A

p
mikrofalowej
o
P
12 blanched before freezing and cooked
–20
9.26 ±0.051
3.1 ±0.15
1.5 ±0.12
4.6 ±0.26
blanszowany przed mro eniem
i ugotowany
–30
9.20 ±0.046
3.8 ±0.17
1.8 ±0.08
5.6 ±0.25
cooked before freezing and prepared
–20
11.13 ±0.061
2.4 ±0.10
1.3 ±0.06
3.7 ±0.14
in microwave oven
gotowany przed mro eniem
–30
11.23 ±0.061
2.9 ±0.14
1.5 ±0.08
4.4 ±0.20
i przygotowany w kuchence
mikrofalowej


LSD, ? < 0.01

0.109
0.25
0.23
0.42
NIR, ? < 0,01
*Mean value of four samples and standard deviation.
*?rednia dla czterech oznacze? i odchylenie standardowe.
Technologia Alimentaria 7(2) 2008

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28
W. Kmiecik ...
Table 5. Content of chlorophyll in Brussel sprouts prepared for consumption depending on pre-
treatment before freezing and temperature of frozen storage, in fresh matter
Tabela 5. Zawarto?? chlorofilu w kapu?cie brukselskiej przygotowanej do konsumpcji w zale no?ci
od obróbki przed mro eniem i temperatury sk?adowania, w ?wie ej masie
Storage
Chlorophyll, mg·100 g-1*
temperature
Dry matter
Chlorofile, mg·100 g-1*
Stage of
Material
°C
g·100 g-1*
estimation
Materia?
Temperatura
Sucha masa
Etap bada?
sk?adowania
g·100 g-1*
a
b
a + b
°C
Before
raw – surowy
–
18.11 ±0.132 4.0 ±0.05
1.8 ±0.19
5.8 ±0.24
freezing
after blanching – blanszowany
–
16.14 ±0.097 3.6 ±0.21
1.6 ±0.10
5.2 ±0.28
Przed
mro eniem after cooking – gotowany
–
17.15 ±0.097 3.6 ±0.10
1.5 ±0.05
5.1 ±0.13
0
blanched before freezing and cooked
–20
16.54 ±0.107 3.5 ±0.10
1.5 ±0.14
5.0 ±0.22
blanszowany przed mro eniem
i ugotowany
–30
16.75 ±0.128 3.5 ±0.10
1.5 ±0.13
5.0 ±0.22
cooked before freezing and prepared
–20
18.48 ±0.116 3.5 ±0.05
1.6 ±0.17
5.1 ±0.21
in microwave oven
gotowany przed mro eniem
–30
18.56 ±0.108 3.5 ±0.05
1.6 ±0.14
5.1 ±0.17
i przygotowany w kuchence
mikrofalowej


c
i
a
n
y
o 4
blanched before freezing and cooked
–20
16.47 ±0.130 3.0 ±0.18
1.5 ±0.06
4.5 ±0.10
t
i
o
p

s
p
blanszowany przed mro eniem
m
o
s
u

d
i ugotowany
–30
16.75 ±0.139 3.3 ±0.19
1.4 ±0.05
4.7 ±0.22
n
i
u
n
a
r

c
o
cooked before freezing and prepared
–20
18.46 ±0.127 3.3 ±0.10
1.5 ±0.15
4.8 ±0.16
w

f
o
t
o
in microwave oven
g
o
g
gotowany przed mro eniem
–30
18.63 ±0.112 3.5 ±0.08
1.6 ±0.13
5.1 ±0.19
r
i
n
y
a
r
z
i przygotowany w kuchence
p
r
e
mikrofalowej

p
d
e
)

i


p
s
)

a
n
s
i
?
c
i
e 8
blanched before freezing and cooked
–20
16.33 ±0.093 2.6 ±0.10
1.3 ±0.10
3.9 ±0.16
t
h
n
blanszowany przed mro eniem
o

(
m
i
u
i ugotowany
–30
16.70 ±0.096 3.0 ±0.21
1.3 ±0.06
4.3 ±0.26
n
e

(
m
a
g
w
y
cooked before freezing and prepared
–20
18.59 ±0.109 3.2 ±0.13
1.4 ±0.13
4.6 ±0.26
r
a
w
in microwave oven
o
r

s
t
o
h
gotowany przed mro eniem
–30
18.74 ±0.120 3.3 ±0.10
1.5 ±0.15
4.8 ±0.24
f
t
e
e
c
r
z
i przygotowany w kuchence
A

p
mikrofalowej
o
P
12 blanched before freezing and cooked
–20
16.34 ±0.108 2.6 ±0.10
1.2 ±0.10
3.8 ±0.17
blanszowany przed mro eniem
i ugotowany
–30
16.78 ±0.102 2.6 ±0.10
1.3 ±0.14
3.9 ±0.24
cooked before freezing and prepared
–20
18.55 ±0.123 3.0 ±0.05
1.4 ±0.13
4.4 ±0.13
in microwave oven
gotowany przed mro eniem
–30
18.66 ±0.109 3.0 ±0.13
1.5 ±0.10
4.5 ±0.22
i przygotowany w kuchence
mikrofalowej

LSD, ? < 0.01

0.207
0.23
0.23
0.39
NIR, ? < 0,01
*Mean value of four samples and standard deviation.
*?rednia dla czterech oznacze? i odchylenie standardowe.
Acta Sci. Pol.

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The effect of pre-treatment, temperature and length of frozen storage ...
29
Statistical analysis
The differences in the content of chlorophyll a, chlorophyll b and total chlorophylls
at the various stages of evaluation were established using single-factor analysis of vari-
ance (ANOVA) on the basis of the Snedecor F and Student’s t tests, the least significant
differences (LSD) being calculated at the probability level ? < 0.01. The Statistica 6.1
program was applied in the calculation. All determinations were carried out in two ex-
perimental replications each in two parallel samples.
RESULTS AND DISCUSSION
In 100 g of the raw material broccoli contained 15.4 mg total chlorophylls, while
green cauliflower and Brussels sprouts contained 12.4 mg and 5.8 mg respectively (Ta-
bles 3-5). The chlorophyll a : chlorophyll b ratio in these vegetables was 1:0.50; 1:0.44
and 1:0.45 respectively.
After blanching the content of both chlorophyll a and chlorophyll b decreased sig-
nificantly, the loss in total chlorophylls compared with the raw material being 18% for
broccoli; 23% for green cauliflower; and 10% for Brussels sprouts. After this process
the chlorophyll a : chlorophyll b ratio was only slightly changed in comparison with the
raw material. After cooking the raw vegetables, the content of chlorophylls a and b was
also statistically lower than that found in the raw material. The losses in total chloro-
phylls were 29% in broccoli; 31% in green cauliflower and 12% in Brussels sprouts.
After cooking, the ratio of chlorophyll a to chlorophyll b was 1:0.49 in broccoli; 1:0.52
in green cauliflower; and 1:0.42 in Brussels sprouts, being no different from that found
in the raw material, apart from the cauliflower. Decreases in chlorophyll content due to
cooking compared with blanching were statistically significant in the case of broccoli
and green cauliflower. However, no significant loss was found in Brussels sprouts de-
spite the fact that cooking took three times as long as blanching (Table 1).
Dong et al. [2004], Heaton et al. [1996] and Canjura et al. [1999] stressed that the
degradation of chlorophylls was brought about by the activity of enzymes and the low
pH of the medium. In the presented experiment the activity of peroxidase, which is the
most heat-resistant indicatory enzyme [Barrett and Theerakulkait 1995], was below 5%
of the initial activity, and the pH of the water varied from 7.3 to 7.7. According to Gun-
awan and Barringer [2000] and Tijskens et al. [2001] a pH of 7-8 is in the optimum
range for the retention of chlorophylls. According to the literature, the losses of chloro-
phylls brought about by thermal processing vary from 12-66% depending on the spe-
cies, the usable part of the plant and the length and temperature of the treatment
[Lisiewska et al. 2004, Negi and Roy 2000, Murcia et al. 2000]. In the presented ex-
periment, with blanching and cooking, for all the vegetables, being carried out at the
same temperature, the difference in the length of thermal processing was determined by
the degree of enzyme inactivation in the case of blanching, and in the case of cooking
by the obtaining consumption consistency. Kidmose and Hansen [1999] also stress that
the loss of chlorophylls was greater in heads of broccoli which were stored before freez-
ing, and increased as the storage temperature of the raw material rose. The time from
harvesting to beginning the technological process of freezing was short, varying from
2 to 4 h depending on the species.
Technologia Alimentaria 7(2) 2008

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30
W. Kmiecik ...
The losses of chlorophylls brought about by blanching parsley [Lisiewska and
Kmiecik 1997]; chive [Kmiecik and Lisiewska 1999]; and dill leaves [Lisiewska et al.
2004] were not significant. In a study by G?bczy?ski [2003] a significant 15% decrease
in chlorophyll content was brought about by blanching leaf blades of beet-leaf. How-
ever, Canet et al. [2005] and Kmiecik and Budnik [1997] found an increase in chloro-
phyll content in French bean and broccoli after thermal processing. These authors ex-
plained that this was only an apparent increase due to considerable leaching of soluble
constituents. According to Premavalli et al. [2001], in six out of seven species of green
vegetables chlorophyll a was more stable than chlorophyll b, while in ground spinach
Canjura et al. [1991] found a 2-6 times greater degradation of chlorophyll a compared
with chlorophyll b. Teng and Chen [1999] reported similar results.
Irrespective of the processing methods applied to the investigated vegetables before
freezing (blanching or cooking) and the temperature of frozen storage (–20°C or
–30°C), a gradual decrease in the content of chlorophylls occurred during the storage
period. Differences between the evaluation periods of 0, 4, 8 and 12 months were sig-
nificant in most analogous samples (the same kind of pre-treatment and the same stor-
age temperature) of broccoli and green cauliflower, while in Brussels sprouts only in
certain samples. In the last species a statistically significant difference for all the sam-
ples was only found after 8 months of storage of frozen products. After 12 months of
frozen storage, the decrease in total chlorophyll content was 14-31% in broccoli; 33-
50% in green cauliflower; and 12-24% in Brussels sprouts in comparison with the mate-
rial directly after freezing. A higher retention of the investigated compounds was found
in samples stored at the lower temperature, with the exception of Brussels sprouts prod-
ucts blanched before frozen storage.
After 12 months of frozen storage, the various samples of vegetables prepared for
consumption, whether cooked in water or heated in a microwave oven, retained 45-66%
of total chlorophylls in broccoli; 30-45% in green cauliflower; and 66-78% in Brussels
sprouts in comparison with the raw material. At this stage of evaluation the chlorophyll
a : chlorophyll b ratio was 1:0.48-1:0.52 in broccoli; 1:0.47-1:0.54 in green cauliflower;
and 1:0.46-1:0.50 in Brussels sprouts, being similar to that in the raw material in the
case of broccoli but slightly lower in the remaining vegetables. According to Teng and
Chen [1999], the ratio of chlorophyll a : chlorophyll b in spinach was subject to slight
variation as the microwave cooking time varied; moreover, these authors cooked fresh
spinach, rather than the frozen product.
As in the research discussed above, the retention of chlorophylls in the different
vegetables species varied during frozen storage and, in general, the authors agree that
the lower storage temperature limited the losses [Dong et al. 2004, G?bczy?ski 1999,
Lisiewska and Kmiecik 1997, Lisiewska et al. 2004]. However, there is no agreement
regarding the magnitude of losses due to the storage period or the distribution of losses
over time. Oruna et al. [1997] reported a rapid decrease in chlorophyll content after the
first month of storing frozen French bean products; thereafter this constituent was sta-
ble. According to Bahçeci et al. [2005], the frozen storage of French beans brought
about considerable losses during the first three months; the losses were limited during
the following three months but after that period their rate increased. However, Labib et
al. [1997] reported that a three-month period of frozen storage did not affect the level of
chlorophylls. The results obtained here cannot be compared with the results found in the
Acta Sci. Pol.

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