THE INFLUENCE OF CULTIVATION CONDITIONS ON THE MYROSINASE ACTIVITY
AND GLUCOSINOLATE CONTENT IN WHITE CABBAGE
Anna Piekarska, Tadeusz Pilipczuk, Barbara Kusznierewicz, Jacek
Namienik, Agnieszka Bartoszek
Faculty of Chemistry, Gdansk University of Technology
“Utilizing white cabbage for the phytoremediation and biofumigation
of soils (AGROBIOKAP)”, co-financed by the European Union from the
European Fund for Regional Development within the framework of the
Operational Program for an Innovative Economy 2007-2013
METHODS
DETERMINATION OF MYROSINASE ACTIVITY Activity of enzyme myrosinase
was determined using pH-stat method. The reaction mixture consisted
of 15 mL 1% NaCl (pH 6.5) and 0,5 g of lyophilized leaves of
cabbage containing myrosinase. After hydrolysis of cabbage internal
glucosinolates and stabilisation of pH of mixture, 0,15 mL of
external glucosinolate (40 mM glukotropaeolin) was added. During
GLS hydrolysis pH decreases. The aim of this method is to keep
constant pH level (6.5) by adding NaOH solution. Myrosinase
activity was determined by measuring the acid release rate by
titrating with 1 mM NaOH with T70 titrator (Mettler Toledo). The
reaction time was 1 hour, the solution was kept at 37oC and gently
stirred.
DETERMINATION OF GLUCOSINOLATE CONTENT Glucosinolates were
extracted from cabbage lyophilisates with hot methanol, then
extracts were poured into ion-exchange columns and washed with 1 mL
of deionized water. Next, a solution of purified sulphatase (0.2
mL) was added into the columns, which were then left to incubate at
room temperature for 12 h. The desulphoglucosinolates obtained were
eluted twice with deionized water (1 mL). The glucosinolates were
determined by reversed-phase HPLC-DAD and gradient elution. The
mobile phases were water (A) and 20% acetonitrile (B); the elution
programme was: 0 min – 100 % A, 20 min –100 % B, 25min – 100 % B,
with a mobile phase flow rate of 1 mL/min. The target analytes were
determined qualitatively by comparing their retention parameters
with those of available standards and quantitatively using an
internal standard (glucotropaeolin).
INTRODUCTION
In the process of biofumigation typically natural compounds present
in Brassicaceae family are used to combat pests and protect crops.
Glucosinolates, sulfur-containing secondary metabolites found in
Brassica plants, are hydrolized by the enzyme myrosinase
(β-thioglucosidase, EC 3.2.3.1) with the liberation of degradation
products such as isothiocyanates, nitriles, thiocyanates and
epithionitriles (fig. 1). Isothiocyanates are the most bioactive
among these compounds and accordingly most efficient in the
suppression of many kinds of herbivores and soil born pathogens.
Other glucosinolates degradation products are much less effective,
therefore it is desirable to direct the glucosinolate hydrolysis in
such a way so as to obtain only isothiocyanates as the end
products. Many factors like environmental conditions may influence
myrosinase activity in cabbage as well as the presence of other
specifier proteins, which promote formation of different products.
All these factors determine the yield of isothiocyanates and in
consequence, the efficacy of the biofumigation process.
CONCLUSIONS
Cultivation conditions and cabbage cultivars determine the content
of glucosinolates and myrosinase activity in Brassica plants
Cultivar of cabbage Kamienna Gowa exhibits the highest content of
glucosinolates, as well as myrosinase activity, irrespective of the
place of cultivation
Even in the same cabbage cultivar grown in various conditions there
are significant differences in glucosinolates profile
The goal of our research is to investigate the influence of a type
of cultivar and cultivation condition on the content of
glucosinolates and myrosinase activity in white cabbage (B.oleracea
var.capitata) grown in the Northern and Southern regions of Poland
(fig. 2). Places of cultivation varied as regards agricultural
aspects: organic and industrial cultivation, soil class, intensity
of sunlight and risk of pest attack.
Fig. 1. Products of GLS hydrolysis
Fig. 3. The content of glucosinolates in cabbages grown in
different localisations
0
2
4
6
8
10
12
0
2
4
6
8
10
DN KG Kraków Mydlnik
TC Strzelno
GLUCOSINOLATE CONTENT
Am- Amager, DN- Ditmarska Najwczeniejsza, KG- Kamienna Gowa, TC-
Tucana, AB- Ambrozja, GR- Gregorian
Fig. 2. Places of cabbage cultivation
ecological cultivation, field is surrouded by forest, away from
roads; soil derived from former greenhouse contaminated with
Hg
conventional cabbage cultivation, surrounded by forests
very polluted area in the middle of Upper Silesian Industrial
Basin
area possibly polluted by industry
CZAPIELSK
STRZELNO
KRAKÓW
SOSNOWIEC
DN KG Kraków Mydlnik
TC Strzelno
Fig. 5. The activity of myrosinase present in cabbages grown in
different localisations
MYROSINASE ACTIVITY
GLUCOSINOLATE PROFILE
Fig. 4. Profile of glucosinolates in cabbages Kamienna Gowa grown
in different localisations
1- glucoiberin 2- progoitrin
5- glucoerucin 6- glucobrassicin
7- metoksyglucobrassicin 8- neoglucobrassicin
Aliphatic GLS
Indolyl GLS
Sosnowiec O!arowice