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Effects of freezing and frozen storage on myrosinase activity from green cabbage Brassica oleracea var. sunta crude extracts were measured. Myrosinase activity was found to be optimal at 7.5 mM ascorbic acid. Freezing at - 20OC lead to approximately 30 % loss in myrosinase enzyme activity when compared to the fresh crude extract. Freezing rate was seen to have an effect on the loss of activity, with slower freezing rates resulting in greater losses than faster freezing rates (P> 0.05). No significant difference (P> 0.05) was found between fresh samples and samples frozen in liquid nitrogen and - 80 OC. The frozen crude extracts were then stored for 60 days under the different freezing conditions. Results showed that the highest reduction (P> 0.05) in myrosinase activity of 50 % occurred at -20 OC after 60 days storage with less than 20 % reduction (P> 0.05) in the other freezing methods studied.
Brassica or cruciferous vegetables such as cabbage, broccoli, cauliflower and Brussel sprouts contain a group of sulphur- containing compounds called glucosinolates. Glucosinolates on their own are non- nutritive compounds but contribute to the health of humans when hydrolyzed to other products (1).
Myrosinase (thioglucoside glucohydrolase EC 3. 2. 3.147, formally EC 188.8.131.52) is the enzyme responsible for the hydrolysis of glucosinolates and is found in all cruciferous vegetables most of which are consumed as part of the human diet (2). It hydrolyses glucosinolates into volatile D- glucose and sulphate products such as isothiocyanates, nitriles or thiocyanates, with the product being produced depending majorly on the substrate and pH of hydrolysis (3). These hydrolysis products contribute both positively and negatively to the characteristics of brassica vegetables. Glucosinolates amongst other compounds such as flavonoids and phenols are responsible for the bitter taste and astringency of brassica vegetables (4) but isothiocyanates are in some cases bitter, but are more often volatile and give the pungent aroma and flavor of brassica vegetables (5). Isothiocyanates in addition to being responsible for aroma and flavor also possess health-promoting properties. The result of a controlled study has shown that isothiocyanates are responsible for the anticarcinogenic properties of brassica vegetables by decreasing damages to DNA in humans (6). Epidemiological studies show that they also help to prevent cardiovascular diseases (7). On the other hand, high amounts of hydrolysis products also have unwanted effects in foods and animal feedstuffs due to their tanginess and also results in unwanted bitterness, strong flavour and taste in the vegetables (7, 8). Experimental studies have shown that high amounts of hydrolysis products results in undesirable goiter activities. A study conducted on rats revealed enlarged thyroids in the rats when fed different cruciferous vegetable seeds for a month (9).
There different types of myrosinase and it vary between brassica vegetables and also differ to some extent in characteristics and activity (8). Myrosinase activity is affected by several intrinsic (ascorbic acid, Magnesium Chloride (MgCl2) ferrous ions) and extrinsic (pH, temperature, pressure) factors. Ascorbic acid and MgCl2 has been shown to increase myrosinase activity at certain concentrations and reduce its activity above those concentrations (10). Studies have also shown that thermal treatment has an effect on myrosinase activity in cruciferous vegetables (9, 11). These studies showed that myrosinase activity increased when temperature was consistently raised to 60 OC with a decrease in activity above it. Myrosinase is inactivated under high temperatures of above 50 OC in red cabbage juice (12). Myrosinase from cabbage has shown to be more stable at low temperatures of about 30 OC when compared to myrosinase from broccoli extracts where myrosinase activity decreases at temperatures above 30 OC and is almost totally inactivated at 50 OC (2). Despite the ability of microorganisms in the human gut to hydrolyze glucosinolates, health benefits derived from the hydrolysis products are more evident when glucosinolates are hydrolyzed by endogenous myrosinase (13). It is, therefore, important to ensure that little or no loss of myrosinase enzyme activity occurs during processing.
Many plant tissues are stored frozen commercially. There are few reports available on the effect of freezing on enzyme activities in various plant tissues. The effect of freezing on allinase enzyme activity in onion extracts and pure enzyme preparations has been studied (14) while the effect of frozen storage conditions on catalase enzyme activity in apple flesh tissues has also been reported (15). Both studies showed that freezing at - 20 OC and - 25 OC respectively led to a substantial decrease in enzyme activity. However, there have been no reports on myrosinase activity after freezing in plant tissues and yet there are two reasons why such study is important. Firstly, in the commercial extraction process for myrosinase enzyme involves freezing the plant tissue in liquid nitrogen and grinding to powder before extracting the enzyme. The crude enzyme extract is further stored in frozen conditions for different times before assays are performed in plant biochemical experiments. Secondly in the freezing of vegetables for commercial distribution and use, it is important to determine whether the freezing procedure has an effect on myrosinase activity in cruciferous vegetable as this will in turn have an effect on the potential health benefit to be derived from the vegetables.
This study was therefore aimed at determining the effect of freezing on the activity of myrosinase enzyme in partially purified green hearted cabbage.
MATERIALS AND METHODS
Sampling and Processing of Cabbage. Green hearted cabbage (B. oleraceae var. sunta) grown on silt loamy soil was supplied by Marshalls Ltd, Boston, Lincs and stored at 4 OC untill processing was complete. The edible portion of the cabbage was cut into small pieces and divided into 50 g portions. The portions were then suspended in 150 ml of Tris- HCL (0.2 mol/L, pH 7.0) containing 0.5 mM EDTA, 1.5 mM DTT (stock solution) and 7.5 g insoluble PVPP. The homogenate was then mixed for 60 s at 4 OC using a blender. The homogenate was then filtered through a threefold cheese cloth to remove the insoluble portion of the cabbage. The filtrate was then centrifuged (15,000 g for 15 min at 4 OC) to remove the remaining insoluble protein and PVPP. The supernatant obtained was further clarified by another centrifugation (30,000 g, 20 mins, 4 OC). The supernatant was saturated at 45 % with (NH4)2SO4, incubated for 20 min, centrifuged (20,000 g, 20 min, 4OC) and resulting pellet discarded. The supernatant was further saturated at 85 % with (NH4)2SO4, incubated for 20 mins and then centrifuged (20,000 g, 20 min, 4OC). The resulting pellet (precipitated protein) was dissolved in Tris-HCL (5 mM, pH 7.5). The resultant crude extract solution was stored at 4 OC for further processing.
Frozen Storage of Crude Extract. The crude extract obtained was divided into aliquots of 170 µl and placed in eppendoff tubes. The myrosinase activity of the fresh non- frozen crude extract was assayed immediately. The remaining tubes were frozen using different freezing methods. One portion was frozen in liquid nitrogen and stored at -80 OC while another portion was frozen and stored at - 80 OC. The last portion was frozen and stored at -20 OC. All portions were stored for 60 days. Tubes were removed and assayed after 24hours, 15 days, 30 days, 45 days and 60 days for myrosinase activity.
Determination of Myrosinase Activity. Myrosinase hydrolyses glucosinolates into D- glucose and sulfates. Therefore, myrosinase activity can be related to the amount of glucose produced in the hydrolysis reaction. Myrosinase activity was determined following the coupled enzyme procedure as described by (16) in which the reaction between myrosinase and sinigrin (substrate) results in glucose formation that is used to convert NADP+ to NADPH. D- Glucose test kit (Cat no. 10 716 251 035, Enzymatic Bioanalysis/ Food Analysis, Boehringer- Mannheim/ R- Biopharm,) was used. The mixture for the reaction consisted of 0.9 ml of 5mM Ascorbic acid, 0.5 ml ATP/ NADP+ solution (test kit solution 1), 10 µl hexokinase/ glucose-6-phosphate dehydrogenase (test kit solution 2), 50 µl crude enzyme extract. The mixture was homogenized and allowed to stand for 5mins at 26 OC and 50 µl 25 mM sinigrin substrate (0.3 g/ml, Sigma- Aldrich, Germany) added. The NADPH being formed was followed spectrophotometrically by reading absorbance at 340 nm at 26 OC for 15 min. Myrosinase enzyme activity was determined by taking the slope of the linear part of the curve of absorbance versus time of reaction. One unit of myrosinase activity is defined as the amount of enzyme that produces 1 µmol of glucose from sinigrin substrate per minute at pH 7.5 and 26 OC.
Effect of Ascorbic acid on green cabbage myrosinase activity. Ascorbic acid concentrations of 0, 2.5, 5, 7.5, 10, 15 and 20 mM were used to test for the effect of ascorbic acid concentration on myrosinase activity. 10 µl hexokinase/ glucose-6-phosphate dehydrogenase, 0.9 ml ascorbic acid, 0.5 ml ATP/ NADP+ solution together with 50 µl of the crude enzyme extract were mixed together and allowed to stand for 5 min at 26 OC. 50 µl 25 mM sinigrin was then added to the mixture and absorbance of the conversion of NADP+ to NADPH was read using a spectrophotometer at 340 nm at 26 OC for 15 min. Myrosinase enzyme activity was determined by taking the slope of the linear part of the curve of absorbance versus time of reaction.
Protein Determination. Protein concentration was measured using the Bradford method (17) with little modifications. The procedure is based on formation of a complex between dye (brilliant Blue G, Sigma- Aldrich) and the protein present in the sample with absorbance read at 595 nm using a spectrophotometer (Biomate 3, Thermo, Electron Cooperation, USA). 1.5 ml of concentrated dye reagent was used. Bovine serum albumin (BSA), (Sigma- Aldrich, UK) was used to construct a standard curve in concentrations ranging from 2.5 - 1.0 mg ml-1 and the protein concentration of the diluted sample calculated from the standard curve obtained.
Statistical analysis. Statistical analysis was conducted using GraphPad Prism 5 (18) software package. One- way ANOVA was used to analyze the data obtained and Newman-Keuls multiple comparison test was used to determine significant differences between means.
RESULTS AND DISCUSSION
Effect of Ascorbic Acid Concentration on Myrosinase Activity. The result for the effect of ascorbic acid concentration on myrosinase activity is as shown in Table 1 and Figure 1. Highest myrosinase activity was found between ascorbic acid concentrations of 2.5 mM and 10.0 mM with optimal activity at 7.5 mM concentration and minimal at 0 mM concentration of ascorbic acid where there was little or no myrosinase activity. Myrosinase activity was found to increase with increase in ascorbic acid concentration until the maximum myrosinase activity was obtained 7.5 mM ascorbic acid concentration, at which point myrosinase activity began to decrease with increase in ascorbic acid concentration. There were no significant differences in myrosinase activity between ascorbic acid concentrations of 2.5 mM, 5.0 mM and 10.0 mM but all other concentrations showed significant differences in myrosinase activity. The result obtained is similar to that reported on the effect of ascorbic acid on red and white cabbage where myrosinase activity was highest at 5 mM and 10 mM respectively (10) and that of myrosinase activity in six partially purified crucifer vegetable extracts (white mustard, radish, brussels sprouts, cauliflower, red cabbage, turnip and watercress) with optimal activity at 5 mM concentration of ascorbic acid (3) except that in this case myrosinase activity was highest at 7.5 mM ascorbic acid concentration. Myrosinase activity was evident at 20 mM ascorbic acid concentration and is in line with results obtained by (3) which show myrosinase activity present at 50 mM, a concentration much higher than that obtained from this study.
From this study, myrosinase activity in green cabbage has been shown to be dependent on ascorbic acid concentration as myrosinase activity was very low at 0 mM of ascorbic acid. This result is consistent with that reported by (20, 10), which showed absence of myrosinase activity in wasabi roots and low activity in red and white cabbages in the absence of ascorbic acid respectively. The reduction in myrosinase activity to about 50 % after addition of 10 mM ascorbic acid is in line with results obtained in isoenzymes were activity was reduces by 50 % at 10 mM ascorbic acid concentration (21). Reduction in myrosinase activity above certain level of ascorbic acid concentrations has also been reported by (10, 20). The reduction in activity can be said to be possibly due to competitive inhibition of ascorbic acid with cabbage myrosinase. Myrosinase activity in partially purified extracts at different ascorbic acid concentrations is dependent on the amount and quantity of activation of a single myrosinase isoenzyme (3). This implies that brassicas which have optimum myrosinase activity at lower ascorbic acid concentrations possess isoenzymes that have compositions that are responsible for speedy activation of individual myrosinase isoenzymes. Decrease of mustard myrosinase has been said to be due to competitive inhibition of ascorbic acid with mustard myrosinase (21). The results obtained above is very important because the ascorbic acid concentration required for optimal myrosinase activity has an effect on flavor production, quality and possible health benefits derived from cruciferous vegetables. 5 mM ascorbic acid, a concentration just below the optimal level was used to determine myrosinase activity in this study.
Effect of Freezing on Myrosinase Crude Extract. Myrosinase activity in crude extracts from green cabbage reduced to approximately 30 % due to freezing as shown in Table 2. Myrosinase activity was highest in samples frozen in liquid nitrogen and stored at -80 OC giving only about 5 % reduction in activity. This was significantly higher (P< 0.05) than the activity of myrosinase in samples frozen and stored at -20 OC, where the activity was the lowest. There was no significant difference found in myrosinase activity between fresh samples, samples frozen in liquid nitrogen and stored at -80 OC and samples frozen and stored at -80 OC. Though the crude extracts frozen and stored at -80OC had higher myrosinase activity when compared to that frozen and stored at -20 OC, no significant difference was found between them after freezing for 24 hours. A significant difference in myrosinase activity was found in samples frozen and stored at -20 OC with fresh samples and samples frozen in liquid nitrogen and stored at -80 OC. No previous studies were found on the effect of freezing on myrosinase from crude extracts of other cruciferous vegetables in the literature, however, a few studies considered effects of freezing on enzymes in other fruits and vegetables. The results obtained from the current study are similar though with inactivation rates to results reported by Gong et al. (15) for the activity of catalase extracted from apple flesh tissue which found a reduction in catalase activity by approximately 80 % when crude extracts from Braeburn apple flesh tissue where frozen and stored overnight at -25 OC. The present results which show that there was no significant difference in the myrosinase activity of the fresh samples with that of the samples frozen and stored at -80 OC is in agreement with the results obtained from the activity of cysteine sulfoxide lyase extracted from broccoli florets frozen and stored at - 80 OC for 24 hours (19). The results of both studies show that loss of enzyme activity is highest when the freezing rate and time is lowest. This goes to say that freezing method has an effect on myrosinase activity. Since the protein content of the frozen samples is still relatively close to that of the fresh sample, it is safe to assume that the loss of myrosinase activity is not a result of protein loss but a result of freezing temperature and rate. Freezing is one of the major methods used for the prolonged storage of plant tissues being consumed and used for laboratory analysis; it can therefore be assumed that it does not have a noticeable effect on protein and most enzyme activity of plant tissues (15).
Results obtained by Wafler (14), showed that slow freezing at -20 OC prior to extraction reduced allinase enzyme activity in onion extracts whereas fast freezing in liquid nitrogen and storing at -80 OC prior to extraction retained enzyme activity; this supports the results obtained from the current myrosinase study. Freezing leads to an irreversible loss of enzyme activity, the effect depending on freezing rate and temperature (22). When materials are frozen, pure water freezes first leading to a concentration of solutes such as enzymes and salts present in the material increasing the pH and ionic concentration of the solution. The increase in pH may dissolve oligometric proteins present in the solution which can lead to inter or intramolecular thiol-disulphide exchanges if the increase in pH is complemented with conformational changes (23). Cabbage myrosinase is known to be active at pH of 6- 8 which is the pH of cabbage. It can therefore be said that freezing of the myrosinase extract may have resulted in conformational changes of the protein and hence a reduction in myrosinase activity with effects greater at -20 OC temperature of freezing compared to freezing in liquid nitrogen and at - 80 OC. Furthermore, freezing damages cell structures of plant tissues due to the formation of ice crystals and the size of the ice crystals is dependent on the rate of freezing. Slower rate of freezing leading to formation of large ice crystals which leads to greater damage to cell structure than the smaller ice crystals formed when plant tissues are frozen fast. Fast freezing removes the heat faster and hence reduces all biochemical activities taking place faster. The lower myrosinase activity observed when extracts were frozen at -20 OC, could have been due to the slower rate of heat removal when compared to the other freezing methods employed. Hence, increasing the loss of enzyme activity.
The results presented above is not enough to conclude that freezing has an effect on myrosinase activity in cruciferous vegetables as myrosinase from different vegetables and varieties may differ in reaction to freezing but it has shown that freezing methods may have an effect on the myrosinase activity in green cabbage. It also shows that freezing of cabbage at -20 OC prior to myrosinase extraction can lead to a reduction in the activity of myrosinase and hence give inaccurate results for myrosinase activity in green cabbage during laboratory analysis. Also, it has shown that slow freezing of cabbage can reduce the possible health benefits to be derived from it. Since results have shown loss in enzyme activity when frozen at -20 OC, freezing of cabbage prior to myrosinase extraction should not be carried out at -20 OC.
Effect of Storage on Myrosinase Activity. The frozen extracts were stored for 60 days at -80 OC and -20 OC respectively and assayed for myrosinase activity every 15 days. The results are as presented in Table 3 and Figure 2. Results obtained showed that there was no significant difference (P> 0.05) over time in extracts frozen and stored at - 80 OC, therefore showing that there was no significant change in enzyme activity of the extracts throughout the storage period, however, it should be noted that samples had a significant reduction (P< 0.05) in myrosinase activity compared to fresh samples after 24 hours (1 day) of frozen storage. Extracts frozen in liquid nitrogen and stored at -80 OC showed significant differences (P< 0.05) between samples during the 60 days storage, however, in this case the samples had not significantly (P>0.05) lost myrosinase activity after 24 hours of frozen storage (1 day) when compared to fresh samples. There was a significant loss (P<0.05) of myrosinase activity after 45 and 60 days of storage when compared to the myrosinase activity 1 day after freezing but no significant loss was evident after 15 and 30 days of frozen storage. Myrosinase activity reduced to about 16 % in the samples after the storage period. There was an appreciable reduction in the myrosinase activity of samples stored at -20 OC over the 60 days period to about 50 %. A significant difference (P<0.05) was observed in myrosinase activity after 45 days of storage when compared to the initial activity after 1 day of freezing. The result for relative activity of myrosinase shows that the enzyme was most stable when frozen and stored at - 80 OC and least stable when frozen and stored at - 20OC over the storage period. The relative activity of extracts frozen in liquid nitrogen and stored at -80 OC compared to extracts frozen and stored at -80 OC was lower but no appreciable difference in values was observed. No literature studies were found to compare results obtained on effect of frozen storage on enzyme extracts but a few studies on effect of frozen storage on enzymes from fruits were found. The results obtained for extracts frozen in liquid nitrogen and stored at -80 OC is in agreement with the results obtained when catalase extracts from selected apple varieties were pre- frozen in liquid nitrogen and stored at -80 OC for 4 weeks. Catalase activity remained stable for the first 2 weeks but about 20 % of activity was lost after the 4 weeks of storage (15). Frozen storage even at very low temperatures, only reduces biochemical reactions to very low levels and does not completely stop it (24). This explains the different levels of loss in myrosinase activity of the crude extracts under different frozen temperatures. The lower myrosinase activity in samples stored at -20 OC implies that biochemical reactions still take place at this temperature which led to reduction in myrosinase activity. Also the larger ice crystals formed at this storage temperature compared to that of the other storage temperatures studied may also be responsible for the loss of myrosinase activity, since it leads to greater disruption in the protein structure of the enzyme. The changes occurring during frozen storage is irreversible and cumulative, and changes in quality of frozen vegetables are slower when stored at temperatures below -20 OC (25). The slight difference in the myrosinase activity for the extracts frozen in liquid nitrogen and stored at -80 OC with that of samples frozen and stored at - 80 OC may be due to fluctuations in the freezing temperature of the extracts. (24) stated that when there is a fluctuation in temperature used for storage, some ice crystals may melt and re- solidify into larger ones and hence increase damage on cell structure. There is an increase in pH concentrations under prolonged frozen storage of products due to reduction in water activity and high concentration of solutes (26). The consistent decrease in myrosinase activity of all the extracts stored might have been due to increase in pH of the extract solution, caused by the increase in solute concentration.
In conclusion, it is not possible to state that storage of myrosinase extracts from other cruciferous vegetables at temperatures of -20 OC has an effect on myrosinase activity, since no previous reports have studied this and freezing and frozen storage may have a different impact on myrosinase extracted from different cruciferous vegetables. However, it can be concluded that storage of green cabbage myrosinase extract at -20 OC reduces its activity by approximately 50 % but that freezing in liquid nitrogen and storing at - 80 OC or freezing and storing at -80 OC has no appreciable effect on myrosinase activity. Therefore, green cabbage myrosinase extract to be used for laboratory analysis should not be frozen and stored at - 20 OC if optimal myrosinase activity is to be achieved. The effect of freezing and storing myrosinase extracts from other crucifers should be studied to determine if freezing and frozen storage has an effect on myrosinase activity. Finally, the effect of frozen storage on myrosinase activity in whole cruciferous vegetables should be studied and it is expected that myrosinase activity will deteriorate on frozen storage and affect the sensory and nutritional quality of the vegetables.
Tris- HCL, Tris- hydrochloric acid; EDTA, ethylenediamine tetraacetate; DTT, dithiothreitol; PVPP, polyvinylpolypyrrolidone; ATP, adenosine triphosphate; NADP+, nicotinamide-adenine dinucleotide phosphate; NADPH, nicotinamide adenine dinucleotide phosphatase; SE, standard error of means.
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TABLE 1: Effects of Ascorbic Acid Concentration on Myrosinase Activity in Partially
Purified Extracts of Green Cabbage.
TABLE 2: Myrosinase Activity in Partially Purified Extracts of Green Cabbage after Freezing at Different Temperatures as compared with a Non- frozen Control sample.
Table 3: Changes of Myrosinase Activity in Partially Purified Extracts of Green Cabbage Stored at Different Freezing Temperatures
Figure 1: Effect of ascorbic acid concentration on myrosinase activity in partially purified myrosinase extracts of green cabbage.
Figure 2: The effect of frozen storage on myrosinase activity in partially purified extracts of green cabbage.
Table 1. Effects of Ascorbic Acid Concentration on Myrosinase Activity in Partially
Purified Extracts of Green Cabbage.
Ascorbic acid Concentration (mM)
Myrosinase Activity (Unit/ml extract) a
1.7 (0.00) a
16.9 (0.00) b
16.9 (0.00), b
30.9 (0.06) c
15.2 (0.00) bd
12.8 (1.70) d
9.3 (0.09) e
a One unit of myrosinase activity is defined as the amount of enzyme that produces 1 µmol of glucose from sinigrin substrate per minute at pH 7.5 and 26 OC.; values (mean ± SE, n= 2) with different letters show statistical difference at P < 0.05.
Table 2. Myrosinase Activity in Partially Purified Extracts of Green Cabbage after Freezing at Different Temperatures as compared with a Non- frozen Control sample.
Myrosinase Activity (units/ml extract) b
Protein (mg/ml) d
Fresh (Non- frozen)
Frozen in Liquid N2and stored at -80OC
20.18 (0.65) a
Frozen and stored at -80OC
18.01 (0.27) ab
Frozen and stored at -20OC
15.18 (1.63) b
a Samples stored for 24 hours. b One unit of myrosinase activity is defined as the amount of enzyme that produces 1 µmol of glucose from sinigrin substrate per minute at pH 7.5 and 26 OC.; values (mean ± SE, n= 2) with different letters show statistical difference at P < 0.05. c (myrosinase activity of frozen sample/myrosinase activity of fresh sample) x 100. d Protein content of myrosinase extract; values (mean ± SE, n = 3).
Table 3. Changes of Myrosinase Activity in Partially Purified Extracts of Green Cabbage Stored at Different Freezing Temperatures
Frozen in liquid nitrogen and stored at - 80 OC
Frozen and stored at - 80 OC
Frozen and stored at - 20 OC
Relative Activity (%) b
Relative Activity (%)
20.18 (0.65) a
19.27 (0.21) ab
17.50 (1.17) ab
16.33 (0.00) b
18.01 (0.27) c
17.09 (0.22) c
16.33 (0.00) c
16.33 (0.00) c
15.17 (1.17) c
15.18 (1.63) d
13.07 (0.03) d
11.67 (1.67) de
8.75 (0.59) e
8.16 (0.00) e
a One unit of myrosinase activity is defined as the amount of enzyme that produces 1 µmol of glucose from sinigrin substrate per minute at pH 7.5 and 26 OC.; values (mean ± SE, n= 2) with different letters show statistical difference at P < 0.05. b (myrosinase activity of frozen sample/myrosinase activity of fresh sample) x 100
Figure 1. Effect of ascorbic acid concentration on myrosinase activity in partially purified myrosinase extracts of green cabbage. Error bars are the standard error of means (n= 2).
Figure 2. The effect of frozen storage on myrosinase activity in partially purified extracts of green cabbage.( ) frozen in liquid nitrogen and stored at -80 OC;( ) Stored at -80 OC; ( ) frozen and stored at -20 OC. Error bars are the standard error of means (n= 2).