Tech Bull Fac Agr Kagawa Univ, Vol 43, No 1, 45-50, 1991
Seasonal Changes in Pectinmethylesterase and Polygalacturonase
Activities in Persimmon Fruit
Toshiyuki MATSUI and Hirotoshi KITAGAWA
This paper describes the seasonal changes in pectin content and pectinmethylesterase (PE) and polygalacturonase (PG) activities in three cultivars of persimmon fruit, Fuyu, Hiratanenashi and Atago The highest PE activity was observed in September in the three cultivars No PE activity was observed a t the mature stage The pattern in the changes of PG activity was the same in the three cultivars
The total pectin (TP) content of 'Hiratanenashi' and 'Atago' was highest in July, while that of 'Fuyu' was highest in October Water soluble pectin (WSP) was lowest in November for 'Hiratanenashi' and 'Fuyu', and in December for 'Atago'
Introduction
Pectic substances are present in the middle lamella of cell and act as cementing materials They are believed to be decomposed into low molecular substances after harvest resulting in an increase in the amount of soluble pectin T P of kiwifruit fell after harvest and WSP slightly rose until 4 days of storage
'"
T P and WSP of persimmon slightly decreased after harvest whereas PG showed an increase after harvest The PGactivity in the soluble and insoluble fractions of persimmon fruit increased moderately a t room t e m ~ e r a t u r e ' ~ ) In this study, the seasonal changes in PE and PG actlvities in relation to T P and WSP contents in three persimmon cultivars, Fuyu, Hiratanenashi, and Atago were investigated 'Fuyu' is the most popular commer- cial cultivar in Japan and is non-astringent, while 'Hiratanenashi' and 'Atago' are astringent types(3)
Tech Bull Fac Agr Kagawa Univ, Vol 43, No 1, 1991
Materials and Methods
1 Materzals
Persimmon fruits were harvested in the fields of Kagawa University located in Nagao-cho, Kagawa Prefecture 'Fuyu', 'Hiratanenashi' and 'Atago' were harvested on the 12th of each month from June to October and 28th of October ('Hiratanenashi') and 26th of November ('Atago') in 1987 Thirteen fruits were harvested a t random every month
2 Enzyme extractzon
(PE)(2) : The fruit pulp was grated using a cooled plastic grater and mixed with 15% polyclar A T (w/w) and chilled water containing 8 8% NaCl and 1% ascorbate, three times the amount of sample The homogenate was extracted for 4h with occasional stirring and filtered through 4 layers of cotton cloth The filtrate was centrifuged a t 20,000xg for lOmin The supernatant was used for enzyme assay
(PG)@) : The fruit pulp was grated using a cooled plastic grater and mixed with 15% polyclar A T (w/w) and chilled water, twice the amount of sample (w/v) The homogenate was filtered through 4 layers of cotton cloth and the filtrate was centrifuged a t 6,000 x g for 10 rnin The residue was rinsed with the same volume of water and then centrifuged in the same way The combined supernatant was dialyzed with 5 mM of sodium phosphate-citrate buffer (pH 7 4) for 12h and the inner dialyzed fraction was designated as soluble fraction (S f ) The residue was re-extracted with 0 2M sodium phosphate-citrate buffer (pH 8 01, twice the amount of sample, containing 5% NaCl for 12h with occasional stirring The resulting homogenate was centrifuged a t 20, 000 x g for 20 rnin The supernatant was designated a s insoluble fraction (Is f ) The extraction was carried out below 4°C
3 Enzyme assay
The extract for PE assay was adjusted to pH 7 5 with 1 N NaOH and the precipitate was separated by centrifugation a t 2,000xg for 10 rnin The temperature was kept below 4°C The clear liquid was used for enzyme assay 'The pectinmethylesterase (PE) assay was carried out a t 25°C for 3 min according to the procedure described by HAGERMAN and A u s r ~ ~ ( ~ ) w i t h some modifications a s described here The assay mixture contained 1 ml of 0 5% (w/v) pectin from apple, 75 , ~ l of 0 01% (w/v) BTB, 0 325 ml of water and 0 1 ml of crude enzyme After 1 rnin preliminary incubation, the reaction was started by the addition of crude enzyme 'The solution was measured a t 620 nm against water and the activity was calculated by using a calibration curve made from commercial PE reagent All solutions (pectin, B'TB, and water) were previously adjusted to pH 7 5 The activity of PE was presented as the amount of acid (,u eq) liberated per rnin per g of fresh weight In the case of PG, the assay medium consisted of 0 2 ml of 0 2 M sodium phosphate-citrate buffer (pH 5 O), 0 1 ml of water and 0 1 ml of crude enzyme The blank contained water instead of substrate The reaction mixture was incubated at 45°C for 15 min After incubation, the reaction mixture was neutral- ized and the concentration of reducing sugar was estimated by the methods of SOMOGYI The enzyme activity was expressed a s the amount of galacturonic acid liberated per rnin per g of fresh weight
4 Determznatzon of total pectzn ( T P ) and water soluble pectzn ( WSP) content
Toshiyuki MATSUI and Hirotoshi KITAGAWA : PG and PE in Persimmon 47
followed by filtration The residue was washed similarly for about 4 times until the washing was free from soluble sugar as tested by Phenol-H2S04 reagent ( G ) Then the residue was washed with a small amount of ethyl ether and dried a t around 60°C
TP : The dried sample (0 5 g or less) was ground with a mortar and pestle with 1 g of sea sand T o the resulting powder 25 ml of 0 05 N HC1 was added and the mixture was boiled for l h under a reflux condenser After rapid cooling with running water, the sample was made up to 50 ml with water and filtered ('1
WSP : The powdered sample (0 5 g or less) was made up to 25 ml with water and was extracted a t 30°C for 2-3h with occasional stirring The mixture was filtered and the filtrate analyzed Pectin was determined as galacturonic acid by the Carbazole-HzSOI method
Results and Discussions 1 Seasonal changes zn wezght of perszmrnon frmzt
Jul Aug Sep Oct Nov Dec Fig 1 Seasonal changes in fruit weight (A) and
pectinmethylesterase activities (B) in the three persimmon cultivars
0,
'Fuyu' ;a,
' H i r a t a n e n a s h i ' ; X , 'Atago' Vertical bars represent the SE : (A)n = 1 3 a n d ( ~ ) n = 3
The weight of persimmon fruit is shown in Fig 1 (A) The average weight of 'Fuyu', 'Hiratanenashi' and 'Atago' a t harvest season was reported to be around 220, 200 and 250 g, respectively (8) For 'Atago', larger fruits were used in this experiment, while for 'Fuyu' and 'Hiratanenashi' average ones were used
48 Tech Bull Fac Agr Kagawa Univ, Vol 43, No 1, 1991
2 Seasonal changes zn pectznmethylesterase and polygalacturonase actzvztzes zn perszmmon fmrt
PE splits the ester linkage between methylesters of galacuturonic acid residue whereas PG splits the glycosidic linkage between adjoining galacturonic acid residues@) Fig 1 (B) shows the seasonal changes in PE activity in 'Fuyu', 'Hiratanenashi' and 'Atago' The highest PE activity was observed on the 12th of September in all three cultivars, while PE activity was almost absent in the three cultivars during the harvest season According to ROUSE et a1 ,(I0) the PE activity of 'Valencia' orange was high during the harvest season, whereas that of 'Silver Cluster' grapefruit'") and k i ~ i f r u i t ' ~ ) showed the same profile as that of persimmon Figs 2 (A)
and 2 (B) show the seasonal changes in PG activity of the three cultivars in the soluble and insoluble fractions,
I I I I I :
Jul Aug Sep Oct Nov Dec Fig 2 Seasonal changes in polygalacturonase
activities in the soluble&) and insoluble (B)
fractions of persimmon fruit Symbols as shown in Fig 1 Vertical bars represent the SE of 3 determinations
respectively PG activity in the soluble fraction of 'Fuyu' was about twice as high as that of 'Atago' a t harvest season 'The profile of PG activity in the soluble fraction of 'Hiratanenashi' was almost the same as that of 'Atago' and that in the three cultivars tended to decrease throughout the experimental period The PG in the insoluble fraction of 'Fuyu' tended to increase throughout the experimental period but that of 'Hiratanenashi' and 'Atago' tended to decrease PG activity was higher in the soluble than in the insoluble fraction in the three cultivars Pears(12) and peaches(13) lack PG activity, whereas PG was found in persimmon and kiwifruit (I4' PE
activity seemed to increase during the growth and development period, whereas PG activity seemed to increase rapidly during maturation Thus, it may be concluded that in the developing fruit PE releases pectic acid from pectin The pectic acid accumulates and serves as substrate for PG to be decomposed at mature stage
Toshiyuki MATSUI and Hirotoshi KITAGAWA : PG and PE in Persimmon
3 Seasonal ~hanges zn total pectzn and water soluble pectzn contents zn perszmmon fmzt
I I I I I
Jul Aug Sep Oct Nov Dec Fig 3 Seasonal changes in total pectin(@ and
water soluble pectin (B) contents of persim- mon fruit Symbols as shown in Fig 1
Fig 3 (A) and (B) show the seasonal changes in T P and WSP contents, respectively, of the three cultivars T P content of 'Fuyu' tended to increase throughout the experimental period but that of other cultivars tended to decrease On the 28th of October, the T P of 'Fuyu' was 2 to 3 times as high a s the WSP content The T P and WSP contents of the two cultivars were highest on the 12th of July In 'Hiratanenashi' and 'Atago' the highest T P and WSP contents coincided with the period of highest PG activity, whereas in 'Fuyu' such was not observed In 'Valencia' orange, the highest PE activity was observed during the period of lowest T P content,(lO) whereas in peach the increasing PG activity paralleled with WSP content during ripening ( I 3 ) The
PG activity of kiwifruit a t harvest'") was not related to T P or WSP contents The results indicate that the decrease in PG activity in 'Hiratanenashi' and 'Atago' was related to the T P and WSP contents but there is no
relation between PE and PG activities, and pectin contents in 'Fuyu'
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50 Tech Bull Fac Agr Kagawa Univ, Vol 43, No 1, 1991
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