全 文 ： ’
一
热带亚热带植物学报 1996 4(4)：50 59
lourrtal川 Tropical and Subtropical Botany
∽
PRo．LoNG涂膜处理对后熟香蕉果实乙烯形成
以及其它有关生理变化的影响
张东林
(中国科学院华南植韧研究所．广州 51o6~o)
Peter C Quantick Peter J Wa ren 4-t．{o~l f ￡、／
{英国亨伯赛^学应用科学技术系食品研究 心 ) ，
．／[一／
摘要 香蕉 (Musa acuminate Colla cv Dwarf Cavendish)果实采后U商业上推荐使用的1 5％ 件o4ong
溶液处理，贮藏于 2O℃和 75％相对湿度下．分别测定果实的 ACC含量、MACC肯量 EFE酶 活性
乙烯释放、叶绿素含量的变化和果实的硬度变化。结果表 明，PRO-LONG处理延缓了香蕉果实果皮的
叶绿索降解、硬度的下降以及乙烯释放的增加．在后熟过程中，处理果实的 ACC含量发生积累．ACC
含量的高峰在乙烯释放高峰和 EFE酶活性高峰之前出现。与对照比较，处理果实 的 ACC含量 和 EFE
酶 活性 的高 峰延 迟 了5d出现。在后 熟过程 中， Pro4ong处理果 肉圆片．其EFE酶 活 眭受部分抑
制(抑制率为 l9．45％ 至 4O 51％)。果实MACC古量在贮藏起l韧处于一个较显著水平，随着后熟的发展
而逐步增加，但与 ACC含量的明显增加相比变化是微小的．我们的研究进一步阐明了 PRO．LONG 涂
膜对香蕉果实后熟的影响主要是通过减少氧的供给，部分地抑制了 EFE酶活性，延缓 丁乙烯 的形成 和
释放，从而延 【乇了后熟过程
若键词 Prodong涂膜；氨基环丙烷援酸 (ACC)，丙 二酰氨基环丙烷羧酸(MACC1， 乙 烯 形 成 酶
(EFE)； 要蔓丛 萝
EFFECTS oF PRo—LoNG CoATING oN ETHYLENE
BIoSYNTHESIS AND OTHER RELEVANT PH YSIoLoGICAL
CHANGES oF BANANA FRUIT DURING RIPENING
Zhang Donglin
{South China Institute Botany，Academia H Ⅱ，Guangzhou 510650j
Peter C QuantIck Pete r J W a rreIq
IFuod Research Centre School Applied Scwnce and Technolooy． University ttumberside)
Abstract Coating banana(Musa acuminata Cola CV．Dwarf Cavendish)
Prodong delayed chlorophyll breakdown in peel，the decrease in firmness
the increase in ethylene production of the whole fruit．During ripening
of ACC content occurred in banana fruit treated with Pro-long．The peak
本文是第一作者在英国亨怕赛太学应用科学技术系食品研究 『，心(Food Rcscarch Centre．School
Technology，University of Humbcrside．G rimsby DN34 5AA UK)作讨刚学者期间所怍的工作
l995 I 2—07收稿 ；】996 05 28悖 回
fruits with I．5％
of the fruit and
the accumulation
in ACC co ntent
of Applied Science and
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第 4期 张东林等：PRO．LONG滁膜处理对后熟香蕉果实乙烯形成 以及其它有关生理变化的影响 5
aDDeared a sho rt time before that in ethylene production and in EFE activity．The peaks
in ACC content and EFE activity of Pro-long treated fruits occurred 5 days later than that
of the contro1．During ripening EFE activity in banana pulp discs treated with Pro-long was
partially inhibited (from 19．45％ to 40．51％)．MACC contents in banana fruit were at
significant levels at the beginning，and then increased gradually with the development of
ripening，but only slightly as co mpared with the marked increase in ACC levels W e suggest
that effects of Pro-long coating on banana fruit during ripening mainly due to affecting
ethylene biosynthesis of the fruit through partially inhibiting EFE activity by reducing the
supplement of oxygen，so as to delay the increase in ethylene production and so extend the
ripening processes．
Key words Pro-long coating； 1-aminocyclopropane carboxylic acid (ACC)=Malonyl_1
aminocyclopropane carboxylic acid(MACC)； Ethylene forming enzyme(EFE)：
Ethylene； Banana frujI
l Introduction
Ripening of climacteric fruits such as banana shows a great increase in respiration rate
and autocatalytic ethylenc production tn these fruits ethylene plays an important role in
the ripening process of fruits．Ethylene is generally considered to be the hormonal regulator
of the ripening of climacteric fruits． Adams and Yang described the pathway of
ethylene production in higher plants as folows：Methionine— S-adenosy1methi0nine(sAM)
一 l-aminocyclopropane．1-carboxylic acid (ACC)一 C2H4⋯ ACC synthase and ethylene
forming enzyme (EFE) are the key enzymes in the pathway ．The content of ACC
is low during the preclimacteric stage of many fruits． but increases greatly during the
climactcric and then decreases in the postclimactedc stage In climacteric fruits，regulation
0f ethylene biosynthesis seems to depend on both ACC and the capability of the tissue
to convert ACC to ethyleneI
．
The ACC level can be regulated by its rate of synthesis
and conversion to ethylene as wel as by jts conjugation to malonyl—ACC (MACC) J
On the other hand． ethylene is ca pable of regulating the activity of EFE ACC
svnthase[~ and M ACC tPansferasc171
．
Pro-long js a coating material for fruits comprising a mixture of sucrose esters of fatty
acids and sodium salt of ca rboxymethylcellulose Coating banana fruit with an aqueous so-
lution of Pro-long has been shown to form a physical barrier to gaseous diffusion through
the stomata on the fruit surface，which are the principal route for gaseous exchange of tile
the fruit tissues with the external atmosphere【8】 Application of Pro-long to C r
treated bananas reduced their rates of respiration and C，Hd P roduction． and dela yed
the decline in skin chlorophyll co ntent these effects were ascribed to the reduced 02 levels
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热带亚热带植物学撒 第 4卷
found in coated fruitst Application of Pro-long to bananas at difierent times relative to ini—
tiat Jon of ripening reduced their respiration rates
， C2H4production and chlo rophyll loss In
addition，other studies have been performed on the efects of Pro-long on ripening process
of banana，babaco and papayal1,1 2] However
， little is known as to how Pro-long coating
affects ethylene biosynthesis so as to delay the ripening process of fruits even though some
applications of Pro-long to fruits weFe doneI This work was designed to examine the effects
of Pro-long coating on biosynthesis of ethylene and other relevant physiological changes in
banana fruits during ripening
2 M aterials and M ethods
2．1 Plant material and treatments
Banana(Musa acuminata Cola cv．Dwarf Cavendish)fruit，green and slightly immature
by commerica l standards，were obrained from Windward Isles through Banana Section of
Geest PLC．The bananas were washed with 1％ sodium hypochloride solution．and allowed
to dry Afterwards．the fruits were dipped into 1
．5％ Proqong coating solution for several
minutes ，and then allowed to dry fo f about l hour．The fruits were stored at 20 ℃ with
75％ relative humidity．
2．2 Determlnations of ethylene production
Fruits were sealed in plastic boxes wi血 septa for 4 hours and then 1ml gas samples
were taken through septa and injected into Shimadzu gas chromatograph with a gas-tight
syringe The Shimadzu gas chromatograph was fitted with a flame ionization detector with
the folowing conditions：AI ，column，oven temperature 90℃，injection temperature I 20℃
and nitrogen ca rrier gas flow rate 40ml min。
2．3 Determination of ACC and M ACC contents
Pulp tissue of banana．1 0g fresh weight，was homogenized in 10ml of 90％ ethanol by
means of an Ystral Gmbh D一780 l Dottingen homogenizer．The shaft was washed with addi—
tional 10rnl of 90％ ethano1．The extract and washings were co mbined and extracted in a
water bath at 70℃ for 1 hour． and then centrifuged at 4000rpm for l 5min． The
supernatant was co llected，and the residue was extracted twice with l0ml of 90％ ethanol
and then centrifuged and all supernatants were co mbined．The supernatant was co ncentrated
~mder reduced pressure at 45℃ by means of CORNING vacuum rotary evaporator to re一
!TIOVe all ethano1 and the extract was mixed with 3rnl of chloroform to remove pigments
．
and then with 5ml of water and was vigorously shaken．ACC in the water phase was assayed
by the m ethod of Lizada and Yang For measuring M ACC co ntent
． an 2ml aliquot
was hydrolyzed in 3tool L HC1 at 100℃ rnr 3 hours to liberate ACC ． Following
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第 4期 张东林等：PR0．L0NG滁膜处理对后熟香蕉果实乙烯形成 及其它有关生理变化的影响 53
neutralization with 3mol L NaOH the resulting hydrolysate was assayed for ACC as des-
cribed above M ACC content was calculated as differenc~s jn ACC content after and before
HC1-hydrolysis
2．4 EFE assays
EFE activity was determined by measuring conversion of administered ACC to ethylene
in vivo 2g of pulp discs，about 3mm thick and 6ram in diameter，were sampled with a No．4
core borer and placed in a 22ml flask．Following addition of 0 5ml of 5mmol L—ACC so．
1ution．they were infused in vacuum for 5mi n Then each flask was sealed with a rubber so．
rum cap for 3 hours and ethylene was determined as above EFE activity was expressed as
ethylene production On the other hand，after infusion in vacuun1．some pulp discs were
co ated with 1．5％ Pro-long co ating solution immediately and then sealed as above for
ethylene determination
2．5 Analysis of chlorophyll
A 4g banana peel sample was homogenized and extracted with 80％ acetone．Ammonium
hydroxide(three to four drops)was added to stabilize the chlorophyl The extract was
filtered with a Buchnet funne1 at reduced pressure．The filtrate was diluted into 50ml The
solution was tead at 645nm and 663nm and the amount of chlorophyll in lhe sample ca1．
culated with the folowing equation：C b nI=(20．2 X AH0+(8．02 x A6j) Micrograms of
chlorophyll per gram flesh weight was calculated by multiplying by the dilution factor．
2．6 Analysis of firmness
The firmness of banana pulp and peel was analyzed by TA-XT2 Texture Analyser with
XTRA Dimension software package(SMS Stable Micro Systems ，Unit 105，Blackdown Ru-
ral Industries，Haste Hil，Haslemere，Surey GU27 3AY，England)
2．7 Statistical analyses
All data are presented as means of measurements from triplicate experiments Analysis
of variance was used for least significant diference at the 5％ leve1．
3 Results
3．1 Efect of Pro-long treatment on chlorophyll contents and firmness
In the beginning stage of storage．the chlorophyll co ntents of the peels in both the con-
trol and Pro-long treatment were high， decreasing slowly with the development of
ripening，and that of Pro-long treatment decreased more slowly than that of the control
(Figure 1)．At day 1 0 of storage，the chlorophyl contents of the control and treatment were
33 52％ and 71．62％ of that at the beginning respectively．Then，they fell to low levels
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热带亚热带植物学报 第 4卷
During the beginning stage of storage，the firmness of peels and pulps in both the con
trol and Pro-long treatment were high．The firmness of peels and pulps in the control
started to decrease quickly after the 8th day，while that in the Pro-long treatment remained
high，starting to decrease by the 1 5th day(Figure 2) The patterns of changes of the
firmness in both the contro1 and the treatmenl are similar．
0L一
0
Days of storage
Fig I Changes in chl⋯ L’ph yl content of banana
fruit pe cl during ripening
— — ．_ Control，
— - 【5％ Proqong
g
g
伽
器
g
Days of storage
Fig．2 Changes in firmness of banana fruit peel and
pulp during ripening
— 1 Control PeeL： — ．_-Co ntrol Pulp，
—
●一 I 5％ Pro4ong Peel； — ●一 l 5％ Pro4ong Pulp
3．2 Effect of Pro-long treatment on ethylene production
In whole fruit，ethylene production in both the control and the Pro-long treatment was
very low before day 4 and day 9 of storage respectively Ethylene production in the control
started to increase greatly after day 4 of storage and reached a peak at day 9 of storage．
while that in Pro-long treatment started to increase after day 9 of storage and then reached
a peak at day 14 of storage(Figure 31 Afterwards，ethylene production in both the co n-
trol and Pro-long treatment decreased quickly．They have similar pattern of ethylene evolu-
tjon
3．3 Eff~ t of Pro．1ong trea tment on ACC and M ACC contents
Changes of ACC and M ACC contents in pulp are shown in Figure 4 and Figure 5
respectively．At day zero of storage，ACC levels in the pulp were very low， and M ACC
contents were at significant levels．After day 5 of storage． ACC co ntent in the contro1
started to increase and reached a peak(23 59nmol g fresh pulp)at day 8 of storage，while
that in the Pro-long treatment started to increase after the 2nd day and increased gradually
and reached a peak (27．53nmol g fresh Pulp)at day 1 3 of storage、Aflerwars．ACC
co ntents in both the control and Pro-long treatment fel1 quickly to 1ow levels．M eanwhile
M ACC co ntents in both the co ntro1 and Pro-long treatment increa sed slowly．After day 5
． 叶 。_荨I 廿
一 上 JJ ∞3
口 c0u = 上A。 0一上_j
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第4期 张东林等：PRO&ONG涂膜处理对后熟香蕉果实己烯形成以及其它有关生理变化的影响 55
of storage． M ACC content in the co ntrol increased gradually and reached up to
5 52nmol g～ fresh pulp at day 1 1 of storage，while M ACC content in the Proqong treat-
ment increased gradually after day 9 of storage and reached up to 5 43nmol g flesh pulp
at day 16 of storage
|
0 5 10 I 5 20
Days of storage
卜 3 Ethylene production of banana fruit
during ripening
— -． Control； I 5％ Pro-long
0
． D ays of storage
Fig 4 Changes of ACC content in banana
fruit pulp during ripening
— -_ Control； I．5％ Pro4ong
3．4 Efect of Pro-long treatm ent oil EFE aedvities
Changes of EFE activities jn pulps of both the control and Pro-long treatment of whole
fruits druing ripening are shown in Figure 6．At day zero of storage，EFE activity was low
r1 8．80nl g-ifresh pulp h-1)．With the development of ripening．EFE activities in both the con·
trol and Proqong treatment increased gra dually，and that in the Proqong treatment in·
creased much lesS than that in the control before day 7 After day 7 of storage，EFE activity
in the co ntrol increased greatly and reached a peak at day 9 of storage，which co incided
with a peak in ethylene production．while EFE activity in the Proqong treatment continued
O 5 10
Days of storage
J 5 L hangc> r M ^( 【 ∞ ntcnt in bamii1a
l1 pulp during ripening
— _ ． Control； —●一 I 5％ Pro4ong
^
_|
^
＼
0 5 10 15
Days of storage
1，ig 6 Changes of EFE (clhvlcrx ibrming enzyme
activities in banana pulp during ripening
— - Control； + I 5％ Pro-long
．¨一～
，
【l【I主 曼 口0【】 P0Jd 兰 g
-
＼f
一 dI； LI 士 Iu一
口0吾nP2 莒 茸
8 6
一d1；厶LI Jj Iu日 一 口0 u二)《
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GO increase slowly before day 12 ofstorage and then increased and reached a peak alday
；4 ofstorage．Then，EFE activitiesin both the controland Pro-long treatmentdecreased to
。
ow levels
Furtherto unde~land theeffectof
Pr040ngcoatingonEFE inbananapulp
discs，chang~ ofEFE activities in ba—
nana pulp discstreated with 1，5％ Pro-
long were measured．The results are
~hown in Figure 7 EFE activities in
banana pulp discs ofboth thecontrol
and treatment increased slowly and
reached a peak at day 9 of storage，
md then they fell quickly to low
levels Throughouttheseprocesses ，EFE
activity in banana pulp discs treated
with15％ Pro-longremainedlowerthan
that in the contr01．EFE activities jn
ihe treated banana pulp discswere re-
luced by from 19．45％ to40．51％ as
compared with the contr01．At day 9，
EFE activity in this treated discs was
reduced by 38．85％ ．
4 Discussion
During ripening ofbanana fruitchlorophyllcontentsin peels of both the controland
Pro-long treatmentdecreased co ntinuously．The rusults shown in Figure l indicate clearly
lhatchlorophyllco ntentin the Prodong treatment decreased more slowly than that in the
control， There js a significant difference between the co ntrol and Pro-long treatment
{PIn the preclimacteric stage，the firmness in peels and pulps of both the co ntroland
Pro-long treated fruitschangedslightly，and then theirfirmnessdecreased quickly fFigure
!)Figure2indicatesclearlythatthePro-longtreatmentdelayedthedecreasesinthefirm．
i!essofpeeland pulp by 5 days Also therearesignificantdifferences in thefirmnessofpeel
andpulpbetwenthecontrolandProdongtreatmentrP<0．05)．
Our data indicate that the Pro．10ng treatment delayed the increase jn ethylene
productionby5days(Figure3)，butthepatternofethyleneevolutioninthePro-longtreat-
meritappeared similar to thatin the control
Days ofstorage
Fig 7 Ethylene forming enzyme aclivitics JIlbanana pulp
discs trealed with l5％ P1040ng coating
r---i Pulp discs+ACC
_ Pulpdiscs+AcC+I5％ Proqong
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第 4期 张东林等 ：PRO-LONG涂膜处理对后熟香蕉果实乙烯形成￡上及其它有关生理变化的影响 57
The fact that the EFE activity increased gradually in the beginning stage indicates that
the EFE system is active during the beginning stage(Figure 6)．The great increase in EFE
activity coincided with that of ethylene production Figure 6 indicates that the Pro·
long treatment delayed the great increase in EFE activity by 5 days EFE is a key enzyme
converting ACC to ethylene[21
． Banks[~31reported that the Km (O2)for the conversion of
ACC to ethylene in banana fruit pulp tissue as estimated by this system is 2 2％ ．ten times
greater than that estimated by previous workers for discs of apple cortex．In o ur study
we applied Pro-long coating to banana pulp discs t0 reduce the supplement of oxygen
thereby inhibiting ErE activity in banana pulp discs by from 1 9．45 to 40 5 1％ daring
ripening (Figure 7)where there is a signifcant diference between the con trol and
Pro-long treatment fP<0．05)．
The ACC levels in pulps of both the control and Pro-long treatment in the beginning
phase were low fFigure 4 Figure 4 shows that there was a slow accumulation of ACC in
Pro—long treated fruit before the great increase of ACC content．The great increases of ACC
content in both the control and Pro-long treated fruit took place a short time before the
peaks of both EFE activities and ethylene production；the increase in ACC level in the
Pro-long treated fruit was delayed by 5 days Afterwards， because of the great increase in
EFE activity，ACC was converted to ethylene and decreased quickly to low levels Also there
is a significant difference in the changes of ACC levels between the control and Pro-long
treatment fP<0．05)We suggest that the folowing sequence of events Occurs during ripening
of banana fruit：ACC synthase is first synthesized producing ACC which is converted to
ethylene by the already existing EFE system 【
．
The ethylene produced stimulates additional
ACC synthase activity and then．when ethylene production reaches a certain threshold level，it
stimulates EFE activity， thereby further increasing ethylene production．Autocatalysis is a
common feature of ripening in climacteric fruitsl。6]and some data indica tes that ethylene en-
hances the induction of both ACC synthase[6；and EFEI5,1 目 in various fruits In our study、
Pro-long co ating was used on banana fruit forming a gaseous barrier oil the surface of the
fru it to reduce the supplement of oxygen，thereby inhibiting EFE activity partly，which re—
suited in the accumulation of ACC content．However，beca use EFE activity was inhibited
only in part，ethylene production co ntinued，and when ethylene production reached a certain
threshold leve1．it stimulated EFE activity so as to increase ethylene production further and
then reached a peak．
It is well acepted that malonylation of ACC may play a role in the regulation of
endogenous ACC level and enhance of ethylene production Preclimacteric banana fruits
co ntain a significant level of MACC (Figure 5)．This indicates that at least part of the ACC
which is synthesized in the course of fruit growth is conjugated to MACC Thus
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热带亚热带植物学报 第 4卷
malorxylation of ACC may participate in regulating ethylene production during the
preclimacteric stage．In contrast．it does not seem t0 play a role in regulating ethylene pro—
ouction at the climacteric stage At this stage，M ACC of both the control and Pro-long
tleated fruit increased only slightly as co mpared with the marked incre3,se in ACC levels
(Figure 4 and Figure 5)．However，MACC content of the control increased earlier than that
0f l 5％ Pro-long treated fruit
Banks【8一 suggested Pro—long coating affects gaseous exchange of banana fruit surface
thereby delaying ripening．Our results further clarify that effect of Pro-long coating on
banana fruit ripening is a complex process，ma inly affecting ethylene biosynthesis of
banana fruit．This is through partly inhibiting EFE activity by reducing the supplement of
oxygen which delays the great increase in ethylene production and so extends the ripening
PFOceSSes ．
References
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第 4期 张东林等：PRO．LONGj象膜处理对后熟香蕉果实己烯形成以及其它有关生理变化的影响 59
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