Post on 03-Feb-2022
730Nippon Shokuhin Kogyo Gakkaishi Vol.37, No.9, 730~736 (1990) 〔Article〕(66)
The Relation of Polyphenols and Polyphenol Oxidase
to the Food Quality of Banana Bud
IKUZO URITANI*, Ma. Gracia L. BAILON**, Joseph L. SAMONTE***,
Angelina M. ALVAREZ**, Mary Ann R. ALMARIO**,
Dulce M.FLORES****, Evelyn MaeT. MENDOZA***
and Virgilio V. GARCIA**
*Laboratory of Biochemistry, Nagoya Women's University,
3-40, Shioji-cho, Mizuho-ku, Nagoya 467, Japan**Institute of Food Science and Technology
, Universityof the Philippines, Los Banos (UPLB), College,
Laguna 4031, Philippines***Institute of Plant Breeding, UPLB
****National Institutes of Biotechnology and
Applied Microbiology, UPLB
Polyphenols in banana buds were found to consist of flavanan tannin (condensed tannin)
as the major component; catechin, its oligomers, dopamine and dopa as minor components.
Total phenol and vanillin-positive phenol were higher in the buds of cultivars Bungulan,
Lacatan and Latundan and lower in the buds of cv. Saba. Polyphenol oxidase (PPO) in cv.
Saba was localized in the male flower; 3 times less in the peduncle and bract; and about 30
and 300 times less in the peel and pulp of the fruit respectively. PPO activity in the male
flower, peduncle and bract was inhibited by 0.13M NaCl about 20 to 30%, while that in the
peel and pulp was inhibited about 40 to 50%. Buds of the other cultivars had similar levels
of PPO which were inhibited about 20% by 0.13M NaCI.The Ki value for NaCl of PPO in
the peel was estimated to be 0.28M. The astringency, bitterness and color of banana buds
were monitored before and after heating, NaCl treatment and squeezing. These are discussed
with reference to the polyphenols and PPO focussing on food quality.
The banana bud (specifically, the banana
male bud) of cv. Saba is a native ingredient
used as a fresh vegetable in various culinary
dishes in the Philippines. It has been exported
to a limited degree to foreign countries as a
novel vegetable in frozen and canned forms1).
The banana bud is a component in the
inflorescence of the banana plant. The male
and female organs of the flower come out of
the plant at some intervals due to the mon-
oecious nature2). The banana bud at the end
of the inflorescence contains the male flower,
peduncle, and bract, as shown in Fig.1. Thebanana bud is commonly called banana blos-
som or heart in the Philippines.Banana buds in most cultivars are regarded
as non-edible materials, and the bud of only
one cultivar named cv. Saba is sold in the
market. This is because the bud of cv. Saba
loses astringency and bitterness by heat
treatment, while those of the other cultivars
remain astringent and bitter even after heat
treatment. These have hindered the utilization
of the buds of other cultivars as food
materials. Thus, a large number of banana
buds is discarded as waste material in thePhilippines especially the major planting areas
around Davao.
It is important to characterize the astringent
and bitter components and to investigate the
effect of cooking or heat treatment on astrin-
gency and bitterness. This will pave the way
(67)URITANI et al.: Polyphenols, phenol oxidase and food quality of banana bud731
Fig.1 Banana plant, bud and its parts
A: banana bud, and B: male flower, C
peduncle and D: bract, shown in the
longitudinal section of the bud.
for utilizing banana buds of cultivars other
than cv. Saba as a fresh vegetable or as a
food ingredient. No studies have been done onthe characteristics of these components in the
banana bud. However, the major polyphenols
in the immature green fruit of banana have
been identified as flavanan tannin (condensedtannin, or proanthocyanidin polymer) , consis-ting of catechin and gallocatechin in the ratio
of about l to 1.3 (MATSU0 and ITOO)3). Theastringent and bitter components in the banana
bud are also suspected to be flavanan tannin
(condensed tannin) similar to that observed inthe fruit.
Besides flavanan tannin, 3, 4-dihydroxypheny-lethylamine (dopamine) has been found to be
present in peel and pulp of the banana fruitand in other parts of the banana plant includ-
ing bract of the bud4). Further, tyrosine and3, 4-dihydroxyphenylalanine (dopa) have beenfound in the peel of the fruits5). It is thuspossible that these components are also presentin every part of the bud .On the other hand , when the banana bud is
cut into pieces and allowed to stand for a
while, the cut surfaces turn brown. When the
pieces are heated, the surfaces turn black. Itis assumed that polyphenols and polyphenol
oxidase (EC 1.14. 18.1, PPO) in the bud take
part in such discoloration. Since the discolora-tion decreases the food quality, it is alsoimportant to elucidate its mechanism.
This paper deals with the characterization,
assay and localization of polyphenols and PPO
in banana buds of both edible and non-edible
cultivars as these relate to food quality includ-
ing flavor (taste) and color. Further, since
table salt (NaCl) is added to the pieces of the
bud prior to cooking as an indigenous prepara-
tive method, the effect of NaCl on food quality
was also investigated with emphasis on the
polyphenols and PPO.
Materials and Methods
Materials
All the chemicals used in the experiment were
reagent grade. Buds of cv. Saba [Musa×
paradisiaca L. (BBB)] were bought from the
public market at Los Banos. Buds of other
cultivars were obtained from the College
Experiment Station, College of Agriculture,
University of the Philippines at Los Banos
(UPLB). The other cultivars used were cv.
Bungulan [Musa×paradisiaca L. (AAA)], cv.
Lacatan [Musa×paradisiaca L. (AA)], cv.
Latundan [Musa×paradisiacaL. (ABB)], cv.
Senorita [Musa×paradisiaca L. (AA)], cv.
Kluai Sa 217 [Musa×paradisiaca L. (AA)]
and cv. Pisang Pilot [Musa×paradisiaca L.
(AAA)].
Banana fruits in the mature green stage (full
three quarters) were also used to compare with
the buds. The cultivars used included cv.
Saba, cv. Lagkitan [Musa×paradisiaca L.
(AAB)] and cv. Latundan.
Sampling for quantitative analysis
The 3 parts of the bud, namely the male
flower, peduncle and bract, were used for the
analysis. The fruit was divided into the peel
and pulp and assayed individually.
For the analysis of polyphenols, 4g of the
sample was homogenized in 20ml of methanol
with adequate amounts of quartz sand using a
732日 本 食品工業学会誌 第37巻 第9号 1990年9月(68)
mortar and pestle in an ice bath. The slurry
was centrifuged and the supernatant was used
for the analysis.
For the determination of PPO activity, 1g
of the sample was extracted with 10ml of
0.1M potassium phosphate buffer (pH6.8)
containing 1% Triton X-100, 0.1% sodium
isoascorbate, 0.5g of Polyclar AT and ade-
quate amounts of quartz sand, using a mortarand pestle in an ice bath. The addition of
Triton X-100 was to solubilize PPO in banana
tissues following the menthod of PALMER6).
The slurry was centrifuged, and 2.5ml of the
supernatant solution was passed through PD-10 column (Sephadex G-25 prepacked column,
Pharmacia) pre-equilibrated with 0.1M potas-
sium phosphate buffer (pH6.8) to remove poly-
phenols by being tightly bound with Sephadex,and 3.5ml of the eluate containing proteins
was collected. Then, 1.0ml of the eluate was
made to 10ml by 0.02M potassium phosphate
buffer (pH6.8) and the diluted solution was
used for the assay of PPO activity.Determination of polyphenols
Total phenol: An aliquot of the methanol
extract was dried in astream of nitrogen gas,
to which was added 2.0ml of distilled water.
Then, the solution was mixed with 2.0ml of 10%
sodium carbonate and 2.0ml of lN phenol
reagent following the Folin-Ciocalteau meth-
od7). The mixture was allowed to stand for 30
min and the absorbance was read at 770nm.
Flavanan tannin (condensed tannin): The
protein precipitation method of HAGERMANand BUTLER8) was used with the followingmodifications. An aliquot (0.20 or 0.60ml) of
the above methanol extract was mixed with 2.0
ml of 0.1% (w/v) bovine serum albumin (BSA)
and allowed to stand for 10min. The precipi-
tate thus formed was collected by centrifuga-
tion and suspended in 1.0ml of water. The
suspension was mixed with 4.0ml of 1% SDS-5%
(v/v) triethanolamine solution and 1.0ml of0.1N ferric chloride solution. The mixture
was allowed to stand for 15 to 30min before
reading the absorbance at 510nm. (+)-Cate-
chin was used as the standard instead of tannic
acid, and the BSA-precipitation procedure was
not made in this case.
Vanillin-positive phenol: To assay for phe-
nols involving flavonoid structure such as cate-
chin, the vani11in-sulfuric acid method9) was
used. The dried material obtained from an
aliquot of the methanol extract was dissolved
in 1.0ml of water. The solution was mixed
with 2.0ml of freshly prepared 1% vanillin in
70% (v/v) sulfuric acid in an ice bath. After
15min, the absorbance of the solution was
read at 500nm.
Thin-layer chromatography: Two ml of
the above methanol extract was dried using a
stream of nitrogen gas. The dried material
was dissolved in 2ml of water. The solution
was twice extracted with 2.0ml of water-
saturated ethyl acetate. The combined extract
was evaporated to dryness by applying nitro-
gen gas, after which 0.2ml methanol was
added. A small volume of the solution thus
prepared was applied to HPTLC plate (Kiesel-
gel 60, 10×10cm, Merck) and developed by
butanol-water-acetic acid (4:1:5v/v) or 2%
acetic acid. Then, the dried plate was sprayad
with phenol reagent of Folin-Ciocalteau meth-
od, vanillin-sulfuric acid reagent, or ninhydrin
reagent. Some authentic samples that included
(+)-catechin, (+)-gallocatechin, dopamine, do-
pa, etc. were co-developed in the plate.
The ethyl acetate extract was also subjected
to total phenol determination as ethyl acetate-
soluble phenol.
Assay for PPO activity
The assay was based on the increase in color
resulting from the oxidation of dopamine by
PPO, following the menhod of PALMER6).The
reaction mixture in the cuvette contained 0.9
ml of 0.1M potassium phosphate buffer (pH
6.8), 1.0ml of 1.9mM dopamine solution, 1.0
ml of water and 0,1ml of enzyme solution
(described above). The reference cuvette con-
tained the same ingredients except for 0.1ml
of 0.02M potassium phosphate buffer, in place
of the enzyme solution. Activity was deter-
mined as the increase in absorbance at 462nm
for lmin at 25℃ lmin after adding enzyme
solution, using a U-3200 spectrophotometer
(Hitachi). Activity was expressed as the value
(69)URITANI et al.: Polyphenols, phenol oxidase and food quality of banana bud733
equivalent to 0.1ml enzyme solution.When the inhibitory effect of NaCl on PPO
activity was investigated, 0.13M NaCl solution
(final concentration) was applied instead ofdistilled water. The Ki value of PPO to NaCl
was determined in the range of 0.067 to 1.3M
concentrations of NaCl.Effect of NaCl-addition and steaming on
palatabilityNaCl-addition and steaming: The banana
bud of cv. Saba was cut into pieces which weredivided into 2 groups. To one group was
added NaCl in the ratio of 1:10 (ratio when
cooked at home). Then, the tissue pieces were
squeezed in cheese cloth by hand, and steamed
for 20min. The other group was also steamed
after squeezing the tissue pieces without addedNaCl. The palatability of both groups was
evaluated before and after steaming by 3 to 5
panelists. Banana buds of cv. Bungulan, cv.Lacatan, and cv. Latundan were cooked in the
same way as in cv. Saba bud (including NaCl-
addition and squeezing), and the palatability
(taste and color) before and after steamingwas compared with that of cv. Saba buds.
Steaming: The buds of cv. Saba, cv.
Latundan, cv. Senorita, cv. Kluai Sa 217 and
cv. Pisang Pilot were cut longitudinally intohalves. One group was steamed for 20min
and the other was not steamed, after which the
taste and color in both groups (male flower,
peduncle and bract) were evaluated.
Results and Discussions
Polyphenols
The polyphenol content of the male flower,
peduncle and bract of cv. Saba bud is shown in
Table1. The male flower contained the high-
est amount while the peduncle and bract had
almost the same level of polyphenols. In the
male flower, vanillin-positive phenol was lower
than flavanan tannin. This may be explained
by the results that when polymeric flavonoids
are reacted with vanillin acidic condition,
flavan-3-ol end group acts more actively than
the inner units of the compounds (BUTLER
etal.)10).
As shown in Table 1, flavanan tannin or
condensed tannin, showing vanillin-positive
reaction, was present in the bud as the main
component, similarly to the results obtainedwith immature green fruit of banana3). The
ethyl acetate-soluble phenol was in the range
of 5 to 8% of total phenol. TLC experiments
showed that the ethyl acetate-soluble phenol
contained dopamine, dopa, catechin and some
other vanillin-positive phenols whose Rf-
values were lower than (+)-catechin, suggest-ing the presence of oligomers of flavan 3-ol.
The male flowers of the buds of cv. Bun-
gulan, cv. Lacatan and cv. Latundan, hadlarger amounts of both total phenol and
vanillin-positive phenol than cv. Saba (Tables l
and 2).
Table 2 Polyphenol of male flowers in
banana buds of some cultivars
(mg/100g)
Table 1 Polyphenol content of the different parts in banana bud of cv.Saba (mg/100g)
734日 本食品工業学会誌 第37巻 第9号 1990年9月(70)
Polyphenol oxidase activity in cu. Saba
The male flower had 3 times higher PPO
activity than the peduncle and bract in the bud
of cv. Saba (Table3).
As indicated in Table 3, NaCl at 0.13M final
concentration inhibited the PPO activity by
about 20 to 30%. The response of PPO to
NaCl differed for the individual parts. This
suggests that PPO exists in the form of isozy-mes, which differ in species and content among
the different parts.
PPO activity of the peel and pulp of cv. Saba
mature green fruit was also investigated. The
activity in the peel was 10 times higher than in
the pulp, but was 9 to 26 times less than in the
parts of bud (Table 3). The activity in theextract of cv. Saba fruit peel was inhibited by
0.13M NaCl 36%. The Ki value for NaCl of
PPO in the peel was estimated to be 0.28M,
equivalent to 1.64% NaCl. When banana budis used for cooking in the home, about 10%
NaCl is added. This is equivalent to 1.71M
NaCl per kg. of banana bud. Assuming that
0.28M of Ki value may apply to PPO in the
bud, the activity in the bud tissue pieces may
be inhibited more than 80 to 85% by NaCl in
the concentration of 1.71M.
Two mechanisms are proposed for the role of
NaCl in improving food quality of banana bud.
The first one is to inhibit PPO activity and the
other is to remove polyphenols by decreasing
the turgor pressure of the cells of the tissue
pieces and squeezing out the juice sufficiently.In fact, when cv. Saba bud was treated as
above and steamed for 20min, the cooked
tissue showed a bright and light reddish color
and lost the astringency and bitterness: The
use of NaCl to inhibit PPO activity in other
food materials has been often appliedl1), but
limited mainly for holding peeled fruits for
processing. The use of NaCl to removeundesirable flavors such as astringency,
bitterness or pungency is also commonly used
in the Philippines for bitter gourd (Momordica
charantia L.) and radish (Raphanus sativus L.)
prior to cooking.Polyphenol oxidase activity in male flowers
of some cultivars
PPO activity in male flowers of the buds of
cv. Bungulan, cv. Lacatan and cv. Latundan
was also investigated. As indicated in Table 4,
the 3 cultivars had less activity than cv. Saba
(see Table 3). The activity was found highestin cv. Bungulan and lowest in cv. Latundan.
NaCl inhibited the activity about 20% at 0.13
M final concentration. As shown in Table 4,
PPO activity in cv. Latundan mature green
fruit (peel and pulp) was much less than that
in male flower, and was similar to cv. Saba
(Table 3).Effect of NaCl-additin and steaming on
palatabilityWhen banana buds of different cultivars were
subjected to NaCl addition and sqeezing, but
without steaming, they showed astringeny and
bitterness. However, in cv. Saba, the degree
was lowest and the unpalatable taste was
greatly decreased aftar NaCl treatment. After20min steaming, astringency and bitterness
disappeared in cv. Saba but not in the other
Table 3 Polyphenol oxidase activity of banana bud and mature green fruit of cv.
Saba and its inhibition by sodium chloride
(71)URITANI et al.: Polyphenols, phenol oxidase and food quality of banana bud735
Table 4 Polyphenol oxidase activity of male flowers in banana buds of some cultivars
cultivars. The unpalatable taste was highestin cv. Bungulan bud, in both before and after
steaming.
Next, the effect of steaming alone on palata-
bility was observed using the buds of somecultivars (see Materials and Methods). As-
tringency and bitterness were present in the 3
parts of cv. Saba bud before steaming. Aftersteaming, such was absent in the peduncle and
bract with a little remaining in the maleflower. In the case of the other cultivars, the
astringency and bitterness of all parts of the
buds were retained even after steaming in equalor less grade, compared with the parts without
steaming, and was highest in male flowers both
without and with steaming.
With regard to color after cooking, the useof NaCl made the cooked buds attractive. The
color turned bright and light reddish during
steaming/cooking and appeared appetizing.
When cooked without NaCl, the color turned to
an unappetizing greyish dark color. Suchdiscoloration is related to the presence of
polyphenols and PPO, as previously mentionedabove.
It is important to elucidate the reasons why
the bud of cv. Saba loses its astringency andbitterness during steaming , but those of othercultivars do not. It is often observed thatmature green banana fruits are astringent, butthe taste disappears or decreases greatly during
heat treatment. This was experienced with
mature green fruits of many cultivars inclu-ding cv. Saba and cv. Lagkitan. According toThe data of LEA et al.12), o1igomers of procy-anidin (flavan-3, 4-diol) are the principle ofbitterness, and the polymers are directly
related to astringency. The effect of heat
treatment may be attributed to polymerization
and insolubilization of condensed tannin,
possibly in combination with proteins andpolysacctarides in the tillues13). Such amechanism may be valid for the disappearance
of astringency and bitterness in the bud of cv.
Saba during heat treatment. The reason whythe astringency and bitterness of the banana
buds of many other cultivars are not notably
reduced when subjected to heat treatment may
be explained by the chemical environments in
the tissues as well as the nature and amounts
of polyphenols present which are different from
the case of cv. Saba.Further studies are needed to fully elucidate
the mechanism for removal of astringency and
bitterness from banana buds.
Acknowledgement
This work was done at the University of the
Philippines, Los Banos, Philippines, under the
Scientific Exchange Program between Depart-
merit of Science and Technology, Philippines
and Japan Society for the Promotion of Sci-
ence, Japan and partly supported by a Grant
from Asian Community Trust, Tokyo. The
assistance of Dr. R.E. CORONEL in the scientific
descriptions of banana cultivars and parts andfor providing the materials for some experi-
ments is greatly appreciated. Further, the
authors wish to express also their gratitude to
Dr. S. ITOO, Kagoshima University, Kago-
shima and Dr R. SAIJ0, National Vegetable
and Tea Experiment Station, Mie Prefecture,for giving them valuable information on
flavanan tannin in persimmon and other fruits
and pure samples of (+)-catechin and other tea
736日 本 食品工業学会誌 第37巻 第9号 1990年9月(72)
catechins, respectively.
References
1) EVANGELISTA, M.E., PARIAN, R.E.G. and
GONZALES, O.N.: NSDS Technology J.
(published by National Science Develop-
ment Board, Philippines), 2, 28 (1977).
2) VALMAYOR, R.V., RIVERA, F.N. and
LOMULJO, F.M.: Philippine Banana Cul-
tivar, Names and Synonyms, IPB Bulletin
(published by Institute of Plant Breeding,
UPLB, Philippines), No.3, p.16 (1981).
3) MATSUO, T. and ITOO, S.: J. Japan. Soc.
Hort. Sci., 50, 262 (1981).
4) GRIFFITHS, L.A.: Nature, No. 4679, 58
(1959).
5) MURATA, T. and Ku, H.S.: J. Jpn. Soc.
Food Sci. Tech., 13, 466 (1966).
6) PALMER, J.K.: Plant Physiol., 38, 508
(1963).
7) FOLIN, 0. and CI0CALTEAU, V.: J. Biol.
Chem., 73, 627 (1927).
8) HAGERMAN, A.E. and BUTLER, L.G.: J.
Agric. Food Chem., 30, 1087 (1982).
9) SONDHEIMER, E.: J. Amer. Chem.Soc., 75,
1507 (1953).
10) BUTLER, L.G., PRICE, M.L, and BROTHER-
TON, J.E.: J. Agric. Food Chem., 30, 1087
(1982).
11) ESKIN, H.A.M., HENDERS0N, H.M. and
TOWNSEND, R.C.: Biochemistry of Foods
(Academic Press, New York), p.215
(1971).
12) LEA, A.G.H. and ARNOLD, G.M.: J. Sci.
Food Agric., 29, 478 (1978).
13) FORSYTH, W.G.C.: The Quality of Foods
and Beverages, Vol.1 Chemistry and Tech-
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G., Academic Press, New York), 377
(1981).
(Received Feb. 27, 1990)
バナナつぼみにおけるポ リフェノール及び
ポ リフ ェノールオキシダーゼと
食品上 の品質 との関係
瓜 谷 郁 三*・ グ レイ シアLバ イ ロン**
ジ ョセフLサ モ ンテ***
ア ンヘ リ-ナMア ルバ レス**
メ リ-ア ンRア ル マ リオ**
ドゥル セMフ ロ ー レス****
エ ヴ ェ リンメ イTメ ン ドーサ***
バ ー ヒ リオVガ ル シ ァ
*名 古屋女子大学生化学研究室
(〒467名 古屋市瑞穂区汐路町3-40)**フ ィ リピン大学 ロスバ ニオス校(UPLB)
食品科学 ・工学研究所
(〒4031 フィリピン カ リジ ラグナ)***UPLB植 物育種研究所
***UPLBバ イオテクノロジー・応用微生物学研究所
バナナつぼみ中のポ リフェノール成分 は主要成分とし
て フ ラバ ナ ンタ ンニ ン(縮 合型 タンニ ン),副 次成分と
して カテキ ン,そ のオ リゴマー,ド パ ミン及び ドパから
成 っていた.数 品種 のつぼみのうち,調 理用バナナであ
る ‘サバ'品 種 のつぼ み において,フ ェノール全量やバ
ニ リン陽性 フェノール量 は最 も少 なか った.ポ リフェノ
ール オキ シダー ゼ(PPO)活 性 の品 種間差異 は著 しく
はな いが,つ ぼみ中の部位 による差 は顕著であり,又 そ
の活性 は果実の場合よ りもかな り大 きか った.つ ぼみの
PPO活 性 は, 0.13M食 塩 で約20%抑 制 された.な
お フィ リ ピンにおい て,‘サバ'品 種 つぼみを加熱 ・調
理 す る際 に,そ の薄片に約10% (1kg当 り約1.71M)
の食塩を加え,汁 液を しぼ りとるが,こ れは食塩により
PPO活 性 を抑制 して加熱後の色を鮮 やかな淡赤色にす
るためで あ り,他 には食塩添加 によ り膨圧を失わせ,渋
味 ・苦味 の原因 となるポ リフェノlル を除 くためである
ことが分か った.他 の品種の場合には,食 塩添加,搾 汁,
加熱 によっても渋味 ・苦味 は十分に除去 されなかった.