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-

nology (Charalambous, G. and Inglett,

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．