全 文 ：41※基础研究 食品科学 2008, Vol. 29, No. 10
Inhibitory Activity of Grapefruit Seed Extract Against Fungi and Its Applica-
tion in Preservations of Grape and Persimmon
XU Wen-tao，HUANG Kun-lun，QU Wei，LIN Xi-jin，DENG Ai-ke，YANG Jia-jia，LUO Yun-bo*
(College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China)
Abstract ：The grapefruit seed extract (GSE), a natural plant extracts from the edible plants used as an extremely potent broad-
spectrum bactericide, and fungicide should be very effective for the fruit preservation. In this study, Grape (Vitis vin f ra L.) and
persimmon (Di spyris kaki L.) easily undergone deterioration, were selected as model fruits. Eight fungi familiar to the rotting
cause of grape and persimmon were used to testify the antifungal activity of GSE. The results of antifungal assay by the agar
diffusion method indicated that GSE can efficiently inhibit the growth of tested fungi. The values of minimal inhibitory
concentration (MIC) also range from 39.06×10-6 t 625×10-6 as demonstrated by the similar results with the data taken from
agar diffusion assay. In the storage application assay of both grape and persimmon, the obvious differences in sensory quality
and microorganism indexes between the GSE treated fruits and control fruits support that GSE has both the strong antifungal
activity and antioxidative activity. Polyphenolic compounds, rich in GSE used as the main effective antioxidative component,
are capable to prevent fungal infection in storage to a large extent and to postpone ripening and aging of fruits effectively by
scavenging the free radical and impacting a series of enzyme relating to postharvest physiological metabolism of fruits, particu-
larly in the key parts liking stem and calyx. The obtained results also indicated that GSE can decrease browning and rot ratio,
prolong the preservation period, ensure the quality of grape and persimmon and can be used as an effective and safe preservative.
Key words ：grapefruit seed extract；pres rvation；antifungal activity；grape； rsimmon
葡萄柚种子提取物对真菌的抑制作用及其
在葡萄和柿子保鲜中的应用
许文涛，黄昆仑，屈 玮，林希瑾，邓爱科，杨加佳，罗云波*
(中国农业大学食品科学与营养工程学院，北京 100083)
摘 要：葡萄柚种子提取物是潜在的广谱性细菌、真菌杀菌剂，应该在果蔬保鲜中具有很好的效果。本研究选
取比较容易受真菌感染的葡萄和柿子作为模式保鲜材料并且测试了葡萄柚种子提取物对8种与这两种水果腐败相关的
真菌的抑制效果。结果表明，这8种真菌的最小抑制浓度分别在39.06×10-6至625×10-6之间不等。果实保鲜结
果也证实了葡萄柚种子提取物对真菌具有很好的杀抑作用，延缓了果实的成熟，并且使保鲜的果实保留了较好的风
味。这些结果可能与葡萄柚种子提取物富含多酚类物质有关，因为多酚类物质具有很强的抗氧化活性。
关键词：葡萄柚种子提取物；保鲜；真菌抗性；葡萄；柿子
中图分类号：TS205 文献标识码：A 文章编号：1002-6630(2008)10-0041-06
收稿日期：2007-07-14
作者简介：许文涛(1979-)，男，讲师，主要从事食品安全检测与食品安全风险评估。E-mail：xuwentao1111@sina.com
*通讯作者：罗云波(1958-)，男，教授，博士，主要从事食品安全生物技术与食品安全。E-mail：lyb@cau.edu.cn
Grapefruit seed extract (GSE) is a commercial product
derived from the seeds and pulp of grapefruit (Citrus paradisi
Macf. Rutaceae). Chemical research revealed the
constituentias of flavoniods [1-2], ascorbic acid, tocopherols,
citric acid [3], l monoids [4-5], sterols and minerals [6] in th
grapefruit seeds and pulp. And GSE also contained large
quantiti s of polyphenolic compounds such as catechins,
epic techin and epocat chin-3-O-gallate, and dimeric, trim-
eri and tetrameric procyanidins [7].
GSE is an extremely potent and effective broad-spec-
2008, Vol. 29, No. 10 食品科学 ※基础研究42
trum bactericide [8-9], fungicide [10], antiviral and antiparasitic [2]
natural extract. GSE is environmentally safe from toxicity to
man and animal within an effectively applied concentration
extent. Inoescu demonstrated that GSE performed well as
the other antimicrobial agents tested on 770 strains of
bacteria, and 93 strains of fungus. The inhibition activity of
GSE to yeast and some yeast-like fungi shows different effi-
cacy according to the strains and is generally weaker than
that of bacteria and fungi [2]. Concerni mechanism of action,
the researchers propounded that GSE could disrupt the bac-
terial membrane and liberates the cytoplasmic content by
inhibiting enzymatic activities [9]. Although the active com-
ponents of GSE in relation to its antimicrobial property are
still incompletely known, polyphenols compounds particu-
larly flavonoid, are considered to play an significant role in
light of the accurate positive correlation between their total
contents and antimicrobial activity of GSE [11]. As antim cro-
bial agent, the most obvious superiority of GSE rests with its
safety better than other chemical synthetic antimicrobial
agent. Tests conducted by the United States Department of
Agriculture (USDA) in the early 1980s confirmed that GSE is
not toxic to animal and has been approved for use by USDA.
Sequentially the safety of GSE was testified by several writers.
Heggers et al. testified that GSE would not be detrimental to
human fibroblast skin cell in an in vitro culture, while still
retained high antimicrobial activity. The results obtained from
J. Juskiewicz et al showed that the addition of 0.1%～0.4%
extract of flavonoid from grapefruit did not affect the diet
intake and body weight gain of rats and slightly increased
the anti-oxidative potential of serum [12]. Today consumers
prefer products without any artificial chemical preservatives
because of the residue and safety problems of synthetic
antimicrobial agents [13-14]. Therefore GSE is of a wide applica-
tion perspective in many fields including agriculture, cos-
metic production, decontaminating water, treating infections
and storage of fruits and vegetables as well.
Grape (Vitis vinifera L.) and persimmon (Dio pyris kaki
L.) are two of the most important fruits in world. But due to
the soft texture and the high water content, these fruits eas-
ily undergo deterioration resulting from physiological
disorder，microorganism infection or mechanical damage,
so preservation is difficult. Under normal atmospheric and
temperature conditions, grapes can be stored for only 2～3
days [15] and persimmon 4～5 days[16]. In order to ensure their
even distribution both in time and in space, preservation of
grape and persimmon is of great concern. The traditional
methods of preserving grape and persimmon are basket
storage, cellar-storage, chemical storage and cold storage.
Most of the storage me hods are selective when kept at low
temperature, but it is not easy to control temperature and
humidi y accurately for marketing. It is well known that varia-
tion of tempera ure ffects respiration, metabolism and
microorganism, growth and consequently, preservation
efficacy. However microorganisms, particularly fungi, exert
harmful effects on quality, safety and shelf life of grape and
persimmon and are most responsible for yield reductions
and a great many losses in the course of storage throughout
the world. The use of antifungal agent is the most effective
method to minimize fungi infection, although the growth of
micro rganisms is easily controlled by chemically synthe-
sized preservatives, such as sorbic or benzoic acids. This
preference has promp ed the application of natural plant ex-
tracts to prevent rot and prolong preservation period of fruits
and vege ables. Th u ed GSE a antifungal agent from the
edible plan s should be superior in comparison with non-
natural antifungal gents.
Our objectives were o evaluate the antifungal effective-
n ss of GSE for the ight fungi familiar in fruit preservation
so as to determin the min mum inhibitory concentration
(MIC) for these eight fu gi. Specifically applied in the
pr servation of grape and persimmon, while results were ob-
served and analyzed. The mechanism between antioxidant
nd antifungal GSE and the postharvest physiological varia-
tions of fruits wer also discussed.
1 Materials and Methods
1.1Selection of fungi and bacteria strains
The eight fungi used as test organisms are: Botrytis
cinerea(CGMCC#3.3789), Alternaria sp. (CGMCC#3.1491),
Stemphyl um lanuginosun (CGMCC#3.4282), Aspergillus
ni er (CGMCC#3.310),Pen.expansum (CGMCC#3.5425),
Streptomyces albussubsp. albus (CGMCC#4.1), Streptomy-
ces griseus subsp. griseus(CGMCC#4.18) and Saccharomy-
ces cerevisiae (CGMCC#2.0401). All of them are closely
correlated to the rot of fruit and vegetable in postharvest
period, especially of grape and persimmon employed in this
study. The culture of each fungi was maintained on potato
dextrose agar (PDA) and stored at 4℃. These fungi were
obt ined from the Institute of Microbiology of Chinese
Acad my of Sciences.
1.2Antifungal assay
GSE was dissolved n the water with 0. 05% (V/V) Tween-
43※基础研究 食品科学 2008, Vol. 29, No. 10
80 as surfactant to make a 10% (V/V) stock solution filtered
and sterilized.
The fungi were swabbed on solidified PDA plates. Each
PDA plate was inoculated with a single identified fungal
isolate. The four sterile metal cylinders 8×6×10 mm in
diameter were placed on the agar and then filled with 50 μl of
0.01%, 0.1%, 1% in 10% (V/V) GSE solution. The same vol-
ume of water containing Tween-80 was also tested as control.
After 2-h diffusion at 4 ℃, all the plates were incubated at 37
℃ for 48 hours. At the end, inhibition zones around each metal
cylinders formed in the medium with their diameters mea-
sured to determine the antifungal effectiveness of diluted
GSE solutions. All the tests were made in triplicate on sepa-
rate assays [17].
1.3Determination of MIC
Broth microdilution assays were performed as previ-
ously described [18]. This test was performed in sterile
cultivation with flat-bottomed 96-well microplates. Stock so-
lution of GSE was diluted (5-fold) with heat-sterilized me-
dium to give the final concentrations. 100μl of th GSE
dilutions were inoculated into the wells with first well of
each row containing the lowest concentration and last well
as the control (GSE-free medium). The working suspension
of the inoculum, adjusted with a spectrophotometer to give
initial numbers ranging from 105 to 106 CFU/ml. Then 100μl
inoculum suspension was added to each well. After
microplates were incubated without agitation at 30℃ for 48
h, spectrophotometric readings (SP) of each well were per-
formed with an automated plate reader (Multiskan MK3,
Thermo) set at 492 nm. SP MIC endpoints were defined as
the lowest GSE concentrations with OD less than or equal to
80% inhibition compared with that produced by the control
tube. Experiments were performed in triplicate.
1.4Application of GSE in preservation of grape and per-
simmon
Grape (Vitis vinifera L.) and persimmon (Dio pyris kaki
L.) were selected as the model plant materials in the preser-
vation with fungi inhibiting experiments in view that they are
easy to be infected by various fungi and led to spoilage. Grape
and persimmon were both obtained from a local farm, and
these species were most popular with consumers.
GSE was dissolved in the water with 0.05% (V/V) Tween-
80 as a surfactant to make a 100×10-6 (0.01% V/V) stock
solution and was filter-sterilized. Water was used as control.
Fresh grape and persimmon without spoilage and browning
appearance were soaked in the above solutions for 5 min
and dried in the air unt solution formed a uniform film on the
surfaces of the fruits. Treated grape and persimmon were
then put into res rvative bag with permeability to air in
which the fruits were capable to maintain natural respiration.
After drying, the treated grape and persimmon were stored
t 27±2 ℃ and 65% RH for different lengths of time. At
regular intervals the fruits were removed and assayed. The
various sensory indexes, microorganism indexes and physi-
ologi al indexes of both treated grape and persimmon and
con r l wer assayed and r corded.
2 Results and Discussion
2.1Antifungal activity of GSE with related active com-
pounds
The results of antifungal activity assays showed that
GSE had the inhibitory effects on the growth of all the tested
fungi strains while GSE antifungal activity differs slightly
with respect to the different strains in each speific concen-
tration of GSE (Table 1). However with the increase of GSE
concentrations, inhibitoty effects were clearer and inhibitoty
zones became larger gradually. At highest concentration of
GSE, the growth of all fungi strains was reduced significantly
and some strains were even inhibited completely. This proved
that the eight tested fungi were susceptible to GSE in differ-
ent degree of concentrations. The response of S ccharomy-
ces cerevisiae is the strongest, while the susceptivity of
Streptomyces griseus subsp. griseusis slightly lower.
Table 1 Mean inhibition zone sizes (mm) of GSE against 8 fungi
sfrains
Average diameter(mm)
Test fungi
0.01%0.10%1 % 10%
Botrytis cinerea9 12 26 35
Alternaria sp. 10 19 30 ＞40
Stemphylium lanuginosun13 19 33 ＞40
Aspergillus niger12 20 35 ＞40
Pen.expansum8 15 32 ＞40
Streptomyces albus subsp. albus4 10 24 38
Streptomyces griseus subsp. griseus5 11 20 33
Saccharomyces cer visiae13 21 38 ＞40
The minimum inhibitory concentration (MIC) is an
impor ant index for application of GSE in postharvest
reatment. Because the fu damental of dose is toxicity, al-
though some antimicrobial agents at high concentration ex-
hibit the strong inhibit ry effect against a wide range of
organism, these concentrations always have the harmful ef-
fects to the fruit itself and heath of consumers. So antimicro-
2008, Vol. 29, No. 10 食品科学 ※基础研究44
bial agent with high MIC is not suitable to be used in
postharvest treatment of fruit because of problem of cost and
safety. The MIC of GSE to the eight fungiis howed in Fig 1.
Fig.1 Mean minimum inhibitory concentration (MIC) of GSE
against 8 fungal species
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In conclusion, the GSE showed antifungal activities
against 8 fungal species at or above 0.01% concentration.
The values of minimal inhibitory concentration (MIC) dem-
onstrated the similar results with the data from agar diffusion
assay. And the MIC of GSE to 8 fungal species is lower than
some chemical antifungal agent such as Nisin (data not
shown). Thereby, it was suggested that the GSE may be used
as antifungal agent to protect fruits against fungal diseases.
2.2Antifungal mechanism of GSE
Microorganisms are inactivated when they are exposed
to factors that substantially alter their cellular structure or
physiological functions. Structural damage includes DNA
strand breakage, cell membrane rupture or mechanical dam-
age to cell envelope. Cell functions are altered when key
enzymes are inactivated or membrane is selectively disabled.
In contrast to bacterial cell and yeast cell, who often exist in
the style of dissociative unicellular state or have no cell wall,
the fungal cells developing from a spore, particularly mold,
form an organic whole and cling close to the surface of fruit.
Therefore the antimicrobial factors including GSE show the
different effectiveness on bacteria, fungi and yeast.
Antifungal action of GSE largely lies on its effect on the
membrane of fungi. Concretely it comes mainly from ability
of antifungal compounds in GSE to act as surfactants
(physical disruption of the membrane). They have an effect
on the fluidity and permeability of the membrane lipids by
disrupting and disorganizing the lipid bilayer-protein inter-
face nspecifically. Thus compartmentalization structures in
th f ngal cell d pending on biomembrane system and some
membrane-bound enzymes and receptors have been im-
pacted to a large extent. So the growth of fungi will be inhib-
ited even completely.
Compared with other chemical synthetic antifungal
agents, GSE showed the better inhibitory effect on the growth
of test fungi in view of synergistic effect of various antifun-
gal compounds such as limomoids, polygodial and
sesqiterpene dialdehyde. The synergistic effect is one of the
most important characteristics exhibited by natural extracts,
increasing their efficacy in contrast to those which could be
obtained with the equivalent amount of the active constitu-
ents alone.
Microorganisms have different intrinsic or natural resis-
tance to a disinfectant, but resistance can also be acquired
by genetic transfer and mutation or by exposure to sublethal
concentrations of the antimicrobial agent. The latter property
can be reversible and is often termed adaptation. For GSE, it
may hinder the developm nt of resistant mechanisms in mi-
croorganisms because of its synergistic effect.
2.3Applica ion effects of GSE in preservations of grape
and persimmon
The sensory evaluation of grape and persimmon treated
with GSE revealed significant different in color, texture, fla-
vor and ast compared with control grape and persimmon.
Fruits treated with GSE had maximum freshness, surface color,
texture, hardness and taste and were best even after 15-day
storage. There were not fungal infection, rot phenomena and
patholog cal changes among fruits treated with GSE. But the
cont o fruits were on the other hand, calyxes of most per-
simmons had fall from the fruit and wound was infected by
fungi obvious y, and pulp tissues exhibited a very soft and
collapsed texture. Almost all control persimmons lost the
edibleness. Calyxe is very mportant for the preservation of
persimmon. W und from dropping of calyxes will arouse
wound respiration, accelerate ripening and aging of fruit and
cause infection by fungi. The storage time and quality of
persimmon mainly depends on whether calyxes drop or not
(Fig.2). For the grape, the stem of grape dehydrated badly
and browned. Some berry in a bunch of grapes dropped
because of rot in calyxes. Most grapes blackened and soften
due to over-ripening d fungal infection (Fig.3). Some
hormo s, especi lly ABA, regulate the formation of abscis-
sion layer in the pos t on of calyxes in many plants. The
variation of hormones content also is important to the
45※基础研究 食品科学 2008, Vol. 29, No. 10
preservation of grape and persimmon [12].
Worth mentioning especially, stem is physiological
active parts with larger respiration intensity than berry and
is the key point of grape storage to the grape [19]. In the Fig.
4, stem of grape treated with GSE and stem of control grape
are shown when they were store at room temperature for 25
days and most berries had been picked off in order to high-
light stem. The white hypha bestrewed on the surface of
stem and berry of control grape and the stem had browned
due to fearful fungal infection. The Fig.5 is the close-up view
of the control. In contrast to berry that is non-climacteric
fruit, grapes stem has the obvious climacteric. In the course
of storage, although berry has not yet produced ETH
mutation, ETH signal from stem could be passed to berry
and lead to variation of respiration intensity in berry. So the
stem is the key point in the storage of grape. The evident
difference in grape stem between treated and control grape
exhibited strong antimicrobial and antioxidant effect of GSE.
The experiment indicated that GSE could decrease
browning and rot ratio, prolong the preservation period and
ensure the quality of grape and persimmon. The application
prospect of GSE in the preservation of grape and persimmon
is very bright, for it has good antimicrobial effectiveness and
Fig.2 Grape treated with GSE and control persimmon after
storage of 5 days at room temperature
A BC
A.Persimmon treated with GSE；B.Control persimmon without any
treatment；C.Calyxes dropping from control persimmon.
Fig.3 Grape treated with GSE and control grape after storage of
7 days at room temperature
A.Grape treated with GSE；B.Control grape without any treatment.
A B
Fig.4 Grape stem treated with GSE and its control after storage
for 27 days at room temperature
A.Grape stem treated with GSE；B.Control grape stem.
A B
Fig.5 Close-up of stem and berry of control grape after storage for
27 days at room temperature
is extracted from natural plant.
2.4Effects of GSE on postharvest physiological varia-
tions of grape and persimmon
GSE showed a good antimicrobial activity in the above
experiment. For the antimicrobial activity of GSE, firstly GSE
solution was able to form a semi-permeable coat, which can
provide protection against fungal infections. Physically, the
coat prevents the osculation between the surface of fruit
and fungi and spore existing in the storage environment.
Chemically, the antimicrobial compounds in GSE inhibit the
growth of fungi and spore in the surface of fruit. This coat
also can reduce the re piration and decrease the loss of wa-
ter from the urface and adjust the postharvest physiologi-
cal metabolism to some extent.
GSE contains a great many of phenolic compounds,
which are the main active compounds for its antioxidant
ac ivity. Abu-Amsha t al. had suggested that the higher the
total polyphenolic content, the greater is the antioxidant ca-
paci y [20]. Once these phen ic compounds diffuse from coat
into the fruit, h y will affect activity of enzymes relating to
postharvest physiological metabolism by scavenging of free
radical that leads to the oxidation of biomolecules and ripen-
ing and agi g of fruit. And the decrease of free radical will
delay activity peak of polyphenol oxidase (PPO) and peroxi-
d se (POD). Sequentially phenolic and pectic compounds in
2008, Vol. 29, No. 10 食品科学 ※基础研究46
the fruit will not degrade when enzyme activity has been
inhibited. Reflected in sense, fruits maintain former firmness,
freshness, colour and texture without browning and softening.
3 Conclusion
Because of strong antifungal activity and antioxidant
avtivity, GSE can decrease browning and rot ratio, prolong the
preservation period and ensure the quality of grape and
persimmon. So it can be used as an effective and preservative.
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