novel edible coating for tropical fruits as an alternative to synthetic fungicide

8/22/2019 Novel Edible Coating for Tropical Fruits as an Alternative to Synthetic Fungicide

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Novel Edible Coating for Tropical Fruits as anAlternative to Synthetic Fungicide

School of Biosciences

TheUniversity of Nottingham

Malaysia Campus

Dr. ASGAR A. WARSI

Asgar.Al i@not t i ngham.edu.my


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Edible coating

The potential of an edible coatings to

maintain the quality and extend shelf-life

of fresh fruits and vegetables , and

prevents microbial storage, which is

extremely important to perishable

horticultural commodities


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Coatings can extend shelf-life andmarketability

Delay ripening of the climacteric fruits

Delay color changes

Reduce weight loss

Maintain texture

Reduce decay

Simple technology

Environmentally friendly


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Materials used as edible coatings

Proteins- soy, milk, corn, wheat, casein

Carbohydrates- cellulose, pectin, starch, gum

Lipids- Waxes and oils- carnauba waxes, vegetables oils

Resin- shellac, wood rosin

Derivatives of acids and polysaccharides

Semperfresh

Chitosan


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Chitosan powder


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Chitosan is a natural biodegradable compound derived fromcrustaceous shells such as crabs and shrimps, whose main

attributes corresponds to its polycationic nature.

Chitosan has been proven to control numerous pre andpostharvest disease on various diseases on varioushorticultural commodities.

It has been reported both soil and foliar plant pathogensfungal, bacterial and viral controlled by chitosan application.

Microscopical observations indicate that chitosan has a directeffect on morphology of chitosan treated microorganisms

reflecting its fungistatic or fungicidal potential.

Ability to induce resistance by eliciting the activities ofantifungal hydrolases and total phenols..


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In addition, chitosan induces structural barriers

for example inducing the lignin material for somehorticultural and ornamental commodities.

Ability to form semi-permeable coating, chitosanextend the shelf life of treated fruits and

vegetables by minimizing the rate of respirationand reducing the water loss.

As a non toxic, biodegradable, edible and

biologically safe material, chitosan has thepotential to become a new class of plantprotectant assisting towards the goal ofsustainable agriculture.


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ANTHRACNOSE INCIDENCE, BIOCHEMICAL CHANGES,POSTHARVEST QUALITY AND GAS EXCHANGE OF

CHITOSANCOATED PAPAYA

Assess the effectiveness of chitosan in controlling postharvest

anthracnose on papaya fruit.

mechanisms involved in controlling anthracnose by chitosan.

Biochemical changes of Eksotika papaya coated with chitosan.

Effects of the coating agent on the physico-chemical characteristics of

Eksotika papaya-II.

Storage life of Eksotika papaya by treatment with a chitosan base

coating agent.

Gas exchange characteristics of chitosan-coated Eksotika-II papaya.


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Papaya

POST-HARVEST DISEASE

Anthracnose

(Alveraz, 1987)

(Paull,1997)

SHORT POSTHARVEST-LIFE

. Water loss

. Accelerated softening

CONTROL

Chemical fungicidesHot water dip

APPROACHES

CA/MALow temperature storage

CHALLENGES

Resistantpathogens

Health hazardsEnvironmentalconcerns


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COMMON SURFACE ROTS OF PAPAYA FRUITS

Anthracnose

Choclate spot

Mycosphaerella lesion

phomopsis lesion

Anthracnose, the majorpostharvest diseaseof papaya caused by

C. g lo e o s p o r io id e s

(Chau and Alvaraz,1987)


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Anthracnose Disease of Papaya

Colletotrichum gloeosporioides


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1.Effect of chitosan concentration on in vitro C.gloeosporioides development

a. Myceliel growth inhibition

b. Conidial germination test

c. Conidial morphology

2. Effect of chitosan concentration on in vivo control ofC. gloeosporioides

a. Disease incidence


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ISOLATE CULTURE FROM INFECTION

Purify by single spore culture on solid

medium (P.D.A)

Prepare medium far control (0+ 0.5% acetic acid)

and treatment (0.25, 0.5,0.75, 1, 1.25, 1.5, 1.75 and 2 % (w/v))

Inoculate 1 cm2 culture from growing

tip of pure culture

Observations on growth rate

% inhibition in radial growth calculated

Myceliel growth inhibition


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A= 0%B= 0.25%

C= 0.5%

D= 0.75%

E= 1%F= 1. 25

G=1. 5%

H=1.75%

I = 2%

A

Effect of chitosan on growth ofC. gloeosporioides on PotatoDextrose Agar (PDA)

B C

D E F

G H I


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Effect of Different Concentrations of Chitosan onMycelial Growth Inhibition After 7 days of Incubation

at 28 2C

0

10

20

30

40

50

60

70

80

90

100

MycelialgrowthInhibition(%)

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

Chitosan Concentration (%)

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

abb

ccdd

e

f

Means with same letters are not significantly different at p 0.05 using LSD


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Conidial GerminationConidial Germination

Cavity Slide Technique

Germ tube length ( Germ tube half the length of conidia)

100 conidia / treatment

Microscopic StudiesMicroscopic Studies

Mycelial growth AbnormalityConidial germination Abnormality


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20

30

40

50

60

70

80

90

100

110

1 hour 3 hour 6 hour 9 hourDuration

Germination

Inhibition(%)

0.50%

1%

1.50%

2%

Effect of different concentrations of chitosan solution in on conidialgermination ofC. gloeosporoides


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Effect of Different Concentrations of Chitosan on Conidial germinationofC.gloeosporoides spores after 7 hours of Incubation

Healthy 0 % 0.5 %

1 % 1.5 % 2 %

AppresoriaShrinkage


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Disease IncidenceDisease Incidence

DI % = Number of Infected Fruits x 100

Total Number of Fruits Assessed

Effect of chitosan coating on i n v i v ocontrol ofC.

g lo e o sp o r io id e s


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Maturity index of Eksotika Papaya

Color index Skin color1 Full green

2 Green with trace of yellow3 More green than yellow

4 More yellow than green

5 Yellow with trace of yellow

6 Fully yellow


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Isolate spores

Dilute in sterile water(Serial dilution)

Maintain required concentration (2105 /ml)

Dip healthy fruit in above concentration (spore) in control

Multiply pure culture in liquid medium


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Disease incidence

Dip in diff.conc. Of chitosan

(0,0.5, 1, 1.5 and 2% w/v)


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0.5% AA0.5% AA0.5% C0.5% C

1% C1% C 1.5% C1.5% C


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0

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8

Storage Duration (Week)

DiseaseInc

idence(%)

T1

T2

T3T4

T5

Effect of Different Concentrations of Chitosan on Disease Incidence of

Anthracnose on Papaya Fruits

T1= 0 %, T2 = 0.5%, T3 = 1%, T4 = 1.5 %, T5 = 2 %

84%

100%

15.2 %

0%


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100 % Anthracnose incidence in

control

7 % Anthracnose incidence in 1.5%chitosan treated papaya

After five weeks of Storage at 12 0 C


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EFFICACY OF CHITOSAN ON THE PRODUCTION OF INDUCIBLE

COMPOUNDS IN PAPAYA FRUITS AS INDICATOR OF THE RESISTANCE

MECHANISM

Work on natural disease resistance (NDR)has led to a remarkable awareness of the

key roles being played by some naturalcompounds in stimulating the defenseresponse in plants. Elicitors of NDR may bebiological, chemical or physical, and may

induce local acquired resistance or systemicacquired resistance


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This study was to determine the role of chitosan inthe induction of inducible compounds such total

phenols,chitinases and -1,3- glucanases activitiesin papaya fruits and their modes of action in thesuppression ofC. gloeosporioides.


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Effect of different concentrations of chitosan on total phenols during storageSampling Time (Days)

0 3 6 9 12 15

TotalPhenols(m

gg-1freshwt)

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

0 %

0.5 %

1 %

1.5 %2 %

a

a

a

b

c

c

a

b

c

d

d

a

b

c

d

d

a

a

b

c

c

a

a

b

c

c


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Chitinase activity in papaya fruits treated with different concentrationsof chitosan and challenge inoculated with C. gloeosporioides. Meanswith same letters within same week are not significantly different at P 0.05 using LSD

Sampling Time (Days)

0 3 6 9 12 15 18

Chitinase(nkatg-1freshwt)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 %

0.5 %

1 %

1.5 %

2 %

a

a

a

aaa

c

c

b

aab

a

a

b

c

c

a

a

b

c

c

a

a

b

c

c


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Glucanase activity in papaya fruits treated with different concentrationsof chitosan and challenge inoculated with C. gloeosporioides. Meanswith same letters within same week are not significantly different at P 0.05 using LSD

Sampling Time (Days)

0 3 6 9 12 15 18

GlucanaseActivity(nkatg-1freshwt)

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 %0.5 %1 %1.5 %

2 %

a

a

bb

a b

aab

b

c

c

c

c

b

a

a

a

b

c

c

a

a

b

c

c

a


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Effect of Chitosan Coating on The Physico

Chemical Properties of Coated Papaya DuringStorage

To determine the effects of coating with Chitosan basedagent on the physico-chemical characteristics of the coated

fruits after and during storage


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Chitosan was dissolved in 0.5% acetic acid and 0.1%tween 80 was added for wettebility. The pH of solutionwas adjusted to 5.6 by adding 2N NaOH .

T1 = controlT2 = 0.5% ChitosanT3 = 1% Chitosan

T4 = 1.5% ChitosanT5 = 2.0% Chitosan

Treatments:Treatments:


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Coated Papaya Fruits (1.5%)

0.5% AA 0.5% C 1% C 1.5% C

Different Concentrations of Chitosan Solution


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Effect of Chitosan Coating on % Weight Loss in PapayaFruits During Storage

0

2

4

6

8

10

12

14

2 4 6

Duration (weeks)

%weightloss

CONTROL

0.5 %

1.0%

1.5%

2.0%

a a a a a

a

b b b b

a

b

dd

c

a aa

aa

a

a

b b b b

b

c

d d


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Effect of Chitosan Coating on Firmness of Papaya Fruits

a

0

2040

60

80

100

120

140

160

Firmness(N

0 2 week 4 week 6 week

Storage Duration

Control

0.5

1

1.5

2

a a a a a a

a

aa

b

b b

c

c

dd

d

c

Means with same letters are not significantly different at p 0.05


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Effect of Chitosan Coating on Total Soluble Solids inPapaya Fruits up to Six Weeks Duration


* * Unacceptable

0

2

4

6

8

10

12

14

TSS0Brix

0 2 4 6Storage Duration

CONTROL

0.5

1

1.5

2

* *

a

a

a

a

a

a

a

b

c

c

a

a

b

b

c

a

b

c


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Effect of Chitosan Coating on Vitamin C in Papaya Fruits upto Six Weeks Duration

70

75

80

85

90

95

AscorbicAcid(mg/100g

)

0 2 4 6

Storage Duration

CONTROL

0.50%

1.00%

1.50%

2.00%

* * Unacceptable

* *

a

aa

a

a

a a

a

b

b

a

b

b b

b

bc

c

c



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Effect of Chitosan Coating on Total Acid (citric acid) inPapaya Fruits

* * Unacceptable

* *


0

0.5

1

1.5

2

2.5

3

%

TotalAcids

0 2 4 6

Storage Duration

CONTROL

0.5

11.5

2

aa

a a a a

a a

b

c

aa

b

c

b

cc

c

**


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A=control ,B=0.5%,C=1%,D=1.5% and E=2%

A B C

D E

Appearance of papaya fruits after five weeks of storage


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Quality After Five Weeks of Storage

Control

2 % Chitosan

1.5% Chitosan


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Observation of Papaya surface under SEMObservation of Papaya surface under SEM


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Scanning electron micrograph (SEMs) of pericarp surfaceof Papaya fruit. (A) show the surface of 1.5% Chitosancoated; noted Chitosan covered Overall pericarp surface(B) show surface of control fruits; Deep cracks on the skin(Arrow)

A B

Scanning electron microscope (SEM) study


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Scanning electron micrograph (SEM) showingthickness of the chitosan film (Arrow)


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Determination of modified atmosphere ofCoated Papaya Fruits

Determination of modified atmosphere ofCoated Papaya Fruits

Objectives

To determine the respiration rate in papaya fruitTo determine the respiration rate in papaya fruit

To determine the internal atmosphere in the cavity ofpapaya fruit

To determine the internal atmosphere in the cavity ofpapaya fruit


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Respiration Rate

Fruits kept in 1 liter air tight jars, sealedAnd incubated for 2 hrs at 10C

1 ml gas sample with drawn from jars using 1mlGas- tight syringe

Gas obtained was injected in to Perkin Elmerthermal conductivity detector gas chromatographequipped with Porapak R (80/100 mesh), usinghelium as carrier gas.

Fruits Incubated for 2 hrs at 10C


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Determination of the internal atmosphere in the cavity ofpapaya fruit

Determination of the internal atmosphere in the cavity ofpapaya fruit

18 Gauge needle was inserted into the cavity of papaya fruit from the stylerEnd of papaya fruit

I ml gas was with drawn and injected in to the gas chromatograph

18- GaugeHypodermicNeedle

Serum Stopper

Hypodermic syringe needle inserted intothe cavity of papaya fruit for removal ofinternal gas sample


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Sampling of Gas from Internal Cavity of Papaya

Injection of Sampled Gas in Gas chromatograph


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Respiration Rate of Coated an uncoated papaya Fruits during storage

T1 = 0 % (Control)T2 = 1 % ChitosanT3 = 1.5 % Chitosan

S torage D uration

0 2 4 6 8 10

CO2mlK

g-1hr-

1

0

5

10

15

20

25

30 T !

T 2T 3


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Storage Duration

0 2 4 6 8 10

CO2

(%)

0

1

2

3

4

5

6

7 T1

T2

T3

Gaseous Atmosphere from the cavity of Papaya Fruit

CO2% in the Cavity of Coated and uncoated papaya fruits



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Storage Duration

0 2 4 6 8 10

O2

%

4

6

8

10

12

14

16

18

20T1

T2

T3

O2 % in the Cavity of Coated and uncoated papaya fruits


O2 concentration in the cavity


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ConclusionConclusion

Chitosan markedly inhibited the growth

ofC. gloeosporioides with greater effect athigher concentration on artificial media


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Spores treated with 2 and 1.5% chitosanshowed shrinkage and shriveling and after 24hrs started to disintegrate

Maximum sporangial germination inhibitionoccurred in 2 % followed by 1.5 , 1, and 0.5 %,showing trend higher the concentration ,higherthe inhibition of spores. There was no inhibitionobserved in control.


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Papaya fruit coated with 1.5 % chitosan hadsignificantly lower disease developmentcompared to control. Fruits in control treatmentrotten completely during 3-5 weeks of storagetime and were not of marketable value.

It is believe that more than 80% ofanthracnose on papaya fruit achieved with

chitosan is sufficiently adequate to considerchitosan as a natural product to controlanthracnose disease.


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SEMs showed that 1.5% chitosan films coveredoverall surface of the papaya fruits. The films could

be retained well up to the end of the storage. whileon the surface of control fruits, deeps cracks wereobserved.

1.5 and 2% chitosan coating significantly retardedthe weight loss Of papaya fruits. Chitosan filmformed on the surface of the fruit delayed migration

of moisture from the fruit into environment


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The total acidity (citric acid) was dependent on

chitosan concentration and storage duration. Therate of citric acid decreases was lower withchitosan concentration except 2%

Carbon dioxide evolution of chitosan coated fruitdecreased gradually during storage and wassignificantly lower in levels than that of non coated

fruits


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Chitosan coating also raised the internal CO2and decreased the internal O2 level with in thefruits, the greater effect was found at 1.5%


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Chitinase, Glucanase and total phenols activitiesof papaya fruits increased during storage. It showedthat chitosan is able induce resistance against

anthracnose disease during storage.

Chitosan coating delayed rate of ripening asindicated by the colour of the fruits.


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0% 1% 1.5%

1%0% 1.5%

Papaya fruits at Ambient Temperature After10 days


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Prospects

Extension of storage upto five weeks wouldfacilitate the export of fruits to longdistance markets by sea and thereby costof export would be reducing making thefruits more competitive in the world

market.


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These effects have been attributed to its direct

antifungal activity, induction of postharvestresistance responses and creation of modifiedatmosphere in papaya fruits.

NutshellNutshell


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Current research

Project: Screening of antagonistic bacteria toenhance the efficacy of chitosan to controlanthracnose disease of papaya.

Optimization of the chitosan coating during

transportation.

Th k f tt ti


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Thank you for your attentio

Have a fruitful day


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Any Queries:

[email protected]

novel edible coating for tropical fruits as an alternative to synthetic fungicide

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