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Effect of Chitosan Coating from Tiger Prawn (Penaeus monodon) Shells on the Shelf

life of Fresh Lanzones (Lansium domesticum Correa)


Introduction

Background of the Study

In seafood industries, shellfish waste management is a huge problem

especially the crustacean sector which lacks cost effective outlets for their waste (Raja et

al 2012). About 45% of processed seafood consists of shrimp, the waste of which

is composed of exoskeleton and cephalothorax (Gortari & Hours 2013).Every year,

some 6 million to 8 million tons of waste crab, shrimp and lobster shells are produced

globally — about 1.5 million tons in South East Asia alone. In developing countries,

waste shells are often just dumped in landfill or the sea. In developed countries, disposal

can be costly (Nature, 2015).

One of the most abundant natural seafood resources in the Philippines is the

province of Roxas City, the Seafood Capital of the Philippines. Chitin or its de-acetylated

derivative chitosan is a valuable product with a number of applications, is one of the main

components of shrimp shells, a waste product of the seafood industry (Microb Cell Fact.

2013).A chitosan coating retarded water loss and the drop in sensory quality, increasing

the soluble solid content, titratable acidity and ascorbic acid content. It also inhibited the

growth of microorganisms. Previous studies reveal that applying a chitosan coating

effectively prolongs the quality attributes and extends the shelf life of particular kinds of

fruits (Journal of Food Engineering, 2007). The coating of chitosan can modify the

internal atmosphere, decrease transpiration, delay ripening fruit while increasing the shelf

life of fruits and vegetables. After applying improved chitosan-based coating, the

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preserving effects were increased in most of the cases compared with single chitosan

coating (RJPBCS, 2014).

This prompted the researcher to conduct a study entitled Effect of Chitosan Coating

from Tiger Prawn (Penaeus monodon) shells on the Shelf life of Fresh Lanzones

(Lansium domesticum Correa) fruits.

2
Statement of the Problem

General Objective

Generally, this study aims to determine if Chitosan coating from Tiger Prawn

(Penaeus monodon) shells can be utilized to improve the shelf life of fresh Lanzones

(Lansium domesticum Correa) fruits.

Specific Objective

To determine the significant difference in the shelf life of Lanzones (Lansium

domesticum Correa) fruits after applying the different treatments of Chitosan Coating

from Tiger Prawn (Penaeus monodon) shells.

Hypothesis

There is no significant difference in the shelf life of Lanzones (Lansium domesticum

Correa) fruits after applying the different treatments of Chitosan Coating from Tiger

Prawn (Penaeus monodon) shells.

3
Conceptual Framework

Independent Variables Dependent

Treatments Variable

Treatment A - 10 mL

chitosan + 15 mL acetic
c
acid

Coating
Treatment B - 15 mL
Number of Days
chitosan + 15 mL acetic
to Rot
acid

Treatment C – 20 mL

chitosan + 15 mL acetic

acid

Figure 1. Number of Days to Rot after Coating the Lanzones (Lansium domesticum
Correa) Fruits with Different Treatments

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Significance of the Study

a. Public. This study will incite the public of alternative solutions to lessen the

demand on artificial shelf life extenders.

b. Fruit Vendors. The desired outcome of this study will be beneficial to the

people who depend on selling fruits as their livelihood.

c. Communities. To support investigations concerning possible health hazards

from artificial extenders for the benefit of general consumption in communities.

d. Department of Agriculture. This study was conducted to improve and in the

promotion of better agricultural development and growth in our country.

e. Farmers and Orchard Owners. This study will helped alleviate the hardship of

availing costly and inefficient artificial extenders that delay shipment of goods to

different markets such as fruits.

Scope and Limitation of the Study

This study was only limited in the use of chitosan coating from tiger prawn

(Penaeus monodon) shells on Lanzones (Lansium domesticum Correa) fruits as a shelf

life extender that were tested for seven (7) days. There were three treatments and a

negative control done in 3 replications: Treatment A – 10 mL chitosan + 15 mL acetic

acid; Treatment B - 15 mL chitosan + 15mL acetic acid; Treatment C - 20 mL chitosan

coating + 15 mL acetic acid; and untreated Lanzones (Lansium domesticum Correa)

fruits as the negative control. The Lanzones (Lansium domesticum Correa) fruits were

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evaluated and checked after applying the different treatments to determine the significant

difference after eight (8) days of observation.

The study was conducted under the supervision of Mrs. Genevieve A. Aponte at

Capiz National High School.

The mean difference was computed. Two-Way Analysis of Variance (ANOVA)

was computed to determine the significant difference among the treatments.

Definition of Terms

Acetate – (Ethanoate) salt or ester of ethanoic acid (acetic acid). The salts are formed by

reacting ethanoic acid with a base, such as a metal hydroxide; the esters by reacting the

acid with an alcohol. (Microsoft Encarta Premium Suite 2005). In this study, acetic acid

is one of the independent variables.

Chitosan – This term means the de-acetylated form of chitin, a polysaccharide found in

exoskeleton of shellfish like shrimp and crabs (Block, 2004). In this study, chitosan is

used to prolong shelf life of Lanzones.

Chitosan coating - extends the shelf life of the fruits and vegetables by minimizing the

rate of respiration and reducing the water loss. Chitosan coating offers a defensive barrier

against bacterial contamination and loss of moisture from the surface of food products,

thus extending their shelf life. With limited increase in the concentration of chitosan

coating, the beneficial effect of chitosan on postharvest life and quality of the food is

enhanced (Block, 2004). In this study, it is used to coat the Lanzones fruits.

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Chitosan Extraction – process used to determine if there is the presence of polymer chitin

or chitosan in a material or substance. In this study, chitosan extraction is used to produce

chitosan from the shrimp shells.

Hydrochloric Acid – this term refers to a Hydrogen Chloride, colorless, corrosive,

nonflammable gas, formula HCl, with a characteristic penetrating, suffocating odor

(Microsoft Encarta Reference Library 2004). In this study, it is used to liquefy proteins

and produce chitosan.

Lye – solid, white material that readily absorbs moisture. It produces extremely corrosive

solutions (World Book 2004). In this study, the processed chitin is boiled in lye to get rid

of the acetate and produced chitosan.

Prawn Shells – is about 2 cm (0.79 in) long, but some shrimp exceeding 25 cm (9.8 in).

Most commonly has an orange transparent shell covering its body (Journal of

Experimental Biology 2004). In this study, shrimp shells are used to produce chitosan.

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Review of Related Literature and Studies

This chapter is made up of three (3) topics, namely: (1) Problems in Preservation,

(2) Lanzones (Lansium domesticum Correa), (3) Tiger prawn (Penaeus monodon) and (4)

Synthesis.

The first topic, Problems in Preservation, discusses the primary complication in

fruit and vegetable preservation within the country.

The second topic, Lanzones (Lansium domesticum Correa), discusses in detail its

description, medicinal properties, and factors involved in its deterioration.

The third topic, Tiger prawn (Penaeus monodon), particularly discusses the

description and the polymers present in the crustacean’s shell.

The fourth topic, Synthesis, provides a summary of all literatures reviewed.

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Problems in Preservation

The problems confronted in food preservation are not simple, for a whole set of

biological factors, prior to the start of the chosen preservation method, can later affect

losses that occur during treatment, storage, conditioning, transport, and distribution.

The most common destructive agents are rodents, insects, and microorganisms

(fungi and bacteria). Man himself, through ignorance and negligence, often handles and

distributes foodstuffs properly, creating conditions which promote physical, chemical or

microbiological changes that make them unsatisfactory consumption.

Although some food may be damaged by chemical changes such as oxidation,

most food is destroyed in storage by spoilage cause by living organisms such as molds,

bacteria, and yeast. Food preservation techniques, therefore, depend upon killing or

inhibiting the growth of these microorganisms (KSHS 2012).

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Lanzones (Lansuimdomesticum Correa)

Figure 2. Lanzones
(Lansuim domesticum Correa)
Photo taken from www.google.com

The lanzones, lansuim, or langsat is not just a delicious treat, but is also quite

healthy. Lanzones are well-known in Southeast Asia. It is recognizable with its yellow to

light brown peel, oval-shaped fruit that grows in clusters. You can cook the fruit, but it is

more common for the locals to consume it raw.

Lanzones are abundant in antioxidants, including vitamin A.This vitamin has a

ton of health benefits, including protecting your eye against macular degeneration and

other vision problems. Lanzones also have other antioxidants that may be able to prevent

health problems. The other antioxidants in the fruit are helpful in keeping the body clean

against free radicals that quicken the rate of aging, but also cause serious illnesses.

Splitting the peel, starts at the end and extends to the stem. This exposes the flesh

to rotting due to the presence of different microorganisms (PCAARRD, 2009).

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Figure 3. Tiger Prawn
(Penaeus monodon)
Photo taken from www.google.com

Tiger prawns have a typical prawn body plan including a head, tail, five pairs of

swimming legs (pleopods) and five pairs of walking legs (pereopods), as well as

numerous head appendages. A carapace (hard exoskeleton) encloses the cephalothorax. A

posterior ridge called the adrostral carina extends from the rostrum to the edge of the

epigastric spine, which reaches to the posterior end of the carapace. (State of New South

Wales through Department of Industry and Investment, 2010). Tiger prawns are

identified by distinct black and white stripes on their backs and tails; on their abdomens,

these stripes alternate black/yellow or blue/yellow. Base body color varies from green,

brown, red, grey, or blue. These prawns are very large, reaching 330 mm or greater in

length and are sexually dimorphic, with females are larger than males. (Dall, et al., 1991;

Environmental Defense Fund, 2011)

The team of researchers discovered a polymer called chitosan, found naturally in

prawn shells, can be used in fruit preservation, shower gels instead of man-made

chemicals(FAO Fisheries and Aquaculture Department, 2012). Chitosan is a chemically

processed form of chitin, a cellulose-like biopolymer found mainly in the exoskeleton of

marine animals such as shrimp, crabs, or lobsters. Chitosan-based coating was concerned

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in recent years owing to its non-toxic, biodegradable, and biocompatible properties (J

Food Process Technol, 2013).

Synthesis

Most fruits and vegetables go bad because of damaged caused by microorganisms

such as bacteria, molds, enzymes or bruising. Microorganisms such as bacteria and molds

release their own enzymes as they grow, produces deterioration through structural decay,

speeding up the spoiling process. Enzymes, which occur naturally in live fruits and

vegetables, are part of the natural aging process. Enzymatic browning leads to

discoloration and, later, spoilage. Bruising alters the exterior of your fruits and

vegetables, which trigger enzymatic reactions (Livestrong, 2015).

Improper shellfish waste management is a huge problem especially the crustacean

sector which lacks cost effective outlets for their waste (Raja et al, 2012). About 45% of

which, consists of shrimp, the waste of which is composed of exoskeleton ad

cephalothorax (Gortari & Hours, 2013). This rapid accumulation of seafood wastes, and

unless acted upon maybe the cause of many diseases and outbreaks throughout the years

such as skin problems, respiratory diseases, or infections. Huge losses of fruits and

vegetables are a matter of grave concern for any country. But this is a general

phenomenon happening in almost every developing country.

Chitosan is one of the main components of shrimp shells, one of the biggest waste

products of the seafood industry (Microb Cell Fact., 2013). A chitosan coating retards

water loss and the drop in sensory quality, increasing the soluble solid content. It also

inhibited the growth of microorganisms. Previous studies reveal that applying a chitosan

12
coating effectively prolongs the quality attributes and extends the shelf life or particular

kinds of fruits (Journal of Food Engineering, 2007).

The study will utilize the cellulose-like biopolymer, chitosan from the shells of

Tiger Prawns (Penaeus monodon), as to help extend the shelf life of Lanzones (Lansium

domesticum Correa). The study will use a modified shelf life test in order to find out a if

there is a significant difference in the shelf life of Lanzones (Lansium domesticum

Correa) fruits after applying the different treatments of Chitosan Coating from Tiger

Prawn (Penaues monodon) shells.

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Materials and Methods

Materials

The materials used in the study were 300 grams of prawn shells, 500 mL

hydrochloric acid, 1500 mL distilled water, 700 grams of lye, 100 mL of acetic acid.

Tools

The tools used in the study were 5 pieces of beakers, plastic basin, mortar and

pestle, platform balance, microgram balance, Erlenmeyer flask, stirring rod, plastic

spoon, 3 plastic containers, strainer, paper and pen. Laboratory gowns, masks and gloves

were also used by the researcher to ensure safety.

Figure 4. Materials and Tools


Photo taken by the Researcher

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Experimental Design

The Completely Randomized Design (CRD) was employed. There were 3

treatments, a positive and negative control all done in 3 replications.

Treatments Replications
1 2Replicatio 3
Treatment A ns

10 mL Chitosan + 15 mL

Acetic acid

Treatment B

15 mL Chitosan + 15 mL

Acetic acid

Treatment C

20 mL Chitosan + 15 mL

Acetic acid

Negative Control

Untreated

Figure 5. Treatments and Replicates

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Procedural Design
Gathering and Preparation of Raw Materials

Cleaning and pulverizing of Tiger Prawn shells

Chitin extraction

Chitosan extraction

Preparation of different treatments

Treatment A Treatment B Treatment C Negative


- 10 mL - 15 mL - 20 mL Control -
chitosan + chitosan + chitosan + Untreated
15 mL 15 mL 15 mL
acetic acid acetic acid acetic acid

Coating the Lanzones with different treatments of


Chitosan coating

Shelf Life Test

Gathering of Data

Statistical Tool

Figure 6. Procedural Design

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General Procedure

Gathering of Materials

Figure 7. Gathering of
Materials
Photo taken by the Researcher

Tiger prawn shells were gathered from Teodoro Arcenas Trade Center (TATC),

Rozas City, Capiz. The prawn shells were cleaned and air dried for 5 days. The prawn

shells were pulverized using mortar and pestle.

Chitosan Extraction

Figure 8. Chitosan Extraction


Photo taken by the Researcher

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Using a grammer 150g of shells were measured and placed in a clean container and was

mixed with 400 mL of 20% NaOH solution and was soaked for 24 hours.

After 24 hours, the mixture was washed and drained using a strainer. The residue

was placed to a clean bottle and added with 50 mL HCl each. It was soaked for 4 hours.

After soaking, the mixture was washed and placed in a clean container. 400 mL of

20% NaOH solution was added and soaked for 2-3 hours. The procedure was repeated

until the shells as soft enough to form a paste-like texture (chitin).

The chitin is again washed and drained thoroughly and were added 300 mL of

40% NaOH solution lye to remove acetate from the molecule. Acid is consumed in the

reaction and the caustic solution is recovered, producing chitosan.

Preparing the Different Treatments

Figure 9. Different Treatments


Photo taken by the Researcher

The chitosanis separately weighed using a grammer. Treatment A- 10mL chitosan

and 15mLacetic acid; Treatment B- 15mL chitosan and 15mL acetic acid; Treatment C-

5g chitosan and 15mL acetic acid and aNegative Control- untreatedthat was labeled

T1/T2/T3/Control.

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Coating of Fruits

Figure 10. Application of Figure 11. Drying of Fruits


Treatments Photo taken by the
Photo taken by the Researcher Researcher

The fruits were first cleaned using a cloth. The different treatments of chitosan

coating were applied to the Lanzones fruits and then left to dry for 30 minutes. The

treated fruits were placed in a properly-labeled open container.

Evaluation of Lanzones (Lansium domesticum Correa) fruits


The fruits were evaluated every day until it reaches its rotting stage. The number
of days to rot was recorded.

Gathering of Data

Each treatment was opened and let out of their individual containers, the

Lanzones fruits were each evaluated and checked, and were returned back to their

respective containers after. The method was repeated for another 7 days, evaluated after

every 24 hours. Every day for eight days, each treatment was observed to determine total

duration of the shelf lifeof each coated fruit from each treatment.

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Statistical Tool

The mean difference was computed. One-Way Analysis of Variance (ANOVA)

was used to determine the significant difference among treatments.

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Results

Table 1. Results of Shelf Life Test on the total duration of the shelf life of
Lanzones (Lansium domesticum Correa) fruits (g) after Chitosan coated
with Varying Treatments of Chitosan from Tiger Prawn (Penaeus
monodon) shells.

Days
Treatments 1 2 3 4 5 6 7 8
No. of Lanzones Mean
A 3 3 3 3 3 3 2.3 1 2.7a
B 3 3 3 3 3 2.7 2 1 2.6a
C 3 3 3 3 2.7 2.3 1 0 2.3b
NC 3 3 2.7 1.7 0.3 0 0 0 1.3c
Mean 3a 3a 2.9a 2.7a 2.3b 2b 1.3c 0.5c

Table above shows thatTreatment A got the highest mean difference of 2.7

followed by Treatment B with a mean difference of 2.6 and Treatment C with a mean

difference of 2.3. The negative control got the lowest mean difference of 1.3.

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Table 2. ANOVA on the number of Days for Lanzones (Lansium domesticum
Correa) to Rot after Applying the Different Treatments

Factor 2
Means:

TA TB TC Control
Day 1 3.0 3.0 3.0 3.0 3.0
Day 2 3.0 3.0 3.0 3.0 3.0
Day 3 3.0 3.0 3.0 2.7 2.9
Factor 1 Day 4 3.0 3.0 3.0 1.7 2.7
Day 5 3.0 3.0 2.7 0.3 2.3
Day 6 3.0 2.7 2.3 0.0 2.0
Day 7 2.3 2.0 1.0 0.0 1.3
Day 8 1.0 1.0 0.0 0.0 0.5
2.7 2.6 2.3 1.3 2.2

3 replications per cell

ANOVA
table
Source SS df MS F p-value
Factor 1 68.33 7 9.762 42.60 1.02E-21

Factor 2 26.83 3 8.944 39.03 1.84E-14

Interaction 22.00 21 1.048 4.57 1.28E-06

Error 14.67 64 0.229


Total 131.83 95

Post hoc analysis

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p-values for pairwise t-tests for
Factor 1
Day 8 Day 7 Day 6 Day 5 Day 4 Day 3 Day 1 Day 2

0.5 1.3 2.0 2.3 2.7 2.9 3.0 3.0


Day 8 0.5
Day 7 1.3 .0001
Day 6 2.0 1.21E-10 .0011
Day 5 2.3 6.79E-13 1.48E-05 .2054
Day 4 2.7 1.55E-16 3.81E-09 .0011 .0369
Day 3 2.9 1.28E-18 2.13E-11 1.48E-05 .0011 .2054
Day 1 3.0 2.71E-19 3.79E-12 3.05E-06 .0003 .0929 .6712
Day 2 3.0 2.71E-19 3.79E-12 3.05E-06 .0003 .0929 .6712 1.0000

Post hoc analysis

p-values for pairwise t-tests for


Factor 2
Control TC TB TA

1.3 2.3 2.6 2.7


Control 1.3
TC 2.3 8.15E-09
TB 2.6 4.71E-13 .0187
TA 2.7 4.24E-14 .0037 .5486

Data showed that factor 1 (No. of Days) has a p-value of 1.02E-21 which is less

than the 5% level of significance. Furthermore, Post hoc analysis revealed that day 1, 2, 3

and 4 is comparable to each other showing no significant difference. Day 6 and 5 are also

comparable, while day 7 and 8 are significantly different among all the days.

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On factor 2 (Different Treatments), the p-value is 1.84E-14, which is also less

than 5% level of significance thus all treatments are significantly different with the

control. Moreover, Treatment A and Treatment B is comparable..

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Discussion

As the ripening process of Lanzones proceeds, there was a decrease in the number

of fruits per treatment at different days of shelf lifeobserved. Results revealed that

Treatment A had the longest duration of shelf life among all the other treatments due to

the balanced concentration of chitosan and acetic acid in the treatment.

Most fruits and vegetables go bad because of damage caused by microorganisms

such as bacteria and mold, enzymatic processes or bruising. Microorganisms speed

produce deterioration through structural decay (Livestrong, 2015). Chitosan has strong

antimicrobial and antifungal activities that could effectively control the growth of

microorganisms that causes fruit decay (RJPBCS, 2014).

Similar to the cellulose, chitosan is a fiber that possesses unique properties

including the ability to form films. This polycationic polymer has specific structure and

properties that contains more than 5000 glucosamine units. Because of positive ionic

charge on chitosan, it has the ability to bind chemically with negatively charged fats,

lipids and bile acids (Sandford, 1992).

Chitosan present in the Tiger Prawn (Penaeus monodon) shells extends the shelf

life of the fruits and vegetables by minimizing the rate of respiration and reducing the

water loss (Block, 2004).Chitosan-based coatings are considered the best edible and

biologically safe preservative coatings for different types of fruits, with functional

advantages, such as slower respiration rates, extended storage periods, firmness retention

and controlled microbial growth (Foods, 2015).

One other factor involved in maintaining the structure of fruits is chitosan coating

contained calcium which demonstrated the best results, probably because calcium may

25
interact with pectic acid in cell walls to form calcium pectate, a compound helpful for

maintaining structure of the fruit (Rolle & Chism, 1987).

Conclusion

1. There is a significant difference in the shelf life of Lanzones (Lansium


domesticum Correa) fruits after applying the different treatments of Chitosan
Coating from Tiger Prawn (Penaeus monodon) shells.

Recommendations

1. Application of Chitosan from Tiger Prawn (Penaeus monodon) shells to lengthen

the shelf life of a specific range of fruits.

2. Development of other usage of the chitosan used in the study.

3. Application of other methods in determining the significant difference.

4. Further study that may enhance the experiment is encouraged.

26
References
De la Cruz, J., (2005). Utilization of Bangus (Chanoschanos) Scales as a Source of
Chitin.
Retrieved last August 1, 2016, from the Science LRC of3
Capiz National High School.

De la Cruz, J.M., (2007). Chitosan: Anticholesterol Agent from Mud Snails


(CerithediaCingulata) Shells in Mice
Retrieved last August 3, 2016, from the Science LRC of
Capiz National High School.

Research Journal of Pharmaceutical, Biological and Chemical Sciences (RJPBCS),


(1999). "Development of Low Molecular Chitosan and Its Silver Nanocomposites for the
Edible Fruits Coating to Improve the Shelf Life Period.". Retrieved last August 2, 2016,
from http://www.RJPBCS.com/chitosan coating.html

J Food Process Technol,(2013).“Application of Chitosan Based Coating in Fruit and


VegetablePreservation: A Review”. Retrieved last August 2, 2016, from
http://dx.doi.org/10.4172/2157-7110.1000227

Lanzones, (2011).Philippine Medicinal Plants.


Retrieved last August 2, 2016, from
http://www.stuartxchange.org/Kolitis

Sheikh, R., (2013)."Review: Chitosan as a Novel Edible Coating for Fresh Fruits ".
Retrieved last August 3, 2016 from https://www.researchgate.net/publication/237080941

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Appendices

28
Appendix A
Raw Data

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Table 1. Total Number of Lanzones (Lansium domesticum Correa) in Each
Replicate of Each Treatment during the Eight (8) Days of Observation

Lanzones

Shelf life (days)


Treatments
1 2 3 4 5 6 7 8

R1 3 3 3 3 3 3 3 2
A R2 3 3 3 3 3 3 2 1
R3 3 3 3 3 3 3 2 0
R1 3 3 3 3 3 3 3 2
B R2 3 3 3 3 3 3 2 0
R3 3 3 3 3 3 2 1 1
R1 3 3 3 3 3 3 1 0
C R2 3 3 3 3 2 2 1 0
R3 3 3 3 3 3 2 1 0
R1 3 3 3 3 1 0 0 0
NCR2 3 3 3 2 0 0 0 0
R3 3 3 2 0 0 0 0 0

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Appendix B
Statistical Tool

31
Null Hypothesis (Ho):

There is no significant difference in the shelf life of Lanzones (Lansium

domesticum Correa) fruits after applying the different treatments of Chitosan Coating

from Tiger Prawn (Penaeus monodon) shells.

Alternative Hypothesis (Ha):

There is a significant difference in the shelf life of Lanzones (Lansium

domesticum Correa) fruits after applying the different treatments of Chitosan Coating

from Tiger Prawn (Penaeus monodon) shells.

Decision:

There is a significant difference in the shelf life of Lanzones (Lansium

domesticum Correa) fruits after applying the different treatments of Chitosan Coating

from Tiger Prawn (Penaeus monodon) shells.

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Table 2. ANOVA on the number of Days for Lanzones (Lansium domesticum
Correa) to Rot after Applying the Different Treatments

Two factor ANOVA

Factor 2
Means:

TA TB TC Control
Row 1 3.0 3.0 3.0 3.0 3.0
Row 2 3.0 3.0 3.0 3.0 3.0
Row 3 3.0 3.0 3.0 2.7 2.9
Factor 1 Row 4 3.0 3.0 3.0 1.7 2.7
Row 5 3.0 3.0 2.7 0.3 2.3
Row 6 3.0 2.7 2.3 0.0 2.0
Row 7 2.3 2.0 1.0 0.0 1.3
Row 8 1.0 1.0 0.0 0.0 0.5
2.7 2.6 2.3 1.3 2.2

3 replications per cell

ANOVA
table
Source SS df MS F p-value
Factor 1 68.33 7 9.762 42.60 1.02E-21

Factor 2 26.83 3 8.944 39.03 1.84E-14

Interaction 22.00 21 1.048 4.57 1.28E-06

Error 14.67 64 0.229


Total 131.83 95

Post hoc analysis

p-values for pairwise t-tests for Factor 1

Row 8 Row 7 Row 6 Row 5 Row 4 Row 3 Row 1 Row 2

0.5 1.3 2.0 2.3 2.7 2.9 3.0 3.0


Row 8 0.5
Row 7 1.3 .0001
Row 6 2.0 1.21E-10 .0011
Row 5 2.3 6.79E-13 1.48E-05 .2054
Row 4 2.7 1.55E-16 3.81E-09 .0011 .0369
Row 3 2.9 1.28E-18 2.13E-11 1.48E-05 .0011 .2054
Row 1 3.0 2.71E-19 3.79E-12 3.05E-06 .0003 .0929 .6712
Row 2 3.0 2.71E-19 3.79E-12 3.05E-06 .0003 .0929 .6712 1.0000

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Post hoc analysis

p-values for pairwise t-tests for Factor 2

Control TC TB TA

1.3 2.3 2.6 2.7


Control 1.3
TC 2.3 8.15E-09
TB 2.6 4.71E-13 .0187
TA 2.7 4.24E-14 .0037 .5486

Interaction Plot by Factor 2


3.5

3.0

2.5
Cell Mean

2.0

1.5

1.0

0.5

0.0
TA TB TC Control

Row 1 Row 2 Row 3 Row 4 Row 5 Row 6 Row 7 Row 8

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Appendix C
Letters and Communications

35

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