Dragon Fruit (Hylocereus undatus) peel extract as an

Alternative component for whiteboard marker ink

Clarence Gio S. Almoite, Gospel Amos R. Azul and Wigan C. Pilien

A. Research Questions

 Is dragon fruit an effective white board marker ink?

 What is the difference of using the dragon fruit peel over using commercially
available ink?
 Is it cheaper to make and is it efficient to use dragon fruit peel?

B.
Fruits and vegetables are in-demand in the market because of its nutritional
value and importance in our daily diet. However, fruits and vegetables are perishable
due to their short shelf life. About 30% of fruits and vegetables are affected or damaged
by insects, microorganisms, pre and post harvesting conditions during transport and
preservation (Vidhyapeeth, 2016). The environmental impact caused by the excessive
quantity of non-degradable waste materials are promoting research and efforts to
develop new biodegradable and consumable packing materials that can be
manufactured with the utilization of environment-friendly raw materials (Avérous et al.,
2001).
Minimum safe low temperature and high relative humidity control are the most
important tools for extending the shelf life in Fruits (Cabezas et al., 1997). Although,
cold storage could extend the shelf life, and help prevent shrinkage and water loss,
extreme cold temperature could result into chilling injury which may cause damage to
plant parts (Yi Wang et al., 1997). Ice crystals formed inside the plant tissues, dehydrate
cells and disrupt membranes that causes the plant to deteriorate faster and die (Ding et
al., 2002).
Edible coatings in post-harvest handling have been particularly considered in
food preservation, because of their capability for improving global food quality (Chillo et
al., 2008). An edible coating or film has been defined as a thin, continuous layer of
edible material formed or placed on or between foods or food components (Bravin et al.,
2006). Edible films can act as mechanical protection, moisture and gas barriers and, at
the same time, can preserve the color, texture and moisture of the coated products.
One of the polysaccharides that can be used to develop edible coatings is chitosan,
which is a biopolymer derived by deacetylation of chitin, a major component of the
shells of crustacean such as crab, shrimp, and crawfish (No et al., 2002).
 Sitsaro, (Pisum sativum L.) and Baguio Beans (Phaseolus vulgaris L.) are grown
extensively in tropical areas and is harvested for its green pods and seeds. It is also
adapted and commercially grown very well in Benguet where the climate is cold
throughout the year. Both crops are considered as high demanded and a money-
making vegetable in the Philippines (Swiader & Ware, 2002). However, Baguio beans
and Sitsaro are susceptible to chilling injury which happens when exposed in extreme
cold temperature and tend to deteriorate faster (Edwin, 2003; Lukatkin et al., 2012)

C. Hypothesis/ Engineering Goals

 The chitosan coating extracted from crab shell wastes has the ability to maintain
the quality of selected bean pods in cold conditions.

 There are significant effects and differences in the initial and final mass of
selected bean pods after applying chitosan coating and storing in low
temperature.

 There’s a significant effect between the increasing concentration of chitosan

coating applied on selected bean pods in its physiological weight loss

D. Materials/Procedures

1. Collection and Preparation of Crab Shell Waste

Crab shell waste will be collected from Farmer’s Market, Cubao. Samples will be
kept chilled in an ice box during transportation to the laboratory. The crab shell wastes
will be cleaned and placed in polyethylene bags because they provide good resistance
against organic solvents, degreasing agents and electrolytic attack. It will be refrigerated
at −20 °C until used.

2. Extraction of chitosan
2.1. Deproteinization
The crab shell wastes will be treated with 4.0 % of Potassium hydroxide (KOH)
solution, with a ratio of ground shell to the solution of 1:20 (w/v), with constant stirring
for 2 hours at 90 °C to remove the protein. The samples will be filtered using a filter
paper and the filtrates will be washed with tap water for 30 minutes until it became
neutral (pH 7). The deproteinized shells will be dried in the oven at 60°C for 24 hours
(Shahidi & Synowiecki, 1991).

2.2. Protein Determination

To determine the protein after deproteinization, shells will be crushed and thoroughly
mixed with 15mL of deionized water. The mixture will bepressed in a clean piece of
fabric and the liquid will be subjected to Biuret test. Two (2) mL of Sodium hydroxide
(NaOH) solution and 1% Copper (II) sulphate solution will be dropped to the liquid
sample. The solution will be placed in a room with ambient temperature for 7 minutes.
If the color of the solution stays at blue, it indicates no proteins are present, if blue turns
to violet, proteins are present, if blue turns to pink, there is a presence of peptides. To
observe the color change, the albumin of a chicken egg will be used as a control.

2.2. Demineralization
Deproteinized crab shell wastes will be demineralized with 2.5 % (w/v) Hydrogen
chloride (HCl), at room temperature (20°C) for 6 hours to remove the mineral content
with a ratio of ground shell to the solution of 1:20 (w/v). The samples will be filtered
using a filter paper and washed for 30 minutes with tap water until it became neutral (pH
7). The demineralized shells will be dried in the oven at 60°C for 24 hrs (Shahidi &
Synowiecki 1991).

2.3. Decoloration and Dewatering

Decoloration will be achieved by treating the samples with acetone for 10 mins
and dried for 2 hours at ambient temperature and the resulting residues will be
removed. The decolorized shells will be washed in running tap water, rinsed, filtered
and dried at 60 °C for 24 hours in the oven to obtain crab chitin (Shahidi & Synowiecki
1991).

2.4. Deacetylation of Chitin

The deacetylation of chitin will be conducted according to the method by Yen et
al. (2009). The chitin obtained will be treated with 40% (w/v) aqueous Potassium
hydroxide (KOH), with a ratio of chitin to the solution of 1:15 (w/v) at 105°C for 2 hours.
Then, the chitin will be filtered using a filter paper and washed with deionized water until
pH neutral (pH, 7) was achieved. The chitosan obtained will be dried at 60°C for 24
hours in the oven.

3. Preparation of Pure chitosan solution

The chitosan solutions will be prepared according to El Ghaouth et al. (1992). An
amount of 20 g chitosan will be dispersed in 900 mL of distilled water to which 50 mL of
glacial acetic acid will be added to dissolve chitosan. The solutions will be centrifuged to
remove undissolved particle. In order to guarantee the stability of the emulsions,
Polysorbate or tween 80 (0.1% v/v) will be added to the solutions to improve wettability
of the solution during coating. (El Ghaouth et al., 1992; Abebe et al., 2017).

4. Preparation of chitosan solution with different concentrations

 Pure chitosan solution will be divided into six (6) different concentrations of 5%,
10%, 15%, 20%, 25 % and 100 %, while controlled set-up will be stored without adding
chitosan.

4. Collection of Sitsaro and Baguio Beans

Sitsaro and Baguio Beans will collected from Farmer’s Market, Cubao. Samples
will be placed in a polyethylene bags to prevent massive exchange of gases. The
beans will be carefully selected to be uniform in appearance (weight and color).

5. Application of Coating material

Sitsaro and Baguio Beans will be sorted in seven (7) different set-ups with five
(5) replicates per set-up and each set- ups will be uniformly dipped for two (2) minutes
in the solution of varying concentrations. Meanwhile, control beans will not be dipped in
the concentration of chitosan solution. The coated vegetables will be placed in a plate
then store in the freezer for 24 hours at 5°C temperature. The stored vegetables will be
weighed and observed its physiological weight loss.

6. Determination of Physiological Weight Loss

After 24-hour cold storage, the weight losses of vegetables will be recorded
(Athmaselvi et al., 2013).

where Mass loss (%) = percentage physiological weight loss, IW=initial weight in
g, and FW-final weight in g at the indicated period.

7. Statistical Analysis
Comparison between groups of different concentrations of chitosan solution and
control group will be analyzed according to the physiological weight loss of Sitsaro (P.
sativum) and Baguio Beans (P. vulgaris). The effects of different treatments will be
compared using Analysis of Variance (ANOVA). Paired sample T-test were done to
compare the effects of different concentrations and the control using Graph Pad
Software.

Table 1: The table for Percentage Weight Loss of Baguo Beans (P. vulgaris) treated
with various concentrations of Chitosan solution after storing in low temperature

Baguio beans (Phaseolus vulgaris )

1 2 3 4 5 AVE SE

Table 2: The table

Control
for PWL
Percentage
Contrations of chitosan solution
IW FW
IW FW
Weight Loss of Baguo
PWL IW FW
IW
Beans
FW
(P. IW
PWL
PWL
vulgaris)
FW
treated
PWL

with various concentrations of Chitosan solution after storing in low temperature

5%
10%
15%
20%
25%
100%
 Sitsaro (Pisum sativum )
1 2 3 4 5 AVE SE
Contrations of chitosan solution IW FW PWL IW FW PWL IW FW PWL IW FW PWL IW FW PWL
Control
5%
10%
15%
20%
E. Risk and safety
25%
100%
 During the collection of crab shell wastes, sharp edges of crab shells might cut
the finger:
- Wear protective garments such as gloves to minimize the risk of being cut.
 Exposure to hazardous chemicals:
- Seek for the attention of guardian or teacher when doing the process to
ensure the safety of researchers.
- Always wear protective clothings such as lab gown, gloves and masks.
 Usage of Centrifuge:
- Seek for the attention of teacher to make sure the correct way of operating it.

F. Waste Disposal
 Used specimens of Baguio beans and Sitsaro will be disposed in a
biodegradable trash bin.
 Used gloves and face masks will be disposed in non-biodegradable trash bin.
 Used chemicals will be placed in a chemical bottle.

G. Bibliography
Burrows, F., Louime, C., & Abazinge, M. Extraction and Evaluation of Chitosan from Crab
Exoskeleton as a Seed Fungicide and Plant Growth Enhancer. American-Eurasian J.
Agric. & Environ. Sci, 103-111. 2007. Retrieved from
file:///C:/Users/Lenovo/Downloads/Extraction-and-Evaluation-of-Chitosan-from.pdf.
Ding, C., Wang, C., Gross, K., & Smith, D. Jasmonate and salicylate induce the expression of
pathogenesis-related-protein genes and increase resistance to chilling injury in tomato
fruit. Planta. 2002. 214(6), 895-901. doi:10.1007/s00425-001-0698-9
Elfalleh, W. Characteristics of Cell Wall Structure of Green Beans During Controlled Freezing
Point Storage. N.d. Retrieved September 17, 2017, from
http://www.tandfonline.com/doi/abs/10.1080/10942912.2014.933437
Shahidi F, Synowiecki J. Isolation and characterization of nutrients and value- added products
from snow crab (Chionoecetes Opilio) and shrimp (Pandalus Borealis) processing
discards. J Agric Food Chem. 1991;39(8):1527–1532. doi: 10.1021/jf00008a032. [Cross
Ref]
Wattada, A., & Morris, L. 2012. Retrieved from: Http://theglobaljournals.com/ijsr/file.php?
val=January_2014_1391510112_0c336_04.pdf International Journal of Scientific
Research, 3(1), 15-17. doi:10.15373/22778179/jan2014/5