Proposal:

Chitosan/D-Glucosamine Nanolaminates for Edible Coating

Applications in Fruits and Vegetable:

Application on Strawberries…

By

Nouran A. El Badawi

Chem 5218

Fall 2014

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1.0 Background:
Nanolaminates are multilayered thin films with final thicknesses of 100 nm or less. Each
layer is attached to the next either chemically (ex. covalent bonding) or physically (ex.
adsorption and hydrogen bonding) 1-2. One of the most common development methods of the
nanolaminates is through layer by layer (LBL) assembly, in which the layers’ deposition is
sequential, thus un-limiting the types of materials to be deposited per layer 1. In addition,
LBL allows a controllable manipulation of conditions affecting the layers’ interface in order
to obtain desirable final thickness and specific properties 3. A naturally occurring example
multilayered assembly is the bone structure. Though they are not thin films, bones are sets of
organized platelets of minerals (calcium based) and organic binders (layers of collagen), with
remarkable difference in properties than their constituting components, which is the general
idea behind assembling a layered composite 4. Nanolaminates technology has large scale
applications in material sciences, engineering, biomedicine, and food industry 1.

Food scientists found a great area of research in using nanolaminates to fabricate edible
coatings for fruits, vegetables, meet, candies, etc… 3. Designed from biodegradable
polysaccharides, proteins, and/or lipids, edible coatings physical properties (permeability and
mechanical strength) are factors of polymeric composites’ design and compounds’
compatibility 5. Not only can they incorporate functional agents such as antioxidants (ex.
vitamin E), antimicrobial agents (ex. chitosan), calcium and essential oils (ex. Turmeric acid),
colorants, and flavors 6-7, they are also effective barriers of moisture, gas, and pathogens 8-9.
This could help in extending products shelf life while preserving their quality especially after
the packaging material is open. This could also increase the environmental friendly approach
of using simpler packaging materials and less chemical preservatives 6,10-11. Furthermore, new
trends in developing nutraceutical based coatings have emerged due to the consumers’
demands of high nutritional values food 9.

One of the most popular types of edible coatings is chitosan that has been extensively studied
on both fruits and vegetables 12. Chitosan, derivative of N-deacetylated chitin polysaccharides
is unique in characters: highly abundant in nature, bio-degradable, non-toxic to humans,
antimicrobial agent against both Gram-positive and Gram-negative bacteria, antifungistatic
properties, and comes from natural origins 12-16. Chitosan edible coatings can be fabricated
using chitosan alone while modifying the preparation conditions 7,10,16-18. Films can also
fabricated using composite blends like with gelatin19, whey proteins 20, essential oils 21-22,
starch 23, and cellulose derivatives 24, or in the form of multilayers like with carboxymethyl
cellulose 25, and beewax 26. The selection of blends, layering, or simple conditions
modification in designing the films is evaluated as a function of the target application,
weather enhanced barrier properties or incorporating an extra nutrition factor to the food
coated.

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An interesting example of a nutrition that is becoming more and more popular is D-
glucosamine, which is the monomer building block of chitosan. Provided as a nutraceutical
dietary food supplement, D-glucosamine has spread into the market as a magical solution for
reduction and possible future protection from osteoarthritis 27-29. Studies suggest that D-
glucosamine can reduce joints pain and help build cartilages when sufficient amounts
reaching the cells (daily intake can be up to 1500 mg of glucosamine) 29. However, as
promising as this may seem, D-glucosamine was never incorporated in food industry, which
makes it an interesting novel area for studying.

2.0 Research Objectives:

This research aims at studying the effect of adding D-glucosamine as nutrition to chitosan
edible coating, as well as studying the effect of varying the films’ preparation conditions on
its morphology, and its performance in terms of extending the shelf life of strawberries
(example of a fruit), while maintaining an acceptable sensory evaluation of the edible coating
shape and taste.

3.0 Research Design:

3.1 Investigating different preparation conditions:

In solvent evaporation method used in preparing chitosan based edible coatings, different
conditions could be varied to study their effect on the film’s morphology and hydrophilicity
including:

a. Chitosan content
b. D-glucosamine content
c. Single layer deposition vs. LBL-deposition
d. Solvent evaporation temperature

Chitosan based edible coatings would be prepared as in literature 16. D-glucosamine different
contents would be adjusted such that the final chitosan/D-glucosamine content in the solution
would always be 2% w/v (0/20, 5/15, 10/10, 15/5, 20/0 g/g in 1 L solution). Strawberries on
which the edible coating would be deposited would be prepared as in literature 17 with a
modification on deposition time and number of layers. The time taken for a single dipping
(single layer formation) would be 1 minute, and up to 3 layers would be studied. Between
every dipping step, 10 minutes of complete solvent evaporation would be carried out. Finally,
the effect of solvent evaporation temperature would be tested. Two temperatures (20 and
40oC) would be evaluated. All test samples would be in 5 replicas to have total samples of
150.

All variation in preparation conditions will be carried out in a randomized manner. A

factorial design using Design-Expert® software version 9 would create the experimental
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sequence. This is necessary since factorial design can predict the interactions between the
preparation conditions, eliminate the ones that don’t have an effect on the experiments, avoid
misleading conclusions, and give the regression equation that can predict the effect of any
further variation of the same conditions 30.

3.2 Investigating the films’ performance:

Several factors have to be studied in order to understand the effect of preparation conditions
on the fruit shelf life, appearance, and taste including:

a. Coatings thickness
b. Coatings loss
c. Weight loss
d. Water vapor transmission (WVT)
e. Respiration rate
f. D-glucosamine leachability
g. Decay evaluation
h. Microbiological analysis
i. Sensory evaluation
j. Statistical analysis

Coatings thickness would be measured by a bench micrometer as described by Geraldine et

al. 11. Coating loss and sample weight loss would be evaluated after the completion of the
storage period using the formulas of Soares et al. 18. Water vapor transmission would be
analyzed using a modified ASTM E96-95 procedure 11, while respiration rate in terms of CO2
production would be evaluated using an Infrared gas analyzer 7. Decay evaluation would be
carried out as described by Wang et al. 17, while microbial analysis would be carried out
using pour plate count agar (PCA) method 31. Sensory evaluation would take place using
untrained panel of 30 people to evaluate taste, texture, brightness, acidity, and odor 21.

A suggested procedure for D-glucosamine leachability tracking is by measuring the samples

pH after blending a constant weight of the sample in deionized water, then a filtration step
using membranes filters (pore size of 0.4 µm) would take place, and the permeate would be
analyzed using Fourier Transformed Infrared spectrophotometer. The procedure would be
repeated over the shelf life.

4.0 Methodology:
Edible films’ morphology is usually the front line that controls the behavior of the coating.
That’s why by modifying preparation conditions, the performance differ. Ideas behind
changing contents, number of layers, and evaporation temperature target films’ porosity and
hydrophilicity. A porous rough topography could create gaps for microbial growth, which is a
general property of polymer based nanocomposites, while increased hydrophilicity increases
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the coated food water vapor intake, reducing its shelf life. The suggested multiple layers
deposition in this research might help in reducing surface roughness, characterized using the
Atomic Force Microscope (AFM) and Scattered Electron Microscope (SEM). However, it
might as well affect hydrophilicity, which is evaluated by contact angle measurements using
the sessile drop technique. As for solvent evaporation temperature, it could become a factor
affecting how rapid the layers deposit, thus directly affecting the morphology and indirectly
affecting the interaction between the monomer D-glucosamine and its polymeric chain
chitosan. This could be easily characterized using X-Ray Diffraction (XRD).

After edible coatings are prepared, a keen evaluation takes place. Coatings thickness
represents money: that is the thinner better performing the film is, the more desirable it
becomes in the industrial market. Such evaluation is also accompanied by coatings loss
investigation. True that biodegradable polymers are desirable, yet, if their life is significantly
shorter than the shelf life of the product, then they become unworthy. Furthermore, the
product weight loss (in this case, strawberries’) is also a significant factor in the films’
performance initial evaluation. Having a fruit maintaining its shape and size over an extended
shelf life is a good indication of the sealing effect of the coating, meaning the product isn’t
losing its moisture content 11.

WVT and respiration rates are excellent indication of the gas barrier behavior of the coatings.
Evaluation of WVT gives a clear idea of the films hyrophilicity, indicating how much water
vapor due to a humid environment would be up-taken. Of course, optimizing the preparation
conditions to reduce such up-take has the upper hand. In return, respiration rates by
evaluating CO2 release using FTIR gas analyzer give an idea of gases permeability from
inside the product. It is important to point out though, that is comes in handy when analyzing
the development of anaerobes in a sealed medium 7.

D-glucosamine leachability is a reflection to how good or bad the monomers interacted with
the polymeric matrix. In a good nanocomposite film, the major focus is on how to mix two or
more materials together and get the maximum interfacial interaction to make used to the
enhance properties produced 32. Theoretically, D-glucosamine and chitosan should highly
interact together since they have the same basic building block: the glucosamine ring.
However, such interactions can vary with preparation conditions as explained earlier, thus
evaluating the leachability becomes necessity.

Decay evaluation and microbiological analysis are interrelated. Attempts to extend the shelf
life of fruits and vegetables using chitosan edible coating were very successful as earlier
mentioned. Some attribute this success to the structure of the building blocks of the polymer:
the glucosamines. In an acidic medium of pH 6 or less, the amino groups become positively
charged. In the presence of a bacterium cell membrane with negative charges, the
electrostatic interaction between the two charges rapture the cell membrane, thus killing the
pathogen. Chitosan is also found to attach itself to metals via chelation, thus inhibiting toxins
production 33.
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As promising as it may seem, chitosan/D-glucosamine edible films won’t find their place in
the market without a well constructed sensory evaluation test. It is a measurement of
consumers’ acceptance to the overall product. Typically, a survey focusing on the product
flavor, aroma, texture and appearance is carried out, and on a scale of 1 to 10 (1 is least
preferable and 10 is most preferable), the product is evaluated.

5.0 Project Duration

This project is expected to take a maximum of 6 months if all materials and characterization
techniques are available, since most of the experimental work can be conducted in parallel.

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Reference:

(1) Decher, G. In Multilayer Thin Films; Wiley-VCH Verlag GmbH & Co. KGaA: 2012,
p 1.

(2) Nussinovitch, A. In Modern Biopolymer Science; Ubbink, S. K. T. N. B., Ed.;

Academic Press: San Diego, 2009, p 295.

(3) Weiss, J.; Takhistov, P.; McClements, D. J. Journal of Food Science 2006, 71, R107.

(4) Ajayan, P. M. In Nanocomposite Science and Technology; Wiley-VCH Verlag GmbH

& Co. KGaA: 2004, p 1.

(5) Falguera, V.; Quintero, J. P.; Jiménez, A.; Muñoz, J. A.; Ibarz, A. Trends in Food
Science & Technology 2011, 22, 292.

(6) Teixeira, J. A.; Vicente, A. A. Engineering Aspects of Food Biotechnology; Taylor &
Francis, 2013.

(7) Simões, A. D. N.; Tudela, J. A.; Allende, A.; Puschmann, R.; Gil, M. I. Postharvest
Biology and Technology 2009, 51, 364.

(8) Embuscado, M.; Huber, K. C. Edible Films and Coatings for Food Applications;
Springer, 2009.

(9) Bhat, R.; Alias, A. K.; Paliyath, G. Progress in Food Preservation; Wiley, 2012.

(10) Moreira, M. d. R.; Roura, S. I.; Ponce, A. LWT - Food Science and Technology 2011,
44, 2335.

(11) Geraldine, R. M.; Soares, N. d. F. F.; Botrel, D. A.; de Almeida Gonçalves, L.

Carbohydrate Polymers 2008, 72, 403.

(12) Elsabee, M. Z.; Abdou, E. S. Materials Science and Engineering: C 2013, 33, 1819.

(13) astro, . . . aul n, E. G. L. r. In The Complex World of Polysaccharides;

Karunaratne, D. N., Ed.; InTech: 2012.

(14) Kong, M.; Chen, X. G.; Xing, K.; Park, H. J. International journal of food
microbiology 2010, 144, 51.

(15) Tripathi, P.; Dubey, N. K. Postharvest Biology and Technology 2004, 32, 235.

(16) Chien, P.-J.; Sheu, F.; Yang, F.-H. Journal of Food Engineering 2007, 78, 225.

(17) Wang, S. Y.; Gao, H. LWT - Food Science and Technology 2013, 52, 71.

(18) Soares, N. M.; Mendes, T. S.; Vicente, A. A. Journal of Food Engineering 2013, 119,
316.

7
(19) Poverenov, E.; Zaitsev, Y.; Arnon, H.; Granit, R.; Alkalai-Tuvia, S.; Perzelan, Y.;
Weinberg, T.; Fallik, E. Postharvest Biology and Technology 2014, 96, 106.

(20) Di Pierro, P.; Sorrentino, A.; Mariniello, L.; Giosafatto, C. V. L.; Porta, R. LWT -
Food Science and Technology 2011, 44, 2324.

(21) Perdones, A.; Sánchez-González, L.; Chiralt, A.; Vargas, M. Postharvest Biology and
Technology 2012, 70, 32.

(22) Azevedo, A. N.; Buarque, P. R.; Cruz, E. M. O.; Blank, A. F.; Alves, P. B.; Nunes, M.
L.; Santana, L. C. L. d. A. Food Control 2014, 43, 1.

(23) de Aquino, A. B.; Blank, A. F.; de Aquino Santana, L. C. L. Food Chemistry 2015,
171, 108.

(24) Gol, N. B.; Patel, P. R.; Rao, T. Postharvest Biology and Technology 2013, 85, 185.

(25) Arnon, H.; Zaitsev, Y.; Porat, R.; Poverenov, E. Postharvest Biology and Technology
2014, 87, 21.

(26) Velickova, E.; Winkelhausen, E.; Kuzmanova, S.; Alves, V. D.; Moldão-Martins, M.
LWT-Food Science and Technology 2013, 52, 80.

(27) Coates, P. M.; Paul, C.; Blackman, M.; Blackman, M. R.; Cragg, G. M.; Levine, M.;
White, J. D.; Moss, J.; Levine, M. A. Encyclopedia of Dietary Supplements (Print); Taylor
& Francis, 2004.

(28) Berning, J. R.; Steen, S. N. Nutrition for Sport and Exercise; Jones & Bartlett
Learning, LLC, 2005.

(29) Moskowitz, R. W. Osteoarthritis: Diagnosis and Medical/surgical Management;

Wolters Kluwer Health/Lippincott Williams & Wilkins, 2007.

(30) Montgomery, D. C. Design and Analysis of Experiments; Wiley, 2013.

(31) Rojas-Graü, M. A.; Tapia, M. S.; Martín-Belloso, O. LWT - Food Science and
Technology 2008, 41, 139.

(32) Bréchignac, C.; Houdy, P. Nanomaterials and Nanochemistry; Springer, 2008.

(33) Dutta, P. K.; Tripathi, S.; Mehrotra, G. K.; Dutta, J. Food Chemistry 2009, 114, 1173.