Active packaging based on chitosan and cinnamaldehyde for meat and fruit

Content of the project: Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde: UV – Vis absorption spectrum and optical transmittance, equilibrium

FACULTY FOR HIGH QUALITY TRAINING

CAPSTONE PROJECT FOOD TECHNOLOGY

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

Ho Chi Minh City, December 2020

ACTIVE PACKAGING BASED ON CHITOSAN AND CINNAMALDEHYDE FOR MEAT AND FRUIT.

Advisor: NGUYEN VINH TIEN, PhD

TRAN THI VAN AN

VU THI THU HUONG

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom – Happiness

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GRADUATION PROJECT ASSIGNMENT

Student name: Tran Thi Van An Student ID: 17116001

Student name: Vu Thi Thu Huong Student ID: 17116012

Major: Food Technology Class: 17116CLA

Advisor: Ph.D Nguyen Vinh Tien

1 Project title: Active packaging based on chitosan and cinnamaldehyde for meat and

fruit

2 Thesis number: 2021 – 17116001

3 Content of the project: Examining the characteristics of chitosan film samples

with varying levels of cinnamaldehyde: UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation-at-break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan – cinnamaldehyde film on meat and fruit

4 Thesis assignment date: 01/2021

5 Thesis submission date: 12/2021

CHAIR OF THE PROGRAM ADVISOR

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First of all, we would like to express our sincere and deep thanks to the teachers in the Department of Food Technology – Faculty of High Quality Education – University of Technology and Education of Ho Chi Minh City for their dedication to convey their knowledge knowledge during the past four years, creating all conditions for facilities and equipment to help us complete the thesis in the best way

In particular, we would like to thank Ph.D Nguyen Vinh Tien who was very dedicated

to help, guide and impart knowledge and experience to help us complete the graduation thesis on time

Finally, we would like to thank our family and friends by our side for supporting us, giving us time to research the topic and wholeheartedly supporting us mentally during the course of the thesis

We have tried to research and research to best complete the graduation thesis, but we are still limited in knowledge and experience, so there are bound to be shortcomings We hope to receive the contributions of teachers and friends to improve the topic

Thank you very much!

COMMITMENT

We hereby declare that all content presented in the graduation thesis is our own work

We hereby certify that the contents referenced in the graduation thesis have been correctly and fully cited in accordance with regulations

17th December,2021 (Sign and write full name)

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ADVISOR'S EVALUATION SHEET

Student name: Tran Thi Van An

Student name: Vu Thi Thu Huong

Student ID: 17116001 Student ID: 171 16012 Major: Food Technology

Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit

Advisor: Nguyen V inh Tien, PhD

EVALUATION

l Content of the project:

Examining the characteristics of chitosan films with 0 – 9% levels of cinnamldehyde: UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation – at – break, water vapor permeability, antioxidant activity,

in vitro antibacterial and antifungal activities, and in vivo antifungal activity on limes

2 Strengths:

A high work load with a wide range of experimental techniques used to characterize the chitosan films incorporated with 0 – 9% cinnamaldehyde In vivo antifungal tests of chitosan – 7 – 9% cinnamaldehyde on limes are promising

3 Weaknesses: modern characterization techniques (XRD, FTIR, TGA, DSC) were not utilized to reveal structural changes in chitosan films after incorporating cinnamaldehyde

4 Approval for oral defense? Approved

5 Overall evaluation: Good

6 Mark: 9.1/10 (in words: nine point one)

Ho Chi Minh City, December 26th, 2021

ADVISOR

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PRE-DEFENSE EVALUATION SHEET

Student name: Tran Thi Van An

Student name: Vu Thi Thu Huong

Student ID: 171 16001 Student ID: 17116012 Major: Food Technology

Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit

Name of Reviewer: Nguyen Quang Duy, M Eng

EVALUATION

1 Content and workload of the project:

Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde:

UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation – at – break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan – cinnamaldehyde film on meat and fruit

2 Strengths:

The project has clear objectives and appropriate methods to achieve the results

The English used in the report is academic and adequate

The project used modern analytical techniques to obtain and analyze the data

3 Weaknesses:

The authors should use the word "investigation" instead of "survey" in the report

The authors should extend the range of CNM concentration

4 Approval for oral defense? (Approved or denied) Approved

5 Overall evaluation: (Excellent, Good, Fair, Poor) Good

6 Mark: 88 (in words: Eighty – eight)

Ho Chi Minh City, December 27th, 2021

REVIEWER

(Sign with full name)

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Independence – Freedom– Happiness

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EVALUATION SHEET OF DEFENSE

COMMITTEE MEMBER

Student name: Vu Thi Thu Huong Student ID: 17116012

Major: Food Technology

Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit

Name of Defense Committee Member

EVALUATION

1 Content and workload of the project:

Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde:

UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation-at-break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan cinnamaldehyde film on meat and fruit

2 Strengths:

3 Weaknesses:

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

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EVALUATION SHEET OF DEFENSE

COMMITTEE MEMBER

Student name: Tran Thi Van An Student ID: 17116001

Major: Food Technology

Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit

Name of Defense Committee Member

EVALUATION

1 Content and workload of the project:

Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde:

UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation-at-break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan cinnamaldehyde film on meat and fruit

2 Strengths:

3 Weaknesses:

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

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Ho Chi Minh City, January 20, 2020 EVALUATION SHEET OF DEFENSE COMMITTEE MEMBER Student name: Vu Thi Thu Huong Student ID: 17116012 Major: Food Technology Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit Name of Defense Committee Member EVALUATION 1 Content and workload of the project Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde: UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation-at-break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan – cinnamaldehyde film on meat and fruit 2 Strengths:

3 Weaknesses:

THE SOCIALIST REPUBLIC OF VIETNAM

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Ho Chi Minh City, December 28, 2021 EVALUATION SHEET OF DEFENSE COMMITTEE MEMBER Student name: Tran Thi Van An Student ID: 17116001 Major: Food Technology Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit Name of Defense Committee Member EVALUATION 1 Content and workload of the project Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde: UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation-at-break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan – cinnamaldehyde film on meat and fruit 2 Strengths:

3 Weaknesses:

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

-

Ho Chi Minh City, January 20, 2020 EVALUATION SHEET OF DEFENSE COMMITTEE MEMBER Student name: Vu Thi Thu Huong Student ID: 17116012 Major: Food Technology Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit Name of Defense Committee Member EVALUATION 1 Content and workload of the project Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde: UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation-at-break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan – cinnamaldehyde film on meat and fruit 2 Strengths:

3 Weaknesses:

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

-

Ho Chi Minh City, January 20, 2020 EVALUATION SHEET OF DEFENSE COMMITTEE MEMBER Student name: Tran Thi Van An Student ID: 17116001 Major: Food Technology Project title: Active packaging based on chitosan and cinnamaldehyde for meat and fruit Name of Defense Committee Member EVALUATION 1 Content and workload of the project Examining the characteristics of chitosan film samples with varying levels of cinnamaldehyde: UV – Vis absorption spectrum and optical transmittance, equilibrium moisture content, water uptake, water solubility, tensile strength, elongation-at-break, water vapor permeability, antioxidant ability, antibacterial and mold resistance of chitosan – cinnamaldehyde film on meat and fruit 2 Strengths:

3 Weaknesses:

CONTENTS

GRADUATION PROJECT ASSIGNMENT 1

ACKNOWLEDGEMENTS 2

COMMITMENT 3

ADVISOR'S EVALUATION SHEET 4

PRE-DEFENSE EVALUATION SHEET 5

EVALUATION SHEET OF DEFENSE COMMITTEE MEMBER 7

CONTENTS 19

LIST OF FIGURES 21

LIST OF TABLES 22

LIST OF ABBREVIATIONS 23

ASTRACT 24

CHAPTER 1: OVERVIEW 25

1.1 Overview about active film: 25

1.1.1 Introduction: 25

1.1.2 Classification: 26

1.1.3 Materials to produce active packaging films: 28

1.2 Overview about Chitosan: 30

1.2.1 Chitosan's origin: 30

1.2.2 Chemical composition of chitosan: 31

1.2.3 Characterizations of chitosan: 32

1.2.4 Applications of chitosan in food industry: 34

1.3 Overview about cinnamaldehyde: 34

1.4 Overview of microbial strain and molds used in this research 36

CHAPTER 2: MATERIALS AND METHODS 41

2.1 Materials, chemistry and equipment: 41

2.1.1 Materials and chemicals: 41 2.1.2 Equipment: 41 2.2 Researching methods: 42 2.2.1 Research diagram: 42 2.2.2 Film preparation: 43 2.3 Content and analytical method: 44 2.3.1 Characterization of cinnamaldehyde - chitosan film: 44 2.4 Statistical processing methods: 50 CHAPTER 3: RESULTS 51 3.1 Appearance of cinnamaldehyde - chitosan film: 51 3.2 Physical properties of films: 52 3.3 Bioactivities assay: 60 CHAPTER 4: CONCLUSION 73 REFERENCES 74 APPENDIX 81

LIST OF FIGURES

Figure 1.1 The chemical composition of Chitin and Chitosan 32 Figure 1.2 Extract CNM from cinnamon (in the bark oil) 35 Figure 1.3 Chemical formula of CNM 35 Figure 2.1 Research diagram 42 Figure 2.2 Diagram of CH - CNM film preparation 43 Figure 2.3 CH films with different CNM concentration (size 20 × 20 mm) 45 Figure 3.1 CH – CNM films 51 Figure 3.2 Schiff base is formed by the nucleophilic addition of the amino group of the CH backbone to the carbonyl group of CNM 52 Figure 3.3 UV – Vis absorption spectrum of chitosan films with various CNM concentration 53 Figure 3.4 Equilibrium moisture content of CH films with various CNM content 55 Figure 3.5 Water uptake of CH films with various CNM content 55 Figure 3.6 Water solubility of CH films with various CNM content 56 Figure 3.7 Tensile strength content of CH films with various CNM concentration 58 Figure 3.8 Elongation-at-break content of CH films with various CNM concentration 58 Figure 3.9 Water vapor permeability as a function of the CNM concentrations of the CH films 60 Figure 3.10 The percentage decrease in DPPH absorbance by time as a function of the CNM concentration of the CH films 61 Figure 3.11 DPPH scavenging activity of CH films at 24h with various CNM concentration 61 Figure 3.12 Effectiveness of CH films at different CNM concentration (0 % CNM, 1 % CNM, 3

% CNM, 5 % CNM, 7 % CNM and 9 % CNM) against Escherichia coli 64

Figure 3.13 The total viable count (TVC) of pork slices during storage at 4°C was affected by CH

- CNM films Unwrapped samples as a control 65 Figure 3.14 Effectiveness of CH films at different CNM concentration (0 % CNM, 1 % CNM, 3

% CNM, 5 % CNM, 7 % CNM and 9 % CNM) against Aspergillus niger 67

Figure 3.15 Effectiveness of CH films at different CNM concentration (0 % CNM, 1 % CNM, 3

% CNM, 5 % CNM, 7 % CNM and 9 % CNM) against Aspergillus fumigatus 69

Figure 3.16 The weight loss of the lemons after 12 – day storage 71 Figure 3.17 Application of CH – CNM films to packaging for lemon fruit during 12 days storage 72

LIST OF TABLES

Table 2.1 Formulation of four types solution 47 Table 3.1 Average percentage of transmittance of CH films with various CNM concentration from 200 – 1000 nm 53 Table 3.2 Result parameters of the effect of CNM content on the properties of CH films 57

LIST OF ABBREVIATIONS

Abbreviations Explanation

A niger Aspergillus niger

A fumigatus Aspergillus fumigatus

E coli Escherichia coli

ASTRACT

This study used an emulsion technique to create chitosan (CH) films (2 % w/v) containing cinnamaldehyde (CNM) (1, 3, 5, 7 and 9 % v/v) After that, take a poll and compare the following characteristics: UV – Vis absorption spectrum and optical transmittant, equilibrium moisture content, water uptake, water solubility, tensile strength (TS) and elongation – at – break (E), water vapor permeability (WVP), antioxidant activity and antimicrobial activity

To evaluate the effect of modifying the CNM content on the characteristics of the membrane, the film was constructed of CH – CNM with fixed glycerol and chitosan concentration The study indicate that increasing the CNM concentration improved tensile strength, antioxidant activity, and antibacterial activity CNM is depicted as having a lower light transmittance It demonstrated a strong capacity to withstand UV radiation and minimize the possibility of spoiling Nevertheless, when the functions of CNM grow from

1 % to 9 %, the transparency (increase in the yellowish tone) decreases Despite the fact that the changes were not significant, the variations in water uptake, moisture content, and elongation to break were recorded Especially, the addition of CNM at high concentration

made the membrane effective against bacteria (Escherichia coli), and anti – fungal (Aspergillus niger, Aspergillus fumigatus) Besides, the application of CH – CNM films on

lemon fruits and pork slices has achieved the expected antibacterial results These findings show that CH – CNM films should be used as active packaging for the preservation of food goods

CHAPTER 1: OVERVIEW 1.1 Overview about active film:

1.1.1 Introduction:

The term "active packaging" was first applied by Labuza in 1987 and can be defined as packaging that performs some desirable function other than actual packaging products [1] The intent is to enhance the shelf life of food while preserving nutritional quality and ensuring their safety, which shows more unilities than simple wrapper Because of advancements in packaging, material science, biotechnology and new consumer needs, active packaging systems are often combined with controlled atmosphere packaging (CAP) or modified atmospheric packaging (MAP) Usually, active packaging is used to prevent moisture loss during food storage and transportation The present day, they are being used widely to ensure the quality of food, inhibit the growth of bacteria organisms that cause food spoilage, increase the shelf life of products, and limit contamination from

surrounding environment [2] This technology is based on the removability of undesired

compounds from foods or its environment such as: moisture, ethylene, oxygen, etc The complex structure of foods can be modified by avitivity of packaging, active substances that are added, specifically These substances are able to control release rates, absorption rates, diffusion rates, etc Normally, active substances are antimicrobial compounds, antioxidants, carbon dioxide, flavors, ethylene, or ethanol depending on particular food [3] Most of the food consumed come directly from nature In addition, the food is being able to be used right after harvesting from the farm in order to reach the hands of consumers, through a time - consuming series of handling, storage, and shipping steps, the product begins dehydration, loss of sensory, taste, nutritional value and lead to spoilage If there is no guarantee of special protection, damage can happen in hours or days, even if this damage is not immediately visible [4]

In most circumstances, the phrases film and coating are used interchangeably to describe how a food's surface is coated with a thin layer of material containing specific ingredients The membrane will be a barrier against transpiration, helping to control physiological and biochemical activities that take place inside food, contributing to maintaining the freshness

of food products during storage [5]

Food wraps should have the following characteristics [6]:

– Help stabilize the structure and prevent mechanical damage during transportation and handling and display

– Do not contain harmful or allergic ingredients

– The good adhesion on food surfaces

– Control the water content of food to maintain the desired moisture content

– Prevent loss or absorption of flavor, nutritional and character-stabilizing ingredients food sensibility

– Resist mold and harmful bacteria

– Maintain or enhance aesthetic and sensory attributes (appearance, taste, etc) of the product

– Easy to manufacture and cost-effective

❖ Carbon dioxide scavengers and emitters

Microorganisms generally inhibited by high carbon dioxide levels because it slows their growth on the surface of meat and poultry and in delaying respiration of fruit and vegetables Carbon dioxide is more permeable than oxygen, that a reason why it usually applies in food packaging Most of the carbon dioxide inside the package is normally permeable through the membrane For packaging which is highly permeable to carbon dioxide, a carbon dioxide emitter system can be needed to slow down respiration and prevent microbial growth The employment of a dual-function system that includes an oxygen filter as well as a carbon dioxide emitter to extend the shelf life of perishable goods [7]

❖ Moisture scavengers

Humidity in food packages is very important and necessary to control to prevent the growth of microorganisms and avoid the formation of mist The lower the water vapor permeability of packaging, the more water accumulation inside the packaging The formation of water inside the food package often occurs due to the respiration of fresh products, and temperature fluctuations in food packages that have pretty high moisture The accumulation of excess water inside the packaging promotes bacteria and mold growth development, leading to reduce quality and reduce shelf life An effective way to control the accumulation of excess water in a food package with a high vapor barrier must use desiccants such as silica gel, natural clay (for example, montmorillonite), calcium chloride,

or other desiccants Silica gel is the most widely used desiccant because it is non-toxic and non-corrosion [7]

❖ Antibacterial membrane

Humans have been developing strategies to preserve food and prevent the growth of harmful microbes since prehistoric times Canning, pasteurization, sterilization, freezing, cooling, drying and fermentation are the main methods of food preservation Nowadays When chemical preservatives are combined with these food preservation techniques, there are benefits in preventing microbial contamination after processing and achieving a stronger inhibitory effect on microorganisms Membranes containing antibacterial substances are of great value in inhibiting the growth of surface microorganisms and prolong shelf life

A multilayer membrane with antibacterial properties usually consists of four layers, including the outer layer, barrier layer, matrix layer, and control layer An antibacterial agent is embedded in the background layer The release of the matrix to the food surface is controlled by the control layer right next to the matrix layer Among the problems encountered in the antibacterial film is an active ingredient that can partially or completely lose its antibacterial properties when it is introduced into the membrane Therefore, polymerization or derivatization of active substances prior to their addition to the polymer

is necessary to increase compatibility between active agents and polymers [7]

❖Other active packaging

Another active packaging that is expected to develop in the future includes antioxidants releasing films, pigment films, light – modulating absorption systems, anti – fog films and non – stick, air – permeable, film – breathable and film containing insect repellent Antioxidant membrane combinations, for example, can be employed to keep fats and oils

flavors developing in foods Han et al proved that antioxidant-infused plastic films may effectively prevent oat oxidation [8]

1.1.2.2 Classification based on materials:

❖ Hydrocolloids

Polysaccharides used for membranes include cellulose, starch derivatives, pectin, seaweed extract and CH [9] The polysaccharide is very hydrophilic, the moisture barrier effect of the polysaccharide coating is negligible Although the polysaccharide coating may not provide a well vapor barrier, it is reported that these coatings can act as retarding agents for loss of moisture from food products [10]

a substrate such as polysaccharide, to provide mechanical strength [10]

❖ Protein membrane

Membranes from a variety of protein sources, such as corn, milk, soybeans, wheat, and whey, have been used for many years In the natural state, proteins usually exist in the form

of fibrous proteins (insoluble in water) or globular proteins (soluble in water and acid, base

or salt solutions) Fibers are formed when fibrous proteins are strongly linked together by hydrogen bonding Hydrogen bonding, ionic, hydrophobic, and covalent (disulfide) bonds all help globular proteins fold into complex globular shapes The physicochemical properties of these proteins depend on the number of amino acid group and their position along the polymer chain The membrane characteristics of several globular proteins have been examined, including wheat gluten, maize zein, soy protein, and whey proteins [10]

1.1.3 Materials to produce active packaging films:

The ingredients used to prepare the membranes are classified into three categories: hydrocolloid (such as proteins, polysaccharides and alginates), lipids (such as fatty acids, acylglycerols, waxes) and synthetic materials combined [9]

❖ Polysaccharides

Cellulose is made up of D – glucose units that are connected by β – 1,4 glycoside linkages Treatment of cellulose with alkali, followed by reactions with chloroacetic acid,

methyl chloride, or propylene oxide to form carboxymethyl cellulose (CMC), methyl cellulose (MC), hydroxypropyl cellulose (HPMC), or hydroxypropyl cellulose (HPC) might enhance its water solubility MC, HPMC, HPC and CMC film forming have good properties, no odor, tasteless, moderate toughness, oil resistance and water solubility Starch is a carbohydrate consisting of many D – glucose units Most starches contain two types of glucose polymers: a linear polymer called amylose and a branched polymer glucose is called amylopectin Starch is commonly used in the food industry It has been used to produce biodegradable membranes that partially or whole polymer because of its low cost and good mechanical properties [10]

High amylose starch, such as maize starch, is a suitable supply of material for manufacturing films, and gelatinized and dried amylose solution can be used to make films Amylose and amylopectin make up roughly 25 % and 75 % of normal maize starch, respectively [9]

❖ Proteins

Gelatin is a protein of animal origin and characterized by its film – forming properties Gelatin helps to protect foods from oxidation caused by oxygen and light It shows the most advantages in low moisture content or oily foods Gelatin is used to package foods that are low in moisture and contain oils that help protect against oxygen and light [11] Gelatin films can be formed from 20 – 30 % gelatin, 10 – 30 % plasticizers (glycerol or sorbitol) and 40 – 70 % water, then they will be dried later In addition, a gelatin film was formed as a coating on the surface to reduce oxidation, moisture [12]

Zein is the most important protein in corn It is a prolamin protein, which can be dissolved in 70 – 80 % ethanol Zein is relatively hydrophobic and this hydrophobic property is related to the amount of non – polar amino acids Zein has good film – forming

properties and can be used as biodegradable film The resulting film is brittle and therefore requires the addition of a plasticizer to increased flexibility Zein film is a relatively good vapor barrier compared to other edible films

1.2 Overview about Chitosan:

1.2.1 Chitosan's origin:

After cellulose, chitin and chitosan are the most abundant naturally occurring polymers

CH is derived from the same sources as chitin Silkworms, insects, honey bees, arthropods, and nematodes are examples of terrestrial creatures Crustacean exoskeletons, shrimp shells, crab, krill, lobster, and other exoskeletons have all been effectively used to extract

CH Apart from Basidiomycetes, CH can be found in mushrooms, fungal cell walls, mycelia, and septa, as well as Zygomycetes and Ascomycetes spores In most situations,

CH is produced by deacetylating chitin To produce the desired result, CH, deacetylation can be done after the shellfish (or any other source) has been decolored Deacetylation is the process of eliminating the acetyl group from a molecule in a variety of methods

❖ Chemical method and enzymatic method

Previously, alkali and acid treatments were used to remove CH from fungi cell walls Internal cells contain many components such as chitin, calcium carbonate, protein, lipids, and colors, etc And CH production follows three important steps: Deproteination; demineralization; and lipid and pigment elimination

Chemical or biological processes, such as microbial fermentation and enzymatic reactions, can be used to extract chitin and then convert it to CH Chemical procedures, on the other hand, produce higher yields

Pre – wash with water: To remove loose prawn meat remnants, lipids, and other

residues, frozen prawn shells were hand washed in hot tap water (about 60oC) or boiling water (around 95oC) while stirring with a mechanical stirrer Finally, they were cleaned in hot distilled water and dried in a 60oC oven to a constant weight They will then be cleaned, dried, and crushed into powder or small particles that can pass through a 60 – 120 m filter

Deproteination: To eliminate any residual proteins and organic compounds, the washed

and dried powdered prawn shells were treated with a 5 % sodium hydroxide (NaOH) solution (w/v 1:8) and refluxed at 60°C for 2 hours After the reaction, the solution was colorful and foamy, therefore the sample was rinsed under water until the majority of the color and foaming vanished and the solution was near neutral Finally, the sample was cleaned with distilled water and dried to a uniform weight in a vacuum oven at 60°C

Decolouration: The shells from the previous state are treated with acetone at room

temperature for 24 hours to remove colour Then they are filtered to the desired size and dried in a vacuum oven at 60°C to an unchanging weight

Demineralisation: To dissolve the calcium carbonate, the material was treated with a

0.5 or 1 % HCl solution (w/v 1:4, 1:10) for 24 hours at 25oC All the precipitants are completely removed by washing under clean water to collect the very light brown powder, chitin Some of the processes (deproteination and decoloration) were skipped in favor of alternative techniques The difference in chitin produced over the original weight of prawn shells is the outcome weight of chitin The resulting weight of chitin is the difference of chitin produced over the initial weight of prawn shells

Deacetylation of chitin: Chemical deacetylation of chitin was achieved by incubating

extracted chitin in a sodium hydroxide (NaOH) solution at elevated temperatures with a 1:5 solid – to – solvent ratio The impacts of a variety of variables on the deacetylation process were studied, including NaOH concentrations (25 and 50 %), temperature (80 and

100oC), and reaction times (2, 5 and 10 hours) Following the reaction, the material was washed several times with distilled water until it reached a pH near to neutral, then dried

in a vacuum oven at 60oC until it reached a constant weight The material was then rinsed multiple times with distilled water until it achieved a pH close to neutral, then dried in a vacuum oven at 60oC until it reached a constant weight [13]

❖ Microwave assisted method

It has been discovered that the three extraction phases of chitin may also be accomplished using a microwave – assisted mechanism, which is both time – saving and eco – friendly method Knidri et al successfully produced CH after deacetylation using microwave irradiation, with an 82.73 % of deacetylation in just 24 minutes, which is quite good when compared to the conventional method, which took 81.5 % of deacetylation in a much longer time of 5 – 10 hours [14]

1.2.2 Chemical composition of chitosan:

The only distinction between CH, cellulose, and chitin is the functional group at the C – 2 position At the C – 2 position, CH possesses an amino group (–NH2), whereas cellulose and chitin have hydroxyl groups (–OH) and N – acetyl amine groups (–NHCOCH3), respectively As indicated in [15, Fig 1.1], CH is a linear – chain polysaccharide made up

of N–acetyl–2–amino–2–deoxy–d–glucopyranose (acetylated unit) and 2–amino 2–deoxy d–glucopyranose (deacetylated unit) linked by – (1,4) –glycosidic bonds [15]

Figure 1.1 The chemical composition of Chitin and Chitosan

CH is a viscosity enhancer that works well The type of acid used as a solvent to dissolve the CH determines the pH of the solution, which impacts the viscosity of the solution The inherent viscosity of CH is critical for its storage and stability Intrinsic viscosity is proportional to the average molecular weight of a polymer and refers to a solution's ability

to become viscous when the correct solvent and temperature are used [16]

❖ Molecular weight

The molecular weight of CH has a significant impact on its physicochemical properties The molecular weight of CH can generally manage the average of all the molecules in the sample Thermal stability, temperature, pH, and mechanical shearing are all factors that influence the molecular weight of CH Viscosity, hydrophilicity, moisture content, thermal characteristics, and stability are all variable between low and high molecular weight CH

❖ Degree of deacetylation

The degree of deacetylation is a very essential attribute of CH since it has a major impact

on practically all of the other properties For biomedical purposes, the optimal degree of deacetylation of CH should be 75 – 98 %, as manufactured by pharmaceutical enterprises The ideal degree of deacetylation of CH for biomedical purposes should be 75 – 98 %, as prepared by pharmaceutical companies

❖ Stability or polymer degradation

The degradation of CH's polymeric chain under diverse conditions determines its stability CH breakdown is influenced by the degree of deacetylation, molecular weight, temperature, moisture content, polydispersity and purity level

❖ Hygroscopicity and swelling ability

CH is a natural polymer that is hygroscopic and pseudo – plastic It can establish hydrogen bonds with the polymeric chain's functional groups, such as –NH2 and –OH with the O – and H – atoms of water molecules, respectively The amount of moisture already present in the sample, as well as the storage conditions, can impact the rate of water absorption When the degree of deacetylation is reduced, the water content of dry CH is shown to increase As the concentration of cross – linking agents rises, CH's swelling property decreases Aside from that, the absorbed water content has influenced viscosity, compressibility, and flow characteristics, resulting in a little drop in tensile strength

1.2.3.2 Chemical properties:

CH is soluble in a variety of organic and inorganic acids with a pH of less than 6.0, forming a non – Newtonian, thin shearing fluid CH, on the other hand, is water and aqueous base insoluble CH is a biopolymer with a reactive site on its backbone that contains – NH2 and – OH functional groups These functional groups, particularly the amino group, are protonated to a greater extent, resulting in additional protons CH is a reactive molecule

1.2.3.3 Biological properties:

CH is generating a lot of buzz because it has several unique features that make it ideal for biomedical applications, such as biocompatibility, biodegradability, and non – toxicity Other qualities have also been documented, including analgesic, anticancer, hemostatic, hypocholesterolemic, antimicrobial, and antioxidant properties [17]

In recent years, the antibacterial activity of CH and its derivatives against a variety of microorganisms, including bacteria, yeast, and fungi, has gotten a lot of attention The suppression of microbial cells by CH has been attributed to two primary mechanisms The

first, because of its polycationic nature, it forms an impenetrable coating around the cell when it interacts with anionic groups on the cell surface, preventing the transfer of essential solutes.) The outer membrane of gram – negative bacteria has been shown to be the location

of action using electron microscopy The permeabilizing action has been reported when

CH is protonated in somewhat acidic circumstances, CH, on the other hand, has a reversible permeabilizing effect

The second approach includes penetrating into the cell nucleus and suppressing RNA and protein creation, which has been observed and demonstrated to be effective against bacteria [18]

1.2.4 Applications of chitosan in food industry:

CH has a wide range of applications in the food sector due to its biological activity and human safety CH's antioxidant and antibacterial characteristics allow it to be used to extend the shelf life of foods, while its outstanding emulsifying properties allow it to be used in a variety of applications CH can be utilized as a functional component against bacteria and can be used to replace synthetic surfactants in food technology, CH can also

be employed as a functional ingredient in the treatment of hypercholesterolemia, hypertension, and inflammation, as well as for the encapsulation of nutrients in the development of functional foods CH's polyelectrolyte and flocculant properties allow it to

be used as a beverage clarifying agent as well as for preliminary purification of drinking water

1.3 Overview about cinnamaldehyde:

Next to black pepper, cinnamon (Cinnamomum zeylanicum, Nees in Wall) is one of the

world's oldest spices Cinnamon is primarily exported in the form of "cinnamon quills'' from Sri Lanka Cinnamon is an unusually intriguing plant in terms of phytochemistry The chemical makeup of volatile oils extracted from the bark, leaf, and root bark varies greatly The principal constituents of each oil are CNM (in the bark oil), eugenol (in the leaf oil), and camphor (in the camphor oil) (in the root-bark oil) [19] Its historical usage included flavoring, food preservation, medications, and even as an embalming ingredient Cinnamon

is nowadays used primarily in foods and fragrances It is used as a flavoring element in a variety of products, including baked goods, beverages, meat meals, cereals, and fruit preparations, and is recognized for conveying a sense of sweetness and warmth in cuisine Perfumes, cosmetics, shampoos, and toilet soap all contain oil extracted from the bark [20]

Figure 1.2 Extract CNM from cinnamon (in the bark oil)

Cinnamon may provide health benefits, according to accumulating data in the scientific literature, particularly in correcting poor blood glucose management caused by type 2 diabetes and obesity (antidiabetic agent) Furthermore, it has cholesterol-lowering characteristics, blood pressure-lowering effects, anti – inflammatory, antioxidant properties, antibacterial properties, and anticancer qualities CNM, a major component of cinnamon, has been studied separately for health advantages similar to those listed above

A yellow oily liquid CNM (C6H5CH = CHCHO) [21] is an organic compound with a strong odor of cinnamon, and sweet taste, occurring naturally primarily as the trans – isomer, a powerful antibacterial molecule found in essential oils like cinnamon and it is the main component of cinnamon [22] CNM makes up to 90 % of the essential oil of cinnamon bark [23] The chemical structure of CNM illustrates in [20, Fig 1.3.]

Figure 1.3 Chemical formula of CNM

Antibiotic resistance to currently available antibiotics has emerged as a worldwide health concern, necessitating the development of new antibacterial strategies There has

been renewed interest in developing antibacterial agents from natural sources, and trans – CNM is one such naturally occurring molecule that has gotten a lot of attention recently Trans – CNM has been proven to have significant antibacterial action, as well as a variety

of other therapeutic qualities, and is a fascinating hit compound from which a number of derivatives have been produced CNM has been shown to exhibit antibacterial activities

against a variety of Gram – positive and Gram – negative bacteria, including Escherichia coli, Bacillus subtilis, Staphylococcus spp., Listeria spp and Salmonella spp., Lactobacillus sakei, Campylobacter jejuni, Vibrio spp., Pseudomonas spp., Porphyromonas gingivalis, Streptococcus pyogenes and Cronobacter sakazakii Influence

of CNM on bacterial ATPases has been studied as a potential mechanism of action for its antibacterial capabilities The capacity of trans – CNM to inhibit membrane bound

ATPases in E coli and L monocytogenes was examined In E coli cell membranes, higher

concentrations of trans – CNM resulted in a higher inhibition of ATPase function [24] Moreover, CNM has been shown to inhibit yeasts and filamentous molds by inhibiting ATPases, altering membrane morphology and integrity, and inhibiting cell wall

biosynthesis [25] Treatment of A flavus with CNM resulted in a decrease in spore

germination as well as significant alterations in the morphology and ultrastructure of hyphae and spores, according to Xie et al [26] CNM had 100 % antifungal indices against

both Coriolus versicolor and Laetiporus sulphureus CNM, given as a gas, was also observed to slow the hyphal extension of A niger Han – Chung et al discovered that CNM had antifungal properties against a variety of harmful fungus (Colletotrichum gloeosporioides, Rhizoctonia solani, Fusarium solani and Ganoderma austral) [27] CNM,

which is found in nature, provides broad-spectrum antifungal activity and is safe for the environment [28] Therefore, they offer a lot of promise for food preservation

1.4 Overview of microbial strain and molds used in this research

 Escherichia coli

E coli, a member of the Enterobacteriaceae bacterial family, is the most common

commensal occupant of humans and warm-blooded animals' gastrointestinal systems, as well as one of the most important pathogens It lives in a mutually beneficial relationship with its hosts as a commensal, and it rarely causes disease It is, nonetheless, one of the most prevalent human and animal infections, as it causes a wide range of diseases in both

humans and animals E coli is an essential host organism in biotechnology because of its

unique qualities, such as simplicity of handling, availability of the entire genome sequence,

and capacity to grow in both aerobic and anaerobic conditions E coli is the most widely

utilized microbe in the field of recombinant DNA technology, and it is used in a wide range

of industrial and medical applications

Prior to the discovery of specific virulence factors in pathogenic strains, E coli was

predominantly categorized based on serologic detection of O (lipopolysaccharide, LPS)

and H (flagellar) antigens E coli strains are classified as pathogenic or nonpathogenic

depending on the kind of virulence factor present and the clinical symptoms experienced

by the host: there are at least seven primary pathotypes for enteric E coli, and three pathotypes for extraintestinal E coli (ExPEC)

E coli with enteropathogenic potential (EPEC) EPEC was the first E coli pathotype to

be identified Despite the fact that significant outbreaks of EPEC – related baby diarrhea have virtually disappeared in developed nations, EPEC remains a major cause of potentially deadly child diarrhea in impoverished countries The methods by which EPEC induced diarrhea were unclear for decades, and this pathotype could only be discovered by O:H serotyping Since 1979, however, major breakthroughs in our understanding of the pathogenesis of EPEC diarrhea have been made, and EPEC is currently one of the best –

understood pathogenic E coli strains [29]

EPEC strains primarily cause diarrhea in children and animals, especially in settings of low cleanliness Hemorrhagic colitis is caused by the bacterium EHEC and is spread through food, also known as HUS EHEC strains release Shiga – like toxins that are similar

to those produced by Shigella dysenteriae, making them the most virulent diarrhoeagenic

E coli strains discovered to date ETEC are the most common diseases in people of all

ages that cause mild to severe watery diarrhea in travelers EAEC strains have been associated to diarrhoeal sickness epidemics over the world and have been linked to prolonged diarrhea in humans EAEC is the world's second most prevalent cause of traveler's diarrhea, and it's found in the stomachs of asymptomatic individuals In children

in underdeveloped countries and HIV – positive patients, EAEC is usually linked to diarrhea [30]

The strain used in this experiment is Escherichia coli ATCC 8739 provided by Saigon

as a source of sustenance Filamentous fungus release a variety of enzyme combinations that are specifically tailored to the polysaccharide they encounter [31]

Due to their potential in plant biomass degradation and a variety of industrial

applications, Black Aspergilli species have been isolated from all over the world A niger

can grow in a wide temperature range of 6 – 47°C, but prefers temperatures around 35 –

37°C, the water activity limit for growth is 0.88 A niger can grow in a pH range ranging

from 1.4 to 9.8 These properties, as well as the abundant generation of spores that are dispersed by the air, ensure the species' extensive occurrence, with a higher frequency in

warm and humid environments [32] Aspergilli, like other common soil fungi found in a

variety of settings, can produce a wide range of enzyme combinations and breakdown a

wide range of polysaccharides When you consider that many Aspergilli have high levels

of protein secretion and good fermentability, it is no surprise that this fungus has found applications in industries like food and feed, pulp and paper, biofuels, biodegradable plastics, and textiles, as well as serving as a host for heterologous protein production

Especially, in food industrial, A niger applied in production of citric acid since 1919 and

had a great attribution in food and beverage productions

A niger is typically regarded as a non-harmful organism However, when people are

exposed to large levels of spore dust, hypersensitivity reactions have been observed in a

few occasions A niger is a non – pathogenic fungus found in large quantities in nature Its spores come into contact with humans on a daily basis and do not cause illness A niger

can only enter the human body as an opportunistic invader under rare conditions, and most

of these people have a history of serious illness or immunosuppressive drugs [33] In

foodborne system, Aspergillus niger can be discovered in a variety of plant and processed food products, including grapes, cereals, coffee, and derivative products [34] Aspergillus niger used in this experiment had been isolated and grown for two weeks on potato dextrose

agar ourselves and identified at the Center for Bioscience and Biotechnology of the University of Science

 Aspergillus fumigatus

The fungus Aspergillus fumigatus is part of the Aspergillus genus It is one of the most common Aspergillus species to infect people with weakened immune systems There are around 300 species of Aspergillus in the genus [31] Conidiophores produce thousands of

minute grey – green conidia (2–3 m) that easily get airborne in fungus colonies

The species Aspergillus fumigatus is remarkable in that it can survive temperatures

ranging from 20 to 50oC [35], with conidia surviving at 70oC (158oF) – conditions it frequently finds in self – heating compost heaps Its spores are widely distributed in the environment, and everyone inhales hundreds of spores every day, in healthy persons, the