Luận án tiến sĩ: Preparation and characterization of active coating solution based on chitosan incorporated with natural extracts for preserving harvested agricultural produce

Gần đây, việc kết hợp polyphenol vào màng phủ chitosan dé tăng hoạt tính sinh học của mang đã và đang được nghiên cứu rộng rãi, tuy nhiên chưa có nghiên cứu nào trên sự kết hợp PBLE và S

VIET NAM NATIONAL UNIVERSITY - HO CHI MINH

UNIVERSITY OF SCIENCE

NGUYEN THI THUONG

PREPARATION AND CHARACTERIZATION OF ACTIVE

COATING SOLUTION BASED ON CHITOSAN

INCORPORATED WITH NATURAL EXTRACTS FOR

PRESERVING HARVESTED AGRICULTURAL PRODUCE

DOCTORAL THESIS

VIETNAM NATIONAL UNIVERSITY — HO CHI MINH CITY

UNIVERSITY OF SCIENCE

NGUYEN THI THUONG

PREPARATION AND CHARACTERIZATION OF ACTIVE

COATING SOLUTION BASED ON CHITOSAN

INCORPORATED WITH NATURAL EXTRACTS FOR PRESERVING HARVESTED AGRICULTURAL PRODUCE

Speciality: Theoretical and Physical Chemistry

Code: 9440119

Reviewer 1: Assoc Prof Dr Huynh Dai Phu

Reviewer 2: Assoc Prof Dr Ton That Quang

Reviewer 3: Assoc Prof Dr Hoang Thi Kim Dung

Independent reviewer 1: assessment waiver

SUPERVISOR

1 Assoc Prof Dr Ha Thuc Chi Nhan

2 Assoc Prof Dr Bach Long Giang

HO CHI MINH CITY - 2023

ORIGINALITY STATEMENT

I declare that PhD thesis specialized in Theoretical and Physical Chemistry, with

title “Preparation and characterization of active coating solution based on chitosan

incorporated with natural extracts for preserving harvested agricultural produce” isthe result of scientific work which is that of author alone under the co-supervision

of Assoc Prof Dr Ha Thuc Chi Nhan, University of Science, Vietnam NationalUniversity, Ho Chi Minh City, and Assoc Prof Dr Bach Long Giang, Nguyen TatThanh university.

The results of the thesis provided here are completely honest, correct, and do notplagiarize from domestically and internationally published works

First and foremost, I offer my sincerest gratitude to my supervisors Assoc Prof Dr.

Ha Thuc Chi Nhan and Assoc Prof Dr Bach Long Giang who have beensupporting me throughout the course of my research with their patience, kindness,valuable advice, guidance, encouragement and comments

I would like to thank Nguyen Tat Thanh university which supporting me chemicals,equipment, and funding Besides, I would like to acknowledge all members in theInstitute of Technology Application and Sustainable Development and Hi-techInstitute — Nguyen Tat Thanh University for supporting me

Additionally, I would like to thank Dr Bui Thi Phuong Quynh at Faculty of

Chemical Engineering and all my friends at the Center for German-Vietnamese

Technology Academy, Ho Chi Minh University of Food Industry for providing mewith the identification and characterization of natural extracts

I would like to thank Vingroup Innovation Foundation (VINIF) and Viet

Nam National University Ho Chi Minh City which offered scholarship for me toafford sample analyses Also, I would like to thank grants from Kurita Water and

Environment Foundation (KWEF) and Youth Development Science andTechnology Center - Ho Chi Minh Communist Youth Union and Department of

Science and Technology of Ho Chi Minh City

Finally, an honorable mention goes to my family and friends, withoutwhose understanding and support this thesis would not have been completedsuccessfully

CHAPTER 1 LITERATURE OVERVIEW LH HH ngư 3

1.1 Effect of important factors effect on shelf life of the harvested fruits and

1.4.1 Overview of polyph€nOlÌ - -c + x11 1v vn ng ng rưy 15

1.4.2 Interaction between chitosan and polyphenols ‹ -«+<s 18

1.4.3 Functions and advantages of edible films/coatings 20

1.5 Recent reports on chitosan-based active films incorporated with naturalCXÍẨTACÍ LG LG SĐT 10 50k E050 0 6k EEED 22

1.6 Introduction of natural €X{TAC( << << 211111 eeeeeeeeeeeeeees 25

1.6.1 Piper Đefle ÌL Ă SG TH kg ng 25

1.6.2 Sonneratia ovata (SOnn€rafiaC€A€) cv kg 26

1.7 Overview of Banana (Musa acuminate) and current research on preservative

1.7.1 Composition of bana1na eseeeeeeceeeesceeceseeseeseeseesecseeseeeeseeeeaeeaeees 27

1.7.2 Changes in color and physiochemical properties of banana duringTIPCNING 108 29

1.7.3 Recent reports on the extending the shelf life of banana 36

1.8 Overview of King orange and its DfeS€TVAfIOT - 55-55 +<++se+ssss+ 37

1.8.1 Development and biochemical composition of King orange 37

1.8.2 Changes in color and physiochemical properties of King orange during

0901115207777 Ö 39

1.8.3 Recent reports on extending the shelf life of oranges - 42

CHAPTER 2 EXPERIMENT AND METHODS - c5 se csscssssessee 43

2.1 Chemicals and mat€TI14ÌS s1 E119 93 91 9v vn ng cry 43

2.2 Extraction, identification and characterization of natural extracts 45

2.2.1 EXtraction DTOC€SS SH SH HH rưy 45

2.2.2 Characterization of chemical constituent - 55555 «<< £+xx48

2.2.3 Determination of moisture, ash degree, and extraction yield 49

2.2.4 _ Determination of total polyphenol and flavonoid contents 50

2.2.5 Evaluation of antibacterial and antioxidant activities of extract 31

2.3 Preparation of coating solution and film oo lees ceeeseeeeeeeeenees 52

2.4 Characterizations of coating ẨiÏIm - s6 + SsseEsesereeeeeesereresre 54

2.4.1 SEM, color, UV-vis, and optical properties - - ««-s«+<<+ 54

2.4.2 ATR-FTIR and XR - G9 TH ghi ưn 55

24.3 (TGA v 55

2.4.4 Contact angle, moisture, solubility, and swelling -«< + 56

2.4.5 Water vapor permeability sgk key 56

2.4.6 Mechanical belhaVIOT - «- «s1 vn ngư 57

2.4.7 Antibacterial and antioxidant activities c5 cssxseeeerse 57

2.5 Release assays of polyphenols from as-prepared active films 58

2.6 Physiochemical determination of coating soÏutIOn - « «<+<x<++ 59

2.7 Evaluation of coating efficiency on banana and orange peel 60

2.8 Application of coating solution to preserve banana and King orange 60

2.8.1 Observation of peel COLOUL cceesceesscestecesceeeeeesneceseeceseeesaeessaeenaees 62

2.8.2 Determination of titratable total soluble sugar, acidity, and pH 62

2.8.3 Determination of Weight ÏOSS - sgk key 63

2.8.4 Determination respiration TAf - - 5 + + SH HH net 63

2.8.5 Determination vitamin CC - «+ x1 ng rưy 63

PB ốc na ca 64

CHAPTER 3 RESULTS AND DISCUSSION - Lee, 65

3.1 Characterization of chitosan film incorporated with P betel extract 65

3.1.1 Identification and charaCf€T1ZAfIOII - Gv ngư 65

3.1.1.1 Moisture, ash degree, and extraction y1eÌd - «« -««+<<e+s+ 65

3.1.1.2 Total polyphenol content ccc eesceeecccesseceneeeeeeeeseeceseeesaeeeseeeenees 66

3.1.1.3 Characterization of phenolic compounnds - ‹«-s-««++<s<+++ 66

3.1.1.4 Antibacterial and antioxidant properties - «5525 << + << <+++ 67

3.1.2 Properties of coating filMS - s6 vn nườn 69

3.1.2.1 The appearance of the modified chitosan films 69

3.1.2.2 ATR-FTIR analyses - - Ăn 1x HH kg re, 71

3.1.2.3 X-ray diffraction anaÌySIS s cv vn HH ng ng re 73

3.1.2.4 Mechanical properties - G5 1H kg ng key 74

3.1.2.5 Thermogravimetric Analysis (TGA) cà seeirey 75

3.1.2.6 Wettable pFOD€TẨI€S - Ăn HH ng HH ng 71

3.1.2.7 Light transmittance of the Íilim - - «+ «+ Essseeeskeree 79

3.1.2.8 Antimicrobial and antioxidant activities - c5 sve 80

3.2 Characterization of chitosan film incorporated with Sonneratia ovata leaf

OXULACE ee 6-35 83

3.2.1 Identification and charaCf€T1ZAfIOII - Gv vn nên 83

3.2.1.1 Moisture, ash degree, and efẨICI€TCy - - 6+ sersee 83

3.1.2.2 _ Total phenolic and flavonoid content -««+<-«<s<<c++ 84

3.1.2.3 Evaluation of chemical compounds - - «+ s «<< ss+seexs+ 85

3.1.2.4 Antibacterial and antioxidant properties - « -«<++<<+++ 85

3.2.2 Properties of CS-SOE coating TÏIm§ - s5 c xxx sseersseeree 87

3.2.2.1 SEM evaluation cv HH HH ng, 87

3.2.2.2 ATR-FTIR anaÌySIS - - G25 11+ 1E vn HH nh ngư 88

3.2.2.4 TGA anaÏyS€S Ăn TH TH HH HH kg ky 90

3.2.2.5 Tensile mechanical property - 5 5- + x tk seeeeeeeeeerseesere 92

3.2.2.6 Optical properties - Gv SH HH rưy 94

3.2.2.7 Moisture, solubility and swelling degree and water vapor

3.2.2.8 Antibacterial activity ch kg ky 96

3.3 Comparative characterization and release study of chitosan film addivitedwith Piper betel L extract and Sonneratia ovata leaf eX{raCf - -«++ 99

3.3.1 Characterization of ÍIÏ1TS - - << << << << E1 EEEEEEEEEEkkkkEEEEEkEE 3353333552 99

3.3.1.1 Visual appearance, surface morphology, and color of film 99

3.3.1.2 ATR-FTIR and XRD analyses 5 5 1+ svEssesrsersrree 101

3.3.1.3 ThermostabilIfy «nh nh HH HH nhà 103

3.3.1.4 Film thickness, mechanical properties, and water vapor transmission

¬— 105

3.3.1.5 UV-protection property -ĩ ng nrnràt 108

3.3.1.6 Antibacterial and antioxidant properties - -. 5< c+<<<+<c<<«2 109

3.3.2 Release of polyphenOÌS + c 11v vn ng giết 114

3.4 Physicochemical properties of coating soÏufIOn - «+-«<+<e<<s«2 122

3.5 Application for banana preservation 25555 + +vvEEeeeereerereee 123

3.5.1 Coating efficiency of coating sỌutIOn ««++«s<++s++seessss 123

3.5.2 Morphology of uncoated and coated banana peeÌs - 125

3.5.3 Changes in the quality of coated bananas - «<< x+<s«+ 126

3.6 Application for King orange pT€S€TVAfION - 5 55 + xe rerrke 135

3.6.1 Coating efficiency of coating soÏu(IOH «-+-«c<«cesec+seeske 135

3.6.2 Morphology of uncoated and coated orange peels - 136

3.6.3 Changes in the quality of coated OraniØ€S - «+ ssssseesske 137

CHAPTER 4 CONCLUSION Ác vn HH HH ng ng 146

REFERENCES 1.00 ceceeccssecsseescesseesseescecscesseccesseesseessecsaecseessecsaesseesseesaessaesseesseesaees 149

LIST OF PUBLICA TIONS - ĐẶ LH HH HH Hit 167

TRANG THÔNG TIN LUẬN ÁN

Tên dé tài luận án: Nghiên cứu chế tạo chế phẩm bảo quản trái cây có hoạt tính sinh học

trên cơ sở mang chitosan và chiét xuât tự nhiên

Ngành: Hóa lý thuyết và Hóa lý

Mã số ngành: 9440119

Họ tên nghiên cứu sinh: Nguyễn Thị Thương

Khóa đào tạo: 2018

Người hướng dẫn khoa học: PGS TS Hà Thúc Chí Nhân và PGS TS Bạch Long Giang

Co sở đào tạo: Trường Đại học Khoa học Tự nhiên, ĐHQG TP.HCM

1 TÓM TẮT NỘI DUNG LUẬN ÁN:

Chiết xuất lá trầu không (PBLE) có hàm lượng polyphenol cao chứa chủ yếu là axit

gallic, eugenol và hydroxychavicol Và vi vậy, PBLE có hoạt tính kháng khuẩn và kháng

oxi hóa mạnh Trong khi chiết xuất lá ban ôi (SOE) chứa hàm lượng polyphenol thấp hơn

PBLE chứa chủ yếu là luteoin-7-O-glucoside, axit gallic và luteolin Gần đây, việc kết hợp

polyphenol vào màng phủ chitosan dé tăng hoạt tính sinh học của mang đã và đang được nghiên cứu rộng rãi, tuy nhiên chưa có nghiên cứu nào trên sự kết hợp PBLE và SOE vào

màng phủ chitosan Vì vậy, mục đích của nghiên cứu này là đánh giá ảnh hưởng so sánh

của sự kết hợp chiết xuất trầu không (PBLE) và chiết xuất 14 ban ồi (SOE) trén tinh chat co

lý, tinh can hơi nước, tính can ánh sáng tử ngoại, hoạt tính khang khuẩn và kháng oxi hóa của màng phủ chitosan (CS) Những kết quả thu được cho thấy răng sự kết hợp PBLE có

thé cải thiện đáng kể hoạt tính kháng khuẩn, kháng oxi hóa, tính cản UV và tính cản hơi

nước trong khi sự kết hợp của SOE lại tăng tính chất cơ lý và tính én định nhiệt Kết quả còn cho thay sự đồng kết hop SOE va PBLE ở tỉ lệ khối lượng1.4:0.6 và 1:1 vào màng phủ chitosan (CS-SOE!4-PBLE?5) không chỉ cho thay tính cản hoi nước được cải thiện rõ rang trong khi vẫn duy trì tính cản UV, tính ồn định nhiệt, hoạt tính kháng oxi hóa và hoạt tính kháng khuẩn Màng phủ CS-SOE!-PBLE°S cho thấy hiệu quả đồng kết hợp tốt nhất trên việc cải thiện tính chất hóa lý và sinh học Ngoài ra, nghiên cứu sự phóng thích polyphenol cho thấy sự phóng thích polyphenol bị ảnh hưởng bởi môi trường thực phâm mô phỏng (Ethanol 50%: môi trường béo, ethanol 10%: môi trường kiềm, axit axetic 3%: môi trường

axit và HạO) và bản chất của chiết xuất kết hợp Cụ thể, polyphenol phóng thích nhanh

trong môi trường axit Sự phóng thích polyphenol từ mang phủ CS-SOE” chậm hơn so với

sự phóng thích polyphenol từ màng CS-PBLE? trong các môi trường thực phâm mô phỏng

được khảo sát Các kết quả cũng cho thấy các dữ liệu phóng thích polyphenol phù hợp với

mô hình Peleg và Kopcha Các kết quả tính toán từ mô hình Peleg đều cho thấy sự phù hợp với số liệu thực nghiệm Kết quả tính toán từ mô hình Kopcha cũng cho thấy sự phóng thích polyphenol từ màng CS-PBLE? theo cơ chế khuếch tán, trong khi đó màng CS-SOE? thể hiện cơ chế ăn mòn và sự tồn tại cả hai cơ chế khếch tán và ăn mòn đối với sự phóng

thích polyphenol từ màng CS-SOE!*-PBLE®° Kết qua tính toán từ mô hình

Korsmeyer-Peppas cho thấy sự phóng thích polyphenol có thể theo cơ chế quasi-Fickian trong các môi trường thực phẩm Ngoài ra, chế phẩm từ chitosan kết hợp với PBLE va SOE cũng được ứng dụng trong bảo quản chuối Laba (đại diện cho trái cây có đỉnh hô hấp) và cam sành (đại diện cho trái cây không có đỉnh hô hấp) Các tính chất của chế phâm cũng được đánh

giá trước khi ứng dụng trong bảo quản Kết quả cho thấy sức căng bề mặt và độ nhớt của

dung dich chitosan giảm khi thêm SOE và PBLE Ngoài ra, dung dịch SOE’,

CS-SOE!-PBLE! và CS-SOE!*-PBLE°° cho hiệu quả phủ trên chuối Laba tốt hơn những dung dịch khác khi hệ số bám dính và hệ số phân tán cao nhất Kết quả phân tích SEM cũng cho thay sự phân tán của lớp màng phủ CS-SOE?, CS-SOE!-PBLE! và CS-SOE!*-PBLE”* cao

hơn các sự xử lý khác trên chuối Việc sử dụng dung dich phủ CS-SOE!-PBLE?° có thể

bảo quản chuối đến 10 ngày ở 20 °C và 64% RH khi lớp mang phủ này có thé giảm sự hô

hấp ở trái và vì vậy duy trì sự mat khối lượng, thay đối màu sắc vỏ, tổng hàm lượng chat

rắn, và độ axit của chuối Trong khi các dung dịch CS, CS-SOE!-PBLE!, va

CS-SOE!4-PBLE°° cho thấy hiệu quả phủ trên cam sành cao hon so với những sự xử lý khác dựa trên đánh giá hệ số phủ và hệ số bám dính trên vỏ cam Hình ảnh bề mặt và mặt cắt ngang được quan sát thông qua phân tích SEM cũng cho thấy sự phân tán tốt của các dung dịch CS, CS-SOE!-PBLE!, và CS-SOE!-PBLE? cao hơn những dung dịch phủ khác Chế phẩm CS-SOE!*-PBLE”* có thé tăng thời gian bảo quản cam sành đến 29 ngày ở 20 °C và 64%

RH cao hơn 3 lần so với cam không được phủ khi lớp phủ CS-SOE!*-PBLEĐS có thể duy

trì độ mat khối lượng, sự thay đổi màu sắc trên vỏ, tổng hàm lượng đường hòa tan và

vitamin C.

2 NHỮNG KET QUA MỚI CUA LUẬN ÁN:

Luận án chứa đựng nhiều kết quả mới và nội dung chính của luận án được công bố trên các tạp chí khoa học uy tín trên thế giới Trong luận án này, các kết quả mới có thê được liệt kê như sau:

- _ Nghiên cứu kết hợp riêng lẻ chiết xuất lá ban 6i và chiết xuất trầu không trên tính chất sinh học, tính chất cản, tính chất vật lý và tính chất cơ lý của màng phủ chitosan.

- _ Đánh giá sự đồng kết hợp của cả hai chiết xuất lá ban ôi và chiết xuất trầu không lên các tính chất của màng phủ chitosan.

- Nghiên cứu sự phóng thích polyphenol từ màng phủ chitosan hoạt tính chứa chiết xuất ban 6i và chiết xuất trầu không trong các môi trường thực phẩm mô phỏng (Ethanol 95%:

môi trường béo, ethanol 10%: môi trường kiềm, axit axetic 3%: môi trường axit và H2O)

dé dự đoán thời gian sử dụng của màng.

- Ung dụng chế phẩm sinh học từ chitosan kết hợp với chiết xuất ban 6i và trau không

trong bảo quản hai loại trái cây đại diện cho loại trái có đỉnh hô hấp (chuối Laba) và trái không có đỉnh hô hấp (cam sành).

3 CÁC UNG DỤNG/ KHẢ NANG UNG DỤNG TRONG THUC TIEN HAY

NHỮNG VAN DE CON BO NGO CAN TIẾP TỤC NGHIÊN CỨU

Nghiên cứu này cung cấp những kết quả sơ bộ (nghiên cứu thí điểm) trong phạm vi phòng thí nghiệm trước khi được ứng dụng thương mại hóa chế phẩm này để tăng thời gian bao

quản của nông sản sau thu hoạch Trong tương lai, chúng tôi sẽ mở rộng nghiên cứu theo

các hướng sau:

- Huong 1: Nghiên cứu ảnh hưởng độ tan của chitosan và chiết xuất ở các pH khác nhau

trên quá trình tạo mang, tính dong nhat, hiệu quả phủ va tính chat kháng oxi hóa của màng.

il

- _ Hướng 2: Nghiên cứu ảnh hưởng của nhiệt độ và độ âm trên hiệu quả bảo quản nông

sản sau thu hoạch.

-_ Hướng 3: Nghiên cứu tác động của vi sinh vật trên chuối và cam sành được phủ bởi lớp phủ chitosan và chiết xuất trong suốt quá trình lưu trữ.

1H

Supervisor: Assoc Prof Dr Ha Thuc Chi Nhan and Assoc Prof Dr Bach Long Giang

At: VNUHCM - University of Science

1 SUMMARY:

Piper betle L leaf extract (PBLE) with TPC of 457.01 + 11.25 mg GAE/g mainly

containing gallic acid, eugenol and hydroxychavicol exhibit greater antibacterial and

oxidant activities than Sonneratia ovata (Sonneratiaceae) leaf extract (SOE) with TPC of 166.53 + 4.66 mgGAE/g and TFC of 132.33 + 2.823 mgRE/g, which mainly consist of luteolin-7-O-glucoside, gallic acid, and luteolin A plethora of studies on the incorporation

of natural extracts into chitosan film was investigated However, the addition of PBLE and

SOE leaf extracts into chitosan film has not yet been reported Therefore, the current work

aims to evaluate comparative effects of incorporating of PBLE and SOE polyphenols

separately and in combination on the mechanical behavior, water permeation barrier, protective property, and antioxidant and antimicrobial activities of chitosan coating film Results have shown that the single application of PBLE provided blend coating film with

UV-strong antibacterial, antioxidant, UV- and water-barrier properties yet worsened the

mechanical behavior Meanwhile, the incorporation of SOE alone contributed to chitosan

film significant enhancement in mechanical and thermostability properties Therefore, the

co-addition of PBLE and SOE at an appropriate rate not only significantly enhanced the

water permeation barrier but also maintained the UV-protection, thermostability,

antibacterial and antioxidant properties compared to the use of single extract, thus beingconsidered as a potential material for food preservation The CS-SOE!3-PBLE?° coatingfilm presented the best synergistic effect on the improvement of physio-mechanical and

biological properties of the blend films Furthermore, the release study revealed that

polyphenols release was influenced by different food simulants (distillated water, 3%

acetic acid, 10% ethanol, and 50% ethanol, respectively, representing for aqueous, acidic,

alkaline, and fatty foods) and incorporated extract’s nature The polyphenols showed rapid

release in acetic acid environment due to chitosan solubility In the simulated conditions,

the CS-SOE? films exhibited slower release of polyphenols than CS-PBLE? films The

results indicated that the release data were well-fitted with Peleg’s and Kopcha’s models, and the polyphenol release was mainly controlled by quasi-Fickian diffusion mechanism in

all food simulants Furthermore, chitosan coatings supplemented with SOE and PBLE

were also applied for extending the shelf life of post-harvest Cavendish banana (Musa

acuminate) representing the climacteric fruit and King orange representing the

non-climacteric counterpart Present findings also showed that surface tension and viscosity of

CS solution significantly decreased by adding single or combination of SOE and PBLE.

Moreover, CS-SOE’, CS-SOE!-PBLE! và CS-SOE!*-PBLE®* coating solution provided

better coating efficiency on Cavendish banana skin (representing the climateric fruits) as

compared to other fomulations, as envidenced by the highest work of adhesion and

iv

spreading coefficient The coating thickness on fruit skin treated with CS-SOE”,

CS-SOE!-PBLE!, and CS-SOE!*-PBLE°*® was also found in higher values as compared to other

treatment CS-based edible coating co-additivated with SOE and PBLE was found to be

more efficient in banana preservation at 20 °C for 10 d as they may delay respiration rate

and retard banana ripening by maintaining weight loss, color change on peel, total soluble

sugar, and organic acid consumption For King orange preservation (representing the

non-climateric fruits), coating solutions such as CS, CS-SOE!-PBLE!, and CS-SOE!*-PBLE”°

provided higher adhesion and spreading coefficient than CS solution which was singly

incorporated with SOE and PBLE Surface and cross-sectional images of coating on fruit

skins also showed good dispersion and completely sealed stoma of fruits coated by CS,

CS-SOE!-PBLE!, and CS-SOE!*-PBLE®® Coating treatment could offer better storability

of king orange as compared to control sample CS-SOE!*-PBLE”® was found to be the

most efficient in king orange preservation at 20 °C for 29 d convinced by maintaining weight loss, color change on peel, total soluble sugar, and vitamin C.

- The evaluation of co-addition of PBLE and SOE on the properties of chitosan film.

- The release study of polyphenols from active packaging films in different food

simulants (distillated water, 3% acetic acid, 10% ethanol, and 50% ethanol, respectively,

representing for aqueous, acidic, alkaline, and fatty foods) was performed.

- Comparative study on using chitosan coatings co-supplemented with SOE and PBLE

for extending the shelf life of Cavendish banana (representing the climateric fruits) and

King orange (representing the non-climateric fruits), has not found.

3 APPLICATIONS/ APPLICABILITY/ PERSPECTIVE

The present work provides preliminary results (pilot study) in laboratory before

utilizing the new edible coating-based chitosan incorporated with natural extracts for

industrial purposes in enhancing storability of other post-harvest fruit crops In the near

future, we will extend our research to the following topics

- Topic 1: Studying the effect of solubility of chitosan and extract at different pH on the forming process, homogeneity, coating efficiency, and antioxidant property of resulting

film.

- Topic 2: Studying the effect of storage temperature and humidity on the preservation of Cavendish banana and King orange.

- Topic 3: Evaluation of impact to post-harvest pathogens on Cavendish banana and

King orange with and without coating during the storage.

LIST OE TABLES

Table 1 1 Phenolic compounds in Piper betle L oil collected from provinces in

HH 32

Table 3 1 Moisture of leaf DOWT - -Q c 1 tk TH HH ng Hiện 65

Table 3 2 Moisture of PBÌLLE «t1 1 21 9319119119 11g ng Hàng nh nh gưệp 65

Table 3 3 Ash degree of crude PBILIE - + 11113 1119 111911 11 8x ng rey 66

Table 3 4 Characteristic parameters of HPLC analysis of the PBLE 67

Table 3 5 The mechanical properties of CS, PBLE', PBLE7, and PBLE? samples c.ccccccccssscsssssssseseseseseseseecssssesesesesesesescssaauesssesesesesesesesceesueeseseseseseas 75

Table 3 6 Degradative temperature and mass loss of PBLE, CS, PBLE!, PBLEZ, and CS-PBLE? samples cccccccscscssscscseseseseseseseseecscseseseseseseseseacseseseseseeees 77

CS-Table 3 7 The water solubility and swelling degree of CS, CS-PBLE!, CS-PBLE’, E000 :)019060 71000) T8 79

Table 3 8 Results of time-dependent assays for Pseudomonas Aeruginosa 82

Table 3 9 ICso values of CS-PBLE!, CS-PBLE’, and CS-PBLE? samples 83

Table 3 10 Moisture of leaf pOWeT .- 5 5 SH HH HH HH rưệt 84

Table 3 11 Moisture of SOE - cà nxHHHTHnHnHn HH Hàng gưệt 84

Table 3 12 Ash degree of crude SOE - - c2 11231991119 1 9 1v kg cư, 84

Table 3 13 Mechanical behavior of control film, SOE!, SOE? and

Table 3 14 Moisture content, water solubility and swelling degree of control film,

CS-SOE!, CS-SOEZ and CS-SOEỶ - + S32 SESE2E£E£E2EEEeEeEerrkrrkrrrrrrrrrrree 96 Table 3 15 Antibacterial activities of control film, CS-SOE!, CS-SOE? and CS- SOE? against S aureus and P acruginOsa cccccccscsssesesesesesesessescscscscseseseseseeeecsees 98

Table 3 16 TPC and TFC of chitosan film incorporated with SOE 98

Table 3 17 The color (L, a, b), total color difference (AE), and white index (WJ) of

CS, CS-SOE?, CS-PBLE’, CS-SOE!*-PBLE°®, and CS-SOE!-PBLE' films 100

vi

Table 3 18 Thickness, mechanical behavior, and WVTR of CS, SOE?’, PBLE2, CS-SOE!*4-PBLE?, and CS-SOE!-PBLE! films ¿2255552 108

CS-Table 3 19 Antimicrobial activity of chitosan films incorporated with extract

against P aeruginosa the liquid incubation f€SS - 5c s + sssvesssersrsee 112

Table 3 20 ICso values of CS-PBLE!, CS-PBLE’, and CS-PBLE? samples 114

Table 3 21 Total polyphenol compounds (TPs) released, total polyphenolcompounds retained (TPCR), and percentage of polyphenols retention in filmmatrix as immersed in different simulants (E10%: ethanol aqueous solution (10%,v/v), E50%: ethanol aqueous solution (50%, v/v), AA3%: acetic acid aqueous

solution (3%, w/v), and distillated water (H2O)), . - 555 Sex 115

Table 3 22 Kinetic parameters of Korsmeyer-Peppas model (k (h}): rate constant

and n: diffusional exponent) at 25 °C in different solvents (E10%: ethanol aqueoussolution (10%, v/v), E50%: ethanol aqueous solution (50%, v/v), AA3%: acetic acidaqueous solution (3%, w/v), and distillated water (H2O)) «0.0 eeeeeseeceeeeeeneeeeees 118Table 3 23 Kinetic parameters of Peleg’s model at 25 °C in different solvents(E10%: ethanol aqueous solution (10%, v/v), E50%: ethanol aqueous solution

(50%, v/v), AA3%: acetic acid aqueous solution (3%, w/v), and distillated water

(H2O)) Constant kị (min.mgTM!.GAE.g"! polymer) is related to the release rate at the

beginning of the process kz (g polymer.mg!GAE) relates to the asymptotic value

which can be related to the equilibrium value Moo is the inverse of kz and is thenumber of polyphenols released at equilibrium (mg GAE-g-1 polymer) 120

Table 3 24 Parameters of Kopcha model for polyphenolic release from the films in

different food stimulants (E10%: ethanol aqueous solution (10%, v/v), E50%:ethanol aqueous solution (50%, v/v), AA3%: acetic acid aqueous solution (3%,w/v), and distillated Water (H)) - - LG 0111111112211 1192111190 111111111821 ke 122

Table 3 25 Effect of natural extracts on physicochemical properties (surface

tension, viscosity, and pH) of chitosan sỌufIOT8 - - 555k ssesersereeree 123

Table 3 26 Effect of natural extracts on work of adhesion and spreading

coefficient of chitosan polymer solutions on banana skins - «+ «<: 125

vii

Table 3 27 The amount of produced CO; (mgCO2/kg.h) from banana uncoated and

coated with CS, CS-PBLE”, CS-SOE2, CS-SOE!-PBLE!, and CS-SOE!*-PBLE”®

during 10 days storage at 20 °C oo eee eeccescecsseeceneceeneceeeceaeecsaeceseeceanecsaeeesaeeeseeeeaes 130

Table 3 28 Recent reports on using eco-friendly coating for banana preservation

Table 3 30 Amount of vitamin C of orange without coating and coated with CS,

CS-SOE2, CS-PBLE’, CS-SCE!-PBLE!, and CS-SCE'*-PBLE®® at 20 °C during

SUOTAQE oo 144

Table 3 31 National reports on using eco-friendly coating for orange preservation

Vili

LIST OF FIGURES AND SCHEMES

Figure 1 1 Chemical structure of chitin and chitosan «<< <<+<<<++ 13

Figure 1 2 Classification of polyphenols [34] ccccesceesseceeeeeeeeeeteeeeeeenseeesees 16Figure 1 3 Polyphenols free radical scavenging by hydrogen-transfer mechanisms

Figure 1 4 Illustration of bactericidal mechanism of polyphenols against negative and Gram-positive bacteria [3⁄⁄{] eeseseeeseceseeesecseeeeeesesseeeaeenseens 18

Gram-Figure 1 5 Illustration of interaction mechanisms between polyphenols (phenolicacid and flavonoids) with chitosan film (A) and chitosan-starch film (B) [36] 19

Figure 1 6 Phenolic compounds in Piper betel L collected from Hoc Mon, Ho ChiMinh City da 26

Figure 1 7 Ripening stages of banana Stage 1 = thoroughly green peel; Stage 2 =green peel with traces of yellow; Stage 3 = more green than yellow; Stage 4 = more

yellow than green; Stage 5 = yellow peel with traces of green; Stage 6 = thoroughly

yellow peel; Stage 7 = yellow with brown spots [72,73] -.-« -cc<ssee+ssees 29

Figure 1 8 Outline of biochemical pathways likely to be involved in respiration

and carbohydrate metabolism in ripening bananas [74] .-. -««++-«<+>+ 30

Figure 2 1 Sonneratia ovata Ï€AV€S - ng TH HH ng Hy 43

Figure 2 2 Piper betle L Ï€aV€S - c1 1111 1H HH TH ng ng 44Figure 2 3 Visual images for the coating progress of solutions on banana 62Figure 3 1 Chromatogram of the ethanol extract of PBLE -«++-s«++ 67

Figure 3 2 Inhibitive zone diameter of PBLE and ampicillin against S aureus, E.coli, P aeruginosa, and S fyDhiTfHHYÏHH cv ng key 68Figure 3 3 Visual images of BPLE and ampicillin against (a) S typhimurium, (b)

P aeruginosa, (C) S aureus, and (d) E COLi cc ceeccccccescceeesscceeseceesneeeessseeeesnseeeseeeess 68

Figure 3 4 Visual images of CS, CS-PBLE', CS-PBLE’, and CS-PBLE3 coating

FULT 0 — Ò-:-:Õ-4 70

1X

Figure 3 5 Surface morphology of (a) CS, (b) CS-PBLE!, (c) CS-PBLE2, and (d) CS-PBLE? coating films ¿- ¿- 2 S22 232E2E£E2EEEEEEEEEEEEEEEEekekekerrkrrkrrrkrrrrerree 70

Figure 3 6 ATR-FTIR spectra of (a) PBLE, (b) CS, (c) CS-PBLE!, (d) CS-PBLE’,

and (e) CS-PBLE? samples ¿-¿-¿- ¿5+ 2E 32323 StEEEEeEeEeEEEEEEEErrkrrrrerererrrrrree 72 Figure 3 7 XRD diagrams of CS, CS-PBLE!, CS-PBLE7, and CS-PBLE? samples

—— .A 73

Figure 3 8 (a) TGA and (b) DTG of PBLE, CS, PBLE', PBLE’, and PBLE? samples ccccccccssscssssssssesesesesesesescssesesesesesesesescasasussesesesesesesesescensneueeeseseseaess 76 Figure 3 9 The water contact angle of CS, CS-PBLE', CS-PBLE’, and CS-PBLE?

against P aeruginosa after films stored for 30 days at room temperature (27 °C) 82Figure 3 13 Inhibitive zone diameter of SOE and Ampicillin against S aureus andS000) ẲẦ 86Figure 3 14 Visual images of SOE, PBLE, and ampicillin against (a) P

aeruginosa, and (b) S AUTCUS - -.- Ă 111v HH TH HH HH ky 86

Figure 3 15 Visual appearance of control film (a), CS-SOE! (b), CS-SOE? (c) and Ằ® 9501 3 87 Figure 3 16 SEM of control film (a), CS-SOE! (b), CS-SOE? (c) and CS-SOE? (d).

Figure 3 19 Visual appearance and surface morphology of films: CS (al, a2),

CS-SOF? (b1, b2), CS-PBLE? (cl, c2), CS-SOE!*-PBLE®® (d1, d2), and

CS-SOE!-PBLE! (€1, €2)sccsssssscsssssssssssssssesssssssssssssssssssssssssessssusssssssssssssssssesssssssstsuisssusssssse 100 Figure 3 20 ATR-FTIR spectra of films: CS (a), CS-SOE7 (b), CS-PBLE? (c), CS-

SOE!-PBLE° (d), and CS-SOE!-PBLE! (€) c.cccccccccccscssesescseesescseesescseessseseeees 102

Figure 3 21 XRD analyses of CS (a), CS-SOE? (b), CS-PBLE? (c),

CS-SOE!4-PBLE°° (d), and CS-SOE!-PBLE! (e) films c.ccccccccccscssssesescssescscssescsesseseseeeeseaes 103 Figure 3 22 TGA analyses of CS, CS-SOE’, CS-PBLE?, CS-SOE!4-PBLE?, and

Figure 3 23 UV-vis transmittance spectra of CS, SOE’, PBLE”, SOE!4-PBLE°°, and CS-SOE!-PBLE! films c.ccccccccscsssssssesesescsescseseseseeeesesesesesees 109 Figure 3 24 Inhibition zone diameter of CS, CS-SOE?, CS-PBLE’, CS-SOE!4- PBLE®°, and CS-SOE!-PBLE'! films against S aureus, E coli, S typhimurium, and

CS-P ACTUQINOSA 0n Ả 111

Figure 3 25 Antibacterial activity of CS, CS-SOE’, CS-PBLE2,

CS-SOE!4-PBLE?, and CS-SOE!-PBLE'! films against P aeruginosa after films stored for 30

days at room temperature (27 C”) - - + + + x19 191119911 11 HH ng nếp 112

Figure 3 26 Percentage of total phenolic compounds releases from all films

(mCS-PBLE?; ACS-SOE’, eCS-SOE!*-PBLE?5) in different food simulants: 50% ethanol

(a), 10% ethanol (b), 3% acetic acid (c), and water (d) . - -<<<+ 117Figure 3 27 Application of Peleg’s model to experimental data for all films (mCS-

PBLE?; ACS-SOE’, eCS-SOE!4-PBLE?5) in different food simulants: 50% ethanol

(a), 10% ethanol (b), 3% acetic acid (c), and water (d) . - -<<<+ 121

Figure 3 28 Surface images of bananas peel uncoated (a) and coated with CS (b),

CS-SOE” (c), CS-PBLE? (d), CS-SOE!-PBLE! (e) and CS-SOE!4-PBLE?S (f) 126

Figure 3 29 Visual appearance of bananas uncoated and coated with CS, CS,

CS-PBLE’, CS-SOE’, CS-SOE!-PBLE!, and CS-SOE]4-PBLE? during 10 days storage

XI

Figure 3 30 Peel color of banana uncoated and coated with CS, PBLE2, SOE’, CS-SOE!-PBLE!, and CS-SOE!“*-PBLE°* during 10 days storage at 20 °C a-

CS-f means signiCS-ficant diCS-fCS-ference among storage periods (p < 0.05) 129

Figure 3 31 Weight loss of banana uncoated and coated with CS, PBLE’, SOE’, CS-SOE!-PBLE!, and CS-SOE!'*-PBLE”* during 10 days storage at 20 °C a-

CS-f means signiCS-ficant diCS-fCS-ference among storage periods (p < 0.05) 132

Figure 3 32 (a) Total soluble sugar (TSS), (b) Titratable acidity (TA), and (c) pH

of banana uncoated and coated with CS, CS-PBLE’, CS-SOE”, CS-SOE!-PBLE! and CS-SOE!4-PBLE® during 10 days storage at 20 °C a-f means significant

difference among storage periods (p < 0.05) .ccceesceeseeesceseeeeeseeeseeeseenseeseeneennees 134

Figure 3 33 Surface morphology of orange skin without coating (a) and coated

with CS (b), CS-SOEZ (c), CS-PBLEZ (d), CS-SOE!-PBLE! (e), and PBLE®® (Ÿ) St tt v21 21111 11111111111111111111111111111111111111111111111111111 111 C 137

SOE!4-Figure 3 34 Visual images of orange without coating and coated with CS,

CS-SOE’, CS-PBLE”, CS-SOE!-PBLE!, and CS-SOE!4-PBLE? at 20 °C during

JiUr TS 139

Figure 3 35 Cutting visual images of orange without coating (a) and coated with

CS, CS-SOE”, CS-PBLE2, CS-SOE!-PBLE!, and CS-SOE!4-PBLE® at 20 °C for

20 days and CS-SCE!*-PBLE”® at 20 °C for 29 days -¿55cccscsxsccse2 140

Figure 3 36 L value (a) and b value (b) of orange without coating and coated with

CS, CS-SOE?, CS-PBLE’, CS-SOE!-PBLE!, and CS-SOE!4-PBLE®® at 20 °C

during storage a-f means significant difference among storage periods (p< 0.05)

Figure 3 37 Weight loss of orange without coating and coated with CS, CS-SOE’, CS-PBLE’, CS-SOE!-PBLE!, and CS-SOE!*-PBLE°® at 20 °C during storage a-f

means significant difference among storage periods (p < 0.05%) - 141

Figure 3 38 Total soluble sugar of orange without coating and coated with CS,

CS-SOE?, CS-PBLE”, CS-SOE'-PBLE', and CS-SOE!'*-PBLE®® at 20 °C during

storage a-f means significant difference among storage periods (p < 0.05) 142

XH

Elongation at break

Xili

Model parameter for antioxidant activity and is defined as themass of film required to scavenge 50% of DPPH:

Optical densityProtocatechuic acidSonneratia ovata (Sonneratiaceae) leaf extract

Scanning Electron MicroscopyTotal polyphenol content

Total polyphenol compoundsTotal polyphenol compounds retained

Total flavonoid contentThermogravimetric analysisTensile strength

Ultraviolet VisibleX-Ray diffractionWater vapor transmission rate

XIV

Spoilage and oxidation process hold accountable for food deteriorationduring storage and distribution, by causing adverse impacts on food quality andshelf life As a consequence, it may cause economic damage and even negativeimpacts on human health due to infections and/or intoxications Recently, naturalantimicrobials and antioxidants have been considered as an effective way to preventspoilage issues and potential alternatives to toxic chemical preservatives.Polyphenols found in most parts of plants are naturally occurring antimicrobial andantioxidant sources However, the direct use of polyphenols in food preservation islimited by their relatively rapid release and may cause unfavorable reactions withfood components The incorporation of polyphenols into films to produce activepackaging has been considered to be an innovative approach as resulting packagingmaterials allowed to a gradual release of active agents during food preservation

Chitosan, composed of B-(1-4) linked 2-amino-2-deoxy-B-D-glucopyranose,

is an excellent candidate in the food packaging area due to its outstanding forming ability, biodegradability and compatibility, and mechanical behavior

film-Sonneratia ovata (film-Sonneratiaceae), which can be found abundantly along rivers and

canals in Can Gio areas of Vietnam to prevent soil erosion, is traditionally used asfood materials and products Previous reports have shown that the ethanolic extracts

of S ovata leaf are rich sources of phytochemical components, namely phenolics,flavonoids, tannins, alkaloid and saponins P betle is a spice plant widely grown inAsian regions and normally used as folk medicines by local residents Thesignificant medical benefits of P betle leaves have been well-reported in literature,

including antimicrobial, antifungal, anticancer, chemopreventive, antioxidant

activities, and gastroprotective effects Some previous studies revealed that P betleleaves contain rich phenolic content, such as hydroxyl chavicol, 4-chromanol andeugenol, thus exhibiting strong inhibition against both Gram-positive and Gram-negative bacteria A plethora of studies on the incorporation of natural extracts intochitosan film has been conducted However, the addition of P betle and S ovata

leaf extracts into chitosan film has not yet been reported Therefore, the thesis,entitled “Preparation and characterization of active coating solution based on

chitosan incorporated with natural extracts for preserving harvested

agricultural produce” was performed

The current work aims to evaluate comparative effects of incorporation of P.betle extract (PBLE) and S ovata extract (SOE) polyphenols in single and incombination on the mechanical behavior, water permeation barrier, UV-protectiveproperty, and antioxidant and antimicrobial activities of chitosan film In addition,the kinetics of release process and comparison on release properties of SOE and

PBLE polyphenols from films in different food simulants were investigated

Furthermore, chitosan coatings supplemented with SOE and PBLE were alsoapplied for extending the shelf life of the harvested Cavendish banana (Musaacuminate) and King orange, which are known as a climacteric and non-climacteric

fruit, respectively The physiochemical properties (i.e., viscosity, pH, and surface

tension) and coating efficiency (work of adhesion and spreading coefficient) ofblend solution were investigated Furthermore, the comparative study on coatingefficiency and preservative performance of single or combinatory use of SOE andPBLE into chitosan coating was examined The coating thickness and changes invisual appearance, peel color, and physiochemical properties such as respiration

rate, loss of weight, total soluble sugars, titratable acidity, vitamin C, and pH of

uncoated and coated fruits were examined during the storage

CHAPTER 1 LITERATURE OVERVIEW

1.1 Effect of important factors effect on shelf life of the harvested fruits andvegetables

The fresh fruits and vegetables play crucial role in human diet and health asthey provide several essential nutritional values such as vitamins, carbohydrates,and minerals However, it is documented that about 30-60% of their total

production is lost and wasted, mostly due to improper post-harvest handling [1]

Post-harvested losses have become an important challenge due to tremendous lossesand waste of nutrients while more than 820 million people worldwide are sufferingfrom hunger Furthermore, they are the main cause of the economic damage,resulting in complicating the life of farmer and finally, they can contribute to thewasting of other very crucial resources, i.e., energy, soil, water and environmentalproblems Many factors contribute to post-harvested losses and waste such as

microbiology, physiological and biochemical changes, and environment

All fresh horticultural fruits are prone to desiccation and mechanical injurydue to high water content Moreover, they are easily attacked by bacteria and fungiand as a consequence, they are pathological breakdown The internal biologicalfeatures may affect metabolic and biochemical changes, ethylene production [2-4].The biological deterioration rate depends on external environmental factors such astemperature, relative humidity, and atmosphere composition Temperature isconsidered as a crucial environmental factor that contributes to the deterioration ofhorticultural commodities It was reported that temperature significantly affectsrespiratory rate, ethylene production, metabolic change and pathogen development

At a low temperature, many subtropical commodities may be susceptible to temperature injury It is documented that avocado, oranges, and melons have slightinjury at low temperatures and the best storage condition is about 3-7 °C [1].Meanwhile mango, papaya, and banana are highly sensitive to low-temperatureinjury and the best storage condition is about 8-15 °C Chilling injury may exertnegative impacts on commodity surface, ripening, and flavors However, the

low-temperature above optimum may cause severe injury to horticultural fruits andvegetables There is a two- to three-fold increase of 10 °C in deterioration of post-havested comodities at the higher temperature than optimum High temperatures

may result in the change in fruit rippening and sometimes cause acceleration, delay,

or even disruption which depends on the fruit type, temperature and exposingduration

Furthermore, relative humidity (RH) is closely related to moisture content ofthe surrounding air which affects the water loss of fresh horticultural commoditiesand metabolic activities [5] RH is kept the best at 95% to delay fruit ripping andretain the freshness, flavor, decrease in weight and water loss, and to restrain thedevelopment of low-temperature injury symptoms It was documented that thewater loss from fresh fruits (transpiration) may result in the loss of weight at low

RH which deteriorates the visual appearance, texture, flavor, and nutritional values

In contrast, at high RH, it may facilitate decaying development due to the water

condensation on the fruit surface

Atmosphere composition (i.e., O02, CO2, and C¿H¿) is considered as a vitalfactor in the storage of fruits and vegetables It has been manifested that lower O2concentration (1-10%) and higher CO2 concentration (1-20%) than normal aircomposition (78% No, 21% Oz, and 0.03% CO2) may reduce metabolic activity,respiration rate and C2Hy, production [5] Reportedly, the O2 and CO2 concentrations

in fruit are paramount importance to production and action of C2H4 It was foundthat the activity of synthesis and oxidase of 1-aminocyclopropane-1-carboxylic acidwas inhibited at ultra-low O2 and uplifted CO2 concentration and as a consequence,

ethylene production was suppressed The C2H4 is produced by fresh fruits and

vegetables and released into surrounding environment, which surge the respiratoryrate of commodities and negatively affect physical and chemical characteristics(e.g., appearance, flavor, and texture) and ripping [2] The ethylene production rateduring ripping is evaluated by synthesis of 1- aminocyclopropane-1-carboxylic acid(ACC) before converting to C2H4 and controlled by ACC synthesis and ACC

oxidase progress The C¿H¿ decreases the commodity quality such as colordevelopment, softening and decreases soluble sugars, organic acids, aroma, and

pitting, appearance of internal and external browning and tissue breakdown

*% Heat treatment

There are three methods to heat fresh fruits and vegetables such as hot waterdip, vapor heat, and hot air rinse [7] Hot water treatment is employed by dippingcommodities in hot water (50-60 °C) for 10 min to remove many postharvest plantfungi However, dipping time can last up to 1 hour or longer (about 4 days) at below

50 °C Heating treatments may induce chilling injury and postpone the ripening ofpost-harvested fruits through heat inactivation of degradative enzymes Besides,they are able to kill the critical insects and control the onset of fungal decay Infact, postharvest dips in hot water (< 50°C) in 1 hour or more can kill insectsmeanwhile it takes some minutes for antifungal treatment 50 °C [8]

up and investment for new coating equipment, and the lack of edible materials withdesired physical and functional properties as well as the challenges of regulatorystatus for the different coating materials Besides, process parameters, such as themethod of coating and the number of additives, can affect the film barrier properties

and overall quality of the food product One of the successfully commercial

hTM

products is Natureseal and Semperfresh’TM, which maintains colour, texture and

shelf life of a number of fruits e.g banana, apples, pears, carrots, celery, etc

low irradiation (< 1 kGy), the disruption of cellular activity only inhibits budding on

bulbs and roots and delay senescence Medium doses in the range of 1-10 kGycause the reduction in microbial development meanwhile at high doses (> 10 kGy),fungi and bacteria spp and pests are killed [7] It was documented that irradiations

at medium and high doses exert sensory defects which negatively affected visual,texture and flavor of fresh products and evenly accelerated senescence because of

the irreparable damage to DNA and proteins

1.2.2 Chemical treatment

+ Antimicrobial and anti-browning agents

The utilization of antimicrobial and anti-browning agents to remain safety

can be classified into chemical- and bio-based grades [10] Chlorine-based solution,peroxyacetic acid (PAA), organic acids, hydrogen peroxide (H2O2), andelectrolyzed water are grouped in chemical grades Among the above-mentionedgroups, chlorine-based solution (i.e., NaClO) is commonly used for the purpose ofdisinfecting fresh fruits and vegetables because of its potential oxidizing capacityand low cost However, the antimicrobial efficacy of this agent mainly depend on

chlorine level and it is warned that high chlorine level negatively impact on tasteand odor of treated fruits and vegetables It is also reported that inhibitoryefficiency of chlorine-based solution against foodborne pathogens has been limited.

More importantly, the possible formation of carcinogenic chlorinated compounds

due to the use of chlorine may result in new regulatory restrictions in the EU PAA

is widely used for preservation of fruits and vegetables due to strong oxidizingwithout harmful by-products It was reported antibacterial effectiveness of PAAagainst E coli and L monocytogenes on apples, strawberries, lettuce, and

cantaloupe HaO2a is used to prolong the shelf life and decrease pathogenic microbial

populations in melons, oranges, apples, tomatoes, grapes, and fresh-cut products

However, the utilization of H2O2 can produce other cytotoxic oxidizing species (i.e.,

hydroxyl] radicals) and the main drawback of H2O2 use is the requirement of longapplication duration and may exert injury on some products Organic acid, ascorbicacid, and calcium-based solutions have been used to reduce enzymatic and non-enzymatic browning, texture deterioration, and bacterial growth on fresh fruits andvegetables It was reported that fresh-cut melon treated with 0.52 mM citric acid for

30s before applying for MAP may inhibit bacterial growth and prevent translucency

and discoloration Other organic acid such as acetic, lactic, and malic acids

combined with MAP showed good inhibition against FE coli and S Typhimurium,

and L monocytogens on cabbage The main limitation of antimicrobial and browning agents is due to the internalization of bacteria and inaccessible sites

anti-within fresh fruits and vegetables

+ Nitric oxide

Nitric oxide (NO), a highly reactive free radial gas, is bioactive signalmolecule for diverse physiological processes in plant species (i.e., fruit ripening andsenescence) [11] It was reported on the importance of NO as an importantendogenous signal in cellular metabolism and in the modulation of hormonalresponses, plant growth and developmental processes For example, endogenous

NO concentrations cause a reduction in maturation and senescence of fresh fruits

and vegetables and thus the utilization of opposite effects to decrease ripeningprocess due to exogenous application NO could reduce ethylene production andsuppress biosynthesis of ethylene due to some mechanisms It was reported that theACC oxidase-NO complex is produced from NO bound to ACC oxidase followed

by the chelation of ACC to form an ACC-—ACC oxidase-NÑO complex and as aresult, ethylene production is decreased NO is directly used by the fumigant orindirectly utilized from the release of sodium nitroprusside, S-nitrosothiols and alsodiazeniumdiolates by dipping technique However, commercial application of NO

in fresh fruit and vegetables depends upon the development of a smart

carrier/controlled release system for NO

* Sulfur dioxide

Sulfur dioxide (SO2) was considered to be efficient in preventing the decay

of table grapes during the storage through weekly fumigation or slow release fromin-package pads containing sodium metabisulfite [12] It was reported that therotting of grapes could be prevented for approximately 2 months at 0 °C by SO2fumigation Recently, SO is still routinely utilized to preserve table grapes

However, there are some side effects from SOz2 treatment on postharvest grape fruits

including berry abscission, destructing the specific flavor of grape, which causehealth risks of consumers, such as dermatitis, flushing, hypotension and abdominalpain Furthermore, SO2 concentration enough to inhibit fungal growth may inflictinjuries on grape fruits and stems meanwhile sulfite residues adversely impact on

produced using corona discharge method through passing a dried, dust-free, oil-free,oxygen-containing gas through two special electrodes producing a high energyelectric field The rapid attack to bacterial cell walls of ozone is considered to bemore efficient as compared to the attack of chlorine to thick-walled spores of plantpathogens at safe doses Ozone is applied due to the contact with water through twomethods such as venturi injection and fine bubble diffusion, depending upon thespecificity of the food processing operation Factors which affect the sanitizingpower of ozone include product nature, delivery techniques to the product, processtemperature, and relative humidity of the storage environment, solution pH, andozone demand of water Smooth surface of products divulges better sanitizing

efficiency with ozone treatment meanwhile the rough surfaces with microbes are

inefficiently exposed to active sites of ozone Delivery methods with higher contacttime of ozone to microbial population result in increasing sanitizing efficiency Inliterature survey, treatment of fruit with ozone at 2.5 ppm can maintain higher total

soluble solids, ascorbic acid, lycopene, and -carotene content, and antioxidant

activity along with reduced weight loss for 10-day storage as compared withuntreated fruits The drawback of ozone use is no penetration on natural opening or

wounds of fruits and vegetables

+ 1-Methylcyclopropene

1-Methylcyclopropene (1-MCP) is one of the synthesis cyclic olefins which

is proved to be active in inhibiting ethylene production [14] 1-MCP plays as acompetitor of ethylene as it can block the access to the ethylene-binding receptors.However, 1-MCP is not stable in liquid medium and the inclusion complex of 1-MCP with a-cyclodextrin can maintain its stability followed by the release of 1-MCP from these inclusion complex in food preservation 1-MCP is used forextending the shelf life of fresh fruits and vegetables such as apple, avocado,banana, broccoli, cucumber, kiwi, and etc However, it is a failure if 1-MCP is used

at an early ripening stage or at high concentration for avocado, banana, pear, andtomato The utilization of aqueous 1-MCP is similar to gaseous 1-MCP in reducing

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ripening of products In recent years, 1-MCP has been approved by the

Environmental Protection Agency regulated by other countries such as US,

Argentina, Brazil, Canada, Chile, New Zealand, and south Africa

+ Controlled atmosphere storage

Controlled atmosphere (CA) storage is closely related to the monitoring and

adjustment of carbon dioxide (CO2) and oxygen (O2) concentration with gas tight

environment at the optimum storage temperature and humidity to extend the shelflife of fresh fruits and vegetables [15] The optimum concentration of CO2 and O2depends on product nature and purpose of CA storage conditions, especially COaconcentration is often higher than O2 concentration in CA environment The mainadvantages of using CA includes delaying senescence related to the changes inbiochemistry and physiology, reduction of ethylene activity at O2 levels < 8% andCO; levels > 1%, decrease in physiological disorders, inhibition to postharvestpathogens However, the drawbacks of CA storage are due to the initiation andaggravation of physiological disorders, irregular ripening of fresh fruits resultingfrom contact to O2 concentration < 2% and CO2 levels > 5% for over than 1 month

The development in off-flavour and off-odour at very low O2 concentration and

very high CO; concentration respectively is related to anaerobic respiration andfermentative metabolism, and finally, increment in susceptible to decay Morenoticeably, the use of CA is limited by the high standard of capital investment toafford high quality storage rooms and maintain and monitor atmospheres As aresult, a large number of fruits are required to fully fill into rooms and long storagetime is needed for high economical investments

+ Modified atmosphere packaging

Modified atmosphere packaging (MAP) involves the packaging of foodproduct in plastic film bags with surrounding modified gaseous atmosphere [16].For passive MAP system, the equilibrium concentrations of CO2z and O2 due to theproduct weight and respiration rate of fruit are affected by temperature, surfacearea, perforations, thickness, and permeable ability to gases of packaging film In

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terms of active MAP system, the introduction of desired atmosphere into thepackage headspace is performed before heat sealing but the final atmosphere will beaffected by similar factors as compared to passive MAP Correct equilibriumatmosphere can extend the shelf life of food products by delaying respiration,senescence, and decay rate In recent years, MAP is also integrated to the adsorption

of Oo, ethylene, moisture, CO2, flavours/odours, and the release of CO,antibacterial and antioxidant agents However, capital intensive and expensiveinvestment of controlled atmosphere storages for preserving bananas is impossiblefor poor countries

+ Plasma

Cold plasma has been considered as a non-thermal disinfection approach toreduce microbial load on fresh food and packaging materials [17] Plasma uses inertgases at room temperature to produce ionized hand highly reactive such as positiveand negative ions, electrons, molecules in non-excited states, free radicals, andphotons The inactivation of biological cells is due to the reaction of plasma species

at different levels which forces permanent changes at the molecular levels andmorphology Primary mechanisms are reported for cell death caused by plasma such

as etching on cell surface caused by reactive species, negative impact onintracellular structure, and disruption of genetic components Furthermore, plasma-mediated treatments to inactivate benefit enzymes result in enhancing the shelf life

of food products For example, surface decontamination is treated by plasma beforeapplying biofilm to them Bioactive compounds degradation is induced by coldplasma due to the synergistic effects of plasma-reactive species and thermal-induced oxidative cleavage pathways However, plasma treatment may causedetrimental effects on food components (i.e lipids and vitamins) and the practicalapplication of this technique still requirea further investigation

1.3 Overview of chitosan

The petrochemical-based polymers were commonly utilized in foodpackaging due to low cost and easy manufacturing; however, such materials

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induced some environmental problems on the ground of long degradation time As

an alternative approach, natural biopolymers, namely proteins, lipids andpolysaccharides, have paid increasing attention to developing biodegradability,biocompatibility and edible food packaging films [18] Among the aforementionedbiopolymers, chitosan is a promising polysaccharide based on its excellent film-forming ability, antibacterial capability, physical property, mechanical behavior,biodegradability and compatibility Chitosan composed of ÿ-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine is a partially deacetylated product of

chitin which extracted from abundant natural resources, i.e shrimp, crab, lobster

and krill shells [19] The presence of active NH2 group on the C2 position of glucosamine units along the backbone (Figure 1.1) was primarily related tobiological activities of the chitosan

Figure 1 1 Chemical structure of chitin and chitosan

The degree of acetylation (DA) and the molecular weight (Mw) has beenconsidered as primary characteristics effecting the physicochemical properties ofchitosan [20] Given this fact, the higher DA is, the higher the amino groups inchitosan are Such active groups are mainly correlated to most of the properties of

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chitosan, namely, solubility, swelling in water, antimicrobial activity, antioxidantactivity, biodegradation and biocompatibility, wherein, the molecular weight wasrelated to viscoelastic properties and film-forming ability of chitosan The highmolecular weight chitosan is likely to produce a more stable solution Moreover, itwas documented that the tensile strength, elongation, moisture adsorption andthermal stability would increase as high Mw value of chitosan [21] The above-mentioned parameters significantly affected to physicochemical and biologicalproperties, i.e mechanical behaviors, barrier properties, thermal stability and

biological activities Reportedly, the tensile strength increased as a function of Mw

due to the structural alternations of chitosan molecules and the free volumereduction of polymer network [22] It was also found that the hydrophilicity andwater vapor permeability rate surged with increasing Mw The intrinsic bactericidalproperty of chitosan strongly depends on the quantity of NH; groups which haveclose correlation with the degree of the deacetylation [23] As reported, theinhibitory efficiency of chitosan against Gram-negative bacteria was highercompared to Gram-positive bacteria because of the electrostatic interaction betweenpositively charged active amine groups and phosphoryl groups in the phospholipid

of the cell membrane [24] Moreover, chitosan is used as nontoxic and safe material

for food preservation when listed in Generally Recognized as Safe (GRAS) by USfood and Drug Administration in 2013 Thus, based on the mentionedcharacteristics, the chitosan has been introduced as a potential candidate for thefabrication of biodegradable and edible food packaging to extend food shelf life andreduce the utilization of chemical preservation Thanks to non-toxic, biodegradable,bio-functional, biocompatible, easy film-forming, inherent antimicrobial andantifungal properties, chitosan based films have attracted substantial attention in avariety of fields ranging from biomedical to packaging technology [25] However,besides the above outlined advantages, a free-standing chitosan film is often brittle,poorly force-resistant and relatively sensitive to moisture In addition, furtherimprovements of its antioxidant and antibacterial properties are essential for active

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packaging and thus becomes a topic of interest over the years To date, manydifferent modification approaches have been proposed, namely the incorporation ofantioxidants, antimicrobial substances and reinforcement agents to either impart orimprove specific properties of chitosan-based films For example, several syntheticantioxidants such as butylated hydroxyanisole (BHA), and butylatedhydroxytoluene (BHT) were tried and showed good results [26] Also, a number ofinorganic fillers were reported to improve the antimicrobial and barrier properties ofchitosan, such as copper oxide [27], titanium dioxide [28], halloysite nanotube [29],sulfur nanoparticles [30] On the other hand, as the utilization of noted syntheticmaterials may hold a potential threat to human health and environment, naturalbioactive additives with pronounced antioxidant and antibacterial activities haveemerged as very effective alternatives A number of natural essential oils whenincorporated into the chitosan matrix proved to produce impressive performance butthe use of essential oils unfortunately faces disadvantages related to the problem ofdurability, difficulty in control of release rate, and sensitivity to environmentalfactors [31,32] Recently, polyphenolic compounds extracted from agriculturalcrops were reported to provide a chitosan matrix with desired antimicrobial andantioxidant functions Simultaneously, they could also alter mechanical properties

of the films through the formation of hydrogen bonds, electrostatic interactions,ester linkages and crosslinking effects, and also, in some cases, behave as aplasticizer to promote the flexibility of polymeric chains

1.4 Introduction of polyphenol and interaction between of chitosan andpolyphenols

1.4.1 Overview of polyphenol

In the past few decades, polyphenols have been drawn increasing attentiondue to the abundance in nature and its antioxidant, antibacterial, anti-inflammatoryand antiviral activity It is documented that the polyphenols can be divided intothree main classes [33], including flavonoids, stilbenoids and phenolic acids, asshown in Figure 1.2 Where the flavonoids consist of an oxygenated heterocyclic

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ring (C) between two phenyl rings (A and B) with a 15-carbon skeleton (C6—C3—C6backbone) structure, phenolic acids are benzoic acid, hydroxycinnamic acid andStilbenoids are composed of diphenylethene polyphenols with a 14-carbon skeleton(C6—C2-C6 backbone) structure.

Figure 1 2 Classification of polyphenols [33]

H-atom transfer and

proton-Electron transfer - proton transfer

coupled electron transfer

(under alkaline conditions) Aduct formation

(in radiolitic solutions)

Recently, the introduction of polyphenols into chitosan films has gained fromfood packaging application due to their natural antioxidant and antibacterialactivity Reportedly, the inhibitory efficiency of polyphenols against oxidativeprocesses in foods is accordance with their reactive species scavenging activity andthe mechanism was followed as equation: Ar-OH + R-: — ArO- + RH [33].However, many other mechanisms were also proposed due to their structure (thenumber and position of hydroxyl groups on phenyl rings) and polyphenols scavengefree radical by H-actom transfer from the active OH groups to the free radicals

(Figure 1.3) For instance, it was found in the relationship between the structure

and HO- scavenging activity in flavonoids that the positions of hydroxyl groupsstrongly effect the antioxidant activity compared with the total number of phenolichydroxyl groups [34] An ortho-dihydroxyl group on the A-ring (on 5, 6 positions)

in baicalin exhibited stronger HO- scavenging ability than meta-dihydroxyl group

on the A-ring (on 5, 7 positions) in lysionotin and matteucinol Furthermore, it isdocumented that the position of meta-hydroxyl groups on the A-ring and ortho-hydroxyl groups on the B-ring may cause an effective HO- scavenging

In addition to antioxidant activity, the bactericidal activity is an important property of the phenols Until now, the mechanism of bactericidal activity ofpolyphenols has not completely known It is presumed that the structural diversity(backbone structure, number and position of hydroxyl groups) of phenolic classes

bio-significantly affects the antimicrobial activity and the interaction between bacteria

and polyphenol as illustrated in Figure 1.4 The interaction between thepolyphenols and bacterial cell wall, membranes and enzymes via non-specificforces, 1.e hydrogen bonding, covalent bond formation, hydrophobic effects andlipophilic forces, prevents the protein transport and caused the death of bacteria[33] In brief, the antioxidant and antimicrobial properties of polyphenol are twoimportant features in the incorporation into chitosan film in order to controlpathogenic growth and prolong the shelf-life of food

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