Nghiên cứu chế tạo màng chitosan nano bạc và bước đầu thử nghiệm trong bảo quản xoài cát hòa lộc TT TIENG ANH

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: Food Technology Code: 62 54 01 01 NGUYEN HUYNH ĐINH THUAN DEVELOPMENT OF CHITOSAN-BASED NANOSILVER FILMS AND PRELIMINARY APPLICATION FOR STORAGE OF HOA LOC MANGO Can Tho, 2021 THE DOCTORAL THESIS WAS COMPLETTED AT CAN THO UNIVERSITY Supervisor: Assoc.Prof Ly Nguyen Binh, PhD The doctoral thesis was evaluated by The Board of Examiners at the basic level Meeting at: ……………………… At … … on the date … … … giờ, ngày 17 tháng 11 năm 2018 Reviewer 1: TS Đáinng Reviewer 2:.T S Đống Thị Anh Đào Confirmation of review by the Chairman of the Board Libraries for more information on the thesis: Learning Resource Center - Can Tho University National Library of Vietnam LIST OF PUBLICATIONS (1) Nguyen Huynh Dinh Thuan, Ly Nguyen Binh, 2018 Research was to create chitosan film of deacetyl 80% and increase the shelf life of Hoa Loc mango Science and Technology Journal of Agriculture and Rural Development, 8/2018, ISSN 1859-4581 (2) Nguyen Huynh Dinh Thuan, Ly Nguyen Binh, Nguyen Ngoc Tuan, Nguyen Thi Hong Anh, 2020 A research on the preparation of nano silver from AgNO3 and effects of the content of silver nano in chiosan-nano silver film Viet Nam Trade and Industry Review, 24/09/2020 ISSN 0866 – 7756, (3) Nguyen Huynh Dinh Thuan, Ly Nguyen Binh, Nguyen Ngoc Tuan, 2021 Research effects of concentration and deacetyl on creativity films of chitosan from shell shrimps Science and Technology Journal of Agriculture and Rural Development, No 10, 05/2021, ISSN 1859-4581 (4) Nguyen Huynh Dinh Thuan, Ly Nguyen Binh, Nguyen Ngoc Tuan, Duong Quoc Dat, Pham Thi Quyen, 2021 A study on the quality change of Hoa Loc mango which is coated by the chitosannano silver film with a 70% deacetylation degree during the storage Viet Nam Trade and Industry Review, 04/2021 ISSN 0866 – 7756 (5) Coordinator of university-level research project Decision of acceptance and liquidation of the contract for scientific research and technology transfer No 1428/QĐ-ĐHCN of the Rector of the Industrial University of Ho Chi Minh city dated 15/06/2018 on the recognition and acceptance of the results of the implementation of the scientific project “Development of chitosan-based nanosilver films and application for storage of Hoa Loc mango” Nguyen Huynh Dinh Thuan, Dam Sao Mai, Nguyen Thi Mai Huong, Pham Thi Quyen, Lam Gia An Chapter 1: INTRODUCTION 1.1 Thesis Rationale Vietnam has a lot of potentials to develop fruit production and export thank to favourable conditions in terms of climate, land, increasing production level of farmers and the market for products in both the country and the world is still open Currently, the annual fruit harvest is very large but the export capacity is small, which are the limitations in the technology of preserving and processing vegetables of our country For fruits to truly become of high economic value, proper preservation technology is required (Gupta et al., 2015) Hoa Loc mango is considered an important fruit that accounts for the majority of the market share of domestic fruits and exports, which have high nutritional value, but for mangoes to be truly an economic value, it is necessary to have appropriate preservation technology because in fresh fruits in addition to nutritional ingredients such as vitamins, sugar, minerals high water content and strong breathing, so mangoes are very vulnerable to damage due to unfavourable storage conditions Furthermore, anthrax caused by Colletotrichum gloeosporioides and withering caused by Fusarium equiseti also affects the quality of mangoes after harvest and preservation There are many studies to preserve mangoes at home and abroad with a variety of methods and often combine with chemicals for preservation However, currently on the market, there are many chemicals of unknown origin, so domestic consumers can not help but wonder about chemical residues when buying fruits as well as the strict requirements of the export market, so it more or less affects the quality and ability to consume Moreover, according to the survey results in key mango growing provinces of the Mekong Delta: Vinh Long, Dong Thap and Tien Giang of the United Nations Industrial Development Organization (UNIDO), postharvest losses is up to 26.9% This number is not small as according to the calculations of this organization, the total value of mangoes of Vietnam is up to 490 million dollars per year This is one of the important reasons affecting the expansion of the output of mangoes in the export sector According to UNIDO, there are reasons why post-harvest losses of Mekong Delta mangoes in particular, Vietnam in general, at a high level are due to the technique, time of harvest and transportation post-harvest is not correct; non-bactericidal harmful fruit; not keep the fruit post- harvest at the right temperature and there is no solution to prevent mango pus To contribute to reducing the above obstacles, the study on storage of Hoa Loc mango using films created from non-toxic organic compound chitosan combined with silver nano and low temperature aiming at prolonging the shelf-life, food hygiene and safety for fresh fruits is essential Chitosan is a high molecular bio-compound extracted from shrimp shells and has superior properties over other chemicals used in fruit preservation Chitosan films are anti-steam, resistant to microorganisms, environmentally and human-friendly Silver has been known to have strong antibacterial properties, limiting and destroying the growth of mould, atomic silver at the nanoscale has antiseptic activity increased to about 2050 times that of silver ion (Phu, 2010) This has attracted many interested scientists to study the application of silver nano into practice Currently, the application of chitosan in combination with other components in fruit preservation is highly studied, mainly creating solutions and embedding the fruit directly into it This preparation, when used with advantages, cost of turning, to operate but the disadvantages of the solution not stick evenly, or the thickness of the solution is not enough to affect the quality of the fruit when stored With the study of the application of chitosan and silver nano in the preservation of Hoa Loc mango instead of dipping mangoes into a solution, then form a membrane and then cover the fruit In fact, there has been no research on the use of chitosan films combined with silver nano to preserve mangoes, for that reason: the research work entitled “Development of chitosan-based nanosilver films and preliminary application for storage of Hoa Loc mango” was done to better contribute to post-harvest preservation, to reduce quality and quantity losses of fruits, especially Hoa Loc mango 1.2 Research objectives Survey of factors affecting the ability to make chitosan-based nanosilver films, the antifungal capacity of film at three different degrees of deacetyl (70, 80, and 90 %) From chitosan-based nanosilver films obtained, to conduct research on the storage of Hoa Loc mango at low temperature To compare the effectiveness of mango storage using with chitosan-based nanosilver films at three degrees of deacetylation with some other storage methods 1.3 Research content - To study on preparation of chitosan films using chitosan material with the deacetyl levels of 70, 80, and 90% - To find optimal values of temperature, duration, and concentration of silver nitrate for preparation of nanosilver solution To make chitosannanosilver films using chitosan with deacetyl levels of 70, 80, and 90% - To preliminarily apply the chitosan-based nanosilver films for storage of Hoa Loc mango in comparison with some other preservation treatments 1.4 Scientific and practical significance 1.4.1 Scientific significance The results of the project provide the optimal conditions for creating chitosan films, chitosan-based nanosilver films with three degrees of deacetylation 70, 80, and 90%; Colletotrichum gloeosporioides and Fusarium equiseti resistance of the chitosan films and the chitosan-based nanosilver films The study showed the effectiveness of storage of Hoa Loc mango using chitosan-based nanosilver films in terms of preserving quality attributes of the fruit for 35 days Besides, the study also showed a possibility of storage of mango fruits using the chitosan/chitosan-based nanosilver films of low deacetyl degree compared to the one of high deacetyl degree 1.4.2 Practical significance The creation of chitosan films, chitosan-based nanosilver films, and their applications to preserve agricultural products in general and Hoa Loc mango in particular, is to continue new trends in the current fruit processing and preservation industry Moreover, the research takes advantage of shrimp shell scrap in seafood processing plants to create organic bio-substances with good antifungal activity, non-toxic to the product, not antibiotic residues, chemical residues to replace conventional preservative chemicals, improving consumer safety The results of the project can also contribute to improving the economic efficiency of growers, traders, transporters, and exporters of Hoa Loc mangoes It will generate motivation in mango-growing areas, improving productivity as well as quality for export, sustainable developing Vietnam Hoa Loc mango brand in both domestic and foreign markets Besides preserving mangoes, chitosan-based nanosilver film can also be used for the storage of many other fruits or foods 1.5 New contributions of the thesis This study includes the optimization of biofilm development using chitosan materials at three different deacetyl degrees (70, 80, and 90%) and nanosilver with suitable physical and biological properties applied to the storage of Hoa Loc mango The first finding, this study developed chitosan-based nanosilver films with suitable mechanical properties and water vapor permeability, good antifungal capacity for Colletotrichum gloeosporioides and Fusarium equiseti, highly effective in preserving Hoa Loc mango with film-forming parameters including chitosan-dissolving pH of 3.6 for the chitosan of 70% deacetylation, and the pH of 3.8 for those of 80 and 90% deacetylation; suitable chitosan concentration of 1%; AgNO3 0.005 M nanosilver solution to 1% chitosan solution ratio was 75 µL to 100 mL The second finding, the studies on chitosan previously published mainly focused on the use of chitosan itself for food preservation In this study, chitosan-based nanosilver films were developed aiming at fruit coating The developed chitosan-based nanosilver films showed good antifungal capacity, suitable for the preservation of mangoes (or other fruits) The third finding, using chitosan-based nanosilver film for fruit coating prolonged the shelf-life of Hoa Loc mango up to 35 days while retaining good quality compared to other storage methods in the survey, prolonging the storage time from 5-10 days The fourth finding, in this study, chitosan with low deacetyl level of 70-80% was used to develop chitosan-based nanosilver films The films showed remarkable preservation results for Hoa Loc mango compared to those of chitosan with a deacetyl level of 90% All of this also contributes to the application of chitosan diversity in food preservation today 1.6 The structure of the thesis The thesis consists of chapters with 133 pages: Chapter 1Introduction (page 1÷4); Chapter 2- Literature revews (page 5÷38); Chapter 3- Research methods (page 39÷51 with experiments); Chapter 4- Results and discussions (page 52÷131); Chapter 5- Conclusions and suggestions (page 132÷133) In the main content, there are 28 tables and 99 pictures The article uses 283 references, including 350 English-language and 33 Vietnamese-language references Chapter 2: LITERATURE REVEWS 2.1 Overview of chitosan Chitin and chitosan are a polysaccharide and they are chemically composed like cellulose, except the presence of nitrogen Chitin is a mucopolysaccharide that is abundant in nature and is known as 2acetamido-2-deoxy-β-D-glucose linked together by β-(1-4)-glycoside and can be hydrolyzed by the enzyme chitinase (Muzzarelli, 1973) Chitosan is a derivative of the deacetyl process for removing the acetyl group of chitin in a dense alkaline environment and is replaced by the amino group at the C2 position on the glucan ring Chitosan is made up of D-glucosamin units linked together by bonding β-(1-4)-glycoside, and it can also be called the linear polymer of the units 2-amino-2-deoxy-Dglucopyranose or poly-(1-4)-D-glucosamin (Rinaudo, 2006) The presence of the NH2 group in chitosan makes it have more applications than chitin Chitosan is a special bio-polymer with properties such as biodegradability, biocompatibility, and responsive antibacterial activity used as functional materials (Honarkar & Barikani, 2009) 2.2 Overview of nano, silver nano The term "nano" is coined from the Greek word for dwarf A nanometer (nm) is one-billionth of a meter, or about a hundred thousand in width of a human hair (Ranjan et al., 2014) The concept of nanotechnology was first introduced by Richard Feynman (1959) at the meeting of the Physical Society (Khademhosseini et al., 2006) Since then, nanotechnology has evolved into a multidisciplinary field of applied science and technology Nanotechnology is the ability to work on a scale of about 1-100 nm to design, manufacture and apply material structures, equipment, and systems with new characteristics derived from their nanostructures (Roco, 2003) Among metal nanomaterials, silver nanoparticles have received considerable attention due to their attractive physicochemical properties (An et al., 2008; Sharma et al., 2009) In particular, silver nano has shown to be a promising antibacterial material (Sondi et al., 2004) Silver nanoparticles are also reported to be capable of absorbing and decomposing ethylene, which can effectively contribute to prolonging the shelf life of vegetables (Li et al., 2009) 2.3 Silver nano-preparation The chemical method is the most popular method because of its convenience and simple equipment Silver nano-synthesis typically uses three main components: (1) metal salts, (2) reducing agents, and (3) stabilizers The formation of glue solutions from the process of desalination of silver salts consists of two stages of germination and increased size The initial germination and subsequent growth of the initial crystal germs can be controlled by adjusting the reaction parameters such as reaction temperature, pH, metal salts, reducing agents (NaBH4, ethylene glycol, glucose) and stabilizer (PVA, PVP, Natri oleat) Another method for synthesizing nanoparticles is to use silver nitrate as a metal salt, 8% sodium dodecyl sulfate as a stabilizer, a solution of hydrazin hydrate (2.0-12 mM) and a solution of sodium citrate (1.0-2.0 mM) as a reducing agent The 1.1 mM silver nitrate mixture produces silver nanoparticles measuring 24 nm In addition, silver nitrate is used as a metal salt, NaBH4 as a reducing agent and trinatri citrate as a stabilizer (Jana et al., 2001; Pinto et al., 2010) In it, 100 mL solution containing silver citrate and trinatri citrate is prepared by stirring for 30 seconds The solution is then added to a mM NaBH4 solution and stirred for 60 seconds 2.5 Introduction to mango Regarding the origin, Hoa Loc mango is grown in Hoa Loc commune, Giao Duc district, Dinh Tuong province, which is now Hoa Hamlet, Hoa Hung Commune, Cai Be District, Tien Giang Province, so it is named Hoa Loc mango Tien Giang currently has an Hoa Loc mango cultivation area of over 1.579 hectares, concentrated mainly in the area along the Tien River in Cai Be and Nam Cai Lay districts The annual output is estimated at 35.926 tons, with a yield of 23 tons/ha Cai Be district is the largest concentration of mangoes in Tien Giang province with an area of 821 hectares, accounting for 52% of the total mango growing area in the province Hoa Loc mango growing area is concentrated in 13 communes: Hoa Hung, An Huu, An Thai Trung, Tan Hung, Tan Thanh, My Luong, An Thai Dong, My Duc Tay, My Duc Dong, Thien Tri, Hoa Khanh, Hau Thanh and My Loi A in Cai Be district, Tien Giang province Currently, Hoa Loc mangoes have been granted to meet GlobalGAP and are exported to many countries around the world such as Japan, Korea, Canada, New Zealand, Russia, (Tuan, 2011) 2.6 Overview of colletotrichum and Fusarium fungi The fungus Colletotrichum has a true bacteriophage, consisting of fine, branched, colorless hyphae growth and hyphae septa The mycelium is intercellular and intracellular and each cell has many nuclei, there are many nuclei Endogenous Colletotrichum, which reproduces only asymuously with attached spores, attached spores that grow on the spores stem in the form of a cluster of spores The spores have a flat disc shape, the back has a smooth chalk structure, each cluster of spores consists of a substrate layer, the surface produces transparent spores Spores are singlecelled, sickle-shaped, curved spores Along with spores and stems, there are stiff hairs on each cluster of spores with long, oblong, non-branched, and multicellular structures such as hard silk, and Frost (1964) describes that a few species of Colletotrichum with or without hard hair can be controlled by moisture changes The formation of a large number of spores causes fractures on the host epidermis, under favorable conditions, each spores grow from one to more germ tubes to form the mycelium system Old hyphae sometimes form thick, dark brown, spherical or irregular walls called post-spores (Chlamydospores), it can be at the end or squeeze between the mycels and last for long periods of time and when separated they also sprout to form new mycels Colletotrichum live on land, and saprophytism or parasitism is the main cause of anthrax on many crops Fusarium fungi have many different strains, so the host ability is very wide on many crops such as fruit trees, vegetables, and some ornamental plants, and they can live under different conditions and can survive in the soil for a long time, in plant carcasses under various forms Fungal spores can survive in extreme conditions for a long time and then with favorable conditions, they will grow and spread (Đong, 1986) The fungus causes severe harm on some crops especially on fruit trees such as bananas, mangoes, labels, grapes, guava, citrus, papaya, avocado In addition, there are several strains of Fusarium fungi that cause diseases in humans and animals such as: causes pharyngeal cancer in humans and hemispheric cancers in horses and rats (Thiel et al, 1991) 2.7 Research at domestic and abroad Study on the effect of low-molecular-weight chitosan coating film combined with nano-silver in preserving fresh chicken eggs was done by Hanh et al (2020) Chicken eggs, after being harvested and selected according to color and size, are covered with chitosan film (mW = 10-20 kDa) with 86-90% deacetylation added 1.5 µL silver nanoparticle, the silver particle size is 20-50 nm Chicken eggs after being covered with film and stored for 30 days at 28-30 oC, showed that chitosan films with 1.5% concentration of silver nano-addition gave the highest egg protein content and the least change in albumin pH Van et al (2017) researched the application of chitosan-based nanosilver films in storage to improve the quality productivity of dragon 10 - The thickness of the chitosan film is measured by an electronic Panme ruler with an accuracy of 0.01 μm, assessing the structure by the physicmechanical TPA method through parameters: elongation, traction resistance, elastic module, color sensory assessment, shape, transparency of the membrane Assessing the water vapor permeability of the films Results: - pH value suitable for dissolving chitosan Investigate the effect of concentration and deacetylation on the film forming ability of chitosan Factors: - Deacetyl levels of chitosan, levels: 70, 80 90 (%) - Chitosan concentrations, levels: 0.5, 0.75, 1, 1.25, 1.5 (%) Evaluation criteria: - The thickness of the chitosan film is measured by an electronic panme with an accuracy of 0.01 μm, assessing the structure by the phycicomechanical method through parameters: elongation, traction resistance, elastic module, color sensor assessment, shape, transparency of the membrane Assessing the water vapor permeability of the film Results: - Film thickness, chitosan concentration suitable for film making Investigation of the antifungal activity of Colletotrichum gloeosporioides and Fusarium equiseti of chitosan flims Factors: Deacetyl degree of chitosan 70, 80 90% - Chitosan concentration, levels: 0,5 0,75 1,0 1,25 1,5 - Colletotrichum gloeosporioides and Fusarium equiseti Evaluation criteria: antifungal ring diameter of film Results: antifungal activity of chitosan film 3.2.3 Study on the influence of some factors on the ability to form chitosan-based nanosilver films Objective: Find out the AgNO3 concentration values, the temperature and time to create silver nanoparticles so that the particles have the required size and stability Determine the appropriate AgNO3 concentration of chitosan-nano silver solution to form chitosan-based nanosilver films applied to fruit coating in mango preservation Considering the dispersion of silver nanoparticles and the existence of silver nanoparticles in chitosan films over time Specific content of the study: Investigate the influence of AgNO3 concentration, temperature and stirring time on the ability to form silver nano glue Factors: 14 - AgNO3 concentration, levels: 0,001 0,005 0,010 M - Temperature, levels: 90, 95, 100 oC - Time, levels: 4, 8, 12 phút Evaluation criteria: - Evaluate the dispersion of silver nanoparticles in solution by determining the maximum absorption wavelength of the solution by UV-VIS; Evaluation of the distribution and size of silver nanoparticles in silver nanoparticle solution by TEM Results: AgNO3 concentration is suitable to create silver nano solution Investigate the effect of the amount of silver nano glue used and the deacetylation of chitosan on the ability to form chitosan-based nanosilver films and the dispersion of silver nanoparticles in chitosanbased nanosilver films over time Factors: - The deacetylation of chitosan, including levels of 70, 80 and 90% with the concentration of chitosan obtained in the study content - Amount of silver nano glue used 100 mL of chitosan solution, including levels 50 µL/100 mL (2,5 µM), 75 µL/100 mL (3,75 µM) and 100 µL/100 mL (5,0 µM) - Time to monitor the dispersion of nano silver (days), including levels: 0, 10, 20, 30 and 40 Evaluation criteria: Evaluation of the existence of silver nanoparticles and the dispersion of silver nanoparticles in chitosan films over time by SEM Results: Create the best chitosan-based nanosilver films Antifungal activity of Colletotrichum gloeosporioides and Fusarium equiseti of chitosan-based nanosilver films Factors - Fungus: Colletotrichum gloeosporioides, Fusarium equiseti - Chitosan-based nanosilver films with three degrees of deacetyl: 70, 80 and 90% Evaluation criteria: antifungal diameter Results: Antifungal ability of chitosan-based nanosilver films 3.3.3 Research on preservation of mango - comparing the effectiveness of different types of film coating treatment Objective: Evaluated the ability to preserve Hoa Loc mango with chitosanbased nanosilver films compared with other types of film coating treatment Specific content of the study: Study on the effectiveness of different types of film coating treatment in mango preservation 15 Factors: - Mango samples were treated, including modes: M0: uncoated mangoes and washed with water at room temperature M1: uncoated mangoes and washed with water at 50 oC M2: mangoes coated with chitosan film and washed with water at room temperature M3: mangoes coated with chitosan film and washed with water at 50 oC M4: mangoes coated with chitosan-based nanosilver films and washed with water at room temperature - M5: mangoes coated with chitosan-based nanosilver films and washed with water at 50 oC - Deacetyl level of chitosan, levels: 70, 80 and 90% Evaluation criteria: Every days, determine the loss of mass, hardness, dissolved dry Every days, weight loss, hardness, soluble solids content, total acid content, vitamin C content, total sugar content, fruit skin color, fruit flesh color, respiratory intensity, ethylene production intensity and mango spoilage rate were determined Results: Compare the efficiency of chitosan-based nanosilver films coating treatment with other coating methods Chapter RESULTS AND DISCUSSIONS 4.1 Effect of some factors on the ability of chitosan to form film and antifungal 4.1.1 Effect of pH on the ability to form chitosan films Table 4.1: Effect of pH on the properties of chitosan films at different deacetylation degrees Deacetyl 70% pH 3.4 3.6 3.8 4.0 4.2 80% 4.4 3.4 3.6 3.8 4.0 4.2 Strength (MPa) Elongation (%) Modulus (109N/m2) a 0.503 ± 0.011 3.980a± 0.142 4.849a± 0.085 a a 0.509 ± 0.012 4.227 ± 0.170 3.997b± 0.499 b b 0.466 ± 0.013 2.819 ± 0.582 4.166b± 0.288 Chitosan does not dissolve completely, the film is not smooth Chitosan does not dissolve completely, the film is not smooth Chitosan film appear salt on the surface 0.410b± 0,007 0.410c± 0.007 4.200a± 1.570 a b 0.520 ± 0,020 1.460 ± 0.250 2.170b± 0.440 b a 0.450 ± 0,040 5.490 ± 2.890 1.080c± 0.110 Chitosan does not dissolve completely, the film is not smooth Chitosan does not dissolve completely, the film is not 16 4.4 3.4 3.6 3.8 4.0 90% 4.2 4.4 smooth Chitosan film appear salt on the surface 0.340b± 0.048 8.957c± 0.017 2.150a± 0.030 b b 0.392 ± 0.042 12.100 ± 0.027 1.053b± 0.030 a a 0.465 ± 0.035 13.246 ± 0.030 0.631c± 0.030 Chitosan does not dissolve completely, the film is not smooth Chitosan does not dissolve completely, the film is not smooth Chitosan film appear salt on the surface Note: the numbers followed by the ± sign represent the standard deviation of the mean values; numbers with different exponents in the same column are significantly different (P