MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION THESIS FOOD TECHNOLOGY CHARACTERIZATION OF BACTERIAL CELLULOSE PRODUCED BY GLUCONACETOBACTER XYLINUS USING RICE EXTRACT AS A NUTRIENT SOURCE SUPERVISOR: VU TRAN KHANH LINH STUDENT: NGUYEN THUY THANH HIEN NGUYEN PHAM HUYEN PHUONG SKL 0 0 9 1 5 5 Ho Chi Minh City, August, 2022 HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION PROJECT CODE: 2022-18116015 CHARACTERIZATION OF BACTERIAL CELLULOSE PRODUCED BY GLUCONACETOBACTER XYLINUS USING RICE EXTRACT AS A NUTRIENT SOURCE NGUYEN THUY THANH HIEN Student ID: 18116015 Major: FOOD TECHNOLOGY NGUYEN PHAM HUYEN PHUONG Student ID: 18116030 Major: FOOD TECHNOLOGY Supervisor: VU TRAN KHANH LINH, PhD. Ho Chi Minh City, August 2022 HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION PROJECT CODE: 2022-18116015 CHARACTERIZATION OF BACTERIAL CELLULOSE PRODUCED BY GLUCONACETOBACTER XYLINUS USING RICE EXTRACT AS A NUTRIENT SOURCE NGUYEN THUY THANH HIEN Student ID: 18116015 Major: FOOD TECHNOLOGY NGUYEN PHAM HUYEN PHUONG Student ID: 18116030 Major: FOOD TECHNOLOGY Supervisor: VU TRAN KHANH LINH, PhD. Ho Chi Minh City, August 2022 ii APPENDIX 3: (Graduation Project Assignment) THE SOCIALIST REPUBLIC OF VIETNAM Independence – Freedom– Happiness -------- GRADUATION THESIS ASSIGNMENT Student name: Nguyen Thuy Thanh Hien Student ID: 18116015 Student name: Nguyen Pham Huyen Phuong Student ID: 18116030 Major: Food Technology Class: 18116CLA1 Supervisor: Vu Tran Khanh Linh, PhD Email: linhvtk@hcmute.vn Date of assignment: 14/02/2022 Date of submission: 08/08/2022 1. Thesis title: Characterization of bacterial cellulose produced by Gluconacetobacter xylinus using rice extract as a nutrient source.
Thesis assignment: The research topic “Characterization of bacterial cellulose produced by Gluconacetobacter xylinus using rice extract as a nutrient source” includes 3 main parts: - Determination of the chemical composition of coconut juice and rice extract. - Effects of rice extract as a nutrient source on film-like cellulosic biomass production. - Characterization of BC films obtained from growth culture containing different ratios of rice extract and coconut juice. The content and requirements of the graduation thesis have been approved by the Chair of the Food Technology program Ho Chi Minh City, August 08, 2022 CHAIR OF THE PROGRAM SUPERVISOR (Sign with full name) (Sign with full name) iii ACKNOWLEDGEMENTS First and foremost, we would like to thank Ms.
Vu Tran Khanh Linh who is a lecturer at the Faculty of Chemical and Food Technology for her enthusiastic guidance, instruction, imparting valuable experience, expertise as well as skills required for us to execute experiments efficiently and scientifically. Secondly, our team would like to thank the seniors who assisted us in instructing the microbiological tactics. To successfully finish the research process and the thesis, we would like to send many thanks to all the lecturers at the Department of Food Technology, the Faculty of Chemical and Food Technology, the Ho Chi Minh City University of Technology and Education had actively taught and conveyed basic knowledge to us throughout the four-year course and made it possible for us to study and do research at the institution. Finally, we'd like to thank our family and friends for their constant encouragement and support throughout the years.
The thesis cannot escape flaws due to a lack of experience and knowledge. We look forward to receiving comments from the committee so that we can enhance our topic. Ho Chi Minh City, 08th August, 2022 iv DISCLAIMER We hereby certify that the whole material of this thesis is our own original work. We declare that the contents of the graduation thesis have been accurately and completely cited in line with the requirements.
Ho Chi Minh City, 08th August, 2022 Sign v vi vii viii ix x xi xii xiii xiv xv xvi TABLE OF CONTENTS GRADUATION THESIS ASSIGNMENT. v LIST OF FIGURES. xx LIST OF TABLES .xxi LIST OF ABBREVIATIONS. xxiii Chapter 1: INTRODUCTION.
Research object and scope. 26 Chapter 2: LITERATURE REVIEW. Characteristics of bacterial cellulose. Effects of growth conditions on biosynthesis of bacterial cellulose.
Biosynthesis of BC. Applications of bacterial cellulose. Overview of food packaging from bacterial cellulose. The potential of BC in food packaging application.
BC films produced by impregnation. Films with disassembled BC. 45 Chapter 3: MATERIALS AND METHODS. Microorganism and culture medium.
Production of bacterial cellulose. Experiment 1: Determination of the chemical composition of coconut juice and rice extract. Experiment 2: Effects of rice extract on film-like cellulosic biomass production. Experiment 3: Film characterization.
Dertermination of total solids of rice extract and coconut juice. Determination of total carbohydrate. Determination of total nitrogen. BC production yield.
Field Emission – Scanning Electron Microscopy (FE – SEM). Fourier – transform Infrared Spectroscopy (FT-IR). Film light transmission. Water vapor permeability (WVP).
Statistical data analysis. 64 Chapter 4: RESULTS AND DISCUSSION. Determination of the chemical composition of coconut juice and rice extract. Effects of rice extract on film-like cellulosic biomass production.
Effects of rice extract on total sugar consumption. Effects of rice extract on bacterial cellulose film production. Changes of pH during BC production. Film light transmission.
86 Chapter 5: CONCLUSION AND RECOMMENDATIONS. 89 xix LIST OF FIGURES Figure 2.1 A typical bacterial cellulose G.2 Bacterial cellulose inter- and intra-hydrogen bonding [60] .3 Production of cellulose microfibrils by Acetobacter xylinum [63] .4 Biosynthetic process of Gluconacetobacter xylinus [98].5 Glucose oxidation pathways in G. oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae, Gluconobacter strains [101].6 Schematic diagram of BC culture.7 Schematic process to obtain Microfibrils, Nanofibrils and Nanocrystals from Bacterial Cellulose (BC) .1 Production of BC film .2 Summary of research contents .3 Glucose standard curve .4 Dry cell weight standard curve .5 Linear regression of water vapor permeability .1 Effects of rice extract on total sugar concentration .2 Effects of rice extract on (a) suspended bacterial biomass concentration, (b) cellulosic biomass content at 192h (D1) and (c) bacterial cellulose yield coefficient.3 Result of pH values during fermentation of G.4 Purified BC and FE-SEM images of BC samples utilizing 5 fermentation medium: (a) C100; (b) C75R25; (c) C50R50; (d) C25R75; (e) R100 .5 Infrared spectra of bacterial cellulose .6 Five different BC samples .7 Light transmission of BC film samples from 200 to 800 nm .8 Results of porosity.9 Results of water absorption and water solubility .10 Results of water vapor permeability.11 Results of puncture strength. 85 xx LIST OF TABLES Table 2.1 Analyzing the differences between cellulose made by bacteria and plant [46] .2 Bacterial cellulose composites and application .1 Maitaining and inoculating medium of G.2 Methods for analyzing the chemical characteristics of rice extract and coconut juice 49 Table 3.3 BC production medium (GM3) .4 Glucose standard curve preparation .1 Chemical composition of rice extract and coconut juice .2 Results of films thickness .3 Characterization of some cellulose unique peaks [219, 220] .4 Results of colorimetry for BC samples .5 Results of moisture content and moisture absorption .6 Results of thickness, tensile strength and elongation at break .7 Results of reusability of BC films.
86 xxi LIST OF ABBREVIATIONS BC bacterial cellulose bioPE biopolyethylene Gluconacetobacter xylinus G.xylinus PLA polylactic acid RH Relative humidity xxii ABSTRACT Bacterial cellulose (BC) is a nanostructured material that is mostly produced by the bacteria Gluconacetobacter. Traditionally, coconut juice has been applied for BC production growing medium. In this study, rice extract or rice wastewater was utilized to investigate the effects of rice extract on BC production in static culture and film characteristics using Gluconacetobacter xylinus (JCM 9730). The rice extract (20.81 g/L total sugar and 0.19% total nitrogen) was replaced the coconut juice (18.86 g/L total sugar and 0.08% total nitrogen) at different ratios of 0:1 (C100), 1:3 (C75R25), 1:1 (C50R50), 3:1 (C25R75), and 1:0 (R100).
After 8 days of fermentation, a BC yield (YBC/S g/g) of 0.11 g/L (C25R75 and R100) were achieved lower than that of C100 (0. Throughout the fermentation process, total sugar consumption and pH dramatically declined due to carbon catabolism and acidic metabolic substances biosynthesis. The obtained BC membranes from all studied cultures medium were then characterized. The light absorption values of all samples in the UV region were less than 8%, and less than 20% for the visible spectrum, which shows that all BC films were good UV resistant.
All samples had a tensile strength value greater than 30 MPa and high values of elongation at break, reflecting good mechanical properties and great elasticity of all BC films. Hydrophilic properties such as moisture, water absorption, solubility, and water vapor permeability were more strongly performed by the BC films produced in mediums with high content of coconut water. According to the reusability test, the C100 membrane can be reused up to 19 times, while the R100 membrane can only be reused 3 times. Besides, all BC specimens can prevent oil permeability because they exhibited no sign of oil absorption during the testing time.
All the above results indicate that the films obtained from the medium with the percentage of rice water from 25% to 75% were best suited for preserving food containing oil with their excellent properties. Keywords: Gluconactobacter xylinus, G.xylinus, bacterial cellulose, rice extract, rice wastewater, coconut juice. xxiii Chapter 1: INTRODUCTION 1. Rationale In today's industrial world, it is undeniable that the relationship between the packaging and food industries is indispensable.
Additionally, packaging also plays a crucial role in many other sectors where almost commodities must be meticulously enclosed in certain shapes before being distributed to the market [1]. The fundamental functions of food packaging are not only to avoid food from being affected by external influences and spoiling agents but also to give customers detailed information about the product as well as the nutritional components in the product [2]. Generally, food packaging aims to cost-effectively contain foods, satisfy industry standards and consumer expectations, guarantee food safety and nutritional values, and minimize adverse effects impact on the environment. Plastics have remained the preferred material in food packaging, where they have attributed to the majority of plastic waste contaminating the environment [3].
Therefore, there is a need for the creation of alternative materials that can perform the same function as conventional plastics. With advanced science and technology, various new types of packaging materials have been born, including bio-based and biodegradable packaging materials. They are produced from natural, renewable, and recyclable resources such as bio-polymers (polysaccharides, protein, etc) [4], bioplastics (polylactic acid (PLA), biopolyethylene (bioPE), polyhydroxyalkanoates, etc) [5], and biomass (bacterial cellulose). These ‘green’ packaging materials can be self-degradable and diminish environmental issues [6].
Among these, cellulose-based packaging and wrapping films and coatings are commercially appealing due to their compatibility with a variety of food items [7, 8]. Many studies have recently proven that bacterial cellulose (BC)– a type cellulose originated from Gluconacetobacter xylinus bacteria - offers numerous advantages in food packaging applications [9-11]. Compared to plant cellulose, BC has smaller diameters (approximately 25 nm – 100 nm), hence it is considered as cellulose-based nano-fiber [9]. Despite having the same molecular formula (C6H10O5)n as plant celluloses, BC lacks lignin, hemicelluloses, and pectin; hence, it’s purification is a simple, low-energy procedure, whereas plant cellulose purification typically requires harsh chemicals [9].
Furthermore, with remarkable properties such as high degree of polymerization, good barrier and tensile features, biodegradability, and biocompatibility with different food product kinds [12, 13], BC is expected to become an eco-friendly packaging material in the near future. Bio-cellulose application research has been steadily gaining traction in Vietnam in recent years although products from BC have been manufactured and applied in a wide range of fields 24 for years [14, 15]. However, there are currently no commercially available BC films for food packaging [12], as a consequence of costly production and low yield [16]. The media and process parameters have been regularly optimized by researchers in an effort to increase BC yields and facilitate effective BC production.