MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, August, 2022 SKL 0 0 9 1 5 5 SUPERVISOR:
INTRODUCTION
Rationale
In today's industrial landscape, the connection between the packaging and food industries is essential, as packaging is vital across various sectors where products must be securely enclosed before market distribution The primary functions of food packaging include protecting food from external influences and spoilage, while also providing consumers with important product information and nutritional details Ultimately, food packaging seeks to contain products cost-effectively, meet industry standards and consumer expectations, ensure food safety and nutritional integrity, and reduce environmental impact.
Plastics dominate food packaging but contribute significantly to environmental pollution This highlights the urgent need for alternative materials that can replace conventional plastics Advances in science and technology have led to the development of bio-based and biodegradable packaging options made from renewable resources like bio-polymers, bioplastics, and biomass These eco-friendly materials are designed to be self-degradable, addressing environmental concerns Cellulose-based packaging, in particular, is gaining commercial interest due to its compatibility with various food products.
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]
Bacterial cellulose (BC) is a cellulose-based nano-fiber with diameters ranging from 25 nm to 100 nm, distinguishing it from plant cellulose Although BC shares the same molecular formula (C6H10O5)n as plant celluloses, it is free from lignin, hemicelluloses, and pectin, allowing for a simpler and more energy-efficient purification process compared to the harsh chemical methods required for plant cellulose With its high degree of polymerization, excellent barrier and tensile properties, biodegradability, and biocompatibility, BC is poised to become a sustainable packaging material Research on BC applications has been increasing in Vietnam, although commercially available BC films for food packaging are still lacking due to high production costs and low yields Researchers are continuously optimizing media and process parameters to enhance BC production, which can be derived from various carbon and nitrogen sources, including fruit juices and vegetal extracts Currently, coconut water is the preferred substrate for BC production in Vietnam, but its limited availability due to climate change highlights the need for alternative sources, such as molasses.
Rice extract, a milky-colored by-product rich in carbohydrates and vitamins obtained from washing or soaking rice in industrial meal kitchens, has not been widely utilized for bacterial cellulose (BC) production despite its favorable qualities and low cost Research on rice extract is limited, focusing primarily on its chemical structure, morphology, and thermogravimetric properties in medical applications This highlights the need for further investigation into the potential uses of rice extract in various fields.
The study titled “Characterization of bacterial cellulose produced by Gluconacetobacter xylinus using rice extract as a nutrient source” explores the potential of using rice extract as a substitute for coconut juice in the cultivation of bacterial cellulose (BC) This research focuses on characterizing the properties of BC to evaluate its suitability for food packaging applications Additionally, the study highlights the innovative use of rice extract, an industrial food waste, in producing BC, thereby contributing to sustainable practices in the food packaging industry.
Research objectives
The research topic “Characterization of bacterial cellulose produced by
Gluconacetobacter xylinus using rice extract as a nutrient source” aimed to investigate the effects of growing media containing rice extract on the production of bacterial cellulose films by
G xylinus Furthermore, the characteristics of the films, and also their reusability were analyzed, providing technical parameters that are compatible with packaging for oil and oily food.
Research object and scope
The research object is bacterial cellulose films produced by Gluconacetobacter xylinus (JCM
9730) using media containing different ratios of rice extract and coconut juice.
Research content
The study consists of 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.
Scientific objective
The research findings are anticipated to significantly influence in-depth studies by establishing optimal conditions that enhance the collection of biomass carbon (BC) and improve the properties of cellulose-based films.
Practical objective
The project anticipates opening up the possibilities of bacterial cellulose film, an environmentally friendly material that can be made into biodegradable food packaging for a particular type of food.
LITERATURE REVIEW
Bacterial cellulose
Cellulose is the most common and affordable carbohydrate polymer globally, primarily sourced from plants and their byproducts However, extracting pure cellulose requires hazardous chemical processes to separate it from hemicellulose and lignin The rising demand for plant cellulose derivatives has led to increased wood usage, contributing to deforestation and environmental concerns Interestingly, various bacteria can also produce cellulose, a discovery made by Dr Brown in 1886 when he observed a jelly-like structure in acetic fermentation that resembled plant cellulose Bacterial cellulose (BC) boasts a crystallinity of up to 95%, compared to just 65% for plant cellulose, and is non-toxic, biodegradable, and chemically stable Following this, Teodula K Africa created the first bacterial cellulose product, nata de coco, in 1949 as an alternative to the traditional Philippine Nata de pina Recent studies have confirmed that bacterial cellulose is used to form the gelatinous pellicle in Nata, with the only commercially available form of BC currently being found in the Philippines' nata de coco.
Acetobacter sp is known for its ability to synthesize bacterial cellulose, a process first described by Schramm and Hestrin Their research established a standard medium for A xylinum, significantly advancing the field of bacterial cellulose production.
Bacterial cellulose exhibits a variety of morphologies, structures, and characteristics, leading to diverse applications Among the bacteria capable of producing cellulose, Gluconacetobacter xylinus (G xylinus) stands out for its high yield and is widely utilized in industrial cellulose production As classified in the Bergey taxonomy (2005), the genus Gluconacetobacter is part of a specific family.
Acetobacteraceae, with about 17 species [47] However, according to Yamada et al., in this genus, there are genotype and phenotypic differences between species, the representative group
Gluconacetobacter liquefaciens is motile and produces brown pigmentation, unlike its representative group, Gluconacetobacter xylinus, which does not exhibit these traits In 2012, Yamada et al reclassified the genus Gluconacetobacter into two new genera, with Komagataeibacter being one of them.
Komagataeibacter xylinus and Gluconacetobacter liquefaciens are key bacteria in the production of bacterial cellulose (BC) G xylinus is a gram-negative, obligate aerobic bacterium that thrives in highly acidic environments, with optimal culture conditions at temperatures between 25 and 30 °C and a pH range of 3 to 7 This bacterium forms transparent thin layers on the surface of liquid media, while its colonies on solid media appear spherical, measuring approximately 1 to 3 mm in diameter.
Figure 2.1 A typical bacterial cellulose G xylinus: (a) The cellulose pellicle formed in the broth;
(b) The morphologies of the Cel+ and Cel- (right) G xylinus colonies [50, 51]
Bacterial cellulose (BC), a natural polymer produced by acetic acid bacteria, has gained significant interest for its applications in biotechnology and medicine While both plant and bacterial cellulose share the same molecular formula (C6H10O5)n, they differ greatly in physical properties BC is entirely pure and forms nanofibrils directly from cells, unlike plant cellulose, which requires pretreatment to break down its complex structure These nanofibrils can be converted into macro fibers that are stronger than steel, offering a sustainable alternative to synthetic fibers derived from fossil fuels Composed solely of D-glucopyranose units linked by β-1,4 glycosidic bonds, BC's fibrous network features a three-dimensional arrangement stabilized by hydrogen bonds, resulting in a film with high surface area and porosity Consequently, BC exhibits remarkable properties, including a unique nanostructure, high water retention, elevated polymerization degree, exceptional mechanical strength, and significant crystallinity.
Figure 2.2 Bacterial cellulose inter- and intra-hydrogen bonding [60]
Prior studies had clearly demonstrated that BC and its derivatives have significant potential and a promising future in a variety of disciplines such as the biological, electronic and food industry [61,
Table 2.1 Analyzing the differences between cellulose made by bacteria and plant [46]
Properties Bacterial cellulose Plant-based cellulose
Size of fibers (nm) 20-100 micrometer scale
Acetobacter xylinum produces two types of cellulose: the thermodynamically stable cellulose II and the ribbon-like cellulose I During this process, protofibrils from the glucose chain are released through the bacterial cell wall, aggregating to form nanofibrils that create a web-shaped network structure of bacterial cellulose (BC) This cellulose is characterized by a high porosity and an abundance of hydroxyl groups on its surface, contributing to its hydrophilicity, biodegradability, and capacity for chemical modification A comprehensive explanation of the BC synthesis mechanism can be found in the work of Chawla et al (2009).
Figure 2.3 Production of cellulose microfibrils by Acetobacter xylinum [63]
Effects of growth conditions on biosynthesis of bacterial cellulose
The selection of bacterial sources significantly impacts bacterial cellulose (BC) synthesis, but the culture medium plays a crucial role as well Key ingredients in culture media, particularly carbon and nitrogen sources, along with pH-buffering salts, directly influence the quantity and quality of cellulose production Despite this, optimal conditions for BC production remain undetermined, as metabolic preferences can vary among species within the same genus.
Sugar is a crucial carbon source for bacterial cellulose biosynthesis, with sucrose, glucose, and fructose being the primary sugars used Different bacterial strains have varying requirements for carbon sources, which in turn affects their cellulose production For instance, glucose is commonly utilized in the production of cellulose by A xylinum species Research indicates that the choice of carbon source significantly influences cellulose formation, highlighting its importance in industrial bulk production.
The HS medium developed by Schramm & Hestrin in 1954, which includes components like 2% glucose and 0.5% peptone, is more costly for cellulose production compared to sugar-based mediums Coconut water, recognized for its isotonic properties and high sugar content, has been identified as a suitable environment for the growth of the "Nata de coco" bacterium since the 1960s This bacterium, known as Acetobacter xylinum, facilitates the spontaneous production of nata de coco at the interface of coconut water and air Nata de coco, a popular Filipino delicacy, has also gained traction in various Asian countries, leading to increased research on using coconut water as a culture medium for bacterial cellulose.
Acetobacter xylinum strain is primarily cultivated in coconut water, which, while a sweet and refreshing drink, limits biomass yield due to its dependence on natural conditions Coconut water serves multiple purposes, including traditional medicine, a medium for microorganism growth, ceremonial gifts, and conversion into vinegar or wine Research by Rohaeti et al highlights rice wastewater as a potential source for bacterial cellulose (BC) production, as it contains starch, protein, minerals, and vitamin B, providing essential nutrients for A xylinum By combining rice wastewater with sucrose, a cellulose yield of 7.6 g/L was achieved, resulting in a cellulose with a high crystalline percentage of 73%, demonstrating mechanical and thermal stability This research aims to identify alternative BC culture mediums that offer higher biomass yields at stable or lower costs compared to coconut water.
Recent research has focused on replacing traditional carbon sources for bacterial cellulose (BC) production with alternative mediums derived from food and agricultural waste, aiming to reduce costs Various cellulosic wastes, such as food processing effluents and molasses, have been explored alongside conventional sources like coconut juice, leading to increased BC yields Studies have also investigated the use of rice extract and wastewater as substrates for BC growth Rohaeti et al characterized films produced from rice water using techniques like FTIR, SEM, XRD, and TGA-DTA Additionally, the antibacterial properties of BC and its composites were tested against Staphylococcus aureus, although the study did not evaluate factors such as solubility, water absorption, or oil permeability.
BC coatings have been explored in various studies for their applications and properties Pham T K D investigated the production of BC films from rice water, noting that the sugar content in the medium was still high, and highlighted BC's potential as a drug delivery system for ranitidine Additionally, N X Thanh assessed the release of curcumin from BC cultured in rice water Trinh V.T et al examined the use of BC from rice water as packaging to preserve Ham Yen oranges, although their study focused on limited properties such as chemical structure, mechanical strength, and antibacterial characteristics.
Nitrogen is a vital nutrient for the production of bacterial cellulose, as it supports the growth and multiplication of microbial cells The foundational medium developed by Hestrin and Schramm, which includes 0.5% yeast extract and 0.5% peptone, is widely utilized in research Various research teams have made slight modifications to the proportions of these nitrogen sources, such as yeast extract and tryptone Among these, corn steep liquor (CSL) has proven to be the most effective source Numerous studies have highlighted CSL's significant influence on media used in various biotechnological applications.
Methionine and glutamate are key amino acids recognized as essential nitrogen sources Research by Matsuoka et al indicates that methionine is particularly crucial, contributing to 90% of cell growth and cellulose formation compared to media lacking this amino acid.
Research indicates that the production of cellulose is supported by vitamins such as pyridoxine, nicotinic acid, p-aminobenzoic acid, and biotin, although the effects of pantothenate and riboflavin remain inconclusive Contrary to these findings, Fiedler et al suggest that vitamins do not enhance cellulose synthesis Additionally, Fontana et al explored the use of plant extract infusions, particularly black tea, to stimulate cellulose formation; however, issues with the quality of the cellulose produced led to its discontinuation for Biofill@ development.
Obligate aerobic bacteria from the genera Acetobacter and Komagataeibacter spp utilize two primary metabolic pathways: the pentose phosphate pathway for carbohydrate oxidation and the less active glycolysis pathway These bacteria also possess enzymes from the Krebs cycle, which facilitate the oxidation of organic acids and their biosynthetic derivatives Notably, the biochemical pathways and regulatory mechanisms involved in cellulose biosynthesis from the A xylinum strain have been extensively researched.
In the bacterial cytoplasm of *G xylinus*, the production of cellulose begins with nucleotide-activated glucose molecules During laboratory fermentation, glucose serves as a key substrate for this biosynthesis, which occurs in four main steps involving specific biocatalysts: (1) glucokinase converts glucose into glucose-6-phosphate; (2) phosphoglucomutase transforms glucose-6-phosphate into glucose-1-phosphate; (3) UDP-glucose pyrophosphorylase changes glucose-1-phosphate into uridine diphosphate glucose (UDP-glucose); and (4) cellulose synthase utilizes UDP-glucose to produce glucan chains These fibrils are then exported from the cell by cellulose export components, facilitating the assembly and crystallization of cellulosic chains.
Figure 2.4 Biosynthetic process of Gluconacetobacter xylinus [98]
The enzyme glucose oxidase converts glucose into gluconate, leading to the production of 2- and 5-ketogluconate acids when G xylinus is grown in glucose- or sucrose-based media This process, facilitated by glucose dehydrogenase (GDH), results in a decrease in pH and a reduced yield of bacterial cellulose (BC).
Figure 2.5 Glucose oxidation pathways in G oxydans [100] G oxydans is a gram-negative bacterium belonging to the family Acetobacteraceae, Gluconobacter strains [101]
Static cultivation is a widely used method for producing bacterial cellulose (BC), where the culture medium is inoculated in shallow trays, allowing BC pellicles to form and float due to CO2 bubbles produced by bacteria The efficiency of BC production is influenced by the air/liquid contact area, with cultivation typically lasting between 5 to 20 days until the BC sheet nearly fills the tray This method enables the BC membrane to adopt the shape of the substrate, which is particularly beneficial in regenerative medicine when specific shapes are required While this characteristic has not been extensively explored for food applications, it holds potential for creating uniquely shaped products similar to nata, catering to diverse consumer needs, including those of children.
This method, illustrated in Figure 2.6, utilizes shallow bottles or trays filled with liquid growth media for bacterial culture Over time, a gelatinous bacterial cellulose (BC) pellicle forms and floats on the surface of the media, ultimately encasing the bacteria within it.
Figure 2.6 Schematic diagram of BC culture (A) Conventional static culture and (B)
Static culture methods are often time-consuming and yield low productivity, which can hinder industrial applications The cellulose membrane formed in these cultures tends to trap bacteria, restricting their access to oxygen and leading to a gradual decline in nutrient concentration, ultimately limiting bacterial cellulose (BC) production An effective solution to this problem is the fed-batch culture technique Research by Shezad et al (2009) indicates that fed-batch cultivation can increase yields by two to three times compared to traditional batch methods by introducing new alternative media during the cultivation process This intermittent feeding creates a critical distance between the old air-liquid contact and the BC pellicle, allowing newly formed BC pellicles to develop at the new air-liquid interface rather than stacking on top of existing ones, facilitating layer-by-layer growth.
Overview of food packaging from bacterial cellulose
2.3.1 The potential of BC in food packaging application
In today's market, there is a growing consumer demand for natural foods that adhere to high standards of quality and safety To preserve the freshness of fruits and vegetables, innovative food packaging solutions have been developed, utilizing materials such as alginate, cellulose, chitosan, carrageenan, and pectins Among these, cellulose-based films and coatings are particularly valuable due to their compatibility with various food products Research indicates that these packaging solutions effectively reduce moisture loss and minimize oil absorption in fried foods While bacterial cellulose (BC) offers numerous advantages for food packaging, its use in pure form presents challenges, leading to the development of various modifications to improve its properties.
The promising application of bacterial cellulose (BC) has garnered significant interest for developing biodegradable materials, primarily by utilizing BC nanofibers or nanocrystals as reinforcing agents in various matrices Additionally, BC can serve as the main matrix material, undergoing modifications to enhance its functionality for food preservation BC composites are effectively applied to different food types to impart antibacterial and antioxidant properties.
Table 2.2 Bacterial cellulose composites and application
No Application field Function Types of food References
6 Methyl cellulose Enhancing shelf life Egg [159]
7 Methyl cellulose Enhance bioavailability Vitamin C [160]
2.3.2 BC films produced by impregnation
BC membranes possess an intricate structure with numerous voids, allowing for the entrapment of various components Research on food films has explored the impregnation technique; however, this method, while technically simple, is not ideal for continuous film production or for creating coatings on food surfaces.
Combining bacterial cellulose (BC) with other polymers can enhance specific properties of the resulting material For example, researchers have developed BC-chitosan films by incorporating BC into a chitosan solution, leveraging chitosan's antibacterial properties.
[162] In addition to displaying activity against Gram-negative and Gram-positive bacteria, the resulting composite film also had a strikingly greater elastic modulus than neat BC films.
Cross-linking is an effective method for improving the tensile and barrier properties of packing sheets Proteins, due to their highly nucleophilic amino groups, are more involved in cross-linking than polysaccharides In 2012, researchers coated bacterial cellulose (BC) sheets with gelatin and immersed them in various protein cross-linking agents to initiate cross-linking reactions This process led to the formation of compact interpenetrating networks of BC and gelatin, particularly at higher gelatin concentrations, which enhanced the tensile strength of the composite sheets.
Bacterial cellulose (BC) has been used to progressively apply antimicrobial agents on food surfaces, extending the microbial food stability in the process.
There was a development of antimicrobial BC membranes by nisin-impregnating BC sheets
The immobilization of nisin, identified through Fourier-transform Infrared Spectroscopy (FT-IR), occurs due to the interaction between nisin's amine groups and the carboxylic acid groups of bacterial cellulose (BC) This immobilization demonstrated effectiveness against both Gram-negative and Gram-positive bacteria Nguyen's team successfully applied similar films to vacuum-packed frankfurters, effectively inhibiting bacterial growth (Nguyen et al., 2008).
The production of active sausage casings involved coating BC tubes with -polylysine (-PL)
Composite casings not only exhibit excellent tensile and barrier properties but also possess antibacterial activity that extends the shelf life of sausages Additionally, these casings maintain thermal stability, effectively inhibiting S aureus activity even after autoclaving at 121°C for 30 minutes.
BC membranes infused with antimicrobial peptides demonstrated superior efficacy in inhibiting the growth of Listeria monocytogenes compared to free bacteriocins, highlighting the protective role of BC for these peptides.
Jebel and Almasi (2016) developed multilayer films featuring an antibacterial layer sandwiched between two outer layers to control the release rate of ZnO nanoparticles The active layer was coated with wet BC membranes, dried, and then infused with a nanoparticle dispersion This incorporation of ZnO nanoparticles not only imparted antibacterial properties to the films but also enhanced their tensile strength and decreased water vapor permeability.
Antioxidants, oxygen scavengers, and ethylene absorbers are among the many active substances that can be incorporated into biodegradable (BC) membranes, alongside antimicrobial agents These compounds serve as carriers that help enhance food stability by combating various stages of degradation.
Ul-Islam et al (2012) successfully developed bacterial cellulose (BC) nanocomposites incorporating montmorillonite (MMT), despite the uncommon practice of using impregnation to reinforce BC membranes This innovative approach enhances the tensile strength and thermal stability of the resulting nanocomposite material.
Poly(L-lactic acid) (PLLA) reinforced with bacterial cellulose (BC) was developed by submerging BC sheets in chloroform-dissolved PLLA, followed by drying to remove the solvent The study revealed that the resulting transparent films exhibited a tensile strength that was double that of pure PLLA, along with enhanced crystallinity in the nanocomposites.
Bacterial cellulose (BC) is widely used in packaging production due to its ability to be converted into various forms such as microfibrils (BCMFs), nanofibrils (BCNFs), and nanocrystals (BCNCs) This versatility facilitates the production of continuous films or coatings, making it more suitable for industrial applications The large surface area of BC enhances physical interactions, particularly hydrogen bonding, which improves the structural integrity of polymers As a result, this leads to superior barrier, mechanical, and thermal properties.
A suspension of BCMFs or BCNFs in aqueous solution (referred to as a "BC slurry") was noted resulting in the obtention process of pure BC films, according to other authors [173].
Figure 2.7 Schematic process to obtain Microfibrils, Nanofibrils and Nanocrystals from
BCNFs feature a long, network-like structure with diameters ranging from 40 to 70 nm Their production involves mechanical methods, including high-pressure homogenization, grinding, and refinement techniques.
Most BCMFs are classified as nanofibrils since their diameters or widths are often in the submicrometer range, and they comprise nanostructures as a primary component [175, 176]
The primary method for isolating bacterial cellulose nanocrystals (BCNCs) is acid hydrolysis, although enzymatic hydrolysis is also viable Cellulose microfibrils consist of chains of cellulose crystals arranged in paracrystalline regions Acid treatment leads to the formation of rod-shaped cellulose nanocrystals by potentially removing these paracrystalline areas The average dimensions of BCNCs obtained through acid hydrolysis are approximately 20±5 nm in diameter and 290±130 nm in length It is important to note that the terms BC nanocrystals and BC nanowhiskers are often used interchangeably.
Summary
In conclusion, bacterial cellulose (BC) has diverse applications across various sectors, including fashion, cosmetics, medical, and food industries To reduce manufacturing costs, researchers are exploring alternative culture media, such as rice water, which is an industrial byproduct While some studies have focused on the medical applications of BC derived from rice water, there is a lack of research on its use in preserving oily foods Additionally, the optimal coconut water to rice ratio for producing the best BC film remains unexamined.
MATERIALS AND METHODS
Materials
The strain Gluconacetobacter xylinus (JCM 9730) used in this study was derived from the strain collection of the Department of Biotechnology – Ho Chi Minh City University of Technology
Matured coconut juice, originated from Phuc An coconut barn, Ben Tre Province, was purchased from Thu Duc market (83 Vo Van Ngan Street, Linh Chieu Ward, Thu Duc District,
Rice extract was collected from a local kitchen in Ho Chi Minh City (246 Cao Dat Street, Ward
1, District 5, Ho Chi Minh City)
Coconut juice and rice extract used for all batch fermentation were homogenized and freezing stored on refrigerator to ensure even media when using
G xylinus was maintained on GM1 medium with the composition shown in Table 3.1 One milliliter of the strain was preserved in a 2 mL eppendorf as a stock culture and frozen at – 40 o C
Table 3.1 Maitaining and inoculating medium of G xylinus
The pH of the medium was adjusted to 5.00 by concentrated acetic acid
To produce the necessary amount of cellulose, G xylinus bacteria were pre-cultivated through two fermentation stages The first stage involved transferring 1 mL of stock culture into 9 mL of GM2 and incubating it in a test tube at 30°C for 72 hours In the second stage, 10 mL from the test tube was transferred to an Erlenmeyer flask containing 100 mL of GM2, which was then covered with cotton wool and stirred at 200 rpm for another 72 hours at 30°C Prior to inoculation, all culture media, containers, and tools were sterilized at 121°C for 15 minutes.
All chemicals utilized in this study were sourced from Bach Khoa Limited Liability Company and Hoa Nam Chemicals – Laboratory Equipment Company Limited, both located in Ho Chi Minh City.
Production of bacterial cellulose
Figure 3.1 Production of BC film
After pre-cultivating in an Erlenmeyer flask, the broth's biomass was measured at 600 nm, yielding a concentration of 6 mg/L Subsequently, 10 mL of the pre-culture medium was centrifuged at 5000 rpm for 15 minutes to separate the biomass The bacterial cells were then transferred to 100 mL of aseptic GM3 medium and incubated for 3 to 5 days at 30 to 35 °C For optimal production of bacterial cellulose (BC) films, G xylinus was cultured in 300 mL of GM3 for 8 days The resulting bacterial cellulose was washed with water to eliminate residual sugar and then boiled in a 0.25M NaOH solution at 80 °C for 1 hour to remove any attached bacterial cells This purification step enhanced color uniformity and eliminated metabolites and residues from the culture medium The films were subsequently washed with deionized water to ensure complete removal of alkali, achieving a neutral pH Finally, the wet BC film was dried at 80 °C until a constant weight was attained.
Experimental design
Figure 3.2 Summary of research contents
3.3.1 Experiment 1: Determination of the chemical composition of coconut juice and rice extract
The purposes of this experiment were to identify and control the quality of input materials by analyzing the chemical composition of rice extract and coconut juice
The total solids, pH, total carbohydrates and total nitrogen of rice extract and coconut juice were all determined (Table 3.2)
Table 3.2 Methods for analyzing the chemical characteristics of rice extract and coconut juice
Total solids Loss-on-drying method AOAC Method 990.19, 990.20 pH pH-meter HI991003
Total carbohydrates Phenol-Sulfuric Acid
Total nitrogen Kjeldahl nitrogen Method Nielsen, S S (2017) [188]
3.3.2 Experiment 2: Effects of rice extract on film-like cellulosic biomass production
Extrinsic factors play a crucial role in influencing the morphology of the nanocellulose network and the rate of cellulose production Additionally, the components of the growing media significantly impact the rate, yield, and structural integrity of cellulose in bacterial cellulose (BC) production.
This study investigates the effects of different nutritional sources, specifically rice extract and coconut juice in various ratios, on the production of film-like biomass.
Rice extract was used to replace coconut juice in BC production according to Table 3.3:
Table 3.3 BC production medium (GM3)
The pH of the medium was adjusted to 4.00 by concentrated acetic acid
*Note: The coconut juice was substituted by rice extract at 0% (C100), 25% (C75R25), 50% (C50R50), 75% (C25R75) and 100% (R100)
The cell cultures were incubated statically at room temperature for 8 days During this period, liquid culture samples were periodically collected to measure suspended biomass and total sugar concentrations Each experiment was conducted in triplicate to ensure accuracy.
Bacterial cellulose (BC) holds significant promise in the food packaging sector, yet its characteristics and limitations have not been thoroughly explored This study aims to investigate the diverse physical and chemical structures and properties of bacterial cellulose produced in 3D matrix or sheet form.
Cutting the BC film into pieces of the appropriate size for each method of analysis then take measurements and record the results a Film thickness
A digital caliper (D0022-06; CMart, Taiwan) with 0.001 mm of precision was used to determine the thickness of the films (conditioned at 75% RH for 24h) b Morphology
Field Emission – Scanning Electron Microscopy (FE-SEM) is a sophisticated technique for capturing the microstructure images of the materials [190] c Chemical structure
The Fourier – transform Infrared Spectroscopy (FT-IR) analysis method scans test materials and examines chemical characteristics using infrared light d Film color
Color of the BC film was evaluated using a colorimeter (KONICA MINOLTA, Model CR-
Film light transmission was determined by using a spectrophotometry (UH5300 Spectrophotometer, Hitachi, Japan) f Barrier properties
The hydrophilicity of BC films was evaluated by assessing their moisture content, moisture absorption, water solubility, water absorption, water vapor permeability, and porosity Additionally, the impermeability of BC to oils and fats was also examined, along with its mechanical properties.
Mechanical testing of bacterial cellulose samples was conducted to evaluate their properties, focusing on tensile strength, elongation at break, and puncture resistance Additionally, reusability testing was performed to assess the durability of the samples.
Reusability is an essential aspect of packaging that has often been overlooked in previous studies on biopolymer composites (BC) This research focused on BC films derived from growth cultures with varying ratios of rice extract and coconut juice to evaluate their potential for oil packaging applications.
Analytical methods
3.4.1 Dertermination of total solids of rice extract and coconut juice
“Total solids” is a term used to describe the dry matter that remains following a moisture analysis The experiment was carried out using the AOAC 990.19, 990.20 method
Weigh 5 g of the liquid sample in a test tube and gently heat it on a hot plate to evaporate most of the water Subsequently, place the samples in a Memmert UF260 forced draft oven at 105°C for 3 hours.
The total solids of the sample were calculated using the following equation:
% Total solids = 100 −weight of H 2 O in sample weight of wet sample × 100 3.1
The pH was measured using a digital pH meter (HI9124, HANNA Instruments, USA) in accordance with AOAC method 981.12 [191] Standard buffer solutions (pH 4.0 and 7.0) were used to calibrate the pH electrode
Under strong acidic conditions and heat, carbohydrates, including simple sugars, oligosaccharides, and polysaccharides, undergo reactions to produce furan derivatives These derivatives then condense with phenol, resulting in stable yellow-gold compounds that can be detected using spectrophotometry.
Glucose standard solution (100 mg glucose/L)
Phenol, 80% wt/wt in H2O: adding 20g deionized distilled water to 80 g of redistilled reagent grade phenol (crystals)
Standard curve of different concentrations (0; 20; 40; 60; 80; 100 μg glucose/2 mL) was prepared in test tubes by 1:1000 dilution with distilled water
Table 3.4 Glucose standard curve preparation μg glucose/2 mL
0 20 40 60 80 100 mL glucose stock solution 0 0.2 0.4 0.6 0.8 1.0 mL distilled water 2.0 1,8 1.6 1.4 1.2 1.0
Adding 0.05 mL 80% phenol and 5.0 mL H2SO4 to each tube from the table with a total volume of 2 mL The test tube should receive a quick addition of the sulfuric acid reagent To achieve good mixing, direct the acid stream against the liquid surface rather than the test tube's side The heat generated when H2SO4 is added to an aqueous sample is what drives these reactions As a result, sulfuric acid addition rates must be standardized Placing tubes in a 25°C bath for 10 minutes after letting them stand for 10 minutes The absorbance was then measured at 540 nm using a Double Beam Spectrophotometer (UH5300, Hitachi, Japan) after cooling to room temperature Each concentration was done in triplicate
Figure 3.3 Glucose standard curve Procedure
1.0 mL of the sample which was made into 1:1000 dilution was pipetted into a test tube, then adding 1.0 mL of distilled water and 5.05 mL of phenol – sulfuric acid reagents After allowing tubes to stand for 10 minutes, place them in a 25°C bath for 10 minutes At 540 nm, the absorbance was measured The data received was calculated following the standard curve: y = 109.57x; R 2 0.9942 (Figure 3.3) and expressed as g/L sample
The Kjeldahl technique is used to determine the nitrogen concentration in a sample, allowing for the estimation of protein content based on a specific protein-to-nitrogen ratio This method involves three key stages: digestion, distillation, and titration During digestion, organic nitrogen is converted to ammonium at approximately 370°C with the help of a catalyst In the distillation phase, the sample is made alkaline with NaOH, and nitrogen is released as ammonia (NH3), which is then captured in a boric acid solution Finally, the amount of ammonia nitrogen is quantified through titration with a standard hydrochloric acid (HCl) solution.
Concentrated H2SO4, pure HClO4, K2SO4, CuSO4, TiO2
NaOH 40%: 40g of NaOH was dissolved in distilled water to make 100 mL of solution
0.1N NaOH solution: dissolving 0.4g NaOH into distilled water to make 100 mL of solution
0.1N H2SO4 solution: dissolving 0.27 mL H2SO4 into distilled water to make 100 mL of solution y = 109.57x R² = 0.9942
G luco se co ncent ra tio n (μ g g luco se/2 m L )
Phenolphthalein 1%: dissolving 1g phenolphthalein into 50 mL ethanol, then adding 50 mL of distilled water
In the digestion process, 2 mL of the liquid sample is weighed and placed into a Kjeldahl tube, followed by the gradual addition of 10 mL of concentrated H2SO4 To enhance digestion, a catalyst mixture is introduced, consisting of 5.0 g K2SO4, 0.15 g CuSO4, and 0.15 g TiO2 K2SO4 raises the boiling point, while CuSO4 and TiO2 serve to catalyze the reaction effectively.
Protein distillation was performed using a semi-automatic nitrogen distiller BUCHI Distillation Unit from Switzerland The complete digested sample solution was transferred from the Kjeldahl flask to a 100 mL volumetric flask and diluted to the mark with water A 10 mL aliquot of the test solution was taken from the volumetric flask and placed into the sample tube, which was then inserted into the system Nitrogen distillation was conducted until all NH3 was completely liberated from the sample container.
Titration: Titrating each sample and blank with the NaOH 0.1N
The percent nitrogen of triplicate liquid samples was calculated:
N: nitrogen content (%) a: volume of 0.1N H2SO4 to absorb NH3 (mL) b: volume of 0.1N NaOH used for titration (mL) m: mass of sample to be digested (g)
V: total volume of the digested solution (100 mL) v: volume of inorganic distillation solution (10 mL)
0.0014: amount of nitrogen (g) for 1mL H2SO4 0.1N
K: NaOH concentration correction factor 0.1N (the titration factor is considered to be 1 if using a standard tube)
The yield ratio YBC/S (g/g) of cellulosic biomass to total sugar consumption was estimated using the equation [192]:
3.3 where D1 is cellulosic biomass content (g/L) - the cellulose was removed from the liquid culture and dried at 80 o C (UF260, Memmert, Germany) until constant weight b Suspended cells growth
Suspended G xylinus cell development was monitored using an ultraviolet-visible spectrophotometer to determine optical density (OD) at 600 nm (UH5300, Hitachi, Japan) The
OD600 was used to calculate the concentration of suspended bacterial biomass using the formula from dry cell weight standard curve (Figure 3.4):
Dry cell weight, D2 (g/L) = 0.3665 × OD600 (R 2 = 0.9875) where D2 is suspended cell biomass (g/L)
Figure 3.4 Dry cell weight standard curve 3.4.6 Film thickness
Each film specimen was measured at five locations—one at the center and four at the edges—to calculate the average thickness (Haghighi et al., 2021) The results are presented as the mean of the measurements with standard deviation (SD), based on tests conducted in pentadruples (n = 5) The relationship is described by the equation \$y = 0.3665x\$ with a coefficient of determination \$R² = 0.9875\$.
Su sp en d ed ce ll c o n ce n tr atio n ( g /L )
3.4.7 Field Emission – Scanning Electron Microscopy (FE – SEM)
An electron microscope functions on principles similar to those of a light microscope, but it uses energetic electrons instead of visible light as its source To minimize the interference of gas molecules on the electron beam and the secondary and backscattered electrons used for imaging, Field Emission Scanning Electron Microscopy (FE-SEM) is typically performed in a high vacuum environment The Zeiss Crossbeam is a notable example of this technology.
In this study, a 340 was employed to capture microstructure images and analyze the specimens Before conducting the morphological examination, the specimens, excluding the powder, were cut into smaller pieces measuring approximately 1 cm × 1 cm and coated with platinum to enhance electrical conductivity.
BC samples were analyzed using a FE-SEM S4800 (Hitachi, Japan) at the Saigon Hi-Tech Park Training Center in Tan Phu Ward, District 9, Ho Chi Minh City, following a drying process at 80°C for 24 hours.
In order to increase the effectiveness of observation, an ion coating machine attaches the sample to a platinum-coated carbon
Parameter: cccelerating voltage: 5.0 kV, magnification: 10 000 – 60 000, resolution: 1 –
Calculation: high-definition photos that are subsequently captured were used to observe and calculate average diameter of fibers by ImageJ 1.48 software
3.4.8 Fourier – transform Infrared Spectroscopy (FT-IR)
The FT-IR method is based on the investigated sample's absorption of infrared radiation An
IR beam is produced by the FT-IR spectrophotometer and emanated from a burning black-body source [193] The interferometer is where the spectral encoding happens after the beam enters it
An interferogram, resulting from the recombination of beams with different path lengths in an interferometer, demonstrates constructive and destructive interference As the beam enters the sample compartment, it is absorbed by the sample at specific frequencies unique to it, as indicated by the interferogram Radiant energy data is recorded at precise moments, enabling the plotting of changes in radiant energy over time.
The FT-IR analysis of BC membranes, dried at 80°C for 2 hours, was performed using a Jasco FT-IR-4700 instrument from Japan The data was collected across a frequency range of 4000 to 400 cm\(^{-1}\) [195], and the spectral measurement results were graphically represented using Origin 2021 software.
Before measuring colors, the colorimeter is calibrated using a reference white standard (L* 90.65, a* = 0.83, b* = -3.7) To ensure accurate measurements, the colorimetric aperture is placed vertically on the surface of the BC film, minimizing light scattering Each BC sample's color is assessed at five different locations on one surface, and the average values from three replicates are computed.
*Note: L* - lightness (0 =black, 100 =white), a* (-a* to greenness, +a* to redness) and b* (-b* to blueness, +b* to yellowness)
The parameters L* (lightness), a* (redness/greenness), and b* (yellowness/blueness) were recorded as the results Using the following the equation, the total color difference (E*) was calculated:
The color difference, represented as ∆E ∗, is calculated using the formula \$\Delta E^* = \sqrt{(\Delta L)^2 + (\Delta a^*)^2 + (\Delta b^*)^2}\$, where L*, a*, and b* indicate the discrepancies between the color parameters of the samples and a reference white plate used as the film background Average values for each film were determined by taking ten readings from various positions on both the top and bottom sides of the films.
Statistical data analysis
To assess the differences in average responses among treatments, one-way ANOVA tests were conducted using Minitab software (Minitab 19, Ink, USA) and Microsoft Excel 2016 Tukey’s multiple range test was employed to identify significant differences, with a significance level set at p < 0.05 Results are presented as mean ± standard deviation (SD).