EFFECT OF PLASTICIZERS ON CHARACTERISTIC OF BACTERIAL CELLULOSE-ALGINATE-GELATIN COMPOSITE FILMS Good morning everybody. My name is Sutasinee Seetabhawang and I would like to present in subject “Effect of plasticizers on characteristic of bacterial cellulose-alginate-gelatin composite films. PRESENT BY: SUTASINEE SEETABHAWANG ADVISOR : ASSOC.PROF. MUENDUEN PHISALAPHONG, Ph.D. Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Thailand

BACKGROUND Biodegradable films for food packaging have attracted and interested because they could enhance food quality and reduce pollution of traditional plastic films. Background of this work In packaging application, composite film for food has attracted and interested because it could enhance food quality and reduce pollution of traditional plastic films. In order to overcome the problem associated with brittle films after drying, plasticizers are used to improve the film’s properties. The addition of plasticizer can reduce inter-chain interactions, increase film flexibility, reduce brittleness and prevent the shrinkage during handling and storage. In order to overcome the problem associated with brittle films after drying, plasticizers are used to improve the film’s properties. The addition of plasticizer can reduce inter-chain interactions, increase film flexibility, reduce brittleness and prevent the shrinkage of the films.

OBJECTIVES 1. To develop composite films from the blends of bacterial cellulose (BC), alginate (A) and gelatin (G), we called “BC/A/G” This work aims 1. To develop composite films from the blends of bacterial cellulose (BC), alginate and gelatin called as “BC/A/G” 2. To investigate the effects of the blend composition and type of plasticizer on the film characteristics 3. To determine the physical, chemical and mechanical properties of the films for further application as packaging materials 2. To investigate the effects of the blend composition and type of plasticizer on the film characteristics 3. To determine the physical, chemical and mechanical properties of the films for further application of packaging materials

INTRODUCTION 1. BACTERIAL CELLULOSE (BC) BC is a linear polysaccharide of glucose. Next, I would like to introduce the 3 key components of the films: The first material is bacterial cellulose. Bacterial cellulose is linear polysaccharide of glucose with formula of (C6H10O5)n as shown below and it can be synthesized by Acetobacter xylinum and is nearly-purified cellulose. The main functional groups of BC are ether group (C-O-C) and water (O-H-O). It can be biosynthesized by Acetobacter xylinum. (Brown, 1996)

INTRODUCTION PROPERTIES OF BC Strength High purity (~99.9% cellulose) High water holding capacity Superior water resistance High mechanical strength Properties of BC, the strength of BC includes high purity about 99.9% cellulose, high water holding capacity, high hydrophilicity, superior water resistance, and high mechanical strength but it is poor ability of rehydration after drying due to its high crystallinity. Weakness Poor ability of rehydration after drying due to its high crystallinity (Chiaoprakobkij et al., 2011; Brown, 1992; White et al., 1989)

INTRODUCTION 2. SODIUM ALGINATE (A) A is a linear polysaccharide copolymer of (1-4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) monomers. The second material is sodium alginate. Sodium alginate is a linear polysaccharide copolymer of (1-4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) monomers with the formula of (C6H7O6Na)n as shown below and it can be isolated from algae and seaweed. The main functional group of sodium alginate is ester group (–COO). It can be isolated from algae and seaweed. (Pinto Cruz et al., 2004, modified)

INTRODUCTION PROPERTIES OF A Properties Well-characterized hydrogel forming Water resistance and high mechanical properties when crosslinked by divalent cations such as CaCl2 Thickener and Stabilizer agent Non-toxicity Properties of Sodium alginate It forms well-characterized hydrogel with water resistance and high mechanical properties by adding divalent cations as crosslinked agents such as CaCl2. Moreover, it is also thickener and stabilizer agent, and non-toxicity.

INTRODUCTION 3. GELATIN (G) G is a water soluble protein. Solubility at temp. > 45˚C It is a thermo-reversible structure. Setting at temp. < 30 ˚C The last one is gelatin. Gelatin is a water soluble protein, which is produced by hydrolysis of animal collagen with formula of (C102H151N31O39)n as shown below. It is a thermo-reversible structure that has solubility at temp. higher than 45˚C and has setting at temp. less than 30˚C. The main functional groups of gelatin are –NH group of amide and partial amine and C-N group. .   (Peña et al., 2010)

INTRODUCTION PROPERTIES OF G Strength High gel formation High transparent Non-toxic High film-forming capacity Properties of gelatin, the strength of gelatin includes high gel formation, high transparent, non-toxic and high film-forming capacity but it is poor mechanical properties and poor water resistance. Weakness Poor mechanical properties Poor water resistance (Schrieber and Gareis, 2007)

Additive Compounds EXPERIMENTAL STUDY Materials in this work are bacterial cellulose, sodium alginate, gelatin type B which they are main material for film preparation. Glycerol and sorbitol are plasticizer and Cacl2 dihydrated is cross-liking agent. CaCl2.2H2O D-sorbitol powder (S) Glycerol (Gly)

1. Preparation of BC slurry EXPERIMENTAL STUDY 1. Preparation of BC slurry Unpurified BC Crushed and homogenized purified BC Experimental method Step1 Purification of BC Unpurified BC was washed with water and soaked in 1% w/v sodium hydroxide for 24 h. After that it was washed again with DI water for neutralization (pH7) so we got purified BC. Soaked in 1 %w/v sodium hydroxide for 24 h Washed with DI water until pH = 7 BC slurry

2. Preparation of A solution Sodium alginate of 2% (w/w) EXPERIMENTAL STUDY 2. Preparation of A solution Sodium alginate of 2% (w/w) DI water Step 3 Preparation of Sodium alginate solution Sodium alginate of 2% (w/v) was dissolved in distilled water with constant stirring at room temperature for 2 days to form gel-like solution.

3. Preparation of G solution EXPERIMENTAL STUDY 3. Preparation of G solution Gelatin of 15% (w/v) DI water Step 4 Preparation of Gelatin solution Gelatin powder from porcine skin 15% (w/v) was hydrated with distilled water at room temperature and heated up to 50±5 °C with constant stirring until completely dissolved. At 50±5˚C

4. Preparation of BC/A/G Films EXPERIMENTAL STUDY 4. Preparation of BC/A/G Films Vary1: 60/10/30 Vary2: 60/20/20 Vary3: 60/30/10 BC slurry A solution G solution Added plasticizer (Gly, S, and GS) at a ratio of 0-3 g per 10 g gelatin solution Step 5 Preparation of BC/A/G films The BC slurry was mixed with the alginate and gelatin solutions at different ratios to form BC-A-G blend mixtures. After that glycerol, sorbitol and the mixture of glycerol and sorbitol were added at the ratio of 0-3 g per 10 g gelatin solution. The mixture was thoroughly stirred at 50±5 °C until the homogeneous mixture was formed. Homogenous mixture At 50±5˚C

4. Preparation of BC/A/G Films (Cont.) EXPERIMENTAL STUDY 4. Preparation of BC/A/G Films (Cont.) Air-dried at room temperature subsequently it was poured into polystyrene Petri plates and was incubated at room temperature for 2 days to form films with thickness 50±10 µm. After that it was crosslinked with 1% (w/v) CaCl2 dihydrated for 1 h and was rinsed with DI water to remove the excess chlorides before the cross-linked gel was air-dried at room temperature. Crosslinked with 1%w/v CaCl2 solution for 1 h Incubated at room temperature to form films

RESULTS AND DISCUSSION 1. Fourier Transforms Infrared Spectroscopy (FTIR) O-H-O (1613-1650 cm-1) -NH (1535-1541 cm-1) A)60/10/30 B)60/20/20 C)60/30/10 D)60/20/20Gly E)60/20/20S F)60/20/20GS 3404 2934 1650 1424 1026 1536 3400 2925 1613 1059 1423 2924 1035 1614 3401 2928 1431 1535 2927 1635 3393 2933 1649 1034 1541 1241 1316 1243 1250 No. 1 FTIR analysis: Lines A, B and C refer to BC/A/G films at a ratio of 60/10/30, 60/20/20 and 60/30/10, respectively. Lines D, E and F refer to BC/A/G at ratio of 60/20/20 with Gly, S and the mixture of glycerol and sorbitol addition. It was found that: The films with adding plasticizers exhibited the characteristic absorption bands with no appearance of new peaks. The comparison between types of plasticizers on BC/A/G films indicated that the films with Gly adding tended to increase in amplitudes of the G characteristic absorption band and free water peaks. These shifts could be attributed to intermolecular interactions between the hydroxyl group of BC, the ester group of A and amide group of G.

2. Water Absorption Capacity (WAC) RESULTS AND DISCUSSION 2. Water Absorption Capacity (WAC) A) B) Results and Discussion No. 1 Effect of PLASTICIZERS ON Water Absorption Capacity (WAC) of the FILMS On Figure A, glycerol was used as a plasticizer at a ratio of gelatin to glycerol at …………….. On Figure B, sorbitol was used as a plasticizer ……….and on Figure C, the mixture of glycerol and sorbitol was used at a ratio of ………….. The water content was calculated using the following equation. [Click!] The results indicated that WAC increased with the increase of plasticizer content. However, excess of plasticizer at a ratio of G solution to plasticizer more than 2:10 (w/w) caused the decrease of the WAC, which should be due to the migration of plasticizer from the re-swollen film surface. For the effect of type of plasticizers, WAC of the films plasticized with S had a greater WAC than that plasticized with the mixture of glycerol and sorbitol (GS) and Gly, respectively. C) Condition: the BC/A/G films at ratio of 60/20/20 at room temp. Wh = Wt. of hydrated films Wd = Wt. of dried films

RESULTS AND DISCUSSION 3. Mechanical Strength A) Condition: Dry state 181.5 No. 2 Effect of Plasticizer on Mechanical strength of the films in dry state: Figure A is for TS Figure B is for EB Yellow bar is for Nonplasticizer; Blue bar is for the films with glycerol; Green bar is for the films with sorbitol and the red one is for the films with mixed glycerol and sorbitol These results indicated that the films at a ratio of 60/20/20 (w/w) showed superior mechanical properties. The maximum TS and EB were at 181.5 MPa and 2.8%, respectively. There was no significant difference between the mechanical properties of the dried films with and without addition of plasticizers. B) 2.8

RESULTS AND DISCUSSION 4. Mechanical Strength A) Condition: Wet state No. 2: Effect of plasticizers on Mechanical strength of the films in wet state Figure A is for TS Figure B is for EB These results indicated that TS of the re-swollen films was reduced with the addition of plasticizer and TS of the films plasticized with Gly was more than the one with GS and S, respectively. It was found that plasticizers helped to improve flexibility of the films, in the wet state, resulting in enhanced EB. The EB of the films plasticized with Gly was more than the one with GS and S. B)

CONCLUSIONS The composite film with BC/A/G at a ratio of 60/20/20 has superior mechanical properties for both dry and wet states. Glycerol is a suitable plasticizer for BC/A/G films because it improves the weakness of the films. The optimal ratio of glycerol to gelatin solution is 2:10 (w/w). From all of results could conclude that The composite film with BC/A/G at a ratio of 60/20/20 has superior mechanical properties for both dry and wet states. Glycerol is a suitable plasticizer for BC/A/G films because it improves the weakness of the films with the optimal ratio of glycerol to gelatin at 2:10 (w/w).

ACKNOWLEDGMENTS Integrated Innovation Academic Center: IIAC Chulalongkorn University Centenary Academic Development (Project Code: CU56-AM01) (Project Code: AM667A) I’m grateful for the financial support from Integrated Innovation Academic Center and Chemical Engineering Research Unit for Value Adding of Bioresources. Chemical Engineering Research Unit for Value Adding of Bioresources

for your kind attention Thank you for your kind attention

Why do you choose the 3 key components? Bacterial cellulose is high biocompatibility and ultrafine-fiber network stacked; therefore, it can be reinforced to increase good mechanical properties in this material for packaging. Sodium alginate can help good mixing between BC and gelatin because it is thickener and stabilizer agent. Moreover, it reduce the swelling and the hydrophilic properties of the films after it was crosslinked with CaCl2 solution so it can reduce. Gelatin could be added to provide the necessary workability to composite packaging film and the supplement of gelatin to enhance the rehydration abilities and mechanical properties of bacterial cellulose has been reported.

Why do you use gelatin type B I used to trial for comparison between gelatin type A and type B, I found that gelatin type B is less bubble than that type A and it has flexibility more than that type A, so I will choose it.

Transparency of the films Without the addition of the plasticizer, it was found that the BAG film at a ratio of 60/10/30 was the most transparent. Transparency of the BAG films was increased with increasing gelatin content. The surface roughness and film thickness might also affect transparency of films Transparency: (A) Bacterial cellulose; (B) alginate; (C) gelatin; (D) B/A at a ratio of 60/40; (E), (F) and (G) BAG without plasticizer at a ratio of 60/10/30, 60/20/20 and 60/30/10, respectively.

Why do you use G from porcine skin Gelatin World Market 2009 Generally, Beef sources carry a higher risk than from pork and also bones carry a higher risk than skins Isoelectric point, PI High quality : good setting Bones have a lot of contaminate Gelatin type A Gelatin type B PI = 7.0–9.0 PI = 4.8–5.2 Prepared by acid extraction Prepared by alkali extraction pH = 3.8-5.5 pH = 5.0-7.0 Viscosity (mPa.s) = 15-75 Viscosity (mP.s) = 20-75 Ash (%) = 0.3-2.0 Ash (%) = 0.5-2.0 (Gelatin Manufacturers Association of Asia Pacific)

Why do you use Calcium ion Mechanism of sodium alginate and calcium ions (which holds a charge of +2) knocks away two sodium ions (each holding a charge of +1). Generally, Beef sources carry a higher risk than from pork and also bones carry a higher risk than skins Isoelectric point, PI High quality : good setting Bones have a lot of contaminate

The mechanical of alginate cross-liked Schematic representation of the egg-box association of the poly-L-guluronate sequences of alginate crosslinked by calcium ions. The figure shows conversion of random coils to buckled ribbon-like structures which contain arrays of Ca2+ ions. The magnitude figure shows the proposed stereochemistry of Ca2+ ion complexation. The oxygen atoms involved in the coordination sphere are shown as filled circles (Rees, 1981, modified).

RESULTS AND DISCUSSION 1. Fourier Transforms Infrared Spectroscopy (FTIR) -NH -COO -OH O-H-O A)60/10/30 B)60/20/20 C)60/30/10 D)60/20/20Gly E)60/20/20S F)60/20/20GS 3404 2934 1650 1424 1026 1536 3400 2925 1613 1059 1423 2924 1035 1614 3401 2928 1431 1535 2927 1635 3393 2933 1649 1034 1541 1241 1316 1243 1250 No. 1 FTIR analysis: Lines A, B and C refer to BC/A/G films at a ratio of 60/10/30, 60/20/20 and 60/30/10, respectively. Lines D, E and F refer to BC/A/G at ratio of 60/20/20 with Gly, S and the mixture of glycerol and sorbitol addition. It was found that: The films with adding plasticizers exhibited the characteristic absorption bands with no appearance of new peaks. The comparison between types of plasticizers on BC/A/G films indicated that the films with Gly adding tended to increase in amplitudes of the G characteristic absorption band and free water peaks. These shifts could be attributed to intermolecular interactions between the hydroxyl group of BC, the ester group of A and amide group of G.

Basic of materials Bacterial cellulose is a linear polysaccharide of glucose with formula of (C6H10O5)n. Sodium alginate is a linear polysaccharide copolymer of (1-4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) monomers with the formula of (C6H7O6Na)n Gelatin is a water soluble protein with formula of (C102H151N31O39)n