1. >>0 >>1 >> 2 >> 3 >> 4 >> Development and Characterization Chitosan blended Bacterial cellulose Film Proposed by Mr.Kampole Intasorn 5470120921 Thesis Advisor :Associate Professor Muenduen Phisalaphong, Ph.D. Chulalongkorn University

2. >>0 >>1 >> 2 >> 3 >> 4 >> Contents 1 2 Introduction Theory and principles Objectives and research scope Material and Methods Result and discussion 5 Conclusion 1 3 2 4 5 6

3. >>0 >>1 >> 2 >> 3 >> 4 >> Introduction Plastic waste problem Shalleh et al.,2007

4. >>0 >>1 >> 2 >> 3 >> 4 >> Introduction Food packaging

5. >>0 >>1 >> 2 >> 3 >> 4 >> Introduction plastic waste

6. >>0 >>1 >> 2 >> 3 >> 4 >> Introduction To solution the problem

7. >>0 >>1 >> 2 >> 3 >> 4 >> Theory and principles Chitosan Figure 1 Structures of cellulose, chitin and CH (Muzzarelliry.,1973).

8. >>0 >>1 >> 2 >> 3 >> 4 >> Theory and principles Chitosan Figure 2 preparation of chitosan from chitin (Muzzarelli., 1977).

9. >>0 >>1 >> 2 >> 3 >> 4 >> Theory and principles Antimicrobial of Chitosan Figure 3 Antimicrobial of chitosan (Chen et al,1998)

10. >>0 >>1 >> 2 >> 3 >> 4 >> Theory and principles Bacterial cellulose(BC) Figure 4 Structure of Cellulose (Nishiyama et al.,2002). Figure 5 Bacterial Cellulose (http://agsci.psu.edu/magazine/articles/2010/winter-spring/building-a-better- bandage)http://agsci.psu.edu/magazine/articles/2010/winter-spring/building-a-better- bandage

11. >>0 >>1 >> 2 >> 3 >> 4 >> Theory and principles Bacterial cellulose(BC) Figure6 SEM micrograph of structure Bacterial Cellulose (Phisalaphon,et al., 2007) Figure7 Size of synthetic and naturally occurring fiber (Yoshinaga et al, 1997).

12. >>0 >>1 >> 2 >> 3 >> 4 >> Objective 1.To develop chitosan/BC composite film (CBC) 2.Characterize for its mechanical and antimicrobial properties.

13. >>0 >>1 >> 2 >> 3 >> 4 >> Research scope Chitosan/BC (CBC) composite films are fabricated by casting method at room temperature (30°C±5°C). Characterization of CBC is as follows: Mechanical property Antimicrobial property

14. >>0 >>1 >> 2 >> 3 >> 4 >> Material and Methods Material 1. Chitosan ;Seafresh Chitosan Powder 85% DAC and Molecular weight 200,000. 2. Bacterial Cellulose (BC); The A. xylinum strain was isolated from nata de coco.

15. >>0 >>1 >> 2 >> 3 >> 4 >> Material and Methods Figure8 Chitosan powder Figure9 nata de coco.

16. >>0 >>1 >> 2 >> 3 >> 4 >> Material and Methods Preparation of Chitosan solution Chitosan (1%, w/v) was mixed in acetic acid solution (1% v/v) pH was adjusted to 5 with 1N NaOH

17. >>0 >>1 >> 2 >> 3 >> 4 >> Material and Methods Preparation of BC slurry Washed by DI water And Spun

18. >>0 >>1 >> 2 >> 3 >> 4 >> Material and Methods Blending Then mixed with BC slurry at various weight ratio (Chitosan:BC) 10:2 to10:10. Moreover, Glycerol (2%v/v) was used to be the plasticizer for these mixture Stirred overnight

19. >>0 >>1 >> 2 >> 3 >> 4 >> Material and Methods After the solution was homogenous mixture, poured into Petri plate and dry with incubator at 35 °C for 5 days.

20. >>0 >>1 >> 2 >> 3 >> 4 >> Material and Methods Figure 10 Product

21. >>0 >>1 >> 2 >> 3 >> 4 >> Result and discussion Fig11. Tensile strength(MPa)

22. >>0 >>1 >> 2 >> 3 >> 4 >> Result and discussion Fig12. Elongation at break(%)

23. >>0 >>1 >> 2 >> 3 >> 4 >> Result and discussion Fig 13. %Water adsorption capacity

24. Result and discussion Table 1: Antimicrobial activities of CBC films against E. coli and S. aureus Micro- organism SampleThe Number of bacterial % reduction E. coli Control a 3.8 x 10 5 - Chitosan Mw.200,000 3.5 x 10 5 7.89 a Control b 2.2 x 10 5 - Chitosan:BC 10:6 6.5 x 10 5 0b0b S. aureus Control c 4.2 x 10 5 - Chitosan Mw.200,000 2.9 x 10 5 30.95 c Control d 3.2 x 10 5 - Chitosan:BC 10:6 2.0 x 10 5 37.50 d a-d refer to different control inoculums.

25. >>0 >>1 >> 2 >> 3 >> 4 >> Conclusion  The mechanical properties of the CBC films depended on the weight ratio of BC.  The water absorption capacity of the film was maximal at 548.2% obtained from the composite film with the ratio of chitosan : BC at 10:6.  The CBC films were able to significantly inhibit S. aureus growth.

26. >>0 >>1 >> 2 >> 3 >> 4 >> Thank you for your attention

27. >>0 >>1 >> 2 >> 3 >> 4 >> Why do you use Chitosan Mw.200,000? To compare 3 Mw. 30,000 80,000 and 200,000 The results showed that mw.200,000 is The most appropriate in term of strength. Jeremy L. Chen and Yanyun Zhao Reported that tensile strength of high mw Chitosan films higher than that of low mw

28. >>0 >>1 >> 2 >> 3 >> 4 >> How to calculate % reduction? Where A = CFU/mL for the flask containing the treated substrate after the specified contact time. C = CFU/mL for the flask containing the untreated substrate after the specified contact time.(Control) Ex chitosan film inhibits E.coli

29. >>0 >>1 >> 2 >> 3 >> 4 >> What is %DAC mean? The term “degree of deacetylation” is used to represent the amount of N-glucosamine units in molecules. If the degree of deacetylation is higher than 50%,it will be classified as chitosan. In contrast, when the deacetylation is lower than 50%, the term chitin is used (Krajewska, 2004).

30. >>0 >>1 >> 2 >> 3 >> 4 >> Why CBC films can’t inhibit E.coli growth? CompositionGram positive.Gram negative. Peptidoglycan.60-100%.5-20%. Amount of lipid.0.2%.10-20%. Polysaccharide.35-60%.15-20%. Cell wall12 The cell wall of gram-negative and gram-positive bacteria have a different key for this Ref:http://www.medtechzone.com/data/bac/bacteria.php, Andrew P. Desbois & Valerie J. Smith, Appl Microbiol Biotechnol (2010) 85:1629–1642 Lian-Ying Zheng*, Jiang-Feng Zhu Carbohydrate Polymers 54 (2003) 527–530

31. >>0 >>1 >> 2 >> 3 >> 4 >> Why the number of bacterial more than control ? CBC cannot able to inhibit the growth of bacteria, So bacteria can growth on CBC film.

32. >>0 >>1 >> 2 >> 3 >> 4 >> Why CBC films inhibit S.aureus more than pure Chitosan film? The value of the test is not very different. So we can not conclude that the BC supplement can increase the S.aureus inhibition. We try to experiment more.