Effect of chitosan purifying methods on inhibition activity toward pathogenic bacteria

Abstract

Chitosan is well-known to possess antimicrobial activity, however, its activity depends on its size and preparation methods. The aim of this study is to investigate the purification process of chitosan, previously prepared from chitin extracted from shimp by products using biological method to ensure antimicrobial activity of the chitosan preparation. Crude chitosan after chitin deacetylation was subjected to furification using two following methods: Method 1: Crude chitosan was soaked in water at 80°C, washed, completely dissolved in 1% acetic acid solution, and then was filtered to remove impurities. Solution after filtration was precipitated with  NaOH 10% at pH= 8. The precipitate obtained was washed until neutral pH and centrifuged at 9000 rpm. The pellet was dried by using freeze-drying method (temperature -20°C, 4 days). Method 2: Crude Chitosan was soaked in water at room temperature, washed, completely dissolved in 1% acetic acid solution, and then was filtered to remove impurities. Solution after filtration was precipitated with  NaOH 10% at pH= 8. The precipitate obtained was washed until neutral pH and centrifuged at 9000 rpm. The pellet was dried by using oven dry (50°C temperature for 1 day). Freeze-dried and conventionally oven-dried chitosan was tested against  Salmonella and Escherichia coli. The Minimum Inhibitory Concentrations (MIC) of freeze-dried chitosan against Escherichia coli and Salmonella were at the value of 1000 ppm, wheres the MIC of oven-dried chitosan were at the value of 250 ppm. Result showed that purification process of chitosan affected the antimicrobial activity of chitosan toward test pathogenic bacteria. This outcome indicated potential of using cost-effective purifying method of chitosan from chitin biologically extracted from shrimp byproduct to obtain an effective antibacterial agent in food industry.

Keyword: Chitosan, purifying method, Antimicrobial activity, Minimum Inhibitory Concentrations.

1. Introduction

Chitosan is a deacetylated product of chitin. It has gained attention of the scientific community due to its functional properties such as film-forming capabilities, mineral – binding properties, hypolipidemic activity, biodegradability, antimicrobial activity, immunoadjuvant activity, acceleration of wound healing, and eliciting of phytalexins. Recently, reseach has shifted and focused on the possibility of developing chitosan as a natural preservative. Numerous studies on bactericidal activity of chitosan have been carried out and reviewed. The goal of this study was to evaluate the influence of  chitosan purifying methods extracted from shrimp shells by biological methods [1, 2, 3].

2. Materials and Methods

2.1. Materials

Crude chitosan was previously prepared using biological method in School of Biotechnology and Food technology, Ha Noi University of  Science and Technology (Le Thanh Ha, unpublished data). In brief, shrimp byproduct was undergone demineralization, deproteinization, deacetylation and subjected to purifying steps described in section 2.2.1. Two bacteria Salmonella enterica subsp enterica serovar Typhimurium ATTC 14028^TM and Escherichia coli ATTC 25922 was used as indicators for antimicrobial assays.

2.2. Methods

2.2.1. Chitosan purifying methods and preparation of chitosan solution

Crude chitosan after the deacetylation, was subjected to purification using two following methods. Method 1: Crude chitosan was soaked in water at 80°C, washed, completely dissolved in 1% acetic acid solution, and then was filtered to remove impurities. Solution after filtration was precipitated with  NaOH 10% at pH= 8. The precipitate obtained was washed until neutral pH and centrifuged at 9000 rpm. The pellet was dried using freeze-drying method (temperature -20°C, 4 days) [4]. Method 2: Crude chitosan was treated similarily to method 1, except for soaking at room temperature and drying using conventional oven at 50^oC.  Then chitosan was brought to determine viscosity and degree of deacetylation. In the preparation of chitosan solution, 1% (w/v) chitosans were dispersed in a acetate buffer (pH 5- 6). After stirring, the solutions were autoclaved at 120^oC for 15 min.

2.2.2. Determination of degree of deacetylation (DDA)

Degree of acetylation (DA) of chitosan was determined by UV spectrophotometry method using dual standards [5]. In brief, Chitosan sample was dissolved in 0.1 M HCl at concentration of 120mg/l. Absorbance was measures at wavelength of 201 nm (A). The DA of  the  sample (m milligram of chitosan in V liters solution) was caculated as followed:

DA = (161.1 .A.V – 0.0218m) / (3.3615m – 42.1.A.V)  (%)

DDA = 100 - DA  (%)

2.2.3. Determination of viscosity

Chitosan was dissolved in acid acetic 1% to obtain 1% chitosan solution. Viscosity was measured using viscosimeter "LVT Dial reading Viscosity'' and expressed in centipoise (cps).

2.2.4. Determination of antimicrobial activity

Test bacteria were incubated in Meat Peptone broth (MP) which contained peptone 10g; meat extract 3g; NaCl 5g in distilled water 1000 ml.

The effect of chitosan on the growth of bacterial cultures was determined by micro broth dilution techniques. Each bacterium was cultured at initial concentration of 10⁴ CFU/ml in a series of tubes containing  MP broth medium and 125 ppm, 250 ppm, 500ppm, 1000 ppm, 2000 ppm, or 4000 ppm of chitosan. All bacteria were incubated at 37^oC, 24 hour. After 24 hour, the presence of microorganisms was verified by  plating on MP agar [6]. Minimal inhibitory concentrations (MIC) were determined as the lowest concentration of chitosan at which microorganisms could not grow [3].

3. Results and Discussion

Chitosan purified by method 1 showed DDA of 95% and viscosity of 50cps. Chitosan purified by method 2 showed DDA of  92% and viscosity of 80cps.

The antimicrobial activity of chitosan purified by method 1 was demonstrated  in Table 1 and Figure 1.

Table 1.  Antimicrobial activity of chitosan purified by method 1 against Salmonella and E.coli

(-) Negative growth ; (+) Positive growth

The Minimum Inhibitory Concentrations of chitosan purified by method 1 (50cps, DDA 95%) on Escherichia coli and Salmonella were at the same value of 1000 ppm.

The antimicrobial activity of chitosan purified by method 2 was demonstrated  in Table 2 and Figure 2.

Table 2. Antimicrobial activity of chitosan purified by method 2 against Salmonella and E.coli

(-) Negative growth ; (+) Positive growth

The Minimum Inhibitory Concentrations of chitosan purified by method 2 (80cps, DDA 92%) on Escherichia coli and Salmonella were at the same value of 250 ppm.

Many hypotheses have been proposed to explain the mechanism of antimicrobial effects of chitosan. One of hypotheses is that the mechanism involves interactions of chitosan positively charged molecules with negatively charged constituents of microbial cell walls and membranes interrupting normal cell metabolism [7, 8, 9].

In both chitosan purifying methods, there were differences in two stages- soaking and drying. In chitosan preparing process, after deacetylation of chitin using NaOH, a small amount of NaOH probably remained in the product. In purifying method 1, the crude chitosan was soaked in the water at 80⁰C, which could have facilitated further deacetylation proposally due to the remained NaOH. More over, chitosan was formed by link β-(1-4) glucozit; at high temperature of 80^oC this link could be hydrolysed in alkaline medium [10, 6] forming chitosan with lower viscosity. In contrast, in method 2, chitosan was soaked in water at room temperature and oven dried at low temperature (50^oC), therefore, deacetyl degree was slightly lower and viscosity was higher than that of method 1.

Previuos studied suggested that antimicrobial activity of chitosan depends on degree of deacetylation as well was molecular mass, represented by viscosity [11, 12, 13]. Chitosan with medium molecular mass (or medium viscosity) showed better antimicrobial activity against certain microorganisms [14, 15]. In our study, chitosan purified by both methods had high degree of deacetylation (greater than 90%), therefore both showed good antimicrobial activity. However, chitosan purified by method 2 demonstrated better activity against indicative bacteria probably due to its lower temperature of purifying stage resulting in medium viscosity. This outcome suggested potential use of conventional method for chitosan purification to obtain chitosan with antimicrobial activity.

4. Conclusion

This study indicated that cost-effective purifying method of chitosan from chitin biologically extracted from shrimp byproduct to obtain an effective antibacterial agent in food industry is worth to be considered for scale-up biological chitosan production processes.

5. References

1. Feradoon Shahidi, et al, 1999, Food application of chitin and chitosans. Trends in Food Science and Technology 10, 37-51.

2. Fernandez – Kim, B.S, 2004, Physicochemical and Functional properties of crawfish chitosan as affected by different processing protocols. Seoul National University.

3. Monarul Islam MD, et al, 2011, In vitro antibacterial activity of shrimp chitosan against Salmonella paratyphi and Staphylococcus aureus. Journal of Bangladesh Chemical Society, Vol.  24(2), 185 – 190.

4. Trang Sĩ Trung, Vũ Ngọc Bội, Phạm Thị Đan Phượng, 2007, “Nghiên cứu kết hợp enzyme protease trong công nghệ sản xuất chitin từ phế liệu đầu vỏ tôm”. Tạp chí khoa học – công nghệ Thủy sản số 3, trang 11 – 17.

5. Dashang Liu, et al, 2006, Determanition of the dgree of acetylation of chitosan by UV spectrophotometry using dual standards. Carbohydrate Research 341, 782 – 785.

6. Lin Jiang, 2009, Comparision of disk diffusion, agar dilution, and broth microdilution for antimicrobial susceptibility testing of five chitosan. Fujian Agricultural and Forestry University, China.

7. Helander IM., et al, 2011, Chitosan disrupts the barier properties of outer membrane of Gram – negative bacteria. International Journal of Food Microbiology 71, 235 – 244.

8. Monarul Islam MD, et al, 2011, In vitro antibacterial activity of shrimp chitosan against Salmonella paratyphi and Staphylococcus aureus. Journal of Bangladesh Chemical Society, Vol. 24(2), 185 – 190.

9. Rhoades J. and Roller S., 2000, Antimicrobial Actions oDegraded and Native Chitosan against Spoilage Organisms in Laboratory Media and Foods. Applied and Enviromental microbiology 66, 80 – 86.

10.Nguyễn Thị Huệ, Khiếu Thị Tâm (2005). “Nghiên cứu phản ứng thủy phân chitosan bằng acid photphoric”. Tạp chí khóa học ĐHQGHN – KHTN&CN, trang 91-96.

11. Liu X.F., et al, 2001, Antibacterial action of chitosan and carboxymethylated chitosan. Journal of Applied Polymer Science 79, 1324 – 1335.

12. Morimoto M. and Shigemasa Y., 1997, Charaterization and bioactivities of chitin and chitosan regulated by their degree of deacetylation. Kobunshi Ronbunshu 54, 621 – 631.

13. Simpson B.K., et al, 1997, Utilization of chitosan for preservation of raw shrimp. Food Biotechnology 11, 25 – 44.

14. Gerasimenko D.V., Avdienko I.D., et al, 2004, Antibacterial effects of water – soluble low – molecular – weight chitosans on diffirent microorganisms. Applied Biochemistry and Microbiology 40, 253 – 257.

15. Zheng L. Y. and Zhu T.F., 2003. Study on antimicrobial activity of chitosan with different molecular weights. Carbohydrate Polymers 54, 527 – 540.

ẢNH HƯỞNG CỦA PHƯƠNG PHÁP tinh sẠCH CHITOSAN ĐẾN HOẠT TÍNH KHÁNG VI SINH VẬT

GÂY BỆNH

Nguyễn Thị Đoàn, Trịnh Văn Châu, Hồ Phú Hà

Viện Công nghệ Sinh học và Công nghệ Thực phẩm, Đại học Bách khoa Hà nội

Tóm tắt

Chitosan được biết đến với khả năng kháng vi sinh vật, tuy nhiên, hoạt tính của nó phụ thuộc vào kích thước và phương pháp sản xuất. Mục đích của đề tài này là nghiên cứu ảnh hưởng của quá trình tinh sạch chitosan từ chitin chiết xuất từ vỏ tôm bằng phương pháp sinh học. Chitosan thô thu được sau khi deacetyl hóa chitin, làm sạch theo hai phương pháp: Phương pháp 1: Chitosan thô ngâm trong nước ở 80^oC, sau đó hòa tan hoàn toàn vào dung dịch axit axetic 1%, tiến hành lọc để loại bỏ tạp chất; dung dịch sau khi lọc được kết tủa bằng NaOH 10%, pH 8; kết tủa thu được rửa trung tính, ly tâm ở 9000 vòng/phút thu chitosan kết tủa, tiến hành sấy kết tủa bằng phương pháp sấy đông khô (nhiệt độ -20^oC, trong 4 ngày). Phương pháp 2: Chitosan thô ngâm trong nước ở nhiệt độ thường, sau đó hòa tan hoàn toàn vào dung dịch axit axetic 1%, tiến hành lọc để loại bỏ tạp chất; dung dịch sau khi lọc được kết tủa bằng NaOH 10%, pH 8; kết tủa thu được rửa trung tính, ly tâm ở 9000 vòng/phút thu chitosan kết tủa, tiến hành sấy kết tủa bằng phương pháp sấy đối lưu trong tủ sấy (nhiệt độ 50^oC trong 1 ngày). Chitosan thu được từ 2 phương pháp trên đem thử nghiệm khả năng kháng lại Salmonella và Escherichia coli. Nồng độ ức chế tối thiểu (MIC) của chitosan làm sạch theo phương pháp 1 đối với Escherichia coli và Salmonella đều là 1000 ppm, chitosan làm sạch theo phương pháp 2 đối với Escherichia coli và Salmonella đều là 250 ppm. Kết quả cho thấy làm sạch chitosan  theo phương pháp 2 có hoạt tính kháng vi sinh vật cao hơn. Như vậy sử dụng phương pháp tinh sạch chitosan đơn giản tiết kiệm (ngâm ở nhiệt độ thường và sấy đối lưu bằng không khí nhiệt độ 50^oC) hoàn toàn có thể bảo đảm khả năng kháng vi sinh vật nhằm ứng dụng chitosan trong bảo quản thực phẩm.

Từ khóa: Chitosan, phương pháp tinh sạch, Họat tính kháng vi sinh vật, Nồng độ ức chế tối thiểu.