Effectiveness of Edible Film Chitosan from Waste White Shrimp
(Litopenaeusvannamei) in Reducingcolonization of Porphyromonas Gingivalis Bacteria: In Vitro Research
Harun Achmad1, Asdar Gani2, ArniIrawaty Djais2, Lenny Indriani Hatta3,Irene Edith Rieuwpassa4,AndiYayangAdhaAprilia Monry5
1Department of Pediatric Dentistry, Faculty of Dentistry, Hasanuddin University, Indonesia
2Department of Periodontology, Faculty of Dentistry, Hasanuddin University, Indonesia
3Department of Dental Material, Faculty of Dentistry, Hasanuddin University, Indonesia
4Department of Oral Biology, Faculty of Dentistry, Hasanuddin University, Indonesia
5Clinical Dental Student, Faculty of Dentistry, Hasanuddin University, Indonesia E-mail Correspondence: harunachmader@gmail.com
ABTRACT
Background: Chitosan is a biopolymer that usually extracted from the shells of crustaceans such as crabs, shrimp, and lobster. The advantages of chitosan as an antimicrobial are abundant availability, low production costs, excellent biodegradability, and biocompatibility. Chitosan can show antibacterial activity against bacteria that cause periodontitis, one of which is Porphyromonasgingivalis. Objective: This research aims to observe and identify the effectiveness of chitosan edible films in inhibiting the growth of Porphyromonasgingivalis bacteria. Method:
This study uses a laboratory experimental research type using the Post-test only with control group research design. In this study, six repetitions were carried out in 8 treatments with six repetitions each, namely chitosan extract with concentrations of 1%, 2%, and 3%, edible chitosan film 1%, 2%, and 3%, positive control (edible film metronidazole), as well as the negative control.
The measuring instrument used in this study is the calipers in millimeters (mm). Results: Least Significance Different (LSD) result shows some significant differences in inhibiting the growth of Porphyromonasgingivalis bacteria (p<0.005) between each treatment. Conclusion: Edible chitosan film from white shrimp (Litopenaeusvannamei) scalp waste can inhibiting the growth of Porphyromonasgingivalis bacteria.
Keywords:Edible Chitosan Film, Porphyromonasgingivalis, Antimicrobial.
INTRODUCTION
Periodontal tissue is the supporting network of teeth consisting of the gingiva, cementum, periodontal ligament, and alveolar bone. Periodontal disease is a pathological condition affecting the periodontal tissue.1,2
Based on the results of the Household Health Survey of the Ministry of Health of the Republic of Indonesia in 1995, the prevalence of periodontal disease patients in Indonesia (by measuring tartar and calculus) reached 42.8%.3Periodontitis is characterized by the presence of a periodontal pocket that forms in the gingival groove area when the groove is more than 4 mm deep.4,5
Periodontal disease is often associated with microbial infections caused by biofilms, plaques and calculus. Common bacteria that cause this disease are Aggregatibacteractinomycetemcomitans, Porphyromonasgingivalis and Fusobacteriumnucleatum.
A number of publications state that microorganisms from the subgingivalmicrobiota, in this case gram-negative anaerobes, are a major contributing factor for chronic and aggressive periodontitis.
A study has identified several periodontal pathogens, one of which is Porphyromonas gingivalis.6,7,8 The advantages of chitosan as an antimicrobial are that it is abundantly available in nature, low production costs, good biodegradability, biocompatibility, and bioresobsibility, as well as sufficient chemical modification.9
Seeing these problems, one of the breakthroughs made in this study was to use chitosan edible film from shrimp head waste as an antimicrobial to inhibit the growth of Porphyromonasgingivalis. Edible film preparations have several advantages, namely being biodegradable, edible (biocompatibility), and having antimicrobial activity.10
MATERIALS AND METHODS
This study uses a laboratory experimental research type. This study also used a post test only research design with control group design. The research was conducted at the Phytochemical Laboratory of the Faculty of Pharmacy, Hasanuddin University, Laboratory of Biology and Microbiology, College of Pharmacy. The study was conducted in July - Completed in 2019. The samples from this study were Porphyromonas gingivalis bacteria provided from the Microbiology Laboratory of the Hasanuddin University Faculty of Medicine and white shrimp scalp waste (Litopenaeus vannamei) obtained from the white shrimp cultivation development center (Litopenaeus vannamei) .
The inclusion criteria of this study were the cultured Porphyromonasgingivalis bacteria.
Then, white shrimp scalp waste (Litopenaeusvannamei) chitosan edible film preparation. The exclusion criteria of this study were contaminated Porphyromonasgingivalis bacteria. Then, the waste of white shrimp scalp (Litopenaeusvannamei) from the edible film of chitosan has been contaminated.
Chitosan Extract Demineralization
The finely ground shrimp head waste powder is put into an erlenmeyer flask that has been given a stirrer stone, then 1.5 M HCl is added (measured using a measuring cup and a beaker with a ratio of 1:15 (w/v), then stirring using a stirrer. After that, the shrimp shell waste powder and 1.5 M HCl solution were heated at a temperature of 60-70oC for 2 hours while stirring at a speed of 100 rpm using a stirrer engine.
Deproteination
Furthermore, the waste powder to the filtered shrimp is then added with NaOH 4% ratio of 1:10 (w/v) (measured using a measuring cup and beaker). After that, the heating is done at a temperature of 60-70oC for 2 hours at a speed of 100 rpm using a stirrer engine. Perform re- filtering using filter paper, funnel and Erlenmeyer flask. The precipitate obtained is rinsed several times then the pH is calculated until a normal pH is obtained (pH 7).
Deacetylation
The final stage in the manufacture of chitosan extract is through the deacetylation process.
The chitin obtained at the deproteination stage was then added with NaOH 60% ratio of 1:20 (w/v) then heated to 65ºC for 2 hours. The results obtained are then filtered using a funnel and filter paper. The precipitate is then rinsed with distilled water to a neutral pH (pH 7).
Chitosan Edible Film
The chitosan extract that has been obtained previously will be made in edible film preparations with various concentrations (1%, 2% and 3%). Making chitosan edible film begins with dissolving the modified starch in distilled water. The solution was heated at 60°C to 70°C and stirred on a hot plate stirrer to form a thickened solution. Glycerol and HPMC were added to distilled water and then added with the chitosan extract 1%, 2%, and 3% respectively, then stirred until gelatinized and homogeneous.
Inhibition Test and Observation of Zone of Inhibition
1. Prepare the tools and materials (paper disk, negative and positive controls, chitosan extract, chitosan edible film with a concentration of 1%, 2% and 3%, and prepare pure isolates of Porphyromonasgingivalis bacteria that have previously been inoculated by swabbing on MHA medium).
2. Next, the inhibitory power test will be assessed by inserting a paper disk in each sample that has been given different treatments (6 treatments). Place aseptically (do not tear).
3. Incubate the petri dishes at 37°C for 24-48 hours. Incubation was carried out without turning the petri dishes.
4. Measure the inhibition zone around the paper disk for each treatment. Observe the zone of inhibition every 6 hours to see the activity of these bacteria qualitatively and take measurements using a caliper as quantitative data. The clear zone formed is measured using a caliper, the measurement is carried out by measuring three sides of
the clear zone, namely horizontally, vertically, and obliquely. The sizes obtained are then averaged. The diameter of the clear zone is in millimeters (mm).
RESULTS
Inhibition test in this study was carried out with 6 repetitions of the 8 treatments obtained by different inhibition zones. The results showed that the results of the normality test using Shapiro-Wilk obtained a value of p>0.05, namely p=0.062, which means that the data was normally distributed so that it was continued with the parametric statistical test, namely One Way Anova. Anova test is used to see the difference between one treatment and another.The results of the measurement of the inhibition zone diameter of each petri dish can be seen in table 1 below Table 1.Results of Inhibition Zone Diameter Measurement.
No Treatment P Total Average
Repetition to-
I II III IV V VI
1 Edible 3% 10,53 10,46 12,2 12,85 10,46 11,86 68,36 11,39 2 Edible 2% 11,91 12,56 11,41 11,88 11,13 11,15 70,04 11,67 3 Edible 1% 11,8 10,95 12,34 10,39 9,11 10 64,59 10,76 4 Extract 1% 7,72 7,33 7,58 7,52 6,86 7,24 44,25 7,37 5 Extract 2% 8,97 9,53 8,21 7,54 9,38 7,76 51,39 8,56 6 Extract 3% 8,36 8,97 7,88 9,13 9,49 10,63 50,93 8,48 7 Control + 10,9 11,05 12,65 7,83 10,33 11,18 63,94 10,65 8 Control - 9,93 9,95 8,26 8,35 9,9 10,35 56,74 9,45
The test results in Table 1 for the extract test material concluded that the addition of 2%
chitosan extract after averaging out of 6 repetitions the result was 8.56 mm and became the treatment with the greatest inhibition of the chitosan extract category while the addition of 1%
chitosan extract and 3% after averaging out of 6 repetitions, the results were 7.37 mm and 8.48 mm, respectively. These three results fall into the moderate inhibition zone category because they only range from 5-10 mm.
For chitosan edible the concentration of 1% was 10.76 mm and for chitosan edible film with a concentration of 3% the inhibition zone was 11.39 mm. For negative control, it shows a size of 9.45 mm, indicating that the inhibition zone of negative control is the smallest zone of inhibition, while the positive control using metronidazole has an inhibition of 10.65 mm.
It can be concluded that the 1% concentration of chitosan edible film has a minimum inhibition zone while the 2% concentration of chitosan edible film has a maximum inhibition zone compared to other treatments. The five chitosan edible films were in the strong category because
they ranged from 10-20 mm. Furthermore, the normality test data is processed using the Shapiro - Wilk test method.
Table 2. Results of Normality Test Data Processing
Data Normality Statistic Df P Value
Inhibition 955 48 0,062
*Shapiro Test – Wilk
From table 2 above, it is known that the results of the normality test in the Saphiro-Wilk column the probability value of the data obtained is p=0.062. The probability value can be said to be normally distributed if p>0.005, so the data in table 2 is said to be normally distributed, so that it can be continued for the One Way Anova parametric test.
Table 3. One Way Anova Parametric Test Results
The One Way Anova test is carried out to see the differences in each treatment and show the results obtained are significant or insignificant. From the table above, the value of p=0.000<0.05, then Ho is rejected, which shows a significant difference in each type of treatment so that it can be concluded that the inhibition test carried out can inhibit the growth of the Phorphyromonas gingivalis bacteria.
Furthermore, the further difference test (Post Hoc Test) uses the Small Significant Difference (LSD) test or better known as the Least Significant Differences (LSD) test. This
Group Data
Mean SD
Edible 2%
11,68 0,56
Edible 1 %
11,39 1,05
Edible 3%
10,77 1,19
Negative Control
9,46 0,91
Positive Control 10,66 1,58
Extract 1% 7,38 0,31
Extract 2% 8,57 0,85
Extract 3% 8,49 0,89
P Value 0.000*
method uses the LSD value as a reference in determining whether the average of two or more treatments is statistically different or not. The LSD test results can be seen as follows:
Table 4.LSD Test Results
Group Mean
Difference
P value
Edible 2%
Edible 1
%
0,28 0,621 Edible 3% 0,91 0,117
Negative control
2,22 0,000 Positive
control
1,02 0,080 Extract
1%
4,30 0,000 Extract
2%
3,11 0,000 Extract
3%
3,19 0,000 Edible
1 %
Edible 3% 0,63 0,276 Negative
control
1,94 0,002 Positive
control
0,74 0,202 Extract
1%
4,02 0,000 Extract
2%
2,83 0,000 Extract
3%
2,91 0,000
Edible 3%
Negative control
1,31 0,027 Positive
control
0,11 0,850 Extract
1%
3,39 0,000 Extract
2%
2,20 0,000 Extract
3%
2,28 0,000
In Table 4, there is a significant difference between the 2% concentration of chitosan edible film with negative control, 1%, 2%, and 3% extracts. The negative control saw a significant difference to the 1% extract, the positive control saw a significant difference towards the 1%, 2%, and 3%
extracts, the 1% extract saw a
Negative control
Positif control
-1,20 0,041
Extract 1% 2,08 0,001 Extract 2% 0,89 0,125 Extract 3% 0,97 0,096 Positive
control
Extract 1% 3,28 0,000 Extract 2% 2,09 0,001 Extract 3% 2,17 0,000 Extract
1%
Extract 2% -1,19 0,043 Extract 3% -1,11 0,057 Extract
2%
Extract 3% 0,08 0,893
significant difference against the 2% extract, then for the 2% extract it looked insignificant against the 3% extract.
DISCUSSION
Both lactate chitosan and carboxymethyl chitosan show significant antimicrobial activity against Aggregatibacter actinomycetemcomitans and Porphyromonas ginigvalis and can be used as potential antimicrobials in periodontal therapy. The inhibition zone obtained by both variants of chitosan was statistically significant (p<0.01) against microorganisms, thus proving the antimicrobial ability of chitosan.11,12,13
In its activity, amino groups of chitosan when in contact with protonated physiological fluids and bind to anionic groups of microorganisms will cause agglutination of microbial cells and inhibit the growth of these microbes. When interacting with bacterial cells, chitosan will increase the transfer of Ca++ from the membrane anionic site resulting in cell damage.14
Chitosan has been able to show low toxicity and the development of resistance has not occurred, and chitosan is able to show anti-inflammatory activity by modulating PGE2 levels through the JNK pathway which can be useful in the prevention or treatment of periodontal inflammatio.15
Edible films can be made from chitosan which can be used as an antimicrobial agent. It has been studied and proven in recent years the mechanism of antimicrobial action of chitosan that in its activity, chitosan involves the interaction between positively charged chitosan molecules and negatively charged microbial cell membranes.15
Strong effect of chitosan on periodontal pathogens through inhibition of biofilm formation with registered activity even after 168 hours and against multiple species biofilms, thus indicating that chitosan can disrupt bacterial coaggregation. In this study, chitosan extract was made which will later be made into edible film. Extract of white shrimp scalp waste chitosan (Litopanneaus vannamei) was prepared by dissolving the extract with each concentration of 1%, 2%, 3% using a 1% acetic acid solution and showed the ability to attract the active substances contained in it.15 Chitosan gel without metronidazole did not form a clear zone in the Mueller Hinton Agar medium, so it can be concluded that the chitosan gel was not effective in inhibiting the growth of the bacteria Porphyromonas gingivalis, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans.16,17
CONCLUSION
Based on the results and observations made in this study, it can be concluded that the edible film of white shrimp scalp waste chitosan (Litopenaeus vannamei) has antimicrobial properties in inhibiting the growth of Porphyromonas gingivalis bacteria.
REFERENCES
1. Suryono. (2014).Periodontic basic surgery. Ed. 1. Yogyakarta – Deepublish. p. 2.
2. Rebecca EF, Lakshmi CW, Robert NP. (2009). The gingipains: scissors and glue of the periodontal pathogen, Porphyromonas gingivalis. Future Microbiol; 4(4): 471.
3. Depi P, Peni P, Tantin E. (2011). Oral hygiene status and periodontal health of patients who came to the periodontia clinic at the Dental Hospital of Jember University for the period August 2009 - August 2010.Stomatognatic (JKG); 3(8): 183.
4. Paula JPC, Gloria IL, Patrice G, Vincent M 1, Zohreh TS, Jaime EC, Martine BM. (2009).
Distribution of Porphyromonas gingivalis Fima genotype. Biomedical;29 :299
5. Banun K, Peni P, DesiSS. (2010). The biochemical detection of Porphyromonasgingivalis clinical isolate from subgingival plaque of chronic periodontitis patients using API 20A.
Journal of the Indonesian Dentists Association: 3(59) : 110-1
6. Nurul A, Irma E, Harry A. (2017). The effectiveness of metronidazole gel based chitosan inhibits the growth of bacteria Aggregatibacteractinomycetemcomitans, Porphyromonasgingivalis, Fusobacteriumnucleatum (In vitro). International Journal of Applied Dental Sciences ; 3(2) : 30-1
7. Putri H. (2010). Potential of chitosan as an anti-bacterial cause of periodontitis. UI Journal for the Nation Series on Health, Science, and Technology; (1) : 14.
8. Achmad H, Djais AJ, Petrenko EG, Larisa V, Putra AP. 3-d printing as a tool for applying biotechnologies in modern medicine. International Journal of Pharmaceutical Research, 2020. 12(4), pp. 3454-3463.
9. Achmad H, Djais AI, Jannah M, Huldani, Putra AP. Antibacterial chitosan of milkfish scales (Chanoschanos) on bacteria porphyromonasgingivalis and agregatibacteractinomycetescommitans. Systematic ReviewsIn Pharmacy, 2020. 11(6), pp. 836-841.
10. Achmad H, Djais AI, Syahrir S, Fitri A, Ramadhany YF. A literature us regarding the use of herbal medicines in pediatric dentistry. International Journal of Pharmaceutical Research. 2020. 12,PP. 881-897.
11. Achmad H, Djais AI, Syahrir S, Fitria A, Ramadhany YF. Impact Covid-19 in pediatric dentistry: A literature review. International Journal of Pharmaceutical Research, 2020.
12,p.830-840.
12. Djais AI, Achmad H, Dewiayu D, Sukmana BI, Huldani. Effect of Combination of Demineralization Freeze Dentin Matrix (DFDDM/0 and Moringaoleifera lam osteoprotegerin (OPG) and receptor activator of nuclear factor kappa Bligand (RANKL) as a marker of bone remodeling. Systematic Reviews in Pharmacy. 2020. 11(6), pp.771-779.
13. Nurhayati, Agusman. (2011). Chitosan edible film from shrimp waste as environmentally friendly food packaging. Squalen ; 1(6): 38.
14. Paranjyothy MV, Deepak P. (2016). Antimicrobial activity of water soluble chitosan lactate and carboxymethyl chitosan on Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Research Journal of Pharmaceutical, Biological and Chemical
Sciences; 7(1): 137, 139.
15. Edward JD, Marni K, Riardi PD. (2012).Isolation of chitin and chitosan from shrimp shell waste. BIAM Magazine; 12(1): 33, 37.
16. Adriana LM, Mona EP. (2014). Antimicrobial activity of chitosan edible films. Human Resources Development Operational Programme: 1.
17. Eduardo MC. (2014). Antimicrobial effect of chitosan against periodontal pathogens biofilms. SOJ Microbiology & Infectious Diseases; 2(1): 5.
