THE POTENCY OF CHITOSAN AS AN ELICITOR ON ANTIBACTERIAL ACTIVITY OF Streptomyces sp. GMR-22 AGAINST HISTAMINE-PRODUCING BACTERIA

Streptomyces is a Gram-positive bacteria that produces the largest secondary metabolite compounds. The results of whole-genome sequence analysis showed that Streptomyces can carry more than 30 Biosynthetic Gene Clusters (BGC) encoding secondary metabolites that have the potential to be explored in the exploration for new bioactive compounds. However, not all BGC can be expressed in the laboratory scale and requires a specific activation method. This study aims to explore the potential of chitosan as an elicitor compound to activate and or increase the antibacterial activity of Streptomyces sp. GMR-22 was tested against histamine-producing bacteria (HPB) Morganella morganii TK7 and Citrobacter freundii CK1. Chitosan was added to the fermentation medium with the final concentration of 250, 500, and 750 μg/ml while without the addition of chitosan used as control. Fermentation was carried out for 10 days at room temperature, with constant agitation 200 rpm. The supernatant was separated by centrifugation at 3500 rpm for 15 minutes, then fractionation with ethyl acetate, concentrated by vacuum rotary evaporator, and freeze-dried. The test for antibacterial activity was carried out by the microdilution method with an extract concentration of 100 mg/ml. The test results of the microdilution method showed that the addition of chitosan successfully increases the antibacterial activity with the highest activity shown by the water fraction of 250 μg/ml addition of chitosan which effective in inhibiting the growth of Morganella morganii TK7 and Citrobacter freundii CK1 by 97,29% and 97,92% respectively.


Streptomyces is a group of filamentous
Gram-positive bacteria belonging to the Actinomycetes phylum.
These bacteria can be found in nature in various types of habitats such as the sea, soil, and in symbiosis with insects, plants and marine sponges (Cheng et al., 2015).
Streptomyces is one type of potential bacteria that has produced nearly two-thirds of the antibiotic compounds that have been discovered and used to date (Procópioa et al., 2012). Several types of secondary metabolite compounds produced by Streptomyces include antifungal, anticancer, antiviral, and other antibiotics (Azerang & Soroush, 2017).
One of the Streptomyces species that has a large potential for bioactive compounds production is Streptomyces sp.  which was isolated from soil samples in Wanagama Forest, Gunungkidul, Yogyakarta (Nurjasmi et al., 2009). Several studies have been conducted to detect the presence of bioactive compounds produced by Streptomyces sp. GMR-22 includes antifungal activity (Alimuddin et al., 2011) and antiviral (Mentari et al., 2019). Streptomyces coelicolor A3 (2) was reported to have 29 types of BGC (Bentley et al., 2003), but only 6 types of BGC were expressed on a laboratory scale (Rutledge & Challis, 2015). The other gene clusters that are not expressed named as silent or cryptic.

Preparation of chitosan solution
Preparation of chitosan stock solution was carried out by dissolving 0.5 g of commercial chitosan powder into 100 ml of glacial acetic acid solution (1% (v/v)), then the solution was homogenized for ± 1 hour.
The chitosan solution was then sterilized using an autoclave at a temperature of 121 o C for ± 10 minutes. The chitosan is then stored in the refrigerator at 4 o C.

Preliminary research
The preliminary research was done to observe the minimum inhibitor concentration with a speed of 100 rpm. The sample was allowed to reach a volume of 1-2 ml, then transferred to a 5 ml vial tube. The extract will then be dried using a freeze dryer.

Antibacterial activity assay
Microdilution assay was conducted according to Balouiri et al. (2016). HPB

Preliminary test
Determination of the concentration of chitosan that will be used as a treatment for the fermentation of Streptomyces sp. GMR-22 was done through a preliminary test. In addition, it is also assumed that there has been a change in pH during the mixing process of chitosan solution with TSB medium which affects the protonation mechanism of chitosan, which results in weakening of the antibacterial activity produced by chitosan. Chitosan has a pKa value of around 6.2 -7.0 which causes chitosan to dissolve in dilute organic acids (Zargar et al., 2015). In this condition, the chitosan amine group will undergo protonation so that it is positively charged to become NH3 + ions which will affect the antibacterial activity of chitosan (Zargar et al., 2015). Increasing the pH value due to mixing chitosan with TSB medium resulted in chitosan not being protonated, so that the antibacterial activity of chitosan did not tend to affect the growth of Streptomyces sp.

GMR-22. Streptomyces is also reported to
have the ability to utilize chitin and its derivatives as a nutrient source by producing chitinase and chitosanase. Streptomyces is able to produce the chitosanase which has a role to hydrolyze the long chain of chitosan into its oligomers so that it can be transported into cells and is useful as a source of carbon and nitrogen (Ghinet et al., 2010).
Based on Table 1   Based on this results, chitosan concentration of 250 µg/ml, 500 µg/ml, and 750 µg/ml were further used as treatment.

Characteristics of secondary metabolite compounds of Streptomyces sp. GMR-22
The fermentation process was carried out using 3 treatments of chitosan  Furthermore, the extract is freeze dried to get the dry extract.
Based on Table 2, it can be seen that the appearance of ethyl acetate extract is generally similar in the whole treatment which will produce yellow to orange crystals after freeze dried with a production capacity of 0.10 -0.33 g/l. In water extract, the resulting compound is more yellowish to    et al., 2015).
To confirm that the antibacterial activity of the extract produced during the fermentation process in the addition of chitosan treatment did not come from the activity of chitosan, an antibacterial activity test of chitosan was also carried out which can be seen in Table 4. When compared with the results of the antibacterial activity described in Table 3, it can be seen that the antibacterial activity which has antibacterial activity.