94 American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) ISSN (Print) 2313-4410, ISSN (Online) 2313-4402 © Global Society of Scientific Research and Researchers http://asrjetsjournal.org/ Analysis of Protein Content, Spectrophotometry FT-IR, and Antibacterial Effects of Earthworm (Eudriluseugenia) Suandy a *, Ermi Girsang b , Ali Napiah Nasution c , I Nyoman Ehrich Lister d a,c,d Master in Biomedical Sciences Faculty of Medicine, Universitas Prima Indonesia, Medan, North Sumatera b Faculty of Public Health, Universitas Prima Indonesia, Medan, North Sumatera a Email: suandy76@gmail.com Abstract This research to compare effectiveness of coelomic fluid and extract ethanol of Eudriluseugeniae against Staphylococcus aureus and Escherichia coli as antibacterial. Suggest that protein on Eudriluseugeniae have antibacterial activity.Experimental laboratory with analytical statistic Mann- Whitney with following step of research: (1) Determination of earthworm, (2) Cold shock methods modification to get coelomic fluid, (3) Maceration ethanol 70 % of Eudriluseugeniae.(4) Determination protein content with Kjehdahl methods, (5) Identification absorbance infrared by spectrophotometry FT-IR .(7) Antibacterial assay using disc diffusion against Staphylococcus aureus and Escherichia coli.The result of this study showed that protein content of coelomic fluid (1.875 %)were higher than ethanol extract (0.437%) of Eudrilus eugenia. Coelomic liquid had no functional group in the chemical compound. However, the ethanol extract showed some functional group includes aliphatic, ethanol, and Amide A group. The antibacterial effect against Escherichia coli and Staphylococcus aureus from ethanol extract or coelomic liquid of Eudriluseugeniae were similar at equal concentration. It due to p value of analysis was lower than 0.05.Coelomic liquid and ethanol extract had similar antibacterial effects against Staphylococcus aureus and Escherichia coli. Keywords: Eudriluseugeniae; coelomic fluid; Ciprofloxacin; Staphylococcus aureus and Escherichia coli. ------------------------------------------------------------------------ * Corresponding author. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2020) Volume 63, No 1, pp 94-101 95 1. Introduction Topical, oral, and injection antibiotic widely used and uncontrolled, which caused resistant of bacteria through the drug, impairment of normal flora, increased morbidity, cost, and period of antibiotic usage. Sri SelviaNingsih, N.K., et.al. (2016) and Chudlori, B. and his colleagues (2012) reported Staphylococcus aureuswere more sensitive against imipenam than ciprofloxacin, while Escherichia coli as negative gram bacteria which often found in most of Pus in Regional Public Hospital of dr. Moewardi 75% were resistant against ciprofloxacin. These defensive responds were caused by acquisition and transfer of resistant plasmid which was part of intrinsic resistant mechanism. It was found especially in flourquinolon such as ciprofloxacin, some type of beta lactam such as penicillin, and tetracycline. Other study show that there were plasmid gene and chromosome mutation in gyrA and parC gene on Escherichia coli which were resistant through many type of antibiotic that usually used in Urinary tract infection therapy[1–4]. Earthworm have been know as food and traditional drug material, which has some health benefits [5,6], such treat fever, anaesthetic, detoxification, treat hypertension, joint inflammation and itch [7]. These health benefits due to enzymes and peptide which are have antibacterial, antioxidant, and anti-inflammatory activity [8]. Coelomic liquid and ethanol extract of Eudriluseugenia has anti bacteria and antifungal effect due to amount of 40 protein, nitrogen, lipid and peptide enzyme (lysosim and fetidin). They are act as humoral and cellular immunity which responsible for cytolytic, proteolytic, antimicroba, hemolitic, and mitogenic[9,10]. Based on the information above, this study aimed to investigate antibacteria effect of Eudriluseugenia for Staphylococcus aureus as gram positive bacteria and Escherichia coli as gram negative bacteria. 2. Methods Figure 1: Research Procedure 2.1. Materials Eudrilus Eugenia, Isolated colonies of Staphylococcus aureus and Eschericia coli, plastic wrap, Ethanol 70%, aquadest, cotton swab, cotten, Nutrien broth, MHA (Muller Hiton Agar), Alcohol 96%, syringe 1 cc, Acceptible American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2020) Volume 63, No 1, pp 94-101 96 Disc Ciprofloxacin 5 µg OXOID. 2.2. Colletion of Samples Eudriluseugenia (African Earthworm)were gotten from livestock in JalanSagu XI Medan Tuntungan which were chosen health and matur earthworm. 2.3. Preparation of Samples Amount of 500 g Eudriluseugenia which had been cleaned and washed by aquedest was put in to funnel glass using filter paper. The funnel glass was directed in to 500 ml Erlenmeyer, and top of the funnel glass was cover by plastic wrap. These was saved in refrigerator for 4-5 hours, after that it was gotten coelomic liquid of Eudriluseugeniae. The remain of Eudriluseugeniae was put in to 500 ml beaker glass and dried by Blast Air Oven Yenaco YNCOV 53L at 60 o C for 4 days, followed by stirring twice in a day using stirrer bar. After that it was gotten 73.01 gram of dry extracts. The dry extract was macerated using 250 ml 70% ethanol followed by stirring until it was homogen in the dark jar which had been filled by silica gel for 3 days. Furthermore, it was filter using funnel glass and filter paper twice, and the ethanol extract was the last filtrate, the residue of filtration was macerated again using 125 ml 70% ethanol. In the purpose of determination of Protein content and Chemical Structure by Spectrophotometry FT-IR, ethanol extract of Eudriluseugenia was form in to pasta using Water Bath Constant Temperature at 75 o C for 4 days. After that, each 0.05 gram of pasta form was diluted twice using aquadest and methanol (1:1) On the other hand, coelomic liquid and ethanol extract of Eudriluseugeniae was diluted using aquabidest for antibacterial assay. The dilution was result various concentration of coelomic and ethanol extract includes 25%, 50%, and 75%. 2.4. Determination of Protein Content Amount of 2.5 ml coelomic liquid and diluted ethanol extract was identified by kjehdahl distillation methods. Amount of protein content were express in percent (%). 2.5. Identification of Chemical Structure using Spectrophotometry FT-IR Amount of 2.5 ml coelomic liquid and diluted ethanol extract from the earth worm were identified by Spectrophotometry FT-IR. 2.6. Preparation of Media There were two types of media which are used in this study included nutrion broth and Muller Hinton Agar (MHA). Nutrient broth was made by mixing 0.4 gram powder of nutrient broth into 50 ml aquabidest, while American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2020) Volume 63, No 1, pp 94-101 97 MHA was made by mixing 17 gram of MHA powder into 500 ml aqubidest. Each of media was stirred by hotplate stirrer untill the media became homogen and then they were autoclave for 15 minutes at 121 o C. 2.7. Preparation of Bacteria Culture Specimen Each colony of bacteria from the pure isolated bacteria of Staphylococcus aureus and Escherichia coli was put by needle ose and culture into Nutrient broth which had been filled into test tube and incubated for 24 hours at 37 o C. 2.8. Antibacterial Assay using Disc Diffusion Methods The bacteria which had been cultured in Nutrient broth was piped by micropipet into petridish. After that MHA was filled into the petridish and mixed it. At the same time, each concentration of ethanol extract and coelomic liquid which had been diluted was piped in to disc difusion. For the last, each of disc diffusion was put into MHA in petridish, and they were incubated in 37 o C for 3 days. After 3 days, the diameter of zone inhibition (clear zone) in MHA was measured using vernier calliper in milimeter (mm). 2.9. Data analysis All data from diameter of zone inhibition was expressed as Median (Range) for each concentration of sample. While Amount of protein content was expressed in percent. However the result of Analysis of Spectrophotometry FT-IR was expressed as description in a table. Furthermore diameter of zone inhibition at equal concentration between coelomic liquid and ethanol extract was analysed using Mann-whitney Test which P value less than 0.05 were considered statistically significant. 3. Result and Discussion 3.1. Identification of Earthworm (Eudriluseugeniae) Earthworm was identified in LaboratoriumSistematikaHewanDepartemenBiologiFakultasMatematikadanIlmuAlam (No. Letter: 038/UN5.2.1.11/KRK/2019) at 9 Januari 2019. The result of the identification was shown in the table 1 and table 2 below. Table 1: Identification from Specific Properties of Eudriluseugeniae No. Species Specific Properties 1. Eudriluseugeniae Length of body was 10 cm. Shape of body was flat and tip tailed. Head to clitellum distance was 3-4 cm. The body had 135-350 of segments and had clitellum at 30th- 32nd segments. It has metameres which are group of all body segments. There were two reproduction organ (hermaphrodite). There were not skleton but had coelomic liquid which were act as hydrostatic frame. The body has reddish-brown colored. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2020) Volume 63, No 1, pp 94-101 98 Table 2: Taxonomy of Eudriluseugeniae Kingdom Animalia Phylum Annelida Class Clitellata Ordo Haplotaxida Family Eudrilidae Genus Eudrilus Species Eudriluseugeniae Eudriluseugeniae or african earthworm has high content of protein [5]. Ethanol extract and coelomic fluid of Eudiluseugeniae has antibacterial and antifungal effect as nature product content [8,9]. However, based on Chauhan and his colleagues (2014) reported that extract of Eudriluseugenia does not have antibacteria effect against Eschericia coli and any types of fungal[10]. 3.2. Determination of Protein Content Determination of protein content from the coelomic liquid and ethanol extract in percent are shown in table 3 below. Table 3: Amount of Protein Content in Coelomic liquid and Ethanol Extract Eudriluseugeniae No Sample Level of Protein 1. Coelomic fluid 1.875 2. Ethanol extract 0.437 Based on table 3 above, amount of protein content in coelomic liquid is higher than ethanol extract. Hrenzjak and his colleagues (1991) reported same result that there was higher protein content in the coelomic liquid due to glycoprotein G90. Other protein might be found in earthworm which had antibacterial effect. In Pheretimajavanica, there wereprotein which had molecular weight between 7.0 kDa – 55kDa. These proteins usually had tryptophan as monomer (amino acid) [11,12]. 3.3. Identification of Chemical Structure Using Spectrophotometry FT-IR The results of identification of chemical structure using spectrophotometry FT-IR are shown in table 4 below. American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2020) Volume 63, No 1, pp 94-101 99 Table 4: The result of Spectrophotometry FTIR from Coelomic Liquid and Ethanol Extract Eudriluseugeniae No. Sample Absorption Bands Peak Type of Bond Conclusion 1. Coelomic Liquid None or Minimal - - - 2. \ Ethanol extract 3300-3311 cm-1 Moderate Stretching Vibration Ethanol group 2925.8 cm -1 Moderate Stretching and Bending Vibration (-NH2) Amide A Group (Primer) 2885.248 cm -1 Moderate Stretching and Bending Vibration CH Aliphatic group 1606.77 cm -1 Moderate Stretching Vibration (C=C) Alipatic system group 1442.75 – 1398.78 cm -1 Moderate Stretching Vibration (-CH3) 1099.43-1025.64 cm -1 strong Stretching Vibration (C- O-C) antisymmetric Based on the table 4 above, coelomic liquid had none or minimal absorbance which meant there no functional group in the chemical compound. However, the ethanol extract showed some functional group includes aliphatic, ethanol, and Amide A group. Amide A group showed presence of amino acid as monomer of Protein. The result of Spectrophotometry showed as oppose to result of determination protein contents. Method which was used to determination of protein content had some disadvantages. Some compound which are not protein also analyse such as purine, pyrimidine, vitamin, large amino acid, or creatinine [13]. 3.4. Antibacterial Assay using Disc Diffusion The result of Mann-whitney test for Diameter of Zone Inhibition from coelomic liquid and ethanol extract was shown in table 5 and 6 below. Table 5: Comparison of Antibacterial Effect of Ethanol Extract and Coelomic Liquid from Eudriluseugenia against Escherichia coli Concentration Diameter of Zone Inhibition P Value Ethanol Extract [Median (R)] Coelomic Liquid [Median (R)] 25% 0 (0) 6.6 (0) 0.083 50% 7.18 (0.15) 6.85 (0.1) 0.121 75% 9.25 (0.70) 7.2 (0.2) 0.121 Positive control 19.58 (2.58) - Negative Control 0 (0) - American Scientific Research Journal for Engineering, Technology, and Sciences (ASRJETS) (2020) Volume 63, No 1, pp 94-101 100 Table 6: Comparison of Antibacteria Effect of Ethanol Extract and Coelomic Liquid from Eudriluseugenia against Staphylococcus aureus Concentration Diameter of Zone Inhibition P Value Ethanol Extract[Median (R)] Coelomic Liquid[Median (R)] 25% 6.7(0.20) 8 (0.2) 0.121 50% 7.15 (0.10) 10.73 (1.55) 0.121 75% 7.45 (0.10) 13.75 (2.7) 0.121 Positive control 25.53 (1.22) - Negative Control 0 (0) - Based on table 5 and 6 above, the antibacterial effect against Escherichia coli and Staphylococcus aureus from ethanol extract or coelomic liquid of Eudriluseugenia were similar at equal concentration. It due to p value of analysis was lower than 0.05. According to Greenwood, Slack, and Peutherer (2002) classification which are shown in table 7 below. Antibacterial activity of ethanol extract at various concentration were weak against Escherichia coli or Staphylococcus aureus. While coelomic liquid had weak antibacterial activity against Escherichia coli but had sufficient antibacterial activity at higher concentration against Staphylococcus aureus. Table 7: Classification of Greenwood, Slack, and Peutherer (2002) Diameter of Zone Inhibition Inhibition of Growth (Antibacterial activity) > 20 mm Very Potent 16-20 mm Potent 10-15 mm Sufficient < 10 mm Weak 4. Conclusion Protein content of coelomic liquid were higher than ethanol extract of Eudriluseugenia. Chemical compounds which were contained in ethanol extract of Eudriluseugenia had ethanol, aliphatic, and amide A compound. 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