antibacterial activity of enzymatic hydrolysis of virgin coconut oil

International Journal of PharmTech Research CODEN (USA): IJPRIF ISSN : 0974-4304

Vol.6, No.2, pp 589-599, April-June 2014

Antibacterial activity of Enzymatic Hydrolysis of Virgin

Coconut oil against Salmonella

Elysa*, Urip Harahap, and Jansen Silalahi

Department of Pharmacology, Faculty of Pharmacy, University of Sumatera Utara, Jl. Tri Dharma No. 5, Pintu 4, Kampus USU, Medan, Indonesia, 20155.

*Corres. author: [email protected] Phone number: +62 821 6651 2555

Abstract: Virgin Coconut Oil (VCO) is a high-value coconut product, because it has the medical potential such as antibacterial, antiviral, and antifungal activity of its medium chain fatty acids (MCFAs), particularly lauric acid (C12: 0) in its monoglycerides form (monolaurin or ML). The aim of this study was to examine the in-vitro and in-vivo antibacterial activity of the enzymatic hydrolysis of VCO (VCOH) which generates a combination of lauric acid and monolaurin against Salmonella. The in-vitro test was conducted by diffusion agar method using the paper discs diameter 6 mm (Oxoid) by observing the zone of inhibition. The best inhibitory zone in the in-vitro test, then used for investigated in-vivo antibacterial activity of VCOH against Salmonella was examined in a Salmonella typhimurium infection mice mus musculus model. The results of in-vitro antibacterial activity of VCOH against Salmonella typhi (ATCC 00786) and Salmonella typhimurium (ATCC 14028) at concentrations of 200, 400, 600, 800, 1000 µL/mL was found to have zone of inhibition that significantly increased which directly proportional with increased concentration. Furthermore, the study of in-vivo antibacterial activity of the VCOH was found to have decrease the number of colonies of bacteria in the feces of mice in the group of mice that were given VCOH. Although the clinical signs and histological damage were rarely observed in treated mice with non-hydrolyzed VCO and VCOH. The untreated controls and comparison of chloramphenicol controls showed signs of lethargy and histological damaged in the liver and spleen when compared with the controls group. Key Words: virgin coconut oil, enzymatic, antibacterial, Salmonella.

INTRODUCTION

Salmonella is a gram-negative bacteria that can infect humans and animals cause significant morbidity and mortality worldwide. Salmonellosis is an infection caused by the bacterial pathogen Salmonella, a endemic disease in Indonesia, which is also a major problem in some developing countries, including Indonesia. The chain of transmission of Salmonellosis associated with the main source of transmission of raw meat or poultry and its products which come in contact with ready-to-eat foods cause foodborne disease through cross-contamination. These bacteria are pathogenic to humans and animals when contaminated by mouth, will cause enteric disease, so it is included into the group bacteria enterobacteriaceae.6,26,27

Elysa et al /Int.J.PharmTech Res.2014,6(2),pp 589-599.

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This becomes concern for related parties concerned and World Health Organization (WHO) to overcome Salmonellosis. Treatment of Salmonellosis commonly used antibiotics in addition to correcting dehydration and electrolyte disturbances. In Indonesia, chloramphenicol is still drug of choice used to treat salmonellosis. The high use of antibiotics has increased the incidence of bacterial resistance, so that the infection continues to thrive in the worldwide, including in Indonesia.6,27 Therefore, it is necessary to find a safer alternative therapies such as monolaurin and lauric acid which obtained from the hydrolysis of virgin coconut oil (Virgin Coconut Oil, VCO) to overcome pathogenic microorganisms.

Lauric acid is a medium chain fatty acids (MCFAs) that contains 12 carbon atoms. This acid is bound in the form of triglycerides in the VCO. In the human body, triglycerides in the VCO is converted into monoglycerides and lauric acid, which have antibacterial, antiviral, and antifungal activity.7,25

Antibacterial ability of the VCO has been investigated and the results showed that the VCO against growth of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris, Bacillus subtilis, Listeria monocytogene, Staphylococcus agalactiae, maupun Helicobacter sp.8,13,15,17,18,24 Other research declared that partial hydrolysis of VCO can increase the zone of inhibition of bacterial growth, either hydrolysis with lipase (Lipozyme TM IL) or by saponification (NaOH)3. Permata (2012) in her study using this both methods to see the effect of the antibacterial activity of partial hydrolysis of the VCO, the best result was shown by hydrolysis with enzymatic method with the incubation time of 12 hours.19

The increase of incubation time of enzymatic hydrolysis is proportional to the increase of free fatty acid content in the VCO and an increased of antibacterial activity; whereas the study of Hasibuan (2012), hydrolyzed VCO by the saponification method to see its in-vitro antibacterial activity against pathogenic bacteria (Salmonella) and probiotics, showed that VCO and hydrolysis results in the whipe cream still maintaining the life of probiotics (L.casei Shirota strain), whereas pathogenic bacteria (Salmonella typhi) cannot grow/die.14

The purpose of this study was to investigated the in-vitro antibacterial activity of the enzymatic hydrolysis of VCO and compared its with the in-vivo against Salmonella and to ensure safety and effectiveness. In this study, hydrolysis of VCO used enzymatic method that different from Permata (2012), by prolong the incubation time in order to obtained the optimum VCO to hydrolyze with of fatty acid value is constant and obtained the endpoint enzymatic reaction. Furthermore, the results of examined the in-vitro antibacterial activity of enzymatic hydrolysis of VCO against Salmonella using agar diffusion method. In in-vivo was examined in a S.typhimurium infection of male mus musculus mice model, by observing the clinical signs and the number of colonies was counted in the feces of mice and histopathological changes in liver and spleen tissue.

MATERIALS AND METHODS

Apparatus

Laminar Air Flow Cabinet (ESCO-EFA-4UDR-W; Astec HLF 1200L), shaker (N-Biotek), ultrasonic cleaner (Soltec), autoclave (Express), incubator (Memmert), spectrophotometer (Dynamica), oven (Fisher), colony counter (Stuart), colony counter (Stuart), and other laboratory glassware (Pyrex).

Materials

The chemicals used in this study are pro-analyst quality product by E. Merck (Germany) that is ethanol, n-hexane, sodium hydroxide, tris-(hydroxymethyl) aminometan (TRIS-buffer), hydrochloric acid, calcium chloride, sodium sulfate anhydrous, potassium biftalat, phenolphthalein indicator (1% in alcohol) and Lipozyme ® TL IM (Novozymes) .

Salmonella typhi (ATCC 00786), and Salmonella typhimurium (ATCC 14028) were bacteria used in this studies.

Bacterial growth media used were nutrient agar (Oxoid)4, nutrient broth (Oxoid)4 and Mueller-Hinton agar (Oxoid)4. paper-disc diameter 6 mm (Oxoid)4. Reference standard for in-vitro assay used were disc chloramphenicol (30µg/disc) (Oxoid)4, whereas for the in-vivo assay used was a reference standard BPFI chloramphenicol (BPOM, Medan). The in-vivo study used male mus musculus mice strain. The sample of VCO that used in this study was Palem Mustika VCO-Virgin Coconut Oil, produced by Siti Nurbaya, West Sumatra.


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Methods

There were three stages conducted in this study, ie the process of enzymatic hydrolysis of the VCO, the in-vitro, and in-vivo test results of the antibacterial activity of the enzymatic hydrolysis of VCO (VCOH).

Enzymatic hydrolysis of the VCO

VCO was hydrolyzed by enzyme lipase (Lipozyme® TL IM) which is active at the position of sn-1,3 for 18 hours with every 2 hours interval observation time of fatty acids value. 30 grams of oil (VCO) weight into the Erlenmeyer then added 30 ml of distilled water, 12.5 ml of 0.063M CaCl2, 25 ml of buffer Tris-HCl 1M pH 8, 500 grams of Lipozyme TL IM, shaken for 10 minutes at a speed of 200 rpm. Subsequently incubated at 55oC, and shake the mixture every hour for 10 minutes at a speed of 200 rpm.3,22 Observation of the increase of fatty acid value every 2 hours up to 18 hours.

The in-vitro test

VCOH which has the optimum fatty acid value, then investigated the in-vitro antibacterial activity of VCOH against Salmonella by diffusion agar method using the paper discs diameter 6 mm (Oxoid)4 by observing the zone of inhibition. Antibacterial activity test was carried out on the non-hydrolized VCO, and the hydrolyzed VCO (VCOH) with concentration of 200 µl/ml, 400 µl/ml, 600 µl/ml, 800 µl/ml, and 1000 µl/ml, respectively. Antibacterial activity of the VCOH compared with standard discs chloramphenicol (30 µg).20

The in-vivo test

The in-vivo test procedures in this study was conducted in previously described by Gi Choi, et al., (2011) with some modifications.12 The best inhibitory zone in the in-vivo test, then used for investigated in-vivo antibacterial activity of enzymatic hydrolysis of VCO against Salmonella was examined in a S.typhimurium infection mice mus musculus model, clinical signs were observed and counted the number of colonies of S.typhimurium from feces and histopathological changes in liver and spleen tissue. As many as 30 male mus musculus mice (± 20 g) aged between 5-6 weeks were used for all in-vivo experiments. They were kept in a temperature-controlled room under a 12 hours light and 12 hours dark cycle. Animal had free access to commercial solid food (T79-4P) and water ad libitum, and were acclimatized for at least 1 week prior to beginning the experiments. Mice were grouped into 5 groups, each group consisted 6 mice, ie. Control group or Normal (SI-CON), Salmonella-infected group (SI), Salmonella-infected group + chloramphenicol (SI-Chlor), Salmonella-infected group + VCO (SI-VCO), Salmonella-infected group + enzymatic hydrolysis of VCO (SI-VCOH).

The SI, SI-Chlor, SI-VCO, and SI-VCOH groups exclusively were then inoculated using gavage needle orally with approximately 106 CFU of S.typimurium in a 0,1 mL volume. One hour after infection, animals in the SI-Chlor, SI-VCO and SI-VCOH groups were orally 1 ml (1000µl/ml) VCO and VCOH; and chloramphenicol 300 µg/ml, whereas SI-CON and SI groups animals were not. Fecal samples were then collected 0, 1, 2, 3, 4, 5, 6 days after the bacterial suspensions were administered and the numbers of bacteria per gram were determined. Aliqouts (100 µl) of fecal suspensions were serially diluted in Phosphate-buffered Saline (PBS) and then plated on Salmonella-Shigella agar plates (Oxoid), which were subsequently incubated overnight at 36-370C. Typical colonies were then counted using colony counter (Stuart) on plated that contained between 30-300 colonies. At day 4 post-infection, the mice were sacrificed, and tissue specimens of the liver and spleen organs were transferred to 10% buffered neutral formalin for histopathologic examinations and then processed using standard procedures.

Data were analyzed statistically with analysis of variance (ANOVA; α 0.05), followed by Duncan's Post Hoc test to see the difference in the average value significantly between treatment groups.

RESULTS AND DISCUSSION

Hydrolysis enzymatic of the VCO

Enzymatic hydrolysis of VCO process using enzymatic methods performed during 18 hours of incubation with the interval observation time of fatty acid value every 2 hours, to determine the optimum incubation time for enzymatic hydrolysis of VCO using Lipozyme TL IM to achieve a constant fatty acid value, which is no longer


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increased the amount of free fatty acids. Data and graphs of fatty acid value of enzymatic hydrolysis of VCO can be seen in Table 3.1 and Figure 3.1.

Table 3.1 Fatty acid value of hydrolysis enzymatic of VCO

Figure 3.1 Relationship graph of incubation time with fatty acid value of hydrolysis enzymatic of VCO Fatty acids value is a measure of the amount of free fatty acids contained in the oil or fat. Enzymatic hydrolysis of the VCO will generate 2 molecules of free fatty acids and 1 molecule of 2-monoglycerides from each triglyceride molecule that contained in the VCO.2,10,17,18 Lipozyme TL IM lipase is an enzyme that is classified into 1,3-regioselective, worked specifically hydrolyzed fatty acids in triglycerides moleculs at the position of sn-1 and sn-3. It also resembles the action of the enzyme lipase in the human gastrointestinal tract.1

In Table 3.1 and Figure 3.1 it can be seen that the constant of fatty acids value was obtained during the incubation after 14 hours. In the graph (Figure 3.1) of incubation time with fatty acid value of enzymatic hydrolysis of VCO was formed sigmoid curve, because the onset of enzymatic reactions increased fatty acid value significantly after incubation over 2 hours and the end point was reached when the enzymatic reaction

Samples Incubation time (hours)

Mean ± SD Fatty Acid Value

n=3 (mg KOH/g Oil)

non-hydrolyzed VCO - 0.3767 ± 0.0569 0 4.0333 ± 0.3602 2 7.3633 ± 0.5390 4 20.8333 ± 1.4202 6 45.4833 ± 1.7471 8 95.9867 ± 1.6129 10 122.2767 ± 4.4251 12 133.4867 ± 2.3280

14 140.3167 ± 0.7697

16 140.8667 ± 1.2751

Enzymatic Hydrolysis of VCO

18 142.1733 ± 1.3405


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incubation time for 14 hours. There is no increase significantly of free fatty acid after 14 hours incubation time that indicated with a linear line in the graph (ANOVA; α 0.05, followed by Post Hoc Duncan test).

It was meant that the fatty acid at the position sn-1,3 of triglycerides have been hydrolyzed completely perfect, so there is no increase in the number of free fatty acids and the constant of fatty acid values was obtained after 14 hours, then the optimum incubation time to obtained the endpoint of enzymatic reaction (Lipozyme ® TL IM) to hydrolyzed VCO is 14 hours.

The results of enzymatic hydrolysis of the VCO will produce 2-monoglycerides and free fatty acids, which are dominated by monolaurin and lauric acid which is known to be most active as an antimicrobial. Lauric acid (C12) has a greater antiviral and antibacterial activity than other medium-chain triglycerides such as caprylic acid (C8), capric acid (C10), or myristic acid (C14).10,17,18

In-vitro antibacterial activity of VCO and enzymatic hydrolysis of VCO

The test result of in-vitro antibacterial activity against Salmonella thypi and Salmonella typhimurium was shown in Figure 3.2, Figure 3.3 and Table 3.2.

Figure 3.2 The zone of inhibition of hydrolysis enzimatic of VCO against Salmonella typhimurium Note: A= zone of inhibition of ethanol against Salmonella typhimurium B= zone of inhibition of VCO and hydrolysis enzimatic of VCO against Salmonella typhimurium C= zone of inhibition of chloramphenicol against Salmonella typhimurium

VCO

1000 200


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Figure 3.3 The zone of inhibition of hydrolysis enzimatic of VCO against Salmonella typhi Note: A= zone of inhibition of ethanol against Salmonella typhi B = zone of inhibition of VCO and hydrolysis enzimatic of VCO against Salmonella typhi C = zone of inhibition of chloramphenicol against Salmonella typhi

Table 3.2. The in-vitro antibacterial activity of hydrolysis Enzimatic of VCO against Salmonella typhimurium and Salmonella typhi

Mean ± SD Zone Inhibition (mm)

n=3 Samples Concentratio

ns S.typhimurium S.typhi

Ethanol - 6.000 ± 0.0000 6.000 ± 0.0000

VCO - 7.533 ± 0.4274 7.583 ± 0.2858

200µl/ml 10.533 ± 0.7448 10.300 ± 0.7537 400µl/ml 11.383 ± 0.4535 10.783 ± 0.4708 600µl/ml 12.133 ± 0.7033 11.083 ± 0.7548 800µl/ml 12.983 ± 0.7026 12.250 ± 0.5244

Enzimatic Hydrolysis of

VCO

1000µl/ml 14.333 ± 0.6623 13.900 ± 0.3033

Chloramphenicol 30 µg/disc 10.167 ± 0.4227 20.167 ± 0.6947


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Table 3.2 shows that there is no significant difference between the antibacterial activity of VCOH against Salmonella typhimurium and Salmonella typhi. In addition, It also showed that the inhibition zone was increased significantly which directly proportional to the increased in the concentration of VCOH (ANOVA; α 0.05, follow by Post-Hoc Duncan test). This is consistent with previous research which states that the results of the hydrolysis of VCO gave inhibition zone against Salmonella typhi.14

The antibacterial activity of chloramphenicol against Salmonella typhi has a greater inhibitory zone compared with Salmonella typhimurium. This indicates that Salmonella typhimurium has been resistance to chloramphenicol. Antibacterial activity of chloramphenicol is by binding to the bacterial 50S ribosomal subunit and directly inhibit peptidyl transferase activity thereby disrupting protein synthesis. Microorganisms that have been resistance to chloramphenicol will reduce membrane permeability, mutation of the 50S ribosomal subunit, and elaboration of chloramphenicol acetyltransferase. This Chloramphenicol acetyltransferase (enzyme inactivator) which produced by plasmid (that also codes for resistance to other drugs) because it has resistance genes which encode these enzyme so that acetylated chloramphenicol prevents chloramphenicol from binding to the 50S ribosomal subunit of bacteria, thus it cannot inhibit the protein synthesis.5

In-vivo antibacterial activity of VCO and hydrolyzed VCO

The in-vivo antibacterial activity of enzymatic hydrolysis of VCO (VCOH) was examined using a S. typhimurium infection mice model. Brieftly, mice were infected with 106 CFU of S. typhimurium in 0.1 mL volume using oral sonde in per oral (po). One hour later, the mice were orally administered VCOH.12 The number of colonies of S. typhimurium in the feces of mice per group presented in Table 3.3 Figure 3.4.

Table 3.3 Effects of treatment with enzymatic hydrolysis of VCO on fecal shedding (CFU g-1) of S. typhimurium by mice Group Day-0 Day-1 Day-2 Day-3 Day-4 Day-5 Day-6 CON 0 0 0 0 0 0 0

CON 0 0 0 0 0 0 0

CON 0 0 0 0 0 0 0

CON 0 0 0 0 0 0 0

CON 0 0 0 0 0 0 0

CON 0 0 0 0 0/* dead dead SI 0 5.8 x 103 3.3 x 103 1.6 x 104 3.2 x 105 6.5 x 105 dead SI 0 8.2 x 103 1.7 x 104 3.6 x 105 4.8 x 105 8.2 x 105 dead SI 0 1.6 x 105 1.9 x 104 5.3 x 105 dead dead dead SI 0 1.1 x 104 1.3 x 104 4.6 x 105 1.4 x 106 dead dead SI 0 1.3 x 104 1.7 x 104 1.8 x 105 5.1 x 105 1.2 x 106 dead SI 0 9.4 x 103 1.3 x 104 2.3 x 104 2.2 x 105/* mati dead SI-Klor 0 4.7 x 103 3.7 x 103 1.4 x 104 1.8 x 104 2.1 x 104 1.7 x 105 SI-Klor 0 2.3 x 103 4.2 x 103 6.4 x 103 1.7 x 104 2.0 x 104 2.6 x 104 SI-Klor 0 3.4 x 103 6.2 x 103 5.2 x 103 7.8 x 103 1.7 x 104 2.4 x 104 SI-Klor 0 1.8 x 103 4.3 x 103 1.1 x 104 2.6 x 104 2.1 x 105 dead SI-Klor 0 9.6 x 103 1.5 x 104 2.3 x 104 2.4 x 104 1.6 x 105 1.7 x 105 SI-Klor 0 4.0 x 103 6.8 x 103 1.8 x 104 2.0 x 104/* dead dead SI-VCO 0 2.3 x 103 2.1 x 103 1.3 x 103 1.1 x 103 1.0 x 103 9.6 x 102 SI-VCO 0 5.1 x 103 3.2 x 103 1.8 x 103 1.5 x 103 1.3 x 103 1.1 x 103 SI-VCO 0 2.6 x 103 2.4 x 103 1.2 x 103 1.2 x 103 1.0 x 103 9.8 x 102 SI-VCO 0 1.7 x 104 6.5 x 103 4.9 x 103 4.1 x 103 3.5 x 103 2.9 x 103 SI-VCO 0 3.1 x 103 2.6 x 103 2.2 x 103 2.0 x 103 1.7 x 103 1.3 x 103 SI-VCO 0 6.6 x 103 5.0 x 103 4.2 x 103 3.1 x103/* dead dead SI-VCOH 0 3.0 x 103 2.8 x 103 9.1 x 102 8.2 x 102 7.6 x 102 6.4 x 102 SI-VCOH 0 9.3 x 103 6.2 x 103 2.6 x 103 1.1 x 103 1.0 x 103 8.2 x 102 SI-VCOH 0 2.2 x 103 4.3 x 103 1.3 x 103 9.4 x 102 8.1 x 102 6.7 x 102 SI-VCOH 0 8.3 x 102 2.7 x 103 9.6 x 102 8.7 x 102 6.5 x 102 5.2 x 102 SI-VCOH 0 9.8 x 103 4.3 x 103 8.7 x 102 5.1 x 103 4.3 x 103 2.6 x 103 SI-VCOH 0 2.4 x 103 1.2 x 103 9.7 x 102 9.2 x 102/* dead dead

* = At day 4 post-infection, one mice from each group were sacrificed, and tissue specimens of the liver and spleen organs were used for histopathologic examinations


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Figure 3.4 Relationship graph effects of treatment VCOH on fecal shedding of S.typhimurium (CFU g-1) by mice

/ * = SI graph continues to rise reaches mean of 1.8 x 105 CFU g-1(day 3), 4.4 x 105 CFU g-1(day 4), dan 8.9 x 105 CFU g-1(day 5) (not shown)

The number of colonies on fecal shedding (CFU g-1) of S. typhimurium by mice was 0 CFU / gram. It shows that all mice were healthy and homogeneous. At day 1 post-infection, all mice in each group were shed viable of S. typhimurium in feces, with the feces of mice in the CON group is 0 CFU g-1, SI group being found to contain bacteria at a concentration of 5.8 x 103 to 1.3 x 104 CFU g-1, SI-Chlor group being found to contain bacteria at a concentration of 1.8 x 103 to 9.6 x 103 CFU g-1, SI-VCO group being found to contain bacteria at a concentration of 6.6 x 103 to 1.7 x 104 CFU g-1, and SI-VCOH group being found to contain bacteria at a concentration of 2.2 x 103 to 9.8 x 103 CFU g-1. At day 6 post-infection, the number of colonies of S. typhimurium in the feces of mice in the CON group is 0 CFU g-1, SI group is 6.5 x 105 to 1.2 x 106 CFU g-1, SI-Chlor group is 2.6 x 104 to 1.7 x 105 CFU g-1, SI-VCO group is 9.8 x 102 to 1.1 x 103 CFU g-1, and SI-VCOH group is 6.4 x 102 to 2.6 x 103 CFU g-1. In addition, at day 6 post-infection, none of the mice in the CON group, SI-VCO group, and SI-VCOH group died, whereas all six mice in group SI group died and in the SI-Chlor group one of the six mice died at the end of the study. The test results of the in-vivo antibacterial activity presented in Table 3.3 and Figure 3.4 show that the number of bacterial colonies of Salmonella typhimurium in the feces of mice on SI-VCO and SI-VCOH group has decrease. It means that the SI-VCO and SI-VCOH group have antibacterial activity against bacterial pathogens Salmonella. The test results of antibacterial activity on SI-VCO groups in in- vitro and in-vivo studies showed different results; The in-vitro study showed that VCO do not have antibacterial activity, as it has a small inhibitory zone, whereas the in-vivo studies showed antibacterial activity, because the number of bacterial colonies of Salmonella typhimurium in the feces of mice has decrease. It is caused VCO in the in-vivo assay in mice body will hydrolyzed by the enzyme lipase which is also found in the digestive systems of mice to produce free fatty acids and monolaurin which have antibacterial activity, while the VCO testing in-vitro there is no role of the digestive enzymes so it does not provide antibacterial activity, whereas the in-vitro assay there was no role of enzymes digestion in it so that it does not provide antibacterial activity. The results of histopathological examination in the liver and spleen of mice infected with Salmonella typhimurium presented in Figure 3.5 and Figure 3.6.


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Figure 3.5 Histopathological changes in liver of mice in CON (a), SI (b), SI-Klor (c), SI-VCO (d), dan SI-VCOH (e). Haematoxylin and Eosin (HE) stained. 100x magnification.

Figure 3.6 Histopathological changes in spleen of mice in CON (a), SI (b), SI-Klor (c), SI-VCO (d), dan SI-VCOH (e). Haematoxylin and Eosin (HE) stained. 100x magnification.

a b c

d e

a

e d

c b


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There were found histological damage in the SI and SI-Chlor group. There was histological damages that occurs in the liver, appears on the wall of the central vein that is not intact because the endothelium cells undergo to lysis, the limits between liver cells (hepatocytes cells) look irregular, and there is congestion in the liver sinusoid cavity. In addition, it is also found a few small necrotic lesions nodules in the liver parenchyma (Kupffer cell hyperplasia) (Figure 3.5). The histopathology of spleen in SI and SI-Chlor group occurs extensive hemorrhagic necrosis indicated by infiltration of red blood cells in the red pulp with multiple apoptotic cells in the white pulp. In contrast, on SI-VCO and SI-VCOH group do not seem significant histological damage on liver and spleen of mice compared with KN (Figure 3.6).

CONCLUSION

In the present study, the VCOH showed that the optimum hydrolysis time of Lipozyme TL IM to hydrolyzed VCO is 14 hours. In in-vitro, antibacterial activity has increased significantly comparable to the increase in concentration of VCOH. There were no significant differences of antibacterial activity of VCOH between S.typhi and S.typhimurium. In in-vivo studies showed that the VCOH have inhibition indicated by the decrease in the number of bacterial colonies feces of mice in the group of mice that were given with the test substance.

Antibacterial activity of free fatty acids (FFA) and monoglycerides (MG) that contained in this enzymatic hydrolysis of VCO was not yet fully understood.9,21,23 In this regard, there are several hypotheses of previous researchers who conclude that the FFA and MG showed antibacterial activity through several mechanisms, that is interfere with the plasma membrane lipid bilayer, disrupting the electron transport chain, inhibits the activity of the enzyme, disrupt nutrient uptake, increased peroxidation and auto-oxidation of toxic decomposition products or spontaneous bacterial cell lysis.9,21

More appropriate mechanisms from the various mechanisms above is to disrupt the plasma membrane lipid bilayer of bacteria because FFA is a weak acids which pH is a major determinant of the effectiveness of antibacterial effects because it can affect the concentration of undissociated acid. Antimicrobial activity of organic acids is indisputable at low pH. It has been assumed that undissociated forms of organic acids easily penetrate the lipid membrane of the bacterial cell and dissociate within the cell. Consequently, it will disrupt of lipid neutral pH of the cell cytoplasm, resulting in impaired of cellular energy production and damage the outer membrane or cytoplasmic membrane, inhibiting the synthesis of macromolecules or denaturation of proteins and DNA.23 The mechanism of antibacterial activity of monolaurin which is a lipophilic substance, also similar with FFA because there are also increase in the permeability of the bacterial membrane thus potentially as an antimicrobial.22 Monolaurin and free fatty acids showed no adverse effects on the normal microbial flora in the digestive tracts but much more potent against bacterial pathogens.14 It can be concluded that the results of the enzymatic hydrolysis of the VCO can inhibit the growth of Salmonella in in-vitro and in–vivo studies.

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antibacterial activity of enzymatic hydrolysis of virgin coconut oil

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