Identification of Food Natural Antimicrobe Compound from Waru

Leaves (Hisbicus tillacaeus L.) Extract by GC-MC

Dewi Sartika, Samsu Udayana Nurdin, Neti Yuliana, Susilawati and Wahyudi

THP Department, Faculty of Agriculture, University of Lampung, Indonesia

Keywords: A Natural Antimicrobial, Agent Chicken Meat, Escherichia coli, Waru Leaves.

Abstract: Chicken is the most common source of animal protein in Indonesia due to its high protein content and low

price. However, because of was not a standardized process, chicken meats sold in the traditional market is

mostly contaminated by phatogen bacteria such as Escherichia coli. Previous research indicated of waru

(Hibiscus tillaceus L.) inhibited Escherichia coli growth. Therefore in this research, had aim 1) to study

whether the antimicrobial activity of extracts of waru/hisbiscus by GCMS, 2) to find out the activity of waru

that have antimicrobial activity against Escherichia coli contaminant. The results showed that the

antimicrobial activity of waru leave extracts depends on the respective proportion in the blend. Increasing

antimicrobial activity was observed when waru leaves extract concentration was an increase in the mixture.

The results of the research showed that content of the leaves of waru was dominated by Phytol (38%); methyl

ester (22,1%); Pentadecanoic acid,14-methyl-Squalene (6,58%); Bis (2-Ethylhexyl) phthalate (5,94%); and

ester compound, that had as an antimicrobe potency.

1 INTRODUCTION

The microbes that often contaminate meat or high

protein food are pathogenic microbes, such as,

Escherichia coli and Salmonella sp. (Sartika et al.,

2019). Contamination of pathogenic microbes can

cause various diseases, such as fever, typhoid,

diarrhea, etc. or often referred to as a foodborne

disease. Djafaar and Rahayu (2007) explained that

Escherichia coli contamination can cause denatured

protein on meat and produce toxin compounds. These

toxin compounds can cause several cases of

foodborne disease, one of them as diarrhea (Jawetz et

al., 1995). Escherichia coli or pathogenic microbes

contamination needs to be inhibited to reduce the

number of pathogenic microbes contamination and

prevent damage to chicken meat.

Contamination of pathogenic microbes on high

protein foodstuffs, meat, generally, can be inhibited

by cooling treatment. The other inhibition microbe

treatments, such as the addition of salt, sugar, acid,

and preservatives with synthetic or chemical

preservatives (Usmiati, 2010). However, the methods

that use preservatives with synthetic or chemical

preservatives can have a serious impact on

consumers. So, need a natural preservative method

that has a low risk of health. Therefore, it is necessary

to develop more effective antimicrobials such as

natural antimicrobials in inhibiting pathogenic

microbes contamination on meat. Natural

antimicrobials are recommended because not cause

side effects or negative effects. The natural

preservative method can use by a natural antimicrobe

from natural resources.

Indonesia is a country that has various natural

resources, such as waru (Hisbicus Tillacaeus L.).

Waru is a popular plant in Indonesia. The leaves of

waru were presumed can act as a natural

antimicrobial because contain antimicrobial

compounds. The waru leaves contain saponins,

flavonoids, tannins, and polyphenols. Meanwhile,

teak leaves contain flavonoids, saponins, tannins,

katekat tannins, quinones, steroids/triterpenoids

(Hartati et al., 2007). The antimicrobial can be

explored from environmental such as dragon fruit

leather (Sartika et al., 2019); plant extract (katalinic

et al., (2006); kapok bananas (Ningsih and Nurmiati

(2013); gambir (Pambayun et al., (2007); red rosella

(Putri et al., (2006) and bacteriophage (Sartika et al.,

2002).

Antimicrobial compounds from waru leaves can

be isolated through extraction processes such as

maceration, soxhlet process, percolation, reflux, and

Sartika, D., Nurdin, S., Yuliana, N., Susilawati, . and Wahyudi, .

Identiﬁcation of Food Natural Antimicrobe Compound from Waru Leaves (Hisbicus tillacaeus L.) Extract by GC-MC.

DOI: 10.5220/0010529300003108

In Proceedings of the 6th Food Ingredient Asia Conference (6th FiAC 2020) - Food Science, Nutrition and Health, pages 69-74

ISBN: 978-989-758-540-1

so on. Maceration is an extraction method that is

simple, easy to perform, and does not require high

costs. The macerated extract of hibiscus leaves was

effective in inhibiting the growth of Bacillus subtilis

and Escherichia coli. In the fact, the content of waru

leaves does not found exactly. So, this research aimed

was to find the waru content with use by GCMS.

MATERIAL

AND

2.1 Material

Materials used in this research were waru leaves that

were collected from Lampung Timur of Lampung

province, ethanol 70%, polar dissolvent, aqua

distillate, and alcohol 70%. type of Equipment used

included knife, basin, blender, filter paper, macerator,

beaker tube, Erlenmeyer tube, vacuum rotary

evaporator, scale tube, stirrer, Gas Chromatography-

Mass Spectrometry (GC-MS).

2.2 Study Area

Waru leaves were obtained from Sribahwono Village,

Bandar Sribhawono District, East Lampung Regency,

Lampung Province. Selected samples of

waru/hisbiscus that were not too old, not too young,

healthy, and not moldy were included (Figure 1).

Figure 1: Location of Desa Sribahwono indicating the

sampling sites of Hibiscus tiliaceus L.; 5°18' 40.76" S | 105°

44' 31.24" E.

2.3 Method

This research was conducted in two stages. The first

stage was the preparation and extraction process of

waru leaves sample at The Laboratory of Chemistry,

Faculty of Sciences at Lampung University. The

second stage of the research was to test the chemical

compounds that contained the waru leaves by using

Gas Chromatography-Mass Spectrometry (GC-MS)

which was conducted in the Integrated Laboratory of

Lampung University.

2.3.1 Powder Making and Leaf Extract

(Ningsih et al., 2013)

The leaves are cleaned first using clean water to

remove dirt, then cut into small pieces. Furthermore,

it is oven using 500C temperature for 24 hours. The

use of temperature and time of the oven is intended to

prevent the active compound in the leaves from being

damaged (Putri et al., 2014). After that, the dry leaves

are ground using a blender to produce leaf powder.

Then the leaf powder is sieved using a 40 mesh sieve

to obtain a uniform leaf powder. Sembiring et al.

(2006) reported that 40 mesh powders can produce a

high yield of active substances after the extraction

process. The powdered leaves of waru and teak leaves

that have been obtained are then mixed according to

the proportions for each treatment. After that, the

powder was immersed in boiling distilled water (1000

C) for 10 minutes. The use of boiling distilled water

(1000 C) as a solvent aims to improve the solubility

of the extract (Pambayun et al., 2007). Furthermore,

it is filtered using filter paper to obtain leaf extract.

2.3.2 Total Microbial Decrease Test

The total microbial reduction test was carried out in

several stages. First, the Escherichia coli bacteria

were rejuvenated (Suwandi, 2012) and made a

turbidity standard of 0.5 Mc Farland (Sutton, 2011).

Then the bacterial suspension was made, where the

bacterial suspension was compared to the standard

0.5 Mc Farland using a spectrophotometer with a

wavelength of 600 nm. After that, total Escherichia

coli testing was carried out on fresh fillet chicken

meat, tested the antimicrobial inhibition of each

treatment using disc paper (Suwandi, 2012), and

decreased total Escherichia coli in chicken meat.

2.3.3 Data Analysis

Antimicrobial inhibition test data, total reduction test

for Escherichia coli in chicken meat, test for phenol

content of chicken meat, and test for the degree of

acidity (pH) were further tested with the descriptive

method. Then the test data for the application of

antimicrobial activity and sensory tests were further

tested using the Microsoft Excel application (Anova

and LSD).

6th FiAC 2020 - The Food Ingredient Asia Conference (FiAC)

3 RESULTS AND DISCUSSION

3.1 Waru Characteristic by GC-MS

Method

The preparation process of this research was the waru

leaves were taken in the morning, washed, and dried

to remove dust on the material (figure 2). It was

shredded into smaller sizes. Then, the small slice of

waru was dried to remove water content using an

oven. The waru leaves dry powder, 500 grams of dry

weight, was taken to make extraction by using ethanol

70% solvent.

Figure 2: Extraction of Waru leaves.

The step of Chemical Compounds research was 3

μl of the sample (waru leaves extract) which had been

washed with an ethanol solvent that was taken by

using a gas chromatograph inlet. The sample was

injected into a gas chromatograph by the following

conditions: Gas Chromatograph with AutoSampler

(Agilent Technologies 5973 N) and Mass Selective

Detector 5873 I; Capillary Column (Innowax) with

dimension of 60 m length, 0.2mm wide, 0.25 mm film

thickness; 290

C injector temperature; 290

temperature detector; temperature program of

C(150 minutes) - 290

C (20 minutes); carriage gas

– Helium 1 ml/min with the constant flow; 1uL Split

(ratio 50:1) injection volume; ethanol solvent. The

analysis of chemical compounds contained in waru

leaves extract was conducted by using Gas

Chromatography-Mass Spectrometry (GC-MS).

Chromatograph results are presented in Figure 3.

Figure 3: Chromatograph graphic of the waru leaves

extract.

The result of chromatograph graphic showed that

the waru had characteristic content from high to low

rating, as follows, Phytol (38,70%); Pentadecanoic

acid,14-methyl-,methyl ester (21,11%); 9,12,15-

Octadecatrienoic acid,methyl-ester, (Z,Z,Z)- (8,24%);

8,11-Octadecadienoic acid, methyl ester (5,59%). The

other compound of waru leaves content by GCMS was

summarized completely in Table 1.

Table 1: Waru Chemical compounds.

The Chemical Compound Area

Area

1. 4-((1E)-3-Hydroxy-1-

)

-2-methox

henol 16.978 1,39

3,7,11,15-Tetramethyl-

2-hexadecen-1-ol 9.722 0,80

2H-Pyran,2-(7-

heptadecynyloxy)tetrahydro- 7.685 0,63

3,7,11,15-Tetramethyl-

2-hexadecen-1-ol 6.447 0,53

11-Hexadecenoica cid,

methyl este

2.510 0,21

Pentadecanoic acid,14-

methyl-,methyl este

258.045 21,11

n-Hexadecanoic acid

58.626 4,80

n-Hexadecanoic acid

8.092 0,66

Heptadecanoic

acid,meth

l este

12.992 1,06

8,11-Octadecadienoic

acid,meth

l este

68.372 5,59

9,12,15-

Octadecatrienoic

acid,meth

l ester,

(

Z,Z,Z

)

- 100.746 8,24

Phytol

472.954 38,70

Octadecanoic

acid,methyl este

29.407 2,41

9,12,15-

Octadecatrienoic acid,2,3-

dihydroxypropyl ester,

(Z,Z,Z)- 5.547 0,45

9,12-Octadecadienoic

acid,meth

l ester,

(

E,E

)

- 2.505 0,20

Pentatriacontane 5.673 0,46

Cedran-diol,8S,14-

2.878 0,24

Bis(2-ethylhexyl)

hthalate 72.592 5,94

Squalene

80.480 6,58

3.2 Antimicrobial Inhibition

The results of the research showed that the proportion

of hibiscus leaves gave an effect on the inhibition

zone width. The increase of concentration would

increase the zone inhibition. The highest of inhibition

Identiﬁcation of Food Natural Antimicrobe Compound from Waru Leaves (Hisbicus tillacaeus L.) Extract by GC-MC

zone was reached at the 25% (4,567 ± 0,320) and the

lowest was at the 0% (1,278 ± 0,298) concentration

level. The following figure describes that the effect of

hibiscus extract concentration (Figure 4).

Figure 4: The effect of the hibiscus extract on Escherichia

coli inhibition.

The research result showed that the concentration

of waru/hisbiscus leaves affected inhibition zone

width, which described that waru was effective against

Escherichia coli bacteria. The best treatment was

reached out at a 25% concentration level This

happened probably because the highest of waru

concentration had the highest antimicrobial

compounds. According to Oktavia (2018), hibiscus

leaf extract at a concentration of 25% has a flavonoid

level of 21.7398%. Meanwhile. Reducing Escherichia

coli by Waru or hisbiscus on chicken meat meat.

The total yield of Escherichia coli in chicken

fillets from Pasar Tempel, Rajabasa Raya, Bandar

Lampung was 2,775 ± 0.775 x 104 colonies/gram.

This case indicates that Escherichia coli

contamination on the fillet chicken meat upper than

the maximum contamination limit set by SNI-7388-

2009 (1x101 colony/gram). Escherichia coli

contamination can be caused by the unhygienic

handling process. Therefore, Escherichia coli

contamination in chicken meat can be prevented by

performing hygienic handling with the best possible

process or added preservative.

The results of the analysis of variance showed that

the proportion of the mixture of hibiscus leaves and

teak leaves had a significant effect on the total

reduction of Escherichia coli bacteria. The results of

further testing with the least significant difference

(LSD) at the 5% level showed that the control

treatment was not significantly different from the

W6J6 treatment (0% of waru leaves and 25% of teak

leaves), but was significantly different from other

treatments. The treatments of W3J3 (15% waru

leaves and 10% teak leaves), W4J4 (10% waru leaves

and 15% teak leaves), and W5J5 (5% waru leaves and

20%) were not significantly different. Then the W2J2

treatment (20% waru leaves and 5% teak leaves) was

significantly different from the W1J1 treatment (25%

waru leaves and 0% teak leaves) and the two

treatments were significantly different from other

treatments. The graph of the effect of mixing the

extract of hibiscus leaves and teak leaves on the

decrease in total Escherichia coli.

Figure 5: The effect of the extract of hibiscus/waru leaves

on the Escherichia coli total.

Figure 5 described that the highest proportion of

hibiscus/waru leaves gave an effect to the Escherichia

coli total was highest too. Thus, the results of the total

microbial reduction test were in line with the

antimicrobial inhibition zone test. The concentration

level at 25% hibiscus leaves treatment showed the

highest total reduction of Escherichia coli on chicken

meat with a decreasing percentage of 76.12%.

Meanwhile, the resulting research showed that the

lowest treatment (0%) tend to decrease of Escherichia

coli on chicken meat was lowest too (log E. coli total

was 6.59%). So, Extract waru leaves can be

categorized as an antimicrobial or a bacteriostatic

compound. According to Pelezar and Chan (1988),

antimicrobials can work bacteriostatically (inhibit

microbial growth) and bactericidal (kill microbes).

Characteristics of Escherichia coli colonies that grow

on Eosin Methylene Blue (EMB) media are described

in Table 1.

Table 2: Characteristics of Escherichia coli colonies

growing on Eosin Methylene Blue (EMB) media.

Treatment

Colony Characteristics of

Escherichia coli

Control

Darkcore purple with metallic green

color. Grows on the base, middle,

and surface of the media

25%

Dark purple core. Grows on the base

of the mediu

20%

Dark purple core. Grows in the

middle, and base of the mediu

15%

Dark purple core. Grows in the

middle, and base of the mediu

10%

Darkcore purple with metallic green

on the base, middle, and surface

Darkcore purple with metallic green

on the base, middle, and surface

6th FiAC 2020 - The Food Ingredient Asia Conference (FiAC)

In Table 2, it can be seen that the characteristics

of Escherichia coli colonies that grow on Eosin

Methylene Blue (EMB) media are different. The

results showed that the addition of hibiscus leaf

extract with a greater proportion could prevent the

formation of the metallic green color of Escherichia

coli colonies on Eosin Methylene Blue (EMB) media.

This is evidenced by the growth of the Escherichia

coli bacteria forming colonies with dark purple cores

without metallic green on EMB media treated by a

25% waru leaves, 20% waru leaves, and 15% hibiscus

leaves. While the EMB medium was treated with 0%

waru leaves, 5% waru leaves, 0% waru leaves formed

Escherichia coli bacteria colonies which are dark

purple with a metallic green color. According to

Connie et al. (2015) on EMB media, Escherichia coli

bacteria can ferment lactose quickly and produce

acid. As a result of acidic conditions, eosin will

change color from clear to dark purple which is

usually accompanied by metallic green. This is

supported by the statement by Molita (2017) which

states that bacteria that ferment lactose from EMB

media produce colonies with dark cores with black

dots and a metallic green sheen. Escherichia coli

colonies that did not form a metallic green color on

EMB media were treated with the extract with more

mixture of leaves indicating that the lactose

fermentation process was not running optimally.

4 CONCLUSIONS

The Extract waru leaves had an antimicrobe potency

and can be categorized as an antimicrobial or a

bacteriostatic compound, it can be seen on the

resulting test using by GC-MS and inhibition zone

method. The GC-MS test result showed that the waru

had characteristic content from high to low rating, as

follows, Phytol (38,70%); Pentadecanoic acid,14-

methyl-,methyl ester (21,11%); 9,12,15-

Octadecatrienoic acid,methyl ester,(Z,Z,Z)- (8,24%);

8,11-Octadecadienoic acid,methyl ester (5,59%). The

highest proportion of hibiscus/waru leaves gave an

effect to the Escherichia coli total was highest too.

Thus, the results of the total microbial reduction test

were in line with the antimicrobial inhibition zone

test. The concentration level at 25% hibiscus leaves

treatment showed the highest total reduction of

Escherichia coli on chicken meat with a decreasing

percentage of 76.12% (inhibition zone method).

ACKNOWLEDGEMENTS

The authors would like to thank Kemenristek DIKTI-

BRIN.

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6th FiAC 2020 - The Food Ingredient Asia Conference (FiAC)