Storage Duration Effect of Kelor Leaf (Moringa oleifera) Extracts

with Methanol against Growth of Streptococcus agalactiae and

Escherichia coli Caused Mastitis in Dairy Cattle

Puguh Surjowardojo

, Rachmad Dharmawan

, Rifai

and Ike Ambarwati

Lecturer at Animal Science Faculty, Brawijaya University

Student at Animal Science Faculty, Brawijaya University

Keywords: Antimicrobial, Mastitis, Storage, Inhibiting Capability

Abstract: The research aimed to determine the storage duration effectiveness of Moringaoleiferaleaf extract to inhibit

the growth of Streptococcus agalactiaeand Escherichia coli causemastitis on dairy cows. The materials

were Streptococcus agalactiae and Escherichia coli from Bacteriology Laboratory Agriculture Faculty,

Brawijaya University counted as 108 CFU/ml and Moringa oleifera leaf. This research method was an

experiment Completely Randomized Design 5 treatments and 5 replications. Storage duration treatment was

P0 (control), P1 (2nd day), P2 (4th day), P3 (6th day), P4 (8th day) on the same concentrations 70%. The

variable measured was the diameter of the inhibition zone. The data analyzed using ANOVA followed by

the Duncan test. The results showed that Moringa oleifera leaf extract had difference highly significant

capability to inhibit the growth of Streptococcus agalactiae and Escherichia coli (P < 0.01). The Capability

of Moringa leaf extract to maintain bacterial growth inhibition until day 2 for Streptococcus agalactiae and

Escherichia coli. Maximum storage time until day 2 to maintain the effectiveness of Moringa leaf extract in

inhibiting the growth of Streptococcus agalactiae and Escherichia coli.

1 INTRODUCTION

Mastitis is an inflammation of the udder gland in

dairy cows. Mastitis is caused by injury to the nipple

or udder tissue so that it is infected by

microorganisms (Surjowardojo, et al. 2016), mastitis

can also be transmitted to other livestock.

Surjowardojo (2011) mastitis can reduce milk

production by 4.4 - 8.3 lt /day or 28.4% - 53.5% of

total production. The decrease in production is

directly proportional to the level of mastitis, so the

higher the rate of mastitis the greater the decrease in

milk production.Manifestations of mastitis infection

can be divided into two types namely clinical and

sub-clinical. Supar and Ariayanti (2008) subclinical

mastitis caused by pathogenic microorganisms

including Staphylococcus aureus, Streptococcus

agalactiae (Tuaskal, etal. 2012), Escherichia coli and

Corynebacterium bovis. Hameed and Korwin-

Kossakowska (2006) mastitis bacteria are dominated

by Staphylococcus aureus, Streptococcus

dysagalactiae, Streptococcus agalactiae and

Streptococcus uberisand Coliform bacteria

especially Escherichia coli (Supar and Ariayanti,

2008) and Klebsiella. It has an impact on reducing

production in large numbers while the treatment of

these infections is difficult to carry out to

completion and requires a large cost in its

operations.Surjowardojo, etc.(2016) teat dipping is a

method of preventing mastitis infection by dipping

the nipple in antiseptic post milking.Moringa

(Moringa oleifera) is a native plant of Indonesia that

can be used as medicine. Moringa oleifera leaves are

a natural ingredient of antibiotics because they have

active compounds, including flavonoids (Widjawati,

et al.) saponins, tannins, alkaloids, and terpenoids

(Yudistira, et al. 2013). Moyo (2012) Moringa

oleifera leaves have antimicrobial activity against

some Gram-negative bacteria including Escherichia

coli. Moringa oleifera leaves contain secondary

metabolites such as essential oils, polyphenols, and

saponins which have potential as antibacterial and

antifungal. Fuglie (2001) added that the saponin

content is 5%, tannins are 1.4% and triterpenoids are

5%. Tannins, polyphenols, and saponins have been

known to damage bacterial cells by inhibiting

152

Surjowardojo, P., Dharmawan, R., Rifai, . and Ambarwati, I.

Storage Duration Effect of Kelor Leaf (Moringa oleifera) Extracts with Methanol against Growth of Streptococcus agalactiae and Escherichia coli Caused Mastitis in Dairy Cattle.

DOI: 10.5220/0009587901520156

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 152-156

ISBN: 978-989-758-483-1

protein synthesis and damaging cell

membranes.Moringa oleifera leaf extract has

potential as an antimicrobial the bacteria

Streptococcus agalactiae and Escherichia coli, it is

necessary to investigate the effect of the storage time

of Moringa oleifera leaves extract with methanol as

a solvent.on the growth of Streptococcus agalaciae

and Escherichia coli bacteria causing mastitis in

dairy cows.2 Manuscript Preparation

We strongly encourage authors to use this document

for the preparation of the camera-ready. Please

follow the instructions closely in order to make the

volume look as uniform as possible (Moore and

Lopes, 1999).

2 MATERIAL AND METHODS

2.1 Material

The materials were Streptococcus agalactiae and

Escherichia coli which were produced by the

Bacteriology Laboratory majoring in Plant Pests and

Diseases, Faculty of Agriculture, Brawijaya

University. The instruments were Moring aoleifera

leaf extract, analytical scales (acc.0.1 mg), oven,

grinder, 1-liter Erlenmeyer, measuring cup, rotary

evaporator, funnel Buchner, vacuum pump, shaker

incubator, filter paper. The instruments of bacterial

inhibition tests were Petri dishes, test tubes, Spertus

/ Bunsen lamps, autoclaves, incubators, Erlenmeyer

flasks, 500 mL measuring cups, micropipettes,

tweezers, calipers, stirrers, magnetic stirrers, label

paper, tissue, plastic wrap, L glass sticks, aluminum

foil, Cork borer. Moringa oleifera leaf extract with

96% methanol solvent Moringa oleifera leaves are

obtained from Mr. Juma'il's garden in Panarukan,

Kepanjen, Malang. Mac Conkey Media Agar,

MRSA media, 96% p.a methanol, 70% alcohol, and

Moringa oleifera leave powder.

2.2 Method

This research method was completely randomized

design (CRD) with 5 treatments and 5 replications.

The treatment used is the storage time of

Moringaoleifera leaves methanol extract at a

concentration of 70%. With the following

conditions:P0 = Storage day 0, P1= Storage day 2,

P2= Storage day 4, P3= Storage day 6, P4= Storage

day 8.

2.3 Procedure

1. Making Simplisia Moringa oleifera Leaves

2. Moringa oleifera leaves Extraction

3. Making 70% Moringa oleifera leaves Extract

Solution Concentration according to Manu (2013).

4. Making Mac Conkay Agar (MCA) Media

anonymously (2011).

5. Making Media de Mann Rogosa Sharpe Agar

(MRSA) according to Anonymously (2011).

6. Inhibitory testing accordingly (Kasogi et al,

2014):

Warbung, et al (2014) the formula for calculating the

inhibition zone is as follows:

Note:

d1 = vertical diameter of the clear zone on the

media.

d2 = horizontal diameter of the clear zone on the

media.X = well hole (5 mm).

Susanto, etal. (2012) inhibitory zones can be

categorized as follows, for diameters> 20 mm are

categorized as highly strong, 11-20 mm are

categorized as strong, 6-10 mm are categorized as

moderate and <5 mm are categorized as weak.

2.4 Variable

The variables were diameter of inhibitory zone in

the form of clear area on the surface of the medium

between the extract of Moringaoleifera leaves and

Streptococcus agalactiae and Escherichia

colibacteria.

2.5 Data Analysis

The research method used was a completely

randomized design experimental method with 5

treatments and 5 replications. The results of the data

obtained were processed using ANOVA followed by

Duncan's Multiple Range Test is performed.

Storage Duration Effect of Kelor Leaf (Moringa oleifera) Extracts with Methanol against Growth of Streptococcus agalactiae and

Escherichia coli Caused Mastitis in Dairy Cattle

153

3 RESULTS AND DISCUSSION

3.1 Inhibition Zone of Streptococcus

agalactiae Bacterial

The results of the research of the effect of the

storage duration of Moringa oleifera leaves

methanol extract on the bacteria Streptococcus

agalactiae are shown in Table 1.

Table 1: Inhibitory zone diameter in Streptococcus

agalactiae bacteria.

Treatment Inhibition Zone Categories

T0 (Control) 22.53 ± 0.42

Highly strong

T1 (Days-2) 22.24 ± 0.29

Highly strong

T2 (Days-4) 21.62 ± 0.49

Highly strong

T3 (Days-6) 20.76 ± 0.25

Highly strong

T4 (Days-8) 20.34 ± 0.58

Highly strong

Note: Different superscripts in the same column show

significant between treatments (P <0.01).

Extract storage of days 0 days-2 showed that the

first and second mean values were not significantly

different, and were significantly different from the

4th, 6th and 8th-day treatments. Storage of methanol

extract of Moringa oleifera leaves is recommended

until the 2nd day (P1). Al-Zubaydiet al. (2009) that

flavonoids have broad antibacterial activity because

of their complex ability to extracellular and soluble

proteins as well as to precipitate proteins on the cell

walls of the bacterium Kiarostami et al, (2010). In

addition, these phenol compounds tend to form

hydrogen bonds with cell wall proteins so that they

can destroy cell membranes in bacteria. The graph of

the reduction in diameter of the inhibition zone of

methanol extract of Moringa oleifera leaves is

shown in Figure 1.

Figure 1: Inhibition zone of Streptococcus agalactiae.

Inhibition zone of Streptococcus agalactiae

bacteria during storage has decreased, especially

temperature factors. If the extract is stored at room

temperature, the extract will quickly evaporate and

cause the effectiveness of the extract to decrease

bacterial growth. This is in accordance with Siswadi

(2002) Decreasing the effectiveness of antimicrobial

compounds is influenced by many factors including

the type, age and state of microbes, concentration of

antimicrobial substances, temperature and contact

time, as well as the physicochemical properties of

the substrate such as pH, water content and surface

tension, number of components existing and other

factors. Klimczak (2006) andSuhartatiket al. (2012)

that the higher the storage temperature, the lower the

flavonoid and phenolic content of the extract. The

storage of plant extracts affects uterine activity and

depends on extraction temperature and storage

temperature.

3.2 Inhibition Zone of Escherichia coli

Bacterial

The results of the study of the effect of the storage

duration of Moringa oleifera leaves methanol extract

on Escherichia coli are shown in Table 2.

Table 2:Inhibitory zone diameter in Escherichia coli

bacteria.

Treatment Inhibition Zone Categories

T0 (Control) 22.04 ± 0.59d

Highly strong

T1 (Days-2) 21.50 ± .49cd

Highly strong

T2 (Days-4)

20.59 ±

0.74bc

Highly strong

T3 (Days-6) 19.59 ± 0.64b

Strong

T4 (Days-8) 18.13 ± 0.46a Strong

Note: Different superscripts in the same column show

significant between treatments (P <0.01).

Extract storage of days 0 days-2 shows the first

and second mean values were not significantly

different and were significantly different from the

treatment on days 4, 6 and 8. Storage of methanol

extract of Moringa oleifera leaves is recommended

until the 2nd day (P1). The inhibitory ability of

Moringa oleifera leaves methanol extract against

Escherichia coli is weaker than that of Streptococcus

agalactiae. The capability of methanol extract of

Moringa oleiferaleaves at a concentration of 70% is

quite strong. Given the cell wall of gram-

negativebacteria is more complex than the structure

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

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of gram-positive bacteria. This is consistent with the

explanation Suwito (2010), that gram-negative

bacteria have a cell wall that consists of three layers.

With a more complex structure of gram-negative

bacteria according to Harijani (2009), making

antibiotic compounds more difficult to enter the cell.

In addition to the effect of shelf life, the decrease in

inhibition is also due to other factors such as type,

age, antimicrobial concentration, microbial state,

and physicochemical properties such as pH, water

content (Siswadi, 2002). A graph of the decrease in

the inhibition zone diameter of methanol extract of

Moringa oleiferaleaves is shown in Figure 2.

Figure 2: Inhibition zone of Eschrichia coli.

Compounds contained in Moringa leaf extracts

such as flavonoids, saponins, and tannins have a

function in damaging cell walls in bacteria. This is

in accordance with the opinion of Khasanah, etc.

(2014) Cell walls are the main target of being

attacked by antibacterial substances contained in the

Methanol extract of Moringa leaves, making it easier

for tannins, saponins and flavonoids to enter the cell

membrane. Cell walls are not selectively permeable

so that these compounds are easily penetrated

through the cell wall which will cause disruption of

the integrity of the bacterial cell wall. The ability of

flavonoids as an antibacterial is able to stick to

bacterial cell walls and disrupt bacterial membranes,

so bacteria become lysis and die. The ability of

flavonoids to provide antibacterial effects includes

inhibiting the function of cytoplasmic membranes,

inhibiting nucleic acid synthesis, and inhibiting

antibacterial activity by inhibiting energy

metabolism, flavonoids inhibit oxygen consumption

by interfering with the electron transport chain

respiration. Permatasari, et al (2013) added that

Saponin is included in the antibacterial group which

interferes with the permeability of microbial cell

membranes. Causing damage to cell membranes and

causing the release of various important components

from the microbial cells, namely proteins, nucleic

acids, nucleotides, and others.

REFERENCES

Al-Zubaydi, Sami R., Al-Hmdany, Maetham A., And

Shyma'a J. Raesan. 2009. Antibacterial Effect Of

Some Medicinal Plant Extracts Against Some

Pathogenic Bacterial Strains. The 2nd Kurdistan

Conference on Biological Science.

Anonymous.2011.MicrobiologyLaboratoryEquipment.//al

atalatlaboratorium.com/LaboratoriumMikrobiologi/ma

c-conkey-agar.November 1, 2019.esJ.Duhok Univ.:

12, 244-249.

Fuglie, L. J. 2001. The Miracle Tree (The Multiple

Attributes of Moringa). Senegal: CWS Dakkar.

Hameed, S. and Korwin-Kossakowska. 2006. Public

Health Hazard Due To Mastitis In Dairy Cows.

Animal Science Pap Reports. 25(2):65-74.

Harijani N., M. Selomashar and B.C Tehupuring,. 2009.

The Effects of Bacteriocin Giving by

Pediococcuspentosaceus Lactic Acid Bacteria as

Nipples for Sub-Clinical Mastitis. Vol. 2(2): 257-268

Kasogi, I., Sarwiyono, and P Surjowardojo. 2013.

Methanol extract of cherry leaves (Muntingiacalabura

L) as a natural antimicrobial against Staphylococcus

aureus bacteria in dairy cows in the ngantang area,

Malang. Brawijaya University Malang

Khasanah, I., Sarwiyono and P. Suryowardojo. 2014.

Ethanol Extract of Kersen Leaves (Muntingiacalabura

L.) As Antibacterial Against Streptococcus Agalactiae

Causes Subclinical Mastitis in Dairy Cattle.Tropical

Animal Production. 15(2): 7-14.

Kiarostami, Kh.,Mohseni, R., Saboora, A. 2010.

Biochemical Changes of Rosmarinus Officinalis

Under Salt Stress. Journal ofStress Physiology &

Biochemistry.6: 114 122.

Klimczak, I., Maecka, M., Szlachta, M., Gliszczyn, A.

2006. Effect of Storage on The Content of

Polyphenols, Vitamin C and TheAntioxidant Activity

of Orange Juices. Elsevier. 20(4): 313-322.

Manu. 2013. Antibacterial Activity of Beluntas

(Plucheaindica L) Ethanol Extract Against

Staphylococcus aureus, bacillus subtilis and

pseudomonas aeruginosa. Surabaya university

scientific journal 2 (1): 1-10

Moyo 2012. Antimicrobial activities of Moringaoleifera

Lam leaf extracts. African Journal of Biotechnology

11 (11): 2797-2802.

Permatasari, G. A. A. A., Besung I. N. K. danMahatmi H.

2013. Inhibition Power of Soursop LeavesAgainst the

growth of Escherichia coli bacteria. Indonesia

MedicusVetreinus. 2(2): 162-169.

Siswadi, I. 2002. Study of Antimicrobial Activity of

Andaliman Fruit Extract (Zanthoxilumacanthopodium

D.C) Against Microbial Pathogens and Food

Destruction. [Thesis].Faculty of Agricultural

Technology. Bogor Agricultural Institute.

Storage Duration Effect of Kelor Leaf (Moringa oleifera) Extracts with Methanol against Growth of Streptococcus agalactiae and

Escherichia coli Caused Mastitis in Dairy Cattle

155

Suhartatik, N., Karyantina, M., Mustofa, A., Cahyanto, M.

N., Raharjo, S., and Rahayu, E. S. 2013. Stability of

Anthocyanin Extract of Glutinous Rice (Oryzasativa

var. Glutinosa) Black During the Process of Heating

and Storage. Agritech Journal, Vol. 33 (4): 384-390.

Supar and T. Ariyanti. 2008. Subclinical Mastitis Control

Study in Dairy Cattle. Indonesian Center for

Veterinary Research, Bogor.

Surjowardojo, P. 2011. Rate of Occurrence of Mastitis

with Whiteside Test and FriesienHolstein Dairy Cow

Milk Production.Tropical Animal Production. 12(1):

46-55

Surjowardojo, P. T. E. Susilorini, and V. Benarivo. 2016.

The Impact Of ManalagiApel Skin (Malus Sylvestris

Mill) On The Growth Of Escherichia Coli And

Streptococcus Agalactiae Causing Mastitical Causes

In Dairy Cow. 17(1): 11-21.

Susanto, D., Sudrajat and Ruga, R. 2012. Study of Active

Ingredients of MerantiMerah (ShorealeprosulaMiq) as

a Source of Antibacterial Compounds. 11 (2): 1-5.

Suwito and Widodo. 2010. Monitoring of Salmonella Sp

and Escherichia Coli in Animal Feed Materials

Salmonella sp and Escherichia coli monitoring in the

animal feed ingredients. Farm bulletin. 34 (3): 165-

168.

Tuaskal, B. J., S. Estuningsih, F. H., Pasaribu and I. W. T.

Wibawan. 2012. Orientation Dose of Streptococcus

agalactiae Irradiation for Sub Clinical Mastitis

Vaccine Material in Dairy Cattle. Scientific Journal of

Isotope and Radiation Applications 8 (2): 83-88

Warbung, Y. Y., Vonny, N. S. W., dan Jimmy. 2014.

Inhibition of Sea Sponge Extract. In Dairy

Cows.Repository.Faculty of Animal Husbandry

University of Brawijaya. Malang.

Yudistira, F. A., S. Murwani and P. Trisunuwati. 2013.

Antimicrobial Potential of Moringaoeifera Leaf Water

Extract Against Salmonella Enteritidis (SP-1-PKH) In

Vitro. Veterinary Medicine Program, University of

Brawijaya. Malang.

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