Isolation and Characterization of Lactic Acid Bacteria from Goat

Milk Kefir and Coconut Milk Kefir

Reno Permatasari Pasaribu, Emma Zaidar and Rumondang Bulan

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Kefir, Goat Milk, Coconut Milk, Antimicrobial Activity.

Abstract: Research about isolation and identification of LAB from goat milk kefir and coconut milk kefir has been

completed. The making of goat milk kefir was used 3% kefir granule (HK) and 5% kefir granule (KK) from

total volume of goat milk. As well as coconut milk kefir was used 3% kefir granule (HS) and 5% kefir

granule (KS) from total volume of coconut milk. The kefir beverage, conducted quality analysis with result

pH 4.2% – 4.6%, water content 89.5% (HK), 89.1% (KK), 79.2% (HS) and 78.9% (KS). Protein content

3.3% (HK), 3.1% (KK), 1.7% (HS) and 1.6% (KS), fat content 3.58% (HK), 15.2% (HS), 3.61% (KK), dan

15.8% (KS), ash content 0.79% (HK), 0.54% (HS), 0.81% (KK) and 0.9% (KS). Antibacterial test was

conducted by observing the clear zone for 48 hours. Using Escherichia coli and Staphylococcus aureus as

the test bacteria, resulted that sample HK showed the highest level of resistance compare to other samples.

Total plate count of LAB showed that sample KK has the highest number of LAB counts for 1.7 x 10

Morphology characterization of those colony are round shape, convex elevation, off-white and also gram

positive bacteria with six isolate rod shape cell, and two isolate coccus shape cell. The catalase and oxidase

test showed that all isolate are negative catalase and negative oxidase.

1 INTRODUCTION

Kefir is a symbiotic cultures from many

microorganism such as lactic acid bacteria and yeast

that taken shape of grains (Bensmira & Jiang, 2012).

“Milk” kefir have white/ yellowish in color

(Magalhães et al., 2011). In other hand “water” kefir

have color more brownish (Laureys & De, 2014)

.Both type of kefir has anti-inflammatory, anti-fungi,

immunomodulatory dan hipocholesterolemic

character (Hsieh et al., 2012).

Kefir grain that we used in this research is

obtained from kefir grain seller in Tanggerang,

Indonesia. Using this kefir grain, we can make milk

kefir and water kefir beverage. “milk” kefir is a kefir

beverage that’s made from mixture of kefir grains

and milk. While “water” kefir is a beverage that’s

made from mixture of kefir grain and other liquid

except milk, such as water, coconut milk, coconut

water, fruit juice, etc (Anonim, 2013).

The goat milk produced has superior nutritional

content, besides that the fat and protein in goat milk

is more easily digested and its vitamin B1 content is

higher than cow's milk. (Mustofa, 2009). Another

advantage of goat milk compared to cow's milk is

not causing allergic reaction compare to cow’s milk.

Therefore, goat milk can be chosen as an alternative

drink for people who are allergic to cow's milk.

While coconut milk is a food that has a high

content of water, protein and fat as well as cow's

milk. The advantage of coconut milk from cow's

milk is that it does not contain lactose as in cow's

milk so that coconut milk can be consumed by

sufferers of lactose intolerant. Protein in cow's milk

has the potential to cause a higher allergy than

protein in coconut milk. (Gea & Sebayang, 2016).

Lactic acid bacteria and their derivatives are able

to prevent the emergence of various diseases such as

preventing enteric pathogenic bacteria, reducing

cholesterol levels in the blood, preventing colon

cancer, anti-mutagen, anti-carcinogenic and

increasing endurance (Suryono, 2003).

Permatasari Pasaribu, R., Zaidar, E. and Bulan, R.

Isolation and Characterization of Lactic Acid Bacteria from Goat Milk Keﬁr and Coconut Milk Keﬁr.

DOI: 10.5220/0010133300002775

In Proceedings of the 1st International MIPAnet Conference on Science and Mathematics (IMC-SciMath 2019), pages 59-64

ISBN: 978-989-758-556-2

2 MATERIAL AND METHOD

2.1 Making Goat’s Milk Kefir

Fresh milk is pasteurized, which is heated at 60-65 °

C for 30 minutes, then cooled to room temperature

(± 28 ° C), pasteurized milk is put in 3% and 5%

grains of kefir and stirred evenly. Furthermore,

allowed / incubated for 20-24 hours (overnight) at

room temperature (25-37

C) so that the fermentation

process takes place. When the milk has coagulated,

strained the milk using a plastic strainer to get the

kefir grains back (Usmiati, 2007).

2.2 Making Coconut’s Milk Kefir

Grated old coconut to separate the coconut meat

from the coconut shell. Then add hot water (90-

C) to the shredded coconut meat with ratio of

water to coconut meat is 1.5: 1. The mixture of

coconut meat and hot water is allowed to stand for

one minute, then the mixture is blended for 5

minutes. After that, pour the mixture into a

cheesecloth and filter coconut meat from coconut

milk. Coconut milk that has been obtained is

pasteurized by heating coconut milk at 85-90

C for

15 seconds. After pasteurization, cool the coconut

milk to room temperature of 28

C, then add kefir

grains as much as 3% and 5% and stir evenly.

Furthermore, allowed / incubated for 20-24 hours

(overnight) at room temperature (25-37

C) so that

the fermentation process takes place. When the

mixture has coagulated, strained the coconut milk

using a plastic strainer to get the kefir grains back

(Anonim, 2013).

2.3 Quality Analysis of Probiotic

Beverages (Kefir)

Analyses of pH, water content, protein content, fat

content and ash content of each sample were

performed according to the standard method by SNI

01-2891-1992.

2.4 Antibacterial Activity Test of Goat

Milk Kefir and Voconut Milk Kefir

Mueller Hinton Agar was poured as much as 15 ml

into a petri dish, after it was left to solidify, streak

Escherichia Coli and Staphylococcus aureus

inoculums into different petri dishes. Inoculum

scraping is done continuously. Dip sterile paper

discs into each sample and inserted into a petri dish.

Incubated in an incubator at ± 35

C for 18-24 hours.

Furthermore, measured the diameter of the clear

zone around the paper disk with calipers.

2.5 Isolation of Lactic Acid Bacteria

from Goat Milk Kefir and Coconut

Milk Kefir

Total plate count of lactic acid bacteria

A total of 1 gram of kefir sample was diluted in 9

mL MRS Broth, vortexed until homogeneous. A

total of 0.1 mL of the dilution results was put into an

eppendorf tube containing 0.9 mL of MRS Broth

media. Make the dilution up to 10-6 dilution. At the

last dilution, 0.1 mL was taken and then sprayed

onto the MRS agar medium and flattened with a

hockey stick. Then put in anaerobic jar and

incubated for 48 hours at 37

C. Calculate the total

growing colonies using the CFU / g formula

(Purwati et al., 2005).

Isolation of lactic acid bacteria

Using a sterile spoon and curd aluminum foil

weighed as much as 1 g sample, then dissolved with

9 ml of MRS Broth solution in a test tube, then

vortex until homogeneous. This result is called

dilution 1, put into an anaerobic jar, then incubated

for 24 hours in an incubator at 37ºC. After 24 hours,

take 1 ml and put into a test tube containing 9 ml of

MRS Broth solution, then vortex until

homogeneous. Do this serial dilution until 7 times.

From the last dilution 100 µl sample is taken and

planted with the spread method on petridish which

contains MRS Agar media, then flattened with a

hockey stick that was previously sterilized with

alcohol and burned with Bunsen and then aerated.

The inoculum was stored in an anaerobic jar then

incubated in an incubator for 48 hours at 37

In order to purify the colony, using the streak

method, by using an ose needle take one colony and

then streak on to the new MRS Agar media

simultanously, and then incubated for 24 hours at

37ºC. (Purwati et al., 2005).

2.6 Identification of Lactic Acid

Bacteria from Goat Milk Kefir and

Coconut Milk Kefir

Macroscopic Identification

This identification activity was carried out on BAL

isolates which observe the shape, color, and edges,

lactic acid bacterial cells (Purwati et al., 2005).

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

Gram staining

Colonies that characterize LAB are stained with

gram as follows: 1) Bacteria and bacteria are taken

flattened on a glass object (preparation) that has

been cleaned with aquadest, 2) then dried on a

bunsen or dryer, 3) dripped with dye violet crystals,

4) then wait for 1 minute for the dye to seep in by

bacteria, 5) then rinse with running water and drop

with a complex iodine solution, then wait for 1

minute, then rinse with running water, 6) wash with

alcohol by means of dipping into dilute alcohol, 7)

drops with safranin dye, then waited 30 seconds, 8)

after it is dried and examined under a microscope

(Dwidjoseputro, 1989).

Catalase test

The procedure performed in this catalase test is first

to apply a small amount of bacterial isolate on a

glass object. Then add a few drops of H

, if

necessary stir using a toothpick. After that positive

catalase was observed by the presence of many air

bubbles and negative catalase was shown by the

absence of air bubbles.

Oxidase test

The procedure used in this oxidase test is to use a

sterile swab, a little isolate is taken from the agar

medium. Then one drop of reagent was added to the

isolates in the swab, after which it was observed that

a positive reaction was marked by changing the

bacterial isolate to a purple color.

3 RESULT AND DISCUSSION

3.1 Quality Analyses for Goat Milk

Kefir and Coconut Milk Kefir

From the producing goat milk kefir and coconut

milk kefir, the quality analyses can be seen on the

Table 1 and Table 2.

Table 1: Quality analyses for kefir goat milk.

Test Bacteria Sample Antimicrobial Index

24 hrs 48 hrs

E. coli HS 0.1 0.11

HK 0.37 0.37

KK 0.18 0.18

KS 0.06 0.06

S. aureus HS 0.35 0.35

HK 0.51 0.6

KK 0.6 0.63

KS 0.25 0.28

Table 2: Quality analyses of coconut milk kefir.

Sample pH Water

Content

Protein

Content

Fat

content

Ash

content

(3%

kefir

grain)

4.6 79.2 1.7 15.2 0.54

(5%

kefir

rain

)

4.3 78.9 1.6 15.8 0.59

From this table we able to see the pH of 3% kefir

grain is different with 5% kefir grains. The 3% kefir

grain is a little bit higher pH than the 5%, we believe

the differences between this pH is because the

fermentation process that takes place. The more

lactic acid that produce by lactic acid bacteria, the

acid it become, thus the 5% kefir grain have lower

pH.

The water and ash content of all samples shows

that the water and ash level does not cross the

existing standard of SNI 7552:2009. The protein

content shows that the more kefir grains we add it

tends to have lower protein content. (Handoyo &

Morita, 2006) stated that protein will be degrade or

hydrolyzed during the fermentation process.

The fat content of coconut milk kefir is

significantly higher than the goat milk kefir. The fat

content of coconut milk vary between 0.9 – 34.7 %

depends on the variety of the coconut and level of

ripeness. The more ripe the coconut, the more higher

the fat content (Gea & Sebayang, 2016). Therefore

after the fermentation process with kefir grains, the

fat content of coconut milk kefir will still be higher

than the goat milk kefir.

3.2 Antibacterial Activity Test of Goat

Milk Kefir and Coconut Milk Kefir

The results of bacterial growth inhibition test

indicator with disc diffusion method showed the four

samples demonstrate the ability to inhibit bacterial

growth indicator, characterized by the formation of a

clear zone around the paper discs with different

sizes.

Table 3: Antimicrobial index.

Sample pH Water

Content

Protein

Content

Fat

content

Ash

content

HK (3%

kefir

rain

)

4.5 79.2 3.3 3.58 0.79

KK (5%

kefir grain)

4.2 78.9 3.1 3.61 0.81

Isolation and Characterization of Lactic Acid Bacteria from Goat Milk Keﬁr and Coconut Milk Keﬁr

In this study two species of pathogenic bacteria

is Staphylococcus aureus and Escherichia coli

(collections of Microbiology Laboratory of the

University of North Sumatra) was used. Based on

this data we able to see that the clear zone on Gram

positive bacteria is more higher than Gram negative

bacteria.

This is because the difference between the

peptidoglycan walls between this bacteria. Gram

positive bacteria only have one peptidoglycan walls,

whereas Gram negative bacteria have outside cover

membrane that protect cytoplasm and peptidoglycan.

3.3 Total Plate Count of Lactic Acid

Bacteria

In this study, TPC of LAB colonies were calculated

in coconut milk kefir and goat milk kefir. It aims to

calculate the number of lactic acid bacteria colonies

that exist at each concentration. After lactic acid

bacteria were grown on MRS agar, the total colony

count was calculated using the CFU / mL formula.

Table 4: Total plate count of lactic acid bacteria.

Sample TPC

HS 5.2 x 10

HK 9.3 x 10

KK 1.7 x 10

KS 1.2 x10

3.4 Isolation of Lactic Acid Bacteria

from Goat Milk Kefir and Coconut

Milk Kefir

Isolation of lactic acid bacteria from the sample

begins by growing LAB on the selective medium,

namely de Mann Rogosa Sharpe (MRS) broth. MRS

broth is called a selective medium because it

contains optimum nutrients and pH for LAB growth.

LAB enrichment with MRS broth was continued

with multilevel dilution aimed at reducing the

number of LAB in the medium making it easier to

isolate bacteria. Serial dilution carried out until 10

-7

also using MRS broth medium. The results of the

dilution were planted into the MRS agar medium

and incubated for 48 hours. After incubation, LAB

colonies will appear on the MRS medium, the white

or yellowish white.

The LAB isolation results are then purified into

new media so that pure isolates are obtained.

Figure 1: The results of isolation of lactic acid bacteria.

3.5 Morphologycal Identification of

Lactic Acid Bacteria

Based on the identification of the shape of the LAB

colony, the appearance of the LAB colony on MRS

agar is circular, milky white that does not change the

color of the MRS agar (brownish yellow), with

smooth edges and convex elevation (Figure 1).

LAB colonies on MRS agar found in this study

are in accordance with research conducted by

(Komang, 2005) stating that morphological

characterization of LAB isolates based on color

indicates that the colonies are milky white with a

round shape.

Gram stain test

Gram stain testing on a single colony (LAB). Figure

2 shows the results of Gram staining of LAB

bacteria isolated from goat milk kefir and coconut

milk kefir with concentrations of 3 and 5%. From

these four samples, 8 isolates were taken. Of the

eight isolates available, there were six isolates in the

form of bacilli, and two isolates in the form of cocci,

and the eight isolates were gram-positive bacteria

that were purple.

Figure 2: Gram staining (gram positive) from LAB (a)

rod-shaped, and (b) coccus-shaped.

It can be seen the results of Gram stain tests and

morphological observations under microscope with

a magnification of 100x of 8 LAB isolates.

IMC-SciMath 2019 - The International MIPAnet Conference on Science and Mathematics (IMC-SciMath)

Table 5: Gram stain and morphological test results of LAB

isolates.

Isolates Gram Morphology

HK1 + Basil

HK2 + Kokus

KK1 + Basil

KK2 + Basil

HS1 + Basil

HS2 + Basil

KS1 + Kokus

KS2 + Basil

Based on the identification of the shape of the

LAB colony, the appearance of the LAB colony on

MRS agar is circular, milky white that does not

change the color of the MRS agar (brownish

yellow), with smooth edges and convex elevation

(Figure 1).

Catalase test

Catalase test was conducted to determine the

presence of the enzyme catalase in bacterial isolates

which were tested using hydrogen peroxide reagents

). Bacteria that have the catalase enzyme are

able to break down H

into H

O and O

. Based on

the test results (Figure 3) below shows that the eight

isolates showed negative results on the catalase test.

This is the evidenced by the absence of air

bubbles formed when the isolate was added to

hydrogen peroxide. This is in accordances with

Djide & Sartini (2008) whose suggested that the

results of biochemical tests in the form of catalase

tests on lactic acid bacteria showed negative results.

Figure 3: Catalase test on LAB isolates.

Oxidase test

In the oxidase test, all 8 isolates shows no sign of

color change. Therefore all 8 isolates are negative

oxidase. The result can be seen on figure 4.

Figure 4: Oxidase test on LAB isolates.

Based on this result, LAB cultures were not able to

carry out the oxidation Gaby Hadley reagent, to

yield the purple compound. This might be due to the

fact that the isolates didn’t possess the cytochrome c

(which means isolates might be anaerobic organism)

that enables them to use free oxygen in their energy

metabolism (Thakur et al., 2017).

4 CONCLUSIONS

The quality analyses of kefir goat milk and kefir

coconut milk shows that the quality meets the

standard requirements of SNI 7552: 2009.

Variations in the concentration of grains of kefir

against goat milk kefir and coconut milk kefir affect

the total lactic acid bacteria present antimicrobial

values. Where the highest type of kefir has a total

lactic acid bacteria is goat milk kefir with 5% kefir

grain concentration that is equal to 1.7 x 10

. Goat

milk kefir and coconut milk kefir can be used as

antibacterial because it can be seen in kefir with

variations in the concentration of kefir grains

affecting antimicrobial values.

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