antibacterial effect of lactic acid bacteria and lab extract on oral

APEC Youth Scientist Journal Vol.6 / No.2

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ANTIBACTERIAL EFFECT OF LACTIC ACID BACTERIA

AND LAB EXTRACT ON ORAL BACTERIA

∗ Na Hyun LEE

1

1Cheongshim International Academy, San 102, Songsan-ri, Seorak-myeon, Gapyeong-gun,

Gyeonggi-do, Korea

ABSTRACT

Dental cavities are one of the most prevalent health problems worldwide, that 60%

to 90 % of school children and nearly 100% of adults have dental cavities according to WHO.

A recent study suggests that dead lactic acid bacteria prevent dental cavities by decreasing

the number of plague bacteria. For further investigation, an experiment was conducted to

observe the antibiotic effect of various types of lactic acid bacteria against cavity-causing

oral bacteria. The result was applied to rabbit by feeding rabbit food covered with lactic acid

bacteria, which showed a decrease of number of plague bacteria in the mouth.

1. INTRODUCTION

Dental cavity is one of the most prevalent health problems worldwide, and is in fact

increasing due to unhealthy diet, tobacco use, harmful alcohol use and poor oral hygiene.

According to World Health Organization (WHO), 60% ~ 90 % of school children and nearly

100% of adults have dental cavities globally. In order to prevent dental cavities, constant

tooth care is needed to maintain low fluoride level in the oral cavity [1-4]. Yet, constant care

is hard, especially for the children to maintain oral sanitation and prevent cavities. In US,

over 19% of children aging between 2 to 19 have untreated cavities [2]. To help children to

prevent dental cavities, a new approach for effective prevention is needed.

∗ Correspondence to : Na Hyun LEE ([email protected])


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Many studies are done to find a new optimistic way of preventing dental cavities [5-

7]. Recently, suggestions have been made that dead lactic acid bacteria have an effect on

decreasing plague bacteria and preventing cavity. It is said that a certain type of lactic acid

bacteria called Lactobacillus has an effect of inhibiting the growth of oral mutans that causes

cavities in vitro. [8-9].

This study is conducted to observe the antibiotic effect of various types of lactic acid bacteria

against cavity-causing oral bacteria and to apply the result to daily life to decrease the high

cavity rate. Different types of oral bacteria and lactic acid bacteria were prepared and were

co-cultured to see antibiotic effect of lactic acid bacteria against oral bacteria. Then, the most

effective lactic acid bacteria was given to rabbit by feeding it with rabbit food covered with

the lactic acid bacteria to see the decrease of number of oral bacteria in the mouth.

2. MATERIALS AND METHODS

2.1.Preparing culture media

23g of NA(Nutrient Agar) powder, 8g of NB(Nutrient Broth) powder, 36g of

EMB(Eosin Methylene Blue) agar powder, 70g of MRS agar powder were each mixed with

1L of distilled water. All the mixtures were sterilized in 121℃, 1.2 atm, for 15 minutes in

autoclave. 20ml of NA, EMB agar, and MRS agar liquid were poured into each petri dish to

be cooled into agar medium. 40ml of NB and MRS broth were put into the test tubes

respectively and were stored in 4℃ for next experiment.

2.2.Cultivation of oral bacteria after fermented food consumption

2 pieces of Kimchi, a pack of cup yogurt, a cup of milk, and a piece of natural cheese

were selected for foods with lactic acid bacteria. And as control group, 2 pieces of Chinese

cabbage, a cup of apple juice, a pack of soybean milk, and 2 pieces of tofu were prepared.

Three women at the age of 14, 17, and 45 participated for the test. The food materials were

paired into four groups: Kimchi and Chinese cabbage, yogurt and apple juice, milk and

soybean milk, cheese and tofu. Each food was intake after an hour of duration after brushing

teeth. Then, oral bacteria were collected through process of rubbing teeth with cotton swab

and were smeared on NA plate. The plates were incubated in room temperature for 3 days.

The amount of bacterial colony grown was compared in the previous pairs.


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2.3.Cultivating lactic acid bacteria

Probiotics1, drinking yogurt

2, Activia cupped yogurt

3, and Korean traditional

fermented foods- Doenjang, Sikhae, and Chunggukjang- were prepared. 2g of probiotics

powder was mixed with 8ml of MRS broth, and 1ml of yogurt, Doenjang, Sikhae, and

Chunggukjang were mixed with 9ml of MRS broth. These were spread and streaked on NA

and MRS agar and were cultivated in temperature of 37℃. Figure 1 shows the cultivation of

lactic acid bacteria.

Figure 1: Cultivating Lactic Acid Bacteria

2.4.Cultivating oral bacteria

Oral bacteria were gathered from three sources: a person without cavity, a person

with cavity, and distribution from KTCT (Korean Collection for Type Cultures). Considerable

amount of saliva was collected from donors respectively. The saliva was mixed with 1ml of

NB and 5ul of the mixture was streaked on NA and MRS Agar. The mixture were cultivated

in temperature of 37℃. Streptococcus mutans (KCTC 5124) stock solution was streaked on

NA and MRS agar, and was inoculated in NB and MRS broth. They were incubated at 37℃.

2.5.Classification and isolation of bacteria

Bacteria colonies were classified according to the following categories: size, color,

opacity, form, elevation, and edge. One colony was collected and was mixed with 100ul of

MRS broth. 10ul of the mixture were spread and cultured on NA and MRS agar over three

times. DNA of lactic acid bacteria from probiotics and oral bacteria that grew well on both

1Chong Kun Dang, Korea 2오직우유100%를유산균으로발효한요구르트 (Only milk 100% lactic acid bacteria fermented yogurt), 파스퇴르

(Pasteur co. Korea) 3DanonePulmuone, Korea


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NA and MRS agar were analyzed through 16s rRNA method by Macrogen.

2.6.Co-cultivating lactic acid bacteria with oral bacteria

10ul of E. faecium stock solution mixed with each oral bacteria were spread on NA

and MRS agar and was cultivated in temperature of 37℃. E. faecium from different sources

and B. sonorensis were cultured in 40ml of MRS broth. They were sterilized at 121℃ and

1.2 atm, for 15 minutes in autoclave and were centrifuged in speed of 3000 rpm for 5 minutes.

10ml of collected supernatant were mixed with 10ul of S. mutans and were cultivated in 37℃.

The absorbance of the samples was measured with UV spectrophotometer of UV wave length

of 630nm. Then, the samples were observed through microscope with magnification of 100x

and 400x.

Figure 2: S.mutans Distributed from KCTC

2.7.Applying on Living Organism

E. faecium was grown in liquid media and was centrifuged in speed of 3000 rpm. The

supernatant was thrown away and the E. faecium were mixed with 5ml of water and was

mixed with rabbit food and was given to rabbit. Then with cotton swab, oral bacteria were

collected before rabbit ate the rabbit food mixed with E. faecium and after. The oral bacteria

were cultivated at 37℃ for a day. The process is shown in Figure 3.

Figure 3: Feeding Rabbit a Rabbit Food Mixed with Lactic Acid Bacteria


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3. RESULTS AND DISCUSSION

3.1.Observing Antibiotic Effect of Fermented Foods against Oral Bacteria

Kimchi, Chinese cabbage, yogurt, apple juice, milk, soybean milk, cheese and tofu

were eaten and oral bacteria were observed. Kimchi, a Korean traditional food, is a fermented

Chinese cabbage seasoned with red pepper powder, garlic, ginger, and salt. When Chinese

cabbage was eaten, great amount of oral bacterial colonies were observed, however, when

kimchi was eaten, the number of oral bacterial colonies found was lower than Chinese

cabbage. To confirm that lactic acid bacteria in kimchi decreased the number of oral bacteria,

dairy foods were compared with non-dairy foods. The result showed that, when milk, yogurt,

and cheese were eaten, fewer oral bacteria were found than when soybean milk, apple juice,

and tofu were eaten. The result shows that existence of lactic acid bacteria hinder the number

of oral bacteria as it is shown in Figure 4.

Figure 4: Comparing Growth of Oral Bacteria of Dairy Products and Non-Dairy Products

3.2.Culturing Lactic Acid Bacteria

Probiotics, Activia, Pasteur yogurt were cultivated in NA, MRS agar, and EMB agar

by spreading and streaking. Lactic acid bacteria that grow well in both NA and MRS agar

were needed. Although majority of lactic acid bacteria are gram positive, EMB agar media

were used to test if there is any gram negative bacteria. The result showed no lactic acid

bacteria grown on EMB agar as it is shown in Figure 5. In probiotics group, similar shaped

colonies were observed in both NA and MRS agar media. In Activia group, colonies were

found in NA only. In Pasteur group, colonies were appeared in both NA and MRS agar, but

the shapes were different.


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Figure 5: Lactic Acid Bacteria Grown in Different Culture Media

3.3.Classifying and Culturing Oral Bacteria

Oral bacteria were collected from 6 people; 2 people had carious tooth and 1 person

had periodontitis. Initially 7 kinds of oral bacteria colonies were classified, but after 3 times

of isolation culture, 8 kinds of oral bacteria were selected as Figure 6. The 8 kinds of bacteria

named A to H were cultured on NA and MRS agar media. Oral bacteria A, B, C, E, and F

grew on both NA and MRS agar media, and oral bacteria D, G, and H only grew on NA

media. Figure 7 shows the oral bacteria grown in MRS and NA media.

Figure 6: 6 Different Oral Bacteria Samples


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Figure 7: Oral Bacteria Grown in MRS and NA Media

S. Mutans from KCTC were cultured in NA, MRS agar, and EMB agar media. The

shape of colonies is shown in Figure 8.

Figure 8: Different Features of S. Mutans Depending on the Culture Media

3.4.Analyzing DNA of bacteria.

Lactic acid bacteria from probiotics and bacteria that grew well on both NA and MRS

agar were sent to Macrogen for their DNA analysis. The analysis report is as Table 1. Oral

bacteria A and B were unable to be analyzed because they were not completely isolated. DNA

of lactic acid bacteria from probiotics synchronized with DNA of E. faecium.

Table 1: Analysis Report

Name Read Length

(Normal)

Read Length

(Q16)

Read Length

(Q20) GC Content

seanggunjae_contig_1 1487 1401 1401 53.597848016139885

seanggunjae_F 908 889 888 53.85462555066079

seanggunjae_R 752 746 745 53.32446808510638


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Table 2: BlastN Report - Query name : seanggunjae_contig_1 - Query length : 1487

Query Subject Score Identities

Start End Description AC Length Start End Bit Raw EV Match Total Pct.(%) Strand

1 1487

Enterococcus

faecium strain

IDCC 2103 16S

ribosomal RNA

gene, partial

sequence

EU003447.1 1519 16 1502 2747 1487 0.0 1487 1487 100 Plus/Plus

1 1487

Enterococcus

faecalis gene for

16S rRNA, partial

sequence, strain:

JCM 20313

AB507170.1 1508 16 1502 2741 1484 0.0 1486 1487 99 Plus/Plus

1 1487

Enterococcus

faecium gene for

16S rRNA, partial

sequence, strain:

NRIC 0114

AB362603.1 1555 17 1503 2741 1484 0.0 1486 1487 99 Plus/Plus

1 1487

Enterococcus

faecalis gene for

16S rRNA, partial

sequence, train:

NRIC 0112

AB362601.1 1555 17 1503 2741 1484 0.0 1486 1487 99 Plus/Plus

1 1487

Enterococcus

faecium strain

HNN29 16S

ribosomal RNA

gene, complete

sequence

FJ378684.1 1519 16 1502 2736 1481 0.0 1485 1487 99 Plus/Plus

1 1487

Enterococcus

faecium strain SF

16S ribosomal RNA

gene, complete

sequence

AY675247.1 1553 16 1502 2736 1481 0.0 1485 1487 99 Plus/Plus

1 1487

Bacterium Te58R

16S ribosomal RNA

gene, partial

sequence

AY587827.1 1521 18 1503 2736 1481 0.0 1485 1487 99 Plus/Plus

1 1487

Enterococcus lactis

strain CK1025 16S

ribosomal RNA

gene, partial

sequence

AY683836.2 1526 11 1497 2736 1481 0.0 1485 1487 99 Plus/Plus

1 1487

Enterococcus lactis

strain CK1114 16S

ribosomal RNA

gene, partial

sequence

AY902459.2 1510 11 1497 2736 1481 0.0 1485 1487 99 Plus/Plus

6 1487

Enterococcus

faecium gene for

16S rRNA, partial

sequence, strain:

NBRC 100602

AB681208.1 1486 1 1482 2734 1480 0.0 1481 1482 99 Plus/Plus

3.5.Co-Cultivating Oral Bacteria and E.faecium

Oral bacteria and E. faecium were co-cultivated to see antibiotic effect of lactic acid

bacteria against oral bacteria. The oral bacteria were collected from human saliva mixed with

NB (Nutrient Broth), streaked on medium and cultivated. Total six types of bacteria were

identified, and each was co-cultivated with E. faecium. After a week, the growth of bacteria

was random, had no certain pattern. Therefore, it was concluded that living lactic acid

bacteria (E. faecium) do not have a clear antibiotic effect against oral bacteria.

3.6.Co-Cultivating Lactic Acid Bacteria with Oral Bacteria

Lactic acid bacteria from Doenjang, Sikhae, Chunggukjang, probiotics were prepared

to see antibiotic effect of dead lactic acid bacteria. A well-known cavity- causing bacteria, S.


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mutans, had been distributed by the KCTC. The lactic acid bacteria were mixed with media

and were sterilized. Then, it was cultivated with S. mutans. According to the result in Figure 9

and Figure 10, the number of S. mutans grown without sterilized lactic acid bacteria was

much higher than S. mutans grown with sterilized lactic acid bacteria. Among the 5 kinds, E.

faecium had the best antibiotic effect. Therefore, it was concluded that sterilized lactic acid

bacteria decreases number of cavity- causing oral bacteria, S. mutans, thus having possibility

of preventing cavity.

Figure 9 : Result of co-culture of Lactic Acid Bacteria and S.mutans; Yellow Arrow shows

microscopic form of S.mutans

Figure 10 : Light Absorbance after Cultivating S. Mutans

0

0.5

1

1.5

2

2.5

3

1st culture re-culture

abso

rban

ce

MRS control E.faecium M (Probiotics)

E.faecium M whole (Probiotics) E.faecium M (Chunggukjang)

E.faecium M (Sikhae) B.sonorensis M (Doenjang)


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3.7.Applying the results

In order to confirm whether lactic acid bacteria prevents cavity, lactic acid bacteria

were fed to a rabbit. Oral bacteria of rabbit were collected and were cultivated before and

after it ate lactic acid bacteria- covered rabbit food. As it is shown in Figure 11, the sample

collected after lactic acid bacteria were eaten showed more bacterial colonies than sample

collected before. However, majority of the bacterial colonies in the sample collected before

turned out to be E. faecium. Oral bacteria were found much less in the sample compared to

the sample collected before lactic acid bacteria were eaten. To conclude, lactic acid bacteria

do have an antibiotic effect against oral bacteria, which leads to prevention of oral plaque.

Figure 11: Oral Bacteria of Rabbit before and after Eating Lactic Acid Bacteria

Contained Food

4. CONCLUSION

Lactic acid bacteria do have an antibiotic effect against oral bacteria including

S.mutans. When oral bacteria were co-cultivated with living lactic acid bacteria, their number

changed in miniscule and random way. However, with dead lactic acid bacteria, the number

of S.mutans decreased harshly compared to the number of S.mutans grown in MRS control.

Therefore, dead lactic acid bacteria were observed to have an antibiotic effect against oral

bacteria which leads to prevention of cavity.

There are many other ways that lactic acid bacteria can be used to improve human

life. Further study is planned to investigate benefits of lactic acid bacteria other than

prevention of cavity.


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5. REFERENCES

[1] World Health Organization (WHO) (2012) Oral Health, Fact sheet N°318

[2] Centers for Disease Control and Prevention (CDC) (2011) Untreated Dental Caries

(Cavities) in Children Ages 2-19, United States, CDC Features

[3] WHO (2001) Water Sanitation and Health (WSH), Programmes and Projects

[4] Robert A. Bagramian et al. (2009) The global increase in dental caries. A pending

public health crisis, American Journal of Dentistry, 21(1)

[5] Shanmugam KT et al. (2013) Dental Caries Vaccine? – A Possible Option? , Journal

of Clinical and Diagnostic Research 7(6) 1250-1253

[6] Ji Hoe Kim et al. (2002) Antibacterial Activity of Sea-mustard, Laminaria japonica

Extracts on the Cariogenic Bacteria, Streptococcus mutans, J. Korean Fish. Soc.

35(2), 191-195

[7] Ok Mi Kim et al. (2003) Antibacterial Activity of Vinegars on Streptococcus mutans

Caused Dental Caries, Korean Journal of Food Preservation 10(4), 563-568

[8] Caterina Holz et al. (2013) Lactobacillus paracasei DSMZ16671 Reduces Mutans

Streptococci: A Short – Term Pilot Study, Probiotics & Antimicro. Prot. 5:259-263

[9] Pamela Hasslof et al. (2010) Growth inhibition of oral mutans streptococci and

candida by commercial probiotic lactobacilli – an in vitro study, BMC Oral Health

10:18

Na Hyun LEE

antibacterial effect of lactic acid bacteria and lab extract on oral

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