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APEC Youth Scientist Journal Vol.6 / No.2
- 206 -
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])
APEC Youth Scientist Journal Vol.6 / No.2
- 207 -
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.
APEC Youth Scientist Journal Vol.6 / No.2
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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
APEC Youth Scientist Journal Vol.6 / No.2
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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
APEC Youth Scientist Journal Vol.6 / No.2
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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.
APEC Youth Scientist Journal Vol.6 / No.2
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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
APEC Youth Scientist Journal Vol.6 / No.2
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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
APEC Youth Scientist Journal Vol.6 / No.2
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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.
APEC Youth Scientist Journal Vol.6 / No.2
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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)
APEC Youth Scientist Journal Vol.6 / No.2
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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.
APEC Youth Scientist Journal Vol.6 / No.2
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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