INHIBITORY GROWTH EFFECT OF PEDIOCOCCUS ACIDILACTICI AND

LEUCONOSTOC MESENTEROIDES ISOLATED FROM KIMCHI ON

STREPTOCOCCUS PYOGENES AND STREPTOCOCCUS PNEUMONIAE

BANGALANDO, MARYJUNE
CAHILO, AVE MARIE
KUSHIDA, CAROL DIAMOND
MALBUYO, DIVINE GLORY P.
PALENCIA, RHEA MAE
SIENES, HAZEL NOREEN

BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY

SILLIMAN UNIVERSITY
DUMAGUETE CITY

MARCH 2017

CHAPTER I

THE PROBLEM AND ITS SCOPE

Background and Rationale of the Study

Nowadays people are getting serious infections brought about by Streptococcus
Pneumoniae (S. pneumoniae) and Streptococcus pyogenes (S. pyogenes). S. pneumonia is a
gram- positive, capsulated bacteria which are found as normal flora in the upper respiratory tract
but it is also the common cause for Pneumonia and meningitis. S. pyogenes on the other hand, is
also found in the upper respiratory tract which commonly causes pharyngitis, tonsillitis,
rheumatic fever and even skin infections like Erysipelas. Normally S. pneumonia and S.
pyogenes could be inhibited by taking in 0.03g/ml or less of penicillin but it was seen that
isolates were developing biologic resistance thus requiring penicillin concentrations to increase
10 to 50 times ( Scott, 2007).
According to Alano, (2010), Philippines is listed as one of the top 10 countries with the
highest number of pneumonia cases and accounts 34% of deaths among children less than 5
years old. Mortality rate of 50 deaths per 100,000 and an incidence rate of 2,237 cases per
100,000. Majority of the victims are children who are below 5 years old and from poorer
communities. A study was conducted that aimed to determine the etiology of different serious
infections among Filipino infants age below 90 days and during the 2 year period, 2053 infants
were screened and 873 had signs and symptoms of the infectious illness. 81 infants died and

some of the organisms that caused the infections were recognized as S. pneumonia and S.
pyogenes (US National Library of Medicine National Institutes of Health, 1999).
Rheumatic fever is the leading cause of heart disease and in the Philippines it has accounted
54.9 % of children with cardiac disease and 46.6% among adults, resulting to an average
prevalence of 47.0%. Patients who are admitted to the hospitals due to heart disease accounts
13.8% of medical case and is steadily increasing year after year (Alimurung, 1962).
S. pneumoniae and S. pyogenes have been one of the causes of morbidity among Filipinos
and it have developed the ability to mutate and grow biologic resistance to antibiotics, thus
becoming a serious concern in the developing countries such as the Philippines. Fortunately, a
recent study proved that lactic acid extracted from kimchi has a strong anti-pathogenic activity
and results show that it has inhibited Clostridium perfringes after 2 days, Staphylococcus aureus
and Salmonella typhymurium disappeared after 4 days and E. coli has been inhibited after 5 days
(Rheem, et al., 2011).
According to Kwon, et al., (2001), Pediococcus acidilactici an active microorganism that is
isolated from kimchi has displayed anti-bacterial effect against most gram-positive and gramnegative bacteria. Leuconostoc mesenteroides which makes up majority of the lactic acid bacteria
during fermentation of kimchi has also some potential in inhibiting bacterial growth.
According to the study done by Bajpai, et al. (2016), the lactic acid bacterium L.
mesenteroides isolated from kimchi displayed significant antibacterial effects against foodborne
pathogenic bacteria.

With this antimicrobial potential found in the biological compounds and functional
components in lactic acid bacteria isolated from kimchi, the researchers want to find out if it can
inhibit the growth of S.pneuomoniae and S.pyogenes in order to aid in the development of
pharmaceutical and antimicrobial drugs against Streptococcal infections that have been a
growing health concern among Filipinos.
Statement of the Problem
This study will seek to explore and assess kimchi and its functional properties for
antimicrobial activity on pathogenic bacteria like S. pneumoniae and S. pyogenes. The study will
be guided by the following questions:
1. What are the factors considered during kimchi fermentation?
2. How to partially isolate and characterize the lactic acid bacteria isolated from kimchi
3.

used in the study with antimicrobial properties?

How will the considered factors during kimchi fermentation affects the increasing

growth pattern of lactic acid bacteria?

4. Which among the lactic acid bacteria isolated from kimchi, Pediococcus acidilactici and
Leuconostoc mesenteroides can successfully inhibit the pathogenic bacteria?
5. What will be the effect of Pediococcus acidilactici and Leuconostoc mesenteroides
isolated from homemade kimchi on S. pneumoniae and S. pyogenes at varying
concentrations?
6. What will be the effect of Pediococcus acidilactici and Leuconostoc mesenteroides
isolated from commercialized kimchi on S. pneumoniae and S. pyogenes at varying
concentrations?
7. At which concentration (50%, 75%, 100%) will the pathogenic bacteria begin to be
susceptible?
8. At what concentration level of the specific lactic acid bacteria isolated from kimchi will
the pathogenic bacteria begin to be killed?

9.

Which quality of kimchi exhibits greater inhibitory growth effect to S. pyogenes and S.
pneumoniae, the homemade kimchi or the commercialized kimchi?

Objectives of the Study

This study will focus on the assessment of the inhibitory growth effect of Pediococcus
acidilactici

and

Leuconostoc

mesenteroides

against

Streptococcus

pneumoniae

and

Streptococcus pyogenes. Thus, to attain these aims, the study will seek to:
1. Determine the factors considered during kimchi fermentation.
2. Partially isolate and characterize the lactic acid bacteria isolated from kimchi used in the
study with antimicrobial properties.
3. Determine how the considered factors during kimchi fermentation will affect the growth
pattern of lactic acid bacteria isolates.
4. Determine which among the lactic acid bacteria isolated from kimchi, Pediococcus
acidilactici and Leuconostoc mesenteroides, can successfully inhibit the pathogenic
bacteria.
5. Determine the effect of Pediococcus acidilactici and Leuconostoc mesenteroides isolated
from homemade kimchi on S. pneumoniae and S. pyogenes applied at varying
concentrations.
6. Determine the effect of Pediococcus acidilactici and Leuconostoc mesenteroides isolated
from commercialized kimchi on S. pneumoniae and S. pyogenes applied at varying
concentrations?
7. Determine the Minimum Inhibitory Concentration (MIC) of Pediococcus acidilactici and
Leuconostoc mesenteroides on the growth of Streptococcus pneumoniae and
8.

Streptococcus pyogenes.
Determine the Minimum Bactericidal Concentration (MBC) of Pediococcus acidilactici
and Leuconostoc mesenteroides on the growth of Streptococcus pneumoniae and
Streptococcus pyogenes.

9.

Compare the antimicrobial activity of lactic acid bacteria isolated from homemade
kimchi and lactic acid bacteria isolated from commercialized kimchi.

Significance of the Study

It is evident nowadays that the current health negative trends raises a growing health
concern and call for new strategies and prevention towards infectious diseases such as
developing antimicrobial in a natural approach since microorganisms recently have the ability to
resist antibiotics, develop immunity, and mutate to a new generation. With this, the researchers
come up with an idea of arriving at a solution that needs further studies like the antimicrobial
activity of kimchi. Kimchi was chosen for this study primarily because of its functional
properties responsible for health benefits (Heejae et al., 2010) and its lactic acid bacteria isolates
contributes to a wide range of inhibitory growth activity against Gram-positive and Gramnegative bacteria (Kwon et al., 2001).
If these lactic acid bacteria isolated from kimchi will be proven to have an inhibitory growth
effect on Streptococcus pyogenes and Streptococcus pneumoniae, then these will be potentially
be used in the food and feed industries as natural biopreservatives, as a starter culture in yogurt
fermentation and probiotic application to humans or livestock. This study will be conducted to
determine the efficacy and effectiveness of these lactic acid bacteria isolated from kimchi to
inhibit the growth of pathogenic bacteria that cause diseases among Filipinos.
The results obtained will be helpful in discerning what quality of kimchi exhibit greater
inhibitory growth effect on pathogenic bacteria and through this study, it will be established that
kimchi and its biological compounds and functional components has antimicrobial properties as
claim by previous studies. Knowledge of the biological compounds and functional components
contained in this fermented vegetable product will be expected to be useful in the development

of various industrial, pharmaceutical and antimicrobial drugs in treating Streptococcal infections

that has shown stagnation of prevalence rate in Philippines and in destroying other diseasecausing microorganism.
Moreover, this study will serve as a guide to future researchers who are interested in
expanding the health benefits and antimicrobial effects of lactic acid bacteria isolated from
kimchi on pathogenic bacteria. The concepts and methodologies encompass by this study will be
a theoretical foundation in building literature and in discovering new strategies for approaching
the same research proposals prior to existing research studies.

Scope and Limitations of the Study

Primarily, this study will focus to determine the inhibitory growth effect of Pediococcus
acidilactici and Leuconostoc mesenteroides isolated from kimchi on Streptococcus pyogenes and
Streptococcus pneumoniae using the Zone of Inhibition Test or the Kirby-Bauer Method. Before
isolating lactic acid bacteria from kimchi, there are some important factors during kimchi
fermentation that shall be achieved such as the salt concentration, temperature, inhibitory raw
materials and natural preservatives used to attain the taste and better quality of kimchi and avoid
fermentation defects like over-ripening or excessive acid production. Moreover, Pediococcus
acidilactici and Leuconostoc mesenteroides will be isolated from home-made and commercial
kimchi using MRS agar or nutrient agar for plating and will be morphologically and
biochemically identified by its characteristics, staining and biochemical procedure. This study
will compare the antimicrobial activity of homemade Kimchi versus commercialized Kimchi and
will determine its minimum inhibitory concentration at varying concentrations of secretions,
100%, 75% and 50% to Streptococcus pyogenes and Streptococcus pneumoniae.

Kimchi is a Korean traditional well-known side dish characterized as a lactic acid

fermented vegetable product based on napa cabbage/Chinese cabbage along with its various
ingredients and fermentation activity that is used as a good source of isolating potentially
beneficial lactic acid bacteria. Since there are many genus and species of lactic acid bacteria and
bacteriocin-producing strain that will be isolated from kimchi, this study will utilize the most
dominant and major microorganisms responsible for kimchi fermentation. Only Pediococcus
acidilactici and Leuconostoc mesenteroides and not its bacterial strain will be tested because of
their domination during the optimum- ripening stage of kimchi and it will be limited to these
lactic acid bacteria because of insufficient funds to finance for bacterial strains analysis from
isolates and time unavailability. Moreover, numerous research studies have proven that lactic
acid bacteria isolated from kimchi exhibited a wide range of inhibitory growth activity against
Gram positive bacteria like Staphylococcus aureus, Listeria monocytogenes, Streptococcus
agalactiae and Gram negative bacteria such as Pseudomonas aeruginosa, Salmonella
typhimurium and Escherichia coli. With this, the study will use pathogenic bacteria that have not
been studied specifically the bacteria that causes Streptococcal infections such as Streptococcus
pyogenes associated with rheumatic fever and Streptococcus pneumoniae associated with
bacterial pneumonia which can lead to possible health complications among Filipinos and it will
be limited to these pathogenic bacteria because of limited sources, insufficient funds and time
unavailability.

Operational Definition of Terms

The terms, concepts and keywords involved in the study will be define in this section to
promote better understanding and absolute clarity.

Antimicrobial Activity- It is defined as the ability of the functional properties of lactic

acid bacteria isolated from fermented kimchi such as Pediococcus acidilactici and
Leuconostoc mesenteroides to destroy or inhibit the growth of extracellular bacterial
pathogens like Streptococcus pyogenes and Streptococcus pneumoniae at varying
concentration as determined by the presence of zone of inhibition which serves as the
independent variable of the study. The increasing growth pattern of lactic acid bacteria
considering the important factors during kimchi fermentation will promote a significant

inhibitory growth effect on bacterial pathogens.

Factors of Kimchi Fermentation- is defined as the characteristics that will influence

the taste and quality of kimchi during its fermentation. The factors to be considered will
be the days it takes to ferment, temperature, salt concentration, inhibitory compounds

used in raw materials and pH.

Kimchi- The term refers to the Korean traditional well-known side dish characterized
as a lactic acid fermented vegetable product based on napa cabbage/Chinese cabbage
along with its various ingredients, and important factors of fermentation activity is used
as a good source of isolating potentially beneficial lactic acid bacteria like Pediococcus
acidilactici and Leuconostoc mesenteroides. Kimchi that will be use in this study refers to
homemade and that will be cook by the Kushida family at the fermented temperature of
5C- 15C after 6 to 30 days and it also refers to commercialized kimchi which will be
bought at Fish153 Korean grocery store which was fermented 2-3 days.

Lactic Acid Bacteria (LAB) - In the context of the study, it is defined as group of
important bacteria isolated from fermented Kimchi at varying concentration using MRS
agar or nutrient agar for plating which will serve as the dependent variable of the study.
The major genus and species of lactic acid bacteria that will be isolated are Pediococcus
acidilactici and Leuconostoc mesenteroides of which they will be morphologically and
biochemically identified by its characteristics using plate culture technique, staining
using Gram stain and biochemical procedures that is further subjected to inhibit the

growth of bacterial pathogens.

Minimum Inhibitory Concentration (MIC) - is defined as the lowest concentration of

lactic acid bacteria isolated from kimchi that prevents the growth of pathogenic bacteria.
It will be determined with the use of a two-fold serial dilution. It will be done by using 12
tubes with equal amounts of a sterile diluent such as Mueller-Hinton broth. 10 tubes will
be contained with broth and the lactic acid bacteria isolates at varying concentration of
50%, 75% and 100%. The two tubes left will serve as positive and negative control.
Moreover, the tube which obtained the most MIC will be subjected for Minimum

Bactericidal Concentration.
Minimum Bactericidal Concentration (MBC)- is defined as the lowest concentration
that results in microbial death in the Broth Dilution Method. It will be determined with
the use of series of steps undertaken after the Minimum Inhibitory Concentration test has
been completed. It will be done by streaking the tubes with the most MIC to the Mueller-

10

Hinton Agar (MHA) plate for MBC, then it will be incubated for 24 hours. After

incubation, the growth of colonies will be counted.

Pathogenic Bacteria- It is defined as group of bacteria that causes diseases once it has
gained access to the body and a factor that continue to raise health concerns among
Filipinos. The pathogenic bacteria that will be use in the study are Streptococcus
pyogenes and Streptococcus pneumoniae which is responsible for streptococcal infections

that can lead to possible health complications.

Zone of Inhibition- The test that will be use to measure the effectiveness of the lactic
acid bacteria isolates to inhibit microbial growth and is exhibited by the formation of a
clear circular space. The space created will be due to the application of Pediococcus
acidilactici and Leuconostoc mesenteroides on a plated medium surrounded with colonies
of Streptococcus pyogenes and Streptococcus pneumoniae.