Effect of Electromagnetic Radiations on Lactobacillus species

(Dissertation II)
Submitted to:
LOVELY PROFESSIONAL UNIVERSITY
(Department of Biotechnology)
For partial fulfilment of Masters of Technology (Biotechnology)
(2012-2014)

Submitted By:
Shreyas K. Dhuliya
Registration No.11203491

Under the guidance of:

Dr. Akshay Garg
(Assistant Professor)
Department of Biotechnology and Biosciences
LOVELY PROFESSIONAL UNIVERSITY
November 2014

1
2
 Abstract

Effect of different types of radiation on different life forms has been studied extensively. In
present study the Lactic acid bacteria which is also part of normal human flora and also of
economic significance in industries was used. Radiation of 6.41 & 7.43 GHz used for the
study were given by Klystron test bench (J band). After radiation of the broth containing
irradiated bacteria, OD at 600 nm were taken at interval of 30 minutes. Assay of lactic acid
was also performed on both the control and irradiated sample at 6.41 GHz &. Direct radiation
to MRS agar plates spread with sample was given followed by Gram staining to visualise any
changes in cell morphology at microscopic level. Result suggest that there was slight
decrease in cell no, colony no and kinetics of cell growth followed by radiation. The % of
lactic acid was also affected at minute level.

3
 ACKNOWLEDGEMENT

Apart from the efforts by me, the success of this project depends largely on the encouragement and
the guidance of others. I take this opportunity to extend my esteemed regards to those people who
have been instrumented in the successful completion of my project.
A formal statement of acknowledgement is hardly sufficient to express my regards to personalities
who have rendered their valuable support, help and guidance to meet this end. To begin with I offer
my thanks to the almighty and my beloved parents who have always encouraged and support me.
First of all a special thanks to my university Lovely Professional University , Punjab for giving me
the opportunity to carry out this project as an integral part of our M.Tech Biotechnology.
I am very thankful for inspiration from my guide Dr. Akshay GargI and entirely indebted to Dr
Leena Parihar (Supervisor) for his valuable and constant assistance, cooperation, guidance,
listening to our queries and correcting the various documents of me with attention and care during
the entire period of my work. It is due to his immense knowledge the expertise that has encouraged
me to start with a challenging and prestigious project.
I would also like to thank for support of M.Sc student Nazaruddin and M.Tech student Saquib Khan
for his support in carrying out work in the laboratory.
I feel privileged to offer my sincere thanks to Dr. Neeta Raj Sharma (HOS) and Mr. Himanshu
Singh (HOD) for providing a wonderful opportunity that has brought a revolutionary change in my
life.
With all these people it would have possible for us to successfully complete our project.

Shreyas Dhuliya

4
 DECLARATION

I hereby declare that the project work entitled Effect of Electromagnetic Radiation on
Lactobacillus species is an authentic record of my own work carried out at Lovely
Professional University, Phagwara as requirements of dissertation project for the award of
degree of M. Tech. in Biotechnology at Lovely Professional University, Phagwara under
the guidance of Dr. Akshay Garg, Assistant Professor, L.P.U., Phagwara during August,
2014 to November, 2014. Taking all the responsibility regarding anti-plagiarism drive by our
institute, I hereby declare that no previously published document has resemblance with my
work to be identified as under act of plagiarism. All the writing and work in this thesis is
mine. Wherever I have borrowed material from other sources, I have diligently acknowledged
the source of the borrowed material.

(Shreyas Dhuliya)

Date: November, 2014 Reg. no: 11203491

5
 CERTIFICATE

This is to certify that Shreyas Dhuliya has partially completed M.Tech dissertation titled
Effect of Radiation on Lactobacillus species under my guidance and supervision. To the best
of my knowledge, the present work is the result of his original investigation and study. No part
of the dissertation has ever been submitted for any other degree or diploma.
The dissertation is fit for the submission and the fulfilment of the conditions for the award of
M. Tech. Biotechnology.

Date: ______________ Signature of Advisor

Name:

6
 Table of content

Sr No. Chapter Page No.

1 List of Tables 5

2 List of Figures 6

3 Introduction 7

4 Review of Literature 10

5 Rationale or Scope of study 14

6 Objective of the study 15

7 Materials 16

8 Research Methodology 17

9 Results and Discussion 19

10 Conclusion and future scope 22

11 References 23

12 Appendix I 25

7
 List of Tables

Sr No. Title of the Table Page No.

1. Table 7.1. Indicated overall OD Values 19
for Radiation experiments conducted
on Lactobacillus fermentum.

2. Table 7.2. Total Titrable Acid (% Lactic 20

Acid)

8
 List of Figures

Sr No. Figure title Page no.

1 Figure 1.1. Schematic representation of electromagnetic 7
wave

2 Figure 1.3. (a). Lactobacillus fermentum colony after 16

streaking

3 Figure 1.3. (b). Lactobacillus fermentum colony 40X 17

4 Figure 6.1. (a) radiation on flask containing broth culture 17

5 Figure 6.1. (b) Radiation to petriplate. 17

6 Figure 5.1. (a) Klystron microwave test bench 16

7 Figure 5.1. (b) Spectrophotometer 16

8 Figure 7.1: A graphical representation of all values given 19

in table 7.1

10 Figure 7.2. (a) control sample cells without radiation. 20

11 Figure 7.2. (b) Radiation period 10min at 6.41 GHz 20

12 Figure 7.2. (c) Radiation period 10min at 6.41 GHz 20
13 Figure 7.2: (d) Radiation period 5 min at 7.43 GHz 21
14 Figure 7.2. (e) Radiation period 10min at 7.43 GHz 21

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 1. Introduction

There are number of evidences that suggest that there is direct or indirect effect of
radiation on human health and other forms of life as well. Among type of radiation the
most common is non-ionising radiation, as it plays a major role in data transmission and
communication technologies like mobile phone, Wi-Fi, blue tooth and satellite phones.
We are constantly living in matrix of this radiation. The focus of the present study is to
check the effect of non-ionising radiation specifically radio waves of frequency (6.41 &
7.43GHz) on a strain of Lactobacillus fermentum which is a probiotic bacteria and
found in human and animal normal flora. The bacterium is of significance in medicine,
industry and research.

1.1. Electromagnetic radiation

Figure 1.1. Schematic representation of electromagnetic wave

[Photo taken from: http://www.nd-ted.org/EducationResources/CommunityCollege/ RadiationSafety/ theory/nature.htm]

The electromagnetic waves increasing in frequency from Extremely Low Frequency

and Very Low Frequency (ELF/VLF), through Radio Frequency (RF) and Microwaves,
to Infrared (IR) light, Visible Light, Ultraviolet (UV) light, X-rays, and Gammarays
togather form and array i.e called electromagnetic spectrum. (Ali Zamanian and Cy
Hardiman 2005).It has one electrical component and one magnatic component and like
10
the light it is of dual nature wave and particle.

1.2. Probiotic

The living microorganism which when administered in adequate amount, confer health
benefits to host. They are living bacteria with health promoting activities. They balance
and stabilise the immune system and relive the symptoms of various diseases. They aslo
prevent pathogen colonisation of gut. Both E. et al., (2010). There are so many more
application and benefits of probiotics and their mechanism of action in each case is yet
to be studied further. For example they up regulate certain genes e.g. hBD-2 via
induction of pro inflamatory pathways which results in antimicrobial peptide defencing
secretion. M. Schlee et al., (2008).

1.3. Lactobacillus fermentum

It is a probiotic bacteria, gram positive, non-motile, catalase negative, non-spore

forming and rod shaped. When observed in microscope figure1.3. (b) . Colony
appearance is glistering white on MRS Agar plate shown Figure 1.3. (a), observed cell
at 40X resolution in microscope. They ferment hexose sugars to produce primarily
lactic acid.

Figure 1.3(a). L. fermentum colony after Figure 1.3. (b) gram stain L.
streaking. fermentum 40X

11
1.4. Cell growth studies:

Microbial growth in a culture follows particular pattern of Lag phase, Log Phase,
Stationary phase and death phase depending upon the species and the type of media
used. The relation between specific growth rate and substrate concentration can be
established during log phase of growth. It is one of the most important tool for
microbiology, biotechnology and other branches of biology.

12
 2. Review of Literature

2.1. Effects of radiation on microbes

Woo et al., (2000) studied the damage of bacterial cell walls (Escherial coli and Bacilus
subtilis). It results in increase in DNA and protein amount and reduction in viable count
number based on final temperature of final suspensions. But there was no change in the
cell density. To investigate the effect of microwave heating on intracellular components,
cells that were microwave heated upto 70C were examined using transmission electron
microscope. Both type of bacteria show dark spot in their cytoplasm. However dark spot
were absent in the untreated cells, suggesting that dark spots were the result of
microwave heating. Moreover sodium dodecyl sulphate (SDS) easily lysed the
microwave injured E.coli cells, but causing no damage to B. subtilis.

Cabuk et al., (2008) exposed the suspension of Escherichia coli ATCC 25922, Bacillus
cereus NRRL 3711 and Staphylococcus aureus ATCC 25923 to microwave radiation.
The pure bacterial strains were incubated at 37C for 24 hr in the nutrient broth at
neutral pH. The effect of microwave radiation was studied at different times, different
initial bacterial concentrations and different power. Maximum efficiency of microwave
was observed at an exposure time of 60 sec, 1.0 108 initial bacterial cells and a power
of 900W for E.coli cells. E.coli was found to be most sensitive bacteria to microwave
radiation. Microwaves produced lethal effect on the examined bacteria by heat
generated during microwave exposure.

Ahmed et al., (2013) studied the effect of gamma radiation on bacterial microflora
associated with human amniotic membrane. This membrane is used for the treatment of
damaged ocular tissues and other skin injuries. They collected 25 amniotic sacs to
determine the quality of human amniotic membrane and to detect the radiation decimal
detection dose (D10 values). Cultural, Morphological and biochemical characteristics
were tested for radiation sensitivity in an increasing dose of radiation from 1 to 10 kG y.
There occurs a decline in bio burden with increase in the radiation dose. Staphylococcus
spp. was found to be most frequently isolated bacterial contaminant in tissue samples.
Streptococcus spp. was found to have a bio burden level of 1000 and was resistant to
13
radiation dose. The study proved that lower level of radiation dose is suitable for the
sterility of allograft and a dose of 25 KGy is essential for the sterilisation of allograft.
Thereby this study was important for the processing of amniotic membranes.

2.2. Importance of Lactobacillus fermentum

Mikelsaar M. and Zilmer M., (2009) studied the characteristics of probiotic

Lactobacillus fermentum strain ME-3DSM-14241 based on regulation of WHO. This
strain has effective anti-oxidative properties. They worked on the identification and
molecular typing of this strain and checked its safety assessment by testing on animals
and volunteers. They found out that this strain has high antimicrobial and anti-oxidative
properties against intestinal pathogens. Eradication of salmonellas and the reduction of
liver and spleen granulomas in Salmonella typhimurium infected mice treated with the
combination of ofloxacin and Lactobacillus fermentum strain ME-3 proved the efficacy
of antimicrobial and antioxidative probiotic. This strain was found to have some health
promoting effects also when used in foodstuffs. This probiotic improved the
antioxidative activity of sera and the composition of low density lipid particles (LDL)
and post prandial lipids.

Abedin et al., (2013) tested the anti-fungal activity of Lactobacillus fermentum against
Aspergillus niger, Aspergillus flavus and Candida albicans. The antifungal activity was
tested by using dry weight, disc diffusion and micro broth dilution methods. They found
that growth of A.niger and A. flavus was inhibited by 31 g lysate protein/disc but C.
Albicans was inhibited at 62 g lysate protein/disc. The highest yield of 10 g dry
biomass/L of Lactobacillus fermentum was found at a pH of 6.8 in a medium of wheat
bran, corm steep liquer and yeast extract. Upto 150 g lysate/ml showed no hemolysis.
The result showed that A. flavus and C. albicans were more susceptible than Aspergilus
niger in terms of lethal effect of lysate. They obtained 5 protein peaks during
fractifaction of Lactobacillus fermentum.

Heinemann et al., (2006) worked on the purification and characterisation of surface

binding protein from Lactobacillus fermentum RC-14 that inhibit adhesion of
Enterococcus faecalis 1131. The protein with high anti adhesive properties against

14
Enterococcus faecalis was purified using a quantitative method. By using two step
method (Size Exclusion Chromatography and Ion Exchange Chromatography) , an
anti-adhesion component was purified. The N-terminal sequence of 29-kDa protein
was identical to that of collagen binding protein from L. reuteri NCIB11951 and also
exhibit close relation with a basic surface protein from Lactobacillus fermentum
BR11. They conclude that this anti-adhesive property can be used as protection against
uropathogens.

2.4. Research investigations

2.4.1. Cell growth studies

Okpokwasili and Nweke et al., (2005), studied the microbial growth and utilisation of
environmental contaminants as substrates. This paper examined the microbial
utilisation of, and growth on, organic chemicals. Many methods including oxidation,
Precipitation , Ion Exchange etc. are used to remove the organic and inorganic matter
from aquous solution. It also examine the various kinetic model applied in the
prediction of microbial removal of organic contaminants. Many of the microbial
growth and biodegradation kinetic Model has been developed and processed which
include Andrews, Monods, Bungays weighted model, general substrate inhibition
model (GSIM) and sum kinetic model. However there is a need to extend such studies
to pilot scale applications. They conclude that in order to manage the industrial
effluents these processes should be modelled in an appropriate way.

Ammerman et al., (1984) checked the ability of marine bacteria to grow in particle
free un-enriched seawater and to study their growth. The inoculum was prepared by
filtering sea water through a 0.6 m Nuclepore filter. Growth on dissolved organic
matter was demonstrated by parallel increase in cell number, ATP and bio volume.
Doubling time of sea water was found to be comparable with bacterial assembly in
natural environment. They found out that for at least some of the bacteria, the growth
condition were similar to those of sea and there occur the synthesis of new biomass
from un-supplemented seawater which is confirmed by the increase in cell bio volume.
During the log phase the average cell number becomes more than double and then
returned to near initial levels. Hence sea water cultures were found to be suitable to
study the growth parameters because of similar growth conditions to that of free living
bacteria in sea water.
15
Liu et al., (2013) studied the growth kinetics of Cancer Stem Cells Populations in both
in vivo and in vitro by using mathematical models. The population of cancer stem cells
vary in different cells and tissues and is associated with breast cancer. After studying
the mathematical models they conclude that both division rates and self-renewal
probabilities need a negative feedback mechanism to maintain the balance between
Cancer Stem cells and non-stem cancer cells. Over expression of HER2 promotes
either symmetric division of increase proliferation rate of CSCs which cause the
expansion of cancer stem cells (CSC). Their model study suggested that CSC targeted
therapy are less effective in shrinkage of tumour size but are more effective in the long
term suppression of tumour growth there by predicting the efficacy of anticancer
therapy.

2.4.2. % Lactic acid

Total titrable acidity of fermentation broth is used as one of the parameter to check the
effect in various studies recent and earlier.

A study conducted by Vamanu E. et al.,(2009) to check alternative mediat to MRS used

TTA as one of the measure.

An another study by Vamanu A. et al.,(2008) for development of symbiotic product

based on honey, pollen and lactic acid bacteria used TTA as measure which is
expressed as % lactic acid.

Mousani et al., (2010) used TTA as one of the parameter in their study of fermentation
of pomegranate by probiotic lactobacillus bacteria.

16
 3. Rationale and Scope of The Study

1. What could be possible negative or positive effect on growth, the positive effect can
be of importance from industrial point of view.

2. To check if any changes in capacity of the bacteria to produce lactic acid.

3. Observe any morphological changes both macroscopic and microscopic with

radiation frequency and time variation.

17
 4. Objectives of the Study

1. To check the effect of radiation on growth of Lactobacillus fermentum.

2. To check the effect of radiation on capacity to produce Lactic acid.

3. To observe the morphological changes in individual cell and colony after radiation.

18
 5. Materials

5.1. Media

MRS Agar

MRS broth

5.2. Reagents/ chemicals

0.1 M NaOH

Phenoptheline indicator

5.3. Glasswares

Petridishes

Testtubes

Conical flasks

Burette

5.4. Instruments

Radiation apparatus klystron microwave test bench (j-band)

Spectrophotometer

Figure 5.1(a) Klystron microwave test bench (b) Spectrophotometer

19
 6. Research Methodology

6.1. Bacteria Preparation:

Cultures for Lactobacillus fermentum 9748 were obtain from MTCC. Cultures were
stored at -200 C for in MRS broth containing 10% glycerol. Experiments were carried
out at 370 C temperatures and growth period for bacteria was 24 hours prescribed.
Microscopic observation after gram-staining generated violet coloured rod shaped gram
positive bacteria.

6.2. Radiation by klystron test bench (J-band):

Radiation to flask containing MRS broth and Lactobacillus fermentum was provided by
keeping flask under machine for desired time period. A cap of other bottle was used to
slightly tilt the flask. Radiation to MRS agar plates spread with Lactobacillus
fermentum was given directly by keeping it close to radiation apparatus.

Figure 6.1(a) radiation on flask containing (b) Radiation to petriplate.

broth culture

6.3. Study of cell growth

The 4 conical flasks each containing 50 ml fresh MRS media, were inoculated with 2ml

20
of fresh culture prepared from microbial colony the previous day. One for control and
others were prone to radiation at 3 min, 5 min 10 min respectively. OD was taken at
600nm at interval of 30 minutes.

Five plates containing MRS agar were spred with 10l of sample and prone to 6.41 GHz
& 7.43 GHz radiation for different time interval of 0 minute, 3 minute, 5 minute and 10
minute. After 24 hours of incubation period at 37 the samples were taken out and
observed for macroscopic changes in colony characteristics and microscopic changes in
cell morphology with gram staining.

6.4. Total Titrable Acidity expressed in % Lactic acid

The broth culture of Lactobacillus fermentum carrying each 2 ml of culture in 3

different conical flask containing 50 ml of fresh MRS broth were kept in rotating
incubator for 24 hours to produce lactic acid. One for control i.e. without radiation, one
which was prone to radiation for 5 minutes and the third was kept in radiation for 10
minutes. The assay of lactic acid was performed on each of them after 24 hours and 3
titration readings were taken. The fact that 0.1N of 1ml HCl corresponds to 0.009008g
of lactic acid taken in to account. (Vamanu et al., 2009)

Titration Reading * Molarity of 0.1M NaOH * 0.009008 * 100

----------------------------------------------------------------------------
0.1 X Taken sample weight

21
 7. Results and Discussion

7.1. Cell growth

S. Time Control 6.41GHz 6.41GHz 7.43GHz 7.43GHz
No [min' Sample for 5 for 10 for 5 for 10
s] O.D mins O.D mins O.D mins O.D mins O.D
values values at values at values at values at
at 600nm 600nm 600nm 600nm
600nm
1 0 0.013 0.013 0.013 0.013 0.013
2 30 0.014 0.013 0.014 0.012 0.011
3 60 0.032 0.027 0.028 0.027 0.026
4 90 0.080 0.078 0.073 0.059 0.039
5 150 0.142 0.132 0.118 0.097 0.069
6 180 0.196 0.183 0.157 0.133 0.102
7 210 0.252 0.220 0.196 0.174 0.138
8 240 0.286 0.264 0.225 0.200 0.176
9 270 0.328 0.296 0.265 0.234 0.199
Table7.1. Indicated overall OD Values for Radiation experiments conducted on
Lactobacillus fermentum.

Figure 7.1. A graphical representation of all values given in table 7.1

Growth of Lactic acid bacteria was affected by EMR. With increase in time the growth
in all samples of bacteria was found lesser in comparison to control. With increase in
frequency and time of exposure gradual lesser growth was recorded in Lactobacillus
fermentum.

22
7.2. % Lactic Acid

Sr. No Frequency control Radiation Period

5 min 10 min
1. 6.41GHz 6.30% 5.40% 5.40%
2. 7.43 GHz 6.30% 5.40% 5.40%
Table 7.2: Total Titrable Acid (% Lactic Acid)

The % Lactic acid was determined after 24 hour to be 6.30% in control. The entire
radiation sample showed decreased in % lactic acid assay without variation of time and
frequency.

7.3. Colony Morphology

With increase in time duration the colony number and morphology of colony changed.
Control showed much growth and with the passage of time at same frequency the size
of colony reduced and growth also reduced.

7.4. Cell morphology

Figure 7.2. (a) Control sample cells without radiation.

Figure 7.2. (b) Radiation period 5 min (c) Radiation period 10 min at
at 6.41GHz 6.41GHz

23
 Figure 7.2. (d) Radiation period 5 min (e) Radiation period 10min
at 7.43GHz at 7.43 GHz

The microscopic photograph taken after the radiation indicates the change in cell shapes
which are primarily of rod shaped changes its shape to oval.

24
 8. Conclusion and Future Scope

Results are revealing less effects on Electromagnetic Radiation on lactobacillus fermentum

on growth, microscopic and macroscopic changes in morphology and lactic acid production
capacity.

%Lactic acid was determined and it is found that not much effect with variation in time and
frequency. No variation was recorded in bacterial cells exposed. % Lactic acid found to be
reduced in comparison to control in all the cases.

Colony morphology and microscopic structure of bacteria was also changed in comarison to
control. All the radiated samples were showing very small colonies with less than 1mm
diameter. Specifically microscopic structure of bacteria radiated for 10 min in 7.43 Ghz was
showing enlarged bacterial cells may be due to building of cell wall. Cells were appearing
oval in shape around double in comparison to control sample.

The probiotic activity of the bacteria of acid tolerance, bile salt tolerance and ability to
remove cholesterol can be studied after radiation and compared with control. A.Al-saleh et
al., (2006)

Antagonistic growth properties of Lactobacillus fermentum could also be altered as it has

inhibitory effect on periodontal pathogens including Strptococcus mutans, Porphyromanas
gingivalis. Chen L. et al., (2012)

25
 9. List of References/Bibliography

1. A. Al-Saleh; A. A. M. Metwalli and H. M. Abu-Tarboush (2006) Bile Salts and

Acid Tolerance and Cholesterol Removal from Media by some Lactic Acid Bacteria
and Bifidobacteria. J. Saudi Soc. for Food and Nutrition. Vol. 1, No. 1

2. Abedin R.M, El-Aassar S.A, Bahloul Y.E, Hameid H.K (2013) Medical Improtance
of Lactobacillus fermentum lysate as a bioactive agent against some pathogenic
candida and Aspergillus strain. African Journal of Microbiology Research 7: 4817-
4827.

3. Ahmed K.T, Atique F.B, Asaduzzaman S.M, Hasan K.N (2013) Effect of Gamma
Radiation on Bacterial Microflora Associated with Human Amniotic Membrane.
Biomed Res Int 586561.

4. Ali Zamanian and Cy Hardiman (2005) Electromagnetic Radiation and Human

Health: A Review of Sources and Effects. EMR & HUMAN HEALTH.

5. Ammerman J.W, Fuhrman J.A, Hagstrom A, Azam F (1984) Bacterioplankton

Growth In Sea-water: Growth Kinetics and Cellular Characteristics in Sea water
Cultures. Mar Ecol Prog Ser 18: 31-39.

6. Both E., Gyorgy E, Csaba Z., Szabo K., Tamas E., Abraham B., MIKLOSSY I.,
Lanyi S. (2010) Acid and bile tolerance, adhesion to epithelial cells of
probiotic microorganisms U.P.B. Sci. Bull.Series B, Vol. 72, Iss. 2.

7. Chen L., Tsai H., Chen W., Hsieh C., Wang P., Chen C., Wang L., Yang C. (2012)
In Vitro Antagonistic Groth Effects of Lactobacillus fermentum and Lactobacillus
Salivarious and their Fermentive Broth on Peridontal Pathogens. Brazilian Journal
of Microbiology 1376-1384.

8. Ewe J. A., Ong J. S., Nadiah W., Abdullah W., Alias A K., Liong M. T. (2014),
Potential ramifications of the effects of sub-lethal ultraviolet B-radiation on the
subsequent three subcultures of Lactobacillus fermentum BT 8219 during
fermentation in biotin-supplemented soymilk and their probiotic properties. Ann
Microbiol.

9. Gedikli S, Tabak O, Tomsuk O, Cabuk A (2008) Effect of Microwave on Some Gram

Negative and Gram Positive Bacteria. Journal of Applied Biological Sciences 2: 67-

26
 71.

10. Heinemann C, Vileg J.E.T, Janssen D.B, Busscher H.J, Mei H.C, Reid G (2006)
Purification and Characterization of a surface-binding protein from Lactobacillus
fermentum RC-14 that inhibit adhesion of Enterococcus faecalis. FEMS
Microbiology Letters 190: 177-180.

11. Liu X, Johnson S, Liu S, Kanojia D, Yue W, Singh U.P, Wang Q, Wang Q, Nie Q,
Chen H (2013) Nonlinear Growth Kinetics of Breast Cancer Stem Cells:
Implications for Cancer Stem Cells Targeted Therapy. Scientific Reports 2473.

12. M. Schlee, J. Harder, B. Koten, E. F. Stange, J. Wehkamp and K. Fellermann (2008)

Probiotic lactobacilli and VSL#3 induce enterocyte b-defensin 2. linical and
Experimental Immunology, 151: 528535.

13. Mikelsaar M., Zilmer M., (2009) Lactobacillus fermentum ME-3- An Antimicrobial
and Antioxidative Probiotic. Microb Ecol Health Dis 21: 1-27.

14. Okpokwasili G.C, Nweke C.O (2005) Microbial Growth and Substrate Utilization
Kinetics. African Journal of Biotechnology 5: 305-317.

15. Singh R., Sivasubramani K., Jayalakshmi S., Satheesh S. and Selvi C. (2013)
Isolation and production of bacteriocin by marine Lactobacillus fermentum.
J.Curr.Microbiol.App.Sci 2(4): 67-73

16. Vamanu E. (2009) Studies regarding the production of probiotic biomass from
Lactobacillus plantarum strains. Archiva Zootechnica 12:4, 92-101,

17. Woo I.S, Rhee I.K, Park H.D (2000) Differential Damage in Bacterial Cells by
microwave radiation on the basis of cell wall structure. App Environ Microbiol 66:
2243-2247.

18. Z. E. Mousavi, S. M. Mousavi, S. H. Razavi, Z. Emam-Djomeh, H. Kiani (2011)

Fermentation of pomegranate juice by probiotic lactic acid bacteria. World Journal of
Microbiology and Biotechnology. Volume 27, Issue 1, pp 123-128

27
 10. Appendix - I

Composition of MRS Broth

Ingredients Quntity (gms)

Peptone 10.0

Meat extract 10.0

Yeast extract 5.0

D-glucose 20.0

Tween 80 1.0

K2HPO4 2.0

Sodium acetate 5.0

Tri-ammonium 2.0
citrate

MgSO4.7H2O 0.2

MnSO4.4H2O 5.0

MRS agar
MRS agar prepared by adding 1.5% agar agar in MRS broth (prepared above)

28