Clemson UniversityTigerPrints

All Theses Theses

12-2014

The Effects of Probiotics Supplementation onHealth Using Caenorhabditis elegans as a ModelSystemMiranda KleesClemson University, mklees@g.clemson.edu

Follow this and additional works at: https://tigerprints.clemson.edu/all_theses

Part of the Microbiology Commons

This Thesis is brought to you for free and open access by the Theses at TigerPrints. It has been accepted for inclusion in All Theses by an authorizedadministrator of TigerPrints. For more information, please contact kokeefe@clemson.edu.

Recommended CitationKlees, Miranda, "The Effects of Probiotics Supplementation on Health Using Caenorhabditis elegans as a Model System" (2014). AllTheses. 2053.https://tigerprints.clemson.edu/all_theses/2053

THE EFFECTS OF PROBIOTICS SUPPLEMENTATION ON A HEALTH USING

CAENORHABDITIS ELEGANS AS A MODEL SYSTEM

A Thesis

Presented to

The Graduate School of

Clemson University

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Biological Sciences

by

Miranda L. Klees

December 2014

Accepted by:

Dr. Min Cao, Committee Chair

Dr. Yuqing Dong

Dr. Thomas A. Hughes

ii

ABSTRACT

The “Western Diet,” prominent among developed nations, often refers to a

diet rich in meat proteins and refined sugars. Western society may in part be plagued with

obesity and obesity related diseases due to a diet enriched with glucose. With an

increasing glycemic index observed in Western society, it comes to no surprise that

obesity, diabetes, heart disease, and other obesity related diseases are on the rise. The

Centers for Disease Control and Prevention describes obesity as an epidemic, affecting

more than 35% of U.S. citizens. Shortened lifespan and increased susceptibility to

pathogens are associated with these diseases and linked to increased consumption of

foods high in sugar. In order to reverse these observed effects, the healthful benefits

probiotics have on immune system stimulation, restoration of shortened lifespan induced

by a high glucose diet, and the beneficial effects of probiotics in combination of

cranberry extracts in the models system Caenorhabditis elegans were investigated. An

exciting benefit of implementing C. elegans in such studies is the innate immunological

response and aging pathways homology with humans. By targeting these pathways, C.

elegans provides an economical model system that provides application in humans. Here,

we investigated the effects probiotics have on high glucose diet in C. elegans. Consistent

with previous studies, when C. elegans was supplemented with each probiotic strain

tested, lifespan extension was observed. Interestingly, the reversal of glucose induced

shortened lifespan and the extension of lifespan in combination of cranberry extracts was

strain dependent. The impact that probiotics have on the reversal of detrimental effects

associated with a high glucose diet, including protection against known pathogens and

iii

immunological response pathways targeted by probiotics, is currently under

investigation.

iv

DEDICATION

I would like to dedicate my work to my family, most importantly my parents.

Their love and support through failures and accomplishments have shaped me into the

woman I am today. The memory of my mother has been a major driving force for my

accomplishments, all the while striving to be the woman she shaped me to be. I would

also like to dedicate my work to my brothers and sisters and stepmom: Jacob, Marshall,

Tateum, Pyper, and Robyn for all of their support during my journey through graduate

school.

v

ACKNOWLEDGEMENT

I would like to thank my advisor Dr. Min Cao for her invaluable support and

guidance in developing this project. I would also like to thank the rest of my committee

members Dr. Yuqing Dong and Dr. Thomas Hughes for their support and guidance in all

of my decisions and work progress.

I would also like to thank my lab mates for helpful discussions and experimental

advice and assistance: Daniel Pederson, Sujay Guha, Ethan Wilson, Joseph Angeloni,

Ojas Natarajan, Jessica Dinh, and Xiaxiao Wang. Lastly, I would like to thank my

undergraduate research advisor Dr. Joseph Flaherty for all of his support along the way

and sparking my interest in research that led to the pursuit of my master’s degree.

vi

TABLE OF CONTENTS

Page

TITLE PAGE .................................................................................................................... i

ABSTRACT ..................................................................................................................... ii

DEDICATION ................................................................................................................ iv

ACKNOWLEDGEMENT ............................................................................................... v

LIST OF TABLES ........................................................................................................ viii

LIST OF FIGURES ........................................................................................................ ix

LIST OF ABBREVIATIONS .......................................................................................... x

CHAPTER

1. INTRODUCTION .......................................................................................... 1

Diet ............................................................................................................ 1

Obesity ....................................................................................................... 2

Obesity and Microbiome ........................................................................... 3

Obesity and Immune Response ................................................................. 4

Caenorhabditis elegans as a Model System .............................................. 4

Probiotic Supplementation ........................................................................ 6

Cranberry and Application ........................................................................ 7

Significance ............................................................................................... 8

2. MATERIALS AND METHODS .................................................................. 10

Bacterial Strains and Worm Strains ........................................................ 10

Preparation of CBE ................................................................................. 10

Preparation of Media ............................................................................... 11

Growth Curve .......................................................................................... 11

Lifespan Assay ........................................................................................ 12

3. RESULTS ..................................................................................................... 14

Experimental Design ............................................................................... 14

Effects of D-glucose and CBE on bacterial growth ................................ 15

Effect of probiotics on lifespan ............................................................... 17

vii

Table of Contents (Continued)

Effect of D-glucose on lifespan ............................................................... 19

Probiotics reversal effects of glucose induced shortened lifespan ......... 20

Effects of probiotics in combination with CBE ...................................... 23

4. DISCUSSION ............................................................................................... 26

5. FUTURE DIRECTIONS .............................................................................. 31

REFERENCES .............................................................................................................. 33

viii

LIST OF TABLES

Table Page

3-1 Lifespan extension via probiotics ................................................................ 18

3-2 Lifespan restoration via probiotics .............................................................. 22

3-3 Lifespan extension via probiotics + CBE .................................................... 24

ix

LIST OF FIGURES

Figure Page

1-1 C. elegans life cycle ........................................................................................... 5

3-1 Experimental design diagram .......................................................................... 14

3-2 Growth curves of probiotic strains .................................................................. 16

3-3 Lifespan curves of each probiotic tested ......................................................... 17

3-4 Lifespan curve of control with glucose ........................................................... 19

3-5 Lifespan curves with and without 2% D-glucose ............................................ 21

3-6 Lifespan curves with and without 2mg/mL CBE ............................................ 25

x

LIST OF ABBREVIATIONS

Bc ........................................................................................................ Bacillus coagulans

Bs ............................................................................................................. Bacillus subtilis

CBE ...................................................................................................... Cranberry Extracts

CFU ................................................................................................. Colony Forming Unit

ddH2O .......................................................................................... Double-Distilled Water

E. coli OP50 ................................................................................... Escherichia coli OP50

FUDR ........................................................................................ 5-Fluoro-2′-deoxyuridine

IL ....................................................................................................................... interleukin

LAB....................................................................................... Lactic Acid Bacilli/Bacteria

LB ...................................................................................................... Luria-Bertani Broth

Ld .............................................................................................. Lactobacillus delbrueckii

Lf ................................................................................................ Lactobacillus fermentum

Lp ............................................................................................. Lactobacillus planatarum

LPS ..................................................................................................... lipopolysaccharides

MRS ........................................................................................ Lactobacilli MRS Medium

NGM ...................................................................................... Nematode Growth Medium

NO .................................................................................................................... nitric oxide

p38 MAPK ..............................................................p38 mitogen-activated protein kinase

qRT-PCR......................................... Quantitative Real Time Polymerase Chain Reaction

spp .......................................................................................................................... species

subsp ............................................................................................................... sub-species

TNFα ...................................................................................... tumor necrosis factor alpha

1

CHAPTER ONE

INTRODUCTION

I. DIET

The “Western Diet,” prominent among developed nations, often refers to a diet

rich in meat proteins and refined sugars. Western society may in part be plagued with

obesity and obesity related diseases due to a diet enriched with glucose. An increasing

glycemic index has been observed in western society, and this increase has been linked to

obesity, diabetes, and heart disease. The Centers for Disease Control and Prevention

describes obesity as an epidemic, affecting more than 35% of U.S. citizens. Though

obesity is a complex trait influenced by diet, developmental stage, age, physical activity,

and genetics, the recent epidemic is probably reflective of environmental and lifestyle

changes, with diet being a major influence 1

. Diet, from a scientific perspective, can be

described as the sum of food consumption by an organism. As humans, we have the

ability to consciously decide what foods we consume; however, proper nutrition involves

consumption of essential vitamins and minerals needed for metabolic processes. Much of

our food energy intake is consumed in the forms of carbohydrates, proteins, and fats. The

dietary decision of a healthful balance plays a major role on quality of life and health and

longevity. Therefore, diet not only affects metabolic activities and overall health, but it

also affects lifespan.

2

II. OBESITY

As previously mentioned, obesity is a complex characteristic influenced by many

factors including but not limited to diet, lifestyle, and genetics. The phenotype is

responsible for 6-10% of national healthcare costs in the United States; in addition to, a

lifetime of medical costs that are $10,000 greater in obese patients in comparison to non-

obese. Researchers argue that the rise in obesity is the result of an increase in sedentary

lifestyles 2, 3

while others argue that the culprit is over-consumption and gluttony 4, 5

. In

the 1960s, the average American weighed 168 pounds; today, that average has increased

nearly 15 pounds. Since the 1970s, obesity rates have doubled according to the Centers

for Disease Control and Prevention. Medications, cosmetic surgeries, and diet may be

methods for short-term treatment of obesity; however, long-term treatment involves a

lifelong commitment to healthy diet habits and increased physical activity. Still, most

people who previously suffered from obesity have relapses within 5 years of surgeries or

other methods of weight loss.

Obesity is characterized by excess body fat accumulation as a result of increased

energy intake compared to expenditure to the point that it becomes detrimental to health.

When more calories are consumed than expended, the resulting phenotype is weight gain.

Increased body fat proportionally increases the risk for chronic diseases such as diabetes,

heart disease, and cancer. Not only are these chronic diseases associated with obesity, but

bacterial diversity shifts are also observed in these patients 6, 7

. Other side effects of

obesity include immune system failures and increased sensitivity to pathogens.

3

III. OBESITY & THE MICROBIOME

The gut is home to a vast array of microorganisms that aid in digestive health,

immune stimulation, and provide a competitive environment to prevent colonization of

many harmful pathogens. With the alteration of diet associated with Western society, the

microbial community is severely impacted within the gut. The human body and

gastrointestinal tract are colonized at birth, and continues to shift based on factors, such

as breastfeeding, diet, and environmental exposure, until the microbial community

becomes stabilized around 3 years of age 8

. The human gut is largely comprised of

Actinobacteria, Firmicutes, and Bacteriodetes 9

. The functions of the gut microbiota

obtained throughout our lifespan are essential to the metabolism of xenobiotic

compounds, amino acids, and carbohydrates. The balance of bacteria within the gut can

be rapidly altered by dietary shifts. For example, when mice were fed a high-fat diet, the

gut microbial composition shifts were observed in 24 hours 10-13

. When altered gut

microbiota, resultant of a high-fat diet, were transferred to germ-free mice, the subjects

experienced increased adiposity 12

, consistent with the suggestion that adiposity and

obesity are linked to microbiota 14

. Specifically, a reduction of Bacteroidetes was

observed in obese subjects 10, 15-17

; however, not all studies consistently observed this

reduction 18

. When obesity is induced via diet the resulted mice experienced an increase

of Eubacterium dolichum, a member of the Firmicutes phyla 12

and a decrease in

Bifodobacterium, a member of the Actinobacteria phyla 17

. Energy restriction and

increased physical activity have also been linked to changes in the gut microbiota 19, 20

.

These microorganisms provide necessary enzymes involved in utilization of non-

4

digestible carbohydrates, involved in cholesterol reduction, important for biosynthesis of

vitamins K and B 21, 22

, and involved in deconjugation and dehydroxylation of bile acids

which, necessary in solubilizing and absorbing lipids 23

. Factors other than diet also

played a crucial role in microbiota, including but not limited to, gut microbiota shifts in

obese subjects which were dependent on age 24

.

IV. OBESITY & IMMUNE RESPONSE

A high-fat diet is associated with chronic low-grade inflammation and increased

susceptibility to infection 25

. In obese patients, the innate immune system responds by

increased macrophage infiltration in adipose tissue and production of inflammatory

adipokines 26

. Increased plasma lipopolysaccharides (LPS) levels were observed with

obesity 17, 27

, activating the synthesis of inflammatory cytokines TNFα. Chronic activation

of the innate immune system results in increased risk for development of obesity; dietary

compounds may in part be responsible 26

.

V. CAENORHABDITIS ELEGANS AS A MODEL SYSTEM

C. elegans is a favorable model system for studying aging and immune response

for many reasons: 1) ease of maintenance, 2) homological pathways, including aging and

immunity, with humans, 3) transparent, 4) economically favorable, and 5) relatively short

lifespan. C. elegans offers a faster, efficient analysis of human related diseases while

allowing us to infer evolutionary history of immune components to study conserved traits

and obtain information about probable functions. Similar to humans, many pathogens

5

have the ability to colonize the digestive tract of C. elegans, giving rise to an ideal model

to study pathogenesis. However, many pathogens may exhibit other modes of infection in

the worm, adhering to the cuticle or producing lethal toxins 28, 29

. We are able to

determine the pathology of such pathogens by determination of a decrease in lifespan.

Though much less complex than the mammalian immune response, C. elegans

immune defenses similarly consists of an innate immune response in which it is solely

reliant. In its natural habit, the

soil-dwelling nematode feeds on

bacteria and has frequent

encounters with pathogens. Most

pathogens infect the worm via

ingestion and then establish in

the intestine where the majority

of the damage occurs. In

response to invasive pathogens

the worms have developed

defenses that include a

behavioral response, physical barriers, and physiological response. As the first line of

defense, behavioral responses must be employed to reduce the number pathogen

encounters. However, exposure is inevitable and the worm must elicit physiological

response to prevent colonization of the gut during the process of feeding. The p38

mitogen-activated protein kinase (MAPK) pathway and the insulin-like receptor pathway

Figure 1-1. C. elegans life cycle at 25°C (artwork from http://www.scq.ubc.ca/genetic-studies-of-aging-and-longevity-in-model-organisms/).

6

are two pathways involved in immune response, longevity, and mammalian homology.

These two pathways interact and respond to a vast array of stress conditions 30

. The p38

MAPK pathway is activated in response to pathogens such as Pseudomonas aeruginosa

and Salmonella enterica 31, 32

, as well as in response to other pathogens and other

stressors such as acute dehydration. The insulin-like receptor pathway is also important to

the response to P. aeruginosa, Enterococcus faecalis, and Staphylococcus aureus 33

. This

pathway also has complex interactions with other regulatory elements such as the heat

shock factor-1 (HSF-1) and the superoxide dismutase-3 (SOD-3).

C. elegans possesses a complex and interactive innate immunological response

that has been extensively studied 30, 34

. The advantages of using this experimental system

as a means to study application in humans is evident through the extensive study of these

evolutionarily conserved pathways.

VI. PROBIOTICS SUPPLEMENTATION

A probiotic can be defined as any microorganism believed to provide health

benefits upon consumption. The most common types of probiotics found in our diet are

Bifidobacterium and lactic acid bacteria. Several strains of lactic acid bacteria, mainly

Lactobacillus spp. have been shown to induce longevity 35

as well as alleviating the

effects of infection caused by Vibrio parahaemolyticus, Salmonella typhimurium,

Salmonella enteritidis, Staphylococcus aureus, and Enterococcus faecalis35-38

.

Bifidobacteria have the ability to increase lifespan in C. elegans via the p38 MAPK

pathway, a homologous innate immunological pathway with humans 39

. However, the

7

pathway in which each probiotic targets may be dependent on the strain of probiotic and

the infectious strain of bacteria. Kim et al. suggested that Lactobacillus acidophilus had

minimal effects on Gram-negative bacteria Pseudomonas aeruginosa and Salmonella

enterica Typhimurium. On the contrary, L. acidophilus provided protection from

Enterococcus faecalis and Staphylococcus aureus infection while targeting the p38

MAPK pathway suggested during Gram-positive immune response in C. elegans 37

. The

aforementioned probiotics and the probiotics utilized in this study are often consumed in

yogurts, fermented foods and dietary supplements. More interestingly, previous studies

suggest that probiotics have the ability to enhance the immune response in not only C.

elegans but also in a higher organism model system, mice 37, 40-42

.

VII. CRANBERRY & APPLICATION

The probiotics targeted in this study are often consumed in yogurts,

fermented foods, and dietary supplements. Though probiotic supplementation is an

emerging area of research, little is known about the benefits they may have on

healthspan and lifespan, particularly when supplemented with nutraceuticals.

Previous studies suggest that probiotics have the ability to enhance the immune

response in not only C. elegans but also in mice 37, 40-42. Numerous studies have also

linked nutraceuticals to longevity and stress resistance 43, 44. Still, little is known of

the benefits of synergistic supplementation. For years, the North American cranberry

(Vaccinium macrocarpon) has been ingested to alleviate the effects of a urinary tract

infection by preventing adherence of bacteria to host’s cells 45

. However, there is no

8

evidence suggesting that cranberry is actually effective in treating or even preventing a

urinary tract infection 46

. Preliminary data suggests components in cranberry may prevent

plaque formation on teeth 47

, kill Helicobacter pylori (the causative agent of stomach

cancer and ulcers) 48

, and even prevent tumor development 49

. Research suggests that

cranberry provides many nutritional and healthful benefits, owing to its antioxidant,

antimicrobial, anti-apoptotic, anti-aging, anticancer, anti-inflammation, and enhancement

of endothelial functions 49

. Recently published research from our lab suggests that

cranberry, when studied in the soil dwelling nematode model organism C. elegans,

effectively extends lifespan and promotes healthy aging while elevating resistance to

certain stressors, including bacterial infection 44, 50

.

VIII. SIGNIFICANCE

Previous research suggests that probiotics such as Lactobacillus isolates can

control and alleviate the effects of pathogenic infections; however, much still needs to be

studied. C. elegans provides an effective model of study of pathogenesis with application

in humans 35-38, 40

. Another exciting benefit of implementing C. elegans in such studies is

the homology in humans in the innate immunological response pathways and aging

pathways. The optimal combination of probiotics that successfully stimulates the immune

system has yet to be studied. If we are able to determine a favorable combination of

probiotics that have the ability to reverse the effects of a high glucose diet while

promoting healthy aging and aiding in immune stimulation, we may be able to further

provide insight into the effects that a high-glucose diet has on overall health and how to

9

reverse such detrimental effects. Studies can then be carried out in a higher organism

such as mice to further confirm implication in supplementing a particular combination of

probiotics in our diet.

10

CHAPTER TWO

MATERIALS & METHODS

I. BACTERIAL STRAINS AND WORM STRAINS

L. fermentum (ATCC 9338), L. planatarum (ATCC 8014), L. delbrueckii (ATCC

7830), and B. coagulans were all acquired from VWR. B. subtilis CU1065 was a lab

strain originally acquired from Cornell University. E. coli OP50 was acquired from lab

stocks. Lactobacillus strains were cultured anaerobically in 5% CO2 on MRS agar at

37°C for 48 hours. Bacillus spp. were cultured aerobically on LB agar at 37°C overnight.

E. coli OP50 was cultured on LB+streptomycin (100μg/mL) at 37°C overnight.

Bacillus spp. and E. coli OP50 overnight cultures for lifespan assays were

inoculated in LB broth and incubated at 37°C shaking at 150 rpm. Lactobacillus strains

were inoculated in MRS broth and incubated at 37°C stationary in 5% CO2.

N2 worms were acquired from the Caenorhabditis Genetics Center (CGC).

Worms were maintained on nematode growth medium (NGM) seeded with E. coli OP50

at 25°C, and transferred to fresh plates every two days.

II. CBE PREPARATION

The cranberry extracts, HI-PAC BL DMAC 5, used in this study were obtained

from DECAS cranberry products, Inc. and were received in the powdered form. For

working stock solutions, CBE was dissolved in ddH2O at a concentration of 5% and final

concentration used was 0.2% (2mg/mL).

11

III. PREPARATION OF MEDIA

NGM for maintenance was prepared via standard protocol in 60mm plates. E. coli

OP50 was dropped on the center of the plates the night prior to transfer of worms.

NGM+glucose plates were prepared via standard protocol with the addition of 2% D-

glucose final concentration in 60mm plates. Bacterial strains were dropped onto the

center of the plates 2 hours prior to worm transfer.

NGM-FUDR 35mm plates were used for lifespan assays. Five times concentrated

bacterial cultures were dropped in 100μL aliquots onto the center of plates 2 hours prior

to worm transfer. D-glucose was added with a final concentration of 2% to NGM-FUDR

plates prior to pouring and 5mL aliquots were added to each plate. For CBE testing, CBE

was dissolved in water and dropped and spread confluent onto 5mL NGM-FUDR the

night prior to start of assay.

IV. GROWTH CURVE

A single colony of each strain was inoculated from a freshly streaked plate into 5

mL MRS for Lactobacillus strains, LB for Bacillus strains, and LB+strep (100μg/mL) for

E. coli OP50. Lactobacillus spp. were grown statically overnight at 37°C in 5% CO2. E.

coli OP50 and Bacillus strains were grown under aerobic conditions overnight at 37°C.

On the next day, the overnight cultures were transferred into sterile 15 mL centrifuge

tube and centrifuged at 4000 rpm for 10 min at room temperature. Cell pellets were

resuspended in 10 mL NGM liquid medium. Aliquots of 50 μl were plated from the

following dilutions 105

and 106 onto designated agar plates. This was the CFU for T=0.

12

Resuspended cultures were transferred in 3 mL aliquots into each glass test tube, and then

desired concentrations of glucose were added to corresponding tubes. Tubes were

incubated under aerobic conditions at 37°C. Lactobacillus tubes were kept at 37oC CO2

incubator. At each time point (T=2, 4, 12, 24, 48 hr), serial dilutions were performed and

dilutions to be plated were estimated based on cultural density. Aliquots of 50 μL were

plated on corresponding plates. CFU/mL were calculated the next day using the standard

plate count method.

V. LIFESPAN ASSAYS

Lifespan assays were carried out at 25°C. Worms were synchronized by

transferring 20 gravid worms to 60mm NGM plates seeded with E. coli OP50 2 days

prior to the start of assays. Worms were allowed to lay eggs for 5 hours, and then parent

worms were removed from plates, leaving only synchronized eggs. Worms were then

incubated until L4 stage. Overnight cultures of Bacillus spp. and E. coli OP50 were

concentrated by centrifugation and removing 80% of supernatant. Cultures were then

resuspended in remaining medium. Lactobacillus overnight cultures were concentrated

via centrifugation and removing 100% of supernatant. Cultures were resuspended in 1

mL of M9 buffer. Aliquots of 100μL were dropped on the center of NGM-FUDR 35mm

plates with and without 2% D-glucose and allowed to dry for 2 hours. Worms in L4 stage

were transferred to assay plates; 30 worms per plate. Plates were subsequently checked

daily and dead worms were counted and recorded. Day of transfer was defined as zero.

Statistical analysis was carried out through SPSS software under Kaplan-Maier lifespan

13

analysis. P-values were determined via log rank test, and P<0.05 was accepted as

statistically significant.

14

CHAPTER THREE

RESULTS

I. EXPERIMENTAL DESIGN

Previous studies showed that 2% glucose significantly reduced lifespan in C.

elegans51

. Consistent with this studied, 2% D-glucose significantly decreased the lifespan

of N2 worms; therefore, 2% D-glucose was used to represent a high glucose diet.

Similarly, previous studies from our lab determined 2mg/mL was the ideal concentration

of CBE supplementation to extend lifespan and modulate the immune response 44, 50

.

Based on previous studies, a series of experiments were employed to determine the

effects that probiotics had on reversing the effects of a high glucose diet in C. elegans

and the effects probiotics have on lifespan in combination with CBE (Figure 3-1).

Figure 3-1. Diagram of the experimental design of lifespan assays carried out at 25°C

with different diets.

L4 worms were transferred

Control-OP50

No supplements

2% D-glucose supplemented

2mg/mL CBE supplemented

Test-with probiotics

No supplements

2% D-glucose supplemented

2mg/mL CBE supplemented

15

II. EFFECTS OF 2% D-GLUCOSE AND 2mg/mL CBE ON BACTERIAL GROWTH

Glucose induced shortened lifespan was first observed in C. elegans (Table 3-2).

Dietary restriction is a well-documented cause of lifespan extension in the worm model.

To determine if 2mg/mL CBE and/or 2% D-glucose would affect bacterial growth,

growth curves using the standard plate count method were performed. Each strain was

treated with either 2% D-glucose or 2mg/mL CBE for up to 72 hours, growth was not

significantly inhibited (Figure 3-2 A-F).

16

Figure 3-2. Growth curves of each probiotic with and without 2% D-glucose and with

and without 2mg/mL CBE in NGM broth. A) E. coli OP50 B) B. subtilis C) B. coagulans

D) L. fermentum E) L. planatarum F) L. delbrueckii.

0 20 40 60 80

Log 1

0(C

FU

/mL

)

Time (hours)

E. coli OP50

10

9

8

7

A.

0 20 40 60 80

Log10(C

FU

/mL

)

Time (Hours)

Bs

9 8 7 6

B.

0 20 40 60 80

Log10(C

FU

/mL

)

Time (Hours)

Bc

9 8 7 6

C.

0 50 100

Log10(C

FU

/mL

)

Time (Hours)

Lf 9

8

7

6

5

D.

0 50 100

Lo

g10(C

FU

/mL

)

Time (Hours)

Lp

7

9

10

9

8

7

6

E.

-20 30 80

Log10(C

FU

/mL

)

Time (Hours)

Ld 9

8

7

6

5

F.

17

III. EFFECT OF PROBIOTICS ON LIFESPAN

Previous studies suggested that probiotic strains significantly extend lifespan in C.

elegans; however, the observable extension has also been suggested to be dose

dependent39-42, 52

. The five strains tested in this study all extended lifespan when

supplemented during L4 stage of worms (Figure 3-3, Table 3-1). B. subtilis fed worms

had one day lifespan extension compared to the control. B. coagulans fed worms had 4

days lifespan extension compared to the control, L. fermentum had 3 days lifespan

extension, L. planatarum had 1.5 days extension, and L. delbrueckii had 2 days lifespan

extension.

Figure 3-3. Probiotic strains extended lifespan of N2 worms compared to E. coli OP50 at

25°C. L. planatarum exhibited greatest lifespan extension with mean lifespan of 16.042

days.

18

Table 3-1. Effect of probiotics on lifespan of N2 worms at 25°C

Probiotic strains extended lifespan compared to E. coli OP50, consistent with previous

research. P-value <0.05 considered significant. Data shown are representative of one

round of lifespan assays.

Diet Mean±SE (Days) N P-value % Increase

of Lifespan

E. coli OP50 13.442±0.244 95 - -

B. coagulans 17.261±0.336 92 <0.05 28.41%

L. fermentum 16.663±0.213 92 <0.05 28.45%

L. planatarum 15.085±0.249 82 <0.05 12.22%

L. delbrueckii 15.480±0.307 75 <0.05 15.16%

E. coli OP50 12.737±0.459 99 - -

B. subtilis 14.468±0.369 109 <0.05 13.6%

19

IV. EFFECT OF D-GLUCOSE ON LIFESPAN

Consistent with previous studies, when glucose was supplemented at a

concentration of 2% into the worm diet, lifespan was significantly reduced. C. elegans’

lifespan was significantly reduced 15.7% as compared to the control strain without

glucose (Figure 3-4, Table 3-2).

Figure 3-4. Glucose significantly reduced lifespan of N2 worms under normal lab

conditions at 25°C. Lifespan was reduced by 2 days, P<0.05. Data shown are

representative of one round of lifespan assays.

20

V. PROBIOTICS REVERSAL EFFECTS OF GLUCOSE INDUCED SHORTENED

LIFESPAN

As previously mentioned, each probiotic strain extended lifespan, and glucose

was determined to reduce lifespan. When probiotics were supplemented with glucose,

reversal of the glucose induced shortened lifespan was dependent on the strain of

probiotic. Though lifespan was not fully restored, three of the five strains exhibited some

level of lifespan restoration. The control strain exhibited a 15.7% decrease in lifespan. B.

subtilis only exhibited a 5.3% decrease, B. coagulans had an 11.39% decrease, L.

fermentum had a 16.1% decrease, and L. delbrueckii had an 8.7% decrease. L.

planatarum exhibited the most dramatic decrease of 64.6% reduction of lifespan (Figure

3-5, Table 3-2).

21

Figure 3-5. Lifespan curves of N2 worms fed on each probiotic with or without 2% glucose supplemented into diet at 25°C.

A) Control E. coli OP50 B) B. subtilis fed worms C) B. coagulans fed worms D) L. fermentum fed worms E) L. planatarum

fed worms F) L. delbrueckii fed worms.

A. C. B.

D. E. F.

22

Table 3-2. Lifespan of N2 worms on high glucose diet and probiotics at 25°C

N2 lifespan was shortened with all strains when supplemented with glucose. Lifespan

restoration was observed in Lactobacillus delbrueckii, Bacillus subtilis, and Bacillus

coagulans. Worms fed Lactobacillus planatarum exhibited an extreme decrease in

lifespan when supplemented with 2% D-glucose. Data shown are representative of one

round of lifespan assays.

Diet Mean±SE (Days) N P-value % Decrease

of Lifespan

E. coli OP50 13.442±0.225 95 - -

OP50+2% glucose 11.333±0.207 69 <0.05 15.70%

B. subtilis 13.477±0.202 86 - -

Bs+2% glucose 12.759±0.138 87 <0.05 5.30%

B. coagulans 17.261±0.221 92 - -

Bc+2% glucose 15.301±0.142 73 <0.05 11.39%

L. fermentum 16.663±0.262 92 - -

Lf+2% glucose 13.986±0.240 73 <0.05 16.10%

L. planatarum 15.085±0.273 82 - -

Lp+2% glucose 5.342±0.130 73 <0.05 64.60%

L. delbrueckii 15.48±0.256 75 - -

Ld+2% glucose 14.135±0.318 74 <0.05 8.70%

23

VI. EFFECTS OF PROBIOTICS IN COMBINATION WITH CBE

Previous studies in our lab have determined that the optimum concentration of

cranberry supplementation was 2mg/mL. At this concentration C. elegans’ lifespan is

extended significantly without affecting bacterial growth of different strains such as

Vibrio cholerae and E. coli OP50. When CBE was supplemented at the time of exposure

to probiotics, C. elegans lifespan was extended (Figure 3-6 A-E). We can also conclude

that when C. elegans is fed probiotics in succession with CBE supplementation, lifespan

was significantly extended in comparison without CBE treatment. L. planatarum only

exhibited bacterial extension effect, and lifespan was not further extended when 2mg/mL

CBE was supplemented into the diet (Figure 3-6 A-E).

24

Table 3-3. Lifespan of N2 worms fed on probiotics and CBE diet at 25°C

N2 lifespan was increased with all strains when supplemented with CBE except L.

planatarum. Lifespan extension was observed in Lactobacillus delbrueckii, Lactobacillus

fermentum, Bacillus subtilis, and Bacillus coagulans. Data shown are representative of

one round of lifespan assays.

Diet Mean±SE (Days) N P-value % Increase

of Lifespan

E. coli OP50 12.737±0.459 99 - -

B. subtilis 14.468±0.369 109 <0.05 13.6%

B.s.+2mg/mL CBE 15.359±0.106 64 <0.05 20.6%

B. coagulans 15.295±0.181 88 <0.05 20.1%

B.c.+2mg/mL CBE 16.136±0.130 81 <0.05 26.7%

L. fermentum 13.44±0.511 90 <0.05 5.5%

L.f.+2mg/mL CBE 15.575±0.320 87 <0.05 22.3%

L. planatarum 14.9±0.194 100 <0.05 17%

L.p.+2mg/mL CBE 14.693±0.300 75 <0.05 15.4%

L. delbrueckii 14.364±0.459 66 <0.05 12.8%

L.d.+2mg/mL CBE 15.742±0.304 66 <0.05 23.6%

25

Figure 3-6. N2 lifespan curves carried out at 25°C with and without 2mg/mL CBE and probiotics. A) B. subtilis B) B.

coagulans C) L. fermentum D) L. planatarum E) L. delbrueckii.

A.

C.

B.

E. D.

26

CHAPTER FOUR

DISCUSSION

Over a century ago, a Russian scientist by the name of Élie Metchnikoff first

hypothesized that probiotics could beneficially modify the gut flora by replacing harmful

pathogens. Recently, manufacturers’ marketing of foods containing beneficial probiotics

has fueled the renewed interest in probiotics research. However, the consumption of

probiotics as a means to provide health benefits can be traced back to the Greeks and

Romans through the consumption of fermented food products such as cheeses and other

dairy products.

The modern hypothesis, adapted from Metchnikoff’s findings, stating that

probiotics and microbiota have major influences on human health, has further evolved

with the recent increase in consumer awareness. Consumers and researchers have

expanded the research interests to target the use of oral pill supplementation of probiotics

into diet and some have even experimented with fecal transplantations as a means to

combat digestive disorders. Of particular importance are the LAB strains. Lactobacillus

is a genus of Gram-positive facultative anaerobes or microaerophilic rod-shaped bacteria.

Their production of lactic acid from the conversion of lactose and other sugars, create an

acidic environment, prohibiting the growth of some harmful pathogens. The lower pH

also provides an environment for a balanced ecosystem in the gut. Metabolism and

metabolite production by Lactobacillus spp. are not only species dependent but also

dependent on the strain and/or subspecies.

27

In combination with previous research of beneficial supplementation of probiotics

as a means to extend lifespan in C. elegans and research suggesting a high glucose diet

shortens lifespan in C. elegans, the reversal effects of probiotics supplementation on a

high glucose diet were studied. Consistent with previous research, the probiotic strains

used in this study significantly extended lifespan when supplemented individually (Figure

2-3).

Lactobacillus planatarum are heterofermentative bacteria producing D-lactic acid,

L-lactic acid, and acetic acid through the Embden-Meyerhoff pathway and are also

known to produce hydrogen peroxide to eliminate other competitive bacteria. They are

found in fermented plant matter such as pickles and kimchi and the most common

bacteria used in silage. Supplementation of L. planatarum may provide anti-inflammatory

effects via reduction of IL-1β, IL-6, and C-reactive protein (CRP), immune response

components involved in inflammation, while increasing IL-10 53

. L. planatarum has the

ability to extend lifespan in C. elegans (Figure 3-3, Table 3-1); however, when glucose is

supplemented into C. elegans diet along with L. planatarum, the shortened lifespan

induced by glucose is worsened dramatically (Figure 3-5E, Table 3-2). The exhibited

shortened lifespan could be a result of a multitude of reasons ranging from metabolites

produced by the bacteria to protective compound produced by the bacteria such as

hydrogen peroxide. Similarly with glucose, L. planatarum fed worms had a slight

reduction in lifespan when supplemented with CBE (Figure 3-6D). L. planatarum fed

worms exhibited a 17% increase in lifespan; however, when supplemented with CBE,

there was only a 15.4% increase in lifespan (Table 2-3).

28

Lactobacillus delbrueckii are homofermentative bacteria that produce lactic acid.

They are comprised of four subspecies separated by their metabolism. L. delbrueckii is

commonly found in yogurts and cheeses. Worms fed on L. delbrueckii exhibited

extension in lifespan (Table 3-1), and when supplemented with a high glucose diet, the

probiotic restored lifespan compared to the control. The control exhibited a 15.7%

decrease in lifespan when fed on a high glucose diet; however, when the worms were fed

a high glucose diet with L. delbrueckii, lifespan was only reduced 8.7%. The mechanism

for restorative properties has yet to be determined. L. delbrueckii subsp. bulgaricus

produces nitric oxide (NO) 54

. NO has previously been shown to extend lifespan in C.

elegans 55

. NO is responsible for mucus generation, motility, and water and electrolyte

transport. More interestingly, when L. delbrueckii lifespan extension properties were

further enhanced when supplemented with CBE, with an increase from 12.8% lifespan

extension to 23.6% lifespan extension compared to the control (Table 3-3, Figure 3-6E).

L. fermentum are heterofermentative bacteria. Although L. fermentum may be

responsible for dental caries, it can also be utilized as a probiotic. The probiotic is

commonly found in sourdough breads and other fermented plant products. Some strains

have the ability to metabolize cholesterol 56

. Though this could be a possible cause of

lifespan extension in C. elegans (Table 3-1), the mechanism of lifespan extension has yet

to be studied. Though worms fed L. fermentum exhibited lifespan extension, when

supplemented with a high glucose diet, the reduction of lifespan was nearly the same as

the control worms fed on E. coli OP50 (Figure 3-5D, Table 3-2). On the other hand, when

the probiotic was supplemented with CBE, lifespan was further extended from 5.5% to

29

22.3%. Interestingly, this combination may provided insight into the combination of

probiotics and fruit extracts commonly supplemented together in the diet.

B. subtilis are spore forming obligate aerobes, commonly found in the human gut.

Because of the spore forming abilities that allow the bacteria to survive under harsh

conditions, the bacteria have become targets for probiotic supplementation in pasteurized

foods and beverages. B. subtilis have the ability to extend lifespan in C. elegans through

nitric oxide production 55

. Here, B. subtilis had the ability to reverse the effects of a high

glucose diet in C. elegans, though not fully. B. subtilis fed worms exhibited only a 5.3%

decrease in lifespan when supplemented with glucose; whereas, the control strain had a

15.7% decrease in lifespan. More interestingly, when worms were fed B. subtilis in

combination with CBE, their lifespan was further increased from 13.6% to 20.6%.

B. coagulans are spore forming facultative anaerobes, and are used as a probiotic

in livestock feed. B. coagulans have the ability to extend lifespan in C. elegans (Table 3-

1), and have some restorative properties when supplemented with a high glucose diet. In

worms fed B. coagulans and a high glucose diet, lifespan was only decrease 11.39% as

compared to the 15.7% decrease in lifespan exhibited in the control. Lifespan in worms

was also further extended when supplement in combination with CBE, extending lifespan

from 20.1% to 26.7%.

In this study, we observed the ability of probiotics to reverse the effects of a high

glucose diet in C. elegans. Owing to C. elegans’ pathway homology in humans, our

findings can provide insight into the possible reversal effects of probiotic

supplementation in humans. As previously mentioned, obesity and other diseases

30

associated with a high glucose diet have become a major issue in developed nations. The

insulin-like signaling pathway involved in aging and innate immune response in C.

elegans is among the pathways of homology in humans.

Insulin, a hormone involved in regulating metabolism and absorption of glucose

in mammals, and the homolog IGF-1 have been shown to regulate lifespan in many

organisms and is involved in other cellular processes such as stress response 57-59

. The

FOXO transcription factor DAF-16 in the insulin/IGF-1 signaling pathway and decreased

activity of heat shock transcription factor HSF-1 reduced the rate of tissue aging,

resulting in increased lifespan 60, 61

. The DAF-16 and HSF-1 transcription factor may be

inhibited by a high-glucose diet. A downstream gene encoding a glycerol channel aqp-1

was also involved in lifespan reduction induced by a high glucose diet 51

. In mammals,

aquaporin glycerol channels are repressed by insulin 62, 63

.

Our findings may also have implications in prevention and/or cure of obesity

related diseases such as type 2 diabetes, tumor formation and heart disease. Studies could

be further carried out in a higher organism such as mice. Because of the pathway

homology including insulin/IGF-1-mediated signaling (IIS) pathway, p38 kinase

pathway, and other immunological/stress response pathways between C. elegans and

humans, these findings may be of high human health importance. In addition these

findings may lead to relatively inexpensive and highly accessible prevention or treatment

for infection.

31

FUTURE DIRECTIONS

Through execution of these objectives, we were able to provide more insight into

the effects probiotics have on overall health with possible application in humans. In

addition, our findings may provide insight into future studies such as probiotics’ effect on

the microbiome. Based on our preliminary data that 1) each probiotic strain extends

lifespan significantly compared to the control, and 2) with the exception of L. planatarum

and L. fermentum, each strain restores lifespan when exposed to a high glucose diet,

though not fully, we propose to test the possible pathway involvement in this observed

protection. Because of the pathway homology including insulin/IGF-1-mediated

signaling (IIS) pathway, p38 kinase pathway, and other immunological/stress response

pathways between C. elegans and humans, we intend to further test the pathways

involved in the alleviated effects of a high glucose diet triggered by probiotic

supplementation. The transcription factor of the IIS pathway DAF-16 is required for

longevity in C. elegans 60

. The DAF-16 target sod-3 encodes a superoxide dismutase

involved in stress response 64

. And the downstream gene of DAF-16, aqp-1, which

encodes for a protein in the aquaporin glycerol channel, may be involved in glucose-

induced decreased lifespan in C. elegans. Similarly in mammals, aquaporin glycerol

channels are repressed by insulin 62

. We can test these pathways via qRT-PCR. If these

genes are involved in the regulation of restored lifespan, we would expect to see

increased expression in daf-16, sod-3, and aqp-1 in worms fed on probiotics compared to

E. coli OP50 and increased expression in worms fed probiotics in the presence of

32

glucose. Finally, we would expect to see down-regulation of these genes when worms are

fed our control strain E. coli OP50 with glucose.

Successful determination as to which pathways are involved in probiotic mediated

restoration of lifespan will provide insight into the mechanism with application in

humans and potentially lead to further studies in a higher organism.

33

REFERENCES

1. Bleich S, Cutler D, Murray C, Adams A. Why is the developed world obese? Annu

Rev Pub Health 2008;29:273-95.

2. Heini A, Weinsier R. Divergent trends in obesity and fat intake patterns: The American

paradox. Am J Med 1997;102(3):259-64.

3. Philipson T. The world-wide growth in obesity: An economic research agenda. Health

Econ 2001;10(1):1-7.

4. Cutler D, Glaeser E, Shapiro J. Why have americans become more obese? Journal of

Economic Perspectives 2003;17(3):93-118.

5. McCrory M, Suen V, Roberts S. Biobehavioral influences on energy intake and adult

weight gain. J Nutr 2002;132(12):3830S-4S.

6. Turnbaugh P, Ley R, Hamady M, Fraser-Liggett C, Knight R, Gordon J. The human

microbiome project. Nature 2007;449:804-10.

7. Waldram A, Holmes E, Wang Y, Rantalainen M, Wilson I, Tuohy K, McCartney A,

Gibson G, Nicholson J. Top-down systems biology modeling of host metabotype-

microbiome associations in obese rodents. J Proteome Res 2009;8(5):2361-75.

8. Yatsunenko T, Rey F, Manary M, Trehan I, Dominguez-Bello M, Contreras M, Magris

M, Hidalgo G, Baldassano R, Anokhin A, et al. Human gut microbiome viewed

across age and geography. Nature 2012;486:222-7.

9. Greiner T, Backhed F. Effects of the gut microbiota on obesity and glucose

homeostasis. Trends in Endocrinology and Metabolism 2011;22(4):117-23.

10. Turnbaugh P, Hamady M, Yatsunenko T, Cantarel B, Duncan A, Ley R, Sogin M,

Jones W, Roe B, Affourtit J, et al. A core gut microbiome in obese and lean twins.

Nature 2009;457:480-4.

11. Hildebandt M, Hoffmann C, Sherrill-Mix S, Keilbaugh S, Hamaday M, Chen Y,

Knight R, Ahima R, Bushman F, Wu G. High-fat diet determines the composition of

the murine gut microbiome independently of obesity. Gastroenterology

2009;137(5):1716-24.

12. Turnbaugh P, Bäckhed F, Fulton L, Gordon J. Diet-induced obesity is linked to

marked but reversible alterations in the mouse distal gut microbiome. Cell Host

Microbe 2008;17:213-23.

34

13. Walker A, Ince J, Duncan S, Webster L, Holtrop G, Ze X, Wu VCH. Dominant and

diet responsive groups of bacteria within the human colonic microbiota. ISME J

2011;5(2):220-30.

14. Backhed F, Ding H, Wang T, Hooper L, Koh G, Nagy A, Semenkovich C, Gordon J.

The gut microbiota as an environmental factor that regulates fat storage. Proc Natl

Acad Sci 2004;101:15718-23.

15. Furet J, Kong L, Tap J, Poitou C, Basdevant A, Bouillot J, Mariat D, Corthier G,

Doré J, Henegar C, et al. Differential adaptation of human gut microbiota to bariatric

surgery-induced weight loss: Links with metabolic and low-grade inflammation

markers. Diabetes 2010;59(12):3049-57.

16. Ley R, Turnbaugh P, Klein S, Gordon J. Microbial ecology: Human gut microbes

associated with obesity. Nature 2006;444:1022-3.

17. Cani P, Amar J, Iglesias M, Poggi M, Knauf C, Bastelica D, Neyrinck A, Fava F,

Tuohy K, Chabo C, et al. Metabolic endotoxemia initiates obesity and insulin

resistance. Diabetes 2007;56:1761-72.

18. Duncan S, Lobley G, Holtrop G, Ince J, Johnstone A, Louis P, Flint H. Human

colonic microbiota associated with diet, obesity, and weight loss. Int J Obes

2008;32(11):1720-4.

19. Nadal I, Santacruz A, Marcos A, Warnberg J, Garagorri J, Moreno L, Martin-Matillas

M, Campoy C, Martí A, Moleres A, et al. Shifts in clostridia, bacteroides, and

immunoglobulin-coating fecal bacteria associated with weight loss in obese

adolescents. Int J Obes 2008;33(7):758-67.

20. Santacruz A, Marcos A, Wärnberg J, Martí A, Martin-Matillas M, Campoy C,

Moreno L, Veiga O, Redondo-Figuero C, Garagorri J, et al. Interplay between

weight loss and gut microbiota composition in overweight adolescents. Obesity

2009;17(10):1906-15.

21. Gill S, Pop M, Deboy R, Eckburg P, Turnbaugh P, Samuel B, Gordon J, Relman D,

Fraser-Liggett C, Nelson K. Metagenomic analysis of the human distal gut

microbiome. Science 2006;312(5778):1355-9.

22. Hooper L, Midtvedt T, Gordon J. How host-microbial interactions shape the nutrient

environment of the mammalian intestine. Annu Rev Nutr 2002;22:283-307.

23. Ridlon J, Kang D, Hylemon P. Bile salt biotransformations by human intestinal

bacteria. J Lipid Res 2006;47(2):241-59.

35

24. Murphy E, Cotter P, Healy S, Marques T, O'Sullivan O, Fouhy F, Clarke S, O'Toole

P, Quigley E, Stanton C, et al. Composition and energy harvesting capacity of the

gut microbiota: Relationship to diet, obesity and time in mouse models. Gut

2010;59(12):1635-42.

25. Sanz Y, Santacruz A, Gauffin P. Gut microbiota in obesity and metabolic disorders.

Proc Nutr Soc 2010;69(3):434-41.

26. Zeyda M, Stulnig T. Adipose tissue macrophages. Immunol Lett 2007;112(2):61-7.

27. Cani P, Delzenne N. Interplay between obesity and associated metabolic disorders:

New insights into the gut microbiota. 2009;9(6):737-43.

28. Bolm M, Jansen M, Schnabel R, Chhatwal G. Hydrogen peroxide-mediated killing of

Caenorhabditis elegans: A common feature of different streptococcal species.

Infect.Immun. 2004;72(2):1192-4.

29. Darby C, Hsu J, Ghori N, Falknow S. Caenorhabditis elegans: Plague bacteria

biofilm blocks food intake. Nature 2002;417:243-4.

30. Schulenburg H, Kurz C, Ewbank J. Evolution of the innate immune system: The

worm perspective. Immunological Reviews 2004;198:36-58.

31. Aballay A, Drenkard E, Hilbun L, Ausubel F. Caenorhabditis elegans innate immune

response triggered by Salmonella enterica requires intact LPS and is mediated by a

MAPK signaling pathway. Curr Biol 2003;13(1):47-52.

32. Kim D, Feinbaum R, Alloing G, Emerson F, Garsin D, Inoue H, Tanaka-Hino M,

Hisamoto N, Matsumoto K, Tan M, et al. A conserved p38 MAP kinase pathway in

Caenorhabditis elegans innate immunity. Science 2002;297(5581):623-6.

33. Garsin DA, Villanueva JM, Begun J, Kim DH, Sifri CD, Calderwood SB, Ruvkun G,

Ausubel FM. Long-lived C. elegans daf-2 mutants are resistant to bacterial

pathogens. Science 2003;300(5627):1921.

34. Antebi A. Genetics of aging in Caenorhabditis elegans. PLoS Genetics

2007;3(9):1565-71.

35. Yang A, Jin C, Gao L, Fang W, Gu R, Qian J, Jiao X. Alleviating effects of

Lactobacillus strains on pathogenic Vibrio parahaemolyticus-induced intestinal fluid

accumulation in the mouse model. FEMS 2012.

36

36. Wang C, Wang J, Gong J, Yu H, Pacan J, Niu Z, Si W, Sabour P. Use of

Caenorhabditis elegans for preselecting Lactobacillus isolates to control Salmonella

typhimurium. Journal of Food Protection 2011;74(1):86-93.

37. Kim Y, Mylonakis E. Caenorhabditis elegans immune conditioning with the probiotic

bacterium Lactobacillus acidophilus strain NCFM enhances gram-positive immune

response. Infection and Immunity 2012;80(7):2500-8.

38. Ikeda T, Yasui C, Hoshino K, Arikawa K, Nishikawa Y. Influence of lactic acid

bacteria on longevity of Caenorhabditis elegans and host defense against Salmonella

enterica serovar enteritidis. Applied and Environmental Microbiology 2007:6404-9.

39. Komura T, Ikeda T, Yasui C, Saeki S, Nishikawa Y. Mechanism underlying

prolongevity induced by bifidobacteria in Caenorhabditis elegans. Biogerontology

2013;14:73-87.

40. Lee J, Yun H, Cho K, Oh S, Kim S, Chun T, Kim B, Whang K. Evaluation of

probiotic characteristics of newly isolated Lactobacillus spp.: Immune modulation

and longevity. International Journal of Food Microbiology 2011;148:80-6.

41. Pagnini C, Saeed R, Bamias G, Arseneau K, Pizarro T. Probiotics promote gut health

through stimulation of epithelial innate immunity. PNAS 2010;107(1):454-9.

42. Perdigon G, Maldonado Galdeano C, Valdez J, Medici M. Interaction of lactic acid

bacteria with the gut immune system. European Journal of Clinical Nutrition

2002;56:S21-6.

43. Fitzenberger E, Deusing D, Wittkop A, Kler A, Kriesl E, Bonnlander B, Wenzel U.

Effects of plant extracts on the reversal of glucose-induced impairment of stress-

resistance in Caenorhabditis elegans. Plant Foods Hum Nutr 2014;69(1):78-84.

44. Guha S, Cao M, Kane R, Savino A, Zou S, Dong Y. The longevity effect of cranberry

extract in Caenorhabditis elegans is modulated by daf-16 and osr-1. Age

2013;35:1559-74.

45. Howell A. Bioactive compounds in cranberries and their role in prevention of urinary

tract infections. Mol Nutr Food Res 2007;51:732-7.

46. Walker E, Barney D, Mickelsen J, Walton R, Mickelsen RJ. Cranberry concentrate:

UTI prophylaxis. J Fam Pract 1997;45:167-8.

47. Steinberg D, Feldman M, Ofek I, Weiss E. Effect of a high-molecular-weight

component of cranberry on constituents of dental biofilm. J Antimicrob Chemother

2004;54:86-9.

37

48. Burger O, Weiss E, Sharon N, Tabak M, Neeman I, Ofek I. Inhibition of Helicobacter

pylori adhesion to human gastric mucus by a high-molecular-weight constituent of

cranberry juice. Crit Rev Food Sci Nutr 2002;42:279-84.

49. Liu R. Potential synergy of phytochemicals in cancer prevention: Mechanism of

action. J Nutr 2004;134:3479S-85S.

50. Dinh J, Angeloni J, Pederson D, Wang X, Cao M, Dong Y. Cranberry extract

standardized for proanthocyanidins promotes the immune response of

Caenorhabditis elegans to Vibrio cholerae through the p38 MAPK pathway and

HSF-1. Plos One 2014;9(7).

51. Lee S, Murphy C, Kenyon C. Glucose shortens the lifespan of C. elegans by

downregulating DAF-16/FOXO activity and aquaporin gene expression. Cell

Metabolism 2009;10:379-91.

52. Zhao Y, Zhao L, Zheng Z, Fu T, Guo H, Ren F. Lactobacillus salivarius strain

FDB89 induced longevity in caenorhabditis elegans by dietary restriction. Journal of

Microbiology 2013;51(2):183-8.

53. Vilahur G, Lopez-Bernal S, Camino S, Mendieta G, Padro T, Badimon L.

Lactobacillus planatarum CECT 7315/7316 intake modulates the acute and chronic

innate inflammatory response. European Journal of Nutrition 2014.

54. Coskun S, Aslim B, Yuksekdag Z. Effect of two strains of Lactobacillus delbrueckii

subsp. bulgaricus on nitric oxide generation and antioxidant status of rat small

intestine. Med Chem Res 2010;19:1082-91.

55. Gusarov I, Gautier L, Smolentseva O, Shamovsky I, Eremina S, Mironov A, Nudler

E. Bacterial nitric oxide extends the lifespan of C. elegans. Cell 2013;152(4):818-30.

56. Pereira D, Gibson G. Cholesterol assimilation by lactic acid bacteria and

bifidobacteria isolated from the human gut. Applied and Environmental

Microbiology 2002;68(9):4689-93.

57. Barbieri M, Bonafè M, Franceschi C, Paolisso G. Insulin/IGF-I-signaling pathway:

An evolutionarily conserved mechanism of longevity from yeast to humans. Am J

Physiol Endocrinol Metab 2003;285(5):1064-71.

58. Katic M, Kahn C. The role insulin and IGF-1 signaling in longevity. Cell Mol Life

Sci 2005;62(3):320-43.

59. Kenyon W, Thomas S, Johnson E, Pallen M, Spector M. Shifts from glucose to

certain secondary carbon-sources result in activation of the extracytoplasmic

38

function sigma factor sigmaE in Salmonella enterica serovar typhimurium.

Microbiology 2005;151:2373-83.

60. Garigan D, Hsu A, Fraser A, Kamath R, Ahringer J, Kenyon C. Genetic analysis of

tissue aging in Caenorhabditis elegans: A role for heat-shock factor and bacterial

proliferation. Genetics 2002;161(3):1101-12.

61. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. A C. elegans mutant that lives

twice as long as wild type. Nature 1993;366(6454):461-4.

62. Kishida K, Shimomura I, Kondo H, Kuriyama H, Makino Y, Nishizawa H, Maeda N,

Matsuda M, Ouchi N, Kihara S, et al. Genomic structure and insulin-mediated

repression of the aquaporin adipose (AQPap), adipose-specific glycerol channel. J

Biol Chem 2001;276(39):36251-60.

63. Kuriyama H, Shimomura I, Kishida K, Kondo H, Furuyama N, Nishizawa H, Maeda

N, Matsuda M, Nagaretani H, Kihara S, et al. Coordinated regulation of fat-specific

and liver-specific glycerol channels, aquaporin adipose and aquaporin 9. Diabetes

2002;51(10):2915-21.

64. Barsyte D, Lovejoy D, Lithgow G. Longevity and heavy metal resistance in daf-2

and age-1 long-lived mutants of Caenorhabditis elegans. FASEB 2001;15:627-34.