influences of probiotics and prebiotics on the microbiome...

Influences of Probiotics and

Prebiotics on the Microbiome:

A Case for Lactose Intolerance

M. Andrea Azcarate-Peril, Ph. D. University of North Carolina at Chapel Hill

Disclosures

M. Andrea Azcarate-Peril, PhD, has disclosed that she has

served as a consultant for Ritter Pharmaceuticals.

Contact:

https://www.med.unc.edu/microbiome/

[email protected]

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facilities and expertise to characterize complex microbial

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Core manager

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Associate

Jason Arnold

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Dan Moore

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https://www.med.unc.edu/microbiome/

mailto:[email protected]

Vitamins and

supplements Age Diet

Host genetic

background Surgical

interventions

Antibiotics

Pathogens

Probiotics

Fecal

microbiota

transplantation

Breast milk

Bacteriophages

Fiber

Nutraceuticals Meet Precision Medicine: Microbiome Modulators

Prebiotics

Any compound, microorganism, or

environmental factor that generates a compositional or functional modification

of the microbiome

Vitamins and

supplements

Age Diet

Host genetic background

Surgical

interventions

Antibiotics

Pathogens

Probiotics

Fecal microbiota

transplantation

Breast milk

Bacteriophages

Fiber

Nutraceuticals Meet Precision Medicine: Microbiome Modulators

Prebiotics

ISAPP definition 2017

ISAPP definition 2017

SCFAs

HDAC inhibition

Energy

GPR signaling

Liver

Prebiotics: A selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health (ISAPP, 2017)

β (1-4) Galacto-oligosaccharides (GOS)

Added to infant formulas to mimic

the beneficial effects of human

breast milk oligosaccharides

Generic formula is β (1-4)

[DGalactose]n-D-Glucose where

n ranges between 3 and 10 sugar molecules

Products of metabolism include SCFAs, lactate, and gases

Mean proportions of the three

individual samples taken

during the treatment periods

for each subject

Representative sequences of all OTUs

detected that were significantly affected by

GOS in individual subjects

Consumption of GOS Results in a Highly Specific Bifidogenic Response in Humans

Davis et al. 2011.

β (1-4) Galacto-oligosaccharides (GOS)

Modified from Misson et al., 2016

+

H20

-galactosyl

Acceptor Transgalactosylation

Hydrolysis 𝛃-galactosidase

𝛃-galactosidase

𝛃-galactosidase

Sporobolomyces singularis

Bruno-Barcena and Azcarate-Peril. J Funct Foods. 2015; Dagher et al. AEM. 2013.

Heterologous Expression of a Bioactive β-Hexosyl-Transferase, an

Enzyme Producer of Prebiotics, from Sporobolomyces singularis

1E-3

0.01

0.1

1

Rela

tive a

bundance (

%)

Control GOS90

Day 7

GOS90

Day 14

R NR.

GOS90 Day 14

Bifidobacterium

*

Pure GOS Enhance Bifidobacterium and Its Metabolic

Activity in the Mouse Gut Microbiome

Monteagudo-Mera et al. 2016.

Common symptoms of lactose intolerance include:

• Bloating, a feeling of fullness or swelling

• Pain

• Diarrhea

• Gas

• Nausea

What is lactose intolerance?

• Lactose in dairy products is usually digested in the

small intestine by the enzyme lactase.

• If small intestine does not produce enough lactase,

lactose cannot be broken down and goes to the colon

where bacteria will break down the lactose generating

fluid and gas, and causing symptoms

Source: NIH NIDDK

Glucose

Galactose Lactose

Lactase-phlorizin hydrolase

(LPH)

Lacta

se e

xpre

ssio

n

Time

Lactase persistence (LP)

Lactase deficience –

Lactose intolerance

birth weaning

Intestinal Lactase and Lactose Intolerance

Distribution of LP and the −13910*T Allele in the “Old World”

Ingram et al. 2008; Gerbault et al. 2011

Family: Glycos_transf_2 (PF00535)

Bacterial 𝛃-galactosidases in the Gut

Baseline

Lactose Challenge

Post-

Treatment

Lactose

Challenge

Follow-up

Lactose

Challenge

35-Days GOS

GOS

Placebo

(Sweetose)

Lactose/Dairy

consumption

Encouraged

Lactose

Avoidance Diet

Screening

Day 0 Day 36 Day 66 30 Days

(Stop Treatment)

Can Pure GOS Increase the Numbers of Lactose-Metabolizing Bacteria?

Savaiano et al. 2013.

Can Pure GOS Increase the Numbers of Lactose-Metabolizing Bacteria?

Savaiano et al. 2013.

Dairy Consumption Impacted the Overall Composition and Diversity of

the Gut Microbiome of Lactose Intolerant Individuals

Azcarate-Peril et al. 2017.

GOS Impacted Selected Taxa of the Gut Microbiome



GOS Increased Abundance of Bifidobacterium Species

Taxa Significantly Associated with Specific Symptoms

M. Andrea Azcarate-Peril et al. PNAS. 2017;114:E367-E375.

Interindividual

variation

Intra-species

genomic and phenotypic variability

What Factors Impact GOS Response?

RE

SP

ON

DER

S N

ON

RESP

ON

DER

Host Genetic Variation Correlated with Bacterial Taxa

• By mining the shotgun metagenomic data from the Human Microbiome Project for host DNA reads, gathered

information on host genetic variation for 93 individuals for whom bacterial abundance data were also available.

• Found 83 associations between genetic variation in host coding sequence and abundance of specific microbial

taxa (genome-wide false discovery rate Q-value <0.1).

Relative abundances of the heritable genus Bifidobacterium are associated with genetic variants in the genomic locus containing

the gene LCT

Blekhman et al. 2015; Goodrich et al. 2016.

Interindividual

variation

Intra-species

genomic and phenotypic variability

What Factors Impact GOS Response?

RE

SP

ON

DER

S N

ON

RESP

ON

DER

Arnold et al. 2018a

Variability in Galacto-Oligosaccharide Utilization by Intestinal Lactobacillus rhamnosus

A

A

A

A

B B

C B B

C

B B

C C C C

Arnold et al. 2018b


bgl PTS IIB p-βgal PTS IIA

⍺-gal


PTS IIB bgl PTS IIA p-βgal

βgal-3 PTS IIB PTS IIC PTS IID PTS IIA

βgal-Large Subunit βgal-Small

Subunit

βgal AMC143_βgal: BT102_02540

AMC143: NA LGG: NA Lc705: NA

AMC143: BT102_00105 LGG: LGG_RS03055-03070 Lc705: Lc705_RS02930-02945

AMC143: BT102_01370-01385 LGG: NA Lc705: Lc705_RS01460-01475

AMC143: BT102_01435-01460 LGG: LGG_RS01605-01625 Lc705: Lc705_RS01540-01560

AMC143: NA LGG: NA Lc705: NA Lc705 Plasmid: Lc705_p00050

AMC010: NA LGG: LGG_RS02250 Lc705: NA

Absent 98.2% Absent

Absent Absent Absent 99.6%


99% 98.8% 100%

Absent* Absent* Absent

99.2% 99% 100%

99.3% 99% 99.9%

97.7% 98.7% 100%

96.4% 97.5% 99.7%

97.4% Absent 100%

98.9% Absent 99.9%

98.1% Absent 100%

97.5% Absent 100%

99.8% 98.7% 100%

98.4% 98.6% 99.9%

98.1% 98.2% 100%

99.7% 98.8% 100%

Absent* Absent* Absent*



Absent Absent Absent

AMC010 – lac1: BVG98_13950-13965

AMC010 – lac2: BVG98_15885-15910

AMC010 – lac3: BVG98_10115-10130

AMC010 – lac4: BVG98_10195-10220

AMC010 – lac5: BVG98_07735-07750

Arnold et al. 2018b

A

A

A

A

B B

C B B

C

B B

C C C C

Arnold et al. 2018b



⍺-gal


PTS IIB bgl PTS IIA p-βgal

βgal-3 PTS IIB PTS IIC PTS IID PTS IIA

βgal-Large Subunit βgal-Small

Subunit

βgal AMC143_βgal: BT102_02540

AMC143: NA LGG: NA Lc705: NA

AMC143: BT102_00105 LGG: LGG_RS03055-03070 Lc705: Lc705_RS02930-02945

AMC143: BT102_01370-01385 LGG: NA Lc705: Lc705_RS01460-01475

AMC143: BT102_01435-01460 LGG: LGG_RS01605-01625 Lc705: Lc705_RS01540-01560

AMC143: NA LGG: NA Lc705: NA Lc705 Plasmid: Lc705_p00050

AMC010: NA LGG: LGG_RS02250 Lc705: NA

Absent 98.2% Absent



99% 98.8% 100%

Absent* Absent* Absent

99.2% 99% 100%

99.3% 99% 99.9%

97.7% 98.7% 100%

96.4% 97.5% 99.7%

97.4% Absent 100%

98.9% Absent 99.9%

98.1% Absent 100%

97.5% Absent 100%

99.8% 98.7% 100%

98.4% 98.6% 99.9%

98.1% 98.2% 100%

99.7% 98.8% 100%




Absent Absent Absent

AMC010 – lac1: BVG98_13950-13965

AMC010 – lac2: BVG98_15885-15910

AMC010 – lac3: BVG98_10115-10130

AMC010 – lac4: BVG98_10195-10220

AMC010 – lac5: BVG98_07735-07750

Arnold et al. 2018b

Genomic and physiological characteristics of strains impact GOS utilization

Inactivation of p-𝛃gal_3 Abolished Growth and Utilization of

GOS and Lactose by AMC143 at 24 Hours

Arnold et al. 2018b

Conclusions

• The nature and purity of the prebiotic matters.

• Pure GOS has a marginal effect on overall gut microbiome composition and diversity

• They increase the abundance of intestinal lactose-metabolizing, beneficial bacteria.

• Lactose-metabolizing bacteria remove undigested lactose from the gut, thus reducing

the gas production and water secretion that produce symptoms.

• Increased bifidobacteria is inversely correlated with pain and cramps associated with

lactose intolerance.

• Host genotype impacts abundance of gut bifidobacteria potentially modulating

prebiotic response.

• Response to GOS can vary due to intra-species genetic and phenotypic differences

of intestinal bacteria.

Todd Klaenhammer

NC State University

Andrew Ritter

Ritter Pharmaceuticals

Dennis Savaiano

Purdue University

Scott Magness

UNC Chapel Hill

“The dependence of the intestinal microbes on the food

makes it possible to adopt

measures to modify the flora in

our bodies and to replace the harmful microbes by useful

microbes”

E. Metchnikoff, Optimistic studies

New York: Putman’s Sons, 1908, 161-183

Jose Bruno Barcena

NC State University

Acknowledgements

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