Maja Gajić1, Nikoleta Lugonja2, Emilija Brdarić3, Maja Tolinački3, Aleksandra Nikolić3, Vladimir P. Beškoski4

1Faculty of Biology, University of Belgrade, Serbia, 2Institute of Chemistry, Technology and Metallurgy, University of

Belgrade, Serbia, 3Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia, 4Faculty of

Chemistry, University of Belgrade, Serbia

Prebiotics are defined as non-digestible ingredients of food that canarrive to the gastrointestinal tract unchanged, selectively stimulate thegrowth and activity of bacterial species (probiotics), and thus attempt toimprove host health [1]. Different carbohydrates as lactulose,fructooligosaccharides and galactooligosaccharides, have positive effecton growth of Bifidobacterium and Lactobacillus [2,3]. However, effects ofmicrobial exopolysaccharides such as levan and pullulan on their growthremains unexplored.

The aim of this study was to investigate the effects of differentcarbohydrates (CHs) on the growth of selected bacterial species:Bifidobacterium bifidum, Bifidobacterium breve and Lactobacillusrhamnosus, as well as on their consortium.

Investigation of the effects of tested CHs on artificial consortiumcomposed of three commercial bacterial stains: Bifidobacterium breveATCC 15700, Bifidobacterium bifidum ATCC 11863 and Lactobacillusrhamnosus ATCC 7469. was carried out by comparing the growth ofbacteria in a consortium with different carbon sources. Five modifiedsubstrates were prepared by replacment of mains sorce of sugars with:GOS, lactulose, pullulan, levan, pectin and digested milk from mothers ofpremature born babies (MMPB). In addition, starch was excluded fromthem too. After 72 hours incubation, under anaerobic conditions,biochemical changes (such as pH value) were examined in the testedsubstrates. Moreover, the growth of bifidobacteria and lactobacilli, andtheir consortium on substrates with different carbohydrates sources wasanalyzed by determination of CFU/mL and by Denaturing Gradient GelElectrophoresis.

Introduction and Aim

1. Gibson, G. R., & Roberfroid, M. B. (1995). Dietary modulation of

the human colonic microbiota: introducing the concept of prebiotics.

J Nutr, 6(125), 1401-1412

2. Roberfroid, M., Gibson, G. R., Hoyles, L., McCartney, A. L., Rastall,

R., Rowland, I., . . . Meheust, A. (2010). Prebiotic effects: metabolic

and health benefits. Br J Nutr, 104 Suppl 2, S1-63.

doi:10.1017/S0007114510003363

3. Wegh, C. A. M., Schoterman, M. H. C., Vaughan, E. E., Belzer, C.,

& Benninga, M. A. (2017). The effect of fiber and prebiotics on

children's gastrointestinal disorders and microbiome. Expert Rev

Gastroenterol Hepatol, 11(11), 1031-1045.

doi:10.1080/17474124.2017.1359539

References

• Levan showed the highest prebiotic potential compared to other

tested carbohydrates.

• Pullulan showed the positive effect only on the growth of L.

rhamnosus.

• Pectin had lower effect on colony growth, as well as pH decrease

compared to other substrates.

• MMPB showed stimulating effect on growth of tested strains.

• In vitro test can quickly and objectively determine the bifidogenic

effect of different carbohydrates.

• Clinical tests are necessary to determine the acceptance and

biological value of potential prebiotics

Conclusion

Figure 5. Grafic representation of bands on DGGE gelFigure 4. DGGE gel

Results

Legend:

A – B. bifidum

B – L. rhamnosus

C – B. breve

D – Without CHs t0h

E – Without CHs t72h

F – Lactulose t72h

G – GOS t72h

H – MMPB t72,h

I – Levan t72h

J –Pullulan t72h

1

2

3

4

5

6

7

9

10

11

12

13

14

15a b c d e f g h i j

Figure 3. Influence of selected CH on L. rhamnosus growth

during anaerobic fermentation - The figure shows the CFU/mL

ratio on the logarithmic scale, in t0h and t72h

Figure 1. Change in pH value in suspension of

consortium treated by milk from mothers of

premature bornes babies

Figure 2. Changes in pH values in suspension of

consortiums treated by different CHs

As it can be seen from figure 1, compare to t0h (pH 6,8 ± 0,2),

the highest decrease of pH value (t72h) is observed in artificial

consortium with MMPB. Compare to other CHs (figure 2) the

pH of artificial consortium with pullulan decreased to 4.67,

while pH values in consortiums with pectin and levan have

decreased only to 5.29 i 5.53 followingly.

The highest increase in the number of colonies of L.

rhamnosus has been observed in the consortium with

levane, from 1.5x105 to 6.9x106 CFU/mL (figure 3). L.

rhamnosus number also increased after pullulan

fermentation by artificial consortium (2.95x106 CFU/mL) as

well as after fermentation of MMPB, while the increase was

smaller in pectin.

Change in the number of microorganisms

pH

Investigation of the effect of selected carbohydrates on the growth of

Bifidobacterium bifidum, Bifidobacterium breve and Lactobacillus rhamnosus

Materials and methods

0

1

2

3

4

5

6

7

pH

Valu

e

Tested CHs

MMPB

t 0h

t 72h

0

1

2

3

4

5

6

7

8

pH

va

lue

Tested CHs

Different CHs

t 0h starting value

t 72h Without CHs

t 72h Lactulose

t 72h GOS

t 72h Levan

t 72h Pullulan

t 72h Pectin

1,E+00

1,E+01

1,E+02

1,E+03

1,E+04

1,E+05

1,E+06

1,E+07

Without CHs Lactulose GOS MMPB Levan Pullulan Pectin

Tota

l n

um

ber

of

L. rh

am

nosu

s(l

og

CF

U/m

L)

Tested CHs

Lactobacillus rhamnosus

t 0h

t 72h