123 
 

Journal homepage: www.fia.usv.ro/fiajournal 
Journal of Faculty of Food Engineering,  

Ştefan cel Mare University of Suceava, Romania  
Volume XII, Issue 2 – 2013, pag. 123 - 129 

 
 
 
 

ANTI M IC ROB I AL ACTI VIT Y OF THE PR OB IOT IC S TR AI N 

LACT OB A CILLUS D ELBRU E CKI I SS P. BULG A R IC US GB OF HUM AN 

ORIGI N AGA INS T PA THO GENS  

 
*Rositsa DENKOVA1, Zapryana DENKOVA2, Velichka YANAKIEVA2, Svetla ILIEVA1  

 
1Sofia University "St. Kliment Ohridski", Faculty of Biology, Department of Biotechnology, 

rositsa_denkova@mail.bg  
2University of Food Technology, Department of Microbiology, zdenkova@abv.bg 

* Corresponding author  
Received  May 9th 2013, accepted 15th June 2013 

 
 
Abstract: Probiotics help restore the balance of the gastrointestinal microflora, but only strains with 
certain properties can be included in their formulations. One of the requirements for a strain to be 
probiotic is to exhibit inhibitory activity against pathogens.The antimicrobial activity of the strain 
Lactobacillus delbrueckii ssp. bulgaricus GB against the pathogens E.coli ATCC 25922, E.coli ATCC 
8739, Salmonella abony NTCC 6017, Salmonella sp., Staphylococcus aureus ATCC 25293 and 
Proteus vulgaris J is determined by joint cultivation at 37±1°C.  Lactobacillus delbrueckii ssp. 
bulgaricus GB inhibits the growth of the pathogens - for 60 - 72 hours the numbers of viable cells of 
the pathogens are reduced. It has been shown that the changes in the proportions in the mixed 
populations are due to the accumulation of lactic and other organic acids which acidify the medium 
and change the conditions for the growth of the pathogens, leading to reduction of the number of 
viable cells of the pathogens. The demonstrated inhibitory activity of Lactobacillus delbrueckii ssp. 
bulgaricus GB makes the strain potentially probiotic and after additional studies it can be included in 
the composition of probiotics and functional foods. 
  
Keywords: Joint cultivation, Probiotic, E.coli, Salmonella, Staphylococcus, Proteus 
 
 
 
1. Introduction 
 
Maintaining the balance of the 
gastrointestinal microflora is necessary for 
good health. In order to restore the balance 
of the intestinal microflora it is neccessary 
to consummate food and concentrates 
containing beneficial lactobacilli and 
bifidobacteria, known as functional foods 
and probiotics, respectively. According to 
FAO probiotics are live microorganisms 
that have beneficial effect on the host 
when administered in adequate amounts [1, 
2]. 

The main components of probiotics are 
lactic acid bacteria (Lactobacillus, 
Enterococcus, Pediococcus, Lactococcus, 
Streptococcus, Leuconostoc) and 
bifidobacteria. They are also applied in the 
manufacture of probiotic foods [3, 4, 5], 
the largest proportion being the 
lactobacilli. But only lactobacilli with 
certain properties can be included in the 
composition of probiotics and probiotic 
foods [4, 5]: to be a part of the natural 
microflora in humans and animals; to be 
able to suppress and expel pathogenic and 
toxicogenic microorganisms from the 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XII, Issue 2 – 2013 

 
 

 
Rositsa DENKOVA, Zapryana DENKOVA, Velichka YANAKIEVA, Svetla ILIEVA , Antimicrobial activity of the 
probiotic strain lactobacillus delbrueckii ssp. Bulgaricus gb of human origin against pathogens, Food and Environment 
Safety, Volume XII, Issue 2 – 2013, pag.  123 - 129 
 

124 
 

biological niche; to allow industrial 
cultivation; to have antimicrobial activity 
against conditionally pathogenic, 
carcinogenic and pathogenic 
microorganisms; to have the ability to 
adhere to epithelial cells or cell lines; to be 
able to survive under the conditions in the 
stomach and the intestines (acidic pH in 
the stomach and bile) [8, 9]; to be able to 
reproduce in the intestinal tract; to produce 
antimicrobial substances; to modulate the 
immune response and to be safe for 
clinical and food applications. 
The studies of Saxelin et al., (1996 a, b), 
Donohue & Salminen, (1996), Salminen et 
al., (1998) [3, 4, 5, 10, 11, 12] demonstrate 
the safety of lactic acid bacteria and 
bifidobacteria and strains belonging to the 
genera Lactobacillus, Lactococcus and 
Bifidobacterium most often are assigned 
with GRAS status. 
The purpose of the present paper is to 
determine the antimicrobial activity of a 
probiotic strain of human origin 
Lactobacillus delbrueckii ssp. bulgaricus 
GB against the following pathogens: E.coli 
ATCC 25922, E.coli ATCC 8739, 
Salmonella abony NTCC 6017, Salmonella 
sp., Staphylococcus aureus ATCC 25293 
and Proteus vulgaris J. 
 
2.Materials and Methods  
 
1. Media 
Sterile skimmed milk with titrable acidity 
16-18ºT. Composition (g/dm3): skimmed 
milk powder (Scharlau). Sterilization - 15 
minutes at 118ºC. 
Saline solution. Composition (g/dm3): 
NaCl - 5. Sterilization - 20 minutes at 
121ºC. 
LAPTg10-broth. Composition (g/dm3): 
peptone - 15, yeast extract - 10; tryptone - 
10, glucose - 10. pH is adjusted to 6.6 - 6.8 
and Tween 80 - 1cm3/dm3 is added. 
Sterilization - 20 minutes at 121ºC. 

LAPTg10-agar. Composition (g/dm3): 
LAPTg10-broth + 2% agar. Sterilization - 
20 minutes at 121ºC. 
LBG-agar. Composition (g/dm3): Tryptone 
– 10, yeast extract - 5, NaCl – 10, glucose 
– 10, agar - 20. pH is adjusted to 7.5. 
Sterilization - 20 minutes at 121°C. 
2. Determination of the antimicrobial 
activity against pathogenic 
microorganisms 
To determine the antimicrobial activity of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB against pathogens a 48 hour culture of 
the Lactobacillus strain is used. In the 
mixtures are mixed 0.5 cm3 of the 
suspension of Lactobacillus delbrueckii 
ssp. bulgaricus GB, 0.5 cm3 of the 
suspension of the pathogen and 9 cm3 of 
culture medium (skimmed milk), and in 
the controls of the Lactobacillus strain or 
the pathogen 9.5 cm3 culture medium 
(skimmed milk) is mixed with 0.5 cm3 of 
the suspension of Lactobacillus delbrueckii 
ssp. bulgaricus GB or of the pathogen, 
respectively. Joint cultivation of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB and each of pathogens E.coli ATCC 
25922, E.coli ATCC 8739, Salmonella 
abony NTCC 6017, Salmonella sp., 
Staphylococcus aureus ATCC 25293 and 
Proteus vulgaris J under static conditions 
in a thermostat at 37±1°C for 72 hours, 
taking samples at the 0th, 12th, 24th  36th, 
48th, 60th and 72nd hour is conducted and 
the change in the titratable acidity and the 
concentration of viable cells of the 
pathogen and of Lactobacillus delbrueckii 
ssp. bulgaricus GB is monitored. 
3. Processing of the results 
Data from triplicate experiments is 
processed using the software MS Office 
Excel 2003 and Origin Pro 8.1, using 
statistical functions to determine the 
standard deviation and maximum error of 
assessment in the significance level of α 
<0,05. 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XII, Issue 2 – 2013 

 
 

 
Rositsa DENKOVA, Zapryana DENKOVA, Velichka YANAKIEVA, Svetla ILIEVA , Antimicrobial activity of the 
probiotic strain lactobacillus delbrueckii ssp. Bulgaricus gb of human origin against pathogens, Food and Environment 
Safety, Volume XII, Issue 2 – 2013, pag.  123 - 129 
 

125 
 

3. Results and Discussion 
 
The antimicrobial activity of Lactobacillus 
delbrueckii ssp. bulgaricus GB against the 
pathogens Escherichia coli ATCC 25922, 
Escherichia coli ATCC 8739, Salmonella 
sp. (clinical isolate), Salmonella abony 
NTCC 6017, Staphylococcus aureus 
ATCC 25293, Proteus vulgaris J during 
joint and separate cultivation at 37±1ºC in 
skimmed milk is determined. 
During separate cultivation Lactobacillus 
delbrueckii ssp. bulgaricus GB and the 
pathogens E.coli ATCC 25922 and E.coli 
ATCC 8739 for 12 to 24 hours at 37±1ºC 
accumulate high concentrations of viable 
cells. These high concentrations of living 
cells preserve Lactobacillus delbrueckii 
ssp. bulgaricus GB in a mixed population 
with the pathogen. In joint cultivation of 
the Lactobacillus strain and E.coli ATCC 
25922 at static conditions, an increase in 
the concentration of viable cells of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB is detected, while that of E.coli ATCC 
25922 starts to decrease after the first 12 
hours and at the 60th hour no viable cells of 
the pathogen are established (Fig. 1). 
  

 
 

Fig. 1. Survival of Lactobacillus delbrueckii ssp. 
bulgaricus GB and E.coli ATCC 25922 during 
separate cultivation and cultivation in a mixed 

population at 37±1ºC. 
 

In the study of the inhibitory activity of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB against E.coli ATCC 8739 the 
concentrations of viable cells of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB and of the pathogen E.coli ATCC 8739 
increase in the first 12 hours, which is 
consistent with the results obtained with 
E.coli ATCC 25922. After that the 
concentration of viable lactobacilli cells 
retains while that the pathogen is quickly 
reduced and at the 60th hour there are no 
living cells of the pathogen, similarly to 
the joint cultivation with E.coli ATCC 
25922 (Fig. 1 and Fig. 2). 
 

 
 

Fig. 2. Survival of Lactobacillus delbrueckii ssp. 
bulgaricus GB and E.coli ATCC 8739 during 

separate cultivation and cultivation in a mixed 
population at 37±1ºC. 

 
In tracking the change in the titratable 
acidity it is noticeable that the acidity 
values of both the two controls of the 
pathogens are significantly lower in 
comparison to the control of the 
Lactobacillus strain and of that of the two 
mixtures (Lactobacillus delbrueckii 
ssp.bulgaricus GB and E.coli ATCC 
25922; Lactobacillus delbrueckii ssp. 
bulgaricus GB and E.coli ATCC 8739) 
and the titratable acidity values of the 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XII, Issue 2 – 2013 

 
 

 
Rositsa DENKOVA, Zapryana DENKOVA, Velichka YANAKIEVA, Svetla ILIEVA , Antimicrobial activity of the 
probiotic strain lactobacillus delbrueckii ssp. Bulgaricus gb of human origin against pathogens, Food and Environment 
Safety, Volume XII, Issue 2 – 2013, pag.  123 - 129 
 

126 
 

mixtures are lower than the values of the 
control of Lactobacillus delbrueckii 
ssp.bulgaricus GB for each hour of 
sampling, which shows that the inhibition 
of the pathogen by Lactobacillus 
delbrueckii ssp.bulgaricus GB is due to a 
great extent to the acidification of the 
medium (Fig. 3). 
 
 

 
 

Fig. 3. Change of the titratable acidity of the 
medium during separate cultivation and 

cultivation in a mixed population of 
Lactobacillus delbrueckii ssp. bulgaricus GB and 

E.coli ATCC 8739 or E.coli ATCC 25922 at 
37±1ºC. 

 
During the cultivation of Lactobacillus 
delbrueckii ssp. bulgaricus GB and 
Salmonella sp. (clinical isolate) at 37±1°C, 
an increase in the concentration of viable 
cells of L.d.ssp.bulgaricus GB as well as of 
Salmonella sp. during the first 12 hours is 
observed, after which the concentration of 
viable lactobacilli cells continues to 
increase, while that of the pathogen is 
reduced and by the 72nd hour no living 
cells of Salmonella sp. are defined (Fig. 4). 
 

 
 

Fig. 4. Survival of Lactobacillus delbrueckii ssp. 
bulgaricus GB and Salmonella sp. during 

separate cultivation and cultivation in a mixed 
population at 37±1ºC. 

 
In the joint development of Salmonella 
abony NTCC 6017 and Lactobacillus 
delbrueckii ssp. bulgaricus GB an increase 
of the cell concentration of the 
Lactobacillus strain and of the pathogen in 
the first 12 hours is observed.  
 

 
 

Fig. 5. Survival of Lactobacillus delbrueckii ssp. 
bulgaricus GB and Salmonella abony NTCC 

6017 during separate cultivation and cultivation 
in a mixed population at 37±1ºC. 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XII, Issue 2 – 2013 

 
 

 
Rositsa DENKOVA, Zapryana DENKOVA, Velichka YANAKIEVA, Svetla ILIEVA , Antimicrobial activity of the 
probiotic strain lactobacillus delbrueckii ssp. Bulgaricus gb of human origin against pathogens, Food and Environment 
Safety, Volume XII, Issue 2 – 2013, pag.  123 - 129 
 

127 
 

 
 

Fig. 6. Change in the titratable acidity of the 
medium during separate cultivation and 

cultivation in a mixed population of 
Lactobacillus delbrueckii ssp. bulgaricus GB and 
Salmonella abony NTCC 6017 or Salmonella sp. 

at 37±1°C. 
 
From the 12th to the 60th hour the 
concentration of lactic acid bacteria 
continues to increase while that of 
Salmonella abony NTCC 6017 decreases 
reaching 0 at the 72nd hour as in the study 
of the antimicrobial activity of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB against Salmonella sp. (Fig. 4 and Fig. 
5), which is mainly a result of the 
acidification of the medium, due to the 
accumulation of lactic acid and other 
organic acids (Fig. 6). 
In the separate cultivation of Lactobacillus 
delbrueckii ssp. bulgaricus GB and 
Staphylococcus aureus ATCC 25293 both 
strains accumulate high concentration of 
active cells. During the joint cultivation 
Lactobacillus delbrueckii ssp. bulgaricus 
GB and Staphylococcus aureus ATCC 
25293 a decrease in the concentration of 
the pathogen, starting after 12th hour is 
observed. As in the determination of the 
antimicrobial activity against the two 
strains of Escherichia coli, Salmonella sp. 
and Salmonella abony NTCC 6017, the 
number of living cells of this pathogen is 
reduced completely under the action of 

Lactobacillus delbrueckii ssp. bulgaricus 
GB. At the end of the process no living 
cells of the pathogen are detected (Fig. 7), 
mainly due to the lowering of the pH as a 
result of the increase in the titratable 
acidity (Fig. 8). 
  

 
 

Fig. 7. Survival of Lactobacillus delbrueckii ssp. 
bulgaricus GB and Staphylococcus aureus ATCC 

25293 during separate cultivation and  
cultivation in a mixed population at 37±1ºC. 

 

 
 

Fig. 8. Change in the titratable acidity of the 
medium during separate cultivation and 

cultivation in a mixed population of 
Lactobacillus delbrueckii ssp. bulgaricus GB and 
Staphylococcus aureus ATCC 25293 at 37±1°C. 

 
 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XII, Issue 2 – 2013 

 
 

 
Rositsa DENKOVA, Zapryana DENKOVA, Velichka YANAKIEVA, Svetla ILIEVA , Antimicrobial activity of the 
probiotic strain lactobacillus delbrueckii ssp. Bulgaricus gb of human origin against pathogens, Food and Environment 
Safety, Volume XII, Issue 2 – 2013, pag.  123 - 129 
 

128 
 

In studying the antimicrobial activity of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB against Proteus vulgaris J the 
concentration of viable cells of 
Lactobacillus delbrueckii ssp. bulgaricus 
GB and of the pathogen increases in the 
first 12 hours. After that the number of 
living cells of the Lactobacillus strain 
continues to grow at a slower rate, while 
the cell number of the pathogen is reduced, 
reaching 0 at the 60th hour (Fig. 9). 
 

 
 

 Fig. 9. Survival of Lactobacillus delbrueckii ssp. 
bulgaricus GB and Proteus vulgaris J during 

separate cultivation and cultivation in a mixed 
population at 37±1ºC. 

 
 
For each sampling the acidity of the 
control of the pathogen is lower than that 
of the control of the Lactobacillus strain 
and that of the mixture, which again 
indicates that the inhibition of pathogens 
by lactobacilli is largely a result of the 
decrease of the pH of the medium, 
resulting from the accumulation of organic 
acids produced by the lactobacilli (Fig. 
10). 
 

  
Fig. 10. Change in the titratable acidity of the 

medium during separate cultivation and 
cultivation in a mixed population of 

Lactobacillus delbrueckii ssp. bulgaricus GB and 
Proteus vulgaris J at 37±1ºC. 

 
 
4. Conclusion 
 
The strain Lactobacillus delbrueckii ssp. 
bulgaricus GB inhibits the growth of the 
pathogens E.coli ATCC 25922, E.coli 
ATCC 8739, Salmonella abony NTCC 
6017, Salmonella sp., Staphylococcus 
aureus ATCC 25293 and Proteus vulgaris 
J. In joint cultivation of Lactobacillus 
delbrueckii ssp. bulgaricus GB and all of 
the pathogens the Lactobacillus strain 
retains a high concentration of viable cells, 
while the cell number of the pathogen is 
reduced, the degree of reduction being 
strainspecific and partly due to the change 
in the acidity of the medium as a result of 
the acid production by Lactobacillus 
delbrueckii ssp. bulgaricus GB. The 
antimicrobial activity against pathogens 
makes the tested strain a potentially 
probiotic one, which after further studies 
may be included in the composition of 
probiotic preparations for prophylaxis and 
treatment. 
 
 
 



Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava 
Volume XII, Issue 2 – 2013 

 
 

 
Rositsa DENKOVA, Zapryana DENKOVA, Velichka YANAKIEVA, Svetla ILIEVA , Antimicrobial activity of the 
probiotic strain lactobacillus delbrueckii ssp. Bulgaricus gb of human origin against pathogens, Food and Environment 
Safety, Volume XII, Issue 2 – 2013, pag.  123 - 129 
 

129 
 

 
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