Content uploaded by Ibrahim A. Abd El Gawad
Author content
All content in this area was uploaded by Ibrahim A. Abd El Gawad on Oct 18, 2014
Content may be subject to copyright.
Volume 4 • Issue 5 • 1000303
J Nutr Food Sci
ISSN: 2155-9600 JNFS, an open access journal
Open Access
Research Article
Nutrition and Food
Sciences
Abd El-Gawad et al., J Nutr Food Sci 2014, 4:5
http://dx.doi.org/10.4172/2155-9600.1000303
*Corresponding author: Abd El-Gawad IA, Dairy Science and Technology
Department, Faculty of Agriculture, Cairo University, Giza, Egypt, Tel: 20 2
35676105; E-mail: ibrahim_gawad@hotmail.com
Received July16, 2014; Accepted August 14, 2014; Published August 16, 2014
Citation: Abd El-Gawad IA, El-Sayed EM, El- Zeini HM, Hafez SA, Saleh FA (2014)
Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against Escherichia
coli and Staphylococcus aureus. J Nutr Food Sci 4: 303. doi: 10.4172/2155-
9600.1000303
Copyright: © 2014 Abd El-Gawad IA, et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided
the original author and source are credited.
Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against
Escherichia coli
and
Staphylococcus aureus
Abd El-Gawad IA1, El-Sayed EM1, El- Zeini HM1, Hafez SA2 and Saleh FA2
1Dairy Science and Technology Department, Faculty of Agriculture, Cairo University, Giza, Egypt
2Special Food & Nutrition Department, Food Technology Research Institute, Agriculture Research Center, Giza, Egypt
Abstract
Introduction: Biopreservation systems in food are becoming increasingly interesting for the industry and
consumer.
Methods: Yoghurt milk and soymilk samples were inoculated separately with E.coli or S. aureus immediately
after adding the starter (bidobacteria and/or yoghurt culture) to investigate the antimicrobial activity of probiotic
yoghurt and soy-yoghurt.
Results: Probiotic yoghurt containing Bidobacterium lactis (Bb-12) and Bidobacterium longum (Bb-46)
exhibited a slight pH drop compared with plain yoghurt (without bidobacteria) during the refrigerated storage period.
Plain yoghurt and probiotic yoghurt containing Bb-12 and Bb-46 inoculated with or without test organisms showed
a signicant (P<0.05) increase in lactic and acetic acids than the probiotic soy-yoghurt containing Bb-12 and Bb-46
which produces not only lactic and acetic acids but also formic acid. From the initial count of E.coli, % decreases
were 96.00, 99.43, 99.14, 97.14 and 98.43%, for the plain yoghurt, probiotic yoghurt containing Bb-12 & Bb-46
and soy-yoghurt containing Bb-12 and Bb-46, respectively. E. coli counts were disappeared in probiotic yoghurt,
soy- yoghurt and plain yoghurt after 2, 3 and 5 days of storage, respectively .The decrease percentage for the plain
yoghurt, probiotic yoghurt containing Bb-12 and Bb-46 and soy-yoghurt containing Bb-12 & Bb-46 were 85.62, 93.36,
95.58, 93.36 and 95.58 from the initial inoculum level, respectively. The growth of S.aureus was not detected in the
probiotic yoghurt containing Bb- l 2 and Bb-46 after the 10th day of storage. Low numbers of S.aureus survived in
the plain yoghurt and probiotic soy-yoghurt Bb- 12 and Bb-46, after 15 days of cold storage.
Conclusion: According to this data probiotic yoghurt, Soy-yoghurt and their antibacterial metabolites can be
used to control pathogenic microorganisms
Keywords: Antibacterial activity; E. coli; S.aureus; yoghurt; Soy-
yoghurt; pH; Organic acids
Introduction
e interaction between food and health is a very complex one.
Accordingly, the food industry has unique opportunities to develop
products that are not only nutritional in the traditional sense, but which
have additional activity that can lead to an improved state of health
and well-being and/or reduction in risk disease «functional foods»
Probiotics have been dened as «live microbial feed supplements that
have benecial eects on the host by improving its intestinal microbial
balance» [1]. Bidobacteria are known to exhibit inhibitory eects
on many pathogenic organisms both in vivo and in vitro, including
Salmonella, Shigella, Colstridium, Bacillus cereus, Staphylococcus
aureus, Candida albicans, Listeria monocytogenes, Escherichia coli and
Campylobacter Jejuni [2-4]. Recent studies on probiotics showed that
the fermented products of probiotics possess strong anti-bactericidal
eects against foodborne pathogens [5]. Because bidobacteria has
been associated with health-promoting eects, there has been an
increasing in incorporating this microbial group into dairy and dairy
like foods or supplementing dairy foods with these organisms. e
ultimate intent of this strategy is to provide the gastrointestinal tract of
humans with viable populations of bidobacteria.
Fermented milks containing bidobacteria are made either using
pure strains or in combination with other lactic acid bacteria [6]
recently, soy-yoghurt has been prepared with the fermentation of lactic
acid bacteria [7] or bidobacteria in soymilk [8].
Although, numerous studies have been focused on the antibacterial
activity of yoghurt, still relatively little is known about the potentially
benecial roles of yoghurt and soy-yoghurt containing bidobacteria
with regards to their potential role in inhibiting food-borne pathogens.
erefore, this study was designed to determine the inhibitory activity
of probiotic yoghurt and soy-yoghurt against Escherihia coli and
Staphylococcus aureus during the refrigerated storage.
Materials and Methods
Preparation of yoghurt from bualo milk
Low-fat bualo milk 1.5% (w/w) was inoculated with a 3% (v/v)
liquid culture of Lactobacillus delbrueckii subsp. bulgaricus and
Streptococcus salivarius subsp. thermophilus (Chr. Hansen Laboratories,
Copenhagen, Denmark), and then divided into three portions. No
bidobacteria were added to the rst portion (plain yoghurt). To
the second portion, a 0.07% (w/v) standardized freeze-dried culture
of Bidobacterium lactis Bb-12 was added. To the third portion, a
0.07% (w/v) standardised freeze-dried culture of B. longum Bb-46 was
Citation: Abd El-Gawad IA, El-Sayed EM, El- Zeini HM, Hafez SA, Saleh FA (2014) Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against
Escherichia coli and Staphylococcus aureus. J Nutr Food Sci 4: 303. doi: 10.4172/2155-9600.1000303
Page 2 of 6
Volume 4 • Issue 5 • 1000303
J Nutr Food Sci
ISSN: 2155-9600 JNFS, an open access journal
added. e two strains were obtained from Chr. Hansen Laboratories
(Copenhagen, Denmark).
Preparation of soy- yoghurt
Fresh and non-beany-avor soy milk was prepared according to
Tanteeratarm [9] and divided into two portions for preparation of soy
yoghurts Bb-12 and Bb-46 according the method described by Abd El-
Gawad [8]. e manufacture of soy yoghurts Bb-12 and Bb-46 involved
the addition of gelatin at a level of 1% (w/w) to the soy milk, heating at
95°C for 5 min, and cooling to 37°C. One portion was inoculated at a
level of 0.07% (w/v) with a freeze-dried B.lactis Bb-12 the other portion
was inoculated at a level of 0.07% (w/v) with a freeze-dried B.Longum
Bb-46.
e Pathogenic microorganisms
E. coli and S. aureus (as a liquid culture cultivated in nutrient broth
were obtained from Egyptian Microbial Culture Collection (EMCC) at
Microbiological Resources Center (MIRCEN) Faculty of Agriculture,
Ain Shams University, Cairo, Egypt and Dairy Microbiology
Department, National Research Center, Giza, Egypt, respectively. ese
two test organisms were added separately to pasteurized yoghurt milk
and soy-milk at 45°C immediately aer adding the starter cultures.
Aer incubation all samples (yoghurt & soy yoghurt) were stored at
4 ± 1oC.
Analytical methods
Counts of yoghurt cultures and Bidobacterial: Bacterial counts
of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus
in yoghurt were determined according to Lee [10] in which the yoghurt
sample was added to Lee’s agar and incubated at 43°C for 3 days.
Bidobacteria were enumerated in soy yoghurt, which did not count
in yoghurt cultures, by a poured plate method using Lactobacilli MRS-
agar medium as described by [11] Bidobacteria were enumerated in
yoghurt containing Bb-12 or Bb-46 according to the method of [12]
in which a mixture of antibiotics, including 2 g paromomycin sulfate,
0·3 g nalidixic acid, and 60 g lithium chloride, was dissolved in 1 litre
distilled water, lter-sterilised (0.2 mm) and stored at 4°C until use.
e antibiotic mixture (5 ml) was added to 100 ml MRS-agar medium.
L-Cysteine-HCl 0.5% (Sigma Chemical Co., St Louis, MO, USA) was
also added to decrease the redox potential of the medium. Plates were
incubated at 37°C for 48 h anaerobically.
E. coli count: e E.coli count was estimated by platting suitable
dilution on Macconkey Agar medium (Oxid) as recommended by the
[13] the plates were incubated at 37°C for 24 h.
Staphylococcus aureus count: Determination of S.aureus was
carried out using mannitol salt agar medium as described in [14].
pH value: It was measured by using Orion pH-meter, model 501
at 20°C.
Determination of organic acids by HPLC: Organic acids in
yoghurt and soy-yoghurt were determined using HPLC according to
the method, of [15] with slightly modication as follows;
Extraction of organic acids: 10 g of sample was centrifuged at
10000 rpm for 25 min and the supernatant was ltrated through a 0.2
µm Millipore membrane lter then 1-3 ml was collected in a vial for
injection by auto-injector into HPLC.
Chromatographic separation: HPLC Hewllet Packared (series
1050) equipped with autosampling injector, solvent degasser, ultra
violet (UV) detector set at 210 nm and quaternary HP pump (series
1050), Hewllet Packared soware. e column temperature was
maintained at 35°C. An isocratic separation was carried out with 0.01
N H2SO4 as a mobile phase at ow rate of 1 ml/min. the organic acids
standard (lactic, acetic and formic acid) from Fluka Co, were dissolved
in a mobile phase and injected into HPLC
Statistical analysis: e results were analysis statistically using one
way analysis of variance (version 16.0 SPSS, USA). When there was
statistically signicant dierence post hoc analysis was applied. e
statistical signicance of the data was determined using P values less
than 0.05.
Results and Discussion
Viability of bidobacteria and yoghurt starter cultures
Changes in the population of bidobacteria in probiotic yoghurt
and soy yoghurt inoculated with and without test organisms during
refrigerated storage period are shown in Table 1. ere was a sharp
decline in all treatments (probiotic yoghurt and soy-yoghurt) during the
refrigerated storage period. Bidobacterial population in the probiotic
yoghurt containing Bb-12 & Bb-46 and probiotic soy-yoghurt made
with Bb-12 and Bb-46, decreased by 98.56, 92.00, 96.43 and 98.86%,
without inoculation with test organisms at the end of refrigerated
storage period, respectively. In case of the treatments inoculated with
E. coil the corresponding decrease ratios were 74.22, 99.36, 48.13
and 62.72%, respectively, whereas for the treatments inoculated with
S.aureus the population of bidobacteria decrease by 82.50, 97.96, 84.19
and 99.00%, respectively. e decline of bidobacterial population
during storage may be due to the decrease of pH value and accordingly
increase of acidity as well as their ability to produce organic acids [16].
Maintaining viability of bidobacteria has been a challenge to the dairy
processors because the organism requires low oxidation reduction
potential for growth and is sensitive to low pH [17]. Another study
[18] showed that 14 out of 17 strains lost their viability in fermented
milk in the rst week of storage. Also it is reported that the presence
of yoghurt culture adversely aected the growth of bidobacteria
irrespective of their species [11]. Klaver et al. [18] reported the survival
of only three out of nine bidobacterial strains in the pH range of 3.7 to
4.3. In the studies by Adhikari, Shin, Medina [15,17,19], the population
of bidobacteria with yoghurt starter culture decreased during
refrigerated storage period.
On the other hand, Shin et al. [17] and El-Sayed et al. [20] found
that the soymilk fermented by bidobacteria were rapidly reduced the
survival of bidobacteria during the refrigerated storage period. It could
be seen from the data in Table 1, that the bidobacterial population
was higher in probiotic soy-yoghurt Bb-12 and Bb-46 inoculated with
or without test organisms than the corresponding probiotic yoghurt
Bb-12 and Bb-46 treatments, over the refrigerated storage period. e
increasing of bidobacterial counts in probiotic soy-yoghurt Bb-12
and Bb-46 compared with probiotic yoghurt Bb-12 and Bb-46 may
be due to the presence of oligosaccharides(stachyose and ranose) in
soymilk, which was approach as growth factors for several species of
bidobacteria [8,21]. Although the bidobacterial level in all probiotic
treatments were variable in products investigated, they were always
above 106 cfu / ml until the end of refrigerated storage period (15 days),
which is recommended dose to receive the health benets of these
organisms [6,17].
Table 1 showed the viability of yoghurt culture (Lactobacillus
delbrueckii spp bulgaricus and Streptococcus salivarius spp thermophilus)
Citation: Abd El-Gawad IA, El-Sayed EM, El- Zeini HM, Hafez SA, Saleh FA (2014) Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against
Escherichia coli and Staphylococcus aureus. J Nutr Food Sci 4: 303. doi: 10.4172/2155-9600.1000303
Page 3 of 6
Volume 4 • Issue 5 • 1000303
J Nutr Food Sci
ISSN: 2155-9600 JNFS, an open access journal
to the added acidity, which produced by bidobacteria in the probiotic
products.
Also, it could be seen from the same Table that pH-values in the
probiotic soy-yoghurt made with Bidobactertum Bb-12 and Bb-
46 inoculated with and without test organisms were higher than the
corresponding values in the probiotic yoghurt inoculated with and
without test organisms over the refrigerated storage period. is may
be due to the absence (not added) of yoghurt culture in probiotic soy-
yoghurt.
Lactic, acetic and formic acids contents of probiotic yoghurt
and soy-yoghurt inoculated with test organisms
Changes in the values of lactic, acetic and formic acids contents of
probiotic yoghurt and soy-yoghurt inoculated by E.coli and S.aureus
during refrigerated storage period are illustrated in Table 3a-c
respectively. It could be noticed that probiotic yoghurt containing
Bb-12 and Bb-46 inoculated with or without test organisms showed
signicantly (P>0.05) increase in lactic and acetic acids than the
corresponding: values in plain yoghurt direct aer the incubation
time, and these increasing continued for acetic acid only over the
refrigerated storage period. is nding conrmed the high ability of
bidobacteria and yoghurt starter cultures (Lactobacillis, dulbrueekii
in plain and probiotic yoghurt Bb-12 & Bb-46 inoculated with or
without test organisms, during refrigerated storage period. e initial
number of yoghurt culture was 810.0, 714.5 and 748×108 cfu /ml in
plain yoghurt, yoghurt Bb-12 and yoghurt Bb-46, respectively. e
decrease in the yoghurt culture population in all of these treatments
may be attributed to the decrease in pH (Tables 1 and 2). ough,
these considerable declines, it could be observed that the yoghurt
culture population was maintained above 108 cfu/ml until the end of
refrigerated storage period (Table 1).
pH-values
Changes in pH-values of probiotic yoghurt and soy-yoghurt
inoculated by E.coli and S.aureus during refrigerated storage period
are shown in Table 2. e pH-values of the most of experimental
treatments decrease and consequently the titratable acidity increase
during the refrigerated storage period (Data not shown). e decrease
in pH-values observed in the treatments may be due to metabolic
activity of bidobacteria and yoghurt starter culture as well as test
organisms. As shown in Table 2 the probiotic yoghurt containing Bb-12
and Bb-46 inoculated with or without test organisms exhibited a slight
pH drop direct aer the incubation time and during the refrigerated
storage period compared with plain yoghurt. is may be attributed
Treatments
(a) Count of Bifidobacteria(cfu x 108 /ml) *
Storage period (days)
0** 510 15
Without
inoculation
Inoculated
with
E. col
Inoculated
with
S. aureus
Without
inoculation
Inoculated
with
E. col/
Inoculated
with
S. aureus
Without
inoculation
Inoculated
with
E. col
Inoculated
with
S. aureus
Without
inoculation
Inoculated
with
E. coil
Inocul ated
with
S. aureus
Yoghurt Bb-12 2.09 1.28 2.40 0.91 1.20 2.67 0.28 0.50 1.03 0.03 0.33 0.42
Yoghurt Bb-46 2.50 3.15 2.21 0.35 1.70 3.34 0.24 0.23 0.11 0.20 0.02 0.045
Soy-yoghurt
Bb-12 30.80 4.24 6.64 14.30 3.64 3.05 2.14 2.33 2.33 1.10 2.20 1.05
Soy-yoghurt
Bb-46 88.00 5.58 6.00 13.75 3.18 4.25 1.79 1.73 1.73 1.00 2.08 0.06
(b) Count of yoghurt culture (cfu x 108 /ml) *
Storage period (days)
Treatments
0** 5 10 15
Without
inoculation
Inoculated
with
E. coli
Inoculated
with
S. aureus
Without
inoculation
Inoculated
with
E. coli
Inoculated
with
S. aureus
Without
inoculation
Inoculated
with
E. coli
Inoculated
with
S. aureus
Without
Inoculation
Inoculated
with
E. coli
Inoculated
with S.
aureus
Plain yoghurt 810.0 342.5 650.0 520.0 88.2 107.0 41.2 15.0 103.2 28.0 5.0 75.0
Yoghurt Bb-12 714.5 408.2 300.0 82.5 71.3 73.8 62.0 77.4 41.2 21.0 15.7 31.0
Yoghurt Bb-46 748.0 704.5 200.0 77.8 45.5 117.6 11.4 53.8 48.4 10.0 18.9 20.0
* mean of three replicates
**Direct after coagulation
Table 1: The viable count of bifidobacteria (a) and yoghurt culture (b) in experimental probiotic yoghurt and soy-yoghurt inoculated with Escherichiacoli and
Staphylococcus aureus during refrigerated storage period.
Treatment
pH-values*
Without Inoculation Inoculated with E.coli inoculation with S.aureus
Storage period ( days)
0 5 10 15 0 5 10 15 0 5 10 15
Plain Yoghurt 4.47 ± 0.02 4.14 ± 0.03 3.95 ± 0.01 4.03 ± 0.03 4.35 ± 0.02 4.10 ± 0.02 4.00 ± 0.02 4.03 ± 0.02 4.35 ± 0.02 4.09 ± 0.02 4.01 ± 0.01 4.03 ± 0.02
Yoghurt Bb-12 4.34 ± 0.03 4.07 ± 0.04 3.98 ± 0.02 3.98 ± 0.01 4.25 ± 0.03 4.04 ± 0.02 3.87 ± 0.02 3.97 ± 0.02 4.31 ± 0.04 4.03 ± 0.03 3.83 ± 0.02 3.99 ± 0.02
Yoghurt Bb - 46 4.34 ± 0.03 4.08 ± 0.01 3.95 ± 0.02 3.95 ± 0.02 4.32 ± 0.02 4.07 ± 0.02 3.90 ± 0.02 4.01 ± 0.00 4.28 ± 0.03 4.07 ± 0.03 3.99 ± 0.01 3.99 ± 0.01
Soy yoghurt Bb-12 4.90 ± 0.03 4.84 ± 0.04 4.79 ± 0.01 4.71 ± 0.03 4.97 ± 0.02 4.79 ± 0.02 4.72 ± 0.01 4.76 ± 0.02 4.54 ± 0.03 4.62 ± 0.01 4.61 ± 0.02 4.63 ± 0.03
Soy yoghurt -Bb- 46 4.46 ± 0.02 4.41 ± 0.02 4.34 ± 0.01 4.37 ± 0.02 4.50 ± 0.02 4.37 ± 0.01 4.36 ± 0.03 4.05 ± 0.59 4.35 ± 0.03 4.37 ± 0.03 4.36 ± 0.03 4.35 ± 0.03
* Means values ( ± SD; n=3)
Table 2: Changes in pH –values of probiotic yogurt and soy-yogurt inoculated with Escherichia coli and Staphylococcus aureus during refrigerated storage period.
Citation: Abd El-Gawad IA, El-Sayed EM, El- Zeini HM, Hafez SA, Saleh FA (2014) Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against
Escherichia coli and Staphylococcus aureus. J Nutr Food Sci 4: 303. doi: 10.4172/2155-9600.1000303
Page 4 of 6
Volume 4 • Issue 5 • 1000303
J Nutr Food Sci
ISSN: 2155-9600 JNFS, an open access journal
sub sp. bulgaricus and Streptococcus salivarius sub sp, thermophilus) to
produce such organic acids. Furthermore it could be observed from the
data in the same Tables that inoculation with test organisms (E. coli and
S.aureus) had little eect on the production of these organic acids by
bidobacteria and yoghurt culture.
On the other hand and as shown in Table 3, the probiotic soy-yoghurt
made with Bb-12 and Bb-46 inoculated with or without test organisms
showed signicantly decrease in lactic and acetic acids than the plain
yoghurt and probiotic yoghurt Bb-12 and Bb-46 direct aer incubation
time and aer the refrigerated stroge period. ese dierences may
be due to the presence of yoghurt culture with bidobacteria in the
probiotic yoghurt. It was of considerable interest that the probiotic
soy-yoghurt Bb-12 and Bb-46 and inoculated with or without test
organisms produces not only lactic and acetic acids but also formic
acid, and when these acids are ranked in order of decreasing activity
(as function of concentration mg/100 g), they form the series: lactic>
acetic> formic. is nding is in agreement with that reported by [22]
who found the lactic, acetic and formic acids contents of bidobacteria-
fermented soymilk were 225.7, 187.1 and 20.7 µmol/g respectively. In
contrast, Adhikari et al. [15] decided that the bidobacteria with yogurt
cultures (L.delbrueckii ssp bulgaricus and Streptococcus salivarus sub sp.
thermophilus) produce lactic and acetic acid only.
Eect of probiotic yoghurt and soy-yoghurt on the growth
and survival rate of E. coli:
e growth and survival patterns of E.coli as aected by probiotic
yoghurt and soy-yoghurt during refrigerated storage period are shown
in Figure 1a. It could be seen that the counts of E.coli in all treatments
decreased during the cold storage period. Direct aer incubation time,
the decrease percentage was 96 for the plain yoghurt from the initiate
level (3.5×105 cfu/ml), whereas in the probiotic yoghurt containing
Bb-12 and Bb-46 and soy-yoghurt made with Bb-12 and Bb-46, the
% decrease was 99.43, 99.14, 97.14 and 98.43 respectively. While aer
one day of storage the corresponding decrease were 98.40, 99.96, 99.17,
99.17 and 99.75% for the plain yoghurt, probiotic yoghurt Bb-12
Treatment
Without Inoculation Inoculated with E.coli Inoculated with S.aureus
Storage period ( week)
012012012
Plain Yoghurt 170 ± 6.0rst 510 ± 2.0ghij 571 ± 3.6bcd 538 ± 7.2defg 535 ± 6.08efgh 614 ± 7.2a421 ± 4.6lm 498 ± 10.6ij 568 ± 5.6bcde
Yoghurt Bb-12 556 ± 6.0cdef 481 ± 6.2jk 260 ± 10o414 ± 3.5 m 503 ± 8.9hij 592 ± 5.6ab 435 ± 6.4lm 523 ± 8.5fghi 586 ± 8.0abc
Yogurt - Bb - 46 426 ± 5.3lm 507 ± 5.3ghij 606 ± 5.57a455 ± 755kl 509 ± 6.2ghij 584 ± 5.3abc 440 ± 5.3lm 529 ± 8.7fghi 533 ± 8.7fgh
Soy yoghurt -Bb-12 137 ± 4.6t151 ± 3.0st 152 ± 3.6st 159 ± 4.4lm 139 ± 6.1t157 ± 3.6t145 ± 3.6t147 ± 3.6t156 ± 4.4t
Soy yoghurt -Bb- 46 181 ± 3.6rs 278 ± 4.4no 314 ± 2.0op 198 ± 4.0qr 440 ± 6.6lm 300 ± 3.6n250 ± 8.7op 196.67 ± 3.8qr 216 ± 3.5pq
(a)Lactic acid
Treatment
Without Inoculation Inoculated with E.coli Inoculated with S.aureus
Storage period ( week)
012012 0 12
Plain Yoghurt 188 ± 6.0o205 ± 3.0n222 ± 5.0m281 ± 9.0j291 ± 3.0t104 ± 3.0tu 263 ± 6.0k158 ± 4.0p74 ± 3.0bc
Yoghurt Bb-12 590 ± 4.0a397 ± 5.0f429 ± 7.0e478 ± 5.0c451 ± 4.0d485 ± 7.0b350 ± 4.0h375 ± 4.0g239 ± 5.0l
Yoghurt - Bb - 46 438 ± 10de 430 ± 5.0e474 ± 7.0bc 468 ± 4.0c445 ± 7.0de 481 ± 3.0bc 317 ± 6.0i246 ± 6.0l218 ± 6.0mn
Soy yoghurt -Bb-12 78 ± 7.0vw 32 ± 6.0xy 85 ± 4.0vw 130 ± 3qrs 47 ± 4.0x1.00 ± 0.0z142 ± 7pq 115 ± 3t2.00 ± 0.0z
Soy yoghurt -Bb- 46 108 ± 5.0tu 93 ± 2.0uv 23 ± 2.0yz 119 ± 4.0rst 106 ± 2.0tu 12 ± 1.0z283 ± 4.0j135 ± 6.0qr 113 ± 2.0t
(b)Acetic acid
Treatment
Without Inoculation Inoculated with E.coli Inoculated with S.aureus
Storage period ( week)
012012012
Soy yogurt -Bb-12 29 ± 3.0gh 27 ± 2.0ghi 39 ± 4.0def 33 ± 2.0fg 37 ± 3.0ef 63 ± 2.0b28 ± 2.0ghi 44 ± 3.0cde 88 ± 2.0a
Soy yogurt -Bb- 46 22 ± 2.0hij 43 ± 3.0cde 62 ± 2.0b17 ± 1.0j28 ± 2.0ghi 65 ± 3.0b46 ± 3.0cd 51 ± 3.0c21 ± 3.0ij
(c)Formic acid
a - z Means values ( ± SD; n=3) with unlike subscripts letters were signicantly different (p˂0.05) .
Table 3: Changes in lactic (a), acetic (b) and formic acids (c) content of probiotic yogurt and soy-yogurt inoculated with Escherichia coli and Staphylococcus aureus during
refrigerated storage period (mg/100 g).
Figure 1 (a, b): Effect of probiotic yoghurt and soy-yoghurt on the growth and
survival rate of Escherichia. coli Staphylococcus aureus during storage period.
(a) E. coli
b) S. aureus
Citation: Abd El-Gawad IA, El-Sayed EM, El- Zeini HM, Hafez SA, Saleh FA (2014) Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against
Escherichia coli and Staphylococcus aureus. J Nutr Food Sci 4: 303. doi: 10.4172/2155-9600.1000303
Page 5 of 6
Volume 4 • Issue 5 • 1000303
J Nutr Food Sci
ISSN: 2155-9600 JNFS, an open access journal
and Bb-46 and probiotic soy-yoghurt Bb-12 and Bb-46, respectively.
Interestingly, while E.coli had disappeared aer 5 days m plain yoghurt,
it could not be detected in the probiotic yoghurt and soy-yoghurt aer
2 and 3 days of storage, respectively (Figure 1a).
However, It could be seen from Table 3 that the acetic acid in
probiotic yoghurt Bb-12 and Bb-46 higher than in plain yoghurt,
probiotic soy-yoghurt Bb-12 and Bb-46, direct aer coagulation (0
week), one and two weeks. ese data also showed that the plain yoghurt
produce lactic acid more than probiotic yoghurt Bb-12 and Bb-46. In
contrast, probiotic soy-yoghurt Bb-12 and Bb-46 produced lactic acid
less than plain and probiotic yoghurts. Buchanan and Gibbons [23],
studied the eect of HCl, malic acid, citric acid, acetic acid and lactic
acid on the activity of 9 strains of E. coli and they found that the all of
these strains more sensitive to lactic acid than acetic acid. In addition,
the minimum pH at which all the E.coli strains were able to grow in
adjusted tryptic soy broth (TSB) was 5.5 for acetic acid. Acetic acid was
a more eective inhibitor than HCl at an equivalent pH. e higher pKa
of acetic acid (4.75) compared with other organic and mineral acids are
responsible for the observed greater ecacy of this acid against E. coli
[24]. Oh et al. [25] studied the eect of organic acids on the survival of
E. coli and they found the minimum inhibitory pH of acetic acid and
lactic acid was 5.0 and 4.0, respectively.
Cherrington et al. [26] reported that the antimicrobial eect of
organic acids has been attributed to undissociated acid molecules that
interfere with cellular metabolism or a decrease in biological activity as
a result of pH changes of the cells environment. On the other hand, the
inhibition mechanisms of bidobacteria on E. coli not only depend on
organic acids but also may be on antibacterial substances [27].
Hussein and Kebary [28] found that the immobilized cells of
Bidobacterium bidum and Bidobacterium infants are able to produce
antimicrobial in yoghurt agents, which inhibited E.coli. e present
results showed that the probiotic yoghurt and soy-yoghurt containing
bidobacteria suppressed the E.coli population more eectively than
non-probiotic yoghurt (plain yoghurt).
Eect of probiotic yoghurt and soy-yoghurt on the growth
and survival rate of Staphylococcus aureus
e growth and survival patterns of S.aureus as aected by probiotic
yoghurt and soy-yoghurt during refrigerated storage period are
illustrated in Figure 1b. It could be observed that the counts of S.aureus
in all treatments decreased during the cold storage period. Direct aer
the incubation time, the percentage decrease for the plain yoghurt was
85.62% from the initial inoculation number (4.5×l06 cfu/ml), whereas
the % decrease in the probiotic yoghurt containing bidobacterium Bb-
12 and Bb-46 was 93.36% and 95.58%, respectively. For the probiotic
soy-yogurt made with Bidobacterium Bb-12 and Bb-46, the decrease
percentage was 87.83% and 95.58% aer same period, respectively. e
present results indicated that while S.aureus growth in the probiotic
yoghurt containing Bb-12 and Bb-46 was not detected aer the 10th day
of storage, low numbers of S .aureus were survived in the plain yoghurt
as well as the probiotic soy-yoghurt made by Bb-12 and Bb-46 aer 15
days of cold storage (Figure 1b).
As seen from Figure 1b, this markedly inhibitory eect of probiotic
yoghurt containing Bb-12 and Bb-46 may be due to their ability
to produce acetic acid much more than plain yoghurt and probiotic
soy-yoghurt Bb-12 and Bb-46, direct aer coagulation and during
refrigerated storage period. Minor and Marth [29] reported that the
acetic acid inactivated 99.99% of the S. aureus at pH 4.4 and this nding
was in agreement with our results. Notermans and Heuelman [30]
found that growth of S.aureus occurred at pH 4.6 but not at pH 4.3. is
nding may be explained that S.aureus still detected in probiotic soy-
yoghurt Bb-12 and Bb-46 during refrigerated storage period, where they
have a pH-values still higher than 4.3 aer 15 day (Table 2 and Figure
1b). Dahiya and Speck [31] found that the Lactobacillus delbruecku spp
bulgaricus and Lactobacillus delbrueckii spp Lactis inhibited the growth
of S. aureus. ey proposed that inhibition of S. aureus resulted from the
formation of hydrogen peroxide by certain Lactobacilli. Gilliland and
Speck [32] found that lactic streptococci in milk inhibited Salmonella
and S. aureus, where its inhibition levels were 88.2-93.4% for Salmonella
and 98.1-98.9% for S. aureus. e authors showed also that inhibition
was due partially to organic acids production and partially to small
molecular weight compounds in whey.
It is evident from the present results that the metabolites are
signicantly eective. In addition, e pH – values were positively
correlated with the viability of E. coli and S. aureus in the probiotic
yoghurt and soy- yoghurt containing Bb-12 & Bb-46 (r .421, r .324;
p<0.05, respectively). is can be explained from the fact that the
metabolites produced by the probiotics include bioactive products such
as organic acid, hydrogen peroxide (H2O2) and bacteriocins [33]. It is
reported by Cheikhyoussef et al. [34] that the principal metabolites
of probiotic bacteria are acetic acid and lactic acid in ratio 3:2 and
these acids are responsible for the consequent drop in pH and may be
sucient to antagonize many pathogenic bacteria belonging to both
Gram-positive and Gram-negative bacteria.
Conclusion
From the previous results, we can concluded, that the probiotic
products containing bidobacteria caused antigonistic eects against
foodborne pathogenic bacteria such as E.coli and S. aureus and the use
of probiotic bacteria like bidobacteria in the production of yoghurt
and soy yoghurt restricts or prohibits the growth of these pathogenic
bacteria. Our results suggest that these probiotic bacteria could be used
as a nature biopreservatives in dierent food products.
References
1. Fuller R (1989) Probiotics in man and animals. See comment in PubMed
Commons below J Appl Bacteriol 66: 365-378.
2. Anand SK, Srinivasan RA, Rao LK (1985) Antibacterial activity associated with
Bidobacterium bidum.II Culture Dairy Products Journal, 20: 21-23.
3. Tomoda T, Yasua N, Kageyama T (1988) Intestinal Candida overgrowth
and Candida infection in patients with leukemia: Effect of Bidobacterium
adiminstration. Bidobactrai Microora 7: 71-74.
4. Yildirim Z, Johnson MG (1998) Characterization and antimicrobial spectrum of
bidocin B, a bacteriocin produced by Bidobacterium bidum NCFB 1454. J
Food Prot 61: 47-51.
5. Yesillik S, Yildirim N, Dikici A, Yildiz A, Yesillik S (2011) Antibacterial effects of
some fermented commercial and homemade dairy products and 0.9% lactic
acid against selected foodborne pathogens. Asian J Anim Vet Adv 6: 189-195.
6. Kurmann JA, Rasic JL (1991) The health potential of products containing
bidobacteria. Therapeutic Properties of Fermented Milks Robinson RK 117-
157.
7. Chou CC, Hou JW (2000) Growth of bidobacteria in soymilk and their survival
in the fermented soymilk drink during storage. Int J Food Microbiol 56: 113-121.
8. Abd El-Gawad IA, Hefny AA, El-Sayed EM, Saleh FA (1998) Reduction of
atulence-causing soymilk oligosaccharides by different starter cultures. Food
and Agriculture Organization of the United Nations.
9. Tanteeratarm K, Nelson AI, Wei LS (1993) Manufacturing of bland soymilk. In
Soybean Extrusion and Soymilk Technology, Soy Food Products and Home
Utilization. Urbana-Champaign, IL: University of Illinois, International Soybean
Program (INTSOY).
Citation: Abd El-Gawad IA, El-Sayed EM, El- Zeini HM, Hafez SA, Saleh FA (2014) Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against
Escherichia coli and Staphylococcus aureus. J Nutr Food Sci 4: 303. doi: 10.4172/2155-9600.1000303
Page 6 of 6
Volume 4 • Issue 5 • 1000303
J Nutr Food Sci
ISSN: 2155-9600 JNFS, an open access journal
10. Lee SY, Vedemuthu ER, Washam CJ, Reinhold BW (1973) An agar medium
for the differential enumeration of yoghurt starter bacteria. J Milk Food Tech
37: 272.
11. Samona A, Robison RK (1991) Enumeration of bidobacteria in dairy products.
J Soc Dairy Techon 44: 64-66.
12. Dinakar P, Mistry VV (1994) Growth and viability of Bidobacterium bidum in
cheddar cheese. J Dairy Sci 77: 2854-2864.
13. APHA (1967) Standard method for the examination of dairy products.Amercan
public Health Association. Broadway, New York, USA.
14. Cappuccino JG, Sherman N (1998) Microbiology A Laboratory Manual Fifth
Edition.Rockland community college Suffern, New York.
15. Adhikari K, Mustapha A, Grün IU, Fernando L (2000) Viability of
microencapsulated bidobacteria in set yogurt during refrigerated storage. J
Dairy Sci 83: 1946-1951.
16. Samona A, Robison RK, Marakis S (1996) Acid production by bidobacteria
and yoghurt bacteria during fermentation and storage of milk. Food Microbiol
13: 275-280.
17. Shin HS, Lee JH, Pestka JJ, Ustunol Z (2000) Viability of bidobacteria in
commercial dairy products during refrigerated storage. J Food Prot 63: 327-
331.
18. Klaver FAM, Kingma F, Weerkamp AH (1993) Growth and survival of
bidobacteria in milk. Neth. Milk Dairy J 47: 151-164.
19. Medina LM, Jordano R (1994) Survival of constitutive microora in commercially
fermented milk containing bidobactria during refrigerated storage. J Food
Protection 56: 731-733.
20. El-Sayed EM, Hefny AA, Saleh FA, Abd El-Gawad IA (1998) Bidobacteria as a
starter for the manufacture of soy-yoghurt products.
21. Salminen S, Wright A (1993) Lactic acid bacteria, Marcel Dekker, Inc., New
York.
22. Kikuchi-Hayakawa H, Onodera N, Matsubara S, Yasuda E, Chonan O, et al.
(1998) Effects of soy milk and bidobacterium fermented soy milk on lipid
metabolism in aged ovariectomized rats. Biosci Biotechnol Biochem 62: 1688-
1692.
23. Buchanan RE, Gibbons NE (1974) Bergey’s Manual of Detrminative
Bacteriology, 8`h Edn. Williams and Wilkins, Baltimore.
24. McKellar RC, Knight KP (1999) Growth and survival of various strains of
enterohemorrhagic Escherichia coli in hydrochloric and acetic acid. J Food Prot
62: 1466-1469.
25. Oh DH, Park JH, Park BK (2000) Effect of organic acids on the survival of
Escherichia coli 0157:H7. J Food Sci Nutri 5: 131-135.
26. Cherrington CA, Hinton M, Pearson GR, Chopra I (1991) Inhibition of
Escherichia coli K12 by short-chain organic acids: lack of evidence for induction
of the SOS response. J Appl Bacteriol 70: 156-160.
27. Luo ZL, Xic JZ, Xu SF, Hua ZJ (1998) Study on growth inhibition of pathogens
by Bidobacteriun infantis. China Dairy Industry 26: 3-6.
28. Hussein SA, Kebary KMK (1999) Improving viability of bidobacteria by
microentrapment and their effect on some pathogenic bacteria in stirred
yoghurt. Acta Alimentaria 28: 113-131.
29. Minor TE, Marth EH (1972) Loss of viability by Staphylococcus aureus in
acidied media. I. Inactivation by several acids, mixtures of acids, and salts of
acids. J Milk Food Technol 35:191-196.
30. Notermans S, Heuelman CJ (1983) Combined effect of water activity, pH
and sub-optimal temperature on growth and enterotoxin production of
Staphylococcus aureus. J Food Science 48: 1832-1835.
31. Dahiya RS, Speck ML (1968) Hydrogen peroxide formation by lactobacilli and
its effect on Staphylococcus aureus. J Dairy Sci 51: 1568-1572.
32. Gilliland SE, Speck (2011) Interactions of food starter cultures and food-borne
pathogens: lactic streptococci versus staphylococci and salmonellae. J Milk
Food Technol 35: 307.
33. Hassan Pyar, Peh KK, Min-Tze Liong (2011) Inhibitory Effect of Metabolites
from Probiotics Lactobacillus acidophilus Strains on Growth of Pathogenic
Bacteria. J Pharmacol Toxicol 6: 533-540.
34. Cheikhyoussef A, Pogori N, Zhang H (2007) Study of the inhibition effects
of Bidobacterium supernatants towards growth of Bacillus cereus and
Escherichia coli. Int J Dairy Sci 2: 11.
Submit your next manuscript and get advantages of OMICS
Group submissions
Unique features:
• User friendly/feasible website-translation of your paper to 50 world’s leading languages
• Audio Version of published paper
• Digital articles to share and explore
Special features:
• 350 Open Access Journals
• 30,000 editorial team
• 21 days rapid review process
• Quality and quick editorial, review and publication processing
• Indexing at PubMed (partial), EBSCO, Index Copernicus and Google Scholar etc
• Sharing Option: Social Networking Enabled
• Authors, Reviewers and Editors rewarded with online Scientic Credits
• Better discount for your subsequent articles
Submit your manuscript at: http://www.editorialmanager.com/lifesciences
Citation: Abd El-Gawad IA, El-Sayed EM, El- Zeini HM, Hafez SA, Saleh FA
(2014) Antibacterial Activity of Probiotic Yoghurt and Soy-Yoghurt against
Escherichia coli and Staphylococcus aureus. J Nutr Food Sci 4: 303. doi:
10.4172/2155-9600.1000303
