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HPLC Analysis of lactic acid inhibition of Salmonella typhi and Escherichia

coli responsible of diarrheal diseases in Algeria: Alternative treatment

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 Moroccan Journal of Chemistry
ISSN: 2351-812X
http://revues.imist.ma/?journal=morjchem&page=login
Guetarni & al./ Mor. J. Chem. 5 N°4 (2017) 534-543

HPLC Analysis of lactic acid inhibition of Salmonella typhi and Escherichia

coli responsible of diarrheal diseases in Algeria: Alternative treatment
S.typhi.E.coli.BacLac.

H.Guetarni *

Department of Biology, Faculty of Natural and Life Sciences and Earth Sciences, Djilali Bounaama University,
Khemis Miliana, 44225, Ain Defla, Algeria.

Abstract
Several works were shown the effect in vitro of lactic acid bacteria on pathogenic
strains responsible for human gastroenteritis. This work aims to seek strains of lactic
acid bacteria could inhibit in vitro the growth of pathogenic bacteria involved in
gastroenteritis and analyze organic acids responsible of this inhibition. Interactions in
* Corresponding author: agar medium were conducted between lactic acid bacteria in pure or mixed culture and
kmhg2009@yahoo.fr
pathogenic bacteria (E. coli and S. typhi). The best inhibition zones of E.coli are found
Received 11May 2017, with Lp (diameter = 10 mm) and St*+ Lb*+ Bl (diameter = 10 mm). For S.typhi, the
Revised 25 July 2017, combinations St3 + Lb1+ Bl and St2 + Lp +Bl have the major inhibitory effect
Accepted 28 July 2017 (diameters of 8 mm and 7 mm, respectively). HPLC method confirmed the presence of
lactic acid in bacterial cultures used in this study.

Keywords: HPLC; lactic acid bacteria; Salmonella typhi; Escherichia coli; organic acids.

Mor. J. Chem. 5 N°4 (2017) 534-543

534
1. Introduction
Diarrheal diseases are still today a major public health problem in developing countries. Diarrhea is a common reason
for consultation, especially in industrially developing countries where fecal and food hygiene is precarious [1].
According to the World Health Organization (WHO) and UNICEF, about two billion cases of diarrhea are recorded
worldwide every year and 1.9 million children under 5 die from diarrhea every year, mostly in developing countries.
Among all child deaths from diarrhea, 78% occur in Africa and South-East Asia [2]. These explosive epidemics are
related to many factors: rapid population growth in some countries, economic recession, disasters, natural or man-
made (war, famine, population massacres) [1]. In industrialized countries, the number of deaths from diarrhea patients
is relatively small, but nevertheless diarrhea remains a major cause of morbidity and induces substantial costs of care.
In developing countries, enteric bacteria and parasites are more prevalent than viruses typically with a peak during the
summer months enteropathogenic E. coli (EPEC) causes most often diarrhea in children less than 2 years[2]. Typhoid
fever caused by Salmonella typhi is still a common cause of mortality. In severe cases, this infection is manifested by
high fever of around 40oC [3]. Algeria crossed, for some years now, an epidemiological transition marked by the
persistence of communicable diseases (infectious childhood diseases, waterborne disease, zoonoses), characteristics of
developing countries, which are becoming more an important place in the burden of diseases [4]. It has been
confirmed that different probiotic strains including L. reuteri ATCC 55730, Lactobacillus rhamnosus GG, L. casei
DN-114 001 and Saccharomyces cerevisiae (boulardii) are useful for reducing the severity and duration of the acute
infectious diarrhea in children. The oral administration of probiotics reduced the duration of acute diarrhea in children
by about one day [2]. Thus, the objective of this work was to study in vitro the interaction of lactic acid bacteria with
pathogenic bacteria responsible for gastroenteritis in Algeria (E. coli, S. typhi), in pure culture, mixed or added
bifidobacteria to highlight the best probiotic combinations, and proceeding with the analysis of organics acids in
bacterial cultures by High Performance Liquid Chromatography (HPLC).

2. Experimental
Lactic acid bacteria:
 Streptococcus thermophilus (St1, St2, St3) and Lactobacillus bulgaricus (Lb1, Lb2, Lb3) isolated from raw
cow's milk and identified in the laboratory lactologie, Department of Food Science, University of Chlef.
 Streptococcus thermophilus (St *), Lactobacillus acidophilus (Lb *), Bifidobacterium longum (Bl) and
Bifidobacterium adolescentis (Ba); isolated from stools of children and identified in Molecular Biology
laboratory, University of Bejaia.
 Lactobacillus paracasei1 (LP) isolated from cow's milk and identified in laboratory lactologie, University
Pamplona - Navara Spain.
Both strains are kept frozen at-18ºC in reconstituted milk. A few microliters of bacteria is transplanting in 10 ml of
milk. The incubation is done at:
 45oC for strains Streptococcus thermophilus; Lactobacillus bulgaricus and Lactobacillus acidophilus [5];
 37oC for Bifidobacterium adolescentis and Bifidobacterium longum [6];
 30oC for Lactobaccilus paracasei 1 [6].
After 72h , 1ml of each tube was raised aseptically in 9 ml of MRS broth for Lb.ac, 9 ml of M17 (Terzaghi and
Sandine) broth for Sc.tr , then incubated for 3h. The streptococci was counted in M17 agar [7], the lactobacilli in MRS
(De Man, Rogosa and Sharpe) agar [7] and the bifidobacteria in MRS(De Man, Rogosa and Sharpe) agar cysteine [8]
. The cultures was mixed by using combinations with Streptococcus thermophilus, Lactobacillus bulgaricus,

Mor. J. Chem. 5 N°4 (2017) 534-543

535
Lactobacillus acidophilus and Lactobacillus paracasei 1 and incubated during 48 h at 45oC for Stx + Lb y, at 30oC for
Stx+Lp [9].
After enumeration, the best combinations were supplemented with Bifidobacterium adolescentis or Bifidobacterium
longum and incubated at 37oC for 48 h [9].

Pathogens bacteria:
Two very common bacterial pathogens in Ain Defla were isolated of stools samples in Hektoen agar: Escherichia coli
responsible of diarrhea children and Salmonella typhi of typhoid fever [10]. After culture at 37oC for 24 h, E. coli
colonies are flat, dry, and yellow with salmon center; Salmonella colonies are green with a black center.

Preparation of bacterial culture:

In this study, we inoculate:
 Lactobacillus strains in 5 tubes containing 5 ml of MRS broth;
 Streptococcus thermophilus strains in 4 tubes containing 5 ml M17;
 Bifidobacterium strains in 2 tubes containing 5ml MRS cysteine broth ;
 the best microbial mixed in tubes containing 5ml of MRS broth;
 E. coli and S. typhi into two tubes containing 5 ml of nutrient broth;
We incubated these preparations at 37oC for 24 h.

Agar diffusion method:

Petri dishes containing M17, MRS and MRS cysteine agar were inoculated by a few milliliters of pathogenic bacteria
culture; and they let dry at 37oC for 30 min. After, we deposit on the surface of the agar blotting paper discs
impregnated beforehand by lactic acid bacteria strains and let dried at 37oC for 30 min. Finally, we incubate the Petri
dishes at 37 ° C for 24 to 48 h [11].

Analyses of organics acids by HPLC:

Preparation of Lactic Acid Bacteria-Pathogenic Bacteria(PB) culture: For this step, we inoculated : A tube of 5ml of
MRS broth with 250 μl of Lb. and 250 μl of PB .A tube of 5ml of M17 broth with 250 μl of Sc.tr and 250 μl of PB. A
tube of 5ml of MRS broth with 250 μl of (Stx + Lby) and 250 μl of PB. A tube of 5ml of MRS broth with 250 μl of
(Stx + Lby+ Bifz) and 250 μl of PB. The cultures were centrifuged at 12400 rpm/min for 10 min to remove bacterial
cells from the middle. The supernatant obtained was treated at 100oC/60min, 100oC/30min and 121 oC/15min to
eliminate the bacteriocins[12].

High Performance Liquid Chromatography (HPLC)

Analysis of samples was performed at 25ºC using Waters 1525 binary HPLC pump, Waters 2487 detectors for UV
and Waters for the degasser AF. The elution time is short, it is obtained by applying high pressure of about 300 bars,
with an equal volume of 20.00 μl injection and retention time (RT) equals 40, 00 minutes.
A cartridge filled with silica bonded with octadecyl was previously washed with 10 ml of methanol and then with 10
ml of water. The degassed sample was filtered through a cellulose membrane (pore diameter= 0.45 mm). A syringe
previously rinsed with the sample isused to collect 8 ml. In the chromatograph is injected successively 10μl of
reference solution (lactic acid, acetic acid) and 10μl of the sample prepared by repeating three times the injections in
the same order[12]. Qualitative Analysis: The qualitative analysis is to determine the retention time of each respective
Mor. J. Chem. 5 N°4 (2017) 534-543
536
compound eluted. The references organic acids are separated in order of elution following: lactic acid, acetic acid.
Quantitative Analysis: The quantitative analysis is to measure the peak areas (reference solution and the sample) and
determining the concentration of organic acid in the sample according to the equation:

3. Results and discussion

Interaction pathogenic-lactic acid bacteria:
Among all pure cultures of lactic acid bacteria strains, Lactobacillus bulgaricus 2, Lactobacillus bulgaricus 1 and
Bifidobacterium longum gave the best growth rates estimated at 1,59x108, 1,42 x 108 and 1,25 x 108 bacteria / mL,
respectively, while the strains of Streptococcus thermophilus 2, Streptococcus thermophilus 1, Lactobacillus
paracasei 1, Streptococcus thermophilus 3, Streptococcus thermophilus, Bifidobacterium adolescentis, Lactobacillus
acidophilus and Lactobacillus bulgaricus 3 gave moderate growth rates estimated at 7.6 x 10 7, 6 x 107, 4.51 x 107,
2.69 x 107, 2.11 x 107, 2 x 107, 1.48 x 107 and 1.28 x 107 bacteria / mL, respectively.
A significant growth rates are found with St3+ Lb1, St1+ Lb3, St*+ Lb3, St1 + Lb1 and St*+ Lb* ( 6.4 x 107, 5.9 x
107, 5.6 x 107, 4.8 x 107 and 3.1 x 107 bacteria / mL, respectively). The growth rates of lactic acid bacteria is higher
with the addition of Bifidobacterium longum.

Interaction lactic acid bacteria- E.coli:

In pure cultures, 70% of lactic acid bacteria inhibit E. coli, another inhibition zones are found with mixed cultures
(Tab1, Fig.1, Fig.2). The strain Lb. paracasei 1 and the combination St*+ Lb*+ Bl gaves a great inhibitory effect
(diameter = 10mm) among all cultures.

Interaction lactic acid bacteria -S. typhi:

We recorded the largest inhibition zone with St1 + Lb3 (7mm) (Tab1). For mixed cultures supplemented with B.
longum, the combination St3+ Lb1+ Bl gave better inhibition zone (diameter = 8 mm), then St2 + Lp + Bl (diameter =
7mm) (Fig. 3). The results show that the inhibition observed in the mixed cultures supplemented with Bifidobacterium
longum is greater than those found in mixed cultures supplemented with Bifidobacterium adolescentis.

High performance liquid chromatography (HPLC):

Qualitative Analysis
The two standard injected by spectrophotometer gave two peaks, one for lactic acid (RT = 4.887 min) and one for
acetic acid (RT = 5.344 min) [12]. Qualitative analysis of the lactic acid, gave small differences (D) relative to the
retention time (RT) of the standard lactic acid for all selected bacterial cultures (Tab.2). While qualitative analysis of
acetic acid, gave large differences (D) relative to the retention time of the standard acetic acid to all the selected
bacterial cultures (Tab.3). These results demonstrate that the inhibition of pathogenic strains (E. coli and S. typhi) is
due to the secretion of lactic acid (Fig.4, 5,6,7).

Quantitative analysis:
In bacterial cultures we detect only the lactic acid. According to the results (Tab.4), we can record a quantities of
lactic acid secreted by Lp (0.069 g / L), St*+ Lb*+ Bl (0.075 g / L), St3 + Lb1+ BL (0.070 g / L) and St2 + Lp+ BL
(0.077 g / L).

Mor. J. Chem. 5 N°4 (2017) 534-543

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3.Discussion:
Lactic acid bacteria have the ability to inhibit, in vitro, the action of Gram positive and negative pathogens[13].
Several works were shown the effect of lactic acid bacteria on pathogenic strains responsible for peptic disease.
Guetarni and al., [14] isolated thirty strains of lactic acid bacteria. The bacterial genera found in samples of raw milk
are represented par strains of Lactococcus and Enterococcus. E. faecium (B13) strain showed a highly significant
inhibition. In vivo, H. pylori was successfully detected in the gastric mucosa of BalB/C mouse. E. faecium (B13)
reduced the colonization of H. pylori in the stomach with a rate of 43% in a week. In another study [15]
homofermentative lactic acid bacteria strains were isolated from raw milk and tested for their antagonistic power
against strains of Helicobacter pylori. The search for the antagonist substance was performed following agar and broth
diffusion method. The substances produced by Enterococcus faecium strain (B13) lose their inhibitory activity after
treatment with trypsin and pepsin, but retain the antibacterial activity after heat treatment for 30 min at 60oC, 80oC for
10 min and 100oC for 5 min. This protein is stable in acid and alkaline pH. Lactobacillus acidophilus and
Lactobacillus paracasei in mixed culture have an inhibitory effect on E. coli[16]. Several strains of probiotic bacteria
have been isolated and identified; these strains have an inhibitory effect on E. coli and Salmonella. Two strains of
Lactobacillus paracasei subsp. paracasei , obtained from nine samples of artisanal Caprino Aspromonte cheese, made
from raw goats' milk, have Anti-E.coli effect[17]. Another probiotic bacterium, Lactobacillus acidophilus strain La-5
has been shown to be effective against enterohemorrhagic Escherichia coli (EHEC) O157:H7 [18]. B. longum can
inhibit the binding of E.coli on Caco-2 cultures and destroy Salmonella [19, 20,21]. B. longum correctly resists the
acidity and H2O2 secreted by the yogurt ferments (Sc. thermophilus and Lb. bulgaricus) hence the importance of
employment in fermented milks [22]. In vitro Lactobacillus acidophilus binds to the Caco-2 cells, and if it is
sufficient, it can occupy the field 'and prevent pathogenic bacteria to join [23]. The difference in affinity between Sc.
thermophilus and those of Lb. bulgaricus; Lb. acidophilus and Lb. paracasei 1 recorded in our work, can be explained
by the difference in growth characteristics of Sc. thermophilus and Lactobacillus spp., individual or associatively [24].
The highest growth rate is of Stx + Lby, this may be due to the positive association of lactic bacteria strains in mixed
culture[25] . This positive association can be explained by the synergy between Sc. thermophilus and Lactobacillus
spp., each species produces one or more substances, absent originally in the culture medium, which stimulates growth
of the other species, with an increase of proteolysis by the genus Lactobacillus and a higher production of lactic acid
by the two species [26]. Strains of Sc. thermophilus are often little proteolytic, due to a low level or absence of a
protease of the wall, hence their growth is limited, amino acids and peptides initially presents in the medium being
insufficient to cover their needs [27]. Inhibition of E.coli by mixed culture may be due also to the large production of
acid by Lactobacillus relative to Streptococcus thermophilus [28]. Lactic acid inhibits the intestinal survival of
Salmonella. This acid is solely responsible for the strengthening of the gastric barrier to Salmonella[29]. The
inhibitory activity in vivo, has been proven where treatment with lactobacilli, given during acute diarrhea in children
under three years, showed a reducing of diarrhea of approximately 0.7 days, and stool frequency of 1.6 stools / day
from the second day[30]. It was also shown that the incorporation of Lactobacillus acidophilus in the diet of infants
fed by bottle reduces the incidence of diarrhea and other digestive disorders [31]. The effect of L. acidophilus L1 was
revealed in vivo by injecting a group of mice with L. acidophilus 1 suspension or its infiltrate 30 min after injecting the
E. coli A99. Intraurethrally and the histopathological sections revealed the disappearance of inflammation signs
caused by E. coli A99 when It was injected alone [32]. The antibacterial effects of supernatant culture of
Lactobacillus species, L. casei, L. acidophilus, L. helveticus and L. delbrueckii ssp. bulgaricus isolated from yougourt
samples were stable at different temperatures conditions (56–100oC) for 30 and 60 min; also, it was stable at pH 3–10
[33].
Mor. J. Chem. 5 N°4 (2017) 534-543
538
Table.1: diameters of inhibition zones (mm) of lactic acid bacteria against pathogenic bacteria
Lactic acid bacteria Escherichia coli Salmonella typhi
Pathogenic bacteria
St1 0 0
St2 0 0
St3 5 0
St* 0 4
Lb1 7 0
Lb2 4 0
Lb3 3 ,5 4
Lb* 8 5
Lp1 10 0
Ba 5 0
Bl 6 4
St2+Lp 5 4
St3+Lb1 4 4
St1+Lb3 4 7
St*+Lb3 4 5
St1+Lb1 4 0
St*+Lb* 5 4
St2+Lp+Ba 0 0
St3+Lb1+Ba 0 0
St1+Lb3+Ba 0 5
St*+Lb3+Ba 0 4
St1+Lb1+Ba 0 0
St*+Lb*+Ba 5 4
St2+Lp+Bl 5 7
St3+Lb1+Bl 0 8
St1+Lb3+Bl 5 6
St*+Lb3+Bl 4 6
St1+Lb1+Bl 8 4
St*+Lb*+Bl 10 6

Tab2. Qualitative analysis of the lactic acid

Samples RT (min) D (difference)
E.coli+Lp 4 ,961 0,074
E.coli+ (St*+Lb*+Bl) 4,959 0,072
S.typhi+ (St3+Lb1+Bl) 4,969 0,082
S.typhi+ (St2+Lp+Bl) 4,949 0,062

Mor. J. Chem. 5 N°4 (2017) 534-543

539
These strains may help in the treatment of E. coli O157:H7-associated diseases and can be applied as a biological
preservative in the food industry [34]. Bacterial associations were carried out between H. pylori strain isolated
togastric biopsy from a patient having ulcer and two strains of lactic acid bacteria: Streptococcus thermophilus and
Lactobacillus acidophilus. Supernatants obtained by centrifugation (12400 rpm / min during 10 min) of the bacterial
cultures treated with various temperatures: 100oC / 60 min, 100oC / 30 min and 121 oC / 15 min, are analyzed by
HPLC. Qualitative and quantitative analysis showed the presence of lactic acid in supernatants with a quantity of
0.019 g / L secreted by Streptococcus thermophilus and 0.058 g / L secreted by Lactobacillus acidophilus [13]. In our
study, lactic acid is the major inhibitor substance in vitro of E. coli and S. typhi. Lactic acid is a weak mono, with a
pKa of 3.86. Only the undissociated form (CH3CHOHCOOH) can cross the plasma membrane and dissociate within
the cell, releasing ions that acidify the cytoplasm. In addition to the effect of pH, undissociated acid brought down the
electrochemical gradient of protons, killing sensitive bacteria [35,36, 37] that the introduction of yogurt in the child's
diet suffering from gastroenteritis allows weight gain, decreased hospitalization time and the frequency of watery
stools. According to these studies, the introduction of the yogurt was done after the rehydration period. The use of
probiotics in various health problems is increasingly documented. Yogurt is obtained by the fermentation of lactic acid
in milk by Lactobacillus bulgaricus and Streptococcus thermophilius. Several mechanisms action of probiotics are
proposed such as synthesis of antimicrobial substances, competition for nutrients required for growth of the pathogen,
competitive inhibition of the accession of the pathogen and a change of toxins or toxin receptor [38].

Tab3. Qualitative analysis of the acetic acid

Samples RT (min) D (difference)
E.coli+Lp 4 ,961 0,074
E.coli+ (St*+Lb*+Bl) 4,959 0,072
S.typhi+ (St3+Lb1+Bl) 4,969 0,082
S.typhi+ (St2+Lp+Bl) 4,949 0,062

Fig.1: Interaction of acid lactic bacteria in pure culture with E.coli

Fig.2: Interaction of acid lactic bacteria in mixed culture with E.coli.

Mor. J. Chem. 5 N°4 (2017) 534-543

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Fig.3: Interaction of acid lactic bacteria in mixed culture with S.typhi.

Fig.4: analyse of organic acids in supernatant of culture E.coli+Lp

Fig.5: analyse of organic acids in supernatant of culture E.coli+ St*+Lb*+Bl

Fig.6: analyse of organic acids in supernatant of culture S.typhi+ St3+Lb1+Bl

Mor. J. Chem. 5 N°4 (2017) 534-543

541
Fig.7:analyse of organic acids in supernatant of culture S.typhi+ St2+Lp+Bl

4. Conclusion
The interaction of lactic acid bacteria that have given the best microbial loads after counting and isolated pathogenic
bacteria showed inhibitions in pure and mixed cultures. Lactobacillus bulgaricus 2, Bifidobacterium longum and
Lactobacillus bulgaricus 1, St2 + Lp, St3 + Lb1, St1 + Lb3, St* + Lb3, St1 + Lb1 and St* + Lb* gave high levels of
bacteria among all pure and mixed cultures of lactic acid bacteria enumerated. The addition of bifidobacteria to six
best combinations, counting from mixed cultures, gave higher bacterial loads with Bifidobacterium longum. The
cultures L. paracasei 1 (Lp) and St*+ Lb*+ Bl have a significant inhibitory effect of Escherichia coli. For Salmonella
typhi, the results of interactions showed the best zones of inhibition with St3 + Lb1+ Bl and St2 + Lp + Bl. The
qualitative and quantitative analysis of organic acids by high performance liquid chromatography (HPLC) was
confirmed the presence of lactic acid in supernatants cultures and it may be alone or in combination with other
substances responsible in the inhibition of E. coli and S. typhi.

Acknowledgments:
I express my sincere thanks to all staff quality control laboratories of El Harrach, Algiers, especially Mrs. Bourouis
and Mr.Berghout.
Other thanks go to the microbiology laboratory personnel of hospital Ain Defla, Algeria.

References
[1] M. Dosso, M. Coulibaly, A. Kadio, Place des diarrhées bactériennes dans les pays en développement. Manuscrit
n°PF02. Journée en hommage au Professeur A. Ed. DOIN, 1998.
[2]WHO, La diarrhée aigüe chez les adultes et les enfants: une approche global. 2012.
[3]G.J. Tortora, B.R. Funke, C.L. Case, Introduction à la microbiologie. Ed. Renouveau Pédagogique Inc (ERPI),
2003.
[4]Y. Laid, Dialogue National Interministériel sur le Changement Climatique, Secteur clé: Santé (Adaptation) Alger,
Algérie. PNUD, 2010.
[5]J.P. Larpent, Microbiologie alimentaire. Ed. Lavoisier, Paris, 1997.
[6]L. Sutra, M. Federighi, J.L. Jouve, Manuel de bactériologie alimentaire. Ed. Polytechnica, 1998.
[7]H. Beerens, F.M. Luquet, Guide pratique d’analyse microbiologique des laits et produits laitiers. Ed. Lavoisier,
1987.
[8]J.D. De Man, M. Rogosa, M.E. Sharpe, J. Appl. Bact. 23 (1960) 130-135.

Mor. J. Chem. 5 N°4 (2017) 534-543

542
[9]C.M. Bourgeois, J.P. Larpent, Microbiologie alimentaire : les fermentations alimentaires. Ed. Lavoisier, 1989.
[10]S. Robin, S. Roche, Virologie : les coprocultures. DES de bactériologie, 2002.
[11]J.P. Guiraud, Microbiologie alimentaire. Ed. Dunod, 1998.
[12]H. Guetarni, A. Bensaid, A. Bensoltane, Journal of Biotechnology Letters 3 (1) (2012) 34-36.
[13]M.H. Nahaisi, Appl. Sci. 6 (1986) 153-178.
[14]H. Guetarni, A. Bensoltane, A. Aichour, Z. Benabdallah, I. R. Khouatria, N. K. Khouatria, OnLine Journal of
Biological Sciences. 12 (2) (2012) 38-43.
[15]H. Guetarni, A. Bensoltane, Egypt. J. of Appl. Sci. 29 (1) (2014) 19-30.
[16]M.E. Nader de Macias, N.C. Romero, M.C. Apella, S.M. Gonzales, G. Oliver, J. Food Prot. 56 (1993)401–405.
[17]A. Caridi, J. Ind. Microbiol. Biotechnol. 29(6) (2002) 303-8.
[18]M.G. Medellin-Pena, M.W. Griffiths, Applied And Environmental Microbiology. 75 (2009) 1165–1172.
[19]W.E.V. Lankaputhra, N.P. Shah, M.L. Britz, Milchwissenschaft. 51(2) (1996) 65-70.
[20]C.J. Ziemer, G.R. Gibson, Int. Dairy J. 8 (1998) 473-479.
[21]L. De Vuyst, L. Makras, L. Avonts, H. Holo, Q. Yi, A.Servin, D. Fayol-Messaoudi, C. Berger, G. Zoumpopoulou,
E. Tsakalidou, D. Sgouras, B. Martinez-Gonzales, E. Panayotopoulou, A. Mentis, D. Smarandache, L. Savu, P.
Thonart, I .Nes, Microb. Ecol. Health Dis. 16 (2004)125-130.
[22]N.P. Shah, Milchwissenschaft. 7 (1997) 349-356.
[23]M. F. Bernet, D. Brassart, J. R. Neeser, A. L. Servin, Gut. 35(1994) 483-489.
[24]S. Terre, Techniques Laitières et Marketing. 1006 (1986) 26-39.
[25]M.J. Amorosso, M.C. Mancade Nadra, G. Diver, Le lait . 6(1989) 519-528.
[26]R.C. Kaeffer, P. Mongin, Veille technologique en industrie laitière. Ed. Hygino. 1993.
[27]V.M. Marshall, J Dairy Res. 54 (4) (1987) 559-574.
[28]V.M. Marshall, Perfumer and Flavorist. 7(2) (1982) 27-34.
[29] R.Van Der Meer, I.M.J. Bovee Oudenhoven, Int Dairy J. 8 (1998) 481-486.
[30]C.W. Van Niel, C. Feudtner, M.M. Garrison, D.A. Christakis, Pediatrics. 109 (2002) 678-84.
[31]H. Szajewska, J.Z. Mrukowicz, J. Pediatr. Gastroenterol. Nutr. 33 (suppl. 2) (2001) 17-25.
[32]J. F. Harith, A. Alaa Muyassar, J. of university of Anbar. for pure science. 5 (1) (2011).
[33]A.A.I. Fooladi, M.C. Forooshai, P. Saffarian, R. Mehrab, Journal of Food Safety. 34 (2) (2014) 150–160.
[34]S. Kalyoussef, E. Nieves, E. Dinerman, C. Carpenter, V. Shankar, J. Oh, B. Burd, R.H. Angeletti, K.W. Buckheit,
D.N. Fredricks, R.P. Madan, M.J. Keller, B.C. Herold, PLoS One. 7(11) (2012): e49506.
[35]M. Zagorec, S. Christieans, Flores protectrices pour la conservation des aliments. Ed. Quae, 2013.
[36]G. Boudraa, M. Benbouabdellah, W. Hachelaf, M. Boisset, J.F. Desjeux, M. Touhami, J. Pediatr. Gastroenterol.
Nutr. 33(3) (2001)307-13
[37]N. Pashapour, S.G. Iou, Turk. J. Pediatr. 48(2) (2006)115-8
[38]BTEC, Les soins aux enfants ayant une gastroentérite ; rapport 8. Bureau de transfert et d’échange de
connaissances, Collection des rapports de BTEC, université Laval (2007).

Mor. J. Chem. 5 N°4 (2017) 534-543

543

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