Annals of Agricultural Science (2016) 61(1), 65–75

H O S T E D BY
Faculty of Agriculture, Ain Shams University

Annals of Agricultural Science

www.elsevier.com/locate/aoas

Screening of isolated potential probiotic lactic acid

bacteria for cholesterol lowering property and bile
salt hydrolase activity
M.G. Shehata a,*, S.A. El Sohaimy a, Malak A. El-Sahn b, M.M. Youssef b

a
Food Technology Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological
Application, Alexandria, Egypt
b
Food Science and Technology Dept., Fac. of Agric., Alexandria Univ., 21545 El-Shatby, Alexandria, Egypt

Received 27 January 2016; accepted 6 March 2016

Available online 29 April 2016

KEYWORDS Abstract A total of 142 isolates of lactic acid bacteria (LAB) were isolated from dairy and
Probiotics; non-dairy sources. The LAB isolates were screened for antimicrobial activity. Out of 142 isolates
Lactic acid bacteria; only 68 isolates exhibited antimicrobial activity. Among these isolates, nine showed wide spectrum
Antimicrobial activity; antimicrobial activity as well as good bile salt, acid and phenol tolerance. Seven isolates of the latter
Bile tolerance; ones showed more than 20% cholesterol reduction and an observed bile salt hydrolase (BSH)
Acid tolerance; activity. The promising isolates were identified using phenotypic, biochemical and genetic methods.
Cholesterol removal; Ó 2016 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams
Bile salt hydrolase University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

Introduction and Gibson, 1999). They are non-pathogenic, technologically

suitable for industrial processes, acid tolerance, bile tolerance
In recent years, different investigations support the importance and produce antimicrobial substances (Mojgani et al., 2015).
of probiotics as apart of healthy diet for humans and animals They are classified as generally recognized as safe (GRAS)
and as a way to provide a natural, safe and effective barrier microorganisms because of their long and safe use as starter
against microbial infections (Angmo et al., 2016; Oh and cultures in fermented products.
Jung, 2015). According to the definition by the World Health Nowadays, most probiotic bacteria are belonging to the
Organization (WHO), probiotics are ‘‘live microbial food sup- genera Lactobacillus and Bifidobacterium (Prasad et al.,
plements which, when administered in adequate amounts con- 1998). However, species belonging to the genera Lactococcus,
fer a health benefit on the host” (FAO/WHO, 2001). Among Enterococcus and Saccharomyces (Salminen and von Wright,
the usually used microorganisms, lactic acid bacteria (LAB) 1998; Sanders and in’t Veld, 1999) are also considered as pro-
are regarded as a major group of probiotic bacteria (Collins biotic microorganisms.
According to the guidelines for the evaluation of probiotics
* Corresponding author. Tel.: +20 34593420; fax: +20 34593423. in food reported by a Joint FAO/WHO working group (Vijaya
E-mail address: gamalsng@gmail.com (M.G. Shehata).
et al., 2015), two of the currently most widely used in vitro tests
Peer review under responsibility of Faculty of Agriculture, Ain-Shams
are resistance to gastric acidity and bile salts, as based on both
University. survival and growth studies. Other functional properties used
http://dx.doi.org/10.1016/j.aoas.2016.03.001
0570-1783 Ó 2016 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
66 M.G. Shehata et al.

to characterize probiotics are the production of antimicrobial Isolation of lactic acid bacteria
compounds and cholesterol removal (Park et al., 2007; Xie
et al., 2015). The mechanism through which probiotics may Milk samples were incubated at 30 °C; 37 °C; 42 °C, while
antagonize pathogens involves production of antimicrobial samples of cheese, Zabady and Rayeb were cultured in steril-
compounds such as lactic acid, acetic acid, hydrogen peroxide ized reconstituted skim milk and incubated until coagulation.
and bacteriocins. Coagulated samples were then streaked on over agar surface
Certain studies showed that among the other effects of pro- of MRS medium (De Man et al., 1960) and were incubated
biotic include, consumption of lactic acid bacteria reduced car- anaerobically at 30 °C, 37 °C or 42 °C for 48 h. Boza, calves
riage of pathogens microorganism, decreased certain risk infant faeces and intestine of marine fish samples, were diluted
factors for coronary artery disease, and resulted in a dose- serially from 101 to 107, then 0.1 ml aliquot of the higher
dependent reduction in the symptoms of Irritable bowel syn- dilutions (104 to 107) were spread on to MRS plates and
drome (Vries et al., 2006). It seems that, in the research for incubated at 30, 37 and 42 °C for 48 h.
strains with probiotic potential, food might also be a good White and creamy colonies were picked up randomly and
source of suitable isolates for finding new probiotic strains purified by three successive transfers on MRS medium. The
for functional food products. Several probiotics bacteria are cultures were routinely checked for purity by microscopic
found to produce bile salt hydrolase (BSH) that helps to examination.
reduce serum cholesterol (Miremadi et al., 2014) and hence The pure cultures were characterized using Gram stain, cell
BSH activity is also considered as an additional criterion for morphology and catalase reaction according to standard pro-
the selection of probiotics. The aim of the present study was cedures (Sharpe, 1979). Gram-positive and catalase-negative
to isolate, identify and screen for potential probiotic lactic acid isolates were selected and stored at 80 °C in MRS broth plus
bacteria with high cholesterol capacity and bile salt hydrolase 28% glycerol (El-Soda et al., 2003). The purified cultures were
activity. activated by subculturing twice in MRS broth before use.

Materials and methods Antimicrobial activity assays

Collection of samples Screening of LAB isolates for antimicrobial activity

The Antimicrobial activity spectrums of cells free supernatants
Samples were collected from the normal habitats of lactic acid of (LAB) isolates were determined against ten pathogens
bacteria (LAB) such as raw animal milk, fermented foods (Table 2) using spot-on-lawn method (Barefoot and
(Boza, Zabady, Rayeb), cheese (Ras, Kareish), calves infant Klenhammer, 1983). LAB isolates were cultivated in MRS
faeces and intestinal of marine fish (Table 1). Samples were for 16-18 h with 1% inoculum then cells were removed from
transported to the laboratory in ice box and stored at ffi 4 °C. MRS medium by centrifugation (6500g for 10 min, 4 °C) to
obtain cell free supernatant. Lawns of pathogenic strains were
prepared by adding 0.125 ml (2  107 cell/ml) of 10 diluted
overnight culture to 5 ml of corresponding soft agar (Table 2).
Table 1 Isolation sources of lactic acid bacteria. The contents of the tubes were gently mixed and poured over
Isolation Number of Number Location the surfaces of pre-poured MRS agar plates. Ten microliter of
Sources examined of each cell free supernatant was spotted onto the surface of the
samples isolates soft agar plate and after 24 h of incubation, the plates were
Boza (BO) 2 43 Manfalut City, Assiut
Governorate, Egypt
Tanta city, Gharbia
Governorate, Egypt Table 2 Indicator strains and their growth conditions.
Calves 2 10 Private farm,
infants Damanhour city, Pathogenic microorganisms Medium and growth
faeces (F) Beheira Governorate, temperature
Egypt Bacillus subtilis DB 100 host Nutrient Broth, 37 °C
karish cheese 3 20 Alexanderia, local Candida albicans ATCC MYA-2876 YPDa Broth, 37 °C
(k) markets Clostridium botulinum ATCC 3584 TPGYb Broth, 37 °C
Milk (M) 2 10 Alexanderia, local Escherichia coli BA 12296 isolated by LBc Broth, 37 °C
markets dr sobhy
Ras cheese 3 15 Alexanderia, local Klebsiella pneumoniae ATCC12296 LBc Broth, 37 °C
(R) markets Salmonella senftenberg ATCC 8400 Nutrient Broth, 37 °C
Rayeb Milk 2 19 Alexanderia, local Staphylococcus aureus NCTC 10788 Nutrient Broth, 37 °C
(RM) markets Staphylococcus epidermidis Nutrient Broth, 37 °C
The 2 0 Alexanderia, local Streptococcus dysgalactiae subsp. Nutrient Broth, 37 °C
intestines of markets Equisimilis
marine fish Streptococcus pyogenes Nutrient Broth, 37 °C
(I) a
Zabady (Z) 4 25 Alexanderia, local YPD broth: Yeast peptone dextrose.
b
markets TPGY broth: tryptone–peptone–glucose–yeast extract.
c
Total 20 142 LB broth: Luria-Bertani medium.
Screening of probiotic lactic acid bacteria 67

checked for the appearance of an inhibition zone. Clear zones cultures of LAB isolates were inoculated (1%) into MRS broth
around the spots indicate the antimicrobial activity of isolated with (0.2 and 0.5% v/v) or without phenol. Bacterial cells in
bacteria. the culture broth were measured by reading the absorbance
(A) at 600 nm after 24 h of incubation at 37 °C. The experi-
Quantification of antimicrobial activity ments were repeated twice in duplicate.

Cholesterol assimilation
Antimicrobial activity in the supernatant was determined by
an adaptation of the critical two fold dilution method
(Parente et al., 1994). Each tested isolate was subcultured in Freshly prepared MRS broth, supplemented with 0.3% oxgall
MRS for 24 h, counted and adjusted to give 109 cell/ml. The (Bio Basic Canada INC.) as bile salt and filter sterilized water
culture was used to inoculate (1%) fresh MRS and propagated soluble cholesterol (100 lg/ml), was inoculated with each iso-
for 24 h. Supernatant was obtained by centrifugation (9000g late at 1% level and incubated anaerobically at 37 °C for
for 15 min). Serial two-fold dilutions of supernatant were car- 24 h. After incubation period, cells were removed by centrifu-
ried out in MRS. Activity was quantified by taking the recip- gation (9000g for 15 min) and the remaining cholesterol in the
rocal of the highest dilution that exhibited a clear zone of spent broth was determined calorimetrically using o-
inhibition and was expressed as activity units (AU) per millil- phthalaldehyde method described by Rudel and Morris
itre of culture media. The titre of the antibacterial substance, in (1973). One millilitre of the cell- free broth was added to
AU/ml, was calculated as (1000/d) D, where D is the dilution 1 mL of KOH (33% wt/vol) and 2 mL of absolute ethanol,
factor and d is the amount of supernatant in ll (Parente vortexed for 1 min, followed by heating at 37 °C for 15 min.
et al., 1994). After cooling, 2 mL of distilled water and 3 mL of hexane were
added and vortexed for 1 min. One millilitre of the hexane
Acid tolerance layer was transferred into a glass tube and evaporated in water
bath at 65 °C. The residue was immediately dissolved in 2 mL
of o-phthalaldehyde reagent. After complete mixing, 0.5 mL
Isolates of LAB were propagated twice in MRS broth (1% v/v)
concentrated sulphuric acid was added and the mixture was
for 20 h at 37 °C before experimental use. The cells from
vortexed for 1 min. Absorbance was read at 550 nm (T80
100 ml MRS culture were harvested by centrifugation
UV/Vis spectrometer PG Instruments LDT, United Kingdom)
(4300g, 10 min), and washed three times in phosphate-
after 10 min. All experiments were replicated twice.
buffered saline, pH 7.0. Washed cell pellets were then sus-
pended in (1/10) cultivation volume in the same buffer, hence
Screening of probiotic LAB for bile salts hydrolases activity
obtaining a 10-fold increase in cell density. To 1 ml of the
(BSH)
washed cell suspension, 5 ml of simulated gastric juice and
1.5 ml NaCl (0.5 w/v) were added. Simulated gastric juice
Qualitative determination of bile salts hydrolases activity
was prepared freshly daily by suspending pepsin (3 g/L) in ster-
ile saline (0.5% w/v) and adjusting the pH to 2.0 with concen- The BSH activity was determined as described by Du Toit
trated HCl (Charteris et al., 1998). The materials were et al. (2003). The LAB isolates were grown on MRS agar
vortexed for 10 s and incubated at 37 °C for 3 h. Aliquots of plates containing 0.5% (w/v) taurodeoxycholic acid sodium
0.1 ml were then removed at constant intervals (0, 1, 2, 3 h) salt (TDCA; Sigma, USA) and 0.037% calcium chloride.
for determination of total viable count. Dilutions were made Plates were incubated under anaerobic conditions at 37 °C
(up to 104) and cells were plated in duplicate on MRS agar. for 72 h. The precipitation zone surrounding colonies indicated
Plates were incubated at 37 °C for 72 h before enumeration the bile salt hydrolase activity of bacteria.
(Charteris et al., 1998).
Quantitative determination of bile salts hydrolases activity
Bile tolerance The BSH activity was determined by measuring the amount of
amino acid liberated from conjugated bile salts, by the probi-
otic isolates as described by Tanaka et al. (2000) with several
Bile containing MRS broth was prepared by the addition of
modifications. Briefly, from cultures grown for 20 h at 37 °C,
0.3 (v/v) of bile salt (Bio Basic Canada INC.). The cells from
cells were harvested by centrifugation at 9700g for 15 min,
100 ml (20 h MRS tested culture) were collected by centrifuga-
washed twice with 0.1 M sodium phosphate buffer containing
tion (3400g, 10 min), washed twice in saline (8.5 g NaCl/L) and
10 mM dithiothreitol (DTT), pH 6.8 and re-suspended in the
resuspended in 10 ml MRS broth. This suspension was inocu-
same buffer to obtain a suspension with an optical absorbance
lated (1%) into MRS broth lacking or containing bile salt.
(A600 nm) of 3.0. Cell suspension was sonicated for 60 s. with
After 0, 1, 2 and 3 h of incubation at 37 °C, viable counts on
cooling on ice with two cycles of 16 mm using a sonicator
MRS agar plates and absorbance of the culture at 625 nm were
(Sonics and Materials Inc., Vibro cell), followed by centrifuga-
determined (Matijasic and Rogelj, 2000). Experiments of acid
tion at 9700g for 15 min. The reaction mixture consisted of
and bile tolerance were repeated three times each with dupli-
180 mL of 0.1 M sodium phosphate buffer, pH 6.0, 10 mL of
cate analysis.
a 200 mM appropriate conjugated bile salt, 10 mM DTT and
10 mL of cell-free extract. The reaction mixture was incubated
Phenol tolerance
at 37 °C for 30 min., then a sample (100 lL) was taken and
200 lL of 15% (w/v) trichloroacetic acid was added to termi-
Phenol tolerance experiments were performed as described by nate the reaction. The sample was centrifuged (9700g for
Aswathy et al. (2008) with slight modifications. The overnight 15 min) and 200 lL of the supernatant was added to 200 lL
68 M.G. Shehata et al.

of distilled water and 1.9 mL of ninhydrin reagent (5 mg nin- (Syngene Bio Maging, Canada). The DNA marker 100–
hydrin, 1.2 mL glycerol, and 0.7 mL 0.5 M pH 5.5 sodium 1500 bp (TAKARA BIO INC., Shiga, Japan) was used as
citrate buffer). The mixture was vortexed and boiled for the molecular weight standard.
14 min. After subsequent cooling, the absorbance at 570 nm
was determined using glycine or taurine as standard. One unit Sequencing of DNA. The DNA sequencing reactions were per-
of BSH activity (U/ml) was defined as the amount of enzyme formed using an automated DNA sequencer. Database
that liberated 1 mmol of amino acid from the substrate per searches were performed using the latest release of non-
min. Protein concentration was determined by the Lowry redundant DNA sequence database present at the National
method (Lowry et al., 1951), with bovine serum albumin Centre for Biotechnology Information (NCBI) website located
(Sigma) as standard. All experiments were repeated twice. at: http://www.ncbi.nlm.nih.gov/BLAST (Altschul et al.,
1997).
Identification of the promising LAB isolates
Statistical analysis
Phenotypic characterization
The promising LAB isolates were phenotyped as described in Data were statistically analysed with CoStat software (version
Bergey’s manual of systematic bacteriology (Logan and De 6.303). One-way analysis of variance was used to study signif-
Vos, 2009). The following tests were applied: cell morphology; icant difference between means, with significance level at
growth at 15, 37 and 45 °C; and growth in MRS containing P = 0.05.
2.5%, 4% and 6.5% NaCl. Fermentation patterns were deter-
mined using API 50 CHL and API 20 kits (Biomerieux SA, Results and discussions
France) according to the manufacturer’s instructions.
Screening of LAB isolates for their antimicrobial activity
Molecular identification
Extraction of bacterial DNA. The DNA extraction and purifi- One hundred and forty-two LAB were isolated from various
cation from bacterial isolates were carried out according to the sources (Table 1). All isolated bacteria fit the classification of
procedure described by Cheng and Jiang, 2006. Overnight bac- LAB as Gram-positive, catalase negative (Sharpe, 1979). The
terial cultures were centrifuged individually at 15,000g for antimicrobial activity is one of the most important selection
10 min. Pellets were washed with 400 ll SET buffer (75 mM criteria for probiotic. The LAB isolates were screened for pro-
NaCl, 25 mM EDTA, 20 mM Tris, pH 7.5), then centrifuged duction of antimicrobial agents against 10 pathogens
at 10,000g for 10 min. The pellets were resuspended in TE buf- (Table 2).
fer (Tris-EDTA buffer, pH 8.0), 100 ll tris-saturated phenol Out of 142 LAB isolates only 68 isolates exhibited antago-
(pH 8.0) was added and the suspensions were centrifuged at nistic activity with varying degrees. Out of sixty LAB strains
10,000g for 10 min at 4 °C. Mixtures composed each of isolated from zabady and cheese samples, only thirty-eight
160 ll of the obtained aqueous phase, 40 ll TE buffer and strains showed inhibitory activity against the tested pathogens.
100 ll chloroform were centrifuged at 10,000g for 10 min. No antagonistic activity could be observed for LAB strains
The resulting supernatant (100 ll) was mixed with 40 ll TE isolated from milk and calves infant faeces. The LAB strains
buffer and 5 ll RNase (10 mg/ml) and incubated at 37 °C for that showed inhibitory activity against more than six of the
10 min. Then 100 ll chloroform was added and the mixture tested pathogens were isolated from Boza and Rayeb milk sam-
was centrifuged at 15,000g for 10 min at 4 °C. The purity as ples. On the other hand, LAB isolates obtained from zabady,
well as the yield of DNA in the aqueous phase was assessed Ras cheese and Karish exhibited antagonistic activity only
spectrophotometrically. against three or less of the tested pathogens. Indicator patho-
gens can be organized in descending order according to their
Polymerase chain reaction amplification. Universal primers sensitivity to the tested isolates as follows: Escherichia coli
identifying LAB, designed using the invariant region in the BA 12296 was sensitive to 41 isolates while Staphylococcus epi-
16 s rDNA sequences for LAB (Wang et al., 1996), were dermidis, Salmonella senftenberg ATCC 8400, Staphylococcus
obtained from Sigma Scientific Services Co., Germany. The aureus NCTC 10788, Streptococcus pyogenes, Klebsiella pneu-
reaction mixture (20 ll) consisted of 5 ll colourless GoTaqÒ moniae ATCC12296, Candida albicans ATCC MYA-2876,
reaction Buffer (5), 0.25 ll GoTaqÒ DNA Polymerase Streptococcus dysgalactiae subsp. equisimilis, and Bacillus sub-
(5 u/ll) (Promega, USA), 2.5 ll PCR nucleotide Mix tilis DB 100 were sensitive to 28, 29, 23, 19, 15, 14, 10, 2 of LAB
(10 mM), 1 ll of each primer 5/CGTGCCAGCCGCGGTAA- isolates, respectively.
TACG 3/and 5/GGGTTGCGCTCGTTGCGGGACT All isolates with antimicrobial activity against any of the
TAACCCAACAT 3/) as forward and reverse primers, respec- tested pathogens (68 isolates) were selected for quantitative
tively, 2 ll genomic DNA and 8.25 ll of nuclease-free water. determination of their antimicrobial activities (Table 3).
The PCR amplification was carried out in the thermo cycler Among 68 isolates, only 9 isolates showed wide spectrum
PCR (Santa Clara, California, United States) according to the activity against four tested pathogens at least.
following programme: initial denaturation at 95 °C for 5 min, Among the nine isolates of LAB, only one isolate (RM39)
amplification for 30 cycles [95 °C/40 s (denaturation), showed strong activity of 1600 AU/ml against Klebsiella pneu-
55 °C/40 s (annealing), 72 °C/1 min (extension)], then final moniae ATCC12296 while, four isolates exhibited inhibitory
extension at 72 °C for 10 min. The products were separated activity of 800 AU/ml against each of Escherichia coli
on 1% agarose gel containing ethidium bromide (1 lg/ml), (BO51); Streptococcus pyogenes (RM28); Staphylococcus aur-
then image was taken using gel documentation system eus NCTC 10788 (BO3, RM3), Salmonella senftenberg ATCC
Screening of probiotic lactic acid bacteria 69

Table 3 Antimicrobial activities of cell-free supernatants of 68 selected LAB isolates against various pathogens.a
Isolate no Sym Sources Incubation T (C) Antimicrobial activity against pathogens expressed in AU/mlc
Bac Cand. Clo. E. coli Kleb. Sal. St.au. St.epi St.Py. St. dy.
d b
1. BO3 BO 30 0 0 0 400 0 400 800 200 200 400
2. BO4 BO 30 0 0 0 200 0 200 0 0 0 0
3. BO5 BO 30 0 0 0 200 0 0 0 200 0 0
4. BO6 BO 30 0 0 0 200 0 0 0 0 0 0
5. BO7 BO 30 0 0 0 400 800 200 0 0 0 0
6. BO8 BO 30 0 0 0 200 0 200 0 200 0 0
7. BO12 BO 30 0 0 0 200 0 200 0 200 0 0
8. BO22 BO 37 0 0 0 200 0 200 0 200 0 0
9. BO24 BO 37 0 0 0 200 0 200 0 200 0 0
10. BO27 BO 37 0 0 0 200 0 200 0 200 0 0
11. BO29 BO 37 0 0 0 200 0 200 0 200 0 0
12. BO30 BO 37 0 0 0 200 0 200 0 200 0 0
13. BO31 BO 37 0 0 0 200 0 200 0 200 0 0
14 BO33 BO 37 0 0 0 200 400 400 0 0 0 0
b
15. BO34 BO 37 200 0 0 800 800 400 0 0 0 0
d
16. BO35 BO 37 0 0 0 800 200 800 200 200 0 200
17. BO36 BO 37 0 0 0 400 0 0 200 400 0 0
d
18. BO37 BO 37 800 0 0 400 0 200 200 800 400 400
19. BO42 BO 42 0 0 0 400 200 0 0 400 0 0
20. BO44 BO 42 0 0 0 0 200 400 0 0 400 0
21. BO45 BO 42 0 0 0 200 0 200 200 0 0 0
22. BO46 BO 42 0 0 0 200 0 200 200 0 0 0
23. BO50 BO 42 0 0 0 0 0 200 200 0 200 0
d
24. BO51 BO 42 0 400 0 800 200 200 200 400 200 400
d
25. BO52 BO 42 0 0 0 400 400 0 0 400 0 400
26. Z1 Zb 30 0 0 200 0 0 0 200 0 200 0
27. Z2 Z 30 0 0 200 0 0 0 200 0 200 0
28. Z3 Z 30 0 0 0 0 200 0 0 200 200 0
29. Z4 Z 30 0 0 0 200 0 0 200 0 200 0
30. Z5 Z 30 0 0 0 0 0 0 0 0 200 0
31. Z8 Z 30 0 0 0 0 200 0 0 0 200 0
32. Z9 Z 30 0 0 0 0 200 0 0 0 0 200
33. Z22 Z 37 0 200 0 400 0 0 0 0 0 0
34. Z23 Z 37 0 0 0 0 200 200 0 0 0 200
35. Z25 Z 37 0 0 0 0 0 400 400 0 0 0
36. Z27 Z 37 0 0 0 200 0 0 200 0 0 0
37. Z28 Z 37 0 0 0 0 200 0 200 400 0 0
38. Z39 Z 37 0 200 0 0 0 0 200 400 0 0
39. Z41 Z 42 0 0 0 0 0 200 200 200 0 0
40. Z42 Z 42 0 0 0 200 0 0 200 200 0 0
41. R20 Rb 37 0 0 0 200 0 0 0 200 0 0
42. R22 R 37 0 0 0 200 0 0 200 0 0 0
43. R24 R 37 0 0 0 200 0 0 200 0 0 0
44. R25 R 37 0 0 0 0 200 200 0 400 0 0
45. R26 R 37 0 0 0 0 0 200 0 0 200 0
46. R27 R 37 0 200 0 0 0 0 200 0 0 0
47. R28 R 37 0 200 0 0 0 0 0 0 0 0
48. R34 R 37 0 0 0 0 0 0 200 200 0 0
49. K1 Kb 30 0 0 0 200 0 0 200 0 0 0
50. K2 K 30 0 0 0 0 0 0 0 0 400 0
51. K4 K 30 0 0 0 0 0 0 200 200 0 0
52. K5 K 30 0 200 0 0 0 0 200 0 400 0
53. K6 K 30 0 0 0 200 0 0 0 0 200 0
54. K7 K 30 0 0 0 0 0 200 0 0 200 0
55. K8 K 30 0 0 0 200 0 0 0 0 0 0
56. K9 K 30 0 200 0 0 0 0 400 0 0 0
57. K23 K 37 0 200 0 200 0 0 0 0 200 0
58. K24 K 37 0 0 0 0 0 0 0 0 0 200
59. K26 K 37 0 200 0 0 0 0 0 0 200 0
60. K27 K 37 0 200 0 200 0 0 0 0 0 0
61. K40 K 42 0 0 0 200 0 0 0 0 0 0
(continued on next page)
70 M.G. Shehata et al.

Table 3 (continued)
Isolate no Sym Sources Incubation T (C) Antimicrobial activity against pathogens expressed in AU/mlc
Bac Cand. Clo. E. coli Kleb. Sal. St.au. St.epi St.Py. St. dy.
62. K42 K 42 0 200 0 200 0 0 0 0 200 0
63. K43 K 42 0 200 0 0 0 0 0 0 200 0
64. RM1 RMb 30 0 0 0 200 200 200 0 0 0 0
d
65. RM3 RM 30 0 1600 0 400 200 0 800 200 200 800
66. RM26 RM 30 0 0 0 0 200 0 0 200 200 0
d
67. RM28 RM 30 0 0 0 400 200 800 200 400 800 0
d
68. RM39 RM 30 0 200 0 400 1600 400 200 400 0 400
a
Bac.: – Bacillus Subtilis; Cand.: – Candida albicans; Clo.: – Clostridium botulinum; E. coli: – Escherichia coli; Kleb.: – Klebsiella pneumoniae;
Sal.: – Salmonella Senftenberg; St.au.: – Staphylococcus aureus; St.epi.: – Staphylococcus Epidermidis; Str. pyo.: – Streptococcus pyogenes; St.
dy.: – Streptococcus dysgalactiae subsp. Equisimilis.
b
Sources of isolation: BO: Boza; Z: Zabady; R: Ras cheese; K: karish cheese; RM: Rayeb Milk.
c
Activity units/ml cell-free supernatant was calculated according to the following equation: (1000/d)  D ،Where D is the two-fold dilution
factor, d is the amount of supernatant used.
d
Isolates had antimicrobial activity against 4 or more pathogenic microorganisms.

Table 4 Survival of selected lactic acid bacteria isolates under simulated gastric juice conditions at 37 °C.
Isolates code Mean of viable count (log10 CFU ml1) ± SD* Surviving percentage (%)
Time of exposure (h)
0 1 2 3
Bo 3 8.29 ± 0.41a 7.80 ± 0.14bcd 7.04 ± 0.20cd 6.32 ± 0.25d 76.2
Bo 34 8.31 ± 0.29a 7.91 ± 0.03bc 7.21 ± 0.16bc 6.37 ± 0.08d 76.6
Bo 35 8.17 ± 0.317a 7.60 ± 0.23cde 7.36 ± 0.27bc 7.22 ± 0.28ab 88.3
Bo 37 8.55 ± 0.22a 7.86 ± 0.04bcd 7.51 ± 0.14ab 7.33 ± 0.13a 85.7
Bo 51 8.37 ± 0.25a 7.55 ± 0.07de 7.13 ± 0.10cd 6.45 ± 0.38cd 77
Bo 52 8.37 ± 0.44a 8.33 ± 0.45a 7.57 ± 0.17ab 6.59 ± 0.24cd 78.7
RM 3 8.35 ± 0.34a 7.28 ± 0.17e 6.81 ± 0.19de 6.81 ± 0.19bc 81.5
RM 28 8.41 ± 0.44a 7.58 ± 0.13cde 6.60 ± 0.41e 5.72 ± 0.27e 68
RM 39 8.37 ± 0.28a 8.03 ± 0.20ab 7.85 ± 0.09a 6.48 ± 0.30cd 77.4
abcde
Means in the same column followed by different superscript letters are significantly different (P < 0.05).
BO: – Boza & RM: – Rayeb Milk.
*
Results are expressed as mean ± SD, and each value is the average of three experiments and each was carried out in duplicate.

8400 (RM28). Growth of pathogens is inhibited by the produc- as dietary adjuncts and enables strains to survive for longer
tion of antimicrobial compounds such as organic acids, hydro- period of time in high acid carrier food without larger reduc-
gen peroxide, diacetyl and bacteriocins by LAB as well as their tion in humans (Conway et al., 1987; Prasad et al., 1998).
competition for nutrients (Bezkorvainy, 2001; Tambekar et al., Tolerance to bile salts is a prerequisite for colonization and
2009).The nine promising LAB isolates were examined for fur- metabolic activity of bacteria in small intestine of the host
ther probiotic criteria such as bile, acid and phenol tolerance as (Havenaar et al., 1992). This will help Lactobacillus spp. and
well as their cholesterol removal capacities. Lactococcus spp. to reach the small intestine and colon and
contribute in balancing the intestinal microflora (Tambekar
Tolerance to acid and bile and Bhutada, 2010). All the tested strains exhibited bile toler-
ance with varying degrees. Among the tested LAB isolated in
As probiotics are usually administrated orally, they must have the present study, BO34 isolate demonstrated the highest bile
the ability to survive passage through the stomach and small salt tolerance followed by BO52 isolate (Table 5).
intestine. Therefore, resistance to the low pH of the gastric
juice in the stomach and the bile salt in the small intestine is Phenol tolerance
one of the important selection criteria for probiotic (Olejnik
et al., 2005). In the present study, all the selected LAB isolates For a strain to be a probiotic, it has to survive the action of
were able to survive simulated gastric juice at pH 2 after 3 h of toxic metabolites, primarily phenols, produced during the
incubation (Table 4). They retained varying levels (68–88.3%) digestion process (Hoier, 1992). Some aromatic amino acids
of viability. The highest survival was for BO35 isolate while the derived from dietary or endogenously produced proteins can
least survival was observed for RM28 isolate. be deaminated in the gut by bacteria leading to the formation
Acid tolerance of bacteria is important not only for with- of phenols which have bacteriostatic properties (Suskovic
standing gastric stresses, but also a prerequisite for their use et al., 1997).
Screening of probiotic lactic acid bacteria 71

Table 5 Survival of selected lactic acid bacteria isolates in MRS broth supplemented with 0.3% bile salts after 0, 1, 2 and 3 h at 37 °C.
Isolates code Mean of viable count (log10 CFU ml1) ± SD* Surviving percentage (%)
Time of exposure (h)
0 1 2 3
Bo 34 8.05 ± 0.05a 7.63 ± 0.26ab 7.04 ± 0.21a 6.85 ± 0.10ab 85
Bo 35 7.98 ± 0.43a 6.77 ± 0.41d 6.54 ± 0.21c 5.69 ± 0.32d 71.3
Bo 51 8.25 ± 0.20a 6.81 ± 0.41cd 6.45 ± 0.42c 5.82 ± 0.45cd 69.8
Bo 52 8.41 ± 0.32a 7.98 ± 0.29a 7.24 ± 0.22a 6.85 ± 0.14a 81.4
Bo 37 8.36 ± 0.44a 8 ± 0.12a 7.39 ± 0.1a 6.57 ± 0.22ab 78.5
Bo 3 8.39 ± 0.39a 7.98 ± 0.33a 6.58 ± 0.40bc 6.35 ± 0.33abc 75.6
RM 39 8.2 ± 0.16a 7.36 ± 0.21bc 6.55 ± 0.06c 6.20 ± 0.65bcd 75.6
RM 28 8.19 ± 0.28a 7.51 ± 0.07ab 7.29 ± 0.06a 6.49 ± 0.40ab 79.2
RM 3 8.51 ± 0.46a 7.45 ± 0.54ab 6.99 ± 0.26ab 6.66 ± 0.17abc 78.2
abcde
Means in the same column followed by different superscript letters are significantly different (P < 0.05).
BO: – Boza & RM: – Rayeb Milk.
*
Results are expressed as mean ± SD, each value is the average of three experiments and each was carried out in duplicate.

Fig. 1 illustrates the effect of two different phenol concen- ability of LAB isolates was assessed in vitro in the presence of
trations (0.2% and 0.5%) on the growth of nine LAB isolates oxgall after 24 h of anaerobic growth at 37 °C (Fig. 2). All the
after 24 h of incubation in MRS medium at 37 °C. The exam- nine LAB isolates showed the ability to remove cholesterol
ined LAB isolates showed different degrees of sensitivity from the media. They exhibited varying degrees of cholesterol
towards different concentrations of phenol. The highest toler- lowering ability ranged from 8.4% to 43.5%. The BO37 isolate
ance (95.89%) to 0.2% phenol concentration was demon- manifested superior ability (43.75%) to remove cholesterol
strated for BO37 followed by RM39 (94.1%), while, BO52 from the medium which was significantly higher than those
had the lowest tolerance. At 0.5 phenol concentration, all the of the other examined LAB isolates. The lowest value of
tested LAB isolates exhibited varying relative growth percent- cholesterol assimilation was traced in BO35 isolate. The ability
age ranged between 15.1% and 21.7%. Vizoso Pinto et al. of in vitro cholesterol level reduction in model culture media
(2006) observed varying degrees of sensitivity for four strains has been shown for numerous strains of LAB (Pereira and
of Lactobacillus johnsonii and six strains of L. plantarum Gibson, 2002; Lavanya, 2001; Wang et al., 2012; Miremadi
towards 0.4% phenol concentration while L. plantarum strains et al., 2014). Further studies are required to determine the
were less sensitive. mechanism(s) involved in the removal of cholesterol by those
probiotic LAB isolates.
Cholesterol removal by LAB isolates
Screening of LAB isolates for BSH activity
Hypercholesterolemia (elevated blood cholesterol level) is con-
sidered a major risk factor for the development of coronary Qualitative determination of bile salts hydrolases activity
heart disease. Therefore, lowering the serum cholesterol level The ability of probiotic strains to detoxify bile salt by produc-
is important to prevent the disease. The cholesterol – removing ing BSH enzyme activity has often been included among the

100

90 0.2% phenol

80 0.5% phenol

70
Relative growth (%)

60

50

40

30

20

10

0
RM28 RM39 Bo3 Bo37 Bo34 Bo51 Bo52 Bo35 RM3
Isolates lactic acid bacteria

Fig. 1 Effect of phenol concentration on the growth of LAB isolates. BO: – Boza & RM: – Rayeb Milk.
72 M.G. Shehata et al.

50
45

Cholesterol removal (%)

40
35
30
25
20
15
10
5
0
Bo 3 Bo 34 Bo 35 Bo 37 Bo 51 Bo 52 RM 3 RM 28 RM 39
LAB isolates

Fig. 2 Cholesterol removal by LAB isolates. BO: – Boza & RM: – Rayeb Milk.

Fig. 3 The BSH activity of LAB isolates grown on bile salt – MRS medium as manifested by the formation of precipitation zone around
the colony. The code numbers 1–9 represent the LAB isolates as follows: 1: BO3; 2: BO34; 3: BO35; 4: BO37; 5: BO51; 6: BO52; 7: RM3; 8:
RM28; 9: RM39. BO: – Boza & RM: – Rayeb Milk.

criteria for probiotic strain selection (Noriega et al., 2006). Bile The deconjugation activity of LAB isolates was manifested in
salt hydrolase is an enzyme that catalyses the deconjugation of Fig. 3, and copious amounts of deoxycholic acid precipitated
bile salt to liberate free primary bile acids (Gilliland and Speck, around active colonies and diffused into the surrounding
1977). medium.
When bile salt hydrolase producing LAB isolates were Out of nine LAB isolates previously selected based on their
streaked on MRS plates containing TDCA, the taurine conju- high antimicrobial activity, eight isolates displayed BSH activ-
gated bile acid was deconjugated producing deoxycholic acid. ity to different levels. Four isolates (BO35, BO37, BO52,
Screening of probiotic lactic acid bacteria 73

RM39) exhibited high BSH activity by providing large precip- enzyme activity was present in the supernatants of overnight
itation zones (2.03, 2.45, 2.25, and 1.98 mm, respectively) cultures, while activity was released either by sonication or
around colonies on plate assay. Notwithstanding, the other other cell disruption methods or by lysis in assays performed
four isolates demonstrated low BSH activity by expressing with whole cells due to the lytic properties of the bile salts
small (less than 1.5 mm) precipitation zones. (Lunden and Savage, 1990; Grill et al., 1995; Tanaka et al.,
The presence of bile salt hydrolase (BSH) in probiotics ren- 2000).
ders them more tolerant to bile salts, which also helps to
reduce the blood cholesterol level of the host (Noriega et al., Identification of promising LAB isolates
2006).
Contrary to the results of the present study, Begley et al. Phenotypic characterization
(2006) reported that BSH activity has not been detected in bac- The seven promising isolated strains (BO3, BO34, BO37,
teria isolated from environments from which bile salts are BO51, BO52, RM28, RM39) are Gram positive, catalase neg-
absent. It is worthy to mention that all the eight BSH- ative, non-spore forming and fermenting glucose. All the iso-
positive LAB isolates are not associated with gastrointestinal lates are rod shaped except RM39 and BO37 that are
environment. spherical cells. The physiological and biochemical characteris-
tics of the selected LAB isolates Table 7 are similar to those
Quantitative determination of bile salts hydrolases activity described in Bergey’s Manual of Determinative of Bacteriol-
The highest total BSH activity (3.09 u/ml) towards tauro- ogy (Logan and De Vos, 2009) for the genera Lactobacillus
cholate was displayed by RM39 LAB isolate compared with and Lactococcus. Further, biochemical characterization using
other LAB isolates Table 6. In contrast, the lowest total API 50 CHL for Lactobacilli and API 20 for cocci showed
BSH activity (0.25 u/ml) was demonstrated for RM3 isolate. the similarity in characteristics with the corresponding identi-
Specific activity of BSH did not correlate well with total fied LAB species (Table 8). Bill et al. (1992) and Klinger
BSH activity by most LAB isolates due to varying protein con- et al. (1992) indicated that some commercial identification sys-
tent in cell extracts. The RM39 and RM3 isolates had high tems often yield good results regarding genus identification but
(3.09 u/ml) and low (0.25 u/ml) total BSH activity, respectively they were not fully adequate at the species level.
and exhibited the same trend as well with respect to the specific
activity. Meanwhile, BO37 isolate that had high total BSH
activity (2.47 u/ml) displayed low specific activity (0.85 u/
mg). Similar results were reported by Liong and Shah (2005) Table 7 Physiological characteristics of lactic acid bacteria
for lactobacillus strains towards different bile salts. Several isolates.
studies have indicated that the mechanism for in vitro removal Characteristics LAB isolates
of cholesterol is linked to the bile salt hydrolase activity of pro-
biotic strains (Kimoto et al., 2002; Liong and Shah, 2005). BO BO BO BO BO RM RM
3 34 37 51 52 28 39
Moreover, the decomposition of bile salts by BSH enzyme
would disrupt the formation of the cholesterol micelle which Gram strain + + + + + + +
in turn prevents cholesterol absorption (Klaver and Van der Catalase       
Meer, 1993). production
Glucose       
From the genetic data, it was obvious that BSH is an intra-
fermentation
cellular enzyme. This is consistent with the observation that no Growth:
15 °C +  + + +  +
Table 6 BSH activity of lactic acid bacteria isolates on 37 °C + + + + + + +
45 °C + +  + + + 
sodium taurocholate.a
NaCl
Isolates BSH activityb,c 2% + + + + + + +
Total protein Total activity Specific activity 4% + + + + + + +
(mg/ml) (U/ml) (U/mg) 6.5%   + + +  +

Bo 34 1.22 ± 0.27b 1.64 ± 0.28d 1.34 ± 0.32a BO: – Boza & RM: – Rayeb Milk.
Bo 35 2.62 ± 0.61a 2.31 ± 0.22bc 0.88 ± 0.25cd
Bo 37 2.89 ± 0.26a 2.47 ± 0.45b 0.85 ± 0.09d
Bo 51 1.08 ± 0.44b 0.67 ± 0.34e 0.62 ± 0.5e
Bo 52 2.14 ± 0.89a 1.95 ± 0.15cd 0.91 ± 0.52c Table 8 Identification of LAB isolates by API 50 CHL and
RM 3 0.42 ± 0.49b 0.25 ± 0.15ef 0.59 ± 1.19e API 20 kits.
RM 28 2.54 ± 0.66a 2.25 ± 0.24bc 0.88 ± 0.16cd
Isolates Species identified by API test
RM 39 2.96 ± 0.11a 3.09 ± 0.27a 1.04 ± 0.13b
BO 3 Lactobacillus rhamnosus
BO: – Boza & RM: – Rayeb Milk.
a BO 34 Lactobacillus delbrueckii subsp. bulgaricus
Results are expressed as means ± standard deviation; values
BO 37 Lactococcus lactic subsp. lactis
are means of triplicate.
b BO 51 Lactobacillus paracasei
BSH activity from cell free extracts of lactic acid bacteria iso-
BO 52 Lactobacillus paracasei
lates grown on MRS broth supplemented with 6 mM sodium
RM 28 Lactobacillus gasseri
taurocholate.
c RM 39 Lactococcus lactic subsp. lactis
Means in the same column followed by different superscript
letters are significantly different (P < 0.05). BO: – Boza & RM: – Rayeb Milk.
74 M.G. Shehata et al.

cholesterol removal ability (43.7%) and good BSH activity

(2.47 u/ml). Accordingly, owing to its good probiotic proper-
ties, this strain could be potentially used in functional food
and health products especially where cholesterol reduction in
food is the main target. Further in vivo study is necessary to
prove the hypocholesterolemic effect of the isolated Lactococ-
cus lactic subsp. lactis. Moreover in vitro studies are required
to determine the mechanism(s) involved in the reduction of
cholesterol by such a promising isolate.

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