Department of Microbial Biotechnology, Rajiv Gandhi Institute of IT and Biotechnology, Bharati Vidyapeeth Deemed University 411046, Pune, India*Corresponding author: neeta.bhadekar@gmail.com

Comparative studies on potential probioticcharacteristics of Lactobacillus acidophilus strains

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EurAsian Journal of BioSciences Eurasia J Biosci 7, 1-9 (2013)http://dx.doi.org/10.5053/ejobios.2013.7.0.1

Lactobacilli are often considered to be

commensal or beneficial participants in human

microbial ecology and considerable research is being

carried out on the effects for the use of lactobacilli

as additives in both human and animal diets

(Hummel et al. 2007). In recent years, the probiotic

activity of lactic acid bacteria (Lactobacilli,

Streptococci and Bifidobacteria) has been

emphasized. A number of health benefits have been

claimed for probiotic bacteria and are also being

recommended as a preventive approach to maintain

the balance of intestinal microflora (Shah 2007).

Their beneficial effects on humans include i)

stabilization of intestinal microflora (excluding

colonization of entero-pathogenic bacteria by

adhesion to the intestinal wall and competition for

nutrients) (Denev 2006), ii) reduction of lactose

intolerance (de Vrese et al. 2001), iii) prevention of

antibiotic-induced diarrhoea (Pochapin 2000), iv)

prevention of colon cancer (Wollowski et al. 2001), v)

stimulation of the immune system (Isolauri et al.

2001) etc.

Probiotic microorganisms should express high

tolerance to acid and bile and ability to adhere to

intestinal surfaces to survive in and colonise in

gastro-intestinal tract (GIT). However in vivo testing

is expensive, time consuming and requires approval

by ethical committees. Hence, reliable in vitro

methods for selection of promising strains have

been used by earlier researchers (Jacobsen et al.

1999). One of the unique features of probiotics is

their antibiotic resistance expression and

transferability as there is great concern over

possible spread of resistance determinants to

Received: August 2012Accepted: December 2012

Printed: January 2013

INTRODUCTION

AbstractBackground: Probiotics are live microorganisms present in food and dietary supplements thatbeneficially affect the individual by improving the intestinal microbial balance properties. Theirmarket value and biological potential is enormous because of their health-promoting properties.Therefore, comparative studies on probiotic potential of three selected strains of Lactobacillusacidophilus were carried out during this work.Method: The selected strains of Lactobacillus acidophilus (NCIM 2660, NCIM 2903 and NCIM 2285)were assessed for tolerance to pH (pH 2.5) and oxgall (0.3%), adhesion to Caco-2 cells, antimicrobialproperties against test microorganisms and susceptibility to antibiotics. Results: The distinguished characteristics of strain NCIM 2903 were high acid tolerance,antagonistic activity against all test micro-organisms and adhesion to Caco-2 cells (225±33 cellsadhering to 100 Caco-2 cells). Strain NCIM 2285 exhibited bile tolerance and inability to grow inpresence of five of the antibiotics used. Strain NCIM 2285 indicated moderate adhesion to Caco-2cells. The strain NCIM 2660 was unable to grow in presence of any of the twelve antibiotics. Incontrast, control plates without discs of antibiotics exhibited confluent growth. It was a desirablefeature. However, NCIM 2660 exhibited poor adhesive property. The optimum temperature ofgrowth for all three strains was found to be 37ºC. Conclusions: Significant differences in the probiotic characteristics of the three strains wererecorded. In the cumulative assessment, strain NCIM 2903 could be regarded as the potentialprobiotic contender. These studies will help in selecting the probiotic micro organisms in a morerational manner for further applications.Keywords: Acid and bile tolerance, antibiotic susceptibility, antimicrobial activity, cell adhesion,lactic acid bacteria.

Dixit G, Samarth D, Tale V, Bhadekar R (2013) Comparative studies on potential probioticcharacteristics of Lactobacillus acidophilus strains. Eurasia J Biosci 7: 1-9.

http://dx.doi.org/10.5053/ejobios.2013.7.0.1

Gauri Dixit, Deepti Samarth, Vidya Tale, Rama Bhadekar*

©EurAsian Journal of BioSciences

human pathogenic and opportunistic bacteria

(Ammor et al. 2007).

Lactobacilli are highly competitive largely due to

their applications in the production of fermented

food. They can also produce antimicrobial

substances including bacteriocins that have ability to

inhibit pathogenic and food spoilage bacteria

(Rattanachaikunsopon and Phumkhachorn 2010).

These compounds have shown to exert specific

antagonistic properties against Gram-negative and

Gram-positive pathogens. Adhesion of lactic acid

bacteria (LAB) to mucosal surfaces has been studied

in vitro using Caco-2 cells (Duary et al. 2011).

The aforementioned points indicate that

screening and selection of novel probiotic strains is

critical, thereby depending on definite criteria. Most

efficient strains will be the strains that are robust

enough to survive the harsh physico-chemical

conditions present in GIT.

Lactobacillus acidophilus is a well known and well

studied probiotic microorganism. However, it is now

clear that different strains undoubtedly vary in their

efficiency and probiotic potentials (Ng et al. 2009).

Hence the present study is an effort to give a

comparative account of three strains of L.

acidophilus in the group of probiotic bacteria.

All media components were purchased from Hi

Media, India. All chemicals were purchased from

Merck (India) and all were of A.R. grade.

Bacterial strains

Bacterial cultures were procured from National

Collection of Industrial Microorganisms (NCIM),

National Chemical Laboratory, Pune, India. Three

strains of Lactobacillus acidophilus used for

comparative studies were NCIM 2660, NCIM 2285

and NCIM 2903. They were maintained by

subculturing on MRS (De Man, Rogosa and Sharpe)

agar under microaerophillic conditions. Test

microorganisms viz. Pseudomonas aeruginosa (NCIM

2200), Staphylococcus aureus (NCIM 5021),

Escherichia coli (NCIM 2065), Klebsiella pneumoniae

(NCIM 5082) and a clinical isolate of Salmonella Typhi

(Salmonella enterica enterica, serovar Typhi) were

assessed for experiments of antimicrobial activity.

They were maintained on nutrient agar. The stock

cultures were preserved in 10% glycerol and were

subcultured routinely at the interval of every two

months. The cultures were stored at 4°C between

transfers and were subcultured once before

experimental use.

Epithelial cell line culture

The enterocyte-like Caco-2 cell line was obtained

from the National Centre for Cell Sciences (NC CS,

Pune, India) and grown on Eagle’s Minimum

essential medium (MEM) supplemented with 5.0

mmol L-1 L-glutamine, 20 mmol L-1 D-glucose, 1.0

mmol L-1 sodium pyruvate and 20% heat-inactivated

(30 min; 56°C) foetal calf serum (Gibco, Germany).

For the adhesion assays, cells were prepared on a 25

cm2 plastic tissue culture flask. They were used,

between the 40th and 45th passage after 21 days of

incubation at 37°C.

For all experiments 10% inoculi of late log phase

cultures (equivalent to the 0.5 Mcfarland turbidity

standards) were used.

Effect of temperature on the growth of L.

acidophilus strains

Effect of temperature on viability of L. acidophilus

strains was examined by inoculating them in MRS

broth and incubating at 28ºC, 37ºC and 45ºC. After

the incubation period of 24 h, growth was

determined by measuring the optical density at 600

nm.

Determination of acid tolerance

Percent survival of the three strains was

determined after exposure to pH 2.5 for 2 h and 4 h

at 37°C. For this, overnight grown cultures were

inoculated in MRS broth adjusted to pH 2.5. The

samples were plated onto MRS agar at the end of

exposure time. The plates were incubated for 24 h at

37°C and total viable count was determined. Un-

inoculated broth served as negative control while

set of test organisms inoculated in MRS broth (pH

6.2) was used as positive control.

Determination of bile tolerance

Bile tolerance of L. acidophilus cultures was

examined by inoculating them in MRS broth

containing oxgall (Central Drug House, New Delhi,

India) at the concentrations of 0.2%, 0.3% and 0.5%.

Dixit et al.

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EurAsian Journal of BioSciences 7: 1-9 (2013)

MATERIALS AND METHODS

The control comprised of MRS broth without bile

salt. Bacterial growth was monitored by measuring

absorbance at 600 nm after incubation for 24 h at

37°C.

Antimicrobial activity

Using in-vitro agar well diffusion method,

antimicrobial activity experiments were carried out

(Zinedine and Faid 2007). The activity of

Lactobacillus strains against test microorganisms

mentioned earlier was recorded. Overnight grown

cultures of L. acidophilus strains were inoculated in

the wells of nutrient agar where as test

microorganisms were inoculated by pour plate

technique. The plates were incubated at 37ºC for 24

h. The inhibition zones were measured at the end of

incubation period.

Antibiotic susceptibility assay

A disc diffusion assay was performed to study

antibiotic susceptibility of L. acidophilus strains. The

cultures were inoculated in MRS agar using pour

plate technique. The antibiotics were supplied in the

form of dodeca discs (Hi Media, India) which included

nalidixic acid, norfloxacin, co-trimoxazole,

gentamycin, ampicillin, cephalexin, mecillinam,

chloramphenicol, tetracycline, kanamycin, amikacin

and streptomycin. The zones of inhibition were

measured after incubation at 37ºC for 24 h. The

strains were cultivated on control plates (MRS agar

without antibiotic discs) under identical conditions.

Adhesion assay

The adherence of L. acidophilus strains to Caco-2

cells was examined as described previously by

Jacobsen et al. (1999). The adherent lactobacilli in 25

random microscopic fields were counted for each

test. Bacterial strains were scored as non-adhesive

when fewer than 60 bacteria were present in 25

fields, adhesive with 61 to 100 bacteria in 25 fields,

and strongly adhesive with more than 100 bacteria

in 25 fields.

Statistical analysis

All experiments in the present study were carried

out in triplicates and the results indicate their mean

values. For statistical analysis, the standard errors of

the means were calculated and the means were

tested according to One-way ANOVA test for

significant differences among the samples. A

statistical significance was accepted at significant

level P<0.05.

In this work, in vitro evaluation of certain

properties of L. acidophilus strains important for

their survival in GIT has been carried out.

Effect of temperature on the growth of L.

acidophilus strains

In all the three experiments, effect of tempera-

tures 28ºC, 37ºC and 45ºC on the growth of three

selected strains were found to be significantly

different (P<0.05). Fig. 1 shows the effect of

temperature on viability on L. acidophilus strains. All

strains grew well at 28ºC and 37ºC and their growth

was comparable. None of them could grow at 45ºC.

Considering the physiological temperature of

human body and the results obtained, all further

experiments were carried out at 37ºC.

Determination of acid tolerance

Probiotic bacteria are mostly delivered in a food

system and must be acid and bile tolerant to survive

in the human gastrointestinal tract. The time from

entrance to release from the stomach has been

estimated to be approximately 90 min with further

digestive processes requiring longer residence time

(Berrada et al. 1991). Table 1 shows survival of L.

acidophilus strains after exposure to pH 2.5 for 2 h

and then after 4 h. The survival rates of all three

strains were higher after 2 h incubation period

(P<0.05) than 4 h (P<0.05) at pH 2.5. The percent

survival of strain NCIM 2285 decreased by almost

50% after 4 h as compared to growth after 2 h. In

case of strains NCIM 2903 and NCIM 2660 percent

survival decreased by 21% and 43% respectively. The

results suggested that strain 2903 had better acid

tolerance among the three strains.

Acid tolerance of Lactobacillus strains was also

reported by Jacobsen et al. (1999) who recorded 4

out of 8 strains of L. acidophilus capable of survival at

pH 2.5 for 2 h. Our results indicating the survival of

L. acidophilus (particularly strain NCIM 2903) at pH

2.5 after 4 h were very significant.

Determination of bile tolerance

The ability of Lactobacillus strains to grow in

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Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)

RESULTS AND DISCUSSION

presence of bile salts was studied by growing them

in MRS broth supplemented with different

concentrations of oxgall (0.2 to 0.5%). Bile tolerance

is an important characteristic of probiotic

microorganisms. A concentration of 0.3% oxgall

closely appropriates the bile level found in the

gastrointestinal tract (Goldin and Gorbach 1992).

Fig. 2 clearly reveals that the strains were viable at

different concentrations of bile salt and showed

maximum growth at 0.2% bile. At 0.3% bile

concentration strain, NCIM 2660 exhibited weak

growth whereas at still higher concentration of

0.5%, both NCIM 2660 and NCIM 2903 showed

negligible growth indicating their inability to

tolerate the high bile concentration. Thus, strain

NCIM 2285 was found to be most bile tolerant

among the three strains. The three strains exhibited

significant difference (P<0.05) in their bile tolerance.

Many reports recorded the bile tolerance of lactic

acid bacteria (LAB). However, majority of them

demonstrated bile tolerance upto 0.3%

concentration (Liong and Shah 2005, Mcauliffe et al.

2005). Therefore our results for strain NCIM 2285

are noteworthy taking into account its high bile

tolerance.

Antimicrobial activity

The antimicrobial properties of L. acidophilus

strains varied with the test microorganisms (Table

2). Strain NCIM 2903 had astonishingly antagonistic

property towards test microorganisms taken under

consideration and the zones of inhibition also varied

from medium to large. The other two strains had

antagonistic activity against only 3 out of 5 test

microorganisms. Strain NCIM 2260 showed weak

zones of inhibition against P. aeruginosa and K.

pneumoniae and could not inhibit S. aureus and E.

coli. Strain NCIM 2285 could not inhibit P. aeruginosa

and Salmonella Typhi.

Our results are in line with reports by Jacobsen et

al. (1999). They reported only 3 amongst 8 strains of

L. acidophilus strains showing weak zone of

inhibition against S. aureus. None of them could

strongly inhibit E. coli. Hence our results of strain

NCIM 2903 showing inhibitory activity against all

test organisms are very promising, thereby

emphasizing its probiotic characteristics. LABS have

been shown to possess inhibitory activity towards

the growth of pathogenic bacteria such as Listeria

monocytogenes (Sifour et al. 2012), E. coli, Salmonella

spp. (Hudault et al. 1997, Makras et al. 2006) and

others (Olsen et al. 1995, Coconnier et al. 1997).

The mechanism(s) of antibacterial activity in

probiotic Lactobacillus strains appears to be

multifactorial (Servin 2004) and is due to

bacteriocins and/or organic acid produced by them.

Inhibition of pathogen cultured from pancreatic

necrosis due to the production of organic acids by

multi-species probiotic was noted by Ridwan et al.

(2008). Lactobacilli have been shown to possess

inhibitory activity towards the multiplication of

enteropathogens (Drago et al. 1997). The

antagonistic activity is variable and more strain-

specific (Jacobsen et al. 1999). Bacteriocins

produced by LAB may also prove useful for different

gastrointestinal and uro-genital infection therapies

and also to overcome problem of antimicrobial

resistance (Šušković et al. 2010).

Antibiotic susceptibility assay

The determination of antibiotic susceptibility of a

bacterial strain is an important prerequisite prior to

considering it safe for human and animal

consumption. All the three strains were subjected to

antibiotic susceptibility test. The control plates

showing confluent growth under identical

conditions were used to compare the results of agar

disc diffusion assay. The results are summarised in

Table 3. Inhibition zones were not observed for the

strains NCIM 2903 and NCIM 2285 in presence of

norfloxacin and mecillinam amongst the 12

antibiotics used while zones of inhibition varying

from 2 mm to 15 mm with rest of the antibiotics

were recorded. Strain NCIM 2660 did not show

growth in presence of any of the antibiotics used.

According to earlier reports, specific antibiotic

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Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)

Table 1. Effect of pH (2.5) on survival of L. acidophilusstrains.

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Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)

Fig. 1. Effect of temperature on the growth of L. acidophilus strains.

Fig. 2. Effect of bile concentration on growth of L. acidophilus strains.

Table 2. In vitro antagonistic activity of L. acidophilus strains against test microorganisms.

-- : Absence of zone of inhibition, SD: Standard deviation

resistance traits among probiotic strains may be

desirable (Charteris et al. 1998). Some authors claim

that in cases of co-administration with antibiotics to

prevent and treat intestinal disorders, probiotics

should be resistant to certain antibiotics so as to

survive in the gastrointestinal tract. However, this

opinion is controversial.

Probiotics containing resistance traits may have

negative consequences to human health. The

presence of antibiotic-resistance genes in many LAB,

and the transfer of plasmids and conjugative

transposons (Yoshiyuki et al. 2009) to and from LAB,

have been reported in Lactobacillus species. The

resistance to chloramphenicol, kanamycin,

erythromycin, gentamycin, streptomycin and

tetracycline (also considered in present work) had

shown to be plasmid borne in certain probiotic

cultures (Temmerman et al. 2002, Moubareck et al.

2005, Hummel et al. 2007). Thus, there exists risk

relating to potential transfer of antibiotic resistance

from probiotic strains to other bacteria either

commensally residing in intestine and/or pathogens

is undesirable and detrimental. Studies by Hummel

et al. (2007) demonstrated that resistant genes

might be present in probiotic strains but are silent.

Genetic basis and associated resistance mechanisms

towards some antibiotics are still unknown.

Adhesion assay

The adhesion of 3 different L. acidophilus strains

was studied using Caco-2 cell line as a model for

intestinal epithelium (P<0.05). The adherence of

bacteria was observed microscopically. Table 4

indicates the number of Lactobacilli exhibited in

vitro adherence to Caco-2 cells. All the three strains

exhibited varying degree of adhesion. The most of

the adhesive strain was L. acidophilus NCIM 2903.

Strain NCIM 2285 was moderately adhesive whereas

strain NCIM 2660 was unable to adhere to this

intestinal cellular model. L. acidophilus strains either

unable to adhere to Caco-2 cells or showing weak

adhesion have been reported by Sareml et al. (1996)

and Jacobsen et al. (1999). However one of the

selected strains (NCIM 2903) showing significant

adhesion to Caco-2 cells that would satisfy major

prerequisite as probiotic.

In conclusion, the study reported in vitro methods

that could be exploited for prediction of the survival

potential of Lactobacilli in human GIT since it is

essentially linked to adhesion to Caco-2 cells and

acid and bile tolerance. Thus, the comparison of

potential probiotic traits of examined strains

revealed NCIM 2903 as a promising candidate.

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Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)

Table 3. Antibiotic resistance of L. acidophilus strains using disc diffusion method.

bMeans in the same row followed by superscript letter are significantly different (P<0.05).NG - no growth -- = inhibition zone not observed.C= Chloramphenicol; A = Ampicillin; T= Tetracycline; G = Gentamycin; K= Kanamycin; Co= Co-trimoxazole; Ak= Amikacin; S= Streptomycin;No= Norfloxacin; Nx= Nalidixic acid; Cp= Cephalexin; Mc= Mecillinam.

Table 4. Adhesion of L. acidophilus strains to Caco-2 cells inculture.

*Expressed as the number of lactobacilli adhering per 100epithelial Caco-2 cells ; mean±standard deviations of three assays,counts were carried out on 25 randomized microscopic fields foreach sample per assay.

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Lactobacillus acidophilus Suşlarının Potansiyel Probiyotik EtkilerininKarşılaştırmalı Araştırılması

ÖzetGiriş: Probiyotikler, canlı mikrobik gıda takviyeleridir. İddia edilen sağlık destekleyici özellikleri sebebiyle, muazzam birpiyasa değerine ve biyolojik potansiyele sahiptirler. Bu yüzden bu çalışmada, seçilmiş üç Lactobacillus acidophilussuşunun probiyotik potansiyeli karşılaştırmalı olarak çalışılmıştır.Metot: Seçilmiş Lactobacillus acidophilus suşları (NCIM 2660, NCIM 2903 ve NCIM 2285), pH (pH2.5) ve oxgall (%0.3)toleransları, Caco-2 hücrelerine yapışmaları, test mikroorganizmalarına karşı antimikrobiyal özellikleri veantibiyotiklere hassasiyetleri açısından değerlendirildi. Bulgular: NCIM 2903 suşunun öne çıkan özellikleri; yüksek asit toleransı, bütün test mikroorganizmalarına karşıantagonistik aktivite ve Caco-2 hücrelerine yapışması (100 Caco-2 hücresine 225±33 hücrenin yapışması) idi. NCIM2285 suşu safraya tolerans sergiledi ve kullanılan beş antibiyotiğin varlığında çoğalamadı. NCIM 2285 suşu Caco-2hücrelerine orta dercede yapıştı. NCIM 2660 suşu, antibiyotik diskleri olmayan kontrollerle kıyaslandığında, on ikiantibiyotiğin hiçbirinde çoğalamadı. Bu arzu edilen bir durumdu. Ancak, zayıf yapışma özelliği sergiledi. Her üç suş içinoptimum çoğalma sıcaklığı 37ºC olarak tespit edildi.Sonuç: Üç suşun probiyotik özelliklerinde önemli farklılıklar olduğu bulundu. Son değerlendirmede, NCIM 2903suşunun potansiyel bir probiyotik olarak kullanılabileceği düşünülebilir. Bu çalışmalar, daha sonraki uygulamalarda,probiyotik mikroorgamizmaların daha rasyonel olarak seçilmelerinde yardımcı olacaktır.

Anahtar Kelimeler: Antibiyotik hassasiyeti, antimikrobiyal aktivite, asit ve safra toleransı, hücre yapışması, laktik asitbakterileri.

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Dixit et al.EurAsian Journal of BioSciences 7: 1-9 (2013)