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Screening, selection and characterization of phytic acid degrading lactic acid bacteria

from chicken intestine

Ponnala Raghavendra, Prakash M. Halami

Food Microbiology Department, Central Food Technological Research Institute, Mysore 570020, India

a b s t r a c ta r t i c l e i n f o

Article history:

Received 30 January 2009

Received in revised form 5 May 2009Accepted 6 May 2009

Keywords:

Lactic acid bacteria

Phytic acid

Phytase

Acid phosphatase

Pediococcus pentosaceus

This study was undertaken to screen and select potent phytate degrading lactic acid bacteria and to evaluate

their additional characteristic features. Forty lactic acid bacterial strains were isolated from different sources

and screened for their ability to degrade myo-inositol hexaphosphate or IP6by cobalt chloride staining (plate

assay) method, using calcium or sodium salt of phytic acid as substrate. All the forty isolates were able to

degrade calcium phytate. However, only two Pediococcus pentosaceus strains (CFR R38 and CFR R35) were

found to degrade sodium phytate. These strains showed phytase activity of 213 and 89 U at 50 C, respectively

and poor acid phosphatase activity. These strains were further evaluated for additional characteristic

features. At pH 2,P. pentosaceusstrains CFR R38 and CFR R35 showed 50.7 and 48.5 percentage survivability

after 2 h of incubation respectively and they could also withstand 0.3% ox-bile. These cultures exhibited 54.6

and 44.8% of hydrophobicity to xylene, antibacterial activity against food borne pathogens and possessed

-galactosidase activity. The resistance pattern to several antibiotics was also analyzed. The present study

indicates that thesestrains, having phytate degrading ability and other characteristicfeaturescan be exploited

as starter cultures in fermented foods to improve the mineral bioavailability.

2009 Elsevier B.V. All rights reserved.

1. Introduction

Cereals, legumes, nuts, seeds and tubers are rich sources of

phosphorus in the form of phytic acid (myo-inositol hexaphosphate,

IP6). This molecule is highly charged with six phosphate groups

extending from the centralmyo-inositol ring and is often reported to

be an anti-nutritional factor for humans and animals as it acts as an

excellent chelator of cations such as Ca2+, Mg2+, Fe2+ and Zn2+. It

also complexes the basic amino group of proteins, hindering their

absorption and reducing their dietary availability (De Angelis et al.,

2003; Reale et al., 2004; Kerovuo et al., 1998; Lopez et al., 2000;

Palacios et al., 2005). Phytate is very important to infants, children,

adults and people in clinical situations, but high phytate diet

decreases the retention of calcium and iron signicantly. The

phosphorylation degree of myo-inositol phosphates determines in

which proportion the mineral absorption is inhibited, enhanced or

unaffected. The lower inositol phosphates (IP14) andmyo-inositol on

the other hand are recognized as benecial through different

biological roles. Phytate should be avoided among vulnerable groups

and eliminated by extraneous processing efforts (Reale et al., 2007).

Phytic acid levels may be reduced by phytase [myo-inositol hexakis

(dihydrogen phosphate) phosphohydrolase, EC 3.1.3.8], an enzyme

that catalyzes the sequential hydrolysis of phytate to phosphate andinositol via penta to monophosphates. This decreases or eliminates

the anti-nutritional effect and results in the bioavailability of divalent

cationic essential dietary minerals (Palacios et al., 2008). Phytase

enzyme is widely distributed in nature, like plants, animal tissues and

microorganisms (Lopez et al., 2000). However, phytase activity has

been found to be low in human small intestine showing the highest

activity in the duodenum and the lowest activity in the ileum (Haros

et al., 2007). Microbial sources of phytase arethe most promising ones

for the production of cereal based fermented foods on a commercial

level. The overall activity of these bacteria enhances the shelf life and

nutritional value of the nal products and contributes to their unique

organoleptic properties (Palacios et al., 2005). Sourdough fermenta-

tion was reported to have signicantly reduced the phytate content in

plant-based foods (Reale et al., 2007).

Lactic acid bacteria (LAB) are known as an ingredient of several

traditional fermented foods and dairy products (Reddy et al., 2007).

Most of the LAB isolated from different food fermentations and

ecosystems are shown to possess phosphatase activity with low levels

of activity against phytate (Palacios et al., 2008). Of late LAB isolated

from the gastrointestinal tract (GIT)of animals and humans constitute

an important source of new functional bacteria, which can develop

biological roles during the gastrointestinal transit (probiotics) or

during food processing (Palacios et al., 2008). These organisms should

possess the ability to cross the barriers from mouth to intestine, such

as low pH in the stomach and bile in the duodenum. They should also

adhere to the intestinal micelle and exhibit antagonistic activity

International Journal of Food Microbiology 133 (2009) 129134

Corresponding author. Food Microbiology Department, Central Food Technological

Research Institute, Mysore-20, India. Tel.: +91 821 2517539; fax: +91 821 2517233.

E-mail address:foodmicro@cftri.res.in(P.M. Halami).

0168-1605/$ see front matter 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2009.05.006

Contents lists available at ScienceDirect

International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

mailto:foodmicro@cftri.res.inhttp://dx.doi.org/10.1016/j.ijfoodmicro.2009.05.006http://www.sciencedirect.com/science/journal/01681605http://www.sciencedirect.com/science/journal/01681605http://dx.doi.org/10.1016/j.ijfoodmicro.2009.05.006mailto:foodmicro@cftri.res.in

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against pathogenic microorganisms. The health benets attributed to

probiotic bacteria in the literature can be categorized as nutritional

and therapeutic (Reddy et al., 2007; Famularo et al., 2005). The aim of

the study was to screen and isolate potent IP6 degrading LAB and to

evaluate their characteristic features.

2. Materials and methods

2.1. Materials

MRS(de Mann, Rogosa and Sharpe), broth/agar, brain heart infusion

agar (BHI), calcium phytate, carbohydrate kit, antibiotic octa-discs were

purchased from HiMedia, India. Ammonium molybdate, ammonium

meta vanadate, hydrochloric acid, sulfuric acid sodium chloride, glacial

aceticacid andtrichloroacetic acid were procured from Qualigens,India.

Sodium phytate and ox-bile were purchased from Fluka, USA. X-gal

(5-bromo-4-chloro-3-indolyl--D-galactopyranoside), IPTG (iso-pro-

pyl-thio--D-galactopyranoside), agarose, TrisHCl, (Fluka-Sigma,

USA), ONPG (o-nitrophenyl-L-D-galactopyranoside) cobalt chloride

and ferrous sulphate were purchased from SRL, India. All the chemicals

used were of analytical grade. dNTPs, Taq DNA polymerase, oligonu-

cleotide primers and DNA ladder were purchased from Bangalore

Genei, India.

2.2. Isolation, culture conditions and screening for phytate degrading ability

Intestines from chicken, fresh and salt-water sh and cucumbers

were purchased from local market. Raw cow milk and cow dung were

collected from local areas in and around Mysore, Karnataka, India. The

intestines were dissected and suspended in different concentrations

of sodium chloride for 48 h at room temperature. At an interval of 1 h

to a period of 4 h, samples were serially diluted in saline and

aliquots were plated on MRS agar for the enumeration of LAB. The

representative colonies recovered from high dilution plates were

inoculated in MRSbroth to obtainpure colonies. The catalase property

was done and the morphology was studied by Gram staining

(Zamudio et al., 2001). Gram-positive and catalase negative isolates

were further identied by physiological, biochemical tests andmolecular tools. Phytase studies were carried out by modied MRS

broth (MRS-MOPS), in which inorganic phosphate (KH2PO4) was

replaced by 0.65 g/l of sodium phytate and 0.1 M 3-[N-Morpholino]

propanesulfonic acid (MOPS, SRL, India). The contents of glucose,

yeast extract and beef extract were reduced to 10, 2 and 4 g/l,

respectively to reduce thenal phosphate content and to promote the

enzyme synthesis. MRS-MOPS medium was inoculated with 5% (v/v)

overnight culture propagated in same conditions for two generations

and incubated until the stationary phase of growth was attained (16

24 h). Cells were harvested by centrifugation (8000 rpm for 15 min at

4 C) and washed with 50 mM TrisHCl (pH 6.5). The cell pellet (107

108 CFU/ml) thus obtained was suspended in saline and in 100 mM

sodium acetateacetic acid buffer (pH 5.5). The saline suspension was

used for plate assay method to test for the phytate degrading ability;whereas buffer suspension was used for enzyme activity and bio-

chemical assay (Haros et al., 2005).

2.3. Phytate degradation

Modied MRS medium was used in the study. Substrates (sodium

and calcium phytate) and calcium chloride were dissolved in sterile

water and lter sterilized prior to use. Cells were harvested as

mentioned above and the cell suspension (3 l of 107108 CFU/ml)

thus prepared was used for point inoculation on the surface of the

modied MRS agar and incubated overnight. After the incubation, the

colonies were washed from the agar surface using double distilled

water and petri plates were ooded with 2% (w/v) aqueous cobalt

chloride solution (Bae et al., 1999). After 5 min of incubation at room

temperature the cobalt chloride solution was replaced with a freshly

prepared solution containing equal volumes of a 6.25% (w/v) aqueous

ammonium molybdate solution and 0.42% (w/v) ammonium meta

vanadate solution. After 5 min incubation, the ammonium molyb-

date/ammonium vanadate solution was removed and the plates were

examined for zone of phytate hydrolysis.

2.4. Phytase and acid phosphatase assay

Phytase activity was assayed by measuring the amount of liberated

inorganic phosphate from sodium phytate (Nielsen et al., 2008). One

unit of phytase activity (U) was dened as the amount of enzyme that

produces 1 nmol of inorganic phosphorous per min at 50 C. The

enzymeactivity was determined by incubating a mixture of 250 l cell

suspension with 250 l of 2 mM substrate prepared in 100 mM

sodium acetateacetic acid buffer (pH 5.5) at 50 C for 15 min. The

reaction was stopped by adding 500 l of 10% (w/v) trichloroacetic

acid solution (TCA) (Haros et al., 2005). Blank was prepared by adding

10% TCA solution before the substrate was added. The inorganic

phosphorous released was quantied at 700 nm using the ferrous

sulphateammonium molybdate method (Nielsen et al., 2008).

Acid phosphatase activity was determined using p-nitrophenyl-

phosphate (p-NPP) as substrate (Palacios et al., 2008). The reaction

mixture contained 250 l of 5 mM p-NPP in 100 mM sodium acetate

buffer, pH 5.5, and250 l ofcell suspension.The mixture was incubated

for 15 min at 50 C. The reaction was stopped by adding 500 l of 1 M

NaOH. Blank was simultaneously preparedby addingNaOH beforethe

addition of the substrate. The amount ofp-nitrophenol released was

measured at 405 nm absorbance using a photometer (Shimadzu,

Japan). One unit of phosphatase activity (U) was denedas theamount

of enzyme that produces 1 mol ofp-nitro phenol per min at 50 C.

2.5. Phenotypic identication of LAB isolates

Gram-positive, catalase negative and sodium phytate degrading

isolates were further subjected to physiological, biochemical and

molecular methods. Growth of the cultures at different temperatures

(15, 37and 45 C), pH (3.5, 4, 4.8 and 8.6), salt concentrations (6.5 and10%) and heat tolerance at 65 C (15 and 30 min) and 70 C (15 min)

were studied (Reddy et al., 2007; Jamuna and Jeevaratnam, 2004).

Carbohydrate utilization tests were performed using HiMedia carbo-

hydrate kit. Overnight grown cells were harvested by centrifugation

(as mentioned earlier), washed with saline and optical density of the

cells was adjusted to 0.5 using saline, from which 50 l was inoculated

to the carbohydrate medium.

2.6. Molecular identication of selected LAB isolates

Genomic DNA from the selected LAB was isolated (Halami et al.,

2005). The amplication of 16S rRNA gene of selected LAB isolates

was carried out using polymerase chain reaction (PCR) by forward

(BS F: GAGTTTGATCCTGGCTCA GG) and reverse (BS R: TCATCTGTCC-CACCTTCGGC) oligonucleotide primers at annealing temperature of

48 C. The amplied products (1.4 kb) were puried using the QIAGEN

PCR purication kit (Qiagen, Germany). The puried PCR products were

cloned into pGEMT vector. The plasmid was isolated from the clone and

the insert was amplied using M13 primers. The amplicon was

sequenced at Bangalore Genei (Bangalore, India). The DNA sequences

were analyzed with the Internet BLAST Gene database (http://www.

ncbi.nlm.nih.gov) and the sequence was submitted to GenBank.

2.7. Additional characteristic features of the isolates

2.7.1. Acid tolerance

The acid tolerance of the selected LAB was studied at different pH

as described by Jacobsen et al. (1999). Overnight (16 h) grown

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inoculum was harvested by centrifugation (8000 rpm at 4 C for

15 min). The cell pellet was washed and resuspended in respective

volume of saline and 10% of it was inoculated to the 50 ml MRS broth

where the pH was adjusted to 2, 2.5, 3 and 3.5 with 0.1 N HCl. The

initial bacterial concentration was 107108 CFU/ml and was main-

tained throughout the experiments. Samples were incubated at 37 C

for 4 h. At 2 h interval, 1 ml of sample was withdrawn and serially

diluted (78 folds) with saline, spread on MRS agar plates and

incubated for 24 h at 37 C. The viable colony forming units (CFU)were counted in a colony counter. The survival rate was calculated as

the percentage of colonies grown on MRS agar compared to the initial

bacterial concentration.

kSurvival = log number of viable cells survived CFU=ml

log number of initial viable cell inoculated CFU= ml 100:

1

2.7.2. Bile tolerance

Bile tolerance of the selected isolates was carried out as reported

byGilliland et al. (1984). LAB cultures grown for 16 h were harvested

by centrifuging cells at 8000 rpmat room temperature for 15 min. The

cell pellet was suspended in saline (~108 cells/ml) and 5% of it was

inoculated in 50 ml of MRS broth, which contains 0.3% bile andincubated for 6 h. At every 1 h interval, sample was drawn and optical

density (O.D.) was recorded at 600 nm using UVvisible spectro-

photometer (Shimadzu, Japan). Delay in growth was considered as the

tolerance ability of the LAB to bile salt.

2.7.3. Hydrophobicity

Bacterial adhesion to hydrocarbons (BATH) test was performed

using xylene as a hydrocarbon to assess the adherent ability of the

isolates (Canzi et al., 2005). Cells were washed once with phosphate-

buffered saline (PBS: 140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 2 mM

KH2PO4,pH 7.2) and resuspended in the same buffer and adjusted to

an absorbance (A) of 0.5 at 600 nm. To this an equal volume of xylene

was added. The two-phase system was thoroughly mixed by vortexing

for 3 min. The aqueous phase was removed after 1 h incubation atroom temperature and its A600 was measured. Adhesion percentage

was calculated according to the formula:

Adhesionpercentage = A0 A =A0 T100 2

where A0 and A are absorbance before and after extraction with

organic solvents, respectively.

2.7.4. Antibacterial activity

For the detection of antibacterial activity, agar spot method was

used (Chen et al., 2002). Cells were harvested as described earlier

and the cell suspension (106107 CFU/ml) was prepared. The

suspension (3 l) was point inoculated onto the surface of the

modied MRS agar plate and incubated overnight at 37 C for 24 h.

After incubation, 1 ml of 46 h grown (early log phase) indicator

pathogenic organism was mixed with 7 ml of soft BHI agar (0.8%)

and poured over the spotted agar plates. The plates were furtherincubated at 37 C for 12 h and the zone of inhibition was measured

in mm. The antimicrobial compound was further characterized by

treating the cell free extract with Trypsin for 1 h at 37 C to conform

its proteinaceous nature. The treated samples were analyzed for its

antimicrobial activity againstListeriaby well diffusion assay method

(Halami et al., 2005).

2.8. Presence of-galactosidase activity

The enzyme -galactosidase activity of the selected isolates was

studied as described by Chen et al. (2002)with slight modications.

Twelve hour cultures were harvested by centrifugation, washed with

10 mM sodium phosphate buffer(pH 7.0)and suspended in theZ-buffer

(60 mM Na2HPO4, 40 mM NaH2PO4and 2.7 l/ml -mercaptoethanol).

The reaction mixture containing 100 l of the cell suspension, 900 l of

Z-buffer and 20 l of toluene, was vortexed at high speed for 2 min

followed by incubation at 37 C for 1 h to remove the toluene prior to

assay. Two hundred micro liters of 200 mM ONGP prepared in Z-buffer

was added and the reaction mixture was incubated at 37 C for 30 min.

The reactionwasstoppedby 500l of1 M Na2CO3 and the concentration

of o-nitrophenol (ONP) released from ONPG was determined by

measuring the absorbance at 420 nm using UVvisible spectrophot-

ometer (Schimdzu, Japan).

2.9. Antibiotic susceptibility assay

Antibiotic susceptibility of the selected LAB isolates was determinedaccording to Danielsen et al. (2006). The selected LAB isolates were

harvested as mentioned earlier and the cell suspension (100 l of 106

107 CFU/ml) was pour plated using MRS agar. Antibiotic E-strips

(HiMedia, India) were placed on the surface of the media prior to

solidication and incubated overnight at 37 C. The zone at lowest

concentration of antibiotic givinga complete inhibitionof visible growth

was considered as minimal inhibitory concentration (MIC) (Wright,

2005).

Fig. 1.Zone of phytate hydrolysis by lactic acid bacteria a) calcium phytate hydrolysis b) sodium phytate hydrolysis and c) sodium phytate hydrolysis in the presence of CaCl 2.

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2.10. Statistical analysis

All assays were conducted in triplicate and repeated three times.

The statistical analysis was carried out for the standard deviations by

using Microsoft excel (Version 5.0; Microsoft, Corp; Redmond, WA).

3. Results

3.1. Selection of phytate degrading lactic acid bacteria

Forty LAB cultures isolated from different sources (chicken andsh

intestinal source, raw milk, cow dung and cucumber) were screened

for their phytate degrading ability using modied MRS agar

supplemented with phytate salt (calcium or sodium) (Fig. 1). All the

isolates exhibited calcium phytate (0.65 g/l) degrading ability, but

could not degrade sodium phytate unless supplemented with 0.2%

calcium chloride. However, only two isolates (strains CFR R38 and CFR

R35) (Fig. 2) could degrade sodium phytate even in the absence of

calcium chloride. These two isolates along withLactobacillus rhamno-sus GG and Lactobacillus amylovorus were further evaluated for

quantitative phytase and acid phosphatase activity (activity was

measured in Units per minute per 9 log CFU) at 37 C and 50 C. The

phytase activity ranged from 3 to 213 U (Table 1). Isolates CFR R38 and

CFR R35 showed an activity of 213 and 89 U at 50 C, respectively. The

acid phosphatase activity of the tested cultures was in contrast with

the phytase activity results, highest being in standard reference

cultures (L. rhamnosus GG and L. amylovorus of 15 and 8 U,

respectively), whereas it was negligible in CFR R38 and CFR R35.

3.2. Physiological, phenotypic characterization and identication of

selected isolates

Isolates CFR R38 and CFR R35, which were of chicken intestinal

origin, were found to be cocci by microscopic observation. Growth

characteristics, physiological investigations and biochemical reactions

suggested that the strainsare closely related to Pediococcus spp. The 16S

rRNA gene sequencing conrmed that the isolates CFR R38 and CFR R35

were Pediococcus pentosaceusand were named as P.pentosaceus CFRR38

and P. pentosaceus CFR R35. The isolates were deposited in repository of

Food Microbiology department of the institute. The 16S rRNA gene

sequences were deposited at GenBank under the accession numbers

FJ586350 and FJ889048.

3.3. Characteristic features of phytate degrading LAB

3.3.1. Acid and bile tolerance

The survival of selected LAB cultures studied is given inTable 2. At

pH 2.0 P. pentosaceus CFR R35, P. pentosaceus CFR R38 and reference

strain L. rhamnosus GG showed 46, 48 and 55% survivability,

respectively after 2 h of incubation. When P. pentosaceus CFR R38

andP. pentosaceusCFR R35 were grown in 0.3% bile, it showed 10 and

40 min delay in growth (Table 2), when compared to the strain grown

in normal MRS broth suggesting that they were ox-bile resistant and

tolerant strains. In contrast L. rhamnosus GG ATCC 53510 showed no

growth at 0.3% bile condition.

3.3.2. Hydrophobicity

BATH property of isolates was studied as an index for adhesion

property (Table 2). Xylene was used as a hydrocarbon to study the cell

wall hydrophobicity.P. pentosaceus CFR R35, P. pentosaceus CFR R38

and L. rhamnosus GG showed 54.6, 44.8 and 59% of hydrophobicity

towards xylene, respectively.

3.3.3. Antibacterial spectrum of phytate degrading LAB

The antibacterial property ofP. pentosaceusCFR R35 and CFR R38

along with L. rhamnosus GG ATCC 53510 was tested against well-

known food borne pathogenic organisms as shown inTable 3. All the

isolates exhibited wide spectrum of antibacterial activity. Among the

cultures studied, P. pentosaceus CFR R38 exhibited a good antibacterial

effect againstEscherichia coliMTCC108,Listeria monocytogenesScott A

andSalmonella paratyphi. The inhibition zone was found to be in the

range of 726 mm. The agar well diffusion assay performed for cell

Table 1

Phytase and acid phosphatase activity of lactic acid bacteria.

Bacterial

culture

Phytase activity

at 37 C UYPhytase activity

at 50 C UYAcid phosphatase

activity at 50 C U

CFR R38a 4.4 213 1.9

CFR R35b 12 89 1.05

ATCC 53510c 27 6 15.1

B4552d 15 3 8.1

aPediococcus pentosaceus CFR R38; bP. pentosaceus CFR R35; cLactobacillus rhamnosus GG

ATCC 53510; dLactobacillus amylovorus B4552. YPhytase activity was determined as

release of 1 nm of inorganic phosphate at 37 and 50 C. Acid phosphatase activity was

dened as release of 1 M ofp-nitro phenol at 50 C.

Table 2

Probiotic properties of phytate degrading lactic acid bacteria.

Bacterial

culture

Acid tolerance

(% survivability) (h)

Bile tolerance

(delay# time

in min)

Adhesion

property

(%)

-Galactosidase

activity, (Miller

units (MU))0 1 2

CFR R38a 100 72.33 50.73 Resistant (10) 54.6 580

CFR R35b 100 73.51 48.5 Tolerant (40) 44.8 613

ATCC 53510c 100 77.3 69.3 Non-tolerant (N60) 59 105

aPediococcus pentosaceus CFR R38; bP. pentosaceus CFR R35; cLactobacillus rhamnosus GG

ATCC 53510; #Delayin growth(lagin time toreachthe0.3 ODvalueforisolatesin theMRS

broth with or without bile salts) time (T) in min b15 min (resistant strain), 1540 min

(tolerant strain), 4060 min (weakly tolerant) andN60 min (sensitive strain).

Table 3

Antibacterial activity of phytate degrading lactic acid bacteria.

Bacterial culture ATCC 53510a CFR R38b CFR R35c

Escherichia coliMTCC 108 ++ +++ +++

Bacillus cereusF 4810 + +++ ++

Listeria monocytogenesScott A ++ +++ ++

Yersinia enterocoliticaMTCC 859 ++ +++ ++

Salmonella paratyphi ++ +++ ++

Staphylococcus aureusFRI 722 ++ ++ +

Interpretation of zone diameter of inhibition. +: 1.010.0 mm; ++: 10.020.0 mm and

+++: more than 20.0 mm.a Pediococcus pentosaceusCFR R38.b P. pentosaceusCFR R35.c

Lactobacillus rhamnosusGG ATCC 53510.

Fig. 2.Zone of sodium phytate hydrolysis byPediococcus pentosaceusCFR R38.

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free extracts of the selected isolates showed inhibitory effect against

Scott A, but it was found to lose its antibacterial activity when culture

ltrate was treated with trypsin.

3.4.-Galactosidase activity

Table 2 shows -galactosidase activity of the LAB strains. It was

found that P. pentosaceus CFR R35 and P. pentosaceus CFR R38

exhibited highest -galactosidase activity of 613 and 580 Miller

units (MU), respectively, whereas reference strain L. rhamnosus GG

showed lowest enzyme activity of 105 MU.

3.4.1. Antibiogram

As shown inTable 4, the antibiotic resistance pattern of both the

isolates P. pentosaceus CFR R38and P. pentosaceus CFR R35 wasanalyzed

byE-test method for 9 antibiotics. It was found that the isolates were

sensitive to six antibiotics. However, the minimum inhibitory concen-

tration (MIC) for polymyxin B was more than 256 g.

4. Discussion

There is a pressing need for food grade LAB to be utilized in

fermented food processes in order to promote functional foods or

nutraceutical supplements (Famularo et al., 2005). As a matter of fact,

studies with experimental animals as well as clinical studies have

elucidated that the phytate content of certain foods such as whole

wheat products, wheat bran and soy products is a foremost

determinant negatively governing the nutritional balance of trace

minerals and proteins in subjects on a regular vegetarian diet (Raboy,

2003). There are seldom studies dealing with the role of LAB in

degrading phytic acid (De Angelis et al., 2003).

In this context, two potent sodium as well as calcium phytate

degrading LAB, P. pentosaceus CFR R38 and P. pentosaceus CFRR35 were

selected by qualitative staining method (Bae et al., 1999). Most of the

cerealand pulsesbased foods arerich in calcium phytate. Allthe testedisolates (40) degraded calcium phytate. However, only two isolates

degraded sodium phytate.Available report states that,calcium ionsare

required for the phytase activity in Lactobacillus sanfranciscensis (De

Angelis et al., 2003). In this study ability of LAB for degradation of

sodium phytate in the presence of calcium was studied. Calcium may

not involve in the reaction but it is needed for enzyme activity ( De

Angelis et al., 2003). The phytatedegrading ability of the isolatesmight

be due to the presence of phytase enzyme, and was conned when all

the 40 isolates degraded sodium phytate in the presence of calcium

chloride. It also revealed that the phytate degrading ability of the

bacteria was due to phytase, but not due to acid hydrolysis. A white

precipitate observed during plate assay around the zone of enzyme

specic phytate hydrolysis conned non-specic phytate hydrolysis

(Bae et al., 1999). The selected potent phytate degrading LAB when

subjected to quantitative analysis exhibited 213 and 89 U phytase

activity with poor acid phosphatase activity (Table 1).Palacios et al.

(2008) reported phytate degrading Bidobacterium from chicken

intestinal origin. In the present work screening and selection of

phytate degradingPediococcusspp. from chicken intestinal origin was

demonstrated.Sreeramulu et al. (1996)observed that decrease in

phytate levels was due to the production of extracellular phytase by

Lactobacillus and Streptococcus. They found production of extracellular

phytase by L. amylovorus B4552. In contrast, L. plantarum producednon-specic acid phosphatase and it showed much less specicity

towards sodium phytate (Zamudio et al., 2001).

Selected potent phytate degrading LAB were evaluated for their

additional characteristic features. A probiotic bacteria need to be

resistant to low pH of the stomach and bile salt of the upper

gastrointestinal tract. One of the main criteria for selection is tolerance

toacid. The gastricpH in healthy human isabout 22.5(Fernandez et al.,

2003). Similarly, tested organisms must be able to survive in the

presence of various bile salts. In this study, the tested LAB cultures were

ableto toleratepH 2 (4851%)for2h(Table 2).Jin et al. (1998) reported

that the survival ofLactobacillus acidophilus isolated from chicken

intestine was less than 50% at pH 3. Bile salt plays an important role in

physiological function with respect to the survival of LAB in small

intestine (Yeong-Soo et al., 2002).Gilliland et al. (1984) reported that

0.3% ox-bile is considered to be a crucial concentration to evaluate bile

tolerant probiotic LAB. In the present study,P. pentosaceusCFR R38 was

found to be bile resistant as itsdelayin growthfallsin resistantcriteriaas

perGilliland et al. (1984)protocol.

Bacterial surface properties have been associated with attachment

to a variety of substrates, which in turn is associated with

hydrophobicity (Aswathy et al., 2008). Bacterial adhesion can also

determine the colonization capability of a microorganism. The BATH

test has been extensively used for measuring cell surface hydro-

phobicity inLactobacillusand Bidobacteriumspp. (Marin et al., 1997,

Vinderola et al., 2004). The tested LAB strains possessed moderate

adherence ability (4559%). A wide spectrum of antibacterial activity

was observed against the tested pathogens. The antibacterial activity

observed can be due to acid, hydrogen peroxide or bacteriocins

(Jacobsen et al., 1999; Lin et al., 2007). The proteinaceus nature of theantimicrobial compound was conrmed by the loss of antimicrobial

activity when the culture free extract was treated with trypsin. This

indicates that the antibacterial activity was due to proteinaceus

compounds (bacteriocins) produced by the isolates.

Lactose intolerance is a term used to describe the discomfort that

occurs after digestion of milk. This condition results from insufcient

amount of-galactosidase to digest lactose in the intestines. Because

of discomfort, intolerant people prefer to avoid milk or milk based

products from the diet (Cebeci and Guakan, 2003). In this context

-galactosidase assay was performed. The tested isolates, exhibited

-galactosidase activity. The isolates CFR R35 and CFR R38 found to

possess high enzyme activity.

Antibiotic susceptibility testing of isolated LAB was done byE-test.

Based on European Commission (2005), the cultures were demon-strated sensitive (S) andresistant (R) by observingthe inhibitory zone

against tested antibiotics taking into consideration the clinical break

points presented by the FEEDAP panel (European Commission, 2005).

It is general belief that starter cultures have the potential to serve as a

reservoir of antibioticresistance genes with the risk of transferring the

genes to pathogenic bacteria (Wright, 2005). As the MIC valuesfor the

tested isolates (Pediococcusspp.) were within the range as presented

by the European Commission, it can be concluded that the isolates

do not carry any resistant genes and can be safely be used as starter

cultures.

In conclusion, the isolated strains P. pentosaceus CFR R38 and

P. pentosaceus CFR R35 showed the maximum levels of phytic acid

degrading ability, phytase activity, acid, bile tolerance and hydro-

phobicity towards hydrocarbons with wide spectrum of antibacterial

Table 4

Antibiogram of phytate degrading lactic acid bacteria.

Name of the

antibiotic

Minimum inhibitory concentration (MIC) in g

Pediococcus pentosaceusCFR R38 Pediococcus pentosaceusCFR R35

Inhibitors of cell wall synthesis

Ampicillin 2 2

Cephalotin 4.0 0.5

Inhibitors of protein synthesis

Chloramphenicol 0.5 0.5Gentamycin 2.0 5.0

Erythromycin 0.25 0.25

Tetracyclin 0.01 8

Streptomycin 5.7 30

Inhibitors of cytoplasmic functions

Polymyxin B 32 32

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activity. These observations indicate that P. pentosaceus CFR R38 and

P. pentosaceus CFR R35 have the potential to be used as starter cultures

for developing several fermented cereal foods, thus decreasing

phytate levels and facilitating the bioavailability of minerals.

Acknowledgements

The authors acknowledge Dr. V. Prakash, Director, CFTRI, Mysore,

and Dr. S. Umesh Kumar, Head, Food Microbiology department, CFTRI,for providing the facilities to perform the research and PR acknowl-

edges ICMR, New Delhi, for the fellowship.

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