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International Journal of Biological Macromolecules 64 (2014) 84– 89

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

jo ur nal home p age: www. elsev ier .com/ locate / i jb iomac

ffects of Lactobacillus plantarum immobilization in alginateoated with chitosan and gelatin on antibacterial activity

men Trabelsi, Dorra Ayadi, Wacim Bejar, Samir Bejar,ichem Chouayekh, Riadh Ben Salah ∗

aboratory of Microorganisms and Biomolecules (LMB), Centre of Biotechnology of Sfax, University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax018, Tunisia

r t i c l e i n f o

rticle history:eceived 14 October 2013eceived in revised form 7 November 2013ccepted 27 November 2013vailable online 4 December 2013

eywords:hitosanelatinntibacterial activity

a b s t r a c t

The present study aimed to investigate and evaluate the efficiency of immobilizing the Lactobacillus plan-tarum TN9 strain in alginate using chitosan and gelatin as coating materials, in terms of viability andantibacterial activity. The results indicate that maximum concentrations of L. plantarum TN9 strain wereproduced with 2% sodium alginate, 108 UFC/ml, and 1 M calcium chloride. The viability and antibacterialactivity of the L. plantarum TN9 cultures before and after immobilization in alginate, chitosan-coated algi-nate, and gelatin-coated alginate, were studied. The findings revealed that the viability of encapsulated L.plantarum could be preserved more than 5.8 log CFU/ml after 35 day of incubation at 4 ◦C, and no effectswere observed when gelatin was used. The antibacterial activity of encapsulated L. plantarum TN9 againstGram-positive and Gram-negative pathogenic bacteria was enhanced in the presence of chitosan coating

materials, and no activity was observed in the presence of gelatin. The effects of catalase and proteolyticenzymes on the culture supernatant of L. plantarum TN9 were also investigated, and the results suggestedthat the antibacterial activity observed was due to the production of organic acids. Taken together, thefindings indicated that immobilization in chitosan enhanced the antibacterial activity of L. plantarum TN9against several pathogenic bacteria. This encapsulated strain could be considered as a potential strongcandidate for future application as an additive in the food and animal feed industries.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Lactic acid bacteria (LAB) have received increasing attention inecent research due to their highly valued physiological effects onhe functioning of the intestines and, hence, on human and ani-

al health [1,2]. Most of non-encapsulated LABs are, however, veryensitive to environmental conditions, such as air, moisture, tem-erature, stomach pH, and bile salt solutions [3,4]. Therefore, whenree LABs pass through the stomach after ingestion, almost all ofhem are killed due to the low pH of the gastric juice. Free LABslso have difficulties in maintaining stability during storage andhen used in products that undergo further processing [5]. It isorth noting that lactobacilli, as a major component of the com-

ensal microbial flora in the intestine of both humans and animals

nd the main representatives of probiotic bacteria, might be use-ul candidates in prevention and treatment of infections caused

∗ Corresponding author at: Laboratoire de Microorganismes et de BiomoléculesLMB), Centre de Biotechnologie de Sfax, Route de Sidi Mansour Km 6, BP “1177”,018 Sfax, Tunisia. Tel.: +216 74 87 04 51; fax: +216 74 87 04 51.

E-mail addresses: riadh fss@yahoo.fr, riadh.bensalah@cbs.rnrt.tn (R. Ben Salah).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.11.031

by multi-resistant bacteria thanks to their ability to modulate theimmune responses of the host [6,7] and to protect the host frompathogens by competitive exclusion [8–13]. Accordingly, there is apressing need for the development of protective technological con-ditions to keep the organisms alive and active during processingand storage [14].

Immobilization technology has often been reported to rep-resent one of the most effective ways to increase cell density,protect probiotics during storage and processing, improve resis-tance to contamination, stimulate the production and secretion ofsecondary metabolites, and shield the physical and chemical stabil-ity of the cells [14–16]. Furthermore, encapsulation systems withcontrol-released ability can deliver probiotics to a specific targetand release them at the required time [17]. Encapsulated materialsconsidered as Generally Recognized as Safe (GRAS) ingredients canbe used in food applications [18].

Although several studies used cellulose acetate phthalate [19]gelatin, vegetable gum [20], fats [21], or �-carrageenan [22,23] as

encapsulating agents, alginate remains the most commonly usedbiopolymer for microencapsulation. The advantages of using algi-nate as an encapsulating agent include: non-toxicity, formation ofgentle matrices with calcium chloride to trap sensitive materials

of Biological Macromolecules 64 (2014) 84– 89 85

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Table 1Parameters used for the immobilization of probiotic strain L. plantarum TN9.

Experiments X1 (CFU/ml) X2 (%) X3 (M) Yield (%)

1 109 1 1 132 109 1 1.5 223 109 1 2 294 108 1 1 35.65 108 1 1.5 51.36 108 1 2 68.27 107 1 1 468 107 1 1.5 49.129 107 1 2 56.7

10 109 2 1 51.1311 109 2 1.5 38.1212 109 2 2 42.5313 108 2 1 47.814 108 2 1.5 50.0115 108 2 2 49.1116 107 2 1 51.1117 107 2 1.5 43.218 107 2 2 50.219 109 3 1 020 109 3 1.5 021 109 3 2 022 108 3 1 023 108 3 1.5 024 108 3 2 025 107 3 1 0

2.4.3. Coating with gelatin

I. Trabelsi et al. / International Journal

uch as living microbial cells, simplicity in entrapping living micro-ial cells, and low cost [23,24]. Alginate is also an accepted fooddditive and can be safely used in foods [24–26].

Alginate is mainly extracted from algae and composed of1 → 4)-linked �-d-mannuronic (M) and �-l-guluronic acid (G)esidues. Alginate is biocompatible and can gel at mild conditionn the presence of calcium cations. The formation of alginate beadsan be performed in sterile environments, and virtually any ingre-ient can be encapsulated. Although the ca-alginate gel possesseso toxicity against cells, it is known to be chemically unstable inhe presence of calcium chelators, such as phosphate, lactate oritrate, and of cations, such as sodium and magnesium, which areble to displace calcium. The removal of phosphate from MRS brothr addition of a calcium cation into it was previously reportedo improve bead stability [27]. The change in the composition ofrowth media may, however, affect the growth parameters of theell. Still, however, the chemical stability of alginate beads can benhanced using barium cation as a gelling agent [27]. Neverthe-ess, barium was reported to induce negative effect on cells due tots toxicity to cells [28]. The beads can also be coated with polyca-ions, such as chitosan, gelatin, and poly-l-lysine, to improve beadtability.

The coating of alginate beads and its effectiveness in the pro-ection of probiotic bacteria have been extensively investigated inhe literature. Several studies have previously reported that coatinglginate microcapsules with chitosan improved the stability of algi-ate beads and, hence, improved the viability of the encapsulatedrobiotic organisms [29,30]. It was suggested that degradation ofhitosan occurs by the microflora available in the colon and solubil-zing alginate gel by sequestering calcium ions [31]. Furthermore,nd taking the bio/muco-adhesive properties of natural biopoly-ers into account, chitosan–alginate microparticulated systems

ave a great potential for colon targeting [31–33].Of particular interest, a LAB strain was isolated in a previous

ork [34]. It was identified by the authors as a new Lactobacilluslantarum TN9. Considering the increasing interest in the devel-pment of efficient methods to protect the viability of this strainnd the promising opportunities that immobilization technologyight bring with regard this pressing need, the present study aimed

o investigate the effects of different coating materials, includinghitosan and gelatin, on the viability and antibacterial activity of. plantarum TN9. The enzymatic activity of the L. plantarum TN9train and the biological antibacterial compounds it produced werelso determined and evaluated.

. Materials and methods

.1. Reagents

All reagents used in this work were of analytical grade and pur-hased from Sigma–Aldrich (St. Louis, USA).

.2. Microorganism

The L. plantarum TN9 strain used in this study was previouslysolated in our laboratory from the gastro intestinal tract of chickennd selected for its ability to support adverse conditions duringastrointestinal transit (resistance to acidic pH and bile salts),n important propriety for probiotic strain [34]. This strain wasdentified as L. plantarum TN9 and maintained in a 20% glyceroluspension at −80 ◦C.

.3. Preparation of cultures

The L. plantarum TN9 cells were cultured and subcultured one Man, Rogosa and Sharpe (MRS) agar plates in 100 ml MRS broth

26 107 3 1.5 027 107 3 2 0

under anaerobic conditions at 30 ◦C for 24 h. They were harvestedby centrifugation at 3000 × g for 20 min. The pellets were washedtwice with phosphate buffer saline (PBS) at pH 7 and re-suspendedin appropriate volume of PBS. The cell suspensions were then eitherdirectly used (free cells) in the assays or submitted to microencap-sulation as described in Section 2.4.

2.4. Immobilization technique

2.4.1. Preparation of alginate beadsL. plantarum TN9 cells were encapsulated in a sodium alginate

matrix as described by Sheu and Marshall [24]. All glassware wasautoclaved at 120 ◦C for 20 min prior to use.

Sodium alginate solutions (1%, 2% or 3%) were prepared, ster-ilized by autoclaving (120 ◦C for 15 min) and cooled to 38–40 ◦C.A sodium alginate solution was mixed in distilled water contain-ing: 11% MRS medium, 5% glycerol, 0.9% Xanthan solution, andcell suspension. The mixture was homogenized by vortex and thenpoured dropwise with a syringe in a sterile calcium chloride solu-tion (CaCl2). The droplets immediately formed gel spheres. Thebeads were put in ice for 15 min, washed three times with sterile bi-distilled water, dried at room temperature, and stored at 4 ◦C untilfurther use. The experiments and their corresponding parametersare summarized in Table 1.

2.4.2. Coating with chitosanThe chitosan and sodium alginate solutions were prepared

according to Krasaekoopt et al. [29]. Alginate–chitosan microcap-sules were prepared as follows: the alginate beads were immersedin 100 ml of chitosan solution (0.8%, w/v), shaken on an orbitalshaker at 100 rpm for 40 min for coating, rinsed with sterile waterto remove the excess chitosan, and then dried.

Alginate–gelatin microcapsules were prepared by dissolving10 g of the filtered microspheres in 100 ml gelatin solution (4%,w/v), and the resulting gelatin-coated alginate beads were thenseparated by filtration and rinsed twice with distilled water.

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.5. Antibacterial activity of free cells

The antibacterial activity spectra of the lactobacilli culturesefore and after immobilization into alginate beads, chitosan-oated alginate, and gelatin-coated alginate were determined byhe well diffusion method using the indicator strains. Aliquots60 �l) of lactobacilli overnight cultures or alginate beads con-aining 108 of entrapped L. Plantarum TN9 cells were placed inells punched in cooled agar plates, which were previously tested

gainst pathogenic bacteria (108 CFU/ml), namely Staphylococcusureus ATCC 6538, Listeria ivanovii BUG 496, Escherichia coli ATCC739, and Salmonella enterica ATCC 43972. The plates were incu-ated overnight at 37 ◦C. The sizes of the inhibition zones (mm)ere measured.

.6. Viability of L. plantarum TN9 in refrigerated conditions

The viability of free, encapsulated L. plantarum TN9 in alginate,hitosan-coated alginate, and gelatin-coated alginate were mon-tored by counting the CFU/ml after storage in 4 ◦C. The obtainedapsules were then resuspended in 10 ml of phosphate buffer salinePBS 0.1 M, pH 7), and survivals of non-encapsulated and free L.lantarum TN9 were enumerated at 1, 14, 28, and 35 days. Experi-ents were performed in triplicates.

.7. Release of encapsulated cells

To determine the viable counts of the encapsulated L. plan-arum TN9, 0.1 g of capsules (alginate, gelatin-coated alginate, andhitosan-coated alginate) were re-suspended in 10 ml of PBS, fol-owed by gentle shaking at room temperature. Samples were takent different time intervals (10, 20, 30, 40, 50, 60, and 120 min) toetermine the complete release of encapsulated bacteria by platingn MRS agar. The colony forming units (CFU/ml) were determinedy plating on MRS agar plates and incubation for 48 h at 30 ◦C.ncoated bacteria were enumerated on MRS agar. Peptone wateras used to prepare the serial dilutions. The culture was platedsing the pour plate technique and incubated at 30 ◦C for 48 h.

The immobilization yield (IY), which is a combined measure-ent of the efficacy of entrapment and survival of viable cells

uring the microencapsulation procedure, was calculated as:

Y = N

N0× 100

here N refers to the number of viable entrapped cells releasedrom the microspheres, and N0 to the number of free cells added tohe biopolymer mix during the production of the microspheres.

.8. Study of probiotic properties of the L. plantarum TN9 strain

One of the major probiotic properties of probiotic LAB is theirntibacterial activity against pathogenic bacteria. Accordingly,his activity was analyzed using the agar well diffusion methodescribed by Vinderola et al. [35]. The pathogenic bacteria useds indicators included Gram-negative and Gram-positive strains,uch as S. aureus ATCC 6538, L. ivanovii BUG 496, E. coli ATCC 8739,nd S. enterica ATCC 43972, which were cultured in Nutrition broth.liquots of 10 �l of the cell were spread on the Nutrition agar plates.

Culture supernatants obtained from the 24 h MRS cultures ofhe L. plantarum TN9 strains were filtrated through a 0.22 �more-size sterile filter (Millipore, Bedford, Mass) and 100 �l of theupernatants were dropped into the wells on the Nutrition agar

rilled with a sterile glass tube (6 mm in diameters). The agarlates were incubated overnight at 37 ◦C, and the diameters ofhe inhibition zones on the agar plate were measured. Each assayas performed in triplicate. The nature of the antibacterial activity

ogical Macromolecules 64 (2014) 84– 89

can be explained by the production of several substances such asorganic acids, hydrogen peroxide or bacteriocin.

Therefore, to determine the biological nature of the antibacte-rial compounds produced by the L. plantarum TN9 strain, 1 ml ofthe cell-free supernatant was submitted to different treatments,including a pH adjustment to 6.5 and an investigation of heatsensitivity (121 ◦C for 20 min), catalase (3 h at 37 ◦C at 1 mg/ml),and proteolytic enzymes (2 h at 37 ◦C in the presence of 1 mg/mlof Trypsin or proteinase K). Immediately after each treatment,residual antibacterial activity was determined against the sameindicator strains described in Section 2.5. The untreated cell-freesupernatant served as a control.

Probiotic strains should also be free of undesirable traitssuch as harmful biochemical activities (like �-chymotrypsin, �-glucuronidase, �-glucosidase, and N-acetyl-�-glucosamidase) thathave been associated with intestinal diseases and recognized to beinvolved in generating carcinogens and tumour promoters. For this,the enzymatic activity of the L. plantarum TN9 was assayed usingthe API-ZYM System (bioMérieux, Montalieu-Vercieu, France), asrecommended by the manufacturer. The inocula, 65 �L of McFar-land standard 1 suspension, were deposited in each well. Enzymeactivity readings were taken after 4 h of anaerobic incubation at37 ◦C and expressed as approximate nmol of substrate hydrolyzedduring 4 h of incubation (from 0 to ≥40 nmol), according to themanufacturer’s instructions.

2.9. Statistical analysis

The assays were performed in triplicate. The data collected inthis study were expressed as the mean value ± standard deviation(SD).

3. Results and discussion

3.1. Release time of the encapsulated bacteria

The ionotropic alginate gel formed by Ca2+ cross linking of car-boxylate groups is insoluble in low pH, exposure to neutral pH orhigher solubilizes the alginate. In our study, this pH-dependentbehaviour of the biopolymer used to release the microencapsulatedcell load under the natural conditions found in the small intestine.The releasing ability of L. plantarum TN9 after exposure to PBS pH7 during 10, 15, 25 and 30 min were respectively 38, 86.3, 97 and100% of the initial population found in alginate beads. However,cell number is stabilized and no further reductions were observedafter 30 min.

The findings indicate that this time was sufficient to release thetrapped cells in the beads. In fact, this result is in agreement withthe ones previously reported by Kim et al. [26], stipulating that arelease time of 30 min was sufficient to liberate the total amountof survival bacteria in the beads.

3.2. Optimization of immobilization of L. plantarum TN9

Immobilization using alginate as a support is affected by vari-ous factors. To develop the immobilization method of L. plantarumTN9, with the aim of increasing cell viability, preliminary experi-ments have been made using different concentrations of sodiumalginate, biomass and calcium chloride [36]. The parameters usedare presented in Table 1. The results indicated that the immobi-lization yield ranged between 13% and 68%. The best performance

corresponded to experiment 6 operating at 108 CFU/ml, 2% sodiumalginate, and 1 M CaCl2. Experiments 19–27 were noted to bringrelatively unsatisfactory yields. The viability of L. plantarum TN9after release was attained with a yield of 68%.

I. Trabelsi et al. / International Journal of Biol

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ays of storage at 4 C ( ) free L. plantarum TN9 ( ) encapsulated L. plantarumN9 in sodium alginate, ( ) encapsulated L. plantarum TN9 coated with chitosan

) encapsulated L. plantarum TN9 coated with gelatin.

The results indicated that 2% sodium alginate concentrationave the maximum viability of cells of L. plantarum TN9 com-ared to the other sodium alginate concentrations. These resultsre congruent with those previously described by Goksunger anduvenc [37] who reported on an optimum sodium alginate con-entration of 2%. Similar results were also obtained by Annan et al.38] who reported on a cell encapsulation yield of 41–43% using08–109 CFU/ml. Maria et al. [39] showed that the encapsulationield varies depending on the strain used for the same cell con-entration (108 CFU/ml). Taken together, the results of the presenttudy strongly suggest that the degree of encapsulation efficiencyepends on the species of the strain being used.

.3. Comparison of the viability and antibacterial activity of L.lantarum TN9 immobilized in alginate coated with chitosan andelatin

.3.1. Viability of encapsulated L. plantarum TN9Further assays were performed to evaluate the efficiency of L.

lantarum TN9 immobilization on alginate, chitosan-coated algi-ate, and gelatin-coated alginate in terms of probiotic viabilitynder refrigeration. Fig. 1 presents the survival rates of free andncapsulated L. plantarum TN9 on sodium alginate, chitosan-coated

able 2ntibacterial activity of free and encapsulated L. plantarum TN9 in sodium alginate, chitosiffusion method, represented by the size of the inhibition zones (mm).

Antibacter

Listeria ivaBUG496

Free L. plantarum TN9 0

L. plantarum TN9 encapsulated in sodium alginate 7

L. plantarum TN9 encapsulated in chitosan-coated alginate 13

L. plantarum TN9 encapsulated in gelatin-coated alginate 0

he bold value indicate the best antibacterial activities.

able 3nhibition of L. Plantarum TN9 against pathogenic bacteria by the agar spot test.

Inhibition zone (mm) of indicator strains

Gram-positive bacteria

Listeria ivanoviiBUG496

Staphylococcusaureus ATCC 65

L. plantarum TN9 35 25

ogical Macromolecules 64 (2014) 84– 89 87

sodium alginate, and gelatin-coated sodium alginate during 4weeks of storage at 4 ◦C. The results revealed that the free cells ofL. plantarum TN9 remained viable for two weeks, during which thenumber of colonies increased from 7.95 to 5.53 CFU/ml. In the thirdand fourth weeks, no viable bacteria were observed. After 35 day,the survival of encapsulated L. plantarum TN9 was noted to decreasefrom 7.98 to 1.2 CFU/ml. No survival was, however, observed for thefree L. plantarum TN9 after the same period.

To further increase the stability of cells in encapsulated mate-rials, the beads were coated in polycationic polymers of gelatinand chitosan. The results showed that the coating of beads withchitosan was more effective than with gelatin. After 35 days, thesurvival of L. plantarum encapsulated in chitosan-coated alginatedecreased from 7.9 to 5.8 log CFU/ml. No survival was, however,noted for L. plantarum TN9 encapsulated in gelatin-coated alginateafter the same period.

Taken together, the results showed that chitosan enhanced theviability of the probiotic strain L. plantarum TN9 at refrigerationconditions. The L. plantarum TN9 cells encapsulated in alginatebeads gave slightly better results than the free cells. Moreover,the alginate beads coated with chitosan yielded better results thanthose coated with gelatin. These findings are in agreement withthe results previously reported by Sawaminee et al. [40]. In fact,several studies showed that survival rates during storage were bet-ter enhanced with bacteria encapsulated in alginate microparticlesthan with non-encapsulated bacteria [22,23,41,42]. Koo et al. [43]reported that Lactobacillus bulgaricus loaded in chitosan-coatedalginate showed higher storage stability than the free cell culture.

3.3.2. Antibacterial activity of encapsulated L. plantarum TN9The beads containing entrapped L. plantarum TN9 were sub-

mitted to of the antibacterial activity tests on agar plates usingthe well diffusion method. The free cell of L. plantarum TN9strain demonstrated no antibacterial activity. Small inhibitionzones were, however, noted immediately after L. plantarum TN9immobilization. The immobilization of the L. plantarum TN9 strainwith chitosan-coated alginate improved its antibacterial activity(>10 mm) against S. aureus, S. enterica and L. ivanovii compared togelatin-coated alginate (no inhibition zone) (Table 2).

The antibacterial activity of L. Plantarum TN9 remained

unchanged after immobilization in sodium alginate. An enhancedantibacterial activity was, however, observed in the presence of chi-tosan. This could be attributed to the higher level of cell protectionthat can induce the production of a highly antibacterial substance.

an-coated sodium alginate and gelatin-coated sodium alginate determined by well

ial activity (mm)

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88 I. Trabelsi et al. / International Journal of Biol

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ogical Macromolecules 64 (2014) 84– 89

The immobilization of beads in chitosan-coated alginate led tothe proliferation of L. plantarum TN9 to the surface of the beadsand surface colonization. This enhanced the secretion of antibacte-rial substances, which can be attributed to the fact that chitosan,as a coating material, can protect the viable and biologically activecells in the beads. In fact, María et al. [39] showed that the immo-bilization of Lactobacillus in calcium alginate induced a partial tocomplete loss of lactobacillus antibacterial activity. Other studieshave reported that coating alginate microcapsules with chitosanimproved viability and, hence, the biological activity of the encap-sulated probiotic organisms [29,39].

3.4. Probiotic properties of the L. plantarum TN9 strain

3.4.1. Antibacterial activity of L. plantarum TN9One of the major probiotic properties of LABs is their inhibitory

effects on pathogenic bacterial growth. The pathogenic bacteriaused as indicators in this study included Gram negative bacteria,such as E. coli ATCC 8739 and S. enterica ATCC 43972, and Gram pos-itive bacteria, such as L. ivanovii BUG 496 and S. aureus ATCC 6538.Table 3 shows the inhibitory effects of L. plantarum TN9 against theassayed pathogenic bacteria. The results showed that L. plantarumTN9 exhibited variable degrees of inhibition with regard to the indi-cator bacteria. The L. ivanovii BUG 496 and E. coli ATCC 8739 strainswere, for instance, noted to display higher levels of tolerance to LABinhibition when compared to other pathogenic bacteria, such as S.aureus ATCC 6538 and S. enterica ATCC 43972 (Table 2). In a previ-ous study, Lan et al. [44] reported that the two probiotic strains(Lactobacillus agillis JCM 1048 and Lactobacillus salivarius subsp.salicinius JCM 1230) isolated from chicken were able to inhibit thegrowth of Salmonella sp. with comparable diameters of inhibition.In another study, Musikasang et al. [45] tested the antibacterialactivity of LAB against E. coli, Salmonella sp., and S. aureus andreported that the inhibition diameters fluctuated between 8–25,13–40, and 6–24 mm, respectively.

3.4.2. Enzymatic activitiyTable 4 summarizes the enzyme activities of strain L. plan-

tarum TN9 determined by the API ZYM system. The probioticstrain L. plantarum TN9 was noted to display diverse enzy-matic profiles, moderate-to-high Leu-arylamidase peptidase and�-galactosidase activities, and no �-mannosidase and �-fucosidaseactivities. This strain was also observed to exhibit no harmful activ-ities such as those displayed by �-glucosidase, �-glucuronidase,�-chymotrypsin, and N-acetyl.

3.5. Nature of L. plantarum TN9 antibacterial activity

The production of inhibitory substances, such as organic acids,bacteriocins, or hydrogen peroxide (H2O2), which inhibit undesir-able and pathogenic bacteria, is a desirable property for probiotics[46]. Accordingly, the culture supernatant was submitted to differ-ent treatments, and residual antibacterial activity was determinedto elucidate the type of antibacterial metabolites produced by L.plantarum TN9. The antagonistic activity of L. plantarum TN9 againstS. enterica ATCC 43972 (and the other bacterial indicator strainstested; data not shown) was produced primarily by organic acidrelease, because the inhibitory effect was entirely destroyed byadjusting the pH of the supernatant to 6 (Fig. 2). The antibacterialactivity was insensitive to heat, catalase, Trypsin, and proteinaseK. In fact, several studies have demonstrated that the productionof organic acids was the major factor responsible for the antago-

nistic activity of many probiotic lactobacilli [47,48]. Furthermore,undissociated forms of organic acids are known to penetrate thepathogenic bacteria cell and to dissociate it inside the cytoplasm,thus decreasing the intracellular pH and the accumulation of the

I. Trabelsi et al. / International Journal of Biological Macromolecules 64 (2014) 84– 89 89

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ig. 2. Effect of different treatments on the antibacterial activity of Lactobacillus pla2–6) different supernatant treatments including a pH adjustment to 6 (2), heating

onized organic acid and, eventually, leading to the death of theathogen [49].

. Conclusions

The survival rates and antibacterial activity of free and encap-ulated L. plantarum TN9 in sodium alginate, chitosan-coatedodium alginate, and gelatin-coated sodium alginate during thetorage periods were compared. The results revealed that algi-ate beads coated with chitosan significantly improved the survivalnd antibacterial activity of L. plantarum TN9 compared to alginateeads coated with gelatin, uncoated beads, and free cells. The find-

ngs also indicated that the L. plantarum TN9 strain encapsulatedn chitosan-coated alginate exhibited a wide spectrum of antibac-erial activity, with an inhibition diameter greater than 10 mm,gainst both Gram-positive and Gram-negative pathogenic bacte-ia. Considering the promising properties and attributes of thisncapsulated strain on chitosan-coated alginate, further in vivotudies, some of which are currently underway in our laboratories,re needed to provide further support for its potential as a feedupplement using poultry production tests.

cknowledgments

This work was supported by the Tunisian Government “Con-rat Programme CBS-LMB”. The authors would like to express theirratitude to Mr. Anouar Smaoui and Mrs. Hanen Ben Salem fromhe English Language Unit at the Sfax Faculty of Science for theironstructive language editing and proofreading services.

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