International Journal of Food Microbiology 142 (2010) 242–246

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International Journal of Food Microbiology

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Short communication

An acid/alkaline stress and the addition of amino acids induce a prolonged viability ofLactobacillus plantarum loaded into alginate gel

Antonio Bevilacqua a,b, Milena Sinigaglia a,b, Maria Rosaria Corbo a,b,⁎a Department of Food Science, Faculty of Agricultural Science, University of Foggia, Italyb Food Quality and Health Research Center (BIOAGROMED), University of Foggia, Via Napoli 25, 71122 Foggia, Italy

⁎ Corresponding author. Department of Food Science,University of Foggia, Italy. Tel.: +39 0881 589232; fax:

E-mail address: m.corbo@unifg.it (M.R. Corbo).

0168-1605/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.ijfoodmicro.2010.05.030

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 February 2010Received in revised form 30 May 2010Accepted 31 May 2010

Keywords:Cell immobilizationDeath timeBiomass productionSodium-alginateStressLactobacillus plantarum

This study reports on the investigation on the effects of the conditions used throughout the step of biomassproduction on the survival of Lactobacillus plantarum loaded into alginate gels. L. plantarumwas grown underdifferent conditions (MRS or a laboratory medium–LB2–at acidic or alkaline pHs, with NaCl, phenols,vitamins or amino acids) and immobilized in sodium alginate; cell number was evaluated throughout thestorage and death (δstand) and first-reduction times (δ) were calculated. The storage of alginate gels at 4 °Cprolonged cell viability up to 60 days (ca. 20 days for cells produced in MRS and stored at 30 °C); however, asimilar prolongation was achieved for cells produced in LB2 adjusted to pH 5.0 and 9.0 or added with aminoacids (death timeN50–60 days).

Faculty of Agricultural Science,+39 0881 589231.

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Polymers derived from natural sources are extensively used asbiomaterials and/or as matrices for the micro-encapsulation of activecompounds and cells (Remminghorst and Rehm, 2006). Alginate is apolysaccharide belonging to the family of linear (unbranched), non-repeating copolymers, containing variable amounts of β-D-mannuro-nic acid and its C5-epimer α-L-guluronic acid, linked through a β-1,4-glycosidic bond (Remminghorst and Rehm, 2006). Beads areextremely easy to prepare on a lab scale, through a syringe extruderand a stirred calcium bath and the process could be considered asmild(Gouin, 2004).

Different microorganisms were loaded in alginate in the past:bifidobacteria (Cui et al., 2000; Krasaekoopt et al., 2004), lactobacilli(Boyaval and Goulet, 1988; Gbassi et al., 2009; Goksunger andGuvenc, 1999; Idris and Suzana, 2006; Yoo et al., 1996), lactococci(Klinkeberg et al., 2001), Bacillus amyloliquefaciens (Abdel-Naby et al.,2000),Kluyveromycesmarxianus (Goughet al., 1998) and Saccharomycescerevisiae.

Lactic Acid Bacteria (LAB) are submitted to potential stressfulenvironmental changes in industrial processes as well as in nature,where the ability to respond quickly to a stress is essential for survival(van de Guchte et al., 2002).

To withstand these adverse conditions, LAB develop differentdefenses that permit them to resist harsh conditions and suddenenvironmental changes (van de Guchte et al., 2002); these physio-logical changes are referred to as “stress response” (Streit et al., 2007)and appear when bacteria are exposed to moderate levels of stress(Kim et al., 1999; Panoff et al., 1995; Streit et al., 2007; van de Guchteet al., 2002). They allow the cells to acquire increased tolerance whenthey are subsequently submitted to higher levels of the same stress orof another stress (cross-protection and general stress response—GSR)(Sanders et al., 1999; van de Guchte et al., 2002).

Bacteria can adapt to adverse conditions by modifying the fattyacid composition of membrane or producing specific proteins, like theHSP (heat shock proteins) (Streit et al., 2008; van de Guchte et al.,2002). Some of these proteins are related also with the growth phase(Cohen et al., 2006).

In this paper we report on the survival of a probiotic strain ofLactobacillus plantarum (c19), loaded into alginate gels, focusing onthe effect of the conditions encountered throughout the step ofbiomass production on the survival of the immobilized cells. Thestrain of L. plantarum (c19) used as target was isolated from Italiantable olives “Bella di Cerignola” and characterized in order to assess itsprobiotic (Altieri et al. 2006) and technological features (data notpublished). In particular, the strainwas able to grow in a large range ofpH (4.0–9.0) and survives in brines containing 10% of NaCl; moreover,it exerted a strong antimicrobial activity against a wild strain ofEscherichia coli O157:H7 and showed an interesting adhesionpercentage on IPEC-J2 cell line (ca. 13%). Hereby this isolate is usedas a model for other probiotic strains to point out the conditions for

Table 2Media for cell production in the different assays of this research.

Medium use for biomass production

Assay 1 (shelf life definition)4 °C MRS brotha

15 °C MRS broth30 °C MRS broth

Assay 2 (acid stress and kind of medium)MRS MRS broth (pH 6.2)MRS at pH 5.0 MRS broth acidified to pH 5.0 through HCl 2.0 NLB2 at pH 6.0 LB2 medium reported in Table 1LB2 at pH 5.0 LB2 medium acidified to pH 5.0 through HCl 2.0 N

Assay 3 (other stresses)LB2 Synthetic medium (Table 1)LB2 at pH 9.0 LB2 adjusted to pH 9.0 through NaOH 2.0 NNaCl LB2+5% (w/v) NaClGlycerol LB2+5% (v/v) glycerolPhenols LB2+0.75% (w/v) of p-coumaric and 0.75% of vanillic acidsb

Assay 4 (addition of amino acids and vitamins)LB2 LB2 (Table 1)Amino acids LB2+10% (w /v) of an amino acid poolc

Vitamins LB2+5% (v/v) of a vitamin solutiond

Assay 5 (kind of carbohydrate)Glucose LB2

Lactose LB2+5.0 g/l of lactose (J.T. Baker)e

Sucrose LB2+5.0 g/l of sucrose (J.T. Baker)Maltose LB2+5.0 g/l of maltose (J.T. Baker)

a The biomass was produced in MRS broth; then, alginate gels containing cells werestored at 4, 15 and 30 °C.

b p-coumaric acid, p-hydroxycinnamic acid; vanillic acid, p-hydroxymethoxybenzoicacid. The compounds were purchased from Sigma-Aldrich (Milan, Italy).

c Amino acidic pool: L-arginine monohydrochloride, 5.0 g/l; L-cysteine, 5.0 g/l;L-phenylalanine, 5.0 g/l; L-lysine, 5.0 g/l; glycine, 5.0 g/l; L-hystidine, 5.0 g/l; tyrosinedisodium salt, 2.5 g/l. The amino acids were purchased from Sigma-Aldrich.

d Vitamin solution: pyridoxine hydrochloride, 2.0 g/l; calcium D-panthotenate, 1.0 g/l;nicotinic acid, 1.0 g/l; riboflavin, 1.0 g/l; folic acid, 0.5 g/l.

e Glucose was replaced with the different carbohydrates.

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the maximal retention of viability, as it is well known that a probioticcan exert its beneficial effect in the gut if it is viable and active.

Cells were produced under various conditions, using MRS broth (acomplex medium, considered as the universal substrate for LAB) or arelatively poor medium (LB2); moreover, some stressful conditionswere used throughout this step (acid or alkaline pH), as well as theaddition of some stimulating elements (like vitamins and aminoacids). Then, cells were harvested and loaded into alginate gels,evaluating their viability during the storage and defining the deathtime through some mathematical models.

2. Materials and methods

2.1. Strain

L. plantarum c19, isolated from Italian table olives “Bella diCerignola” (Altieri et al., 2006) and identified through Rep-PCR(repetitive sequence-based PCR) and ITS-PCR (intergenic transcribedspacer PCR) and DNA sequencing (Bevilacqua, 2006), was usedthroughout this study. The strain was stored at −20 °C in MRS broth(Oxoid, Milan, Italy) added with 33% of sterile glycerol (J.T. Baker,Milan).

2.2. Cell production

Themicroorganismwas grown inMRS broth, incubated at 30 °C for24 h, and then inoculated at 7 log(cfu/ml) into aliquots of 250 ml ofMRS broth or the medium LB2, originally described by Liew et al.(2005) and Jyoti et al. (2003) andmodified byBevilacqua et al. (2008b)and Corbo et al. (2008). The media were incubated at 30 °C for 72 h inorder to attain a culture in the stationary phase (ca. 8.5 log cfu/ml).

The composition of the medium LB2 is shown in Table 1.Five different assays were carried out, and a brief description of the

media used for cell production is reported in Table 2.

2.3. Cell immobilization

Twenty milliliters of broth was centrifuged at 1000 g for 10 min;after removing the supernatant, the pellet was suspended in 20 ml ofsterile distilled water (cell suspension). Five milliliters of the cellsuspension was put into 15 ml of sterile distilled water, added with1.2 g of Na-alginate (Fluka, Milan) andmixed gently for 2 min; then, avolume of 0.66 ml of CaCl2 at 6% was added and the gel was mixed for2 min. For each combination, 60 different samples were prepared. Toassess the effect of alginate and kind of medium, two differentcontrols were used, i.e. control 1, consisting of cells grown in thedifferent media, listed in Table 2, and control 2, represented by cellssuspended in distilled water (cell suspension).

2.4. Microbiological analyses

The samples and the respective controls were stored at 4, 15 and30 °C (assay 1) or at 30 °C (assays 2, 3, 4 and 5); the viable cell countof L. plantarum was evaluated periodically through the pour plate

Table 1Composition of basic medium (LB2) used for cell production of L. plantarum c19.

Compound Amount

Glucose (J.T. Baker) 5.0 g/lYeast extract (Oxoid) 2.0 g/lTween 80 ® (Biolife, Milan, Italy) 1 ml/lKH2PO4 (J.T. Baker) 2.7 g/lMgSO4·7H2O (J.T. Baker) 0.2 g/lMnSO4·H2O (J.T. Baker) 0.05 g/l

method on MRS agar, incubated under anaerobic conditions at 30 °Cfor 48 h.

2.5. Statistical analyses and data modeling

All analyses were performed in duplicate over two differentbatches; for each batch the experiments were carried out twice.

Experimental data weremodeled through theWeibull equation, asmodified by Mafart et al. (2002), by means of the software Statisticafor Windows version 6.0 (Statsoft, Tulsa, OK):

log10 N = log10 N0− t=δ� �p ð1Þ

where: δ is the first-reduction time (i.e. the time to attain a 1-logreduction in the population number) (day) and p is the shapeparameter (pb1, downward curve; pN1 upward curve).

Through the parameter of Weibull function, the cell number after30 days of storage was evaluated (logN30).

Then, the equation of Weibull, modified by Bevilacqua et al.(2008a), was used for the evaluation of the death time of thepopulation:

logN.logN0

� �= 1− t=δstand

� �p ð2Þ

where: δstand is the death time (day).The parameters Eqs. (1) and (2), as well as their standard errors

and confidence intervals, were evaluated as follows: first, a fit was runwith the original data; then, using the data point standard deviation,100 additional fits were run on artificial data sets, which were

Fig. 1. Death kinetic of L. plantarum c19 cells immobilized in sodium alginate and storedat 4, 15 and 30 °C. Before entrapping into alginate, biomass was produced in MRS broth.Data are the mean±SD.

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generated by randomly varying the data around the fitted function.From these additional fits, a distribution of values for each parameterwas obtained.

Experimental data and Weibull parameters were submitted to aone-way analysis of variance (ANOVA) and Tukey's test (Pb0.05) topoint out significant differences.

3. Results and discussion

The survival of cells immobilized into alginate gels and stored at 4,15 and 30 °C is reported in Fig. 1; the lines are the best fit of theWeibull equation through the data. The storage temperature affectedstrongly the survival of L. plantarum cells, prolonging both the 1-logreduction (δ) and the death times (δstand); in particular, δ value was5.04±1.98 days at 30 °C and increased to 25.55±4.59 and 26.05±2.55 days at 4 and 15 °C, respectively. Another result of great concernis the prolongation of δstand; this parameter was 20.87±1.20 at 30 °Cand N60 days both at 4 and 15 °C.

As a final step of each assay, L. plantarum population after 30 dayswas evaluated; this break point was chosen as in Italy it's a common

Table 3Weibull parameters (±SE) of L. plantarum c19 population immobilized in sodium alginate aand 5.0.

logN0a δ

Cells loaded into Na-alginateMRS pH 6 (control) 8.81±0.68 5.04±1.98MRS pH 5 9.43±1.23 1.05±1.46LB2 pH 6 7.83±0.28 13.93±1.75LB2 pH 5 8.07±0.32 9.57±3.74

Control 1 (cell cultures)MRS pH 6 (control) 7.85±0.50 6.35±1.57MRS pH 5 8.51±0.55 4.47±1.64LB2 pH 6 8.03±0.33 2.32±0.71LB2 pH 5 7.83±0.27 3.07±0.76

Control 2 (cells suspended in distilled water)MRS pH 6 (control) 7.67±1.44 0.89±1.88MRS pH 5 8.53±0.80 2.06±1.49LB2 pH 6 7.49±0.99 0.67±0.90LB2 pH 5 7,78±0.01 18.10±1.66

a Weibull parameters: logN0, initial cell number (log(cfu/g)); δ, first-reduction time (determination coefficient; logN30, population number after 30 days of storage (log(cfu/g));

b Cell numberbdetection limit (10 cfu/g for gels–1 cfu/ml for controls).c δstandN running time (50 days).

practice to impose a shelf life of 30 days for functional products. L.plantarum was at the undetectable level after 30 days at 30 °C,whereas the population level was ca. 7–7.5 log cfu/g at 4 and 15 °C.

Table 3 reports the fitting parameters of the equation of Weibulland the survival of L. plantarum cells loaded into alginate gels andstored at 30 °C, when the biomass had been produced in MRS broth,the universal medium for lactobacilli, or on LB2, and adjusted to pH 5.0and 6.0. In this assay a result of great interest was the prolongation ofthe death time value, due both to the kind of the medium usedthroughout the step of biomass production and its pH; death timevalue, in fact, was 20.87±1.20 days for the cell produced in MRS andincreased up to 25.56±0.20 days in MRS adjusted to pH 5.0. Thiseffect on the death time, however, could not be regarded assignificant, because death time value was strongly dependent on theinitial population size (8.81 in MRS at pH 6.0 and 9.43 in MRS at pH5.0); otherwise, the effect was highly significant in LB2 medium,acidified at pH 5.0: cells produced under this condition, in fact,experienced a death timeN50 days.

The effect of the production of biomass in the LB2, acidified to pH5.0, was highlighted also by the data recovered in the controls; inparticular, in the control 2 (cells in water) death time was N30 days.

Table 4 reports the results of the survival of the L. plantarum loadedin the gel, after cell production in LB2 medium, containing NaCl,glycerol or some secondary phenolic compounds (p-coumaric andvanillic acids) or adjusted to pH 9.0.

The addition of NaCl or of the phenolic compounds increased thefirst-reduction time (δ) from 7.42±0.52 days to 10.03±0.23 and9.36±0.26 days, respectively. However, δ value could not be usedalone to compare cell resistance, due also to the great variability of theshape parameter (p-factor); this variability could be explained bothby the differences of conditions and by the strong self-correlationwhich occurred between δ and p. In this case the comparison of cellresistance could be achieved better through the death time, whichwas ca. 20 days in the control and increased up to 28–35 days for theimmobilized cells, produced in the substrate containing NaCl, glycerolor the phenolic compounds; moreover, death time was N60 days forcells produced at pH 9.0. As reported for the acid stress, this positiveeffect was confirmed by the data of the controls.

After testing the effect of biomass production under sub-optimalconditions (acidic or alkaline pHs or in the medium containing NaCl,glycerol, p-coumaric and vanillic acids), the cells were produced in LB2mediumenrichedwith amino acids or vitamins and then immobilized;

nd stored at 30 °C. The strain was revitalized in MRS broth and in LB2 medium at pH 6.0

p R logN30 δstand

1.53±0.40 0.961 –b 20.87±1.200.70±0.28 0.973 – 25.56±0.203.48±0.73 0.982 – 25.69±0.701.07±0.38 0.928 4.67 N50c

2.33±0.62 0.976 – 15.44±0.611.89±0.59 0.983 – 13.97±0.630.95±0.12 0.995 – 20.91±0.761.49±0.27 0.996 – 21.18±0.30

0.66±0.43 0.957 – 19.53±3.070.94±0.27 0.975 – 20.24±1.340.64±0.24 0.979 – 20.57±1.340.56±0.10 0.963 5.46 N30

i.e. time to attain a 1-log reduction in cell number) (day); p, shape parameter; R,δstand, death time (day).

Table 4Weibull parameters (±SE) of L. plantarum c19 population immobilized in sodium alginate and stored at 30 °C. The strain was revitalized in LB2 medium, adjusted to pH 9.0, or addedwith 5% NaCl or glycerol or 1.5% of phenols.

logN0a δ p R logN30 δstand

Cells loaded into Na-alginateControl (pH 6.0) 8.06±0.44 7.42±1.52 2.12±0.42 0.974 –b 19.84±0.71pH 9.0 8.27±0.25 4.84±1.97 0.63±0.10 0.977 5.11 N60c

5% NaCl 8.09±0.24 10.03±1.23 2.03±0.24 0.990 – 27.95±0.595% glycerol 8.09±0.86 2.19±2.06 0.75±0.23 0.969 1.02 35.47±2.690.5% phenols 7.93±0.15 9.36±1.23 0.89±0.22 0.983 0.23 22.50±0.88

Control 1 (cell cultures)Control (pH 6.0) 6.50±0.21 4.69±0.70 1.62±0.20 0.996 – 14.87±0.30pH 9.0 6.88±0.13 19.36±1.74 1.72±0.13 0.991 4.76 N605% NaCl 7.28±0.79 1.01±0.53 0.81±0.24 0.980 – 11.84±0.915% glycerol 6.83±0.56 3.53±1.25 1.53±0.40 0.977 – 12.41±0.610.5% phenols 7.67±0.25 5.01±0.23 1.89±0.22 0.983 – 16.02±1.45

Control 2 (cells suspended in distilled water)Control (pH 6.0) 6.10±0.21 2.65±0.56 0.99±0.10 0.997 – 16.57±0.35pH 9.0 5.99±0.57 5.81±0.23 1.25±0.49 0.967 – 24.89±1.975% NaCl 6.55±0.35 3.47±0.99 1.21±0.20 0.991 – 16.44±0.555% glycerol 6.53±0.72 0.83±0.88 0.64±0.21 0.979 – 15.69±1.440.5% phenols 5.59±0.79 4.65±2.54 2.55±2.02 0.976 – 9.35±0.69

a Weibull parameters: logN0, initial cell number (log(cfu/g)); δ, first-reduction time (i.e. time to attain a 1-log reduction in cell number) (day); p, shape parameter; R,determination coefficient; logN30, population number after 30 days of storage (log(cfu/g)); δstand, death time (day).

b Cell numberbdetection limit (10 cfu/g for gels–1 cfu/ml for controls).c δstandN running time (60 days).

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the results are reported in Fig. 2. For the evaluation of the individualconstituents of the growthmedium, like vitamins and amino acids, theuse of a laboratory medium without yeast extract would have beenadvisable; however, preliminary investigations revealed that L.plantarum c19, as well as other lactobacilli isolated from table olives,did not grow in a medium without a complex ingredient, like yeastextract, bacteriological peptone or tryptone.Moreover, the use of yeastextract assured a level of biomass corresponding to at least 8–9 log cfu/ml (Corbo et al., 2008). The production of the biomass in the mediumcontaining vitamins did not exert any significant effect on its viabilitywhen loaded in the gels; otherwise, the addition of amino acidsresulted in a strong prolongation of the viability of immobilized cells(death timeN60 days).

Finally, an experiment was conducted to evaluate if the kind ofcarbohydrate (glucose, lactose, maltose and sucrose) added through-out the phase of biomass production could exert an effect of cellviability into the gel. The results showed that the effect of carbonsource was not significant (data not shown).

Fig. 2. Death kinetic of L. plantarum c19 cells immobilized in sodium alginate and storedat 30 °C. The biomass was produced in LB2 medium added with a vitamin solution or anamino acidic pool. Data are the mean±SD.

As expected, the 1st assay showed that the storage temperatureinfluenced significantly the survival of cells loaded into alginate gels;however, there is an open question, that needs to be investigated: is itpossible to enhance L. plantarum survival and achieve the same resultsof the temperature in terms of prolongation of viability? The approachproposed in this paper was the improvement of the conditions used inthe preliminary phase of biomass production.

Some cultural conditions affected strongly the survival of loadedcells, i.e. the use of LB2 medium, which could be considered as a poorsubstrate if compared to MRS, and strain reviving under acidic oralkaline conditions or with amino acid added.

It is not clear what happened at cell level, probably, cells loadedinto gels are in a condition of starvation. In nature starvation is one ofthe most frequent stresses encountered by bacteria and bacterialgrowth itself contributes to nutrient exhaustion and subsequentstarvation for one or more compounds (van de Guchte et al., 2002). Inaddition, some extreme environmental stress conditions may provokea deprivation of several components, for example, acidic conditionscould decrease the activity of some transporters, thereby decreasingthe availability of essential nutrients (van de Guchte et al., 2002).

If the loading into gels is a kind of starvation stress, the productionof biomass under acidic and alkaline conditions could be referred to asa slight stress (induction) before a kind of extreme condition and it iswell known, as reported elsewhere, that a slight stress could induceresistance (De Angelis and Gobbetti, 2004; Streit et al., 2007).

In the literature there are many papers dealing with this topic.For example, Streit et al. (2007, 2008) reported on the possibilityof inducing crytotolerance by acidification in L. delbrueckii subsp.bulgaricus, due to a slight decrease in unsaturated to saturated andcyclic to saturated membrane fatty acid ratios and a change in proteinmetabolism, with an increase synthesis of stress proteins.

Based on these ideas, what we can suggest is that probably theexposure of L. plantarum to slight stress throughout cell production(acid and alkaline conditions and slight starvation in LB2 medium)activated stress response and induced resistance to the subsequentstarvation into alginate gels.

In conclusion this paper provides some useful results concerningthe viability of L. plantarum loaded into alginate gels. Cell survival wasca. 20 days under ordinary conditions (biomass production in MRSand gel storage at 30 °C); on the other hand, L. plantarum viability

246 A. Bevilacqua et al. / International Journal of Food Microbiology 142 (2010) 242–246

could be increased using either refrigeration or producing cells in arelatively poor medium adjusted to pH 5.0 or 9.0, or added with anamino acidic pool.

References

Abdel-Naby, M.A., Reyad, M., Abdel-Fattah, F., 2000. Biosynthesis of cyclodextringlucosyltransferase by immobilized Bacillus amyloliquefaciens in batch andcontinuous cultures. Biochemical Engineering Journal 5, 1–9.

Altieri, C., Bevilacqua, A., Ouoba, L.I.I., Sinigaglia, M., Jakobsen, M., 2006. Adhesionproperties of lactic acid bacteria, isolated from Italian table olives, andPropionibacteria. Proceedings of 20th International ICFMH Symposium FoodMicro2006 (Bologna, August 29–September 2). Alma Mater Studiorum University ofBologna, Bologna (Italy), p. 348.

Bevilacqua, A., 2006. Phenotypic, genotypic and biotechnological characterization oflactic acid bacteria isolated from Italian table olives “Bella di Cerignola”. PhD thesis,University of Foggia (Italy).

Bevilacqua, A., Cibelli, F., Cardillo, D., Altieri, C., Sinigaglia, M., 2008a. Metabiotic effectsof Fusarium spp. on Escherichia coli O157:H7 and Listeria monocytogenes on rawportioned tomatoes. Journal of Food Protection 71, 1366–1371.

Bevilacqua, A., Corbo, M.R., Mastromatteo, M., Sinigaglia, M., 2008b. Combined effects ofpH, yeast extract, carbohydrates and di-ammonium hydrogen citrate on thebiomass production and acidifying ability of a probiotic Lactobacillus plantarumstrain, isolated from table olives, in a batch system. World Journal of Microbiologyand Biotechnology 24, 1721–1729.

Boyaval, P., Goulet, J., 1988. Optimal conditions for production of lactic acid from cheesewhey permeate by Ca-alginate entrapped Lactobacillus helveticus. Enzyme andMicrobial Technology 10, 725–728.

Cohen, D.P.A., Renes, J., Bouwman, F.G., Zoetendal, E.G., Mariman, E., de Vos, V.M.,Vaughan, E.E., 2006. Proteomic analysis of log to stationary growth phaseLactobacillus plantarum cells and a 2-DE database. Proteomics 6, 6485–6493.

Corbo, M.R., Bevilacqua, A., Mastromatteo, M., Sinigaglia, M., 2008. Influence of thenutrients on growth, biomass production and acidifying ability of Lactobacillusplantarum, isolated from table olives. Advances in Food Sciences 30, 94–100.

Cui, J.-H., Goh, J.-S., Kim, P.-H., Choi, S.-H., Lee, B.-J., 2000. Survival and stability ofbifidobacteria loaded in alginate poly-L-lysine microparticles. International Journalof Pharmaceutics 210, 51–59.

De Angelis, M., Gobbetti, M., 2004. Environmental stress responses in Lactobacillus: areview. Proteomics 4, 106–122.

Gbassi, G.K., Vandamme, T., Ennahar, S., Marchioni, E., 2009. Microencapsulation ofLactobacillus plantarum in an alginate matrix coated with whey proteins.International Journal of Food Microbiology 129, 103–105.

Goksunger, Y., Guvenc, U., 1999. Production of lactic acid from beet molasses by calciumalginate immobilized Lactobacillus delbrueckii IFO 3202 batch and continuous.Journal of Chemical Engineering and Biotechnology 74, 131–136.

Gough, S., Barron, N., Zulbov, A., Lozinsky, V.I., McHale, A.P., 1998. Production of ethanolfrommolasses at 45 °C using Kluyveromyces marxianus IM3 immobilised in calciumalginate gels and polyvinyl alcohol. Journal of Bioprocess Engineering 19, 87–90.

Gouin, S., 2004. Microencapsulation: industrial appraisal of existing technologies andtrends. Trends in Food Science and Technology 15, 330–347.

Idris, A., Suzana, W., 2006. Effect of sodium alginate concentration, bead diameter,initial pH and temperature on lactic acid production from pineapple waste usingimmobilized Lactobacillus delbrueckii. Process Biochemistry 41, 1117–1123.

Jyoti, B.D., Suresh, A.K., Venkatesh, K.V., 2003. Diacetyl production and growth ofLactobacillus rhamnosus on multiple substrates. World Journal of Microbiology andBiotechnology 19, 509–514.

Kim, W.S., Ren, J., Dunn, N.W., 1999. Differentiation of Lactococcus lactis subspecieslactis and subspecies cremoris strains by their adaptative responses to stresses.FEMS Microbiology Letters 171, 57–65.

Klinkeberg, G., Lystad, K.Q., Levine, D.W., Dyrset, N., 2001. Cell release from alginateimmobilized Lactococcus lactis spp. lactis in chitosan and alginate coated beads.Journal of Dairy Science 84, 1118–1127.

Krasaekoopt, W., Bhandari, B., Deeth, H., 2004. The influence of coating materials onsome properties of alginate beads and survivability of microencapsulated probioticbacteria. International Dairy Journal 14, 737–743.

Liew, S.L., Ariff, A.B., Raha, A.R., Ho, Y.W., 2005. Optimization ofmedium composition forthe production of a probiotic microorganism, Lactobacillus rhamnosus, usingresponse surface methodology. International Journal of Food Microbiology 102,137–142.

Mafart, P., Couvert, O., Gaillard, S., Leguerinel, I., 2002. On calculating sterility in thermalpreservation methods: application of the Weibull frequency distribution model.International Journal of Food Microbiology 72, 107–113.

Panoff, J., Thammavongs, B., Laplace, J., Hartke, A., Boutibonnes, P., Auffray, Y., 1995.Cryotolerance and cold adaptation in Lactococcus lactis subsp. lactis IL 1403.Cryobiology 32, 516–520.

Remminghorst, U., Rehm, B.H.A., 2006. Bacterial alginates: from biosynthesis toapplications. Biotechnology Letters 28, 1701–1712.

Sanders, J.W., Venema, G., Kok, J., 1999. Environmental stress responses in Lactococcuslactis. FEMS Microbiology Reviews 23, 483–501.

Streit, F., Corrieu, G., Bèal, C., 2007. Acidification improves cryotolerance of Lactobacillusdelbrueckii subsp. bulgaricus CFL1. Journal of Biotechnology 128, 659–667.

Streit, F., Delettre, J., Corrieu, G., Bèal, C., 2008. Acid adaptation of Lactobacillusdelbrueckii subsp. bulgaricus induces physiological responses at membrane andcytosolic levels that improves cryotolerance. Journal of Applied Microbiology 105,1071–1080.

van de Guchte, M., Serror, P., Chervaux, C., Smokvina, T., Ehrlich, S.D., Maguin, E., 2002.Stress responses in lactic acid bacteria. Antonie van Leeuwenhoek 82, 187–216.

Yoo, I.K., Seong, G.H., Chang, H.N., Park, J.K., 1996. Encapsulation of Lactobacillus caseicells in liquid core alginate capsules for lactic acid production. Enzyme andMicrobial Technology 32, 553–559.