encapsulation of lactobacillus casei

8/12/2019 Encapsulation of Lactobacillus Casei

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ELSEVIER

Encapsulation of actobacillus caseicells in liquid-core alginate capsulesfor lactic acid productionIk-Keun Yoo,* Gi Hun Seong ,* Ho Nam Chang,* and Joong Kon Park*D epart ment of Chemi cal Engineeri ng and Bi oPro cess Engi neeri ng Research Cent er, Kor eaAdv anced i nsti t ut e of Sci ence and Technology (EXI ST), Taej on, Kor ea D epart ment of Chemi calEngineeri ng, Ky ungpook Nat ional U niv ersit y, Taegu, Kor ea

The immobil i zation method of Lactobacillus casei cells was i nvest i gated usin g algin at e capsules t hat possess ani nt erphosi c membrane and a l iqui d core. The capsules w ere ound t o off er more space or cell ul ar grow t h thangel -core beads, w hich result ed in I.5-fold higher cell concentration than in the latter; however, the Ca-alginatestructure was unstable during repeated batch fermentations for lactic acid production. Ba-alginate capsules werechemically and physically more stable than the Ca-alginate capsules in phosphate and lactate solutions. Attemptswere also made to use various hardening agents to stabilize the structure of the Ba-alginate capsules. It wasfound that the treatment with a mixture of chitosan and BaCl, solution gave the best results for hardening.Finally, stable lactic acid production was possible with a productivity of more than 2.7 g l-h- by L. casei cellsimmobilized in chitosan-coated Ba-alginate capsules. The cell leakage from the capsules was maintained rela-tively low during repeated batch fermentations.

Keywords: Lactic acid; barium-alginate capsules; encapsulation; alginate gel stability; Lactobucillus casei

IntroductionGiven the low productivity of batch processes for lactic acidproduction, recent research has focused on increasing thecell concentration in the reactor.14 Cell immobilization isone of the most attractive methods in maintaining high cellconcentration in the reactor and has been extensively stud-ied. Among various cell immobilization methods, entrap-ment in Ca-alginate beads has commonly been used forimmobilization of lactic acid bacteria.4-9 On the other hand,encapsulation of cells in a liquid-core capsule which offersmore space for cellular growth than entrapment will be agood method for a high density culture. In the previouswork of our group, encapsulation of yeast cells in a Ca-alginate capsule based on Nigam et al. l1 was developed;however, Ca-alginate gels are chemically unstable on con-

Address reprint requests to Dr. Ho Nam Chang, Director, Bioprocess En-gineering Research Center, Korea Advanced Inst. of Science and Technol-ogy, Daeduk Science Town, Taejon 305-701, South KoreaReceived 29 June 1995; revised 27 November 1995; accepted 11 January1996Enzyme and Microbial Technology 18: 0 1996 by Elsevier Science Inc.655 Avenue of the Americas, New York, NY 10010

tact with various cation-chelating agents such as phosphate,citrate, and lactate which can cause bead disruption or dis-solution.* Many hardening techniques have been investi-gated to improve the stability of Ca-alginate gels9,* butmost procedures are complicated, time consuming, and/orcostly. Recently, it was reported that barium alginate beadswere chemically and physically more stable in electrolytesolutions than conventional calcium alginate beads.13,14

The aim of this investigation was to compare the methodof entrapment and encapsulation of Lactobacillus casei cellsin Ca-alginate gels. Also, different metal ions other thancalcium and various coating agents were tested to improvethe stability of alginate gel in a lactate solution. Finally, theproduction of lactic acid with chemically stabilized alginatecapsules containing L. casei cells was studied.Materials and methodsMaterialsSodium alginate (Na-alginate) was obtained from Junsei (Tokyo,Japan, Cat. No. 13035-1201). Xanthan gum (Cat. No. G1253),chitosan (Cat. No. C3646), and polyethyleneimine (PEI, Cat. No.P3143) were supplied by Sigma (St. Louis, MO, U.S.). Yeastextract and MRS medium were from Difco (Detroit, MI, U.S.). Allother chemicals were of reagent grade quality.

Enzyme and Microbial Technology 19:426-433, 19960 1996 by Elsevier Science Inc.655 Avenue of the Americas, New York, NY 100100141-0229/96/ 15.00

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Encapsulation of Lactabacillus casei cells in liquid-core: I.-K. Yoo et al.~~crooorga~ism and substrateL. cusei ssp. r~~nosus (ATCC 10863) was used throughout thisinvestigation, The strain was maintained at 4C on MRS-agar. Theproduction medium consisted of 40 ml MRS broth or GY mediumcontaining per liter: 1 g Na-acetate *3H,O; 0.5 g K,HPO,; 0.5 gK&PO,; 0.2 g MgSO, - 7H,O; 0.03 g MnSO, - H,O; and 0.03 gFeSO, *7H,O. Glucose, yeast extract, and CaCo, concentrationswere variable.Immobilization of cellsEncapsulation Cells of L. casei for encapsulation were obtainedfrom culture grown in 50 ml of MRS broth at 42C for 24 h. Thecells were harvested from the fe~entation broth by cen~~gationat 10,000 g for 10 min and washed thoroughly with 0.85% saline.The cells were resuspended in 10 ml of a 1.0% (w/v) sterile CaCl,or BaCl, solution containing 0.15% (w/v) xanthan gum. This cellsuspension with a density of about 0.4 g dry wt. 1-l was droppedthrough a 22G bluntly ended needle into 130 ml of sterile 0.6%(w/v) Na-alginate and 0.1% (v/v) Tween 20 solution stirred by amagnetic bar. Xanthan gum and Tween 20 were used for makingclean spherical capsules. The schematic diagram of the experimen-tal apparatus was the same as that described in our previouswork. The capsules formed were washed with sterile saline toremove excess sodium alginate. The capsules were resuspended invarious coating and hardening solutions stirred with a magnetic barfor 30 min. A 1.0% CaCl, solution was used for hardening ofCa-alginate capsules. The chitosan solution for the Ba-alginatecapsule coating was prepared as described by Yoshioka et a1.l5The coating solutions were discarded and the resulting alginatecapsules (3-3.5 mm diameter) were washed with sterile saline.Entrapment Harvested cells (0.04 g dry weight) were mixed with100 ml of a 1 O% sterile sodium alginate solution. The mixture wasadded dropwise into a sterile 1.0% CaCl, solution at room tem-perature while stirring it continuously. The beads were hardened inthis solution for 1 h. The beads (2-2.5 mm diameter) were thenwashed with sterile saline prior to use to remove excess calciumions and untrapped cells.Fe~e~tation conditionsA 50 ml sample of immobilized cells prepared above was firstgrown in 150 ml MRS broth at 42C for 24 h. Batch fermentationswere then performed in 250-ml Erlenmeyer flasks containing 40ml of medium and 15-40 ml of alginate beads or capsules withimmobilized cells. For the cultures grown in GY medium, 2.5-5 gof CaCO, was added to the flasks to prevent significant decreasein pH by produced lactic acid. The flasks were incubated at 42Cin a shaking incubator (Vision Scientific Co., Seoul, Korea) ad-justed to 100 pm. After each batch fermentation, beads or cap-sules were washed two times with fresh medium for the next batchculture.Analytical rnet~~dsFree cell concentrations were determined by measuring the opticaldensity (OD) of the fermentation broth at 620 mn using a standardcurve to correlate OD and dry cell weight. For the cultures grownin GY medium, the optical density was measured after removingCaCO, particles with 1 HCl. The cell density within Ca-alginatebeads or capsules was determined after dissolving the beads orcapsules in McIkaine buffer (0.2 M Na POd, 0.1 citric acid).The number of viable cells liberated from the Ca-alginate gels wasobtained by plating appropriate dilutions on MRS agar and incu-

bating them at 42C for 36 h. In the case of Ba-alginate capsules,capsules were placed in a 0.5 EDTA solution with continuousstirring and then broken by pinching, since the McIlvaine bufferwas not effective for liquefying Ba-alginate capsules. After cellswere completely released from the ruptured capsules, cell densityinside the capsules was determined by measuring the optical den-sity at 620 nm. The lactic acid concentration was measured byHPLC using an Aminex HPX-87H column (Bio-Rad Co., Rich-mond, VA, U.S.) and a refractive index detector (Hitachi L-6000.Tokyo, Japan). Glucose concentration was determined by enzy-matic assay kits of Glucose-E kits (Youngdong Pharm. Co.. Seoul.Korea).Results are the average of at least three independent trials.Statistical analyses on the data were carried out by using Duncansnew multiple range test.

Results and discussionComparison of encapsulation and entrapment forlactic acid productionLactic acid production by encapsulated cells was comparedwith that by entrapped cells using Ca-alginate gels. Figure1 shows the lactic acid production during the second andthird batch of fermentations in 40 ml of MRS broth con-taining 0.5 g 1-l CaCI, and 15 ml of alginate beads orcapsules with immobilized cells. The addition of CaCl, wasdone to keep the capsules from swelling as reported in ourprevious work. As shown in Figure I, the Ca-alginatebeads showed a little higher but not statistically significant(P > 0.05) rate of lactic acid Production (1.9 1 g 1-l h-) thanthat by the capsules (1.75 gl- h-) during the first 7 h of thesecond batch; however, during the 20 h of the third batch,

00 5 10 15 20

Time h)Figure 1 Comparison of the lactic acid production by Ca-alginate entrapped and encapsulated L. casei cells during thesecond and third batch of fermentations: second batch with cap-sules, 0; third batch with capsules, 0; second batch with beads,

and third batch with beads, W. Bars indicate standard devia-tion from the mean on the basis of four replicate experiments

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Papersthe production rate by the beads (0.625 gl- h-l) signifi-cantly decreased (P < 0.05) compared with that by the cap-sules (0.81 g l-h-l). As reported earlier,1037 this could bedue to the limitation in increasing biomass per unit volumeof the conventional gel-core beads. Figure 2 shows the com-parison of the cell densities within both immobilized ma-trices during repeated batch fermentations in terms of drycell weight per liter of gels and the number of viable cellsper milliliter of gels. As can be seen, there was no increasein dry cell weight within beads during the third batch fer-mentation, while the number of viable cells remarkably de-creased compared with that after the second batch. The celldensity inside the Ca-alginate capsules after the third batchwas about 65 g dry wt 1-l gels, which was 1.5-fold higherthan that of the beads. Until the third batch, the free cellconcentrations were less than 0.15 g dry wt 1-l in bothimmobilized matrices. In the fourth batch, serious leakageof cells was observed in the beads and the free cell concen-tration was more than 0.8 g dry wt 1-l while significant cellleakage was not observed in the capsules yet (0.2 g dry wt1-l). The latter may be due to the barrier effect of the cap-sule membrane. In the gel-core beads formed by the con-ventional method, cells are homogeneously distributed bothnear the surface of and inside the beads at the stage of beadformation. During fermentation, the cells inside the beadsgrow poorer than near the surface of beads due to masstransfer limitation. The cells may then be released into thesurrounding medium once the matrix space near the surfaceof beads has been occupied. On the other hand, the inter-phasic membrane of the capsule can act as a barrier to retainmicrobial cells inside the capsule.

The fermentation using a GY medium containing 50 g 1-lglucose and 15 g 1-l yeast extract was carried out with 15 mlof Ca-alginate capsules. As shown in Figure 3a, the finalconcentrations of lactic acid produced in three repeated fer-mentations were almost the same even though the rate of

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0 1 2 3Repeated batch No.

Figure 2 Comparison of the cell density within Ca-alginatecapsules and beads during repeated batch fermentations: im-mobilized cells within capsules, 0; viable cells within capsules,0; immobilized cells within beads, Cl; and viable cells withinbeads, H

r^g 0.6Fuf 0.4=88 0.2

t 00

Time h)Figure 3 Time profiles of lactic acid production (a) and cellleakage (b) during repeated batch fermentations by Ca-alginate-encapsulated cells: (symbols are for graph a, graph b). Firstbatch (0, 0); second batch (Cl, ; and third batch (A, A). Thestandard deviation from the mean is based on three replicateexperiments

lactic acid production in the third batch was slightly slowerthan the previous ones. During fermentations with MRSmedium, the addition of 0.5 g 1-l CaCl, could prevent thecapsules from swelling and also cell leakage from the cap-sules to some extent. On the other hand, the free cell densityin GY medium containing 0.5 g 1-i CaCl, even after thethird batch significantly increased compared to that after thesecond batch (P < 0.05) as shown in Figure 3b; therefore, itcould be concluded that a higher concentration of lactic acidbrought about a decrease in the stability of Ca-alginatestructure.Enhancement of capsule stability in lactate solutionAlthough there have been numerous reports on the J;%;tion of lactic acid with Ca-alginate entrapped cells, the problem of cell leakage was not solved yet due to thedecalcification of Ca-alginate beads by lactic acid.6V8 Wefirst investigated the stability of alginate capsules gelledwith various metal ions besides Ca2+ in a high concentrationof phosphate and lactate solution. Among the tested metalions such as Ba*+, Fe3+, Mn2+, Zn+, and Ca*+, only the

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20

080; 5 10 15 20 25 30 ;

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Figure 5 Time courses of lactic acid production by chitosan-and BaCI,-coated Ba-alginate encapsulated ceils in media withdifferent initial glucose (a) and yeast extract (bi concentrations(g I-): G 40 and YE 30,O; G 70 and YE 30, Cl; GIOO and YE 30,A; 70 and YE 15, g; and G 70 and YE 5, 0 where glucose is Gand yeast extract is YEthe medium with 5, 15, and 30 g 1-l of yeast extract, re-spectively. Guoqiang et ~1.~ eported that the productivity oflactic acid by L. casei immobilized in Ca-alginate beadswas 1.6 g l-h-l in the medium with 10 g 1-l yeast extract.

Repeated batch fermentations were carried out five timessuccessively using 40 ml of Ba-alginate capsules with im-mobilized cells. As shown in Figure 6, the conversions ofglucose to lactic acid were in the similar range besides thefifth batch when the fermentation medium was replacedwith a fresh one every 24 h. Until the fourth batch, theproductivity of lactic acid during 24 h of fermentation wasmore than 2.7 g l-h-i and no residual glucose was found;however, in the fifth batch, the pr~uctivity was less than2.6 g I-h- and the concentration of residual glucose wasabout 2 g I-. Cell leakage from the capsules was main-tained relatively low during repeated fermentations; theconcentration of free cells after five batches was less than0.28 g dry wt 1-l. By contrast, the concentration of free cellsin the fermentation broth using Ca-alginate capsules with

03Yc7 20 ~t 4

i0.6 sL1 i?0.4 s

OIOO 1 2 3 4 5Repeated batch No.

Figure 6 Lactic acid production (0) and cells leakage (0) duringrepeated batch fermentations by ~hitosan-coated Ba-alginateencapsulated cells in GY media containing 70 g I- glucose and15 g I- yeast extract. Recycling took place every 24 h

immobilized cells was more than 0.55 g dry wt 1-i evenafter three batches as shown in Figure 3b. Boyaval andGoulet6 reported that serious cell leakage due to the decal-cification of Ca-alginate beads was observed during con-tinuous lactic acid fe~en~tion by L. ~e~vetic~~entrappedin Ca-alginate beads. Champagne et al9 found that a doublecoating of poly-r,-lysine and alginate reduced cell leakageby a factor of approximately 50 during milk fermentation byLuctococcus la immobilized in Ca-alginate beads.ConclusionThe entrapment in conventions Ca-alginate beads has beena popular method for immobilization of lactic acid bacteria;however, it seems that there are two disadvantages for lacticacid production using immobilized cells in Ca-alginatebeads. One is the limitation of space for cellular growth dueto gel-core structure and the other is an instability of Ca-alginate structure due to the decalcification of Ca-alginatebeads by lactic acid. In this study, the encapsulation methodin alginate capsules was used for immobilization of L,.caseicells. It was found that the encapsulation method gavehigher cell density and lactic acid productivity compared tothe entrapment. The stability of alginate capsules was alsoimproved using Ba2+ as the gelling agent for alginate in-stead of Ca*+ and a mixture of chitosan and BaCl, as thehardening solution for the preformed Ba-alginate capsules.References

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