Journal of Microencapsulation

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Microencapsulation of L. acidophilus (La-05) and B. lactis (Bb-12) and evaluation of their survival at the pH values of the stomach and in bile

C. S. Fávaro-Trindade a; C. R. F. Grosso a a Department of Food Technology; Department of Food Planning and Nutrition, Faculty of Food Engineering, State University of Campinas, UNICAMP, Campinas, São Paulo, Brazil. Online Publication Date: 01 July 2002 To cite this Article: Fávaro-Trindade, C. S. and Grosso, C. R. F. (2002) 'Microencapsulation of L. acidophilus (La-05) and B. lactis (Bb-12) and evaluation of their survival at the pH values of the stomach and in bile', Journal of Microencapsulation, 19:4, 485 - 494 To link to this article: DOI: 10.1080/02652040210140715 URL: http://dx.doi.org/10.1080/02652040210140715

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j. microencapsulation, 2002, vol. 19, no. 4, 485±494

Microencapsulation of L. acidophilus (La-05) and B. lactis (Bb-12) and evaluation of their survival at the pH values of the stomach and in bile C. S. FAÂVARO-TRINDADE and C. R. F. GROSSO* Department of Food Technology; Department of Food Planning and Nutrition, Faculty of Food Engineering, State University of Campinas, UNICAMP, Campinas, SaÄo Paulo, Brazil (Received 8 August 2001; accepted 6 November 2001 ) Microcapsules were prepared using the probiotic microorganisms Lactobacillus acidophilus (La-05) and Bi®dobacterium lactis (Bb-12) and the spray drying technique and cellulose acetate phthalate as the wall material. This study evaluated the resistance of these microorganisms to drying at three temperatures and also the in vitro tolerance of the free and microencapsulated form to pH values and bile concentrations similar to those found in the human stomach and intestine. With an air entry temperature of 130 °C and exit temperature of 75 °C, the number of viable cells of B. lactis was practically unaltered, whereas the population of L. acidophilus was reduced by two logarithmic cycles. B. lactis was more resistant to the drying process than L. acidophilus under all conditions tested. The morphology of the microcapsules was determined by scanning electron microscopy and the microcapsules presented a rounded external surface containing concavities, a continuous wall with no apparent porosity, average size of 22 mm, moisture content varying from 5.3 to 3.2% and water activity between 0.230 and 0.204. After inoculation into HCl solutions with pH values adjusted to 1 and 2, incubated anaerobically at 37 °C, and plated after 0, 1 and 2 h of incubation, microcapsules were eective in protecting the microorganisms, while the populations of both free microorganisms were eliminated after only 1 h at the acidic conditions. Microencapsulated B. lactis and L. acidophilus, both free and microencapsulated, were also resistant after 12 h to bile solutions. Keywords: Probiotics, Lactobacillus acidophilus, Bi®dobacterium lactis, microencapsulation, spray drying, cellulose acetate phthalate.

Introduction Probiotic cultures have been used in foods as an adjunct, due to the bene®cial eects attributed to them with respect to human health (Sanders 1999). However, in order to exert these bene®cial eects on the host, these organisms must remain viable in the food up to the moment of consumption in large amounts, and must be capable of surviving their passage through the gastrointestinal tract (Playne 1994). The survival of these cultures in foods is open to question and has given rise to much controversy, since some strains are extremely sensitive and intolerant of

* To whom correspondence should be addressed. e-mail: [email protected] Journal of Microencapsulation ISSN 0265±2048 print/ISSN 1464±5246 online # 2002 Taylor & Francis Ltd http://www.tandf.co.uk/journals DOI: 10.1080/02652040210140715


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C. S. Favaro-Trindade and C. R. F. Grosso

acids, oxygen, bile and the presence of other cultures. In addition, these cultures normally contribute little to the sensory quality of the product (Laroia and Martin 1990). The resistance to acid pH values to the presence of bile can vary considerably between dierent strains of the same species, both for Lactobacillus acidophilus and for Bi®dobacterium spp. (Lankaputhra and Shah 1995). Microencapsulation techniques and the immobilization of microorganisms in gels have been used eectively, with the objective of providing protection to the bacteria (Rao et al. 1989, Sheu and Marshall et al. 1993, Sheu et al. 1993, Champagne et al. 1994, Dinakar and Mistry et al. 1994, Amiet-Charpentier et al. 1998, Gobbetti et al. 1998, Khalil and Mansour et al. 1998). Microencapsulation is a process by which particles are formed containing an active ingredient covered by a layer of another material, which provides protection and controlled liberation as well as convenience to the ingredients. The composition of the wall material usually determines the functional properties of the microcapsules (Hegenbart 1993). Cellulose acetate phthalate acts as a barrier to acid pH values, being widely used as a wall material in capsules for the liberation of drugs in the intestine, since it is insoluble in acid media, but soluble at pH values equal to or greater than 6.0 (Malm et al. 1951). Despite this, there are few reports about the use of this polymer as a wall material in the preparation of microcapsules by spray drying. L. acidophilus and Bi®dobacterium lactis immobilized in calcium alginate were not more tolerant to pH 1 and 2 than the same microorganisms in free form (FÂavaro-Trindade and Grosso 2000). However, the microencapsulation of Bi®dobacterium pseudolongum in cellulose acetate phthalate, using coacervation with phase separation, provided a greater survival of this culture during incubation in gastric and intestinal juices (Rao et al. 1989). The objectives of this study were to microencapsulate the probiotic microorganisms L. acidophilus (La-05) and B. lactis (Bb-12) using cellulose acetate phthalate as a wall material by the spray drying technique, at three dierent drying temperatures, and to evaluate the tolerance of these microorganisms in the free and microencapsulated forms to acid pH values and bile concentrations similar to those found in the human stomach and intestine, respectively.

Materials and methods Materials Cellulose acetate phthalate (Eastman Kodak Co., Rochester, NY), maltodextrin (Corn Products International, Mogi GuacËu, Brazil), raftilose1(Orafti, Tienen, Belgium), tween 80 (Synth, SaÄo Paulo, Brazil) and glycerol (Synth, SaÄo Paulo, Brazil). Cultures Lactobacillus acidophilus (La-05), Bi®dobacterium lactis (Bb-12) (both Chr. Hansen, Valinhos, Brazil), in the DVS (direct vate set) form, pure and freeze dried, were maintained in the proportion of 1 g per 150 ml of a sterile solution of 12% reconstituted skimmed milk, at a temperature of ¡ 18 °C.

487


Microencapsulation of La-05 and Bb-12 Table 1.

Formulation used for the preparation of microcapsules of B. lactis and L. acidophilus.

Material

Function

Phosphate buer pH 8 Cellulose acetate phthalate Glycerol Maltodextrin Raftilose 1 Tween 80 Reconstituted milk

Quantity

Solvent Wall material Plasticizer Adjustment of density Fructoligossacharyde Surfactant Carrier of the microorganisms

100.0 10.0 3.5 2.0 1.0 0.1 10.0

(ml) (g) (g) (g) (g) (g) (ml)

Table 2. Operating conditions of the spray-dryer in the microencapsulation process. Parameters Temperature at entrance (°C) Mean temperature at exit (°C) 2 Air pressure (kgf/cm ) Flow rate of peristaltic pump (ml/min) Nozzle diameter (mm)

Conditions 130, 160, 190 75, 100, 120 5 5 0.5

Preparation of microcapsules The microcapsules were obtained by spray drying the formulation shown in table 1, in a spray dryer model SD 04 (Lab-Plant, Hudders®eld, UK), using the operating conditions shown in table 2. Each treatment was processed in triplicate, using collection ¯asks, tubing, spatulas and caps, all sterilized at 121 °C for 15 min. The formulation used was determined in preliminary tests.

Bacterial enumeration Reinforced Clostridial Agar (RCA) (Oxoid, Hampshire, UK) containing 0.01% aniline blue (Nuclear, SaÄo Paulo, Brazil) was used to enumerate B. lactis. L. acidophilus was enumerated in DeMan Rogosa and Sharp Agar (MRS). Both cultures were plated by the pour plate technique. A 2% solution of sodium citrate (Synth, SaÄo Paulo, Brazil) was used to prepare the serial dilutions. The plates were incubated in jars containing Anaerobac (Probac, SaÄo Paulo, Brazil) anaerobiosis generating systems, at 37 °C for 72 h. Plating was carried out in duplicate. To make it possible to quantify the numbers of viable cells in the microcapsules, it was necessary to dissolve the wall material to liberate the microorganisms. This was done by blending the samples with 2% sterile sodium citrate solution for 4 min at high speed, in a Stomacher 400 homogenizer (Seward, UK). The same procedure was used to prepare samples containing microorganisms in the free form.


488


Determination of the eect of drying temperature on the survival of L. acidophilus and B. lactis Counts were made in the spray dryer feed solutions and in the microcapsules minutes before and 1 h after processing, respectively, with the objective of determining the resistance of B. lactis and L. acidophilus at the dierent temperatures used in the process to obtain the microcapsules. Moisture content and water activity The moisture content in the samples with the microcapsules was determined in triplicate using the methodology of AOAC (1995). Water activity was determined in triplicate using the AQUALAB equipment (Decagon Devices, Pullman, WA). Particle size analysis The particle size and distribution was evaluated using the Lumosed PhotoSedimentometer, Anton Paar (model A-8054, Retsch, Haan, Germany), with isobutanol as a sedimentation medium (Synth, SaÄo Paulo, Brazil). Sample size was 30 mg and the analysis time was 40 min. The determinations were made in triplicate. Scanning electron microscopy The morphology of the microcapsules was observed by scanning electron microscopy. The encapsulated samples were ®xed in stubs on a double faced metallic tape and covered with a ®ne layer of gold using a Balzers evaporator (model SCD 050, Baltec, Liechtenstein, Austria) for 120 s, while applying a current of 40 mA. Observations were made using the scanning electron microscope (JEOL, JSM-T300, Tokyo, Japan) at an accelerating voltage of 10 kV (Rosenberg and Young 1993). Survival of bacteria at pH levels similar to those of the human stomach The determination of the eect of pH values similar to those found in the human stomach on free and microencapsulated (spray dried at 130/75 °C) cells of L. acidophilus and B. bi®dum was carried out in triplicate, according to Clark et al. (1993), using distilled water (pH 7.2) as control. Survival of bacteria in bile concentrations similar to those of the human intestine This test was carried out in triplicate using the methodology of Clark and Martin (1994) using Oxgall bile solutions (2 and 4%) (Difco, Detroit, USA) and peptone water as control. Statistical analysis A means dierence analysis was used to check for signi®cant dierences between the values obtained according to Tukey’s Test, with the help of the software STATISTICA 6.0 (Microsoft1 ).

489


Microencapsulation of La-05 and Bb-12 Results and discussion Eect of drying temperature on the survival of L. acidophilus and B. lactis

During vacuum drying and freeze drying processes, some cells undergo injury, others die and some are unaected by the process; injury and death probably being caused by a loss of protein from the cell wall as well as losses of bound water, both of which are extremely important for the maintenance of the structural and functional integrity of biological macromolecules (Brennan et al. 1986). The lower the entrance temperature, the greater the survival of spray dried microencapsulated B. lactis and L. acidophilus, as can be seen in table 3. This same eect was also observed for L. acidophilus (Espina and Packard 1979), for Salmonella (Licari and Potter 1970) and for L. bulgaricus (Teixeira et al. 1995a). The lowest entrance/exit temperature was the least lethal for the microorganisms, the population of B. lactis, showing practically no alteration, going from 1:8 £ 108 cfu/g at the start of the process to 1:3 £ 108 cfu/g at the end. This result was similar to that obtained for L. bulgaricus at an entrance temperature of 200 °C and exit temperature of 80 °C (Teixeira et al. 1995b). The population of L. acidophilus suered a reduction of two logarithmic cycles when the drying temperatures used were 130/75 °C. A similar result was obtained by Espina and Packard (1979), who reported a loss of at least two logarithmic cycles when they dried L. acidophilus in a spray dryer, using an entry temperature of 170 °C and exit temperatures of 75, 80 and 85 °C. B. lactis was shown to be more resistant to the process than L. acidophilus in all the treatments tested, although at the highest drying temperature (190 °C/120 °C, entrance/exit) its population was drastically reduced by three logarithmic cycles. Moisture content and water activity also decreased with increasing drying temperature. The values determined for moisture content and water activity were, respectively, 5.3% and 0.230; 3.8% and 0.226 and 3.2% and 0.204 for the temperatures of 130, 160 and 190 °C. Morphology and size distribution The morphological analysis of the microcapsules, carried out soon after the microencapsulation procedure showed that they had a rounded external surface containing concavities and indentations as a result of the drying process was smooth walled, continuous, without apparent porosity and having a variety of sizes (®gure 1). Table 3. Survival of L. acidophilus and B. lactis during the spray drying process. Drying temperature (°C) Microorganism B. lactis L. acidophilus

Entrance 130 160 190 130 160 190

Mean exit 75 100 120 75 100 120

(cfu/g of solids) Before the process 8

1:8 £ 10 8 1:0 £ 10 8 3:5 £ 10 8 1:2 £ 10 8 5:7 £ 10 8 1:2 £ 10

After the process 8

1:3 £ 10 7 2:5 £ 10 5 7:9 £ 10 6 2:3 £ 10 6 1:5 £ 10 4 1:9 £ 10


490


Figure 1. Micrograph of the microcapsules containing L. acidophilus encapsulated in cellulose acetate phthalate (magni®cation £3000).

Figure 2.

Eect of pH on the survival of free L. acidophilus. & control; & pH 2; & pH 1.

The average size of the particles was 22 mm, with a variation from 5±50 mm. Drying temperature showed no signi®cant eect (p < 0:05) on mean particle size, which were 20.0; 23.1 and 22.6 mm (SD = 2.1, 1.2 and 0.8 mm) at temperatures of 130, 160 and 190 °C, respectively. Survival of bacteria at pH values similar to those of the human stomach As reported previously (FaÂvaro-Trindade and Grosso 2000), free B. lactis and L. acidophilus underwent a reduction of one logarithmic cycle after inoculation at pH 2, and were completely destroyed after 1 h at pH 1, suggesting that the therapeutic bene®ts oered by these microorganisms would be somewhat limited if the stomach pH of the host were near to 1 (®gures 2 and 3). The immobilization of B. lactis and L. acidophilus in calcium alginate gel was not eective in protecting the cells submitted to pH values similar to those of the stomach (FaÂvaro-Trindade and Grosso 2000). It was shown to be eective in protecting B. bi®dum and B. infantis inoculated into mayonnaise (Khalil and Mansour 1998) and Lactobacillus bulgaricus added to frozen dessert (Sheu et al. 1993) and of L. bulgaricus added to ice-cream (Sheu and Marshall 1993).


Microencapsulation of La-05 and Bb-12

Figure 3.

491

Eect of pH on the survival of free L. lactis. & control; & pH 2; & pH 1.

Figure 4. Eect of pH on the survival of microencapsulated L. acidophilus. & control; & pH 2; & pH 1.

Spray-dried microcapsules of cellulose acetate phthalate containing B. lactis and L. acidophilus were eective in protecting both these microorganisms when inoculated into pH values similar to those at the human stomach (®gures 4 and 5). Microencapsulated L. acidophilus suered a reduction of only one logarithmic cycle at pH 1 after 2 h of incubation, and the population of B. lactis was reduced by only one logarithmic cycle immediately after inoculation into pH 1, and between 1±2(h) after inoculation into pH 2. Similar results were obtained by Rao et al. (1989), where B. pseudolongum, microencapsulated in cellulose acetate phthalate by coacervation, survived in greater numbers in the tests with gastric type ¯uids than the same microorganism in the free form. The survival of microencapsulated L. acidophilus and B. lactis in bile concentrations similar to those of the human intestine The results presented in tables 4 and 5, referring to the survival of free L. acidophilus and B. lactis in the presence of bile, con®rm the data reported in a previous paper (FaÂvaro-Trindade and Grosso 2000), where free L. acidophilus and B. lactis were shown to be extremely tolerant to bile, even at 4%, which is considered to be greater than the concentration normally observed in the human


492


Figure 5.

Eect of pH on the survival of microencapsulated B. lactis. & control; & pH 2; & pH 1.

Table 4. Survival of free and microencapsulated B. lactis 12 h after inoculation into bile (cfu/ml). a;b

Bile concentration (%) Condition/time (h) Free/0 Free/12 Microencapsulated/0 Microencapsulated /12

0 (control) 8

3:1 £ 10 8 5:6 £ 10 8 2:1 £ 10 8 4:3 £ 10

2

4 8

8

7:9 £ 10 8 2:5 £ 10 8 9:1 £ 10 8 3:7 £ 10

6:3 £ 10 8 4:7 £ 10 8 3:0 £ 10 8 2:4 £ 10

a

Means with the same letter in the same line are not signi®cantly dierent (p < 0:05). Means with the same letter in the same column are not signi®cantly dierent (p < 0:05). b

Table 5.

Survival of free and microencapsulated B. acidophilus 12 h after inoculation into bile (cfu/ml). a;b

Bile concentration (%) Condition/time (h) Free/0 Free/12 Microencapsulated/0 Microencapsulated /12

0 (control) 6

3:2 £ 10 6 6:7 £ 10 6 7:7 £ 10 6 2:5 £ 10

2

4 6

3:4 £ 10 6 5:4 £ 10 6 2:1 £ 10 6 3:3 £ 10

6

3:2 £ 10 6 4:9 £ 10 6 1:7 £ 10 6 3:9 £ 10

a

Means with the same letter in the same line are not signi®cantly dierent (p < 0:05). Means with the same letter in the same column are not signi®cantly dierent (p < 0:05). b

intestine. These results are in agreement with those of Holcomb et al. (1991), but dier from those reported by Clark and Martin (1994), where the population of B. bi®dum was completely destroyed after 12 h incubation in 2% bile or above. The spray-drying process did not alter the tolerance of B. Lactis and L. acidophilus to the presence of bile, which diers from the results reported by Brennan et al. (1986), in which the drying of L. acidophilus under vacuum and by



493

freeze drying resulted in intolerance to bile, an eect attributed to losses of speci®c components from the cell membrane during the drying processes. After inoculation of the microcapsules into the bile solutions (pH 7), a complete dissolution of the powder was observed, indicating that the wall material and the process used in the preparation of the microcapsules were both adequate with respect to the objectives of the microencapsulated microorganisms passing undamaged through the acid conditions of the stomach, followed by their rapid liberation in the pH of the intestine. Conclusions The survival of spray-dried microencapsulated B. lactis and L. acidophilus was shown to be dependent on the drying temperature used, and lower temperatures resulted in higher survival rates. Microencapsulation by spray-drying using the enteric polymer, cellulose acetate phthalate, as wall material, was shown to be a convenient process to obtain microcapsules, especially when using B. lactis. B. lactis and L. acidophilus, microencapsulated in cellulose acetate phthalate show a good potential for use as dietary aids, since they are resistant to acid and bile solutions and were liberated in conditions similar to those of the intestine. Acknowledgements The authors are grateful for the ®nancial support received from CNPq. The microorganisms and the raftilose1were kindly provided by Chr. Hansen, Brazil and Orafti, Belgium, respectively. References Amiet-Charpentier, C., Gadille, P., Digat, B., and Benoit, J. P., 1998, Microencapsulation of rhizobacteria by spray-drying: formulation and survival studies. Journal of Microencapsulation , 15, 639±659. AOAC, 1995, Ocial Methods of Analysis of AOAC International, 16th edn (Arlington: AOAC), v.2, chap 33, p. 49. Brennan, M., Wanismail, B., Johnson, M. C., and Bibek, R., 1986, Cellular damage in dried Lactobacillus acidophilus. Journal of Food Protection, 49, 47±53. Champagne, C. P., Gardner, N., and Dugal, F., 1994, Increasing of immobilized Lactococcus lactis cultures stored at 4 °C. Journal of Industrial Microbiology, 13, 367± 371. Clark, P. A., and Martin, J. H., 1994, Selection of Bi®dobacteria for use as dietary adjuncts in cultured dairy foods: III Tolerance to simulated bile concentrations of human small intestines. Cultured Dairy Products Journal, 29, 18±21. Clark, P. A., Cotton, L. N. , and Martin. J. H., 1993, Selection of Bi®dobacteria for use as dietary adjunts in cultured dairy foods: II Tolerance to simulated pH of human stomachs. Cultured Dairy Products Journal, 28, 11±14. Dinakar, P., and Mistry, V. V., 1994, Growth and viability of Bi®dobacterium bi®dum in cheddar cheese. Journal of Dairy Science, 77, 2854±2864. Espina, F., and Packard, V. S., 1979, Survival of Lactobacillus acidophilus in a spray-drying process. Journal of Food Protection, 42, 149±152. FAÂvaro Trindade, C. S., and Grosso, C. R. F., 2000, The eect of the immobilization of Lactobacillus acidophilus and Bi®dobacterium lactis in alginate on their tolerance to gastro-intestinal secretions. Milchwissenschaft, 55, 496±499.


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