ARTICLE IN PRESS

International Dairy Journal 14 (2004) 505515

Encapsulation of bidobacteria in whey protein-based microcapsules and survival in simulated gastrointestinal conditions and in yoghurt
Arnaud Picota, Christophe Lacroixb,*
b a ! Laval, Que !bec, PQ, Canada G1K 7P4 Dairy Research Centre STELA, Pavillon Paul Comtois, Universite Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology, ETH Zentrum, LFO F18, CH-8092 Zurich, Switzerland

Received 14 April 2003; accepted 20 October 2003

Abstract Bidobacterium breve R070 (BB R070) and Bidobacterium longum R023 (BL R023) were encapsulated as freeze-dried or fresh cultures in water-insoluble food-grade microcapsules produced by emulsion and/or spray-drying, using milk fat and/or denatured whey proteins as immobilization material. The encapsulation yield differed signicantly according to the method and the strain used. Dispersion of fresh cells in heat-treated whey protein suspension followed by spray-drying was the least destructive immobilization technique tested, with a survival rate of 25.770.1% and 1.470.2% for BB R070 and BL R023, respectively. Viable counts of BB R070 cells entrapped in whey protein microcapsules using this method were signicantly higher than those of free cells after 28 days in yoghurt stored at 4 C (+2.6 log cycles), and after sequential exposure to simulated gastric and intestinal juices (+2.7 log cycles). In contrast, no protective effect of encapsulation was observed with BL R023. Immobilization of probiotic cultures in whey proteinbased microcapsules can increase cell survival when subjected to extreme conditions, making this approach potentially useful for delivery of viable bacteria to the gastrointestinal tract of humans via dairy fermented products. However, technological properties of the strains, and particularly heat resistance, should be taken into consideration for spray-dry encapsulation of sensitive microorganisms. r 2003 Elsevier Ltd. All rights reserved.
Keywords: Immobilization; Whey proteins; Bidobacteria; Gastrointestinal conditions; Yoghurt

1. Introduction Probiotics, dened as living microorganisms which, upon ingestion in certain numbers, exert health benets beyond inherent general nutrition (Guarner & Schaafsma, 1998) have become increasingly popular during the last decade. Yoghurt or yoghurt-like products have been used as the most popular vehicle for incorporation of probiotic organisms, mainly bidobacteria and lactobacilli (Sanders, 1998). It has been recommended that foods containing such bacteria should contain at least 106 live microorganisms per g or mL of product at the time of consumption, in order to produce therapeutic benets (Shah, 2000). Despite the importance of this essential prerequisite, several surveys have shown large
*Corresponding author. Tel.: +41-1-632-4867; fax: +41-1-6321403. E-mail address: christophe.lacroix@ilw.agrl.ethz.ch (C. Lacroix). 0958-6946/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2003.10.008

uctuations and poor viability of probiotic bacteria, and especially bidobacteria, in yoghurt preparations (Shah, Lankaputhra, Britz, & Kyle, 1995; Schillinger, 1999). Several factors have been claimed to affect the viability of Bidobacterium cultures in fermented milk products, including acidity (Klaver, Kingma, & Weerkamp, 1993), pH (Martin & Chou, 1992), concentration of lactic and acetic acids (Samona & Robinson, 1994), hydrogen peroxide (Lankaputhra, Shah, & Britz, 1996), and dissolved oxygen content (Dave & Shah, 1997). Moreover, because viable and biologically active microorganisms are usually required at the target site in the host, it is essential that the probiotic be able to withstand the hosts natural barriers against ingested bacteria. Several studies have shown that many strains of Bidobacterium ssp. intrinsically lack the ability to survive the harsh conditions of acidity and bile concentration commonly encountered in the

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gastro-intestinal tract of humans (Berrada, Lemeland, Laroche, Thouvenot, & Piaia, 1991; Lankaputhra & Shah, 1995). Different approaches that increase resistance of these sensitive bacteria against adverse conditions have been proposed, including appropriate selection of acid and bile resistant strains, use of oxygen-impermeable containers, two-step fermentation, stress adaptation, microencapsulation, incorporation of micro-nutrients such as peptides and amino acids, and sonication of yoghurt bacteria (Shah, 2000). Microencapsulation has previously been reported as a technology that can provide protection to these sensitive cultures from high oxygen levels (Sunohara, Ohno, Shibata, & Seki, 1995), manufacture and storage of yoghurt (Adhikari, Mustapha, Grun, . & Fernando, 2000), freezing (Shah & Ravula, 2000), and during transit through the human gastro-intestinal tract (Wenrong & Grifths, 2000). In almost all cases, gel entrapment using natural biopolymers such as calcium alginate, k-carrageenan and gellan gum has been favoured by researchers. However, although promising on a laboratory scale, the technologies developed to produce gel beads present serious difculties for large-scale production (Poncelet & Neufeld, 1996; Picot & Lacroix, 2003a) such as low production capacity and large bead diameters for the droplet extrusion methods and transfer from organic solvents and large-size dispersion for the emulsion techniques. Moreover, the addition of these polysaccharides is not permitted in yogurts or fermented milk in some European countries. We recently reported the preparation of multiphase water-insoluble food-grade microcapsules designed for cell microencapsulation, using emulsication and spraydrying (Picot & Lacroix, 2003a). Soluble whey protein polymers are used as coating material and form upon rehydration a stabilizing lm around milk fat globules containing micronized powder of freeze-dried bacteria. This two-step continuous technology, which was developed using micronized skim milk powder as model powder for freeze-dried cultures, can generate large quantities of food-grade material at low cost. However, cell survival during the process and its ability to protect the probiotic organisms against adverse conditions in food products and during digestion have not yet been investigated. The aim of the present work therefore was to study the effect of encapsulation in spray-dried whey proteinbased microcapsules on the survival of two strains of bidobacteria, Bidobacterium breve R070 and Bidobacterium longum R023. The effects of different conditions of the microencapsulation technology, i.e. the use of fresh or freeze-dried cultures dispersed in milk fat or that of fresh culture directly added in heat-denatured whey protein polymer dispersion as immobilization matrix, on cell viability after spray-drying were exam-

ined. Finally, viability of bidobacteria encapsulated by the technique that lead to the highest survival rate was evaluated during storage in yoghurt at 4 C and under conditions similar to those encountered in the human gastrointestinal tract.

2. Materials and methods 2.1. Ingredients Whey protein isolate (WPI) containing 97.5% (w/w) protein was obtained from Davisco International (Le Sueur, MN, USA) and used as the immobilizing (method A) or coating (method B and C) agent. Anhydrous milk fat (AMF) containing 99.9% (w/w) lipids was obtained from Ault Foods (Mitchell, ON, Canada) and used as the immobilization matrix in methods B and C. 2.2. Cultures and culture preparations Pure freeze-dried cultures of Bidobacterium breve R070 (BB R070) and Bidobacterium longum R023 (BL R023), with viable cell counts of 3 109 colony forming units (cfu) g1, were kindly provided by the Rosell Institute Inc. (Montreal, QC, Canada). Fresh cultures were obtained after activation by two successive transfers in MRS broth (Lactobacillus broth according to de Man, Rogosa, & Sharpe, 1960) (Rosell Institute Inc., Montreal, QC, Canada) supplemented with 0.05% (w/v) l -cysteine-HCl (MRS-C) under anaerobic conditions. Cultures in late-log phase (2 109 cfu g1) were harvested by centrifugation at 7700 rpm for 10 min at 4 C (Sorvall centrifuge, model RC-5C, rotor GS-3, Newtown, CT, USA), washed twice in sterile 0.9% saline under the same centrifugation conditions, and used in the microencapsulation process. Particle-size reduction (micronization) of powder of freeze-dried bacteria was performed with a spiral jet mill 50 AS (Hosokawa Alpine AG, Augsburg, Germany), using the optimum operating conditions previously determined (Picot & Lacroix, 2003b). The grinding air pressure was set at 4 bar and the product feed rate at 300 g h1, in order to produce micronized powders with a D(v, 0.9) o25 mm. 2.3. Cell microencapsulation Microcapsules were prepared using three different methods, as represented in Fig. 1. Cells were incorporated as fresh culture (methods A and B) or as micronized powder of freeze-dried culture (method C). A late-log phase culture was prepared in 200 mL and 9 L volume for methods A and B, respectively, and the cells were centrifuged and washed as described earlier. The

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METHOD A

METHOD B

METHOD C Micronized powder of freeze-dried culture

Fresh culture

Milk fat (402C)

Heat-denatured 10% w/w WPI solution (402C)

O/W EMULSION (1L IMT Dynamic Loopmixer)

SPRAY-DRYING (co-current two-fluid nozzle Ti=160C - To=80C)

ENCAPSULATED CULTURE POWDER

Fig. 1. Schematic representation of the three encapsulation techniques tested. o/w: oil/water; IMT: International Mixing Technology (Dunkerque, France); Ti : inlet air temperature and To : outlet air temperature.

pellets were resuspended directly in 1000 mL of a 10% (w/w) suspension of whey protein polymers (method A) or in 1500 g of the hydrophobic phase using a Caframo stirrer (Model RZR 50, Caframo Ltd., Wiarton, ON, Canada) at the lowest speed for 1015 min (method B) at 4072 C before spray-drying. These conditions were used to obtain a nal theoretical cell concentration of 5 109 cfu g1 of microcapsules assuming that no viability was lost during the microencapsulation process. For method C, 75 g of micronized powder of freezedried culture was dispersed into 1425 g of the hydrophobic phase using a Caframo stirrer at the lowest speed for 1015 min and kept at 4072 C with stirring before the emulsication step, for a nal theoretical cell concentration of 5 107 cfu g1. Preparation of soluble whey protein polymers, emulsication, and spray-drying were carried out according to the procedures described previously by Picot and Lacroix (2003a). Whey protein solution containing 10% (w/w) solids was prepared in distilled water and kept overnight at 4 C. The solution was adjusted to pH 7.070.1, heat-treated at 80 C for 30 min under agitation in a Groen steam-jacketed cooker/mixer (Model DTA/3, Groen, Jackson, MS, USA), and cooled to 4072 C. The suspension of whey protein polymers was used as resuspending medium for washed fresh cells (method A), or as continuous phase in oil/water (o/w) emulsion (methods B and C). Emulsication was carried out as follows. AMF was preheated at 60 C in a water bath to dissolve all crystals,

and cooled to 4072 C. The probiotic culture, fresh (method B) or freeze-dried (method C), was dispersed into the hydrophobic phase using a Caframo stirrer (Model RZR 50, Caframo Ltd., Wiarton, ON, Canada) at the lowest speed for 1015 min and kept at 4072 C with stirring. The resulting suspension and the heatdenatured whey protein solution were then continuously pumped in a 1L IMT dynamic loop mixer (International Mixing Technology, Dunkerque, France) using a peristaltic pump (Model 503S, Watson-Marlow, Flamouth, England) and a progressive cavity pump (Seepex MD 006, Seepex Inc., Enon, OH, USA), respectively. In agreement with the optimum emulsion conditions previously determined for the production of fat globules ranging from 10 to 50 mm (Picot & Lacroix, 2003a), a 95/5 (w/w) hydrophilic/hydrophobic phase ratio was used and the internal mixing speed was set at 2500 rpm. The total ow rate entering the vessel was set at a low value of 30 kg h1 for the mixer. Once the equilibrium was reached in the dynamic loop mixer, i.e. 10 min after emulsication was started, the exit of the mixer was connected to the feed pump of the spray-dryer. Because the total ow rate entering the mixer was 30 kg h1 and the evaporation rate of the spray-dryer was around 1 kg h1, only part of the emulsion could be processed continuously, the remaining being discarded using a bypass pipe. The suspension of whey protein polymers containing fresh culture (method A) and the emulsions (methods B and C) were dried at an inlet air temperature of 160 C using a laboratory-scale spray-dryer (Minor Mobile portable spray-dryer, Niro Atomizer, Copenhagen, Denmark) equipped with a co-current two-uid nozzle atomizer. Pressure air and air valve of the nozzle were adjusted in order to get a ne and homogeneous spray with a pulverization angle of 30 . The outlet air temperature was stabilized at 80 C by adjusting the feed rate with distilled water. When thermal equilibrium in the drying chamber was attained, the atomizers feed was switched from water to the cell-containing suspension (heat-denatured whey protein solution or emulsion). The outlet temperature was continuously maintained at 80 C by controlling the ow rate. In method A, the cell-containing suspension was kept at 4072 C with stirring during operations. In agreement with the thermotolerance characteristics of the two Bidobacterium strains (Picot & Lacroix, 2003b), the powder particles were collected every 5 min at the bottom of the dryers cyclone and immediately stored at 4 C in airtight glass jars before bacteriological analysis and stability tests. For each cell-immobilization method used, two batches of encapsulated cultures were produced for both probiotic strains. Viable counts of bidobacteria were determined in the feed suspensions before spray-drying and in the resulting powders. Encapsulation yield (EY), i.e. survival rate to

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the immobilization process, was calculated as follows: EY N =N0 100; where N0 is the number of viable bacteria (colonyforming units) per gram of dry matter before drying and N is the number of viable bacteria (colony-forming units) per gram of dry matter in the powder. 2.4. Yoghurt preparation Skim milk powder and distilled water were blended for a total solids content of 15% (w/w). The resulting mix was sterilized at 121 C for 10 min and cooled to 4271 C before inoculation with a commercial freezedried starter culture containing Streptococcus salivarius ssp. thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, and Lactobacillus acidophilus (yogourmets, Lyo-san, Lachute, QC, Canada). The parent mix was divided into three portions. One portion was used as a control (without bidobacteria), and encapsulated and nonencapsulated bidobacteria were added separately to the other two portions. Encapsulated bacteria were produced with method A which gave the highest survival rates. The amount of BB R070 and BL R023 microcapsules added to milk was 0.300.35 g and 6.07.0 g 100 mL1 to give an initial cell concentration of 5 to 6 106 cfu mL1 and 6 to 7 107 cfu mL1, respectively. The different mixes (control, encapsulated, and nonencapsulated) were then dispensed into 120 mL screw-capped glass jars and incubated at 4271 C for 45 h until the pH reached 4.5. Samples were then stored at 4 C for storage studies. All treatments were made in triplicate. 2.5. Tolerance to simulated gastrointestinal conditions To evaluate the survival of encapsulated (method A) and nonencapsulated bidobacteria under conditions that mimic in vivo human upper gastrointestinal transit, an in vitro methodology was developed using a modied version of the method of Gauthier, Vachon, and Savoie (1986). The transit tolerance of the probiotic cultures was determined by exposing the microorganisms at 37 C to simulated gastric juice (pH 1.9) and simulated small intestinal juice (pH 7.5) successively, and monitoring changes in total viable counts in the digestion mixture periodically, without release treatment for encapsulated cells, except at inoculation. Simulated gastric juice was prepared by dispersing pepsin 1:60,000 (porcine gastric mucosa, P7012, Sigma-Aldrich, Oakville, ON, Canada) in 0.1 n HCl and adjusting the pH to 1.9 with 1 n NaOH in order to achieve a nal concentration in the digestion mixture of 0.26 g L1. Simulated pancreatic juice was prepared by dispersing pancreatin 5X (hog pancreas, ICN Nutritional Biochemicals, Cleveland,

OH, USA) in sterile sodium phosphate buffer (0.02 m , pH 7.5) and adjusting the pH to 7.5 with 1 n NaOH in order to achieve a nal concentration in the digestion mixture of 1.95 g L1. Concentrated bile salt solution (150 g L1) was prepared by dissolving a bile extract powder (bile bovine, B3883, Sigma-Aldrich, Oakville, ON, Canada) in distilled water. The resulting suspension was lter-sterilized. The simulated gastric juice, bile salt solution, and simulated pancreatic juice were prepared fresh daily. Fresh cells were obtained after activation and washing in the same way as described earlier, except that the washed cells were resuspensed in sterile 0.9% saline to approximately 1.5 109 cfu mL1. A volume (75 mL) of pepsin preparation (0.277 g L1) was transferred aseptically to a 125 mL Erlenmeyer ask and maintained at 37 C in a magnetically stirred water bath. The washed cell suspension (5 mL) was then added. After 30 min, the reaction was stopped by increasing the pH to 7.5 with 1 n NaOH. A sample of 2.0 mL was withdrawn and kept in ice before determination of viable cell numbers. Five mL of concentrated sodium phosphate buffer (0.5 m , pH 7.5) and 2.0 mL of the bile salt solution were then added. After quickly adjusting the pH to 7.5 and the volume to 90 mL with sterile distilled water, 10 mL of the simulated pancreatic juice were added for a nal volume of 100 mL. At different time intervals (1, 2, 3, and 6 h), 2.0 mL aliquots were removed for bacterial enumeration. The reaction was stopped by placing the samples in ice for 5 min. For encapsulated bidobacteria, 8 g of microcapsules were rehydrated in 50 mL of sterile 30 mm CaCl2 solution for 15 min at 4 C, in order to form the whey protein lm. The resulting dispersion was transferred aseptically to a 125-mL Erlenmeyer ask and incubated at 37 C in a magnetically stirred water bath. After quickly adjusting the pH to 1.9 with 1 n HCl and the volume to 60 mL with sterile distilled water, 20 mL of pepsin preparation (1.040 g L1) were added. The remainder of the procedure was then identical to that described for the nonencapsulated cells. Enumeration of bidobacteria from samples taken during simulated digestion with nonencapsulated and encapsulated cells was carried out by pour plate counts as described below. No dispersion step was performed to release encapsulated cells to estimate the release properties of the microcapsules at different steps of the test. The cell counts were corrected by taking into account the dilutions resulting from pH and volume adjustments of the digestion medium at the end of the gastric phase and before starting the intestinal phase. The bacterial count of the digestion medium at the start of the gastric phase was taken as the baseline and used to adjust all the other counts in order to analyze the changes in cell viability during digestion. All the digestion experiments were duplicated.

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2.6. Analyses 2.6.1. Bacterial enumeration BB R070 and BL R023 were enumerated on MRS-C agar using the pour plate method. The plates were incubated at 37 C for 48 h in anaerobic jars with the AnaeroGen system (Oxoid Ltd., Basingstoke, Hampshire, England). Cell counts were performed in duplicate and means are reported. For enumeration of washed cells dispersed in AMF (method B), 5 g of the microbial suspension was vortexed vigorously for 1 min with 10 mL of sterile 0.1% peptone buffered water and the resulting dispersion was left at 37 C to allow phase separation and cell rehydration in the aqueous phase (about 10 min). The upper phase (milk fat) was discarded and the viable cell number was determined in the lower aqueous phase. Viability of freeze-dried cultures (method C) was assayed prior to incorporation in AMF by dissolving 0.5 g of the micronized powder in 10 mL of sterile 0.1% peptone buffered water. For enumeration of encapsulated cells, 2 g of spraydried microcapsules were rehydrated in 50 mL of sterile 0.1% peptone buffered water and ground in a rollertype jar mill (US Stoneware, Akron, OH, USA) for 1 h at room temperature to release cells from microcapsules. A verication was done with freeze-dried cells added to AMF to ensure that the extraction method did not affect culture viability, and showed that cell recovery after the treatment was high, equal to 10676%. Modied MRS-C agar containing 0.2% lithium chloride and 0.3% sodium propionate was used for selective enumeration of Bidobacterium cultures in yoghurts. For enumeration of encapsulated cells, 2 g of yoghurt was suspended in 50 mL of sterile 0.1% peptone water and treated as described above. For enumeration of nonencapsulated cells, 2 g of yoghurt was diluted in 10 mL of sterile 0.1% peptone-buffered water and mixed uniformly with a vortex mixer at room temperature. Reported plate counts expressed as cfu per gram of yoghurt are means of triplicate analyses. 2.6.2. Moisture content and relative humidity The moisture content of the microcapsules was determined gravimetrically by oven-drying at 100 C over 24 h. Relative humidity was determined at 25 C using a Novasina water activity meter (model Humidat. fkon, Switzerland). Reported IC II, Novasina AG, Pfa data are means of ve replicates. 2.6.3. pH The pH was measured with an Orion 410A pH meter (Orion, Boston, MA, USA). After calibrating, the pH electrodes were sterilized in a concentrated sodium hypochlorite solution (10% w/w) and thoroughly rinsed

with sterile distilled water before use in yoghurts and digestion mixtures. 2.6.4. Microstructure Prior to microscopic observations, microcapsules were rehydrated in a 30 mm CaCl2 solution to form the whey protein lm. Microcapsules were then stained with uorescein isothiocyanate (uorescent protein dye) and nile red (uorescent lipid dye) for observations with confocal laser-scanning microscopy. The confocal laserscanning microscope (LSM 310, Carl Zeiss, Oberkochen, Germany) was equipped with two excitation sources, an Ar-ion laser (488 nm) and an He-Ne laser (543 nm), and with two photomultipliers (2 and 1) which selected emission signals from 515 to 565 nm and from 575 to 640 nm, respectively. 2.6.5. Statistical analysis All statistical analyses were performed using procedures for the general linear model (PROC GLM) of SAS (version 5, SAS Institute Inc., Cary, NC, USA). Signicant differences among treatment means were tested using the LSD multiple comparison test, with a probability level Po0:05:

3. Results and discussion 3.1. Moisture content and relative humidity of encapsulated cell powders The moisture content is of critical importance in dehydrated products, particularly in microencapsulated oil. The moisture content and water activity (Aw ) of the powders manufactured in this study varied from 1.3% to 2.3% and from 0.143 to 0.176, respectively (Table 1). Both parameters were not signicantly affected by the cell immobilization methods tested and the probiotic strain used. The results obtained are in agreement with the values previously reported in a similar system (Picot & Lacroix, 2003a) and usually recommended for good stability of dried cultures throughout storage (Champagne, Mondou, Raymond, & Roy, 1996). Low moisture content and water activity in encapsulated food powder contribute to improved physical and bulk properties (Onwulata, Smith, & Holsinger, 1995). 3.2. Encapsulation yield Spray-drying is one of the oldest and the most commonly used methods of encapsulation in the food industry. Compared with the other conventional microencapsulation techniques, it offers the attractive advantage of producing large quantities of microcapsules in a simple continuous processing operation. However, there are obvious challenges associated with using

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510 A. Picot, C. Lacroix / International Dairy Journal 14 (2004) 505515 Table 1 Encapsulation yield and nal cell concentration of Bidobacterium cultures in the microcapsule powder with residual humidity (RH) and Aw for the three microencapsulation methods tested Culture Bidobacterium breve R070 Methods EY (%) Log cfu g1 RH (%) Aw
ad

Bidobacterium longum R023 C

b

A 25.67 70.12 9.2370.23 1.9570.03 0.1670.00

a

B 10.58 70.68 8.5370.31 1.4070.27 0.1670.00

A
d

B
c

C
e

0.71 70.09 5.4370.05 1.2870.11 0.1470.02

1.44 70.16 7.9370.29 2.0570.17 0.1870.01

0.03 70.00 6.1170.22 2.0770.36 0.1670.06

0.03e70.00 4.6170.11 1.4270.31 0.1470.01

: Means with the same letter are not signicantly different (Po0:05).

spray-drying to dry or encapsulate sensitive probiotic bacteria such as Bidobacterium ssp., including the requirement that the cells tolerate the relatively high levels of shear and the drying conditions used for the process. One objective of the present study was to determine the most suitable way to incorporate probiotic cultures in spray-dried dairy-based microcapsules, in order to obtain powders containing high numbers of viable cells in a form suitable for applications in foods or nutraceutical supplements. Signicant differences for EY were measured for the two Bidobacterium strains and for the three encapsulation techniques used (Table 1). The highest survival rate for each method, in the range from 0.71% to 25.7%, was obtained with BB R070, and might be attributed to a greater thermal tolerance of this microorganism (Picot & Lacroix, 2003a). Dispersion of fresh cells in heattreated whey proteins followed by spray-drying was the least destructive procedure (method A). Using this approach, EY of 25.7% and 1.44%, with cell concentrations in the powder of 1.7 109 and 7 1 8.6 10 cfu g were obtained for BB R070 and BL R023, respectively. A large mortality occurred with method C (freeze-dried bacteria in milk fat) as illustrated by the very low EY (o 1%) and the 2 to 3 log reduction in population recorded for both cultures. Similarly, the use of milk fat as dispersing medium for fresh cells before emulsication and subsequent spraydrying (method B) led to survival rates signicantly lower than those obtained with method A. A decrease of 1 log in the cell concentration was observed for BB R070 (EY=10.6%) whereas EY of BL R023 was very low and identical to that of method C (EY=0.03%). Milk fat and other hydrophobic substances have previously been reported as potential cell immobilization matrices (Kim & Olson, 1985; Modler & VillaGarcia, 1993; Sunohara et al., 1995; Champagne, Raymond, Mondou, & Julien, 1995; Baba, Kubota, Horishita, & Matsunobu, 1996). Diffusion of H+, organic acids, water, and oxygen across membranes of lipid capsules is limited. This property could be particularly interesting for the preservation of viable probiotic bacteria during storage in fermented dairy

products. However, in our study, cell viability was largely affected and not satisfactory following microencapsulation in milk fat by spray-drying. Additionally, homogeneous dispersion of the microorganisms (cell paste and micronized powders of freeze-dried cultures) into the hydrophobic phase before emulsication was very difcult. Shear by atomizing air pressure, heating inside the atomizer, dehydration and thermal inactivation have previously been proposed as possible causes for explaining reduced cell viability after spray-drying (Kim & Bhowmik, 1990; Fu & Etzel, 1995). The extent of survival or destruction of lactic acid bacteria during the process depends essentially upon the temperature-time combinations encountered and upon the heat-resistance of bacterial species (To & Etzel, 1997a). Residual viability can be increased by lowering the outlet air temperature which is generally considered as the major processing parameter affecting number of surviving bacteria (Kim & Bhowmik, 1990). In our particular application, because of the small size of the spray-dryer employed and the relatively low total solids content (between 10 and 15%) of the cell dispersions, it was not possible to achieve complete and satisfactory drying of the feed suspensions at an outlet air temperature lower than 80 C. Losses in cell viability due to cell dehydration have previously been demonstrated for spray-dried starter cultures (Lievense & vant Riet, 1993a, b; Lievense, Verbeek, Noomen, & vant Riet, 1994). In contrast to freeze-drying, the cells experienced both thermal and dehydration inactivation simultaneously during spraydrying. In our study, the use of probiotic strains in a dried form (method C) was attempted to limit the extent of cellular injury and increase the ability of the organisms to withstand high temperatures employed in the process. According to To and Etzel (1997b), dehydration might protect cells from thermal inactivation by decreasing water activity and thus lowering the rate of inactivation reactions. However, as mentioned earlier, using micronized freeze-dried cultures instead of fresh cells resulted in a higher mortality after spray-drying. Methods B and C both required an

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emulsication step during which the wet and dry cultures suspended in AMF, respectively, were dispersed in soluble whey protein polymers. No loss of viability was measured following the emulsication step (data not shown), indicating a negligible effect of shear forces in the mixer on cell survival. A suitable size distribution for the different components of capsules constitutes an essential prerequisite when preparing microcapsules with small diameters. In our particular application, particle-size reduction of powders of freeze-dried cultures was required to allow an efcient coating of cells in the protective hydrophobic phase. We recently showed that micronization resulted in a dramatic decrease in the heat resistance of freezedried bidobacteria and lactobacilli (Picot & Lacroix, 2003b). Thus, the very low survival rates obtained with method C in the present study were probably directly related to a greater thermosensitivity of the two Bidobacterium strains following particle-size reduction of the freeze-dried cultures. 3.3. CLSM observations Figs. 2AC shows CLSM micrographs of microcapsules prepared with the three methods tested. Spherical microcapsules with a diameter ranging from 3 to 75 mm were observed for all samples. A central void characterized the microcapsules prepared with method A (Fig. 2A). For microcapsules produced with methods B and C (Figs. 2B and C), the use of CLSM enabled simultaneous localization of AMF (coloured in red) and whey proteins (coloured in green) using double labelling with specic uorophores for fat and proteins. Milk fat was organized in large droplets completely surrounded by a continuous water-insoluble lm of whey proteins, each of the encapsulated droplets forming an individualized particle. Probiotic bacteria (indicated with yellow arrows) were only observed in the protein matrix of the microcapsules produced by methods A (Fig. 2A) and B (Fig. 2B), suggesting a transfer of fresh cells from the fat to the protein phase for method B. The powder particles of micronized freeze-dried cultures could not be detected inside the fat globules of the microcapsules produced with method C (Fig. 2C), indicating a rehydration phenomenon similar to that of method B. Transfer of the micronized powder of non-fat solids from the hydrophobic to the aqueous phase has previously been observed during the development of the multiphase microencapsulation technology (Picot & Lacroix, 2003a). 3.4. Survival in yoghurt Method A with the highest viability was therefore selected to test the stability of encapsulated bidobacteria in yoghurt and simulated gastrointestinal condi-

Fig. 2. Confocal laser-scanning micrographs of whey protein-based microcapsules produced with method A (A), method B (B), and method C (C). Small dark rods indicated by yellow arrows represent the locations of the bacterial cells within the protein matrix.

tions. Survival of the two Bidobacterium cultures in microcapsules produced by method A was compared with that of nonencapsulated cells during manufacture and storage of yoghurt for 28 days at 4 C (Figs. 3A and B). No signicant differences

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Fig. 3. Changes in pH (n:starter culture; }:starter culture and encapsulated bidobacteria; &: starter culture and nonencapsulated bidobacteria) and viable cell counts (: detection limit of the method) of immobilized ( ) and free cells ( ) of B. breve R070 (A) and B. longum R023 (B) during manufacture and refrigerated storage of yoghurt. Error bars represent standard deviation.

(P > 0:05) in the pH prole and the incubation time to reach 4.5 were found among tested treatments. A gradual decrease in pH (post-acidication) to 3.94.0 for all treatments was observed throughout the storage period of 28 days. Viable counts of nonencapsulated bidobacteria increased slightly during yoghurt manufacture (0.20.3 log cycles). During refrigerated storage, the numbers of both bidobacteria dropped dramatically in yoghurts containing free cells, with a sharp decline of 3 log in population after 1 week at 4 C. From day 7 onwards, nonencapsulated BB R070 showed better survival than BL R023. This difference between the two strains is in agreement with previous ndings of Hussein and Kebary (1999) who noted that tolerance of bidobacteria to yogurt storage conditions was species-dependent. The viable number of free BB R070 cells decreased to about 103 cfu g1 after 28 days at 4 C, whereas BL R023 was not detected after 18 days.

As illustrated in Fig. 3A, survival of BB R070 during fermentation and storage of yoghurt was increased by encapsulation with a loss of viability limited to 2.5 compared with 5.1 log cycles after 4 weeks at 4 C. Higher survival rates during refrigerated storage in fermented dairy products have previously been reported for various Bidobacterium cultures entrapped in kcarrageenan (Dinakar & Mistry, 1994; Adhikari et al., 2000) and alginate (Hussein & Kebary, 1999) beads. Despite the protection provided by microencapsulation, cell mortality occurred at a high rate. Viable counts of encapsulated BB R070 decreased from 5 106 cfu mL1 to about 104 cfu g1 after 28 days at 4 C, which is unsatisfactory considering the recommended level of 106 cfu g1 of product necessary to achieve therapeutic benets. Immobilized probiotic cultures were both added at relatively low inoculation rates (0.55%, w/v) to avoid possible detrimental effects on sensorial and physical characteristics of yoghurt. It can be assumed that the use of higher inoculation rates and the production of microcapsules with a higher nal cell concentration will result in higher numbers of viable cells at the end of the shelf life of the product. In this regard, sensory analyses performed on yoghurts containing various levels of encapsulated bidobacteria are needed in order to determine the upper limit acceptable for consumers. In contrast to BB R070, immobilization in spraydried whey protein microcapsules did not improve the survival of BL R023 during refrigerated storage in yoghurt (Fig. 3B). The viable cell numbers of encapsulated BL R023 declined with counts lower than the detection limit of 2 log cfu g1 after 7 days at 4 C. Moreover, a 3 log reduction in population was observed during yoghurt manufacture. Because the nonencapsulated cells survived well during fermentation and because diffusion through the protein matrix was theoretically very limited and slow, this very high mortality rate was unexpected. A potential drawback of spray-drying as a way of preserving lactic cultures is the high proportion of injured cells resulting from the process. Signicant lag times until onset of pH decrease have been observed with spray-dried starter cultures due to repair of reversible cellular injuries prior to cell growth and lactic acid production (Fu & Etzel, 1995; To & Etzel, 1997a). Teixeira, Castro, Malcata, and Kirby (1995) reported several possible sites in the bacterial cell where damage may occur as a result of spray-drying or heat stress, including cell membrane, cell wall and DNA. If not repaired, such injuries may prove to be lethal to the cell. In addition, microorganisms that have been damaged, but not killed, by exposure to stress often become considerably more sensitive to other types of agents. Increased sensitivity of sub-lethally injured cells to NaCl has previously been associated with cell membrane damage caused by the drying process

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(Brennan, Wanismail, Johnson, & Ray, 1986; Teixeira, Castro, Mohacsi-Farkas, & Kirby, 1997). The rapid decrease in counts of encapsulated BL R023 during yoghurt fermentation and subsequent refrigerated storage might therefore be attributed to a developed sensitivity of the spray-dried cells to low pH and to hydrogen peroxide and organic acids produced by yoghurt bacteria. 3.5. Tolerance to simulated gastrointestinal conditions In order to exert their benecial effects in the host, it is generally accepted that probiotic bacteria must be alive in the product at the time of consumption and also capable of reaching the large intestine in high enough quantities to facilitate colonization and proliferation (Shah, 2000). Changes in viable counts of bidobacteria in the digestion mixture, released from whey protein microcapsules (method A), were monitored during sequential exposure to simulated gastric and intestinal juices and compared with nonencapsulated control (Figs. 4A and B). Inoculation in simulated gastric juice at 37 C resulted in a dramatic decline in viable counts of free BB R070 cells (Fig. 4A), with a 6 log reduction after 30 min at pH 1.9. The number of viable cells remained constant (102103 cfu mL1) during the rst hour of the simulated intestinal phase, and increased signicantly after 3 and 6 h to about 105 cfu mL1. This increase in viable cell counts after 3 h of incubation at pH 7.5 in the presence of pancreatin and bile salts indicated only temporary damage to bidobacterial cells due to the low pH stress. As shown in Fig. 4A, almost no viable organisms were detected following exposure of encapsulated BB R070 to simulated gastric juice for 30 min. The viable number of bidobacteria in the digestion mixture increased substantially from 1.0 101 to 7.2 107 cfu mL1 after 6 h incubation at pH 7.5 in the presence of pancreatin and bile salts, respectively. The cell counts after 1, 3 or 6 h were more than 2 log cycles higher than those obtained with nonencapsulated BB R070. The considerable increase of 3.5 log in population recorded after 1 h of exposure to simulated pancreatic juice cannot reasonably be attributed to only cell multiplication, and probably resulted from a massive release of uninjured bidobacteria from degraded microcapsules and/or recovery of sub-lethally injured cells. Microscopic observations carried out during digestion revealed that the whey protein microcapsules exhibited a resistance to the hydrolytic action of pepsin but were totally digested in the pancreatic medium within 3 h (data not shown). The concept of microencapsulation allows the active core ingredient, or substrate, to be separated from its environment by a protective lm or coating. Such a

Fig. 4. Changes in viable cell counts (: detection limit of the method) of immobilized ( ) and free cells ( ) of B. breve R070 (A) and B. longum R023 (B) during simulated gastrointestinal digestion. No dispersion step was performed to release encapsulated cells, except for encapsulated cell counts at inoculation step. Error bars represent standard deviation. 30peps: 30 min in the presence of pepsin at pH 1.9; 60panc: 60 min in the presence of pancreatin at pH 7.5. Note: numbers represent minutes (30360 min)

separation occurs until a point in time when the release of this functional ingredient is desired. In the case of probiotics, the objective is not only to protect the microorganisms during the food manufacturing process and the passage in the upper part of the gastrointestinal tract, but also to allow a release of viable and metabolically active cells in the intestine (Suita-Cruz and Goulet, 2001). Our data show that encapsulation of bidobacteria in whey protein-based microcapsules lead to a progressive liberation of living cells during

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simulated intestinal phase and therefore preserved viability during simulated gastric phase by providing a protective envelope that acted as a barrier against both acidity and gastric enzymes. Since no treatment was performed to release cells before plate counts, the incomplete digestion of the protein network may have resulted in underestimation of the total viable cell counts (encapsulated and released cells), especially at the beginning of the test. A similar gastroresistance characteristic has recently been reported for whey protein beads by Beaulieu, Savoie, Paquin, and Subirade (2002) who observed a very slight degradation after gastric incubation. The increased stability conferred by microencapsulation in simulated gastrointestinal conditions has previously been reported by several researchers for various strains of Bidobacterium ssp. Wenrong and Grifths (2000) reported a higher survival of B. infantis immobilized in gellan-xanthan beads in low pH environments. Microencapsulation in cellulose acetate phtalate capsules was successfully used by Rao, Shiwnarain, and Maharaj (1989) to increase the viability of B. pseudolongum in simulated gastric and intestinal juices. Our study demonstrated that immobilization in water-insoluble whey protein microcapsules may be an effective way to increase the survival of bidobacteria in the harsh acidic conditions of the human stomach, allowing better colonization of the large intestine by these benecial organisms. After 6 h of incubation at pH 7.5 in the presence of pancreatin and bile salts, viable counts in the digestion mixture of encapsulated BB R070 increased to 1.9 107 cfu mL1, which was 0.6 log cycles lower than the initial bacterial concentration. In contrast to BB R070, entrapment in spray-dried whey protein microcapsules was not as effective in protecting BL R023 during digestion (Fig. 4B). After 30 min in the simulated gastric juice, the viable cell numbers in the digestion mixture of both nonencapsulated and entrapped bacteria rapidly were below 101 cfu mL1 (detection limit of the method). After 6 h of incubation at pH 7.5 in the presence of pancreatin and bile salts, a nal bacterial population slightly higher than 101 cfu mL1 was obtained for nonencapsulated cells, suggesting an intrinsic low resistance of BL R023 to gastrointestinal conditions and particularly to the gastric environment (irreversible cellular damage). On the other hand, viable numbers of bidobacteria in the digestion mixture signicantly increased during incubation in the simulated intestinal juice with the encapsulated treatment, from 1.9 101 to 7.9 103 cfu mL1 after 1 and 6 h, respectively. Bidobacterium ssp. have previously been reported to differ greatly in their susceptibility to low gastric pH and their tolerance to bile salts (Lankaputhra & Shah, 1995; Charteris, Kelly, Morelli, & Collins, 1998).

4. Conclusion Food-grade water-insoluble microcapsules containing sensitive probiotic cultures were successfully prepared using whey protein polymers and spray-drying in a continuous simple processing operation, capable of generating large quantities of material at low cost (method A). The use of probiotic cultures in a freezedried form and their dispersion in hydrophobic material did not improve their viability during spray-drying. However, this latter technique might be suitable for stabilization and/or controlled release of other food ingredients or supplements, including hydrosoluble (minerals, vitamins, etc.) and liposoluble (essential oils, avoring oils, etc.) components. The two Bidobacterium strains selected for this study varied considerably in their ability to survive the encapsulation process, and in the immobilized state, the refrigerated storage in yoghurt and the in vitro digestion test. BB R070 exhibited a high survival rate during spraydrying, and encapsulated cells showed a higher viability than nonencapsulated ones during 28 days storage in low-pH yoghurts and when subjected to conditions similar to those encountered in the human gastrointestinal tract. Therefore, immobilization of bidobacteria in water-insoluble whey protein-based microcapsules can increase their tolerance to high acid environments, making this approach potentially useful for delivery of probiotic cultures to the gastro-intestinal tract of humans. However, our ndings highlight the need to take into consideration the technological properties of probiotic strains with regard to processing, and particularly heat stability, when using spray-dry encapsulation to stabilize sensitive cultures. Future work should include investigations of techniques which could prevent cell damage and optimize viability during the process, such as stress adaptation during cell preparation.

Acknowledgements This research was carried out within the program of the Canadian Research Network on Lactic Acid Bacteria, supported by the National Sciences and Engineering Research Council of Canada, Agriculture and Agri-Food Canada, Novalait inc., The Dairy Farmers of Canada, and Rosell Institute inc.

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