Full Paper Bioscience Microflora

Vol. 15 (1), 9-15, 1996

Effect of Immobilization in Alginate on the Stability of

Freeze-Dried Bifidobacteriumlongum

Claude P. CHAMPAGNE,1 Francine MONDOU,2* Yves RAYMOND1

and Edouard BROCHU2

1 Food Researchand DevelopmentCentre,AgricultureCanada, 3600, Casavant West,St. Hyacinthe,

Quebec J2S8E3, Canada
2RosellInstitute
, 8480, St. Laurent,Montreal, QuebecH2P 2M6, Canada

(Received for publication, February 1, 1996)

Abstract Bifidobacterium
longumRO23 was immobilized in calcium alginate gels and
freeze-dried. The effects of immobilization, storage temperature and composition of
the protective solution were studied. High mortality (two log reduction in viable
count) was observed when a concentrated culture was immobilized and immediately
freeze-dried. Such and immobilization procedure also reduced stability during storage
at 20°C. This feature was strain/species related, as immobilization in alginate beads
was not detrimental to the survival of LactococcuslactisRO58 and Lactobacillusrhamnosus
RO11 to freeze-drying, but influenced negatively Streptococcus
thermophilus
RO57. Alginate
and calcium chloride, used in the immobilization process, were not responsible for the
high mortality of immobilized B. longumRO23. Bifidobacteria grown in the alginate
beads did not produce high post-lyophilization mortality, suggesting that freeze-drying
the culture immediately following immobilization was the cause of the high mortality,
and exposing the cells to a growth medium between immobilization and freeze-drying
may allow recovery. Whey-based protective solutions that are mixed to the alginate
beads were more effective than milk-based solutions in protecting the immobilized cells
against mortality during freeze-drying.

Key words: immobilized cells; drying; whey; milk; lactic acid bacteria

Bifidobacteria-containing products are in-
creasingly popular because of the assumed health
benefits of these cultures (8, 13). Bifidobacteria
cultures are marketed in dry form as food supple-
ments or are incorporated in the dairy food itself.
Food manufacturers do not maintain the bifido-
bacteria cultures themselves, preferring to rely on
specialized suppliers for their cultures. Shipping
and storage of cultures is thus a common practice
with bifidobacteria. Therefore, means of increas-
ing survival during freezing and drying, as well as
increasing stability during storage are of interest
to the culture suppliers.
Immobilization or microencapsulation has
been shown to increase the survival of Lactobacillus
plantarum to freeze-drying (10), as well as to in-
crease the stability of various lactic acid bacteria
during storage (1, 12). Although immobilization
has been shown to increase survival of bifidobac-
teria on exposure to simulated gastric and intes-
tinal juices (15), no data exist on the effect of
immobilization on the survival of bifidobacteria to
freeze-drying and their stability during storage.
The important variations observed in the effec-
tiveness of polysaccharides in improving stability
to freeze-drying (4) warrant a specific study on
bifidobacteria before commercial application can
be attempted.

9
10 C.P. CHAMPAGNE, et al

The aim of this study was to examine the perature (25•Ž).

effect of immobilization of Bifidobacterium longum on 4. Biomass production in alginate beads. The

survival to freeze-drying and the subsequent sta- freshly prepared beads (100 g) obtained from an

bility of the cultures during storage. unconcentrated MRS-grown bacterial suspension

were incubated in 1l of the industrial growth

MATERIALS AND METHODS
medium at 37°C for 17 hr. Agitation at 80 rpm

1. Biological material. Lactobacillus rhamnosus was maintained throughout the fermentation us-

RO11, Bifidobacterium longum RO23, Lactococcus lactis ing a Bellco (Vineland, N J) SciEra System with

RO58 and Streptococcus thermophilus RO57 were carrierTM flasks. This enabled B. longum RO23
ƒÊ

cultures of Rosell Institute Inc. (Montreal).

growth in the beads and the population reached
2. Free cell concentrates. The production of 5.9 •~ 109 CFU/g at 17 hr of fermentation. This

B. longum RO23 cultures in a 19 l Bioengineering culture is characterized by post-immobilization

fermenter (Wald, Switzerland), 15 l working vol-

growth and will be referred to as the "bead-grown
ume, was carried out in an MRS-type growth culture."

medium and under proprietary conditions which 5. Freeze-drying of the free cell concentrate. The

are used industrially by Rosell Inc. The fermen- free cell concentrate was mixed with an equal

tations were stopped at the end of the logarithmic volume of a protective solution composed of 30%

growth phase. The cultures were immediately skim milk (Agropur, Granby), 11.4% sucrose

cooled to 4°C, and concentrated by a factor of 10

(Lantic, Montreal) and 0.8% ascorbic acid (Sigma).
by microfiltration (Amicon 0.1 ƒÊm cut-off; Koch This bacterial suspension was placed in metal trays

Membrane Systems, Philadelphia). Filtration and spread so as to obtain a film approximately

pressures were of 1.7 bar at the entrance and 1.0 0.5 cm thick. The trays were placed in a -40°C

bar at the exit of the cartridge. The concentra- freezer for 18 hr and subsequently freeze-died at

tion operation was accomplished in 30 min. This 30°C for 24 hr in vacuum at a pressure of 20-50

UF-concentrated bacterial suspension will be re- mHg. The powders were then mechanically ƒÊ

ferred to as the "free cell concentrate," and had a

ground (Retsch/Brinkman centrifugal grinding mill)

population of 2.6 •~ 1010 CFU/ml. Biomass pro- and placed in a chamber containing a saturated

duction for the other strains was carried out simi- solution of lithium chloride for 24 hr at 23°C in
larly, the only difference being their respective order to stabilize water activity (aw). Following

industrial growth media (ingredients included glu- freeze-drying, and prior to aw stabilization, the

cose, lactose, peptones, yeast extracts, meat ex- water activity reading was of 0.24. The water

tracts, ammonium citrate, phosphates and other content of the powders adjusted to aw = 0.22 was

salts). 4.8%. The powders were divided into aliquots

3. Immobilization in alginate beads. A solution of 4-5 g which were placed in serum bottles and
composed of 4% (wt/vol) alginate (Grinsted Sobalg capped under atmospheric conditions.

FD 120; Denmark) and 11.4% sucrose (Lantic, In one series of assays aimed at evaluating

Montreal) was sterilized at 115•Ž for 10 min and the effect of immobilization components per se on

supplemented with 0.8% of filter-sterilized ascor- the survival of the bifidobacteria to freeze-drying,

bic acid (Sigma, Mississauga). This solution was two treatments were prepared by adding the fol-

mixed at a 1 : 1 ratio with the cell suspension. lowing ingredients to the free cell concentrate prior

The mix was extruded dropwise in a 0.1 M CaCl2 to mixing with the milk-based protective solution:

(Anachemia, Montreal) solution supplemented with (1) 1% CaCl2 and (2) 0.5% sodium citrate with
5.7% sucrose and 0.4% ascorbic acid. The re- 2% sodium alginate.

sulting beads (1-2 mm in diameter) were kept in 6. Freeze-drying of immobilized cells. The
this solution for 1 hr to allow for hardening . alginate beads were mixed with a protective solu-

These operations were carried out at room tem- tion at a 1 : 1 ratio, and incubated 15 min to al-
 STABILITY OF FREEZE-DRIED
low diffusion of the protective ingredients in the
beads (preliminary experiments had shown that
equilibrium was reached in this time period). The
protective solution had been previously cooled at
4°C and the incubation was carried out at 4°C.
Freeze-drying and aw stabilization was then con-
ducted as described for free cell concentrates.
Following freeze-drying, the awwas 0.26, and water
content was 6.6% following stabilization at aw
= 0.22.
In one series of assays, milk solids were sub-
stituted by whey solids (22% wt/vol) in the pro-
tective solution.
7. Sorptioncurves. Following freeze-drying,
the various powders were further dried by oven
incubation at 80°C for 24 hr. They were then
placed in chambers containing a saturated solu-
on of either lithium chloride (aw= 0.11), sodium
ti
acetate (aw= 0.22) and magnesium chloride (aw=
0.33). The powders were incubated for 72 hr at
25°C in the chambers, following which both aw
and water content were determined.
8. Analyses. For all cultures, 1 g of powder
was rehydrated, for 10 min at 25°C, in 4 ml of a
sterile solution composed of 0.5% meat extract
(Lab-Lemco, Oxoid), 1.5% peptone (Difco, De-
troit, MI) and 1.0% tryptone (Difco). These
rehydration conditions were found to provide high
recovery rates following freeze-drying (5, 6). The
rehydrated bacterial suspension was added to 95
ml of 1% sodium citrate and homogenized in a
Stomacher unit (Seward, London, UK) for 10 min.
This homogenization step was not detrimental to
the cultures' viability (data not shown). Serial di-
lutions were carried out in 0.1% peptone solu-
tions and plating was conducted on MRS agar.
The plates were incubated for 48 hr at 37°C un-
der anaerobiosis. Duplicate counts were con-
ducted at each sampling time.
Water activities were determined on a Rotro-
nic Hygroskopt DT (Huntingdon, NY). Saturated
solutions of lithium chloride (aw= 0.11), sodium
acetate (aw= 0.22) and magnesium chloride (aw
= 0.33) were used to verify the accuracy of the
unit. A Mettler DL 18 Karl Fisher Titrator (Zu-
rich) served to determine water content of the
BIFIDOBACTERIUM 11
freeze-dried powder. A 1-g sample was added to
30 ml methanol and incubated at 25°C for 24 hr.
The standard 1-hr extraction period was insuffi-
cient in our products as at least 8 hr of incubation
was required to obtain a stable reading (data not
shown).
Statistical analyses were carried out using the
SAS (Cary, NC) software. At least two complete
independant replications of the design were car-
ried out. To stabilize the variance and normal-
ize the residuals, CFU counts were transformed
to their base 10 logarithms.
RESULTS AND DISCUSSION
I. WaterContentand aw
The alginate-containing powders had a slightly
higher aw than controls (0.24 vs 0.26) following
the 24 hr/30°C drying period. This is in agree-
ment with Font de Valdez et al (7) who showed
that the composition of the medium influences
the drying curves of cultures. Water content is
an important parameter in the stability of dried
cultures (2, 14). In this study, it was opted to
stabilize the water activity at a given level (0.22)
rather than at a given water content (%), since
Ishibashi et al (9) had shown aw to be a major
factor in the storage stability of bifidobacteria.
This resulted in powders having different water
contents (4.8% for controls and 6.6% with algi-
nate) at a water activity of 0.22. Alginate-con-
taining powders tend to have higher residual
humidity (1) and the sorption curves confirm the
effect of alginate on the water activity-water con-
tent relationship in the cultures (Fig. 1). Alginate
has the property of binding water, which reduced
the increase in water activitywhen water was added
to the culture. For commercial purposes, it would
thus appear preferable to immobilize the culture
in alginate to help stabilize aw if the packaging
does not completely prevent water from entering.
2. EffectofImmobilization on the Concentrated
Cultures
Survival of the concentrated free-cell control
samples of B. longumRO23 to freeze-drying was of
29% (Table 1). However, survival was very low
(0.1%) if the concentrated culture was immobi-
 12 C.P. CHAMPAGNE, et al

lized just prior to freeze-drying. There was no 0.2%, respectively; free-cell controls all had sur-

significant effect of immobilization in alginate dur- vival levels above 20%. Thus, only with B. longum

ing storage at 4•Ž, but this procedure appeared RO23 and S. thermophilus RO57 were there signifi-

detrimental to the culture after 6 months of stor- cantly higher mortality rates to freeze-drying when

age at 20•Ž (Fig. 2). immobilization of the concentrate in alginate was

It was first examined whether this detrimen- conducted. This is in agreement with the results

tal effect of immobilization was strain-related. of Kearney et al (10) and Champagne et al (1)

When carrying out the freeze-drying step imme- who had found that immobilization in alginate

diately following immobilization of concentrated was not detrimental to the survival of lactobacilli

suspensions of Lactobacillus rhamnosus RO 11, Lacto- and lactococci during freeze-drying. These re-

coccus lactis RO58 and Streptococcus thermophilus RO57, sults also demonstrate that the detrimental effect
the respective survival levels were of 47 of immobilization in alginate beads prior to freeze-
, 20 and

drying is not limited to the bifidobacteria and could

be strain-related. Such results show the neces-

sity to test each strain prior to industrial applica-

tion of freeze-drying of immobilized cells .

Attempts were made to determine the reason

for the increased mortality to freeze-drying of B .

longum RO23 due to post-growth immobilization .

When 1% alginate was added to the protective

solution (as well as 0.25% citrate to prevent gelifica-

tion in the presence of the dairy ingredients' cal-

cium) the survival level was similar to that of the

control (Table 1). Stability during 3 months of

storage at 20•Ž was also unaffected by the addi-

tion of alginate/citrate to the protective medium .

Thus, alginate in itself was not found to be toxic

to B. longum RO23. The addition of 0 .5% cal-

Fig. 1. Effect of immobilization in alginate on sorption cium chloride to the protective medium did not
curves of freeze-dried cultures of Bifidobacterium longum
influence survival to freeze-drying, but appeared
RO23.
to be slightly detrimental during storage at 20•Ž
Dotted lines are the 95% confidence limits . Data are means
of two independent assays. (Table 1). Thus, the introduction of calcium chlo-

Table 1. Effects of various treatments on the survival of Bifidobacterium longum RO23 to freeze -drying

and its stability during storage for 3 months at 20•Ž

1All products were established at 100% at the beginningof storage, irrespective of survival to freeze-drying.2C
onstitutes the industrial control.
3a, b, c, d: Values that are followedby the same letter are not judged to be significantlydifferent (p > 0
.05).
 STABILITY OF FREEZE-DRIED BIFIDOBACTERIUM 13

the immobilized concentrated cell suspension.

Increased survival is not the only advantage of
growing the cells in beads. The population in
beads can be up to 30 times higher than in free
cell controls (1), and the centrifugation step is not
required. Therefore, the processing steps may be
reduced.

4. SubstitutionofMilk by Wheyin the ProtectiveSolu-

tion
Survival of the alginate-immobilized cells was
approximately four times higher when the protec-
tive medium was whey-based, as compared to the
cultures freeze-dried in the milk-based medium
Fig. 2. Effect of storage temperature and immobilization (Table 1). This was the case for both the bead-
in alginate beads on the stability of freeze-dried Bifido- grown bifidobacteria and the concentrated cul-
bacterium longum RO23. ture dried immediately following immobilization.
Dotted lines are the 95% confidence limits; for reasons of No significant effect of milk substitution by whey
clarity, only the confidence zone of samples stored at
could be observed, however, during storage. Al-
20•Ž are shown. So as to solely evaluate the effect of

storage, viability was established at 100% at the begin-

ning of storage, irrespective
drying. Data are means
ride in the beads during immobilization may be
partially responsible for reduced stability during
storage at 20°C (Fig. 2), but not of the reduced
viability observed immediately following freeze-
drying. Since none of the ingredients used dur-
ing immobilization were responsible for the 2 log
loss of viability, it was concluded that the timing
of the immobilization process, with respect to sub-
sequent freeze-drying, should be investigated.
3. Effectof the Timingof Immobilization
Bead-grown cultures had approximately 100
times higher survival rates to freeze-drying than
the concentrated B. longumRO23 cells immobi-
lized following growth and concentration (Table
1). Freeze-drying immediately following immo-
bilization thus appears highly detrimental to this
particular culture. Enabling the bacteria to be
exposed to the fermentation medium following
immobilization (bead-grown culture) significantly
increases the cells' capacity to survive freeze-
drying. It remains to be determined what time
period would be required to enable recovery of
though milk can rarely be improved upon as a
of survival rates during freeze- protective medium (3), in this particular applica-
of two independent assays. tion not all the milk components can exert their
protective influence. The caseins form large mi-
celles and such proteins are separated from the
cells inside the alginate beads (18). Whey pro-
teins and lactose, on the other hand, can diffuse
into alginate gels (16, 19) and be useful protectants
for freezing (17) or freeze-drying (11) of lactic cul-
tures. Their contents were consequently adjusted
to be similar in both treatments (30% milk vs 22%
whey). Since lactose and whey protein concen-
trations were similar in whey and milk treatments,
the reason for the increased survival to freeze-
drying with the whey-based medium thus does
not appear to be related to these particular com-
ponents. Although the nature of the beneficial
effect of whey has not been determined, these re-
sults suggest that it is technically advantageous to
substitute milk by whey when considering the
freeze-drying of B. longumimmobilized in alginate
beads. Since whey is less expensive than milk,
this approach is also commercially advantageous.
The slight increases in survival to freeze-dry-
ing and storage stability of the bead-grown immo-
bilized bifidobacteria (whey freeze-drying medium),
compared to the free cell controls (milk freeze-
 14 C.P. CHAMPAGNE, et al

drying medium), are not judged to be significant Benedicte Couespel, Nancy Gardner and Carmelle
when analysed separately. Their combined ef- Perron is gratefully acknowledged. Gratitude is ex-
fect becomes significant, however, and the free- pressed to Dr. J. P. Julien for helpful discussions. This
work was partially funded by the CNRC Industrial
cell control population was 1.0 •~ 108 CFU/g after
Research Assistance Program (IRAP/PARI).
3 months of storage at 20°C while that of the

bead-grown culture was 3.0 •~ 108 CFU/g. REFERENCES

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