Journal of Applied Microbiology 1998, 84, 342–348

Spectrophotometric quantification of lactic bacteria in

alginate and control of cell release with chitosan coating
Y. Zhou, E. Martins, A. Groboillot,1 C. P. Champagne2 and R. J. Neufeld
Department of Chemical Engineering, McGill University, Montréal, Québec, Canada, 1Laboratoire de Microbiologie
du Froid, I.U.T. de Biologie Appliquée, Université de Rouen, Evreux, France, and 2Centre de Recherche et de
Développement sur les Aliments, St Hyacinthe, Québec, Canada
6107/02/07: received 12 February 1997 and accepted 23 May 1997

Y . Z HO U , E . M A RT IN S , A . G R OB OI L LO T, C .P . C H AM PA G NE AN D R. J. N EU FE L D. 1998.
Lactococcus lactis ssp. cremoris was entrapped within a Ca-alginate matrix, and an in situ
spectrophotometric method for monitoring cell population in calcium alginate beads
described. The intracapsular cell population can be estimated by measuring the
optical density of beads containing cells, using cell-free beads as reference, or by
measuring absorbance of a liquified bead suspension. Alginate beads, and beads
coated with chitosan type I, II, and I and II mixtures, were examined for cell release.
Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from
105 cfu ml−1 to 103 cfu ml−1 after 6 h of fermentation. Reuse of chitosan I coated
alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and
initial cell growth within the beads greatly affected cell release. Reducing the initial cell
release would lower the overall levels of cell release throughout the fermentation. Compared
to non-immobilized cultures, a 20–40% reduction in the lactic acid production rate was
observed for alginate beads and chitosan I coated alginate beads, respectively.
This reduction can be compensated for by increasing the intracapsular cell loading
during immobilization, or before the onset of fermentation.

INTRODUCTION in extracapsular populations reaching 108 cfu ml−1 (Prévost

and Diviès 1992). In applications such as cream fermentation
Immobilized cell technology has been widely applied in a
for the production of cultured butter and cottage cheese
variety of research and industrial applications (Groboillot
dressings, the release of cells is undesirable since the residual
et al. 1994). The use of immobilized lactic acid bacteria as
free cells may result in continued acidification during storage.
starter cultures in the dairy industry is one of the potential
Cell release is also problematic in encapsulated biomass pro-
applications (Champagne et al. 1992, 1993, 1994).
duction processes as yields are reduced (Champagne et al.
While numerous immobilization techniques have been
1993). In order to reduce cell release during fermentation,
described, entrapment within synthetic or natural polymers
Champagne et al. (1992) rinsed alginate beads with distilled
remains among the most popular due to its ease and
water, ethanol, Al (NO3)2 or hot CaCl2 before fermentation
simplicity, low cost and gentle formulation conditions ensur-
to kill lactic acid bacteria near the bead surface. Another
ing high retention of cell viability. A popular gel forming
approach was the application of multiple coats of poly L-
material is alginate which is a linear heteropolysaccharide of
lysine and alginate, which reduced cell release by a factor of
D-mannuronic and L-guluronic acid residues. Sodium alginate
10 (Champagne et al. 1992; Prévost and Diviès 1992).
may be ionically crosslinked with multivalent cations (typi-
Chitosan has been used as a membrane forming material in
cally Ca2+) to form gels (Smidsrod and Skjak-Braek 1990).
While high cell loading may be achieved in alginate beads the micro-encapsulation of lactic acid bacteria (Groboillot
(109–1010 cfu g−1 beads), cell release from the gels may result et al. 1993). Chitosan is a cationic polyelectrolyte, which inter-
acts with the polyanionic alginate to give rise to a chitosan
Correspondence to: R.J. Neufeld, Department of Chemical Engineering, membrane coat surrounding an alginate core (Sandford and
Queen’s University, Kingston, Ontario, Canada K7L 3N6. Hutchings 1987). This present study examined the potential
© 1998 The Society for Applied Microbiology

of chitosan in further reducing cell release when applied as a
membrane coat around a gel core containing cells. As well,
an in situ spectrophotometric assay was developed to estimate
the populations of growing lactic acid bacteria in alginate
beads, during consecutive fermentations.
MATERIALS AND METHODS
Bacterial culture
Mesophyllic Lactococcus lactis ssp. cremoris, kindly provided
by Institute Rosell Inc., Montreal, Canada, is used as a starter
culture in the dairy industry. Lyophilized cultures were
stored at – 18 °C. Rehydrated cells (0·1 g 100 ml−1 medium)
were incubated in sterile Bacto-Elliker broth (Difco, Detroit,
MI, USA) for 20 h at 23 °C.
Cell immobilization
A 20 h culture was harvested from 50, 100 and 150 ml Elliker
broth by centrifugation at 4000 g for 15 min at 4 °C, washed
with 0·1% (w/v) sterile peptone (Bacto, Difco Laboratory)
and recentrifuged. The pellets were suspended in 5 ml of
0·1% (w/v) peptone and mixed with 20 ml sodium alginate
(gift from Sanofi Bio-industries) solutions to yield a final
alginate concentration of 2% (w/v). The mixtures were added
dropwise to sterile 50 mmol l−1 CaCl2 through a 0·114 mm
I.D. needle. After 30 min gelification, the 2 mm diameter
beads were rinsed with 0·1% sterile peptone and stored in
peptone at 4 °C. Some beads were prepared by mixing a 5%
(w/v) CaCO3 suspension with 20 ml of 2% (w/v) alginate to
achieve a final concentration of CaCO3 in alginate of 0·5, 1·0,
1·5 and 5% (w/v). These beads were stored in distilled water
at 4 °C.
Milk fermentation was started by the addition of 11–13 g
beads to 100 ml of 9% non-fat milk, previously sterilized at
121 °C for 8 min. Fermentations were performed at 30 °C on
a 100 rev min−1 shaker (Haake SWB 20 ; Montreal, Canada)
for 2–4 h. Some beads were incubated for an additional 4 h
to obtain higher cell loadings. After fermentation, the beads
were washed with distilled water and rinsed with 0·1%
peptone.
Chitosan membrane coating on alginate beads
Alginate beads were coated with either type I (Seacure 123,
Protan, Portsmouth, NH, USA; Lot 10037RG) or type II
(Seacure 123, Protan, Lot 17230123) chitosan. Type I has a
lower molecular weight, thus a lower viscosity (14 mPas in
1% w/v solution) than type II (22 mPas in 1% w/v solution).
Chitosan is generally insoluble at pH levels above 6·5, so an
aqueous solution was prepared by dissolving 0·4 g in 90 ml
distilled water acidified with 0·4 ml of glacial acetic acid to
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 342–348
13652672, 1998, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1046/j.1365-2672.1998.00348.x by Swinburne University Of, Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
I MM OB I LI ZE D LA CT I C A CI D BA CT E RI A 343
achieve a final chitosan concentration of 0·4% (w/v). After
dissolution, 1 mol l−1 NaOH was added to adjust the pH to
between 5·7 and 6. This solution was filtered (Whatman no.
4), and the volume adjusted to 100 ml.
Alginate beads with cells were washed with distilled water
and immersed (about 12 g) in 100 ml of chitosan solution with
gentle shaking for 40 min on an orbital shaker. Coated beads
were then filtered and washed with sterile peptone and stored
in peptone at 4 °C.
Bead dissolution and cell counting
Beads (0·1 g) were liquified in 100 ml of 1% sterilized sodium
citrate solution (pH 6·0) and serially diluted with 0·1%
peptone. Plate counts were conducted in triplicate at each
dilution on Elliker broth supplemented with 1·5% (w/v)
Bacto-agar (Difco). Plates were incubated at 23 °C for 2 d,
and results reported as colony forming units per gram of
beads (cfu g−1). This same plate counting procedure was
used to determine the cell numbers released from the beads
into the culture medium during fermentation.
Spectrophotometric determination of cell loading
Beads (2 g) containing cells were suspended in water within
a 10 mm cuvette and absorbance measured at 560 and 620 nm
using a Varian CARY 1 spectrophotometer (Montreal, Can-
ada) with cell-free beads as reference. Concurrently, 0·1–0·4 g
beads were dissolved in 10 ml of 1% sterile sodium citrate.
The absorbance of this cell-alginate suspension was measured
using liquified, cell-free alginate beads as reference.
Lactic acid determination
Fermented milk (1 ml) was mixed with 0·05 g of 5-sulfo-
salicylic acid crystals for deproteinization. After 1 h at 4 °C,
the reaction mixture was centrifuged at 96 700 g for 10 min
and the supernatant fluid filtered through a 0·2 mm membrane
filter and frozen for storage. Lactic acid was determined
with a Hewlett-Packard HP 1050 HPLC equipped with a
300 mm × 7·8 mm I.D. cation–exchange interaction ION-
300 column and a differential refractive index detector. The
mobile phase was membrane filtered 0·0025 mol l−1 sulphuric
acid. Samples were run with a mobile phase flow rate of
0·3 ml min−1 and a column temperature of 30 °C. Lactic acid
standards had a retention time of 20 min.
RESULTS
Intracapsular cell quantification
Cell loading in alginate beads ranged from 108 to 1010 cfu g−1,
determined by conducting plate counts on released cells
 13652672, 1998, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1046/j.1365-2672.1998.00348.x by Swinburne University Of, Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
344 Y . Z HO U ET AL .

following bead dissolution. This represents an encapsulation

yield of 82%.
Beads which were scanned spectrophotometrically
absorbed strongly in the range 260–280 nm due to cell DNA
and proteins. Visible light obscuration in the visible region
was also evident with increasing intracapsular cell loading
due to growth. As cell growth of Streptococcus cremoris in
liquid media was following at 620 nm in previous studies
(Kanasaki et al. 1975; Lee and Lim 1980; Thatipamala et al.
1991), this wavelength was selected for monitoring intra-
capsular cell concentration in the present study. Alginate did
not absorb at 620 nm. However, reference (cell-free) beads
were loaded with 1% insoluble calcium carbonate to increase
the reference baseline, allowing cell measurements within a
practical working range of 108–1010 cfu g−1. Higher or lower
Fig. 2 Cell absorbance following bead liquefaction with an
levels of cell loading in the beads would require adjustments
equivalent amount of cell free beads used as reference.
in the carbonate content of the reference beads due to partial Liquefaction of () 0·1 g or () 0·4 g of beads
light obscuration of the reference. Alginate does not absorb
within the region 560–620 nm. Alginate beads containing 1%
calcium carbonate were used to construct a calibration curve
of immobilized cell density at 620 nm as shown in Fig. 1. A Abs620 nm  2·3 × 10–8 (cfu g−1)0·68
linear calibration curve was constructed for cell loaded beads Abs620 nm  4·0 × 10–7 (cfu g−1)0·62
yielding the relation: for 0·1 and 0·4 g cell loaded beads, respectively.
−1
Abs620 nm  0·28 ln (cfu g ) – 5·30. Cells were quantified in situ spectrophotometrically for 10
replicate samples at three different wavelengths: 260, 560 and
Beads containing cells were also liquefied in citrate and the 620 nm. The standard deviation was 1·4% at 560 nm and
cell population estimated spectrophotometrically as shown in 620 nm, and 6·5% at 260 nm, indicating a good repro-
Fig. 2. The reference in this case consisted of liquefied 2% ducibility.
alginate beads (0·1 or 0·4 g, respectively). A 0·4 g bead sus-
pension results in a data distribution within 0·05–1 absorb- Milk acidification with immobilized lactococci
ance range, while the absorbance fell between 0·01 and 0·22
for the 0·1 g bead suspension. Linear calibration curves were The time course of a milk fermentation with alginate immo-
constructed, which provided the following relations: bilized cells is illustrated in Fig. 3. Generally, replicate experi-
ments were conducted, but data from one experiment only
are presented in subsequent plots as the results were highly
comparable as may be observed in Fig. 3.
The cell loading in the beads was 109 cfu g−1, and 103
cfu ml−1 cells were initially released to the medium. The
intracapsular cell population increased gradually concomitant
with an increase in the level of free cells. After 2 h, the
intracapsular cell concentration stabilized between 109 and
1010 cfu g−1 beads. Six hours later, the free cell population
approached 1 × 105 cfu ml−1 while the cell concentration
inside the beads was 1010 cfu g−1 beads.
During fermentation, intracapsular cells grew as short
chains, averaging 3·5 cells as observed microscopically, and
produced lactic acid which decreased milk pH to 5·5 after
2·5 h.

Cell release from alginate beads coated with

chitosan

Fig. 1 Absorbance of cell loaded alginate beads against cell free Milk fermentation was conducted with lactococci immo-
reference beads bilized in alginate beads coated with type I chitosan. After
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 342–348

Fig. 3 Milk fermentation with alginate immobilized cells.
Intracapsular cell populations (circles) are compared to the level of
released cells (squares) and milk pH (triangles) throughout
course of fermentation. Clear and dark symbols represent
data from replicate experiments
6 h, extracapsular cell concentrations were 1 × 103 cfu ml−1,
considerably lower than that released from uncoated beads.
Various application levels of chitosan type II coatings (12 g
beads added to 100 ml of 0·4, 0·8 and 1·6% chitosan solutions)
were investigated with the same levels of intracapsular cell
loading in each case. At the onset of fermentation, between
1 × 103 and 1 × 104 cells ml−1 were released into the
medium, with the lowest level of free cells observed in the
case of chitosan II applied at the lowest level (0·4 g). When
testing different types of chitosans (type I, type II, and type
I and II mixtures) applied at the same level, the lowest release
of cells at the onset of fermentation was observed in the case
of chitosan I and chitosan I and II, with approxi-
mately 1 × 102 free cell ml−1 observed free of the alginate-
chitosan beads. After 6 h fermentation, levels of free cells
originating from chitosan I coated beads and chitosan I and
II coated beads were about 103 cfu ml−1, compared to 104 cfu
ml−1 from chitosan II coated beads.
Cell release during consecutive fermentations
Consecutive fermentations were conducted to test the stab-
ility of the beads and membranes, the recovery of immobilized
cell activity, and the level of cell release. After 2 h, the beads
were removed from the milk, rinsed with 1% peptone water,
and introduced to fresh milk. This procedure was repeated
for three or four consecutive fermentations. Figure 4 illus-
trates the levels of free cells released from alginate beads
and chitosan I coated alginate beads during consecutive
fermentations.
The level of free cells released from the beads increased
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 342–348
13652672, 1998, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1046/j.1365-2672.1998.00348.x by Swinburne University Of, Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
I MM OB I LI ZE D LA CT I C A CI D BA CT E RI A 345
Fig. 4 Consecutive fermentations with alginate immobilized
cells. Initial (ž) and final (Ž) intracapsular cell populations
(cfu g−1) are compared to initial () and final () levels of
released cells (cfu ml−1) throughout the course of
fermentation. Consecutive fermentations with chitosan I coated
alginate beads compare initial (R) and final (E) intracapsular cell
populations to initial (r) and final (e) levels of released cells
slightly with consecutive fermentation. Larger differences
(order of magnitude) were observed between initial and final
free cell levels in the first fermentation in comparison to
subsequent fermentations, where the differences were
reduced. After the third fermentation, the level of free cells
originally from chitosan I coated alginate beads was 103 cfu
ml−1, compared to 105 cfu ml−1 from uncoated beads. The
final intracapsular cell loading was approximately 1010 cfu g−1
beads. Chitosan I coating did appear to reduce the level of
released cells during consecutive fermentations.
Effect of intracapsular cell population on initial cell
release
Figure 5 shows the effect of intracapsular cell loading on cell
release within the first 5 min of each fermentation. Both
coated and uncoated alginate beads were examined. An
increase in intracapsular cell concentration resulted in higher
cell release, probably due to a larger cell gradient between
the interior of the beads and the external medium. The release
from uncoated beads was greater than that for chitosan coated
beads, indicating that the chitosan membrane did have a
significant impact on reducing initial cell release from the
beads.
Lactic acid production by immobilized cells
Fermentations were conducted with free and alginate-immo-
bilized cells, inoculated at equivalent levels of 108 cfu ml−1

346 Y . Z HO U ET AL .
Fig. 5 Effect of intracapsular cell population on initial cell release
from alginate () and CaCO3 loaded alginate (ž), and from
chitosan I () and chitosan I and II (r) coated alginate beads
of milk, to compare rates of lactic acid production (Fig. 6).
Reductions in the rate of lactic acid production were observed
with immobilized cells, in comparison to the non-encap-
sulated controls. Taking the slope as the rate of acid
production, reductions of 20% were observed with alginate-
immobilized cells, compared to 30%–40% reductions for
alginate-chitosan immobilized cells. Due to the mass transfer
resistances, the fermentation times to pH 5·5 were 2·5 and
4 h for uncoated and chitosan I coated alginate beads, respec-
tively, compared to less than 2 h in the case of free cells. Mass
transfer resistance for coated beads appears to be higher than
that of uncoated beads. The loss of activity could be recovered
Fig. 6 Lactic acid production by free cells (ž) and cells
immobilized in alginate (), and in chitosan I (), and chitosan I
and II (r) coated alginate beads
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 342–348
13652672, 1998, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1046/j.1365-2672.1998.00348.x by Swinburne University Of, Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
by increasing the cell loading in the immobilized system or
by cell culture to increase intracapsular cell concentration.
DISCUSSION
Immobilized cell concentration estimates are of considerable
importance to the understanding and control of fermentation
processes. Spectrophotometry provides a simple and rapid
method for measuring free cell concentrations in culture
medium. However, gel immobilized cells present a problem
since the gel beads are assumed to interfere with light trans-
mission. Dry weight determination is also problematic due to
the presence of the gel. Estimates of cell populations are
normally determined following partial or complete liquefac-
tion of the gel, followed by plating and colony counting on
agar plates. Due to the time needed for cell separation and
incubation, colony counting is unsuitable for on-line or rapid
cell density estimates. Spectrophotometric methods for cell
population estimation have been used to quantify yeast in
semi-solid media or media with solid components (Wei et al.
1983; Hong et al. 1987; Kennedy et al. 1992). For example,
Wei et al. (1983) measured Streptococcus cerevisiae in a 10%
suspension of solidified gelatin by assuming that the gelatin
and medium components produced no interfering absorb-
ance. Also, proteins (Monshipouri and Neufeld 1991, 1992)
and DNA (Alexakis et al. 1995) have been measured intra-
capsularly by spectrophotometric assay using empty capsules
as reference.
Population levels obtained in beads (1010 cfu g−1) and in
free cells released to the culture medium (103–105 cfu ml−1),
were lower by a factor of 10 than those reported for Lacto-
coccus lactis ssp. cremoris CRA-1 (Morin et al. 1992). Com-
parative assays were therefore carried out with strain CRA-
1, which showed that the strain used in this study simply
gave lower cfu counts (data not shown). This was partially
related to chain lengths of the two cultures. Thus, the lower
cfu counts registered in uncoated alginate beads were related
to strain, and were not due to methodology underestimation.
The mechanism of cell release has been studied recently
(Arnaud et al. 1992; Champagne et al. 1994). Cell growth
often occurs as colonies in the small cavities close to the
surface region of the gel (Audet et al. 1988). Due to the force
exerted by cell growth and shear forces resulting from mixing,
the gel cavities containing the microcolonies are disrupted
and cell release occurs. Cachon et al. (1995) also demonstrated
that 95% of Lact. lactis colonized a 150 mm radius around the
periphery of alginate beads at steady state, contributing to
cell release through cell growth.
The phenomenon of cell release is different from molecular
diffusion. Cell release occurs on the surface of the beads and
is unrelated to the bead pore or cell size. Smidsrod and Skjak-
Braek (1990) reported that the pore size of 2% alginate beads
(0·5–3 mm in diameter) ranges from 5 to 200 nm (0·005–

0·2 mm), much smaller than the size of most microbial cells
(× 0·5 mm), interfering with cell movement. In this study,
cells grew as short chains averaging 3·5 cells. A mechanism
other than cell diffusion or permeation, such as that described
by Audet et al. (1988), is the likely mechanism for cell release.
Lactate has been shown to compete with alginate for the
binding of Ca2+ (Boyaval and Goulet 1988; Morin et al. 1992),
and high levels may cause softening or dissolution of the
beads. This effect was not observed in the present study.
Chitosan is a positively charged polyamine which forms
a semi-permeable membrane around a negatively charged
polymer such as alginate. This membrane does not dissolve
in the presence of Ca2+ chelaters or anti-gelling cations and
thus enhances stability of the gel (Smidsrod and Skjak-Braek
1990), and provides a barrier to cell release. It was observed
(McKnight et al. 1988) that reduced molecular weight
chitosan provides relatively strong and flexible membranes
because the lower molecular weight polymer presumably dif-
fuses more readily into the alginate gel matrix resulting in a
denser membrane. In this study, chitosan I demonstrated
better control of cell release than chitosan II, consistent with
the above explanation.
Chitosan mixtures (types I and II) were as effective as
chitosan type I, most probably due to chitosan type I playing
the dominate role in membrane formation. Membrane coat-
ings consisting of chitosan I, and I and II mixtures, reduced
cell release by two orders of magnitude during 6 h of fer-
mentation. In comparison, poly L-lysine or two-layer alginate
coatings reduced cell release by one order of magnitude
(Champagne et al. 1992). Chitosan I coating used in this study
enables a significant reduction of cell release. In consecutive
fermentations, reduction of cell release by two orders of
magnitude to the third fermentation (103 cfu ml−1 compared
to 105 cfu ml−1 for uncoated alginate beads) was demon-
strated.
Free cells measured in the milk were thought to be due
primarily to cell release from the beads and not to bacterial
growth in the milk. In a typical fermentation, the initial cell
release from the beads was 5·5 × 102 cfu ml−1. As the doub-
ling time of lactic bacteria is approximately 1 h (Cogan 1980),
after 2 h, the cell concentration in milk would reach 2·2 × 103
cfu ml−1 assuming no further cell release from the beads. In
fact, 1·0 × 104 cfu ml−1 were observed. Likewise, after 4 h,
the cell concentration in the milk would have been 8·8 × 103
cfu ml−1 if it were only due to cell growth in the milk.
However, it was found to be approximately 3·0 × 104 cfu
ml−1. For chitosan I coated cell-alginate beads, the initial cell
release was 1·0 × 102 cfu ml−1. After 2 h, the cell population
in the milk due to cell replication should have been 4 × 102
cfu ml−1, the actual level observed. Thus, it appears that no
further cell release occurred after initial cell release.
Champagne et al. (1994) suggested that there is a steady
state for the non-uniform cell growth inside the beads. It was
© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 342–348
13652672, 1998, 3, Downloaded from https://ami-journals.onlinelibrary.wiley.com/doi/10.1046/j.1365-2672.1998.00348.x by Swinburne University Of, Wiley Online Library on [01/08/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
I MM OB I LI ZE D LA CT I C A CI D BA CT E RI A 347
assumed that the cell distribution was uniform following
immobilization, but cell growth is non-uniform following
onset of fermentation. By simulation, Champagne found that
a highly heterogeneous distribution of biomass within the
beads was due to product accumulation rather than to sub-
strate exhaustion. At steady state, the cell growth around the
surface of the bead is balanced by the cell release rate. The
main effect of chitosan coating may then be to decrease the
cell release rate during early stages of fermentation.
Lactic acid production in milk was affected by the immo-
bilization procedure and bead coating. Mass transfer resist-
ance due to the entrapment is thought to be one of the
main factors affecting growth and lactic acid production. The
intracapsular lactic acid accumulation is high, thus inhibiting
further production of lactic acid compared to free culture
fermentations (Champagne et al. 1994).
The pH level is another factor affecting lactic acid
production. The optimal pH is 6·3–6·9 for Streptococcus
cremoris. However, in this study the batch fermentation was
carried out at pH 6·3–5·0. The lower pH would reduce the
rate of cell growth and the production of lactic acid (Bibal
et al. 1988).
Chitosan itself was also observed to inhibit the growth of
lactic acid bacteria, possibly due to binding of chitosan to the
cells (Groboillot et al. 1993). This may explain why cells
within chitosan coated alginate beads had lower acid pro-
ductivity than within alginate beads alone. The membrane
also serves as a mass transfer barrier, potentially reducing
growth and lactic acid production rates.
The reduction in the lactic acid production rate may be
compensated by increasing the cell loading inside the beads,
potentially reaching higher levels (1010–1011 cfu g−1 beads)
than typically encountered in free cell fermentations. Also,
reduced diameter beads have been shown to enhance growth
yield (Cachon et al. 1995), probably due to lower mass transfer
resistance. However, an increase in cell release may occur
due to a higher specific surface area (Champagne et al. 1994)
and surface colonization (Cachon et al. 1995).
ACKNOWLEDGEMENTS
This project was supported by the Québec fonds pour la
formation de chercheurs et l’aide à la recherche and the Natural
Sciences and Engineering Research Council of Canada. The
technical assistance of Nancy Gardner is gratefully acknow-
ledged. The authors also wish to thank Dr Denis Poncelet
for fruitful discussions.
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