Springer 2005
World Journal of Microbiology & Biotechnology (2005) 21:739–746
DOI 10.1007/s11274-004-4735-2
Use of alginate and cryo-protective sugars to improve the viability
of lactic acid bacteria after freezing and freeze-drying
B. De Giulio1, P. Orlando2, G. Barba1, R. Coppola1,3, M. De Rosa4, A. Sada1, P.P. De Prisco1 and F. Nazzaro1,*
1
Institute of Food Science and Technology – CNR, Via Roma 52, 83100 Avellino, Italy
2
Institute of Protein Biochemistry – CNR, Via P. Castellino, 80100 Napoli, Italy
3
Di.S.T.A.A.M. – University of Molise, Via De Sanctis, 86100 Campobasso, Italy
4
Department of Experimental Medicine, Biotechnology and Molecular Biology Section, Medical School – Second
University of Naples, Via Luigi de Crecchio, 80138 Napoli, Italy
*Author for correspondence: Tel.: +39-825299381, Fax: +39-825781585, E-mail: [email protected]
Keywords: Alginate, cryo-preservation, freeze-drying, lactic acid bacteria, trehalose
Summary
In the present paper, the effect of cryo-protective sugars on the survival rate of different strains of Lactic Acid
Bacteria (LAB, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp bulgaricus, Streptococcus salivarius subsp.
thermophilus), after freezing or freeze-drying procedures, was compared. The cells were incubated at 4 C in 32%
final concentration sugar solutions (trehalose, maltose, sucrose, glucose and lactose), and viability was evaluated by
the enumeration of colony-forming units. All sugars tested showed a protective effect on cell viability as compared
to isotonic solution, especially after freeze-drying procedures (nlog c.f.u./ml ranging between 1.16 and 2.08,
P < 0.001). Furthermore, the resistance to different stress agents (lysozyme, pepsin, bile salts) was estimated.
Trehalose was the most effective sugar in preserving bacterial viability [% (log c.f.u. trehalose/log c.f.u. isotonic
solution) ranging between 124 and 175, P < 0.001] although each strain showed a different sensitivity. Finally, the
protective effect of immobilization of LAB in Ca-alginate beads was compared to that exercised by trehalose. The
immobilization induced a good survival rate but lower as compared to the trehalose effect, mainly after freezedrying in the presence of the selective agents [% (log c.f.u. alginate/log c.f.u. trehalose ranging between 81.1 and
94.5, P < 0.0001]. The protective effect of trehalose was evident in particular for Lactobacillus delbrueckii subsp.
bulgaricus in presence of lysozyme. Therefore, because of its chemical inertness and low cost, trehalose could be
easily utilized as excellent bacterial preservative, both to improve the viability of starter cultures and to obtain
probiotic formulations more resistant to a variety of stressful conditions.
Introduction
Lactic acid bacteria (LAB) play a substantial role in
food biotechnology, being involved in many processes of
food fermentation, such as the production of cheese, dry
sausages, wine and sourdough breads. LAB contribute
to the formation of organoleptic and rheological characteristics of these products and inhibit the growth of
undesirable bacteria (Piard & Desmazeaud 1992; Vogel
et al. 1994; Coppola et al. 1998; Caplice & Fitzgerald
1999). Furthermore, many LAB strains form the basis
for a variety of probiotic foods. LAB are defined as
‘Probiotic’ for their beneficial effects on human health
by influencing the microbial balance of the endogenous
intestinal microflora. Species and strains of LAB useful
as probiotics are selected through specific characteristics, such as their resistance to gastric acids and bile
salts, and their capability to adhere and to colonize the
gut mucosa. Industrial use of LAB in food
biotechnology or for probiotic formulations is strictly
connected to the preservation technologies employed to
ensure stable cultures in terms of viability, bacterial
metabolism and technological properties. Different processes, such as freezing (F) and freeze-drying (FD), have
been used to preserve LAB. Freeze-drying, in particular,
is one of the most common processes for the production
of large amounts of concentrated microbiological cultures. However, during this process, bacteria are subjected to adverse conditions, such as water
crystallization and low temperatures, producing a
degree of protein denaturation and bacterial membrane
injury (Visick & Clark 1995), with consequent decrease
in viability, a higher sensitivity to air exposure and loss
of reproductive capability. To prevent or reduce these
adverse effects, many substances are used as cryoprotectives (Carcoba & Rodriguez 2000; Hubalek 2003).
Previous studies have demonstrated that some nonreducing disaccharides, such as sucrose and trehalose,
can be used for the cryo-preservation of micro-organisms (Chavarri et al. 1988). Trehalose (a-D -glucopyr-
740
anosyl-a-D -glucopyranoside) is a natural disaccharide
present in plants and yeast cells, highly soluble in water.
The glycosidic bond, characterized by a very low energy
level (nG < 1 Kcal/mol), renders trehalose among the
most stable sugars present in nature: the molecule does
not dissociate except under extreme hydrolysis conditions (or in the presence of the specific hydrolytic
enzyme trehalase) so, it does not undergo reactions with
proteins and other reactive biomolecules.
Several studies have demonstrated that it can protect
liposomes, isolated biological membranes and cells from
the adverse effects of freezing and freeze-drying, replacing water molecules during dehydration, depressing the
melting temperature of dry lipids, and preserving both
activity and structure of some proteins and biomolecules
(Crowe et al. 1984, 1985; Louis et al. 1994; Leslie et al.
1995).
In recent years, Immobilized Cell Technology (ICT)
has gained impelling momentum in the food and health
industry. Immobilization of cells offers several advantages, including enhanced fermentation productivity,
greater cell stability and, finally, reduced downstream
processing costs due to the easier cell recovery and
recycling (Groboillot et al. 1994; Draget et al. 1997).
ICT was successfully experimented with the production
of immobilized starters in the fermented meat and in
wine industries (Buyukgungor 1992; McLoughlin &
Champagne 1994; Maicas 2001). The main utilization of
this technology is related to the dairy industry. In this
field, ICT is used for starter production, for acidification
of raw milk before ultra-filtration, for inhibition of
psychrophilic bacteria in raw milk, and for yoghurt,
cheese, and cream fermentations (Prevost & Divies 1987,
1992; Audet et al. 1988; Lacroix et al. 1990; Champagne
et al. 1994). Many LAB strains have been immobilized
in various gel matrices such as pectate, dextran, jcarrageenan and calcium alginate (Ca-alginate) beads.
Alginic acid, a polysaccharide from brown seaweeds, is
an unbranched binary copolymer constituted of (1-4)linked b-D -mannuronic acid and a-L -guluronic acid in
which the relative amount and sequence of the two
constituent monomers can show a wide variation
(Smidsrod & Skjak-Braek 1990; Ertesvag & Valla
1998). In the presence of divalent ions (in particular
calcium ions), alginate forms micro-niches or microenvironments capable of protecting the microbial cells
during freezing or freeze-drying and during the subsequent re-hydration (Yabannavar & Wang 1991;
Sodini et al. 1997; Selmer-Olsen et al. 1999; Champagne
et al. 2000). In a previous work (Nazzaro et al. 1999)
was demonstrated the cryo-preservative action of trehalose on some LAB strains. The aim of the present work
is to compare the survival rate of three strains of LAB,
Lactobacillus acidophilus, Lactobacillus delbrueckii
subsp. bulgaricus, Streptococcus salivarius subsp.
thermophilus, frozen or freeze-dried after treatment with
cryo-protective sugars (trehalose, lactose, sucrose,
glucose, maltose) or after immobilization in Ca-alginate
beads. The rate of surviving cells was determined
B. De Giulio et al.
depending on their resistance to different chemical stress
agents, such as lysozyme, pepsin and bile salts that
simulate the effects of the digestive tract.
Materials and methods
Micro-organisms
Lactobacillus delbrueckii subsp. bulgaricus (DSM20081)
and Lactobacillus acidophilus (DSM20079) were grown
in MRS broth (Oxoid SpA Garbagnate Milanese, Italy);
Streptococcus salivarius subsp. thermophilus (from the
strain collection of our laboratory) was grown in M17
broth (Oxoid). The media were prepared according to
the instructions of the manufacturer and sterilized at
121 C for 15 min. After incubation at 42 C for 16 h
(OD620 ¼ 3.5), pellets were harvested by centrifugation
at 15,700 · g for 15 min at 10 C (5415 R centrifuge,
rotor F 452411-Eppendorf AG, Hamburg, Germany),
washed twice and re-suspended to the initial cell
concentration with cold sterile isotonic solution (8.5 g
NaCl/l). All solutions used in our experiments were
previously sterilized and manipulation was carried out
aseptically.
Cell freezing and freeze-drying conditions
The cell suspensions were divided into aliquots and
diluted to one-fifth of the original volume with the
following cryo-protective sugars in isotonic solution
(final concentration 32%): trehalose (British Sugar,
Peterborough UK), sucrose (Carlo Erba Reagenti,
Rodano-MI, Italy), lactose (Oxoid), maltose (AppliChem, Darmstadt, Germany), glucose (Carlo Erba
Reagenti). The mixtures were incubated at 4 C for
2 h and frozen at )80 C or frozen at )80 C and freezedried for 36 h until the achievement of constant weight.
Aliquots diluted to one-fifth of the original volume with
isotonic solution were used as control. Samples were
freeze-dried using a Christ Alpha RVC (Martin Christ
Gefrietrocknungsanlagen GmbH, Osterod, Germany)
apparatus. The freeze-dried samples were kept at 4 C
for 72 h before use.
Immobilzation in calcium alginate
Immobilization was carried out essentially as described
by Cortón et al. (2000) modified as follows: 1 ml of
microbial suspension was mixed with 1 ml of 2% (w/v)
sodium alginate (Sigma, Milano, Italy). The resulting
suspension was dropped (bead drop distance 10 cm)
with a 10-ml syringe into the gelling solution (1 l of
0.05 M CaCl2, in physiological solution). Rounded
beads obtained (average diameter 1.7 ± 0.2 mm, wet
weight 2 mg, volume about 2 ll) were collected by
centrifugation at 4 C for 15 m at 15,700 · g into
polypropylene tubes, frozen at )80 C for 24 h or
741
Lactic acid bacteria preservation
freeze-dried overnight to achieve a constant weight by
using a Christ Alpha RVC apparatus and stored at 4 C
for 72 h before use.
Viable count determination
Bacterial viable counts (c.f.u./ml) were performed
from serial dilutions in isotonic solution. Bacterial
dilutions were plated on medium containing 1.2%
agar (w/v) and grown as above described before
counting. To detect cell injury, fresh, thawed (after
freezing) or re-hydrated (after freeze-drying) samples
were serially diluted in isotonic solution and platecounted in agar supplemented with the following
substances: 0.1–0.5% bile salts, 100 mg lysozyme/l.
Plates were incubated in an anaerobic condition
(Anaerogen, Oxoid) at 42 C for 48 h. For Caalginate-immobilized cells, beads were dissolved in
2% (w/v) EDTA, the suspension was centrifuged and
washed at 15,700 · g and the resulting pellet was resuspended in 1 ml of isotonic solution before plating.
The resistance of free or immobilized cells to gastric
juices was evaluated by the method of Charteris et al.
(1998). Briefly, 1 ml of fresh, thawed, or re-hydrated
cells were added to 5 ml of medium containing 3 g
pepsin/l pH 2.5. After a mixing of 5 s, the suspension
was incubated at 37 C. Samples (100 ll) were withdrawn at different time of incubation (1–180 min) and
plated. Alginate-immobilized cells were directly added
to the simulated gastric juice, being dissolved in 2%
(w/v) EDTA only after the incubation in the medium
described above.
Statistical analysis
All the results presented in this work are the mean of
three independent experiments. Data were expressed as
Mean ± SD. Comparisons between groups were performed by two-way or one-way analysis of variance, as
indicated, and by Tukey test for multiple comparisons.
Statistical significance was set at P < 0.01.
Results and discussion
Table 1 summarizes the data concerning the cryopreservative effect of sugars on the microbial survival
rate, evaluated by the enumeration of colony-forming
units.
All sugars tested, without significant differences,
exhibited a good preservative effect after freezing and
especially after freeze-drying procedures in comparison
to isotonic solution (nlog c.f.u./ml ranging between 1.16
and 2.08, P < 0.001). In particular, no significant
variations (P ¼ 0.118) in viability after freezing and
after freeze-drying were observed for Lactobacillus
delbrueckii subsp. bulgaricus in the presence of trehalose,
thus suggesting that this sugar, for this strain, exhibits a
better cryo-preservative effect.
Within the three LAB strains, a lower cryo-preservative effect after freeze drying for all the sugars tested was
observed for Streptococcus salivarius subsp. thermophilus (freeze-drying survival rate ranging from 63 to 87%
in comparison to before freezing, P < 0.001). For a
discussion of this behaviour see below.
The protective effect of the sugars, after thermal
stress, was more relevant when the cells were exposed to
Table 1. Survival rate of lactic acid bacteria strains to freezing and freeze drying procedures in presence of cryoprotective sugars.
Isotonic solution Trehalose
Maltose
Sucrose
Glucose
(Log c.f.u./ml)% (Log c.f.u./ml)% (Log c.f.u./ml)% (Log c.f.u./ml)% (Log c.f.u./ml)%
Lactose
Pa
(Log c.f.u./ml)%
Lactobacillus delbrueckii subsp. bulgaricus
BF
7.90±0.21 (100) 8.13±0.25 (100)
AF
6.11±0.17* (77) 8.08±0.21 (99)
AFD
5.84±0.19* (73) 7.92±0.20 (95)
Pb
<0.001
0.118
8.13±0.24 (100)
7.90±0.16 (97)
7.36±0.19 (90)
<0.001
8.15±0.18 (100)
7.94±0.18 (97)
7.75±0.20 (95)
<0.001
8.14±0.20 (100)
7.94±0.23 (97)
7.00±0.21** (86)
<0.001
8.13±0.22 (100)
7.92±0.24 (97)
7.60±0.25 (93)
<0.001
Lactobacillus acidophilus
BF
7.54±0.18 (100)
AF
5.54±0.10* (73)
AFD
5.11±0.12* (67)
Pb
<0.001
7.79±0.11 (100)
7.00±0.17 (89)
6.86±0.14 (88)
<0.001
7.54±0.17 (100)
7.10±0.18 (94)
6.78±0.19 (90)
<0.001
7.41±0.18*** (100)
6.90±0.19 (92)
6.55±0.18** (87)
<0.001
7.53±0.21 (100) <0.001
7.37±0.24** (98) <0.001
6.95±0.21 (92)
<0.001
<0.001
7.99±0.22 (100)
7.75±0.27 (97)
6.48±0.20 (81)
<0.001
7.89±0.24 (100)
7.64±0.26 (97)
6.46±0.20 (82)
<0.001
7.95±0.22 (100)
7.74±0.24 (97)
6.54±0.18 (18)
<0.001
7.95±0.24 (100)
7.57±0.22 (95)
6.46±0.18 (82)
<0.001
7.68±0.15 (100)
7.36±0.14** (95)
7.11±0.22 (92)
<0.001
Streptococcus salivarius subsp. thermophilus
BF
7.84±0.25 (100) 7.98±0.23 (100)
AF
7.04±0.19* (89) 7.81±0.23 (97)
AFD
5.01±0.14* (63) 6.97±0.17 (87)
<0.001
<0.001
Pb
0.114
<0.001
<0.001
<0.001
<0.001
Survival rate was expressed as log of colony forming units (c.f.u.)/ml and was reported, for each strain, before freezing (BF), after freezing (AF)
and after freeze-drying (AFD). The results were the mean of three independent experiments. Data were expressed as M±SD. Comparison
between groups by one-way analysis of variance (a=between media and b=between thermal stress comparison).
*P<0.001 isotonic vs. sugars, Turkey test for multiple comparisons.
**P<0.001 vs. all media, Turkey test for multiple comparisons.
***P<0.001 vs. trehalose and maltose, Turkey test for multiple comparisons.
742
B. De Giulio et al.
the action of selective agents, such as lysozyme, pepsin,
and bile salts (that mimicked some events occurring in
the digestive tract) (Table 2). LAB strains utilizable as
probiotics in humans have to survive the low pH of the
stomach and the conjugated bile acids in the duodenum
(Alander et al. 1999). When the bacteria are subjected to
freezing, some membrane regions can present layer
defects with the consequent penetration of molecular
harpoons, constituted essentially by water crystals. So,
freeze-dried bacteria present holes in the membrane and
can be injured by the successive acid pH and bile salts
stresses (Fernandez-Murga et al. 1998; Machado et al.
2004).
In our experiments, L. delbrueckii subsp. bulgaricus
exhibited a greater resistance to 90-min pepsin incubation, when treated with the protective sugars in comparison with isotonic solution (Figure 1). The effect was
especially relevant after freeze-drying, and, among the
cryo-protective sugars tested, trehalose was the most
effective agent (nlog c.f.u. trehalose–sucrose 0.58;
nlog c.f.u. trehalose–lactose 0.94; P < 0.0001). A
comparable effect was observed for L. acidophilus and
S. salivarius subsp. thermophilus (Table 2 panel A).
The effects of lysozyme and bile salts were evaluated
by supplementing the plating media with different
concentrations of these selective agents. For
Table 2. Protective effect of trehalose vs. isotonic solution in presence of stress agents before freezing, after freezing and after freeze drying.
½Log c.f.u. trehalose
ð%Þ
½Log c.f.u. isoton. Sol.
Panel A: Pepsin incubation
Before freezing
After freezing
After freeze-drying
Panel B: Lysozyme treatment
Before freezing
After freezing
After freeze-drying
Panel C: Bile salts treatment
Before freezing
After freezing
After freeze-drying
L. delbrueckii subsp.
bulgaricus
L. acidophilus
S. salivarius subsp.
thermophilus
104±2
115±2**,***
131±5*
112±3***
112±2***
126±1*,***
105±1
108±3***
133±1*
<0.001
133±2***
137±2**,***
163±3*,***
100±7
133±1**,***
152±2*,***
101±3
112±3**,***
130±2*,***
<0.001
132±3***
137±2**,***
175±1*,***
102±1***
133±3**,***
158±2*,***
95±2***
111±2**,***
124±1*,***
<0.001
P
The protective effect of trehalose in the presence of stress agents was evaluated as described in Materials and methods.
In panel A the results about pepsin incubation 90 min, in panel B about lysozyme treatment and in panel C about bile salts treatment were
reported. The results presented in this table were the mean of three independent experiments. Data were expressed as M±SD.
Comparison between groups by two-ways analysis of variance.
*P<0.001 AFD vs. BF and AF; ** P<0.01 vs. BF, Tukey test for multiple comparisons.
***P<0.001 vs. other strains, Tukey test for multiple comparisons.
Figure 1. Pepsin treatment was performed as described in Materials and methods and the enumeration of colony forming units (c.f.u.)/ml was
carried out before freezing (BF), after freezing (AF) and after freeze-drying (AFD). Percent standard deviation did not exceed 5%. Comparison
between media within each thermal stress: F between 3.57 and 9.79, P < 0.001, one-way analysis of variance. *P 0.001 vs. isotonic solution;
§
P < 0.001 vs. trehalose, Tukey test for multiple comparisons.
Lactic acid bacteria preservation
L. delbrueckii subsp. bulgaricus, during exposure to both
bile salts (Figure 2) and lysozyme (Figure 3), the sugars
showed a significant protective effect during the thermal
stress (F between 44.1 and 139.4, P between 0.007 and
0.0001, one-way analysis of variance).
The cryo-protective effect in the presence of lysozyme
and bile salts (Table 2, panel B and C, P < 0.001
between strains comparison) was more evident for
L. delbrueckii subsp. bulgaricus [% (log c.f.u. trehalose/log c.f.u. isotonic solution) 163 and 175 respectively], and L. acidophilus [% (log c.f.u. trehalose/log
743
c.f.u. isotonic solution) 152 and 158 respectively] than
for S. salivarius subsp. thermophilus [% (Log c.f.u.
trehalose/Log c.f.u. isotonic solution) 130 and 124
respectively]. The different behaviour towards different
agents, as observed therefore for micro-organisms of
genus Propionibacterium (Jan et al. 2001; Leverrier et al.
2005), could be ascribed to the differences in the
membrane components for S. salivarius subsp. thermophilus in comparison to the Lactobacillus strains.
So, our data showed that pretreatment with the sugar
solutions provided a general improvement of the cell
Figure 2. The effect of lysozyme was evaluated by supplementing the plating media with this selective agent and by enumeration of colony
forming units (c.f.u.)/ml before freezing (BF), after freezing (AF) and after freeze-drying (AFD). Percent standard deviation did not exceed 5%.
Comparison between media within each thermal stress: F between 44.1 and 139.4, P<0.001, one-way analysis of variance. *P 0.001 vs. isotonic
solution; §P < 0.001 vs. trehalose, Tukey test for multiple comparisons.
Figure 3. The effect of bile salts was evaluated by supplementing the plating media with this selective agent and by enumeration of colony
forming units (c.f.u.)/ml before freezing (BF), after freezing (AF) and after freeze-drying (AFD). Percent standard deviation did not exceed 5%.
Comparison between media within each thermal stress: F between 50.7 and 125.6, P<0.001, one-way analysis of variance. *P 0.001 vs. isotonic
solution; §P < 0.001 vs. trehalose, Tukey test for multiple comparisons.
744
B. De Giulio et al.
viability after thermal stress in the strains under
investigation, and, moreover, a greater resistance to
chemical (bile salts) and enzyme (pepsin and lysozyme)
attacks. The protective effect of cryo-preservative agents
is potentially relevant, in order to use these strains for
probiotic formulations.
Among the different sugars used in our experimentation, trehalose seemed to be the most promising. This
disaccharide, unlike the other sugars, does not produce
moisture during the freeze-drying process. The presence
of residual water crystals could cause some damages to
the bacterial structure during thawing. Trehalose is
capable of preventing the damages occurring to biomolecules during dehydration by replacing the water
molecules; it also forms a thin, glass-like layer which,
enveloping the bio-structures, limits their intramolecular
mobility thus preserving their functional conformations
(Crowe et al. 1988). Some authors (Colaco et al. 1994;
Leslie et al. 1995) have also demonstrated a rapid influx
of trehalose into bacterial cells upon cooling.
In addition, in recent years, some patented biotechnological processes related to trehalose production, have
decreased its price from about 400 $/kg to a cost very
similar to sucrose, thus allowing a wider diffusion on the
world market for this sugar. Sucrose, the other nonreducing disaccharide tested, for its high energy bond
level (nG ¼ 27 kcal/mol) can be easily split to the
reducing monosaccharides glucose and fructose in the
presence of the chemically reactive amino-groups of
proteins, leading to progressive chemical damages due
to browning reactions (Crowe et al. 1988).
Finally, the protective effect of LAB immobilization
in Ca-alginate beads was compared to that exercised by
trehalose. As shown in Table 3, for all the strains tested,
a lower cryo-preservative effect of Ca-alginate ICT,
worsened after freeze-drying, was observed in comparison to trehalose treatment [% (log c.f.u. alginate/log
c.f.u. trehalose) between 80 and 94, P< 0.0001, column
AFD, string ‘without stress agent’]. When cells were kept
together with the selective agents, the greater protective
effect of trehalose was more evident. In particular, this
effect was relevant for L. delbrueckii subsp. bulgaricus
relatively to lysozyme treatment, after freeze-drying
[% (log c.f.u. alginate/log c.f.u. trehalose) 72.7,
P < 0.0001]. However, since alginates are commercially
available with different molecular weight distributions,
content in mannuronic and guluronic acid and purification grade, the results could be ascribed to the choice of
the type of alginate (Diefenbach et al. 1992). In fact,
data obtained with different alginates purified and
characterized in our laboratory for their guluronate
and mannuronate content and molecular weight distribution, suggest that the protective effect should be also
related to the fine chemical structure of the alginate
molecule (Nazzaro et al. 2001). The behaviour of
alginate in comparison to trehalose could be also
ascribed to the different moisture content, present in
the sample. In fact, in relation to the guluronic acid
Table 3. Protective effect of Ca-alginate ITC in comparison with trehalose treatment before freezing (BF), after freezing (AF) and after freezedrying (AFD).
½Log c.f.u. alginate
ð%Þ
½Log c.f.u. trehalose
BF
AF
AFD
P
Lactobacillus delbrueckii subsp. bulgaricus
Without stress agent
96.6±0.5
Lysozyme
94.8±2.4
Bile salts 0.5%
88.2±2.2***
Pepsin 900
94.5±2.8
98.0±1.0***
80.1±1.8**,***
85.4±2.6**
85.0±2.4**
80.3±2.4*
72.7±1.6*,***
78.1±2.0*
85.1±2.6**,***
<0.0001
Lactobacillus acidophilus
Without stress agent
Lysozyme
Bile salts 0.5%
Pepsin 900
99.4±3.3***
85.3±2.7
82.6±2.3**,****
88.9±2.9**
94.4±2.5*,***
84±2.9
84.7±2.3**
82.5±2.7*
<0.0001
87.3±2.8
87.6±3.0
86.6±2.8**
87.4±2.4**
86.1±2.9**
87.6±2.9
79.6±2.9*,*****
81.1±2.8*,*****
<0.0001
99.7±3.5***
85.6±2.5***
92.8±3.2
91.3±3.1
Streptococcus salivarius subsp. thermophilus
Without stress agent
90.5±3.1
Lysozyme
90.6±2.9
Bile salts 0.5%
91.4±3.1
85.4±2.8***
Pepsin 900
The protective effect of Ca-alginate ITC in the presence of stress agents in comparison with trehalose was reported, for each strain, before
freezing (BF), after freezing (AF) and after freeze drying (AFD). The results presented in this table were the mean of three independent
experiments. Data were expressed as M±S.D.
Comparison between groups within each strain by two-ways analysis of variance.
*P between 0.01 and 0.0001, AFD vs. BF and AF; **P<0.01 vs. BF, Tukey test for multiple comparisons.
***P<0.001 vs. other stress agents; ****P<0.001 vs. pepsin; *****P<0.001 vs. without stress agent and lysozyme, Tukey test for multiple
comparisons.
Lactic acid bacteria preservation
content and the immobilization technique used, freezedried preparations of microbial strains immobilized in
Ca-alginate beads, can present a moisture content higher
in comparison to microbial preparations freeze dried in
presence of trehalose, for which it is normally calculated
a moisture value <1%. This could affect the different
answer of bacteria, during freezing and freeze drying
(Champagne & Gardner 2001).
In conclusion, trehalose, in comparison to some common sugars, such as sucrose or maltose, and to calcium
alginate, for its chemical features, low hygroscopicity and
low cost, could be utilized as an excellent bacterial
preserving agent. Trehalose could enhance the performance of starter cultures in the food industry and allow
for the preparation of probiotic formulations more
resistant to the different stress conditions found along
the gastric tract (i.e. bile salts and/or gastric juices).
Acknowledgements
We express our sincerest thanks to Dr Nicola De Marchi
for his excellent comments on an earlier draft of this
paper.
The work was partially supported by: Research
Project cluster 26 ‘Innovative materials’ workpackage
no. 2 from the Italian Ministry of University and
Scientific Research, Research project of Italian Ministry
of University and Scientific Research (art. 51 (9), 27 /12/
97, n. 449). Year 1999. ‘Treatment of highly perishable
fresh foods to guarantee their quality, safety and health
(PROFSICURI)’ and the project ‘Life-Environment’
Bio. Co. Agri n. 377.
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