produce trehalose died much faster than the wild type at 4
C due to lack of these two
genes otsA and otsB. But when the mutant strain of E. coli was transformed with otsA
and otsB genes, the cells acquired the trehalose synthesizing capability with its
viability restored at 4
C (Kaasen et al. 1992). E. coli under cold shock conditions
and as a consequence of the accumulation of trehalose increase the cells viability as
temperature declines to near freezing. Therefore, it may be assumed that increase of
trehalose accumulation in E. coli is probably responsible for higher viability at low
temperature (Kaasen et al. 1992; Mitta et al. 1997).
6.13.1.2 Amino Acid Cryoprotectants
Glycine betaine is a cryoprotectant of bacterial origin and was first demonstrated in
the food-borne pathogen Listeria monocytogenes which survives at low
temperatures and high osmolarity (Angelidis and Smith 2003). Angelidis and
Smith (2003) observed that more than 100 colonies appeared on culture plates
with betaine after 32 days of incubation at 7
C, while there was no growth without
betaine. The exact mechanism of action of glycine betaine is not yet clear. However,
it is thought to function as a chemical chaperone, which prevents the aggregation of
cellular proteins during stress conditions. The possible function of glycine betaine is
to regulate the fluidity of membrane at lower temperatures (Chattopadhyay 2002).
In bacteria proline is an important natural compatible solute during osmotic stress
(Yoshiba et al. 1997; Kempf and Bremer 1998). Higher level of intracellular proline
has been reported in microorganisms to improve freeze tolerance (Morita et al. 2003;
Nanjo et al. 1999). This indicates that proline accumulation might be a general
protective mechanism against freeze stress. In addition, intracellular accumulation of
charged amino acid arginine and glutamate also seems to enhance microbial freeze
tolerance (Shima et al. 2003).
6.13.2 Role of Ice Nucleation in Freeze Tolerance
The term ice nucleation describes the initiation of the phase transition of water from
a liquid to a solid state. When a water sample of moderate size is cooled, it will
normally not freeze at 0
C. If the water is pure, it can be cooled to temperatures near
to À40
C before it freezes. Liquid water at temperatures lower than 0
C is termed
supercooled water and this supercooled state is metastable. To enable ice formation
to take place, water molecules must cluster in an ice-like pattern and this cluster must
reach a critical size. If the initial aggregation of water molecules takes place on a
foreign structure, the process is termed heterogeneous ice nucleation. If the water
molecules aggregate without the help of another structure, the nucleation is termed
homogenous (Lundheim 2002). A structure that organizes water into an ice-like
pattern so that nucleation takes place is called an ice nucleator and responsible for
heterogeneous ice nucleation (Fig. 6.2). However, the term is usually not used for
substances inducing ice nucleation at temperatures lower than À10
C. Many bacteria have ability to minimize freezing injury due to extracellular ice formation and
impose an ice like arrangement on the water molecule in contact with their surface
204
P. K. Mishra et al.
C due to lack of these two
genes otsA and otsB. But when the mutant strain of E. coli was transformed with otsA
and otsB genes, the cells acquired the trehalose synthesizing capability with its
viability restored at 4
C (Kaasen et al. 1992). E. coli under cold shock conditions
and as a consequence of the accumulation of trehalose increase the cells viability as
temperature declines to near freezing. Therefore, it may be assumed that increase of
trehalose accumulation in E. coli is probably responsible for higher viability at low
temperature (Kaasen et al. 1992; Mitta et al. 1997).
6.13.1.2 Amino Acid Cryoprotectants
Glycine betaine is a cryoprotectant of bacterial origin and was first demonstrated in
the food-borne pathogen Listeria monocytogenes which survives at low
temperatures and high osmolarity (Angelidis and Smith 2003). Angelidis and
Smith (2003) observed that more than 100 colonies appeared on culture plates
with betaine after 32 days of incubation at 7
C, while there was no growth without
betaine. The exact mechanism of action of glycine betaine is not yet clear. However,
it is thought to function as a chemical chaperone, which prevents the aggregation of
cellular proteins during stress conditions. The possible function of glycine betaine is
to regulate the fluidity of membrane at lower temperatures (Chattopadhyay 2002).
In bacteria proline is an important natural compatible solute during osmotic stress
(Yoshiba et al. 1997; Kempf and Bremer 1998). Higher level of intracellular proline
has been reported in microorganisms to improve freeze tolerance (Morita et al. 2003;
Nanjo et al. 1999). This indicates that proline accumulation might be a general
protective mechanism against freeze stress. In addition, intracellular accumulation of
charged amino acid arginine and glutamate also seems to enhance microbial freeze
tolerance (Shima et al. 2003).
6.13.2 Role of Ice Nucleation in Freeze Tolerance
The term ice nucleation describes the initiation of the phase transition of water from
a liquid to a solid state. When a water sample of moderate size is cooled, it will
normally not freeze at 0
C. If the water is pure, it can be cooled to temperatures near
to À40
C before it freezes. Liquid water at temperatures lower than 0
C is termed
supercooled water and this supercooled state is metastable. To enable ice formation
to take place, water molecules must cluster in an ice-like pattern and this cluster must
reach a critical size. If the initial aggregation of water molecules takes place on a
foreign structure, the process is termed heterogeneous ice nucleation. If the water
molecules aggregate without the help of another structure, the nucleation is termed
homogenous (Lundheim 2002). A structure that organizes water into an ice-like
pattern so that nucleation takes place is called an ice nucleator and responsible for
heterogeneous ice nucleation (Fig. 6.2). However, the term is usually not used for
substances inducing ice nucleation at temperatures lower than À10
C. Many bacteria have ability to minimize freezing injury due to extracellular ice formation and
impose an ice like arrangement on the water molecule in contact with their surface
204
P. K. Mishra et al.