Ph.D in Biology Azizpour K.
Artemia and Aquatic Animals Research Institute,
Effective factors
for the preparation of
freeze-dried
Probiotic (based on lactic acid bacteria) for application in aquaculture
Abstract
The industrial exploitation of lactic
acid bacteria (LAB) as starter and/or probiotic cultures depends strongly on
the preservation technologies employed, which are required to guarantee
long-term delivery of stable cultures in terms of viability and activity.
Freeze-dried preparations exhibit
advantages relative to preparations made with other techniques in terms of
long-term preservation, coupled with convenience in handling, storage,
marketing and application. Degrees of survival of LAB cultures as high as
possible, during drying and subsequent storage, are thus of nuclear importance,
both technologically and economically.
Introduction
The viability of dried
cultures depends also on the method employed to rehydrate them, as survival is
increased after slow rehydration [3, 4]. Finally, heat shock increases survival
of exponential phase cells during spray-drying. However, exponential phase
cells are not as resistant to spraydrying, even after heat shock, as cells
harvested in the stationary phase [1, 2].
This paper results of the
research done on the improvement of LAB survival during freeze-drying and
subsequent storage, including specific sections on growth factors, sub-lethal
treatments, drying medium, storage and rehydration.
1. Growth factors
Several factors have been identified
which may explain the protection afforded by each of various growth media, e.g.
accumulation of compatible solutes (which involves type of sugar substrate
present and occurrence of osmotic stress, among other factors), production of exopolysaccharides,
and altered fatty acid profile of the membrane. Each of these factors will be
detailed in the following subsections.
1.1. Accumulation of compatible
solutes
1.2. Exopolysaccharide production
1.3. Altered membrane profile
2. Sub-lethal treatments
The relationship between
final pH of growth of Lb. bulgaricus and survival through spray-drying and storage
of the dried cells was evaluated. These authors have shown that, in
noncontrolled pH fermentation runs (bearing a final pH of 4.5), the cells were
more resistant to heat stress, spray drying and storage in the dried state than
those from cultures under controlled pH (set to 6.5). These findings seem to
indicate that acid shock, or adaptation there to may yield cells which exhibit
alternative physiological states, and hence potentially different tolerances to
other stresses.
3. Drying medium
Freeze-drying has for
long been considered as a suitable dehydration process for bacteria, with the
ultimate goal of achieving a solid and stable final formulation. The choice of
an appropriate drying medium is thus very important in the case of LAB, so as
to increase their survival rates during dehydration itself and subsequent
storage. For most LAB cultures of commercial interest for the dairy industry,
skim milk powder is selected as drying medium because it (i) prevents cellular
injury by stabilizing the cell membrane constituents, (ii) creates a porous
structure in the freeze-dried product that makes rehydration easier and (iii)
contains proteins that provide a protective coating for the cells.
3.1. Sugars and sugar derivatives
present in the drying medium
3.2. Other compounds
4. Storage and rehydration
An organism which
survives the various steps of freezing, drying and storage may, nevertheless,
lose its viability during rehydration. Therefore, rehydration is a critical
stepin the recovery of freeze-dried microorganisms, because cells that were
subjected to sublethal injury may not be able to repair said damage if they are
rehydrated under inappropriate conditions. The rehydration solution itself (in
terms of osmolarity, pH and nutritional energy source), as well as the
rehydration conditions (in terms of rehydration temperature and volume) may
significantly affect the rate of recovery to the viable state, and thus
influence survival rates.
5. Practical recommendations for
preparation of freeze-dried LAB
Optimum protocols for the preparation
of freeze-dried LAB vary widely between species, and even between strains.
However, in view of the results reviewed above, a basic (necessarily general)
methodology for the preparation, storage and rehydration offreeze-dried LAB is
suggested.
5.1. Growth conditions
5.2. Drying conditions
5.3. Storage conditions
5.4. Rehydration conditions
Conclusions
The mechanisms underlying damage and
protection by freezing, drying and storage are indeed complex, and not fully
understood to date. However, evidence has been made available which proves that
suitable selection of the composition of the growth and drying media is
essential to afford protection during storage of freezedried cells. In
addition, information on the sites of impact and the nature of the injury
produced by a variety of stressful conditions (e.g. freezing, drying, storage
or rehydration), together with knowledge of the induction of stress proteins
(particularly those which provide resistance during drying and subsequent
storage) are definitively important towards production of dried starter
cultures, which will be characterized by high survival rates even after
extended storage.
References:
1. Rince, A.,
Flahaut, S., & Auffray, Y. (2000). Identification of general stress genes
in Enterococcus faecalis. International
Journal of Food Microbiology, 55,
127–131.
2. Silva, J., Carvalho, A.
S., Domingues, P., Ferreira, R., Vitorino, R., Teixeira, P., & Gibbs, P. A.
(2003). Effect of the pH of growth on the resistance of
Lactobacillus delbrueckii spp. Bulgaricus to stress conditions. Applied
and Environmental Microbiology, submitted.
3. Teixeira, P., Castro, H.,
& Kirby, R. (1995a). Spray drying as a method for preparing concentrated
cultures of Lactobacillus bulgaricus. Journal of Applied Microbiology, 78,
456–462.
4.