Determination of optimum conditions for preservation and reactivation of freeze-dried Acetobacter senegalensis used as vinegar starter

Availability of efficient starters is one of the most important elements during fermentation technology.
Acetic acid bacteria (AAB) face extreme conditions (low pH, high acetic acid concentration (>7%),
ethanol and high temperature) during vinegar fermentation. In spite of introduction of broth media
contained viable AAB as starter by some companies; traditional methods for initiation of vinegar
fermentation are still used even in modern factories, therefore there is a long Lag phase for initiation of
fermentation. In recent studies in CWBI, a kind of lyophilized starter has been produced by using an
isolated thermotolerant bacterium: Acetobacter senegalensis. The aim of this study is to determine the
best media for revitalization and the optimum temperature for preservation of the freeze-dried cells.
To produce biomass, glucose was used as carbon source in batch culture under regulated conditions. The
cells harvested when they reached to stationary phase. Harvested cells suspended in spent growth medium
(final supernatant of fermentation). Maltodextrin (10%), manitol (20%) and spent growth medium were
used as protestants. After freeze-drying they were subjected to storage test at -20° C, 4° C and 35° C for
six months. The viability of cells determined by using spread plate technique using three different media:
YG (yeast extract, glucose) and YGE (yeast extract, glucose, ethanol) and YGEA (yeast extract, glucose,
ethanol, acetic acid).
According to residual viable cells, manitol and maltodextrin showed higher protective functions rather
than spent growth medium (92.3%, 88.2% and 82.1% survival, respectively) during freeze-drying
process. Viability of cells during rehydration is completely dependent to the composition of the broth
media used for this purpose. Adding ethanol (>0.5%) or acetic acid (>1%) to rehydration medium caused
40-45% reduction in viable cell numbers in comparison to YG broth.
The composition of culture media can also influence the growth of bacteria after rehydration. In YG,
much higher cell growth (about 1 log unit) was observed in comparison to YGE and YGEA. Addition of
glucose (20 g/l) to YGE can neutralize the adverse effect of ethanol considerably but it cannot improve
the growth in YGEA. This can be partially explained according to the activity of alcohol dehydrogenase
and acetaldehyde dehydrogenase. It was revealed that freeze-dried cells had very low activities for these
two enzymes.
Analysis of total soluble protein contents of lyophilized cells during storage at different conditions
revealed that the soluble protein content of cells reduced by increasing the storage temperature. At 35° C
after 15 days, there were 4.87 log units reduction in the viability of cells, and also about 14% less soluble
protein was detectable under this condition whereas keeping of cells at -20 or + 4° C had no influences
on protein and viability of cells. Storage of cells at +4° C in darkness for six month showed that about
79.2% and 68.3% viability of cells remained for cells protected by manitol and maltodextrin, respectively.
To assess the real activity of lyophilized cells as starters, they were introduced to broth media (YGE
contained 20g/l of glucose). It was seen that 6 months old lyophilized cells (with maltodextrin or manitol
as protectants) were able to grow and consume 5 v/v of ethanol in 48 hr in the presence of initial acetic
acid concentration (0.5%) at 30° C.
In conclusion, the mentioned methods for preservation and reactivation of freeze-dried Acetobacter
senegalensis can provide a promising tool for decreasing the lag phase of vinegar fermentation.