Browning of freeze-dried probiotic bacteria cultures in relation to loss of viability during storage.

Freeze-dried cultures of Lactobacillus acidophilus (La-5) showed visible brown discoloration even after a short storage at relatively mild conditions (a(w) = 0.22 and 30 degrees C), and the browning processes were found to coincide with bacteria inactivation. It was demonstrated, by using high-pressure treatment for obtaining bacteria samples with different ratios of live/dead bacteria, that death of bacteria is not a prerequisite for the browning processes. Furthermore, it was shown that hydroxymethylfurfural (HMF) (or condensation products of HMF) introduces accelerated viability loss when HMF is added to the freeze-drying medium. Discoloration of bacteria cultures containing only sucrose/maltodextrin or lactose/maltodextrin in the freeze-drying matrices is suggested to be related to various types of nonenzymatic browning reactions, including carbonyl-protein (or carbonyl-DNA) interactions and carbohydrate condensation/polymerization (without involvement of proteins), the latter proceeding at low a(w) following hydrolysis of the peptidoglycan layer in the bacteria cell wall. More than one single type of browning reaction is accordingly concluded to be related to bacteria death, and the loss of viability in freeze-dried bacteria seems to be influenced by oxidation reactions, browning reactions, and the physical instability of the bacteria membrane/cell wall.

Investigation of oxidation in freeze-dried membranes using the fluorescent probe C11-BODIPY(581/591).

Incorporation of the fluorescent probe C11-BODIPY(581/591) in two dried membrane systems, soy bean phosphatidylcholine liposomes freeze-dried in a carbohydrate/protein matrix and Lactobacillus acidophilus (La-5) freeze-dried in a carbohydrate matrix, was successful and could be visualised by Confocal Laser Scanning Microscopy (CLSM). The C11-BODIPY(581/591) probe is a lipid oxidation reporter molecule, which is known to associate with the lipids of biological membranes and exhibit a fluorescence shift from the red range to the green range of the visible spectrum when it is oxidised together with the lipids. The present study is the first to demonstrate that the C11-BODIPY(581/591) probe can be used in dried membrane systems, and that a detection of oxidation is possible by CLSM analysis directly on the dried samples.

Water activity-temperature state diagrams of freeze-dried Lactobacillus acidophilus (La-5): influence of physical state on bacterial survival during storage.

Water activity-temperature state diagrams for Lactobacillus acidophilus freeze-dried in a sucrose or a lactose matrix were established based on determination of stabilized glass transition temperatures by differential scanning calorimetry during equilibration with respect to water activity at fixed temperatures. The bacteria in the lactose matrix had higher stabilized glass transition temperatures for all a(w) investigated. The survival of Lactobacillus acidophilus determined as colony forming units for up to 10 weeks of storage at 20 degrees C for (i) a(w) = 0.11 with both freeze-dried matrices in the glassy state, (ii) a(w) = 0.23 with the bacteria in the lactose matrix in a glassy state but with the bacteria in sucrose matrix in the nonglassy state, and (iii) a(w) = 0.43 with both freeze-dried matrices in a nonglassy state showed that the nature of the sugar was more important for storage stability than the physical state of the matrix with the nonreducing sucrose providing better stability than the reducing lactose.

Storage stability of freeze-dried Lactobacillus acidophilus (La-5) in relation to water activity and presence of oxygen and ascorbate.

Storage stability of freeze-dried Lactobacillus acidophilus was found to depend on water activity (0.11-0.43), oxygen level (atmospheric oxygen level and <4% oxygen compared) and presence of sodium ascorbate (0% and 10% (w/w)). Increasing water activities decreased bacterial survival, and a reduced oxygen level (<4% oxygen) improved the storage stability, which strongly indicates a connection between oxidative reactions and bacterial instability. The detrimental effect of atmospheric oxygen was reduced by including ascorbate in the freeze drying medium. However, when ascorbate was present a pink/red colour was observed on the surface of the dried samples increasing with the water activity and oxygen level. Increased water activity lead to increased browning also for samples without ascorbate. Free radicals were detected in the dried bacteria by ESR spectroscopy (broad single-peak ESR spectra), where the shape and the g-value was found to depend on the presence of ascorbate and the extent of browning. For increasing water activities the content of radicals increased to a certain level, after which it levelled off and/or decreased. The highest concentrations of radicals were detected in the dried bacteria with highest survival for a given water activity, i.e. low oxygen level and presence of ascorbate, pointing towards a role of semi-stable ascorbyl radicals as a "dead end" for otherwise detrimental free radical reactions.

Impact of water activity, temperature, and physical state on the storage stability of Lactobacillus paracasei ssp. paracasei freeze-dried in a lactose matrix.

The aim of this study was to determine whether the combined effect of water activity and temperature on inactivation rates of freeze-dried microorganisms in a lactose matrix could be explained in terms of the glass transition theory. The stabilized glass transition temperature, Tg, of the freeze-dried products was determined by differential scanning calorimetry at two different temperatures, T (20 and 37 degrees C), and different water activities (0.07-0.48). This information served as a basis for defining conditions of T and water activity, which led to storage of the bacteria in the glassy (T < Tg) and nonglassy (T > Tg) states. The rates of inactivation of the dry microorganisms subjected to different storage conditions were determined by plate counts and could be described by first-order kinetics. Rates were analyzed as a function of water activity, storage temperature, and the difference between Tg and T. Inactivation below Tg was low; however, Tg could not be regarded as an absolute threshold of bacteria stability during storage. When the cells were stored in the nonglassy state (T > Tg), inactivation proceeded faster, however, not as rapid as suggested by the temperature dependence of the viscosity above the glass transition temperature. Furthermore, the first-order rate constant, k, was dependent on the storage temperature per se rather than on the temperature difference between the glass transition temperature and the storage temperature (T - Tg).