Charlotte U. Carlsen

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 °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).

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 aw investigated. The survival of Lactobacillus acidophilus determined as colony forming units for up to 10 weeks of storage at 20°C for (i) aw = 0.11 with both freeze‐dried matrices in the glassy state, (ii) aw = 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) aw = 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.

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.

Light sensitivity of elderberry extract. quantum yields for photodegradation in aqueous solution

The quantum yield for photobleaching of a commercial elderberry (Sambucus nigra L.) extract, mainly composed of anthocyanins, has been determined at 25°C for air-saturated 10 mM citrate buffer solutions, using monochromatic light at each of the wavelengths 313 nm, 366 nm, and 436 nm, respectively, in continuous photolysis experiments, in order to provide an objective measure for the sensitivity of this food colorant to ultraviolet and visible light. In the pH region investigated, pH 3.0-3.8, typical of fruit-based products, the quantum yield for photodegradation was found to depend only on the wavelength of irradiation, being 2.1 ±0.2.10-4 mol·einstein-1 at 313nm, 1.6±0.2.10-4mol.einstein-1 at 366 nm, and 2.8±0.6.10-5mol.einstein-1 at 436 nm, whereas solution pH only affects the colour intensity. Results show that light bleaching will be the major destabilising factor for anthocyanin-coloured products in display and that exclusion of uv-light will greatly improve their colour stabilty.

Peptides as antioxidants and carbonyl quenchers in biological model systems

Subjecting selected peptides to in vitro analyses covering their ability to interfere with the lipid oxidation chain reaction as well as to protect proteins from direct and indirect oxidation has provided the basis for a more detailed understanding of peptide-mediated protection in biological systems. The efficiency of peptides as radical scavengers and chain-breaking antioxidants in oxidizing lipid membranes was found to be low. Previous studies on antioxidative activity of peptides tend not to include comparisons with efficiencies of more well-documented antioxidants and/or use irrelevantly high dosages of peptides. The present study demonstrates that the effect of the investigated peptides towards oxidation in biological membrane systems is mainly a protection of vital proteins from being oxidatively modified. This protection is obtained through a prevention of lipid oxidation derived carbonylation (indirect protein oxidation) and through interference with aqueous radical species (direct protein oxidation), and it is only achieved if the peptides are present in high concentrations as sacrificial antioxidants.

Investigation of oxidation in freeze-dried membranes using the fluorescent probe C11-BODIPY581/591

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.