The effect of sodium chloride on molecular mobility in amorphous sucrose detected by phosphorescence from the triplet probe erythrosin B

You, Yumin; Ludescher, Richard D.

doi:10.1016/j.carres.2007.11.005

Cited by 18 publications

(17 citation statements)

References 59 publications

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“…No crystalline material was detected in the Ficoll based formulation. NaCl can form complexes with the hydroxyl moieties in carbohydrates 9,10 . In Ficoll and HEC-based formulations the NaCl to hydroxyl moiety molar ratio was 1:11 and 1:1, respectively, explaining why no crystallization was detected in the Ficoll-based formulation.…”

Section: Discussionmentioning

confidence: 99%

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Wessman

Håkansson

Leifer

et al. 2013

JoVE

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Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled 1 . Video LinkThe video component of this article can be found at http://www.jove.com/video/4058/ Protocol 1. Cultivation and harvest of P. putida 1. Prepare a starter culture of Pseudomonas putida by inoculating 100 ml of tryptic soy broth (TSB) with a colony of P. putida cells cultured on agar supplemented with tryptic soy broth (TSA). Keep the culture at 30 °C on a shaking board set at 130 rpm. 2. After 7 hr transfer an aliquot from the starter culture equivalent to 1/10 of the final volume of the main culture to fresh TSB-medium. Keep the cells at 30 °C on a shaking board at 130 rpm for 16 hr. 3. After 16 hr of growth the cells are harvested by spinning the cell culture at 1,500 x g for 20 min at RT. Decant the supernatant and wash the cells by re-suspending the cell pellet is in a NaCl-solution that is isotonic to the growth medium. In this case a 150 mM NaCl solution was used. The cells are then centrifuged once more and the supernatant is decanted before resuspending the cells in the formulation medium, see step 3.2. Cultivation of other species1. To adapt the protocol to other bacterial species the cultivation and harvest procedures should be changed according to the requirements of that species. To avoid osmotic shock when handling the cells during harvest and the washing step(s), the osmolality of the solutions needs to match that of the growth medium at harvest. Formulation of cells1. Prepare the formulation solutions by weighing out the respective matrix components and dissolve them in water. To achieve isotonic conditions, eit...

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Section: Discussionmentioning

confidence: 99%

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Wessman

Håkansson

Leifer

et al. 2013

JoVE

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“…Data were fit to a stretched exponential, or Kohlrausch-Williams-Watts (KWW) decay model, 44 which has been used to fit the intensity decays of Ery B dispersed in a wide variety of amorphous solid sugars 21,26,32,33,[37][38][39][40] and proteins 23,34-36 :…”

Section: Methodsmentioning

confidence: 99%

“…We have used Ery B phosphorescence to study molecular mobility in a wide range of amorphous biomaterials including sugars, 26,32,33 proteins 23,34-36 and in mixtures. [37][38][39][40] In this report, we analyze recent work from our laboratory using phosphorescence from Ery B to probe molecular mobility and oxygen permeability in amorphous solid films of bovine serum albumin (BSA), 34 β-lactoglobulin (βLg), 23 α-lactalbumin (αLa), 41 glycinin 11S storage protein of soybean (11S) (unpublished data), and porcine high bloom gelatin. 31 These proteins were selected in part because of their relevance to food systems and because they provide a range of size (from 14 to 360 kDa), structure (soluble globular, insoluble globular, and fibrous), and function (transport, storage, and structural).…”

Section: Introductionmentioning

confidence: 98%

Photophysical Probes of the Amorphous Solid State of Proteins

et al. 2010

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The properties of amorphous solid proteins influence the texture and stability of low-moisture foods, the shelf-life of pharmaceuticals, and the viability of seeds and spores. We have investigated the relationship between molecular mobility and oxygen permeability in dry food protein films-bovine α-lactalbumin (α-La), bovine β-lactoblobulin (β-Lg), bovine serum albumin (BSA), soy 11S globulin, and porcine gelatin-using phosphorescence from the triplet probe erythrosin B. Measurements of the phosphorescence decay in the absence (nitrogen) and presence (air) of oxygen versus temperature provide estimates of the non-radiative decay rate for matrixinduced quenching (k TS0 ) and oxygen quenching (k Q [O 2 ]) of the triplet state. Since the oxygen quenching constant is the product of the oxygen solubility ([O 2 ]) and a term (k Q ) proportional to the oxygen diffusion coefficient, it is a measure of the oxygen permeability through the films. For all proteins except gelatin, Arrhenius plots of k TS0 reveal a gradual increase of apparent activation energy across a broad temperature range starting at ∼50°C; this suggests that there is a steady increase in the available modes of molecular motion with increasing temperature within the protein matrix. Arrhenius plots for k Q [O 2 ] were linear for all proteins with activation energies ranging from 24 to 29 kJ/mol. The magnitude of the oxygen quenching constants varied in the different proteins; the rates were approximately 10-fold higher in α-La, β-Lg, and BSA than in 11S glycinin and gelatin. Although the rate of oxygen permeability was not directly affected by the increased mobility of the protein matrix, plots of k Q [O 2 ] versus k TS0 were linear over nearly three orders of magnitude in the protein films, suggesting that the matrix mobility plays a specific role in modulating oxygen permeability. This effect may reflect differences in matrix-free volume that directly influence both mobility and oxygen solubility.

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“…The principle is that phosphorescence spectroscopy of triplet probes can examine the slow mobility motions occurring on the milliseconds to seconds time scale in highly viscous glasses and melts. Ery B is a food grade phosphorescence probe which has been found to be very sensitive to the molecular mobility in amorphous sugars [13][14][15][16][17][18][19][20]22,25,26 and proteins 27,28 . By embedding Ery B within the amorphous matrix, we can collect signals from probe molecules that are surrounded by matrix molecules and respond to the slow modes of mobility in local environments.…”

Section: Glass Transition Temperatures Of Oligosaccharides Asmentioning

confidence: 99%

“…Our studies indicate that matrix composition (concentration of sugars, sugar alcohols, and molecules of different molecular size and structure) has a complex influence on molecular mobility. The dynamic effect of matrix composition varies depending on the properties of the matrix molecules, the concentration of additive, and the temperature [14][15][16][17][18][19][20] . Composite matrixes composed of sucrose and glycerol 16 , of maltose and maltitol 14 , or of lactose and lactitol 15 , for example, all show lower molecular mobility than the corresponding matrixes composed of the pure sugar, while a sucrose matrix containing added biopolymer, either gelatin 19 , xanthan 20 , amylose, or maltodextrin 21 , displayed higher molecular mobility under some concentration conditions than the pure sucrose matrix.…”

Section: Introductionmentioning

confidence: 99%

The Effect of Molecular Size on Molecular Mobility in Amorphous Oligosaccharides

You

Ludescher

2010

Food Biophysics

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The physical properties and especially the molecular mobility of amorphous carbohydrate matrixes directly influence the stability of foods, feeds, and pharmaceuticals and the dessication tolerance of animals and plants during anhydrobiosis. Phosphorescence of the sodium salt of erythrosin B was used to investigate the local molecular mobility in pure amorphous solids of a homologous series of malto-oligosaccharides (maltose, G 2 ; maltotriose, G 3 ; maltotetraose, G 4 ; maltopentaose, G 5 ; maltohexaose, G 6 ; and maltoheptaose, G 7 ); sucrose and maltodextrin DE18 (a hydrolytic fraction of starch) were investigated for comparison. Measurements of the temperature-dependence of the phosphorescence emission energy, an indicator of the extent of local dipolar relaxation, and the phosphorescence emission lifetime, an indicator of the rate of collisional quenching of the excited state by matrix molecules, demonstrate that the local matrix molecular mobility increases with molecular size, and thus, with an increase in T g , in these glucose oligosaccharides. Master curves of the spectroscopic measures of matrix mobility for each oligosaccharide, plotted against T-T g , were not superimposable, suggesting that local properties of the amorphous sugar matrixes, rather than T g per se, influence local matrix mobility. Indicators of spectral heterogeneity also varied with molecular size, indicating that dynamic site heterogeneity also increased with molecular size and thus, T g . These results emphasize the importance of additional research in developing appropriate "molecular rules" for designing amorphous matrix systems with better long-term stability for foods, feeds, or pharmaceuticals.

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The effect of sodium chloride on molecular mobility in amorphous sucrose detected by phosphorescence from the triplet probe erythrosin B

Cited by 18 publications

References 59 publications

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Photophysical Probes of the Amorphous Solid State of Proteins

The Effect of Molecular Size on Molecular Mobility in Amorphous Oligosaccharides

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