In this paper I relate the loss of CFU following a life-threatening treatment to the inactivation of critical components. Equations are used to calculate the loss of CFU following isothermal and temperature-scanning treatments, and the results are discussed in relation to differential scanning calorimetry of bacteria.

Differential scanning calorimetry (DSC) was used to study the thermal stability of native and synthetically cross-linked rat-tail tendon at different levels of hydration, and the results compared with native rat-tail tendon. Three cross-linking agents of different length between functional groups were used: malondialdehyde (MDA), glutaraldehyde and hexamethylene diisocyanate (HMDC). Each yielded the same linear relation between the reciprocal of the denaturation temperature in Kelvin, T(max), and the water volume fraction, epsilon (1/T(max)=0.000731epsilon+0.002451) up to a critical hydration level, the volume fraction of water in the fully hydrated fibre. Thereafter, water was in excess, T(max) was constant and the fibre remained unchanged, no matter how much excess water was added. This T(max) value and the corresponding intrafibrillar volume fraction of water were as follows: 84.1 degrees C and 0.48 for glutaraldehyde treated fibres, 74.1 degrees C and 0.59 for HMDC treated fibres, 69.3 degrees C and 0.64 for MDA treated fibres, and 65.1 degrees C and 0.69 for untreated native fibres. Borohydride reduction of the native enzymic aldimines did not increase the denaturation temperature of the fibres. As all samples yielded the same temperature at the same hydration, the temperature could not be affected by the nature of the cross-link other than through its effect on hydration. Cross-linking therefore caused dehydration of the fibres by drawing the collagen molecules closer together and it was the reduced hydration that caused the increased temperature stability. The cross-linking studied here only reduced the quantity of water between the molecules and did not affect the water in intimate contact with, or bound to, the molecule itself. The enthalpy of denaturation was therefore unaffected by cross-linking. Thus, the "polymer-in-a-box" mechanism of stabilization, previously proposed to explain the effect of dehydration on the thermal properties of native tendon, explained the new data also. In this mechanism, the configurational entropy of the unfolding molecule is reduced by its confinement in the fibre lattice, which shrinks on cross-linking.

The kinetics of unfolding of a collagen-like peptide, (Pro-Pro-Gly)10, has been studied under isothermal conditions to gain a better understanding of the stabilization of the collagen triple helix. The formation process was third-order and relatively insensitive to temperature at concentrations of 1 mg/ml and below, while the unfolding process was first-order and highly temperature-dependent. The helix–coil transition was studied over a range of scanning rates and polymer concentrations, using differential scanning calorimetry and the observations were compared with solutions of an approximate differential equation governing the process. At high concentrations (24 mg/ml) and very low scanning rates (0.025 °C min−1), the helicity, F, approached a quasistatic state in which it reached its equilibrium value at all temperatures. Under these conditions, the temperature at which the endotherm peaked, Tmax, increased with chain concentration but was independent of scanning rate, while (dF/dT)max was dependent on the van't Hoff enthalpy and on the order of the formation process. On scanning from a low to a high temperature (up-scanning) at low concentrations (0.25–1.0 mg/ml) and higher scanning rates (0.1 °C min−1 and above), the peak in dF/dT was taller and narrower than for slow quasistatic scanning. Tmax increased linearly with the logarithm of the scanning rate, and was independent of concentration, while (dF/dT)max was governed by the temperature-dependence of the rate of unfolding. At intermediate scanning rates, two peaks in dF/dT were apparent. One peak was a nascent “quasistatic peak”; the other was a nascent “rate peak”. Comparison of this peptide data with the properties of the collagen denaturation endotherm showed that the collagen denaturation endotherm was determined only by the rate of unfolding, and not by an unobserved equilibrium.

The deletion of the alpha2 chain from type I collagen in the oim mouse model of osteogenesis imperfecta has been shown to result in a significant reduction in the mechanical strength of the tail tendon and bone tissue. However, the exact role of the alpha2 chain in reducing the mechanical properties is not clear. We now report that the stabilizing intermolecular cross-links in bone are significantly reduced by 27%, thereby contributing to the loss of tensile strength and the change in stress-strain profile. We also report that, in contrast to previous studies, the denaturation temperature of the triple helical molecule and the intact fibers are 2.6 degrees and 1.9 degrees C higher than the corresponding tail tendon collagen from wild-type mice. The increase in hydroxyproline content accounts, at least in part, for the increase in denaturation temperature. The alpha2 chain clearly plays an important part in stabilizing the type I collagen triple helix and fiber packing, but further studies are required to determine the precise mechanism.

The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (ϵ) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 ± 0.37) × 10−4 K−1, compared with (7.31 ± 0.42) × 10−4 K−1 for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low ϵ. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.

A review with 130 references covering photochemical damage caused by UV rays in collagen. The damage is known to be related to collagen type and age, and to the nature of the admixtures present in collagen, but the mechanism of the damage has not yet been fully recognized. Collagen's chemical structure is discussed in detail to present collagen properties of various collagen types before and after collagen has been UV-irradiated.

We have proposed that the denaturation kinetics of the characteristic sharp melting point of the collagen molecules is an irreversible rate rather than an equilibrium process as previously believed. This leads to the concept of domains of variable thermal stability along the length of the molecule. We have identified the major thermally labile domains from which the denaturation process is initiated as hydroxyproline deficient sequences of 65, 65 and 59 residues near the carboxy terminus in fibrillar collagen types I, II and III, respectively. These domains differ in that there is a single hydroxyproline in the type II domain and two hydroxyprolines in the type III domain. Similar sized domains are conserved in these collagen types across species including amphibians and invertebrates. The effective size of the domain is reduced in the fibrillar aggregates to 26 residues due to the interaction with adjacent molecules and because of the precise quarter-staggered alignment of the molecules the domains are located in the gap region. This spatial confinement within the lattice of the fibre leads to the significant increase in denaturation temperature of the fibre compared to the molecule. These labile domains have also been located in molecules that form the non-fibrillar type IV basement membrane collagens and the fibril-associated aggregates such as type IX. Based on the location of the different domains in type IX we have proposed a different arrangement of the type IX on the type II fibril. The model stresses the importance of hydroxyproline in stabilising the triple helix and supports the concept of hydrogen-bonded water-bridges originally proposed from X-ray diffraction studies in contrast to other studies indicating water-bridges do not play a role in stabilising the collagen molecule.

Isinglass is widely used commercially to clarify alcoholic beverages by aggregation of the yeast and other insoluble particles. It is derived from swim bladders of tropical fish by solubilisation in organic acids and consists predominantly of the protein collagen. The low content of intermolecular cross-links allows ready dissolution of swim bladder compared to bovine hide which is cross-linked by a high proportion of stable bonds and requires enzymic digestion to solubilise. Isinglass is no longer effective as a clarifying agent if thermally denatured hence the collagenous triple helical structure must be maintained. Thermal denaturation of isinglass occurs at 29 degrees C, compared to 40-41 degrees C for mammalian collagens, primarily due to the lower hydroxyproline content. The hydroxyproline is essential for the formation of H-bonded water-bridges through the hydroxyl group and the peptide chain thereby stabilising the triple helix. Based on the lower enthalpy determined by differential scanning calorimetry we have calculated that the thermally labile domain of the isinglass molecule was 41 residues compared to 66 for mammalian collagen. The fining efficiency was unaffected by pH, chelating agents, detergents and removal of surface proteins from yeast cells. Studies on the mechanism of action of isinglass have shown that higher molecular weight aggregates that increase the length of the collagen molecules (trimers, tetramers, etc.) increase efficiency and that their surface charge are important in the clarification process. By chemical modification, we have shown that blocking positively charged groups had no effect on the fining process, whilst negative charges are clearly essential and that increasing the negative charge by succinylation increases its efficacy. Solutions of bovine hide collagen were shown to be equally effective in refining beers and standard yeast preparations. The higher thermal denaturation temperature, ready availability and reproducibility of bovine collagen preparations gives it considerable advantages over isinglass.

Differential scanning calorimetry has revealed the presence of a new denaturation endotherm at 32 degrees C following UV irradiation of collagen, compared with 39 degrees C for the native triple helix. Kinetic analyses showed that the new peak was a previously unknown intermediate state in the collagen helix-coil transition induced by UV light, and at least 80% of the total collagen was transformed to random chains via this state. Its rate of formation was increased by hydrogen peroxide and inhibited by free radical scavengers. SDS-polyacrylamide gels showed evidence of competing reactions of cross-linking and random primary chain scission. The cross-linking was evident from initial gelling of the collagen solution, but there was no evidence for a dityrosine cross-link. Primary chain scission was confirmed by end group analysis using fluorescamine. Electron microscopy showed that the segment long spacing crystallites formed from the intermediate state were identical to the native molecules. Clearly, collagen can undergo quite extensive damage by cleavage of peptide bonds without disorganizing the triple helical structure. This leads to the formation of a damaged intermediate state prior to degradation of the molecules to short random chains.

Ultrasonic thawing tests were undertaken on frozen meat and fish samples to assess the effectiveness of high intensity ultrasound for thawing frozen foods, previous work having indicated that ultrasonic absorption is greatest near the initial freezing point where most of the freezing occurs. Ultrasonic frequencies from 0.22 to 3.3 MHz and intensities up to 3 W cm−2 were investigated. Overheating near the surface was found to be a problem at high intensities and at high and low frequencies, due to the increase in attenuation with frequency, and the onset of cavitation at low frequencies. Using frequencies and intensities around 500 kHz and 0.5 W cm−2, surface heating was minimized, and beef, pork and cod samples were thawed to a depth of 7.6 cm within about 2.5 h. Thawing times were consistent with those predicted by an ultrasonic thawing model. The effects of muscle fibre orientation and inclusion of a fat layer were found to agree with known attenuation coefficients and characteristic acoustic impedances.

Fibre-forming collagens in dilute solution show highly co-operative helix-coil transitions at temperatures that are remarkably close to the body temperature of the animal from which the collagen was extracted. This close correlation holds across animal Phyla and the transition temperatures, which range from 5 degrees C to 40 degrees C, are adjusted to suit by changing the primary structure, especially the concentration of the water-bridge-enhancing hydroxyproline residue. Fibril-forming collagens are thermally stabilised by fibrillogenesis, which causes a loss of random coil configurational entropy by intermolecular and intramolecular cross-linking and by spacial confinement of the molecule within the lattice of the fibre. But this mechanism cannot apply to the full length of the type IX collagen molecule, since its COL3 arm, according to current models, projects out from the stabilising influence of the type II fibre. In this paper we examine the thermal stability of the type IX collagen molecule and its three triple-helical domains, thereby demonstrating that the COL3 arm is much more stable than the rest of the molecule. At a scanning rate of 60 deg. C/h COL3 exhibited an unfolding endotherm with a tmax at 49.0 degrees C, well above body temperature. Corresponding peak maxima for COL1 and COL2 were seen at 40.6 degrees C and 39.6 degrees C, respectively. The sizes of the thermally labile units of COL1, COL2 and COL3, calculated from the measured activation enthalpies, were 24, 28 and 28 residues, respectively, much smaller than type I (65 residues) because of the relatively short lengths of triple helix to be unfolded. However, unlike type I collagen, no regions of the required size were found completely devoid of hydroxyproline. Consequently, the intrinsic stabilities of these thermally labile units were higher than that of type I with DeltaH updownarrow DeltaS updownarrow for COL1, COL2 and COL3 being, respectively, 385 K, 371 K and 384 K, contrasting with the much lower 349 K of type I collagen. We therefore speculate that the increased thermal stability of the thermally labile units was caused by the presence of the water-bridge-enhancing residue, hydroxyproline. Finally the stabilisation of type IX collagen tissue is considered and an alternative structural organisation of the type IX molecule on the type II fibre is proposed.

The thermal degradation of plasmid pUC18 held at temperatures between 100 and 135 degrees C was examined by measuring the ability of heat-treated plasmid preparations to transform Escherichia coli to ampicillin resistance using electroporation. Substantial protection against loss of transforming ability during heating was provided by concentrations of NaCl between 0.25 and 2.0 mol l-1. For example, the addition of 1.0 mol l-1 NaCl to samples heated at 100 degrees C for 15 min increased transformation frequency about 200-fold compared with samples heated without NaCl. In the presence of 0.5-2.0 mol l-1 NaCl, transforming capacity was not destroyed even by heating at 121 degrees C for 15 min, i.e. after a typical sterilization treatment. These findings may have implications for the safe disposal of genetically modified micro-organisms and recombinant DNA preparations.

The thermal conductivity of starch gels, ranging in water content from 51 to 97% by mass, was measured using the thermal probe method and the thermal conductivity of starch itself was determined using a revised analysis of the null point method of Sakiyama et al. (1993), taking into account the effect of hydration. The thermal conductivity of hydrated starch (mass fraction of water = 0.22) was found to be 0.364, 0.386 and 0.388 W m−1K−1 at 10, 50 and 80 °C, respectively, and its density at 20 °C was estimated to be 1.38 × 103 kg m−3. The corresponding effective thermal conductivities and density of dry starch were estimated as: 0.293, 0.305 and 0.301 W m−1 K−1 and 1.54 kg m−3, respectively. The measured thermal conductivities of starch gels followed quite closely intermediate models such as the Maxwell model or the parallel-series model, but were inconsistent with either the parallel or series models alone.

The initial freezing point of food was estimated from the mole fractions of individual solutes in the aqueous phase (ions, sugars, acids and alcohol), derivable from information given in nutritional tables. Predicted values were compared with experimental measurements taken from the literature. Predicted and experimental values generally agreed to within 1 °C, despite the solute concentrations of the measured foods being assumed to be those of the average compositions given in nutritional tables. The effects of the addition of salt to meat and of dehydration of various foods on freezing point depression, and the effect of temperature on the ice content of frozen foods were also successfully predicted.

Ultrasound velocity, attenuation, and backscattering were measured in vitro in samples of equine digital flexor tendon sandwiched between plane, parallel rexolite buffer rods. The buffer rods were coupled to transmitting and receiving transducers (nominally 10 MHz) mounted in-line and facing one another on the jaws of a digital caliper. Six superficial digital flexor (SDF) tendons and six deep digital flexor (DDF) tendons were measured in three orthogonal directions: along the long axis of the tendon (D), and across the tendon in the dorsal-volar (C), and lateral (L) directions. Substantial anisotropy was apparent in all the measured properties. The velocity data, which in both tendons showed a higher velocity along the fibers than across (e.g., in the DDF tendon at 0 degrees C: 1713 +/- 9 m/s in the D direction compared with 1650 +/- 5 m/s in the C direction), were consistent with a composite comprising stiff fibers embedded in a less stiff medium of lower speed. The apparent backscattering coefficient adjusted for the tissue's frequency-dependent attenuation (e.g., in the C direction of the DDF tendon at 0 degrees C: 7.4 x 10(-3) cm-1 sr-1), was independent of frequency in both transverse directions and larger than that measured along the long axis of the tendon (e.g., in DDF tendon at 0 degrees C: 1.2 x 10(-3) cm-1 sr-1 at 7 MHz) in which direction the apparent backscattering coefficient increased with frequency as f4.0 +/- 1.2. The frequency-independent backscattering was thought to be due to specular reflection from the boundaries between the fascicles, i.e., the bundles of fibers making up the tendon, while backscattering along the axis was due to structures of unknown origin, but of a size much smaller than 45 microns. Attenuation of ultrasound directed along the fibers was higher than that across (at 7 MHz in DDF tendon at 0 degrees C: 58 dB/cm in the D direction compared with 11.3 dB/cm in the C direction). Calculations indicated that the attenuation was primarily caused by absorption rather than scattering.

The velocity, attenuation and apparent backscattering coefficient of 6–11-MHz ultrasound were measured in three orthogonal directions in equine deep digital flexor (DDF) and superficial digital flexor (SDF) tendons at 0°C. Ultrasonic measurements were examined for correlation with tendon water, collagen, DNA and glycosaminoglycans contents, determined by chemical analyses and with structure observed by scanning electron microscopy. The SDF tendon contained more water, more DNA (i.e., more cells), less collagen and less glycosaminoglycans and exhibited lower velocities and attenuations than the DDF tendon. Velocities were governed primarily by the adiabatic bulk modulus and density, perturbed by a highly direction-dependent rigidity. Ultrasound propagating across tendon generated frequency-independent back-scattering which appeared to derive from the large interfaces between the fascicles, while along the fibres backscattering varied as f3.62±0.88 and appeared to derive from small structures such as collagen fibres. The mechanisms by which ultrasound is attenuated by tendon remain unknown.

C.A. MILES, M.J. MORLEY, W.R. HUDSON AND B.M. MACKEY. 1995. When particles in suspension are placed in a stationary ultrasonic field, they form bands of high concentration at half‐wavelength intervals. Several theoretical papers have recently contributed to an understanding of the process but, for particles as small as bacteria, there are substantial discrepancies between the different theories, and a lack of published observations.The threshold amplitudes required to band latex spheres (0.5‐5.0 μm diameter) were measured in the frequency range 1–3 MHz and used to establish conditions, reported here for the first time, suitable for banding and moving vegetative bacterial cells in pure culture. Ultrasonic means for separating and concentrating cream and bacteria at opposite ends of a tube containing a mixture of Escherichia coli and diluted milk are also described and the breakdown of theoretical equations at low particle size is discussed.

This paper shows that the position and shape of the denaturation endothem of collagen fibrils are governed by the kinetics of an irreversible rate process. This was proved by measuring the rate of denaturation in rat tail tendons held isothermally at different temperatures, thereby determining rate constant characteristics such as the activation enthalpy and entropy and predicting endotherm position and shape therefrom. Comparison with actual scanning results showed good correspondence. Isothermal measurements of the rate of collagen denaturation, measured continuously using a calorimetric method, were used to determine rate constants for collagen denaturation in tendons immersed in water and 0.5 M acetic acid. The temperature dependence of the rate constants were fitted to the three rate process models, previously examined theoretically: the D and z formulation, the Arrhenius equation and the absolute rate theory. For example, in water the activation enthalpy was 0.518 (+/- 0.016) Mj mol-1 and the activation entropy 1.485 (+/- 0.049) kj mol-1 K-1, while in acetic acid the corresponding figures were 1.306 (+/- 0.099) Mj mol-1 and 4.142 (+/- 0.323) kj mol-1 K-1. These characteristics are discussed in terms of the thermal activation of a region of the molecule, the co-operative unit. The ratio of the activation enthalpy to the calorimetry enthalpy of denaturation indicated a co-operative unit that was 66 (+/- 5) residues long when fibrils were swollen in acetic and the collagen molecules acted essentially independently. On the other hand the intact fibrils in water gave a co-operative unit of 26 (+/- 1) residues long. The reason for the reduction in size of the co-operative unit is that it is surrounded, and therefore stabilized by other molecules in the fibre. It is interesting to note that the suggested co-operative unit lies almost entirely within the "gap" zone of the collagen fibril in its quarter-staggered arrangement of molecules. We believe that the co-operative unit would be represented by a domain that is free of stabilising hydroxyproline residues. Indeed such a domain exists near the C terminus of the triple helix from Gly877 to Pro941, i.e. 65 residues. In acetic acid, activation is similar to that of collagen molecules in solution. All the inter alpha-chain hydrogen bonds in the co-operative unit are broken and the separate chains in this short region are free to flail around under the action of thermal collisions relatively unimpeded by intermolecular interactions.(ABSTRACT TRUNCATED AT 400 WORDS)