Salt, Alone or in Combination with Sucrose, Can Improve the Survival of Escherichia coli O157 (SERL 2) in Model Acidic Sauces

The commercial production of microbiologically safe and stable sauces containing acetic acid is guided by the Comité des Industries des Mayonnaises et Sauces Condimentaires de la Communauté Économique Européenne's (CIMSCEE) code. The CIMSCEE safety value is calculated using a linear regression equation combining weighted contributions of pH and aqueous-phase concentrations of undissociated acetic acid, NaCl, and sugars. By implication, the CIMSCEE safety equation predicts that increasing concentrations of hurdles will always increase inactivation of the target pathogen. In this study, the time to achieve a 3-log₁₀ reduction of an acid-resistant, acid-adapted, Shiga toxin-producing Escherichia coli (STEC) O157 isolate was determined experimentally for 81 formulations at various pHs and acetic acid, NaCl, and sucrose concentrations in a broth model. The combinations were intended to simulate the aqueous phase of acidic sauces and dressings. Experimental data were fitted to the log logistic model to estimate the time to 3-log₁₀ reduction (t_3D). Comparison of fitted t_3D estimates with CIMSCEE values showed agreement in predicting safety (as defined by CIMSCEE) for the majority of formulations. However, CIMSCEE safety predictions were “fail dangerous” for 13 of 81 formulations. Among these formulations and others, the observed E. coli t_3D initially increased and then decreased with increasing osmolalities (NaCl and sucrose). Relative protection increased with exposure time where the protective effect of NaCl predominated. While commercial acidic sauces are not considered high-risk vehicles for STEC, interactions among hurdles that decrease their combined effectiveness are deserving of further investigation because they may reveal mechanisms of broader relevance in the inactivation of pathogens in foods.     

Escherichia coli and Salmonella enterica Are Protected against Acetic Acid,but Not Hydrochloric Acid,by Hypertonicity

Chapman et al. (B. Chapman, N. Jensen, T Ross, and M. B. Cole, Appl. Environ. Microbiol. 72:5165-5172, 2006) demonstrated that an increased NaCl concentration prolongs survival of Escherichia coli O157 SERL 2 in a broth model simulating the aqueous phase of a food dressing or sauce containing acetic acid. We examined the responses of five other E. coli strains and four Salmonella enterica strains to increasing concentrations of NaCl under conditions of lethal acidity and observed that the average “lag” time prior to inactivation decreases in the presence of hydrochloric acid but not in the presence of acetic acid. For E. coli in the presence of acetic acid, the lag time increased with increasing NaCl concentrations up to 2 to 4% at pH 4.0, up to 4 to 6% at pH 3.8, and up to 4 to 7% (wt/wt of water) NaCl at pH 3.6. Salmonella was inactivated more rapidly by combined acetic acid and NaCl stresses than E. coli, but increasing NaCl concentrations still decreased the lag time prior to inactivation in the presence of acetic acid; at pH 4.0 up to 1 to 4% NaCl was protective, and at pH 3.8 up to 1 to 2% NaCl delayed the onset of inactivation. Sublethal injury kinetics suggest that this complex response is a balance between the lethal effects of acetic acid, against which NaCl is apparently protective, and the lethal effects of the NaCl itself. Compared against 3% NaCl, 10% (wt/wt of water) sucrose with 0.5% NaCl (which has similar osmotic potential) was found to be equally protective against adverse acetic acid conditions. We propose that hypertonicity may directly affect the rate of diffusion of acetic acid into cells and hence cell survival.We previously observed that inactivation of Escherichia coli O157 SERL 2 by acetic acid at adverse pH in a broth model simulating the aqueous phase of acidic sauces and dressings was reduced by the presence of NaCl (4). Specifically, the time to a 3-log₁₀-unit reduction (t_3D) of E. coli SERL 2 as function of NaCl concentration was significantly nonmonotonic; that is, the t_3D initially increased when NaCl was increased (from 1 to 3% [wt/wt] of solution), but the t_3D decreased upon a further increase in NaCl concentration (to 8% [wt/wt] of solution) (4). The statistical significance of this “nonmonotonic” response increased with increasing exposure time from 24 to 72 h (at 23°C), primarily due to a proportionally greater increase in inactivation at 1% (wt/wt) NaCl with increasing treatment time than that which was observed at higher NaCl concentrations (4).The combination of acid and NaCl is a common example of the food industry''s “hurdle” approach, which is used to preserve a large and diverse range of foods, including acidic dressings and sauces, fermented meats, cheeses, and preserved vegetables. Given the widespread use of this hurdle combination in food manufacturing, the first aim of this study was to determine whether the observed protection of E. coli SERL 2 from acid inactivation by NaCl is common among E. coli and Salmonella enterica and at what NaCl concentration maximum protection is achieved. A second aim was to determine whether NaCl protection is specific against acid pH in general or against acetic acid in particular. Third, possible protection against acid inactivation by another osmolyte, sucrose, was assessed to resolve whether the effect is solute specific.When cells are placed in hypertonic environments, plasmolysis occurs as the cytoplasmic volume reduces due to water loss by osmosis. The thin peptidoglycan layer of gram-negative microorganisms is anchored to the cytoplasmic membrane and can be distended by plasmolysis or even ruptured when plasmolysis is more extreme. Decad and Nikaido (5) observed that the cytoplasmic volume in gram-negative microorganisms was reduced to ∼50% at ∼0.3 M NaCl but that the plasmolysis-induced cell wall damage was minimal. At 0.5 M (2.9%, wt/wt) NaCl, however, they observed cell wall damage in a large fraction of cells. Thus, in the experiments described here we explored the mechanism of the protective effect of NaCl, and specifically cell wall damage in E. coli populations simultaneously exposed to NaCl and either acetic or hydrochloric acid (HCl), by enumeration of both injured and noninjured survivors by culture on media with and without bile salts.     

Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7

Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.     

Involvement of surface polysaccharides in the organic acid resistance of Shiga Toxin-producing Escherichia coli O157:H7

In general, wild Escherichia coli strains can grow effectively under moderately acidic organic acid-rich conditions. We found that the Shiga Toxin-producing E. coli (STEC) O157:H7 NGY9 grows more quickly than a K-12 strain in Luria-Bertani (LB)-2-morpholinoethanesulphonic acid (MES) broth supplemented with acetic acid (pH 5.4). Hypothesizing that the resistance of STEC O157:H7 to acetic acid is as a result of a mechanism(s) other than those known, we screened for STEC mutants sensitive to acetic acid. NGY9 was subjected to mini-Tn5 mutagenesis and, from 50,000 colonies, five mutants that showed a clear acetic acid-sensitive phenotype were isolated. The insertion of mini-Tn5 in three mutants occurred at the fcl, wecA (rfe) and wecB (rffE) genes and caused loss of surface O-polysaccharide, loss of both O-polysaccharide and enterobacterial common antigen (ECA) and loss of ECA respectively. The other two mutants showed inactivation of the waaG (rfaG) gene but at different positions that caused a deep rough mutant with loss of the outer core oligosaccharide of lipopolysaccharide (LPS) as well as phenotypic loss of O-polysaccharide and ECA. With the introduction of plasmids carrying the fcl, wecA, wecB and waaG genes, respectively, all mutants were complemented in their production of O-polysaccharide and ECA, and normal growth was restored in organic acid-rich culture conditions. We also found that the growth of Salmonella LPS mutants Ra, Rb1, Rc, Rd1, Rd2 and Re was suppressed in the presence of acetic acid compared with that of the parents. These results suggest that the full expression of LPS (including O-polysaccharide) and ECA is indispensable to the resistance against acetic acid and other short chain fatty acids in STEC O157:H7 and Salmonella. To the best of our knowledge, this is a newly identified physiological role for O-polysaccharide and ECA as well as an acid resistance mechanism.     

Gustatory Responsiveness to food-associated acids in the spider monkey (Ateles geoffroyi)

The gustatory responsiveness of four adult spider monkeys to five food-associated acids was assessed in two-bottle preference tests of brief duration (3 min). The animals were given the choice between a 30 mM sucrose solution and defined concentrations of citric acid, ascorbic acid, malic acid, acetic acid, or tannic acid dissolved in a 30 mM sucrose solution. With this procedure,Ateles geoffroyi was found to significantly discriminate concentrations as low as 5 mM ascorbic acid, citric acid, and acetic acid, 10 mM malic acid, and 0.1 mM tannic acid from the alternative stimulus. With the latter two substances, the monkeys rejected all suprathreshold concentrations tested, whereas with the former three substances, the animals showed an inverted U-shaped function of preference, i.e. they rejected high concentrations, but significantly preferred low but detectable concentrations of these acidic tastants over the alternative sweet stimulus. The results showed (1) the spider monkey to respond to the same range of acid concentrations as other nonhuman primate species; (2) thatAteles geoffroyi, is able to detect food-associated acids at concentrations well below those present in most fruits; and (3) that unlike most other primate species tested so far, spider monkeys do not generally reject acidic tastants but show a substanceand concentration-dependent change in responsiveness that may range from rejection to preference. The results support the assumptions that spider monkeys may use sourness and/or astringency of food-associated acids as a criterion for food selection, and that the gustatory responsiveness ofAteles geoffroyi to acidic tastants might reflect an evolutionary adaptation to frugivory.     

Surface areas of 1-palmitoyl phosphatidylcholines and their interactions with cholesterol.

The activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. was affected by added NaCl in such a way that an initial phase of stimulation was followed by a phase of rapid non-linear decrease in velocity and finally by a phase of slow linear decrease in velocity as the concentration of NaCl was increased. In the presence of 0.014 M-sodium acetate/acetic acid buffer (pH 5.0) there was a slight increase in enzymic activity in the presence of low concentrations of dioxan (up to about 10% dioxan) and a rapid decrease in enzymic activity at higher dioxan concentrations, but both effects were mitigated in the presence of 0.1 M buffer. The order of efficiency of added glucosyl acceptors in beta-glucosidase-catalysed reactions was found to be fructose greater than sucrose greater than glycerol greater than methanol. The enzyme was inactivated by the active-site-directed compound conduritol-B-epoxide; but this inactivation was concentration-dependent, was prevented by 10 mM-glucose, and involved an acidic group with pKa 4.3. A rate equation has been derived on the assumption of a mechanism of action involving a solvent-separated and an intimate glucosyl cation-carboxylate ion-pair intermediate and an alpha-glucosyl enzyme intermediate [Umezurike, G. M. (1981) Biochem. J. 199, 203-209]. Calculations based on the application of the derived rate equation and the calculated kinetic parameters show that the rate equation explains the peculiar properties of beta-glucosidase in the presence of added glucosyl acceptors or of NaCl.     

Amiloride-sensitive and amiloride-insensitive responses to NaCl + acid mixtures in hamster chorda tympani nerve

Component signaling in taste mixtures containing both beneficial and dangerous chemicals depends on peripheral processing. Unidirectional mixture suppression of chorda tympani (CT) nerve responses to sucrose by quinine and acid is documented for golden hamsters (Mesocricetus auratus). To investigate mixtures of NaCl and acids, we recorded multifiber responses to 50 mM NaCl, 1 and 3 mM citric acid and acetic acid, 250 μM citric acid, 20 mM acetic acid, and all binary combinations of each acid with NaCl (with and without 30 μM amiloride added). By blocking epithelial Na(+) channels, amiloride treatment separated amiloride-sensitive NaCl-specific responses from amiloride-insensitive electrolyte-generalist responses, which encompass all of the CT response to the acids as well as responses to NaCl. Like CT sucrose responses, the amiloride-sensitive NaCl responses were suppressed by as much as 50% by citric acid (P = 0.001). The amiloride-insensitive electrolyte-generalist responses to NaCl + acid mixtures approximated the sum of NaCl and acid component responses. Thus, although NaCl-specific responses to NaCl were weakened in NaCl-acid mixtures, electrolyte-generalist responses to acid and NaCl, which tastes KCl-like, were transmitted undiminished in intensity to the central nervous system. The 2 distinct CT pathways are consistent with known rodent behavioral discriminations.     

Protective effects of organic acids on survival of Escherichia coli O157:H7 in acidic environments

Outbreaks of disease due to acid-tolerant bacterial pathogens in apple cider and orange juice have raised questions about the safety of acidified foods. Using gluconic acid as a noninhibitory low-pH buffer, we investigated the killing of Escherichia coli O157:H7 strains in the presence or absence of selected organic acids (pH of 3.2), with ionic strength adjusted to 0.60 to 0.68. During a 6-h exposure period in buffered solution (pH 3.2), we found that a population of acid-adapted E. coli O157:H7 strains was reduced by 4 log cycles in the absence of added organic acids. Surprisingly, reduced lethality for E. coli O157:H7 was observed when low concentrations (5 mM) of fully protonated acetic, malic, or l-lactic acid were added. Only a 2- to 3-log reduction in cell counts was observed, instead of the 4-log reduction attributed to pH effects in the buffered solution. Higher concentrations of these acids at the same pH aided in the killing of the E. coli cells, resulting in a 6-log or greater reduction in cell numbers. No protective effect was observed when citric acid was added to the E. coli cells. d-Lactic acid had a greater protective effect than other acids at concentrations of 1 to 20 mM. Less than a 1-log decrease in cell numbers occurred during the 6-h exposure to pH 3.2. To our knowledge, this is the first report of the protective effect of organic acids on the survival of E. coli O15:H7 under low-pH conditions.     

Expanded models for the non-thermal inactivation of Listeria monocytogenes

Previously developed four-variable response surface models for describing the effects of temperature, pH/lactic acid, sodium chloride and sodium nitrite on the time to achieve a 4-log, non-thermal inactivation ( t _4D) of Listeria monocytogenes in aerobic, acidic environments were expanded to five-variable models that distinguish the effects of pH and acidulant concentration. A total of 18 new variable combinations were evaluated and the inactivation kinetics data appended onto a consolidation of two data sets from earlier studies. The consolidated data set, which included 315 inactivation curves representing 209 unique combinations of the five variables, was analysed by response surface analysis. The quadratic model without backward elimination regression was selected for further evaluation. Three additional quadratic models were generated using the concentrations of undissociated lactic and/or nitrous acids as variables in place of percentage lactic acid and sodium nitrite concentration. Comparison of predicted t _4D values against literature values for various food systems indicated that the models provide reasonable initial estimates of the inactivation of L. monocytogenes. The models based on the concentration of undissociated lactic and nitrous acids support the hypothesis that antimicrobial activity is associated with this form of the compounds. Evaluation of several examples suggests that these models may be useful for predicting the equivalent of the compounds'minimal inhibitory concentrations'for accelerating inactivation under various conditions.     

Depression of gustatory receptor potential in frog taste cell by parasympathetic nerve-induced slow hyperpolarizing potential

Parasympathetic nerve (PSN) innervates taste cells of the frog taste disk, and electrical stimulation of PSN elicited a slow hyperpolarizing potential (HP) in taste cells. Here we report that gustatory receptor potentials in frog taste cells are depressed by PSN-induced slow HPs. When PSN was stimulated at 30 Hz during generation of taste cell responses, the large amplitude of depolarizing receptor potential for 1 M NaCl and 1 mM acetic acid was depressed by approximately 40% by slow HPs, but the small amplitude of the depolarizing receptor potential for 10 mM quinine-HCl (Q-HCl) and 1 M sucrose was completely depressed by slow HPs and furthermore changed to the hyperpolarizing direction. The duration of the depolarizing receptor potentials depressed by slow HPs prolonged with increasing period of PSN stimulation. As tastant-induced depolarizing receptor potentials were increased, the amplitude of PSN-induced slow HPs inhibiting the receptor potentials gradually decreased. The mean reversal potentials of the slow HPs were approximately -1 mV under NaCl and acetic acid stimulations, but approximately -14 mV under Q-HCl and sucrose stimulations. This implies that when a slow HP was evoked on the same amplitude of depolarizing receptor potentials, the depression of the NaCl and acetic acid responses in taste cells was larger than that of Q-HCl and sucrose responses. It is concluded that slow HP-induced depression of gustatory depolarizing receptor potentials derives from the interaction between gustatory receptor current and slow hyperpolarizing current in frog taste cells and that the interaction is stronger for NaCl and acetic acid stimulations than for Q-HCl and sucrose stimulations.     

Inactivation of shiga toxin-producing Escherichia coli (STEC) and degradation and removal of cellulose from STEC surfaces by using selected enzymatic and chemical treatments

Some Shiga toxin-producing Escherichia coli (STEC) strains produce extracellular cellulose, a long polymer of glucose with β-1-4 glycosidic bonds. This study evaluated the efficacies of selected enzymatic and chemical treatments in inactivating STEC and degrading/removing the cellulose on STEC surfaces. Six cellulose-producing STEC strains were treated with cellulase (0.51 to 3.83 U/15 ml), acetic and lactic acids (2 and 4%), as well as an acidic and alkaline sanitizer (manufacturers' recommended concentrations) under appropriate conditions. Following each treatment, residual amounts of cellulose and surviving populations of STEC were determined. Treatments with acetic and lactic acids significantly (P < 0.05) reduced the populations of STEC, and those with lactic acid also significantly decreased the amounts of cellulose on STEC. The residual amounts of cellulose on STEC positively correlated to the surviving populations of STEC after the treatments with the organic acids (r = 0.64 to 0.94), and the significance of the correlations ranged from 83 to 99%. Treatments with cellulase and the sanitizers both degraded cellulose. However, treatments with cellulase had no influence on the fate of STEC, and those with the sanitizers reduced STEC cell populations to undetectable levels. Thus, the correlations between the residual amounts of cellulose and the surviving populations of STEC caused by these two treatments were not observed. The results suggest that the selected enzymatic and chemical agents degraded and removed the cellulose on STEC surfaces, and the treatments with organic acids and sanitizers also inactivated STEC cells. The amounts of cellulose produced by STEC strains appear to affect their susceptibilities to certain sanitizing treatments.     

Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.     

Biological,microbiological and chemical control of the plum mothlaspeyresia funebrana tr. [Lep. tortricidae] in Poland

Résumé Une expérience a été conduite en vue de comparer la lutte chimique, la lutte biologique par entomophages et la lutte biologique par entomopathogènes contre le Carpocapse des Prunes. Le verger choisi comportait plus de 6 000 arbres de 15 ans, pratiquement d'une seule variété. Chaque lot contenait 100 arbres séparés par une zone d'isolement de 50 arbres. Les résultats obtenus dans chacune des 18 formulations testées sont donnés dans le tableau 1 et l'analyse de la signification des différences de production (totale) constatées entre chaque lot est présentée par la figure 2. En conclusion, les auteurs soulignent l'intérêt deTrichogramma cacoeciae qui se révèle un des plus efficaces moyens de lutte contre ce ravageur.      

Establishing equivalence for microbial-growth-inhibitory effects ("iso-hurdle rules") by analyzing disparate listeria monocytogenes data with a gamma-type predictive model

Preservative factors act as hurdles against microorganisms by inhibiting their growth; these are essential control measures for particular food-borne pathogens. Different combinations of hurdles can be quantified and compared to each other in terms of their inhibitory effect ("iso-hurdle"). We present here a methodology for establishing microbial iso-hurdle rules in three steps: (i) developing a predictive model based on existing but disparate data sets, (ii) building an experimental design focused on the iso-hurdles using the model output, and (iii) validating the model and the iso-hurdle rules with new data. The methodology is illustrated with Listeria monocytogenes. Existing data from industry, a public database, and the literature were collected and analyzed, after which a total of 650 growth rates were retained. A gamma-type model was developed for the factors temperature, pH, a(w), and acetic, lactic, and sorbic acids. Three iso-hurdle rules were assessed (40 logcount curves generated): salt replacement by addition of organic acids, sorbic acid replacement by addition of acetic and lactic acid, and sorbic acid replacement by addition of lactic/acetic acid and salt. For the three rules, the growth rates were equivalent in the whole experimental domain (γ from 0.1 to 0.5). The lag times were also equivalent in the case of mild inhibitory conditions (γ ≥ 0.2), while they were longer in the presence of salt than acids under stress conditions (γ < 0.2). This methodology allows an assessment of the equivalence of inhibitory effects without intensive data generation; it could be applied to develop milder formulations which guarantee microbial safety and stability.     

Electrical responses of supporting cells in the frog taste organ to chemical stimuli

1. The mean resting potential of supporting cells in the frog taste organ was -19.1 mV. The supporting cells responded to the four basic taste stimuli with a depolarization but responded to water with a depolarization or a hyperpolarization. 2. The membrane resistances of supporting cells decreased during stimulation with sucrose, NaCl and acetic acid, but increased during stimulation with Q-HCl and water. 3. Reversal potential of the depolarizing response for 0.5 M NaCl in supporting cells was +7.6 mV. The depolarizing responses for Q-HCl and acetic acid were independent of the membrane potential level. 4. These results suggest that the characteristics of taste responses in supporting cells are similar to those in taste cells.     

Acid stability of anti-Helicobacter pyroli IgY in aqueous polyol solution

IgY was separated from a hen's egg yolk that was immunized with Helicobacter pyroli. The anti-H. pyroli IgY activity at acidic pH and the suppressive effect of polyol on acid-induced inactivation of IgY were investigated. Sorbitol and xylitol were used as polyols. IgY was quite stable at pH 5-7. Irreversible inactivation of IgY was observed at pH below 4, and proceeded rapidly at pH below 3. The acid stability of IgY was enhanced in the presence of 30% sorbitol or above. In a 50% aqueous sorbitol solution, an acid-induced inactivation was almost completely suppressed at pH 3. However, the improvement of IgY activity was not observed in the aqueous xylitol solution. IgY showed almost the same activity as native IgY when sucrose was substituted for sorbitol. On the other hand, the xylitol replacement with sucrose did not enhance the acid stability of IgY. The acid-induced inactivation of IgY was related to tryptophyl fluorescence. Fluorescence emission spectra suggested that structural changes near the tryptophan residues may occur under acidic conditions. An increase in sorbitol concentration induced a blue shift. The fluorescence emission of IgY in a 50% sorbitol solution had a peak at 330 nm, which was the same emission peak that was exhibited by native IgY. Sorbitol could, therefore, be used as a good stabilizer of IgY under acidic conditions.     

Changes in the Mark–Houwink hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths

The objective of this study is to explore the cause(s) of changes in the hydrodynamic volume of chitosan molecules in solutions of different organic acids, at different temperatures and ionic strengths. Change in intrinsic viscosity is used as the parameter to elucidate the causes of changes in the hydrodynamic volume of chitosan molecules in these solutions. Results show that the intrinsic viscosity of chitosan decreases in acetic acid or in malic acid over storage time. These decreases are more pronounced in acetic acid solution than in malic acid solution, more significant in higher temperature than in lower temperature solutions, and greater in solutions without NaCl than in solutions containing higher NaCl. The decrease in intrinsic viscosity can perhaps be attributed to the compounded effects of compaction of the chitosan molecules and/or acidic degradation during storage.     

Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.     

CONSÉQUENCE DE LA VARIATION DES EFFECTIFS DES FEMELLES DE DINARMUS VAGABUNDUS,SUR LE PARASITISME DE L'UN DE LEURS HÔTES CALLOSOBRUCHUS MACULATUS

Lorsque 2 femelles ou 3 femelles de l'ectoparasite D. vagabundus parasitent conjointement un même lot d'hôtes l'intensité du grégarisme larvaire s'accroît par rapport à celui observé en présence d'une seule femelle pondeuse. Cela se traduit par une augmentation significative du nombre moyen de descendants des 2 sexes mais la production moyenne en fils est toujours très inférieure à celle des filles. Cependant, lorsque la densité de femelles pondeuses passe de 1 à 3, le rapport des sexes mâles/femelles des descendants évolue vers l'unité traduisant une augmentation plus rapide de l'effectif des fils que des filles.