Effect of pentasodium triphosphate concentration on physicochemical properties, microstructure, and formation of casein fibrils in model processed cheese.

The effects of varying the concentration of pentasodium triphosphate (PP) emulsifying salt [0, 0.6, 1.2, 1.5, and 1.8%, plus 0.9% of a mixture of citric acid (CA) and disodium phosphate (DSP) to adjust cheese pH to 5.85] on rheological, textural, physicochemical, and microstructural properties were studied in a processed cheese model system containing ~20% micellar casein concentrate, ~20% sunflower oil, and ~59% water. Special emphasis was placed on the unique casein fibrils recently described in a comparable processed cheese model system. Our results show that during processing (90°C, 17.37 rpm over 270 min) the apparent viscosity increased more and faster for formulations containing higher concentrations of PP, in analogy to the so-called creaming reaction, a general thickening of the molten cheese mass with prolonged processing. We found that 1.2% PP (plus 0.9% CA-DSP) appeared to be the threshold for the creaming reaction to take place. With increasing PP concentrations, cheese hardness increased in a sigmoidal fashion, and insoluble (protein-bound) calcium concentration decreased exponentially. Light micrographs of samples taken at the end of processing indicated initially large and dense casein aggregates within the matrix that disappeared with higher levels of PP, in parallel with the development of a finer emulsion. With transmission electron microscopy analysis on the same samples, the highly complex restructuring of the casein matrix was evident; casein fibrils had formed de novo at the periphery of the loosening casein aggregates. With higher levels of PP, amorphous areas were observed in place of the dense casein aggregates that appeared progressively void of protein, whereas fibril concentration increased throughout the rest of the matrix. Fibrils progressively attached to the surface of fat globules, thereby emulsifying them. Reverse-phase HPLC analysis of insoluble and soluble fractions indicated κ-casein to be the most likely constituent of the newly formed fibrils. The results of this study suggest that PP induced a concentration-dependent dissociation of caseins (through increased calcium chelation) and further led to their spatial separation. In essence, their chaperone activity was hindered, which resulted in amorphous aggregation on the one hand and fibril formation on the other.

Mechanisms of structure formation underlying the creaming reaction in a processed cheese model system as revealed by light and transmission electron microscopy.

The "creaming reaction," a general thickening of the molten cheese mass during the manufacture of processed cheese, which is often seen to occur in a stepwise fashion, affects the viscosity and texture of the finished product. Thus, this phenomenon is of critical importance for the processed cheese industry, yet mechanisms underlying the structure formation in this surprisingly complex and dynamic food system are only poorly understood. Using a model system consisting of micellar casein concentrate, vegetable oil, water, and a mixture of melting salts, we followed the characteristic viscosity profile with its primary and secondary increase over time. A rheometer equipped with a custom-made cup geometry was used, which served as a mini-reaction vessel to simulate the conditions during the manufacture of processed cheese. The mixture was subjected to constant heat (90°C) and stirring (7.93 rpm), comparable to processed cheese cooking, for up to 410 min. At specific time points, samples were taken, and the micro- and ultrastructure was investigated with light and transmission electron microscopy. Results from our extensive study uncovered the following key steps: (1) a decrease in fat globule size with concomitant increase in the number of fat globules, which were also more evenly distributed; (2) a progressive separation of the casein matrix into fibrillogenic and nonfibrillogenic fractions; (3) formation of fibrils and their higher-order structuring followed by their partial degradation; and (4) increasing interactions of the fibrils with the fat globule surface leading to a higher degree of emulsification. Of these different observations, results indicate that after the caseins dissociated under the influence of the melting salts, protein-protein interactions were the primary driver of the structure formation and thus contributed to the initial viscosity increase. Fat globules were involved in the structure formation at later time points. Therefore, fat-protein interactions in addition to continued protein-protein interactions were assumed to contribute to the secondary viscosity increase. An updated processed cheese creaming model is presented. The use of the term "texturization" instead of "creaming" is proposed.

Progressive ultrastructural changes in the casein matrix during the ripening of inadequately acidified feta cheese.

This study investigated the ultrastructural changes underlying the undesired softening of insufficiently acidified feta cheese during cold storage. Experimental feta cheeses with a range of pH values before brining were manufactured by allowing the cheese blocks to ferment overnight at 3 temperatures (35, 20, and 3°C), which resulted in pH values of 4.80, 4.88, and 5.17, respectively. Cheese blocks were stored in pH-adjusted whey brine solutions for up to 120 d, at which point significant decreases in the cheese firmness were confirmed with compression and shear tests. Samples for transmission electron microscopy were taken during the make procedure, after overnight fermentation, and after 7 and 90 d of cold storage. Increasing the initial pH from 4.80 to 5.17 resulted in a fundamentally different ultrastructure at d 90, with the protein matrix as the continuous phase having markedly decreased density compared with the typically open porous and discontinuous protein matrix of high density in the low-pH control feta cheese. Ultrastructural changes were progressive, and the first signs were evident after only 20 h (the overnight fermentation), when fine, proteinaceous material dissociated from the edges of the casein strands into the serum phase. By d 7, the serum phase was completely filled with the loosely aggregated casein closely surrounding the spheroidal fat globules. A further breakdown of the protein matrix was observed after 90 d, with the complete loss of open porous network structure. Image analysis quantitatively confirmed the progressive and significant decrease in density of the protein matrix. In summary, this is the first study to provide a comprehensive and in-depth view of the progressive and most likely irreversible ultrastructural changes that lead to this textural defect.

Response surface methodology modeling of protein concentration, coagulum cut size, and set temperature on curd moisture loss kinetics during curd stirring.

The effects of the independent variables protein concentration (4-6%), coagulum cut size (6-18 mm3), and coagulation temperature (28-36°C) on curd moisture loss during in-vat stirring were investigated using response surface methodology. Milk (14 kg) in a cheese vat was rennet coagulated, cut, and stirred as per semihard cheesemaking conditions. During stirring, the moisture content of curd samples was determined every 10 min between 5 and 115 min after cutting. The moisture loss kinetics of curds cut to 6 mm3 followed a logarithmic trend, but the moisture loss of curds from larger cut sizes, 12 or 18 mm3, showed a linear trend. Response surface modeling showed that curd moisture level was positively correlated with cut size and negatively correlated with milk protein level. However, coagulation temperature had a significant negative effect on curd moisture up to 45 min of stirring but not after 55 min (i.e., after cooking). It was shown that curds set at the lower temperature had a slower syneresis rate during the initial stirring compared with curds set at a higher temperature, which could be accelerated by reducing the cut size. This study shows that keeping a fixed cut size at increasing protein concentration decreased the level of curd moisture at a given time during stirring. Therefore, to obtain a uniform curd moisture content at a given stirring time at increasing protein levels, an increased coagulum cut size is required. It was also clear that breakage of the larger curd particles during initial stirring can also significantly influence the curd moisture loss kinetics. Both transmission and scanning electron micrographs of cooked curds (i.e., after 45 min of stirring) showed that the casein micelles were fused at a higher degree in curds coagulated at 36°C compared with 28°C, which confirmed that coagulation temperature causes a marked change in curd microstructure during the earlier stages of stirring. The present study showed the dynamics of curd moisture content during stirring when using protein-concentrated milk at various set temperatures and cut sizes. This provides the basis for achieving a desired curd moisture loss during cheese manufacture using protein-concentrated milk as a means of reducing the effect of seasonal variation in milk for cheesemaking.

Influence of protein concentration and coagulation temperature on rennet-induced gelation characteristics and curd microstructure.

This study characterized the coagulation properties and defined the cutting window (CW; time between storage modulus values of 35 and 70 Pa) using rheometry for milk standardized to 4, 5, or 6% protein and set at 28, 32, or 36°C. Milks were standardized to a protein-to-fat ratio of approximately 1 by blending ultrafiltration retentate, skim milk, and whole milk. The internal curd microstructure for selected curd samples was analyzed with transmission electron microscopy and scanning electron microscopy. Lowering the coagulation temperature caused longer rennet coagulation time and time to reach storage modulus of 35 Pa, translating into a wider CW. It also led to a lower maximum curd-firming rate (MCFR) with lower firmness at 40 min at a given protein level. Increasing protein levels resulted in the opposite effect, although without an effect on rennet coagulation time at a given temperature. On coagulation at 28°C, milk with 5% protein resulted in a similar MCFR (∼4 Pa/min) and CW (∼8.25 min) compared with milk with 4% protein at 32°C, which reflects more standard conditions, whereas increasing milk to 6% protein resulted in more than doubling of the curd-firming rate (MCFR = 9.20 Pa/min) and a shorter CW (4.60 min). Gels set at 28°C had lower levels of rearrangement of protein network after 40 min compared with those set at 36°C. Protein levels, on the other hand, had no influence on the levels of protein network rearrangement, as indicated by loss tangent values. The internal structure of curd particles, as investigated by both scanning electron microscopy and transmission electron microscopy, appeared to have less cross-linking and smaller casein aggregates when coagulated at 28°C compared with 36°C, whereas varying protein levels did not show a marked effect on aggregate formation. Overall, this study showed a marked interactive effect between coagulation temperature and protein standardization of milk on coagulation properties, which subsequently requires adjustment of the CW during cheesemaking. Lowering of the coagulation temperature greatly altered the curd microstructure, with a tendency for less syneresis during cutting. Further research is required to quantify the changes in syneresis and in fat and protein losses to whey due to changes in the microstructure of curd particles arising from the different coagulation conditions applied to the protein-fortified milk.

Serum amyloid A (SAA) is an early biomarker of influenza virus disease in BALB/c, C57BL/2, Swiss-Webster, and DBA.2 mice.

Assessment of influenza virus disease progression and efficacy of antiviral therapy in the widely used mouse models relies mostly on body weight loss and lung virus titers as markers of disease. However, both parameters have their shortcomings. Therefore, the aim of our study was to find non-invasive markers in the murine model of severe influenza that could detect disease early and predict disease outcome. BALB/c mice were lethally infected with influenza A(H1N1)pdm09 virus and serum samples were collected at various time points. Enzyme-linked immunosorbent assays were performed to quantify amounts of serum amyloid A (SAA), C-reactive protein, complement 3, transferrin, corticosterone, prostaglandin E2, H2O2, and alpha-2,6-sialyltransferase. We found that SAA was the most promising candidate with levels acutely and temporarily elevated by several hundred-fold 3 days post virus inoculation. Upon treatment with oseltamivir phosphate, levels of SAA were significantly decreased. High levels of SAA were associated with poor disease prognosis, whereas body weight loss was not as a reliable predictor of disease outcome. SAA levels were also transiently increased in BALB/c mice infected with influenza A(H3N2) and influenza B virus, as well as in C57BL/2, Swiss-Webster, and DBA.2 mice infected with influenza A(H1N1)pdm09 virus. High levels of SAA often, but not always, were associated with disease outcome in these other influenza virus mouse models. Therefore, SAA represents a valid biomarker for influenza disease detection in all tested mouse strains but its prognostic value is limited to BALB/c mice infected with influenza A(H1N1)pdm09 virus.

A zanamivir dimer with prophylactic and enhanced therapeutic activity against influenza viruses.

OBJECTIVES: Emerging drug resistance to antiviral therapies is an increasing challenge for the treatment of influenza virus infections. One new antiviral compound, BTA938, a dimeric derivative of the viral neuraminidase inhibitor zanamivir, contains a 14-carbon linker bridging two zanamivir moieties. In these studies, we evaluated antiviral efficacy in cell cultures infected with influenza virus and in mouse models of lethal influenza using H1N1pdm09, H3N2 and oseltamivir-resistant (H275Y) viruses. METHODS: In vitro activity was evaluated against 22 strains of influenza virus. Additionally, in vivo studies compared the efficacy of BTA938 or zanamivir after intranasal treatment. We also tested the hypothesis of a dual mode of action for BTA938 using scanning electron microscopy (SEM). RESULTS: BTA938 inhibited the viruses at nanomolar concentrations in vitro with a median 50% effective concentration value of 0.5 nM. In mouse models, the dimer provided ∼10-fold greater protection than zanamivir. The data also showed that a single low dose (3 mg/kg) protected 100% of mice from an otherwise lethal oseltamivir-resistant (H275Y) influenza virus infection. Remarkably, a single prophylactic treatment (10 mg/kg) administered 7 days before the challenge protected 70% of mice and when administered 1 or 3 days before the challenge it protected 90% of mice. Additionally, SEM provides evidence that the increased antiviral potency may be mediated by an enhanced aggregation of virus on the cell surface. CONCLUSIONS: In vitro and in vivo experiments showed the high antiviral activity of BTA938 for the treatment of influenza virus infections. Moreover, we demonstrated that a single dose of BTA938 is sufficient for prophylactic and therapeutic protection in mouse models.

In vitro activity of favipiravir and neuraminidase inhibitor combinations against oseltamivir-sensitive and oseltamivir-resistant pandemic influenza A (H1N1) virus.

Few anti-influenza drugs are licensed in the United States for the prevention and therapy of influenza A and B virus infections. This shortage, coupled with continuously emerging drug resistance, as detected through a global surveillance network, seriously limits our anti-influenza armamentarium. Combination therapy appears to offer several advantages over traditional monotherapy in not only delaying development of resistance but also potentially enhancing single antiviral activity. In the present study, we evaluated the antiviral drug susceptibilities of fourteen pandemic influenza A (H1N1) virus isolates in MDCK cells. In addition, we evaluated favipiravir (T-705), an investigational drug with a broad antiviral spectrum and a unique mode of action, alone and in dual combination with the neuraminidase inhibitors (NAIs) oseltamivir, peramivir, or zanamivir, against oseltamivir-sensitive pandemic influenza A/California/07/2009 (H1N1) and oseltamivir-resistant A/Hong Kong/2369/2009 (H1N1) virus. Mean inhibitory values showed that the tested virus isolates remained sensitive to commonly used antiviral drugs, with the exception of the Hong Kong virus isolate. Drug dose-response curves confirmed complete drug resistance to oseltamivir, partial sensitivity to peramivir, and retained susceptibility to zanamivir and favipiravir against the A/Hong Kong/2369/2009 virus. Three-dimensional analysis of drug interactions using the MacSynergy(TM) II program indicated an overall synergistic interaction when favipiravir was combined with the NAIs against the oseltamivir-sensitive influenza virus, and an additive effect against the oseltamivir-resistant virus. Although the clinical relevance of these drug combinations remains to be evaluated, results obtained from this study support the use of combination therapy with favipiravir and NAIs for treatment of human influenza virus infections.

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-ß) expression.

The human parainfluenza virus type 3 (HPIV3) phosphoprotein (P) gene is unusual as it contains an editing site where nontemplated ribonucleotide residues can be inserted. This RNA editing can lead to the expression of the viral P, PD, putative W, and theoretical V protein from a single gene. Although the HPIV3 PD protein has been detected, its function and those of the W and V proteins are poorly understood. Therefore, we first used reverse genetics techniques to construct and rescue a recombinant (r)HPIV3 clone with a polyhistidine sequence at the 5' end of the P gene for tagged protein detection. Western blot analysis demonstrated the presence of the P, PD, and W proteins, but no V protein was detected. Then, we functionally studied the D domain of the PD protein by constructing two rHPIV3 knockout clones that are deficient in the expression of the D domain. Results from growth kinetic studies with infected MA-104 and A596 cells showed that viral replication of the two knockout viruses (rHPIV3-ΔES and rHPIV3-ΔD) was comparable to that of the parental virus in both cell lines. However, viral mRNA transcription and genomic replication was significantly reduced. Furthermore, cytokine/chemokine profiles of A549 cells infected with either knockout virus were unchanged or showed lower levels compared to those from cells infected with the parental virus. These data suggest that the D domain of the PD protein may play a luxury role in HPIV3 RNA synthesis and may also be involved in disrupting the expression of beta interferon.

Unique cell wall abnormalities in the putative phosphoinositide phosphatase mutant AtSAC9.

SAC9 is a putative phosphoinositide phosphatase in Arabidopsis thaliana involved in phosphoinositide signaling. sac9-1 plants have a constitutively stressed phenotype with shorter roots which notably accumulate phosphatidylinositol 4,5-bisphosphate and its hydrolysis product inositol trisphosphate. We investigated the primary roots of sac9-1 seedlings at the cytological and ultrastructural level to determine the structural basis for this altered growth. Despite the normal appearance of organelles and cytoplasmic elements, our studies reveal extreme abnormalities of cell wall and membrane structures in sac9-1 primary root cells, regardless of cell type, position within the meristematic area, and plane of section. Cell wall material was deposited locally and in a range of abnormal shapes, sometimes completely fragmenting the cell. Simple protuberances, broad flanges, diffuse patches, elaborate folds, irregular loops and other complex three-dimensional structures were found to extend randomly from the pre-existing cell wall. Abundant vesicles and excessive membrane material were associated with these irregular wall structures. We argue that a perturbed phosphoinositide metabolism most likely induces these observed abnormalities and hypothesize that a disorganized cytoskeleton and excessive membrane trafficking mediate the cell wall defects.