Rennet is a liquid, paste, or powder preparation containing the enzyme, rennin. It is usually prepared by extracting the fourth stomach (abomasum) of milk-fed calves with a sodium chloride solution, and is used in the manufacture of cheese, rennet casein, and various specialty products such as junket or rennet custard. Rennet is probably one of our oldest commercially-used enzyme preparations. Several hundred-thousand gallons of extract are produced each year in addition to large amounts of paste and powder. In spite of its long and extensive use, many properties of rennet, as well as rennin itself, are not fully understood. The milk-clotting action of rennet has been known for centuries, but the nature of this action has never been satisfactorily explained. Many theories of rennin action have been advanced, but none have been soundly established by experimental evidence. Much of the material on rennin in the literature is contradictory. Because of the complexity of milk and the complexity of the clotting reaction, no completely satisfactory method has yet been devised for measuring rennin activity and studying the influence of pH, temperature, and possible activators and inhibitors on the activity of the enzyme itself. While it is known that calcium ions and other di- or trivalent cations are essential for the clotting of milk in the presence of rennin, the function of the ions in clot formation has never been demonstrated. Rennin has some proteolytic activity, but its importance in the proteolytic breakdown of cheese during ripening has been questioned from time to time. These are but a few of the unsolved problems which stand in our way of a better understanding of the nature of rennin and the role it plays in the cheese-making operation.

The objective of this work was to examine the relationship between water mobility and the glass transition behavior of vital and chemically-treated wheat gluten. Dynamic Mechanical Analyzer (DMA) and Differential Scanning Calorimetry (DSC) was used to study thermal transition behavior and water mobility was evaluated by $\sp2$H and $\sp{17}$O Nuclear Magnetic Resonance (NMR). $\sp2$H NMR percent detected intensity reflected the changes occurring in the sample through the glass-rubbery transition (T$\sb{\rm g}$). With the addition of deuterated water, below 0.10 g D$\sb2$O/g total, in the glassy region, detected NMR intensity was almost zero and above 0.26 g D$\sb2$O/g total, in the rubbery region, the detected signal was the maximum. Freezable water occurred at $>$0.18 g water/g total, close to the midpoint of the T$\sb{\rm g}$ region. Fermi's equation was used to develop empirical models relating moisture and temperature with relative stiffness for gluten. The critical moisture content and steepness (a) from the relative stiffness data correlated well with the $\sp2$H NMR detected intensity. Potassium bromate and ascorbic acid-treated gluten showed lower detected signal and higher water sorption than the control. The oxidant-added gluten showed a broader TB (approx. 150$\sp\circ$C) at moisture content $>$0.10 g water/g total as compared against the control (100$\sp\circ$C). The T$\sb{\rm g}$, mechanical, and water vapor barrier properties of wheat gluten films containing glycerin, sucrose, glycerin-sucrose and glycerin-sorbitol were also studied. Glycerin and sucrose showed limited miscibility with each other and displayed two separate transitions. The low-T tan $\delta$ peak height (due to glycerin-rich phase) was found to influence the tensile strength and elongation linearly and the water vapor permeability (WVP) curvilinearly. However, the T$\sb{\rm g}$ did not change with composition and exhibited low correlation with barrier and mechanical properties. Initial addition of glycerin increased the WVP dramatically. The presence of sucrose decreased the WVP (only slightly), but resulted in a rigid and fragile film. Sorbitol resulted in a single effective T at $-$42$\sp\circ$C and intermediate values for tensile strength, percent elongation, and WVP (between the 15:6:0 and 15:4:2 gluten:glycerin:sucrose films). Both low-T$\sb{\rm g}$ and high-T$\sb{\rm g}$ transitions showed strong moisture dependence. Sorbitol-added film, showed a stronger moisture effect as compared to glycerin- and sucrose-added film. The sucrose ester and glycerin-added film showed two transitions similar to the earlier case. The magnitude of the low-T transition was affected by the concentration of the sucrose ester used.

Protein isolates made from low quality muscle foods can be an efficient method to increase the water holding capacity of muscle foods. Increased water holding capacity may improve the sensory values of muscle foods after cooking. Protein isolates from various sources can be utilized to add value and increase water holding of muscle foods. In this study we focused on the mechanism of increasing the water holding of muscle foods after the injection of protein isolate suspensions. Cook yields of muscle samples increased after the injection of these suspensions. Addition of buffering salts and increasing the amount of NaCl in the suspensions increased cook yields further more. Injection material containing protein isolates increased the retention of injected material in the muscle when compared to the injection solutions that did not contain the protein isolate. To reach a certain level of injection, a larger amount of injection solution has to be injected as part of this solution may leak out of the muscle once the injection is complete. When the suspension containing protein isolate is injected into the muscle, retention of the injected material is greater than in the case without the isolate and the injected suspensions stay in the muscle after injection. The increased retention may be due to the suspension having a consistency sufficiently high to resist the pressure of the muscle to force it out. Injected protein isolate suspensions form pockets inside the muscle by deforming the muscle structure and the deformed muscle does not go back to its initial form even after the suspension is removed from the pockets. The injected material flows along the muscle fibers but not through connective tissue. Cuts of muscles with all fibers oriented in parallel fashion will have a better distribution of the injected material than muscle cuts with complex fiber orientation. The consistencies of protein isolate suspensions showed similarities with their solubilities. Both of these properties increased with increase of pH and stayed at a higher level when the pH decreased back to initial pH values from the alkaline side. Solubility and consistency of protein isolate suspensions increased over time.

The chronic diseases such as diabetes mellitus and cardiovascular disease are among the leading causes of death globally. These are strongly associated with obesity as a result of change in dietary pattern towards high calories and reduced physical activity, which now affects both developing and developed countries. Excessive cellular energy from high calorie foods results in incomplete reduction of oxygen resulting in oxidative stress leading to reactive oxygen species (ROS). The harmful effects of ROS can be balanced by the combination of non-enzymatic antioxidants and antioxidant enzymes. Hence, the maintenance of balance between oxidants and antioxidants through well-designed diet that can modulate cellular protection through critical energy and reductant pathways coupled to antioxidant enzymes is essential. Therefore, the aim of this dissertation was to develop strategies for designing diets enriched in phenolic phytochemicals for chemoprevention of diseases. This was achieved through understanding the critical metabolic pathways contributing to cellular protection against oxidation dysfunction-linked chronic diseases. Based on structure-function rationale, the potential antioxidant, anti-hyperglycemia and anti-hypertension functionality of plant foods such as clonal lines of Lamiaceae family, Rhodiola, wine, tea, vegetables (peppers, eggplant and pumpkin) and grains (corn and legume) were investigated. Results indicated that specific phenolic profiles had high anti-hyperglycemia and anti-hypertension potential which generally corresponded to total phenolic content and free radical scavenging-linked antioxidant activity. Dietary chemoprevention of bacterial pathogens such as Helicobacter pylori and Staphylococcus aureus was investigated through inhibition of critical control points that breakdown cellular energy and antioxidant pathways. Results indicated that lemon balm with the highest gallic acid and caffeic acid contents had potent anti-S. aureus activity. L-Lactic acid, a major food ingredient, had high anti-H. pylori activity through inhibition of proline dehydrogenase and catalase. Overall cellular protection through modulation of energy and reductant stimulating metabolic pathways was investigated in model eukaryotes. Rhodiola crenulata with high anti-hyperglycermia activity, induced apoptosis in V14a breast cancer cell line. Further, phenolic phytochemicals from R. crenulata protected Saccharomyces cerevisiae from UV-induced oxidative stress and delayed death. The cellular protection with phenolic phytochemicals was linked to modulation of proline-linked oxidative phosphorylation coupled to pentose phosphate pathway (PPP).

The objective of this research was to extend the functionality of maltodextrin in foods, by forming maltodextrin-surfactant complexes that had novel properties not exhibited by maltodextrin alone. To achieve this objective it was necessary to understand the molecular origin and characteristics of maltodextrin-surfactant interactions. A variety of analytical techniques were therefore used to characterize maltodextrin-surfactant interactions. Initial experiments using isothermal titration calorimetry (ITC) and surface tensiometry showed that an anionic surfactant (SDS) bound to maltodextrin when the surfactant concentration exceeded a critical value (∼0.05 mM). ITC showed that the interaction of SDS to maltodextrin was exothermic, which could have been due to an exothermic coil-helix transition and/or an exothermic binding reaction. Surfactant binding to maltodextrin only occurred when the number of monomers in the maltodextrin chain exceeded ∼24 glucose units. NMR studies showed that the interaction involved carbons 1 and 4 of the D-glucopyranose residues of maltodextrin and the surfactant hydrophobic tail, which suggested the formation of a helical inclusion complex. The effect of surfactant type on maltodextrin-surfactant interactions was also investigated. ITC, surface tension and ultrasonic measurements indicated that the charge on the surfactant head group influenced their binding to maltodextrin. Similar amounts of anionic and cationic surfactant bound to maltodextrin, but a much smaller amount of non-ionic surfactant bound. The ITC indicated that surfactants with longer tail groups bound more strongly to maltodextrin than surfactants with shorter ones. The effect of temperature, pH, and salt concentration on maltodextrin-surfactant interactions was studied using ITC. This study indicated that the enthalpy changes associated with surfactant demicellization were highly temperature-dependent. In contrast, the binding of surfactants to maltodextrin was exothermic and relatively temperature-independent. There was no effect of pH on the binding of surfactant to maltodextrin. In contrast, salt concentration affected both surfactant demicellization and surfactant binding to maltodextrin. A potential application of maltodextrin-surfactant complexes was demonstrated by studying the rheology and thermal behavior of maltodextrin solutions in the presence and absence of SDS. The rheology and thermal properties of maltodextrin solutions changed significantly when surfactant was added, e.g. the viscosity increased, gelation occurred, and a melting transition was observed.

The rheological characterization of semi-liquid foods has encountered two major difficulties: (1) the separation of a layer of fluid of different viscosity at the wall of the rheometer (slippage) and (2) the disruption of the internal structure of the specimen when inserted into the equipment. The increasing utilization of lubricated squeezing flow viscometry in food industry has not only been determined by its relative simplicity but mainly by its ability to overcome the above mentioned problems. Although the mathematical models to describe the lubricated squeezing flow of fluids have been extensively considered, several aspects and possible applications of this methodology remain unstudied. Therefore, this research was focused on evaluating the effect of test conditions, especially the displacement rate, on the measured rheological parameters of several semi-liquid food products. A considerable discrepancy was noted between the observed rate effects and those predicted using a pseudoplastic (power law) model. Consequently, a alternative model was proposed and the effect of the deformation rate was described using the following empirical relationship (Fv1− FR)/(Fv2 − FR) = (V1/V2)m. Since lubricated squeezing flow viscometry barely produces a significant disruption of the sample, it was used to quantify the structural damage caused to a specimen subjected to several disruption mechanisms such as compression and stirring. As expected, compression of the specimen prior to its testing had very little effect on the specimen consistency which supports the view that there was little friction between the Teflon® coated plates and the samples. On the other hand, shearing caused a measurable loss of consistency. In order to study the recovery of the sample consistency after disruption a rest period was introduced and the rheological parameters of the treated samples were determined. Different levels of recovery were observed for different specimens, however in none of the cases a full recovery of the original consistency was observed in the time span considered (1–3 hours). Finally, a relation between the instrumental value of consistency obtained using lubricated squeezing flow and sensory perception was estimated. It was established that squeezing flow is more sensitive to textural changes sensory evaluation and that the methodology was appropriate to determine thresholds for sensory detection.

A new process solubilizes muscle proteins making it possible to separate membranes with their oxidizable phospholipids. Isolated cod muscle membranes sedimented at 4,000 × g for 15 min at pH 5 or below but not at pH 6 or higher. Isolated membranes added to the supernatant of homogenized muscle solubilized at pH 3 and centrifuged at 10,000 × g for 30 min also sedimented at 4,000 × g for 15 min. This was in contrast to the membranes naturally present in solubilized homogenate. This suggests that factors other than viscosity were involved in sedimentation. Most membrane phospholipid in cod or herring muscle homogenates solubilized at pH 3 or 10.5 could not be sedimented by centrifugation at 4,000 or 10,000 × g for 15 min. Addition of two high Mw chitosans (50–190 and 310–375 kDa) increased membrane removal while two low Mw chitosans (1 and 33 kDa) did not. The high Mw chitosans had to be added prior to solubilization of the muscle homogenates to be effective. Calcium chloride, and to a lesser extent MgCl2, aided in membrane removal from muscle homogenates solubilized at pH 3 in the presence of citric acid or malic acid but not lactic acid. Adding the citric acid and Ca 2+ before solubilizing the muscle homogenates was needed for the effect. At 1 mM citric acid, 70–80% of the phospholipid and 25–30% of the protein were removed at 10 mM Ca2+. At 8 mM Ca2+ , citric acid showed an optimal effect on phospholipid removal at 5 mM with 90% of the phospholipid and 35% of the protein removed from homogenates solubilized at pH 3. The percentage of phospholipid and protein removed from muscle homogenates solubilized at pH 10.5 increased with increasing Ca 2+ concentrations at 1 mM citric acid. At 8 mM Ca2+, addition of citric acid at 5 mM improved membrane removal from muscle homogenates solubilized at pH 10.5 to about 80% from 58% in its absence. Ca2+ and citric acid might exert their influence by disconnecting linkages between membranes and cytoskeletal proteins and/or aiding in aggregation of the membranes.

The Layer by layer electrostatic deposition (LBL) technique has been shown to have potential applications in foods such as controlled/triggered release, prevention of lipid oxidation and stabilization of emulsions to processing and storage conditions. Biopolymer properties (e.g., as charge density, molecular weight and conformation) and droplet properties ( e.g., concentration, size and charge) are the most important factors in the production of stable multilayered emulsions using this technique. In this study all natural biopolymers, β-lactoglobulin (Blg), pectin and chitosan have been used to produce multilayer-coated O/W emulsions. Blg was used to produce a primary emulsion with small droplet sizes, then, an anionic biopolymer (pectin) was added to the system to produce secondary emulsions containing droplets coated with an emulsifier-biopolymer (Blg-pectin) layer. The effect of preparation pH, presence or absence of NaCl, and thermal treatment was studied. It was found that mixing the droplets and biopolymer at a pH where they initially had similar charges (pH 7) and then adjusting the pH to values where they had opposite charges (pH 3-4) led to the formation of more stable secondary emulsions than directly mixing the droplets and biopolymer at pH values where they had opposite charges. The Blg-pectin coated droplets in the secondary emulsions were stable to droplet aggregation and creaming at temperatures up to 90°C and NaCl concentrations up to 300 mM. The interaction of Blg and chitosan as a function of pH and chitosan concentration was examined with the expectation of drawing conclusions for their interaction at emulsion droplet surfaces. Chitosan interacted with Blg exothermically to form either soluble or insoluble complexes depending on the pH. At pH values where chitosan and Blg have opposite charges (pH 6 and 7) they interact strongly with each other to form insoluble complexes. Stable Blg/chitosan secondary emulsions however could not be produced due to the narrow range of chitosan concentration (between saturation and depletion concentrations) where this could be achieved. Stability maps are suggested as a strategy to predict areas of possible multilayer formation based on preparation conditions and polyelectrolyte and droplet characteristics. Blg-pectin-chitosan tertiary emulsions were also prepared and found to be stable over a wider range of pH (3-6) which was attributed to the ability of the multi-layered interfaces to either increase the repulsive interactions between the droplets (e.g., steric and electrostatic), decrease the attractive interactions (e.g., van der Waals), and/or to increase the resistance of the interfacial layer to rupture. This study provides valuable information on use of the LBL technique in the area of multilayered food emulsions with a focus on the effects of preparation conditions and biopolymer characteristics on stable emulsion formation and long term stability. Better understanding of these systems provided here may lead to applications in the area of encapsulation and delivery of sensitive active ingredients.

Muscle foods are a major source of food protein in many parts of the world including the United States. Acid and/or alkali solubilization is a recent method developed to separate muscle proteins with good functional properties. The unsaturated lipids of the fish muscle are highly susceptible to oxidative degradation, especially in the presence of hemoglobin. The goal of this research work was to study structural and physicochemical changes of fish membranes that occur during the acid or alkali solubilization procedures. The effect on lipid oxidation and ways to stabilize the membrane lipids against oxidation were also studied. Washed and unwashed cod, as well as cod microsomes were used as model systems for the study. Peroxide value, TBARS and sensory were employed to follow lipid oxidation. Citric acid and calcium chloride were effective in protecting the muscle from lipid oxidation either when used in washing the muscle or when added directly to the minced muscle. The maximum effect was achieved when citric acid and calcium chloride were added in the first washing step. Solubilizing the muscle at pH 3 or 10.8 in the absence of hemoglobin protected the protein isolates from lipid oxidation. However, when the muscle and hemoglobin were together exposed to low pH, oxidation was promoted. Protein isolates prepared by citric acid/calcium chloride-treated washed cod solubilized at pH 3 in the presence of hemoglobin exhibited very good oxidative stability. Part of the protection that citric acid/calcium chloride offered against lipid oxidation was attributed to the ability of citric acid to break down unstable peroxides. Hydrochloric acid was also able to break down formed peroxides to a similar degree as citric acid. At pH 5.3 or lower, 99% of isolated cod membranes sediment at low centrifugation speeds. Isolated membranes that were exposed to pH 3.0 or 10.8 were less susceptible to hemoglobin-mediated lipid oxidation. Cod hemoglobin exposed for 20sec or 20min to pH 3 was rendered less pro-oxidative than the untreated cod hemoglobin. However, when microsomes and hemoglobin were together exposed to low pH, oxidation was promoted. Microsomes prepared in the presence of citric acid and calcium chloride were less susceptible to hemoglobin-mediated lipid oxidation compared to untreated microsomal suspensions. The soluble fraction of the cod muscle (press juice) was able to retard lipid oxidation of washed cod and protein isolate, even when washed cod was solubilized at low pH in the presence of hemoglobin. Press juice retained its anti-oxidative activity even after exposure to low pH. Press juice was able to prevent hemoglobin-mediated, NADH-dependent and Fe-ascorbate mediated lipid oxidation of isolated cod microsomes.

The objective of this study was to investigate the impact of low molecular weight cosolvent systems on the gelling mechanism of globular proteins, including bovine serum albumin (BSA), β-lactoglobulin, (β-Lg), and whey protein isolate (WPI). The influence of sucrose, glycerol, sorbitol, and cosolvent mixtures (0 to 40 wt%) on the thermal denaturation and gelation of bovine serum albumin (BSA) in aqueous solution has been studied. The effect of these cosolvent systems on heat denaturation of 0.5 wt% BSA solutions (pH 6.9) was measured using ultrasensitive differential scanning calorimetry. The unfolding process was irreversible and could be characterized by a denaturation temperature (Tm). As the cosolvent concentration increased from 0 to 40 wt%, Tm also increased depending on the cosolvent system surrounding the protein molecule. The rise in Tm was attributed to the increased thermal stability of the globular state of BSA relative to its native state because of differences in their preferential interactions with the cosolvent system. The change in preferential interaction coefficient (ΔΓ3,2) associated with the native-to-denatured transition was estimated for each cosolvent system. The dynamic shear rheology of 2, 4, and 5 wt% BSA solutions (pH 6.9, 0–200 mM NaCl) was monitored as they were heated from 30 to 90°C, held at 90°C for either 15 or 120 minutes, and then cooled back to 30°C. The impact of each cosolvent system on the gelation temperature was characterized. Depending of the cosolvent system surrounding the protein molecules, the complex shear modulus (G*) of cooled gels either increased or remained constant with cosolvent concentration when they were held at 90°C for 15, 70, or 120 minutes. The turbidity of the same solutions was monitored as they were heated from 30 to 90°C at 1.5°C min−1 or held isothermally at 90°C for 10 minutes. As the cosolvent concentration increased the protein gelation temperature increased, but the isothermal gelation rate at 90°C decreased. The impact of various cosolvent systems on gel characteristics was interpreted in terms of its ability to decrease the protein-protein collision frequency, increase the attractive forces between proteins (at low temperatures) and increase protein thermal stability.