Peggy M. Tomasula

Structure and Function of Protein-Based Edible Films and Coatings

Research and development on films and coatings made from various agricultural proteins has been conducted over the past 20 years, but is of heightened interest, due to the demand for environmentally-friendly, renewable replacements for petroleum-based polymeric materials and plastics. To address this demand, films and coatings have been made from renewable resources, such as casein, whey, soy, corn zein, collagen, wheat gluten, keratin and egg albumen. Those made from agricultural proteins create new outlets for agricultural products, byproducts and waste streams, all of which can positively impact the economics of food processes.

An assessment of pasteurization treatment of water, media, and milk with respect to Bacillus spores

This study evaluated the ability of spore-forming Bacillus spp. to resist milk pasteurization conditions from 72 to 150 degrees C. Spores from the avirulent surrogate Sterne strain of Bacillus anthracis, as well as a representative strain of a common milk contaminant that is also a pathogen, Bacillus cereus ATCC 9818, were heated at test temperatures for up to 90 min in dH2O, brain heart infusion broth, or skim milk. In skim milk, characteristic log reductions (log CFU per milliliter) for B. anthracis spores were 0.45 after 90 min at 72 degrees C, 0.39 after 90 min at 78 degrees C, 8.10 after 60 min at 100 degrees C, 7.74 after 2 min at 130 degrees C, and 7.43 after 0.5 min at 150 degrees C. Likewise, log reductions (log CFU per milliliter) for viable spores of B. cereus ATCC 9818 in skim milk were 0.39 after 90 min at 72 degrees C, 0.21 after 60 min at 78 degrees C, 7.62 after 60 min at 100 degrees C, 7.37 after 2 min at 130 degrees C, and 7.53 after 0.5 min at 150 degrees C. No significant differences (P < 0.05) in thermal resistance were observed for comparisons of spores heated in dH2O or brain heart infusion broth compared with results observed in skim milk for either strain tested. However, spores from both strains were highly resistant (P < 0.05) to the pasteurization temperatures tested. As such, pasteurization alone would not ensure complete inactivation of these spore-forming pathogens in dH2O, synthetic media, or skim milk.

A continuous process for casein production using high-pressure carbon dioxide☆

A continuous process for protein production that uses high-pressure carbon dioxide (CO2) instead of organic acids as precipitant has been used to isolate casein from milk. Central to the process is a pressure reduction stage that follows a reaction/precipitation stage, in which protein produced in the reactor may be removed without depressurizing the reactor and subjecting the products to excessive shear. Two reactor/precipitators were designed and tested. One was a spray reactor in which milk and CO2 were contacted by spraying milk into CO2. The other was a tubular reactor fed a mixture of liquid CO2 and milk. Both reactors gave casein products of good quality, but casein yield from the tubular reactor was greater than that from the spray reactor. The methods presented here may be extended to other processes that currently use organic acids for precipitation or supercritical extraction systems at pressures less than 15000 kPa for continuous transport of solid-liquid mixtures from high-pressure units to atmospheric pressure.

Two Methods for Increased Specificity and Sensitivity in Loop-Mediated Isothermal Amplification

The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.

Effect of homogenization and pasteurization on the structure and stability of whey protein in milk1

The effect of homogenization alone or in combination with high-temperature, short-time (HTST) pasteurization or UHT processing on the whey fraction of milk was investigated using highly sensitive spectroscopic techniques. In pilot plant trials, 1-L quantities of whole milk were homogenized in a 2-stage homogenizer at 35°C (6.9 MPa/10.3 MPa) and, along with skim milk, were subjected to HTST pasteurization (72°C for 15 s) or UHT processing (135°C for 2 s). Other whole milk samples were processed using homogenization followed by either HTST pasteurization or UHT processing. The processed skim and whole milk samples were centrifuged further to remove fat and then acidified to pH 4.6 to isolate the corresponding whey fractions, and centrifuged again. The whey fractions were then purified using dialysis and investigated using the circular dichroism, Fourier transform infrared, and Trp intrinsic fluorescence spectroscopic techniques. Results demonstrated that homogenization combined with UHT processing of milk caused not only changes in protein composition but also significant secondary structural loss, particularly in the amounts of apparent antiparallel β-sheet and α-helix, as well as diminished tertiary structural contact. In both cases of homogenization alone and followed by HTST treatments, neither caused appreciable chemical changes, nor remarkable secondary structural reduction. But disruption was evident in the tertiary structural environment of the whey proteins due to homogenization of whole milk as shown by both the near-UV circular dichroism and Trp intrinsic fluorescence. In-depth structural stability analyses revealed that even though processing of milk imposed little impairment on the secondary structural stability, the tertiary structural stability of whey protein was altered significantly. The following order was derived based on these studies: raw whole>HTST, homogenized, homogenized and pasteurized>skimmed and pasteurized, and skimmed UHT>homogenized UHT. The methodology demonstrated in this study can be used to gain insight into the behavior of milk proteins when processed and provides a new empirical and comparative approach for analyzing and assessing the effect of processing schemes on the nutrition and quality of milk and dairy product without the need for extended separation and purification, which can be both time-consuming and disruptive to protein structures.

Effect of high-pressure processing on reduction of Listeria monocytogenes in packaged Queso Fresco

The effect of high-hydrostatic-pressure processing (HPP) on the survival of a 5-strain rifampicin-resistant cocktail of Listeria monocytogenes in Queso Fresco (QF) was evaluated as a postpackaging intervention. Queso Fresco was made using pasteurized, homogenized milk, and was starter-free and not pressed. In phase 1, QF slices (12.7 × 7.6 × 1 cm), weighing from 52 to 66 g, were surface inoculated with L. monocytogenes (ca. 5.0 log10 cfu/g) and individually double vacuum packaged. The slices were then warmed to either 20 or 40°C and HPP treated at 200, 400, and 600 MPa for hold times of 5, 10, 15, or 20 min. Treatment at 600 MPa was most effective in reducing L. monocytogenes to below the detection level of 0.91 log10 cfu/g at all hold times and temperatures. High-hydrostatic-pressure processing at 40°C, 400 MPa, and hold time ≥ 15 min was effective but resulted in wheying-off and textural changes. In phase 2, L. monocytogenes was inoculated either on the slices (ca. 5.0 log10 cfu/g; ON) or in the curds (ca. 7.0 log10 cfu/g; IN) before the cheese block was formed and sliced. The slices were treated at 20°C and 600 MPa at hold times of 3, 10, and 20 min, and then stored at 4 and 10°C for 60 d. For both treatments, L. monocytogenes became less resistant to pressure as hold time increased, with greater percentages of injured cells at 3 and 10 min than at 20 min, at which the lethality of the process increased. For the IN treatment, with hold times of 3 and 10 min, growth of L. monocytogenes increased the first week of storage, but was delayed for 1 wk, with a hold time of 20 min. Longer lag times in growth of L. monocytogenes during storage at 4°C were observed for the ON treatment at hold times of 10 and 20 min, indicating that the IN treatment may have provided a more protective environment with less injury to the cells than the ON treatment. Similarly, HPP treatment for 10 min followed by storage at 4°C was the best method for suppressing the growth of the endogenous microflora with bacterial counts remaining below the level of detection for 2 out of the 3 QF samples for up to 84 d. Lag times in growth were not observed during storage of QF at 10°C. Although HPP reduced L. monocytogenes immediately after processing, a second preservation technique is necessary to control growth of L. monocytogenes during cold storage. However, the results also showed that HPP would be effective for slowing the growth of microorganisms that can shorten the shelf life of QF.

Effect of heat and homogenization on in vitro digestion of milk

Central to commercial fluid milk processing is the use of high temperature, short time (HTST) pasteurization to ensure the safety and quality of milk, and homogenization to prevent creaming of fat-containing milk. Ultra-high-temperature sterilization is also applied to milk and is typically used to extend the shelf life of refrigerated, specialty milk products or to provide shelf-stable milk. The structures of the milk proteins and lipids are affected by processing but little information is available on the effects of the individual processes or sequences of processes on digestibility. In this study, raw whole milk was subjected to homogenization, HTST pasteurization, and homogenization followed by HTST or UHT processing. Raw skim milk was subjected to the same heating regimens. In vitro gastrointestinal digestion using a fasting model was then used to detect the processing-induced changes in the proteins and lipids. Using sodium dodecyl sulfate-PAGE, gastric pepsin digestion of the milk samples showed rapid elimination of the casein and α-lactalbumin bands, persistence of the β-lactoglobulin bands, and appearance of casein and whey peptide bands. The bands for β-lactoglobulin were eliminated within the first 15min of intestinal pancreatin digestion. The remaining proteins and peptides of raw, HTST, and UHT skim samples were digested rapidly within the first 15min of intestinal digestion, but intestinal digestion of raw and HTST pasteurized whole milk showed some persistence of the peptides throughout digestion. The availability of more lipid droplets upon homogenization, with greater surface area available for interaction with the peptides, led to persistence of the smaller peptide bands and thus slower intestinal digestion when followed by HTST pasteurization but not by UHT processing, in which the denatured proteins may be more accessible to the digestive enzymes. Homogenization and heat processing also affected the ζ-potential and free fatty acid release during intestinal digestion. Stearic and oleic acids were broken down faster than other fatty acids due to their positions on the outside of the triglyceride molecule. Five different casein phosphopeptide sequences were observed after gastric digestion, and 31 sequences were found after intestinal digestion, with activities yet to be explored. Processing affects milk structure and thus digestion and is an important factor to consider in design of foods that affect health and nutrition.

Fractionation of Whey Protein Isolate with Supercritical Carbon Dioxide—Process Modeling and Cost Estimation

An economical and environmentally friendly whey protein fractionation process was developed using supercritical carbon dioxide (sCO2) as an acid to produce enriched fractions of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) from a commercial whey protein isolate (WPI) containing 20% α-LA and 55% β-LG, through selective precipitation of α-LA. Pilot-scale experiments were performed around the optimal parameter range (T = 60 to 65 °C, P = 8 to 31 MPa, C = 5 to 15% (w/w) WPI) to quantify the recovery rates of the individual proteins and the compositions of both fractions as a function of processing conditions. Mass balances were calculated in a process flow-sheet to design a large-scale, semi-continuous process model using SuperproDesigner® software. Total startup and production costs were estimated as a function of processing parameters, product yield and purity. Temperature, T, pressure, P, and concentration, C, showed conflicting effects on equipment costs and the individual precipitation rates of the two proteins, affecting the quantity, quality, and production cost of the fractions considerably. The highest α-LA purity, 61%, with 80% α-LA recovery in the solid fraction, was obtained at T = 60 °C, C = 5% WPI, P = 8.3 MPa, with a production cost of

Preserving viability of Lactobacillus rhamnosus GG in vitro and in vivo by a new encapsulation system

8.65 per kilogram of WPI treated. The most profitable conditions resulted in 57%-pure α-LA, with 71% α-LA recovery in the solid fraction and 89% β-LG recovery in the soluble fraction, and production cost of

Enrichment and Purification of Casein Glycomacropeptide from Whey Protein Isolate Using Supercritical Carbon Dioxide Processing and Membrane Ultrafiltration

5.43 per kilogram of WPI treated at T = 62 °C, C = 10% WPI and P = 5.5 MPa. The two fractions are ready-to-use, new food ingredients with a pH of 6.7 and contain no residual acid or chemical contaminants.