Escherichia coli O157:H7 is an important foodborne pathogen. The majority of outbreaks of this organism have been attributed to ground beef. In order to improve the sensitivity for detection of E. coli O157:H7 in ground beef by the polymerase chain reaction (PCR), sample preparation methods including differential centrifugation, enrichment, immunomagnetic separation (IMS), and DNA extraction were studied. Quantitative detection protocols based on standard PCR, nested PCR and competitive PCR were developed. Using standard PCR enabled detection of 150 colony forming units (CFU) per gram of ground beef without enrichment and 1.2 CFU/g after 4.5 h of enrichment in modified EC broth plus novobiocin (mEC+n) with SLT I and SLT II primers respectively. The quantitative detection range was 150 to 15,000 CFU/g for protocol without enrichment and 1.2 to 19.2 CFU/g for protocol with enrichment. Standard PCR incorporating IMS detected 70 CFU/g with SLT I and SLT II primers without enrichment and 0.7 CFU/g after 6 h of enrichment in Tryptic Soy Broth (TSB) and IMS. The quantitative detection range was 70 to 24,000 CFU/g for protocol without enrichment and 0.7 to 70 CFU/g for protocol with enrichment. A partial digestion of the cell pellets derived from differential centrifugation was required for IMS. DNA extracted from seeded target cells captured by IMB could be used for PCR amplification without further purification. Nested PCR incorporating differential centrifugation and IMS was capable of detecting 2.4 CFU/g with SLT 1 and SLT 2 primers without enrichment. Within the range of 2.4 to 240 CFU/g, a linear relationship between the log CFU of the inocula and the fluorescent intensity of secondary PCR products was achieved by using 25 cycles for both primary and secondary PCR run. Competitive PCR incorporating IMS enabled quantitative detection of 0.5 to 5.5 CFU per gram of ground beef with SLT I and SLT II primers after 6 hours of enrichment using TSB. The numbers of target organisms after enrichment and IMS determined by competitive PCR were highly correlated to those derived from viable plate counts on violet red bile agar.

Chelators are valuable ingredients used to improve the oxidative stability of many processed foods. Caseins and casein peptides have phosphoseryl residues capable of binding prooxidant metals such as iron and may have antioxidant activity. In 5% corn oil-in-water emulsions CPP (25 μM) inhibited the formation of lipid oxidation at both pH 3.0 and 7.0 as determined by lipid hydroperoxides and hexanal. Calcium (0–100 mM) had no influence on the antioxidant activity of CPP. Casein hydrolysates were tested for their antioxidant properties as hydrolysates represent a more economical source of phosphopeptides. Casein hydrolysates were more effective inhibitors of lipid oxidation than CPP at equal phosphorous content (0–310 μM) suggesting that antioxidant properties were not uniquely related to chelating prooxidant metals but also to free radicals scavenging activity. The antioxidant activity of CPP and casein hydrolysates was studied in phosphatidylcholine liposomes. CPP (<1.0 mg/ml) and casein hydrolysates (0.3–1.7 mg/ml) were effective inhibitors of TBARS development when oxidation was promoted by iron. High amounts of CPP (>1.0 mg/ml) were prooxidant while casein hydrolysates were only observed to be antioxidative. In the presence of peroxyl radicals, casein hydrolysates were more effective scavengers than enriched CPP (3–15 mM). When antioxidant activity was evaluated in cooked ground beef, TBARS formation was inhibited 75 and 39% by 0.5% CPP and casein hydrolysates, respectively, after 4 days of storage. The binding interactions of CPP and casein hydrolysates to iron were studied using isothermal titration calorimetry (ITC) with Fe-nitrilotriacetic acid (NTA) as the titrant. The observed binding constants were in the range of 5.8–1.7 × 103 M−1 for CPP and casein hydrolysates. The number of binding sites was below 1.0 suggesting that more than one phosphoseryl group is needed to bind iron or that other amino acids such as glutamic acid might participate in the binding. The interactions seem to be independent of pH since the apparent binding constants were similar between pH 3.0–7.0. These results show that CPP and casein hydrolysates are promising sources of natural antioxidants in foods since they have the potential ability to act as chelators of prooxidant metals and as free radical scavengers.

The pH of beef muscle affected water-holding capacity (WHC) and tenderness. When the pH was brought to 4.0 and below by immersion in citric acid or lactic acid or brought to ∼7.0 after base immersion, WHC and tenderness of beef muscle increased. When the pH was brought back to the isoelectric point of the major myofibrillar proteins after either acid or alkaline treatment (e.g. from ∼3.7 or 7.0 to ∼5.5), most of the weight gained from prior acidic or basic treatment was lost. The Warner-Bratzler shear force values (WBSF) returned at least to the level of the WBSF of the controls after the readjustment of pH to 5.5. This reversibility strongly suggests that tenderization is not caused primarily by proteolysis. It is proposed that myofibrillar structures could swell and disrupt at acid/neutral pH due to electrostatic repulsion. When the pH was brought back near the isoelectric point, the myofibrillar structure might collapse and/or aggregate. Less water could be held with the aggregated proteins than in the initial myofibrillar structure of raw beef. Lactic acid and citric acid produced essentially the same shear force if WBSF values after marination were compared at a similar pH value. CaCl 2 showed a synergistic effect and negative effect, respectively, on the tenderization induced by citric acid and lactic acid. The solubility of beef myofibrillar proteins increased steeply from pH 4.27 to 4.03, where a significant decrease in WBSF was often observed. The solubility of beef muscle protein was below 20% between pH 6.9 and 7.6. The relation of protein solubilization to tenderization observed at acid pH was not seen in the tenderization induced by base treatment. Elastic gels from washed chicken breast muscle with a moisture content of 88% were formed at an acid pH (∼3.7 and lower) and low ionic strength. A decrease of pH from 4.1 to 3.7 or below increased gel elasticity and decreased water loss under pressure. In the presence of NaCl, gels made at pH ∼3.7 had decreased elasticity and increased water loss under pressure compared to those prepared without salt.

Proteins and protein-derived materials, such as enzymatic hydrolysates, have been shown to improve the oxidative stability of food lipids. However, the mechanisms of protein antioxidant action are complex, particularly in heterogeneous, dynamic environments such as food systems. The goal of this research is to provide a fundamental understanding of the various mechanisms by which proteins inhibit oxidative reactions in food lipid dispersions. The presence of low concentrations of the whey protein β-lactoglobulin (β-Lg) present in the continuous phase of oil-in-water emulsions was observed to inhibit lipid oxidation. Furthermore, the antioxidant activity of continuous phase β-Lg was significantly improved by thermal denaturation (95°C; 15 min) at pH 7.0, despite having lower iron binding values and concentration of reactive sulfhydryls than untreated, native β-Lg. The observed enhancement in antioxidant activity of heated β-Lg seems to result from improved solvent accessibility of radical scavenging amino acids, and/or changes in β-Lg's surface activity, leading to increase interactions with lipophilic oxidants. Cysteine and tryptophan residues in native β-Lg dispersed in the continuous phase of emulsions were observed to oxidize prior to emulsified lipid, suggesting that these amino acids possess antioxidant activity. However, methionine residues in β-Lg did not appear to participate in free radical scavenging in this system, despite its reported oxidative lability. This may be due to the nature of the endogenous oxidant (i.e. lipid-derived radical species) or to the fact that β-Lg's methionine residues have low solvent accessibility when the protein is in its native conformation. Finally, it was observed that β-Lg's antioxidant activity is enhanced with enzymatic (e.g. chymotrypsin) hydrolysis. Increased peroxyl radical scavenging activity and iron chelation seem to be responsible for the increased antioxidant activity of hydrolyzed β-Lg. Liquid chromatography - mass spectrometry (LC-MS) was used to assess the oxidative modification of three specific amino acid residues in the selected chymotrypic hydrolysates of β-Lg during the period preceding the formation of lipid oxidation products. Significant oxidation of tyrosine and methionine were observed during this period, suggesting that these residues are capable of free radical scavenging, thereby conferring improved oxidative stability to oil-in-water food emulsions.

Plesiomonas shigelloides has been recognized as a potential human and animal pathogen in the past six decades. In this study, RAPD typing was performed to determine the genetic variability among the isolates of P. shigelloides from different sources. In addition, quantitative methods to detect P. shigelloides based on PCR were developed. Also, factors affecting PCR detection were examined, methods to reduce the inhibition of PCR by PCR inhibitors were tested, and a method for quantitative detection of viable P. shigelloides in a mixture of viable and dead cells was developed. RAPD analysis showed that there was notable genetic variability among most of the strains of P. shigelloides isolates from fresh water, fish and human clinical sources. The isolates from fish had a closer linkage to the human clinical isolates than did the freshwater isolates, suggesting that fish may be the more serious source for potential risk of infection. Using standard quantitative PCR, the lowest level of detection for P. shigelloides, without enrichment, was 60 CFU/g in clams with the treatment of BSA and 200 CFU/g in oysters with the treatment of formaldehyde and BSA respectively. Using conventional quantitative PCR in combination with ethidium bromide monoazide (EMA) enabled quantitative detection of viable P. shigelloides cells in varying ratio's of viable to dead cells at a minimum of 24 CFU per PCR reaction. Competitive PCR was capable of detecting DNA derived from 2.4 × 102 to 2.4 × 105 CFU/g of clam tissue without enrichment, and 4.0 × 101 to 1.2 × 104 CFU/g of clam tissue with 7 hrs. of nonselective enrichment at 37°C respectively. Real-time PCR quantitatively detected 3 to 1.0 × 104 CFU per gram of oyster after 7 hrs. nonselective enrichment. Without enrichment, the minimum detection level was 1.0 × 103 CFU per gram of oyster tissue with the addition of formaldehyde following by the treatment with coated charcoal.

This work focused on the factors that affect the cook yield and moisture retention of different muscle foods at reduced salt concentrations (≤ 150 mM). Before studying the water-holding capacity (WHC) of different muscles, which was evaluated by cook yield and moisture retention upon heating and pressing, solubilization of myofibrillar proteins of chicken and cod muscles in water were first investigated. Certain possible solubility inhibiting (PSI) polypeptides and exposure to low pH affected the water solubility of myofibrillar proteins. The PSI proteins acted like a barrier that prevented the rest of the myofibrillar proteins from disorganization, swelling and final solubilization in water. M-protein (160 kDa), α-actinin (95 kDa), amorphin (87 kDa) and desmin (56 kDa) were tentatively identified as the PSI polypeptides in the chicken breast muscle. Re-addition of these PSI polypeptides to the PSI-extracted chicken mince inhibited the full solubilization of the chicken mince. Exposure of the cod myofibrillar proteins to a low pH could cause protein denaturation and subsequently the loss of extractability in water. M-protein (160 kDa), a 103 kDa polypeptide and α-actinin (95 kDa) were tentatively identified as the PSI polypeptides in cod muscle. By extracting the acidified cod mince with a solution of physiological ionic strength at neutral pH promoted the process to reverse the low pH effect on these three PSI polypeptides, thus the muscle proteins solubility was re-established. The cook yield and moisture retention of the washed chicken mince at low ionic strength (25-150 mM sodium chloride) was governed by the balance between the driving force and the constraints against swelling of the myofibrils. Washing the chicken mince with moderate salt at neutral pH increased electrostatic repulsive force and the osmotic potential of myofibrillar proteins, and also solubilized the constraint components associated with myofibrillar structure. Therefore, improved water-holding capacity was observed. By lowering the pH of chicken mince previously extracted with moderate salt at neutral pH to 6.3, the improved WHC was lost, suggesting that pH might be a dominant factor in controlling WHC. Examining the aqueous phase (press juice) after centrifugation showed that pH adjustment from 5.8 to 7.0 can selectively solubilize some proteins in chicken muscle. The adjustment of pH to neutrality also improved the cook yield and moisture retention of unwashed chicken muscles. It is hypothesized that these polypeptides represent proteins which are soluble in the aqueous phase of the muscle at neutral pH but much less so at pH 6 and their solubilization in situ may be involved in the water-holding properties of the muscle. After pH adjustment, the net negative charges of muscle proteins increased; and also removed some of the polypeptides that hold structural elements like thick filaments and Z-disks together. Removal of these polypeptides from where they are normally located in the muscle cell structure at neutral pH allows the components of the structural elements, for example, myosin of the thick filaments, to be released or to expand and to pull in water by the increase of electrostatic repulsive forces and the entropy of the system. The proteins thus unfold more extensively during heating resulting in a better cook yield and moisture retention upon heating and pressing. Mechanical force such as mincing and homogenizing, can damage the muscle cell structure, thus removing some of the constraints associated with the cell membrane or the linkage between the myofibrils. Therefore, cook yield and moisture retention after heating and pressing were improved in the ground and homogenized muscle.

Abstract not available

Lipid oxidation leads to quality deterioration of foods high in unsaturated fatty acids. Edible oils contain surface active compounds and water that can form physical structures known as association colloids. To better understand the influence of physical structures on the oxidative stability of bulk oils the role of association colloids on lipid oxidation was investigated. The effectiveness of chain-breaking antioxidants at retarding lipid oxidation depends on their chemical properties and physical location within a food. The first study showed that the surface activity and/or polarity of lipid-soluble antioxidant were not the only determinant of their effectiveness in food lipids. In the second study of model association colloids in oils, we found that the size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Phosphatidylcholine decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. The influence of these compounds on physical structures of bulk oil was also confirmed in a study using surface active fluorescence probe. The fluorescence intensity of 5-dodecanoylaminofluorescein (DAF) increased with increasing water concentration in the edible oil. Addition of oleic acid decreased DAF fluorescence due to the ability of the free fatty acid to decrease the pH of the aqueous phase of the bulk oil. Phosphatidylcholine increased DAF fluorescence due to its ability to increase DAF exposure to the aqueous phase. Oleic acid had no impact in the interactions between DAF and water soluble peroxyl radicals while phosphatidylcholine decreased peroxyl radical degradation of DAF. This research established the significance of physical structures of bulk oils on lipid oxidation. Understanding how the physical properties of bulk oils impact lipid oxidation could lead to development of novel antioxidant technologies that help improve the oxidative stability of oils containing increased concentrations of polyunsaturated fatty acids.

Phytophenols are antimicrobials that may inhibit the growth of foodborne pathogens. However, their antimicrobial activity is low because of low water solubility. The objective of this study was to formulate emulsion and microemulsion and test their efficacy as antimicrobial delivery systems. Eugenol was solubilized into cationic-nonionic (Mirenat-N® -T-Maz®80K or LAE-TM) and nonionic surfactant mixtures (T-Maz®80K-Surfynol®485W or TM-S485). Formulation of emulsions using eugenol is challenging due to Ostwald ripening. Eugenol was mixed with hydrocarbons to decrease Ostwald ripening rate using compositional ripening. Physicochemical characterization included surface tension, particle size, charge and solubilization capacity. Antimicrobial efficiency was tested using spot inoculation against four strains of Listeria monocytogenes and Escherichia coli O157:H7. The antimicrobial activity of eugenol micelles in ultra-high temperature pasteurized milk containing different percentages of milk fat was investigated. Nonionic mixed surfactant micelles showed no inhibition against both pathogens but the individual surfactants showed inhibition with 8mM of eugenol. The antimicrobial efficiency of cationic-non-ionic micelles was high since LAE alone inhibited the growth of E. coli O157:H7 and Listeria. Micelles inhibited all microbial the growth with exception of the TM:LAE (5:1) ratio. Addition of eugenol at 3mM inhibited the growth of Listeria and 7 mM inhibited the growth of E. coli O157:H7. When microemulsions were tested in a food system (milk), the antimicrobial efficiency varied depending on the fat level. Microemulsions completely prevented growth of Listeria and E. coli O157:H7 in skim and 2% milk but not in 4% fat in milk. Therefore, food composition, especially fat level, may affect the efficiency of targeting of foodborne pathogens by surfactant-encapsulated antimicrobials. The stability and antimicrobial efficacy of 5% oil-in-water emulsions formulated with eugenol and hydrocarbons was evaluated. Eugenol and lipids were mixed at different eugenol:lipid ratios. Corn-oil emulsions loaded with eugenol were the most stable and inhibited the growth against E. coli O157:H7 strains depending on loading ratio but failed to inhibit growth of Listeria strains. When surfactants are used as transport vehicles, selection of surfactant and mixing ratios is of crucial importance. Microemulsion and emulsions have substantial potential as new preservation systems for foods since they were completely composed of GRAS compounds.

Abstract not available