Marie K. Walsh

Solid-Phase Capture of Proteins, Spores, and Bacteria

ABSTRACT Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 μg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 andB. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25°C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.

Influence of a poly-ethylene glycol spacer on antigen capture by immobilized antibodies

The use of spacers to distance an immobilized antibody from the surface of a support matrix introduces flexibility, which can reduce steric interferences between antibodies leading to a higher antigen capture efficiency. In this paper we investigated the use of a spacer molecule, poly-ethylene glycol (PEG), between the matrix surface and antibodies for the capture of Bacillus globigii, E. coli O157:H7, and ovalbumin. The antigen capture efficiency was determined using a surface ELISA method. Antibodies against the antigens were covalently immobilized either directly or via PEG to glass surfaces using a one-step EDC reaction. The amount of antibody immobilized was determined before blocking the nonspecific binding sites with bovine serum albumin. Antibodies immobilized via a PEG spacer showed a higher capture efficiency compared to direct immobilization, which was more pronounced with large antigens. Antibodies immobilized on glass supports were stable at 65 degrees C for at least 80 min, and the capture efficiency increased with heating at 65 degrees C for 20 min.

Performance of dairy cattle clones and evaluation of their milk composition.

Genetic and phenotypic performance of U.S. Holstein embryo-split and nuclear-transfer clones was documented for yield and fitness traits. For cows, mean genetic superiority based on pedigree was 186 kg of milk, 9 kg of fat, and 7 kg of protein for embryo-split clones and 165, 10, and 8 kg, respectively, for nuclear-transfer clones compared with the population for the same birth year; pedigree advantage for male clones generally was slightly greater. Estimates of genetic merit that considered a clones own performance as well as pedigree merit were slightly lower for embryo-split cows than for their full siblings for yield but not for milk composition (fat and protein percentages), mastitis resistance (somatic cell score), longevity (productive life), or cow fertility (daughter pregnancy rate); no corresponding genetic differences were found for nuclear-transfer cows or for cloned bulls regardless of clone type. For bulls, estimated genetic merit based on daughter yield was more similar for clone pairs with apparent identical genotype than for pairs from the same biotechnology but nonidentical as confirmed by blood typing. Yield deviations were lower for clones than for their full siblings. Milk composition (total solids, fat, fatty acid profile, lactose, and protein) also was compared for nuclear-transfer clones (Brown Swiss, Holstein, and Holstein-Jersey cross) with non-cloned cows and literature values; no differences were found for gross chemical composition of milk. No obvious differences were evident between cloned and non-cloned animals or for the milk that they produced.

Solid-phase capture of pathogenic bacteria by using gangliosides and detection with real-time PCR.

ABSTRACT We developed a method for concentrating pathogens from samples without enrichment. Immobilized gangliosides concentrated bacteria for detection with real-time PCR. A sensitivity of ∼4 CFU/ml (3 h) in samples without competing microflora was achieved. Samples with competing microflora had a sensitivity of 40,000 CFU/ml. The variance was less than one cycle.

Effect of Lactose Monolaurate on Pathogenic and Nonpathogenic Bacteria

ABSTRACT The antimicrobial activities of sucrose monolaurate and a novel ester, lactose monolaurate (LML), were tested. Gram-positive bacteria were more susceptible than Gram-negative bacteria to both esters. The minimal bactericidal concentrations of LML were 5 to 9.5 mM for Listeria monocytogenes isolates and 0.2 to 2 mM for Mycobacterium isolates.

Characterization and expression of the Pseudomonas putida bacterioferritin alpha subunit gene.

The root-colonizing pseudomonad Pseudomonas putida (Pp) appears to produce two subunits, alpha and beta, of the iron-binding protein, bacterioferritin. A gene encoding the alpha-bacterioferritin subunit was located adjacent to the major catalase in Pp. The deduced protein sequence of the Pp bfralpha gene had a very high identity with other alpha-subunits, possessing conserved amino acids responsible for ferroxidase activity. The gene also lacked a deduced methionine at residue 52, associated with heme binding in beta-subunits. An antibody generated toward the Escherichia coli (E. coli) multifunctional single subunit bacterioferritin recognized two proteins in the Pp extract, a 22 kDa protein likely to be a beta-subunit and, to a lesser extent, a 23 kDa band. The 23 kDa band was absent in a Pp mutant in which the bfralpha gene was disrupted. Loss of alpha-bacterioferritin stimulated production of fluorescent siderophore. Growth on media and on root surfaces was not impaired by deletion of the alpha-bacterioferritin. Transcription of bfralpha was independent of the catalase gene and was dependent on iron. The transcript levels from bfralpha decreased in iron deficiency experienced during stationary-phase or upon treatment during growth with an iron chelator.

Use of amino acid analysis for estimating the individual concentrations of proteins in mixtures.

The concentrations of five individual proteins in a mixture were determined from one amino acid analysis of the mixture by solving for each protein using simultaneous equations. Dried casein and whey were separated into five individual protein components using reversed-phase HPLC. Individual proteins were collected and analyzed for amino acid composition. These data were used as standards. Mixtures of purified proteins were analyzed for total amino acid composition and the concentrations of individual proteins in the mixtures were determined by solving simultaneous equations based on the amino acid analysis composition of the standards.

Antilisterial activity of lactose monolaurate in milk, drinkable yogurt and cottage cheese

Lactose monolaurate (LML) was previously found to be an antimicrobial against Listeria monocytogenes in culture medium at concentrations between 3 and 5 mg ml−1. In this study, the microbial inhibitory activity of LML in dairy products inoculated with a 5‐strain cocktail of clinical isolates of L. monocytogenes was investigated. Addition of LML at a concentration of 5 mg ml−1 resulted in 4·4, 4·0 and 4·2 log reductions in 0·5% fat, 1% fat and 3·25% fat milks, respectively; 4·1, 4·4, and 3·5 log reductions in nonfat, 1% fat, and 1·5% fat yogurts, respectively; and 4·0 log reductions in both nonfat and 2% fat cottage cheese. The inhibitory effect of LML was only observed at 37°C and not 5°C. Experiments suggest that both the lauric acid and the esterified lactose moiety of LML play roles in the growth inhibition.

Immobilized enzyme technology for food applications.

Publisher Summary This chapter describes that there are very few examples of commercial processes that utilize immobilized enzymes for food constituent modifications. In order for the immobilized process to be more economical or more useful than the soluble enzyme, either the cost per unit of product must be less or the product formed can only be produced with an immobilized enzyme. The two most successful examples of the use of immobilized enzymes are the production of high-fructose corn syrup and trans-free oils. The use of enzymes for food constituent modification has several advantages over the use of chemicals. The reactions are specific with generally fewer side reactions. Numerous publications over the past few years have focused on the development of immobilized enzymes for future commercial use, yet currently there are few successful examples of immobilized enzymes for food processing despite the advantages associated with immobilized enzyme processing. The immobilized form of glucose isomerase is used for the production of high-fructose corn syrup and immobilized lipases are used for production of diacylglycerols and trans-free fats, and oils. Despite the numerous benefits associated with immobilized enzymes, the economics of the system outweighs most other benefits.

Properties of Extrusion-Expanded Whey Protein Products Containing Fiber

Three insoluble dietary fibers (cellulose, wheat, and oat) were incorporated into an extruded-expanded product containing 32% whey protein at three concentrations (18, 36, and 48%) to determine the influence of fiber on extrudate properties. As the concentration of fiber in the extrudates increased, there was a significant decrease in expansion ratio, air cell size, water soluble carbohydrate, and water solubility index, and a significant increase in extrudate density, breaking force, moisture content, and water absorption index. The main effects observed were attributed primarily to a decrease in the amount of normal cornstarch.