Effect of media, additives, and incubation conditions on the recovery of high pressure and heat-injured Clostridium botulinum spores.

The effect of additives and post-treatment incubation conditions on the recovery of high pressure and heat-injured (i.e., processed at 620 MPa and 95 and 100 degrees C for 5 min) spores of Clostridium botulinum strains, 62-A (proteolytic type A) and 17-B (nonproteolytic type B) was studied. High pressure and heat-injured spores were inoculated into TPGY (Trypticase-Peptone-Glucose-Yeast extract) anaerobic broth media containing additives (lysozyme, L-alanine, L-aspartic acid, dipicolonic acid, sodium bicarbonate, and sodium lactate) at various concentrations (0-10 microg/ml) individually or in combination. The spore counts of high pressure and heat-injured 62-A and 17-B recovered from TPGY broth containing lysozyme (10 microg/ml) incubated for 4 months versus that recovered from peptone-yeast extract-glucose-starch (PYGS) plating agar containing lysozyme (10 microg/ml) incubated under anaerobic conditions for 5 days were also compared. None of the additives either individually or in combination in TPGY broth improved recovery of injured spore enumeration compared to processed controls without additives. Addition of lysozyme at concentrations of 5 and 10 microg/ml in TPGY broth improved initial recovery of injured spores of 17-B during the first 4 days of incubation but did not result in additional recovery at the end of the 4 month incubation compared to the processed control without lysozyme. Adding lysozyme at a concentration of 10 microg/ml to PYGS plating agar resulted in no effect on the recovery of high pressure and heat-injured 62-A and 17-B spores. The recovery counts of high pressure and heat-injured spores of 62-A and 17-B were lower (i.e., <1.0 log units) with PYGS plating agar compared to the MPN method using TPGY broth as the growth medium.

Inactivation of Clostridium botulinum type A spores by high-pressure processing at elevated temperatures.

The effects of high-pressure treatments at various temperature-time combinations on the inactivation of spores of Clostridium botulinum type A strains 62-A and BS-A in phosphate buffer (0.067 M, pH 7.0) and in a crabmeat blend were investigated. The log unit reduction of strain 62-A spores increased significantly as the processing pressure increased from 417 to 827 MPa (from 60,000 to 120,000 lb/in2) at 75 degrees C. The reduction of BS-A and 62-A spores in either medium increased as processing temperatures increased from 60 to 75 degrees C and processing times increased from 5 to 15 or 20 min at a maximum pressure of 827 MPa. Approximately 2- and 3-log reductions of BS-A and 62-A spores, respectively, in phosphate buffer were obtained at the maximum pressure-maximum temperature combination of 827 MPa and 75 degrees C for a processing time of 20 min. Processing for 15 min at the maximum pressure-maximum temperature combination resulted in maximum reductions of 3.2 and 2.7 log units for BS-A and 62-A spores, respectively, in the crabmeat blend. Results obtained in this study indicate that the crabmeat blend did not protect BS-A and 62-A spores against inactivation by high-pressure processing.

Cytokine mRNA expression in leukocytes of efferent lymph from stimulated lymph nodes in pigs.

To test the hypothesis that characteristic cytokine responses occur in stimulated porcine lymph nodes (LNs), lymph node efferent ducts were surgically cannulated. Efferent lymph (EL) leukocytes were collected before and after stimulation of LNs with mitogens [bacterial lipopolysaccharide (LPS) or phytohemagglutinin-P(PHA-P)] and antigens [hen egg white lysozyme (HEWL) or purified protein derivative of tuberculin (PPD)]. Cytokine mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). Interleukin (IL)-1alpha was predominantly produced after all stimuli except for HEWL after which tumour necrosis factor (TNF)-alpha message was dominant. None of the stimuli induced message for IL-2, IL-4 or IL-8. Other cytokine mRNAs were produced in variable amounts and percentage of overall production of each cytokine message was in the following descending rank: LPS: IL-1alpha, TNF-alpha, interferon (IFN)-gamma, IL-10, IL-12-p35, IL-6, IL-12-p40 and TNF-beta; PHA-P: IL-1alpha, TNF-alpha, IL-10, IFN-gamma, IL-12-p40 and TNF-beta; HEWL: TNF-alpha, IL-1alpha, IFN-gamma, IL-10, IL-6, IL-12-p40, TNF-beta and IL-12-p35 and PPD: IL-1alpha, IFN-gamma, TNF-alpha and IL-10. Time course response of cytokines revealed early (IL-1alpha, 10, TNF-alpha) and intermediate (IL-12-p40, TNF-beta, IFN-gamma) responses for PHA-P and early (IL-1alpha, 6, 10, IL-12-p35, IL-12-p40, TNF-alpha), intermediate (TNF-beta, IFN-gamma) and late (IL-1alpha, 6) for LPS. Cytokine mRNA response induced by HEWL was early (IL-alpha, IFN-gamma), intermediate (IL-10, IL-12-p40, TNF-beta), late (IL-1alpha, IL-12-p35) and very late (IL-1alpha, 6, 10, IL-12-p40, TNF-alpha). In Bacillus Calmette-Guérin (BCG) sensitized pigs, stimulation of LNs with PPD induced message for IL-1alpha, 10, TNF-alpha and IFN-gamma which peaked at 24h. Cytokine mRNAs varied by stimulus and differed for antibody and cell-mediated immune response.

Cytokines in Mycoplasma hyorhinis-induced arthritis in pigs bred selectively for high and low immune responses.

Yorkshire pigs were bred selectively for high and low immune responses (H and L pigs, respectively) based on multiple antibody (Ab) and cell-mediated immune response traits. In a previous experiment, generation 4 (G4) pigs of each line were infected with Mycoplasma hyorhinis. High responders had a more rapid and higher Ab response and less polyserositis, but arthritis was more severe in H pigs than in L pigs. To test the hypothesis that line differences were attributable to differential expression of cytokines, M. hyorhinis infection was induced in pigs of G8. Arthritis was more severe clinically (P,

Quantitation of porcine cytokine and beta 2-microglobulin mRNA expression by reverse transcription polymerase chain reaction.

A quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) method was developed to measure pig cytokine mRNA expression. The method utilized an internal control with primer sequences for interleukin (IL)-1alpha, 2, 4, 6, 8, 10, tumor necrosis factor (TNF)-alpha, TNF-beta, interferon (IFN)-gamma and beta-2 microglobulin (beta(2)-m). The control was modified by insertion of sequences for IL-12 (p35 and p40). Pig blood mononuclear cells (BMCs) were stimulated in vitro with phytohemagglutinin-P (PHA-P) or bacterial lipopolysaccharide and cytokine or beta(2)-m mRNA quantified. To evaluate method performance and the use of beta(2)-m as a housekeeping gene (HKG), beta(2)-m mRNA expression was examined. Quantitative analysis was achieved at up to threefold differences between control and target for beta(2)-m. Results were reproducible with coefficients of variations (CVs) ranging between 12.5% and 22.4%. There were no significant differences in beta(2)-m mRNA between treated and untreated cells or between untreated cells of three pigs (p>/=0.05) suggesting that beta(2)-m can be used as a HKG. The method allows quantitation of multiple cytokine mRNAs using a single internal control subjecting target and control to the same conditions throughout the Q-RT-PCR. The system is versatile since the control plasmid can be modified by insertion or deletion of sequences.

Shelf Life and Toxin Development by Clostridium botulinum during Storage of Modified-Atmosphere- Packaged Fresh Aquacultured Salmon Fillets.

Shelf life (onset of sensory spoilage) and the potential for toxin production by Clostridium botulinum type E in retail-type packages of fresh aquacultured salmon fillets packaged in high-barrier film bags under selected atmospheres (100% air, a modified atmosphere containing 75% CO2:25% N2, and vacuum) and stored under refrigeration (4°C) and temperature-abuse conditions (8 and 16°C) were investigated. Chemical spoilage indicators (trimethylamine and surface pH) and microbial populations were compared with sensory spoilage characteristics. Storage temperature influenced the time to onset of both sensory spoilage and toxin development in salmon fillets packaged in all atmospheres. The shelf life of fillets packaged in all atmospheres decreased with increase of storage temperature from 4 to 16°C. Trimethylamine content associated with the onset of spoilage for 100% air-packaged fillets increased as storage temperature increased. However, for modified-atmosphere-packaged fillets, the trimethylamine content associated with the onset of spoilage increased as storage temperature decreased from 8 to 4°C. Surface pH was not a good spoilage indicator for modified-atmosphere-packaged fillets. Toxin development preceded sensory spoilage at 16°C storage for fillets packaged in modified atmospheres. Toxin development coincided with sensory spoilage or was slightly delayed for the fillets packaged in all the atmospheres at 8°C storage. At 4°C none of the fillets packaged in either of the atmospheres developed toxin, even 20 days after spoilage as determined by sensory characteristics.

Construction of an internal control to quantitate multiple porcine cytokine mRNAs by RT-PCR.

A multiple internal control was constructed to be used as an exogenously added control in reverse transcription polymerase chain reaction (RT-PCR) for pig cytokines. It consists of 5' and 3' primer sequences in the order of beta 2 microglobulin (beta 2-m), IL-1, IL-4, IL-6, IL-8, IL-2, IL-10, TNF-alpha, TNF-beta and IFN-gamma. Construction was accomplished by overlapping and extension PCR (OE-PCR) utilizing short oligonucleotides. The primers were designed to give two products of different sizes on co-amplification of control and target RNAs by RT-PCR in a single tube. This permits analysis of message for several cytokines using a single exogenously added competitive template. Incorporated endonuclease sites allow construct modification by oligonucleotide addition.

High pressure studies of energy transfer and strongly coupled bacteriochlorophyll dimers in photosynthetic protein complexes.

High pressure is used with hole burning and absorption spectroscopies at low temperatures to study the pressure dependence of the B800→B850 energy transfer rate in the LH2 complex of Rhodobacter sphaeroides and to assess the extent to which pressure can be used to identify and characterize states associated with strongly coupled chlorophyll molecules. Pressure tuning of the B800-B850 gap from ∼750 cm(\s-1) at 0.1 MPa to ∼900 cm(-1) at 680 MPa has no measurable effect on the 2 ps energy transfer rate of the B800-850 complex at 4.2 K. An explanation for this resilience against pressure, which is supported by earlier hole burning studies, is provided. It is based on weak coupling nonadiabatic transfer theory and takes into account the inhomogeneous width of the B800-B850 energy gap, the large homogeneous width of the B850 band from exciton level structure and the Franck-Condon factors of acceptor protein phonons and intramolecular BChl a modes. The model yields reasonable agreement with the 4.2 K energy transfer rate and is consistent with its weak temperature dependence. It is assumed that it is the C9-ring exciton levels which lie within the B850 band that are the key acceptor levels, meaning that BChl a modes are essential to the energy transfer process. These ring exciton levels derive from the strongly allowed lowest energy component of the basic B850 dimer. However, the analysis of B850s linear pressure shift suggests that another Förster pathway may also be important. It is one that involves the ring exciton levels derived from the weakly allowed upper component of the B850 dimer which we estimate to be quasi-degenerate with B800. In the second part of the paper, which is concerned with strong BChl monomer-monomer interactions of dimers, we report that the pressure shifts of B875 (LH2), the primary donor absorption bands of bacterial RC (P870 of Rb. sphaeroides and P960 of Rhodopseudomonas viridis) and B1015 (LH complex of Rps. viridis) are equal and large in value (∼-0.4 cm(01)/MPa at 4.2 K) relative to those of isolated monomers in polymers and proteins (< -0.1 cm(01)/MPa). The shift rate for B850 at 4.2 K is-0.28 cm(-1)/MPa. A model is presented which appears to be capable of providing a unified explanation for the pressure shifts.

Shelf Life of Modified-Atmosphere-Packaged Fresh Tilapia Fillets Stored under Refrigeration and Temperature-Abuse Conditions.

We investigated the shelf life of fresh Tilapia spp. fillets packaged in high-barrier film under both 100% air and a modified atmosphere (MA) of 75% CO2:25% N2, and stored under refrigeration (4°C) and abuse temperatures (8 and 16°C). The chemical spoilage indicators trimethylamine, K-value, and surface pH, as well as microbial counts, were compared with the sensory characteristics of spoilage. For fillets packaged under 100% air, the shelf life was 9 to 13 days at a storage temperature of 4°C, but decreased to 3 to 6 days at 16°C. However, the shelf life of MA-packaged fillets stored at 4°C increased to >25 days when the lag phase and generation time of the bacteria were extended. MA packaged fillets stored under temperature-abuse conditions (8 and 16°C) had a shorter shelf life. The trimethylamine content associated with onset of sensory spoilage for MA packaged fillets increased as storage temperature increased and differed for each temperature. The surface pH and K-values of MA-packaged fillets were not good indicators of spoilage onset.

A photoinduced persistent structural transformation of the special pair of a bacterial reaction center.

Structural modification of photosynthetic reaction centers is an important approach for understanding their charge-separation processes. An unprecedented persistent structural transformation of the special pair (dimer) of bacteriochlorophyll molecules can be produced by light absorption alone. The nonphotochemical hole-burned spectra for the reaction center of Rhodopseudomonas viridis show that the phototransformation leads to a red shift of 150 wave numbers for the special pair's lowest energy absorption band, P960, and a comparable blue shift for a state at 850 nanometers, which can now be definitively assigned as being most closely associated with the upper dimer component. Additional insights on excited-state electronic structure include the identification of a new state.

Applications of spectral hole burning spectroscopies to antenna and reaction center complexes.

The underlying principles of spectral hole burning spectroscopies and the theory for hole profiles are reviewed and illustrated with calculated spectra. The methodology by which the dependence of the overall hole profile on burn wavelength can be used to reveal the contributions from site inhomogeneous broadening and various homogeneous broadening contributions to the broad Qy-absorption bands of cofactors is emphasized. Applications to the primary electron donor states of the reaction centers of purple bacteria and Photosystems I and II of green plants are discussed. The antenna (light harvesting) complexes considered include B800-B850 and B875 of Rhodobacter sphaeroides and the base-plate complex of Prosthecochloris aestuarii with particular attention being given to excitonic interactions and level structure. The data presented show that spectral hole burning is a generally applicable low temperature approach for the study of excited state electronic and vibrational (intramolecular, phonon) structures, structural heterogeneity and excited state lifetimes.

Composition and Pesticide and Herbicide Residue Analysis of Fresh and 40-Year-Old Pasteurized Blue Crab ( Callinectes sapidus ) Meat.

Fresh and 40-year-old pasteurized blue crab ( Callinectes sapidus ) meat was analyzed for proximate composition, mineral, heavy metal, and amino acid content and volatiles concentration and possible pesticide and herbicide residue levels. Both fresh and 40-year-old meat had similar proximate compositions. The 40-year-old crab meat contained high levels of iron, manganese, copper, and heavy metals compared to the fresh. Recovery of total amino acids was lower from the 40-year-old meat. Aspartic acid, glutamic acid, leucine, lysine, and arginine were the major protein amino acids of fresh and 40-year-old crab meat. The 40-year-old meat contained high concentrations of ethanol and trimethylamine compared to the fresh. No herbicide residues were detected in either of the products. Decomposition products of pesticide DDT were detected at very low levels only in the 40-year-old crab meat.

Foods and Food Ingredients for Prevention of Diarrheal Disease in Children in Developing Countries.

Increased awareness of the morbidity and mortality attributed to diarrheal disease among children under 5 years of age in developing countries has led to a variety of approaches aimed toward prevention. In this review, foods and food ingredients which appear to be most effective in prevention of diarrheal disease in infants are considered. The effect of each of the following potential food or food ingredient categories on the control or prevention of diarrheal disease is discussed: human milk components, antibodies, probiotics, and fermented foods.

Effect of epicondylectomy in early ulnar neuritis treated with steroids.

Sixty-two ulnar nerves belonging to 44 patients with early neuritis were studied to assess the benefits offered by medial epicondylectomy and external decompression in addition to steroid therapy. The patients were randomly allocated to the surgical or the medical group. In those cases where there was bilateral involvement, surgery was carried out only on one side. All cases were assessed prior to treatment, and at predetermined intervals following treatment. This study presents the results after a 12-month follow-up. There was statistically significant improvement in both groups following treatment as assessed by improvement in motor and sensory functions and in the reduction of pain and tenderness. The study, however, failed to demonstrate any added benefit with surgical intervention as compared to steroid therapy alone in the treatment of early ulnar neuritis.

Intracellular glycosidases of human colon Bacteroides ovatus B4-11.

Activity of various glycosidases in the intracellular enzyme fraction of Bacteroides ovatus B4-11 was investigated. During 120 h of incubation at 37 degrees C, ca. 30% of the crude hemicellulose was hydrolyzed by an intracellular enzyme fraction of strain B4-11. Xylose was the major sugar released from crude hemicellulose. Glycosidases (alpha-1,6-glucosidase, alpha-1,4-glucosidase, beta-1,4-glucosidase, and beta-1,4-xylosidase) were induced in B. ovatus B4-11 by crude hemicellulose and heteroxylan. When B. ovatus B4-11 was grown on either crude hemicellulose or heteroxylan, the predominant enzyme in the intracellular enzyme fraction was beta-1,4-xylosidase.

Inhibition of Clostridium botulinum by antioxidants, phenols, and related compounds.

A total of 75 compounds, including antioxidants, preservatives, gallic acid and p-hydroxybenzoic acid esters, hydroquinones, hydroxyquinolines, phenol derivatives, and related compounds, were screened for their antibotulinal activity in prereduced Thiotone-yeast extract-glucose broth. The most effective inhibitors of Clostridium botulinum growth and toxin production were long-chain esters of p-hydroxybenzoic acid and gallic acid, antioxidants, and butylphenol derivatives. The antioxidant nordihydroguaiaretic acid at 100 microgram/ml delayed the growth and toxin production for the entire incubation period (7 days). Other antioxidants, such as butylated hydroxytoluene, butylated hydroxyanisole, and tert-butylhydroquinone were also very effective (at 200 to 400 microgram/ml) for the inhibition of C. botulinum growth and toxin production. Toxin was detected, although no detectable growth was found by daily absorbance measurements, in the prereduced medium containing 50 to 400 microgram of 8-hydroxyquinoline, pentylphenol, tert-pentylphenol, 3,5-ditert-butylphenol, 3,5-ditert-butylcatechol, (2-hydroxydiphenyl)methane, or (4-hydroxydiphenyl)methane per ml.

Legume lipids.

The storage lipids of legume seeds are a major source of dietary fat. As a result of their importance in the food industry, much is known about lipid composition, chemistry, flavor, off-flavor development, and their technological implications in foods of dry, oil-rich seeds such as soybeans and peanuts. Lipids from green pea have also been investigated to some extent. Other food legume lipids have not been studied in any great detail because of their low lipid content and limited or negligible use for oil purposes. Literature on the biochemical, nutritional, and toxicological aspects of lipids from these other legumes is scanty, compared to published reports of seed lipids from soybean and peanuts. Lipids of soybean, peanut, and green pea are reported in this article. Their chemistry, interactions with other constituents, role in flavor development, as well as alterations due to processing and removal of off-flavors are reviewed. The nutritional and toxicological implications of legume lipids from soybean, peanuts, and other food legumes are also discussed.