Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua) protein hydrolysate and peptide fractions.

Mechanically-deboned cod muscle proteins were sequentially hydrolysed using pepsin and a trypsin+chymotrypsin combination, which was followed by passing the digest through a 1 kDa equipped tangential flow filtration system; the permeate (<1 kDa peptides) was collected as the cod protein hydrolysate (CPH). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to separate the CPH into four peptide fractions (CF1-CF4) and their in vitro antioxidant properties investigated. Results showed that most of the peptide fractions (CF2-CF4) displayed significantly higher (p<0.05) oxygen radical absorbance capacity values (698-942 µM Trolox equivalents, TE/g) and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (17-32%) than those of CPH (613 µM TE/g and 19%, respectively). However, the unfractionated CPH displayed improved capability to scavenge superoxide and hydroxyl radicals as well as significantly higher (p<0.05) ferric iron reduction and chelation of iron than the RP-HPLC peptides. The CPH and peptide fractions displayed a dose-dependent inhibition of linoleic acid oxidation.

Interaction of protamine with gram-negative bacteria membranes: possible alternative mechanisms of internalization in Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa.

This study was concerned with the interaction between the cationic antimicrobial peptide, protamine (Ptm) and the cytoplasmic membranes of the gram-negative bacteria Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa. The objective of the study was to explain the observed paradox of internalization without permanent disruption of the cell envelope. We carried out Monte Carlo computer simulation of Ptm in an aqueous environment in the presence of ~100 mM NaCl and model membranes consisting of either (65:35) or (75:25) PE:PG molar ratios. The (75:25) model, representative of the gram-negative cytoplasmic membrane, showed that the Ptm center of mass remained at least 7 nm from the membrane surface leading to the prediction that Ptm would not internalize via disruption of the inner membrane. By using immunoelectron microscopy of Ptm-treated cells, we showed that Ptm internalization to the cytoplasm took place rapidly in the presence or absence of the outer envelope. Ultrastructural examination revealed no obvious morphological changes to cells that were treated with subinhibitory or bactericidal levels of Ptm. Reconstituted phospholipid bilayers were constructed and were unperturbed by Ptm treatment over a wide range of concentrations and applied transmembrane voltages. We conclude that in the cases of the cell envelopes of E. coli, S. typhimurium and P. aeruginosa, Ptm internalized by means independent of the phospholipid bilayer, most likely mediated by one or more membrane proteins such as cation-selective barrel-like proteins. Work is currently underway to test this hypothesis.

Pseudoalteromonas bacteria are capable of degrading paralytic shellfish toxins.

Marine bacterial isolates cultured from the digestive tracts of blue mussels (Mytilus edulis) contaminated with paralytic shellfish toxins (PSTs) were screened for the ability to reduce the toxicity of a PST mixture. Seven isolates reduced the overall toxicity of the algal extract by > or = 90% within 3 days. These isolates shared at least 99% 16S rRNA gene sequence similarity with five Pseudoalteromonas spp. Phenotypic tests suggested that all are novel strains of Pseudoalteromonas haloplanktis.

Bacterial degradation of paralytic shellfish toxins.

Bacteria isolated from the digestive tracts of blue mussels (Mytilus edulis) contaminated with paralytic shellfish toxins (PSTs) were screened for the ability to reduce the toxicity of a PST mixture in vitro. Bacteria were isolated on marine agar and grown in marine broth supplemented with a mussel extract and an algal extract containing PSTs (saxitoxin, neosaxitoxin, gonyautoxins 2 and 3, decarbamoyl-gonyautoxins 2 and 3 and C1/C2 toxins). Toxin levels were measured before and after 5d of incubation, using high performance liquid chromatography (HPLC) and reduction of overall toxicity verified by mouse bioassays. Of the 73 bacterial cultures screened, seven isolates were designated "competent" PST degraders, individually reducing the overall toxicity of the PSTs by at least 90% within 3d. Most isolates degraded 100% of the saxitoxin and neosaxitoxin within 1-3d. In all cases, the overall kinetics of degradation of the toxicities was first order, as were the individual degradation kinetics of most of the individual toxins. This is the first report of nearly complete elimination of PSTs through bacterial action and may perhaps result in the development of a practical means to eliminate or reduce the risk of PSP intoxication associated with shellfish consumption.

Thermal denaturation and aggregation properties of Atlantic salmon myofibrils and myosin from white and red muscles.

Thermal denaturation and aggregation abilities of salmon myofibrils and myosin were studied measuring turbidity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonic acid binding, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking. The thermal behaviors of protein preparation from white and red muscles were compared, and the relationship with thermal gelation properties is discussed. The low gelation ability of salmon muscle proteins was related to a limited extent of protein denaturation and aggregation upon heating. These properties seemed to be carried by myosin molecules as a similar behavior was observed for both myofibrils and myosin preparations. The higher thermal stability observed for red muscle proteins with higher transition temperatures in rheological profiles was related to a shift to higher temperature in denaturation and aggregation processes. The extent of denaturation and aggregation was very similar for both muscle types as was the final rigidity of the gels formed.

Astaxanthin binding protein in Atlantic salmon.

The rubicund pigmentation in salmon and trout flesh is unique and is due to the deposition of dietary carotenoids, astaxanthin and canthaxanthin in the muscle. The present study was undertaken to determine which protein was responsible for pigment binding. Salmon muscle proteins were solubilized by sequential extractions with non-denaturing, low ionic strength aqueous solutions and segregated as such into six different fractions. Approximately 91% of the salmon myofibrillar proteins were solubilized under non-denaturing conditions using a protocol modified from a method described by Krishnamurthy et al. [Krishnamurthy, G., Chang, H.S., Hultin, H.O., Feng, Y., Srinivasan, S., Kelleher. S.D., 1996. Solubility of chicken breast muscle proteins in solutions of low ionic strength. J. Agric. Food Chem. 44: 408-415.] for the dissolution of avian muscle. To our knowledge, this is the first time this solubilization approach has been applied to the study of molecular interactions in myofibrillar proteins. Astaxanthin binding in each fraction was determined using an in vitro binding assay. In addition, SDS-PAGE and quantitative densitometry were used to separate and determine the relative amounts of each of the proteins in the six fractions. The results showed that alpha-actinin was the only myofibrillar protein correlating significantly (P<0.05) with astaxanthin binding. Alpha-actinin was positively identified using electrophoretic techniques and confirmed by tandem mass spectroscopy. Purified salmon alpha-actinin bound synthetic astaxanthin in a molar ratio of 1.11:1.00. The study was repeated using halibut alpha-actinin, which was found to have a molar binding ratio of astaxanthin to alpha-actinin of 0.893:1. These results suggest that the difference in pigmentation between white fish and Atlantic salmon is not due to binding capacity in the muscle, but rather differences in the metabolism or transport of pigment.

Inhibition of foodborne bacteria by native and modified protamine: importance of electrostatic interactions.

Protamine is a naturally occurring cationic antimicrobial peptide (CAP) that has shown some promise for control of microorganisms in food. It was hypothesized that the antibacterial effect is partially due to protamine's electrostatic affinity to the negatively charged cell envelopes of actively growing bacteria. However, nonspecific binding of the CAPs to negatively charged food particles may reduce the effect in food systems. To test the hypothesis, the antibacterial efficacies of native and reduced charge protamines (chemically modified by randomly blocking 10 to 71% of the guanido groups of the arginine residues) were compared in model and food systems. In Tryptic Soy Broth, moderate reductions of charge (<26%) resulted in either a similar or slightly improved antimicrobial efficacy, measured as the minimum inhibitory concentration (MIC) toward 21 food-related bacteria. Further reductions in positive charge led to lower antimicrobial activity. Compared to protamine, the affinity of reduced charge protamines (10 and 20%) for binding to Listeria monocytogenes cells was higher at pH 7 and 8. As perhaps would be expected, L. monocytogenes is most sensitive to modified protamines in this pH range. Protamine with reduced charge (14 and 23%) inhibited growth of L. monocytogenes in milk as well as total bacteria and coliforms in ground beef significantly (P<0.05) better than native protamine, demonstrating that the reduced charge peptides were more inhibitory in these high protein food matrices. Electrophoretic analysis of the 21 bacteria revealed a statistically significant (P<0.01) relationship with antimicrobial activity, where the most negatively charged bacteria were also the most susceptible to protamine. In conclusion, components of food matrices interfered with the antibacterial effects of the peptides, however; these undesirable interferences were reduced by altering the electrostatic properties of protamine.