Vibeke Barkholt

Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification, and meat application experiments

A new culture, Leuconostoc carnosum 4010, for biopreservation of vacuum-packed meats is described. The culture originated from bacteriocin-producing lactic acid bacteria (LAB) naturally present in vacuum-packed meat products. Approximately, 72,000 colonies were isolated from 48 different vacuum-packed meat products and examined for antibacterial activity. Bacteriocin-producing colonies were isolated from 46% of the packages examined. Leuc. carnosum was the predominant bacteriocin-producing strain and Leuc. carnosum 4010 was selected for further experiments because it showed strong antilisterial activity without producing any undesirable flavour components in meat products. For identification of the bacteriocins produced, partial purification was carried out by ammonium sulphate precipitation, dialysis, and cation exchange chromatography. SDS-PAGE analysis revealed two bands with inhibitory activity corresponding to molecular sizes of 4.6 and 5.3 kDa. N-terminal amino acid sequencing showed that Leuc. carnosum 4010 produced two bacteriocins highly similar or identical to leucocin A and leucocin C. Application experiments showed that the addition of 10(7) cfu/g Leuc. carnosum 4010 to a vacuum-packaged meat sausage immediately reduced the number of viable Listeria monocytogenes cells to a level below the detection limit and no increase of L. monocytogenes was observed during storage at 5 degrees C for 21 days. The results presented demonstrate that Leuc. carnosum 4010 is suitable as a new protective culture for cold-stored, cooked, sliced, and vacuum-packed meat products.

High intakes of milk, but not meat, increase s-insulin and insulin resistance in 8-year-old boys

Objective:Our objective was to examine if a high animal protein intake from milk or meat increased s-insulin and insulin resistance in healthy, prepubertal children. A high animal protein intake results in higher serum branched chain amino acids (BCAA; leucine, isoleucine and valine) concentrations, which are suggested to stimulate insulin secretion. Furthermore, milk possesses some postprandial insulinotrophic effect that is not related to its carbohydrate content.Design:A total of 24 8-y-old boys were asked to take 53 g protein as milk or meat daily. At baseline and after 7 days, diet was registered, and insulin, glucose, and amino acids were determined. Insulin resistance and beta cell function were calculated with the homeostasis model assessment.Results:Protein intake increased by 61 and 54% in the milk- and meat-group, respectively. In the milk-group, fasting s-insulin concentrations doubled, which caused the insulin resistance to increase similarly. In the meat-group, there was no increase in insulin and insulin resistance. As the BCAAs increased similarly in both groups, stimulation of insulin secretion through BCAAs is not supported.Conclusions:Our results indicate that a short-term high milk, but not meat, intake increased insulin secretion and resistance. The long-term consequences of this are unknown. The effect of high protein intakes from different sources on glucose–insulin metabolism needs further studying.

Digested Ara h 1 has sensitizing capacity in Brown Norway rats.

Background Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion.

Identification of IgE-binding egg white proteins: comparison of results obtained by different methods.

The binding of IgE to egg white proteins was investigated for 34 sera from adults with a positive case history and/or positive RAST towards egg, and the impact of experimental conditions on IgE binding in commonly used methods was studied. Radioimmunoblotting after SDS-PAGE of both reduced and unreduced egg white extracts showed complex reaction patterns. The results were confirmed by crossed radioimmunoelectrophoresis (CRIE). Radio dot immunobinding was used to investigate the effect of treatment of allergens for SDS-PAGE and to evaluate the other methods. As a conclusion, the use of combinations of at least two methods is recommended for the identification of IgE-binding egg white proteins. Of the 34 sera, 18 reacted with ovotransferrin, 13 with ovomucoid, 11 with ovalbumin and 5 with lysozyme. The amounts of IgE bound to ovalbumin and lysozyme were generally lower than the amounts bound to ovotransferrin and ovomucoid.

Purification of egg-white allergens.

Purification procedures for the four egg‐white proteins ovomucoid, ovotransferrin, ovalbumin, and lysozyme are presented with reference to mechanistic studies at epitope levels of allergic reactions to these proteins. The applied procedures resulted in four preparations containing less than 0.1% contaminating proteins each. The purified protein preparations were characterized by SDS—PAGE and by crossed Immunoelectrophoresis with polyclonal antibodies raised against an egg‐white extract or the purified proteins. The necessity of these well‐characterized proteins in studies on allergic reactions was shown by testing human sera in immunoblots of lysozyme, and by immunoblots of ovomucoid probing with antibodies against the proteins.

The plasma membrane proteome of germinating barley embryos

Cereal seed germination involves a complex coordination between different seed tissues. Plasma membranes must play crucial roles in coordination and execution of germination; however, very little is known about seed plasma membrane proteomes due to limited tissue amounts combined with amphiphilicity and low abundance of membrane proteins. A fraction enriched in plasma membranes was prepared from embryos dissected from 18 h germinated barley seeds using aqueous two‐phase partitioning. Reversed‐phase chromatography on C4 resin performed in micro‐spin columns with stepwise elution by 2‐propanol was used to reduce soluble protein contamination and enrich for hydrophobic proteins. Sixty‐one proteins in 14 SDS‐PAGE bands were identified by LC‐MS/MS and database searches. The identifications provide new insight into the plasma membrane functions in seed germination.

Digested Ara h 1 loses sensitizing capacity when separated into fractions.

The major peanut allergen Ara h 1 is an easily digestible protein under physiological conditions. The present study revealed that pepsin digestion products of Ara h 1 retained the sensitizing potential in a Brown Norway rat model, while this sensitizing capacity was lost by separating the digest into fractions by gel permeation chromatography. Protein chemical analysis showed that the peptide composition as well as the aggregation profiles of the fractions of Ara h 1 digest differed from that of the whole pool. These results indicate that the sensitizing capacity of digested Ara h 1 is a consequence of the peptides being in an aggregated state resembling the intact molecule or that most peptides of the digests need to be present in the same solution, having a synergistic or adjuvant effect and thereby augmenting the immune response against other peptides.

Identification of immunogenic maize proteins in a casein hydrolysate formula

Cows milk‐based formulas used for infants with cows milk allergy are based on hydrolyzed proteins. The formulas that are successful in preventing allergic responses are extensively hydrolyzed. Nevertheless, reactions to such formulas are occasionally reported, and protein material of higher molecular weight than expected has been detected by binding immunoglobulin E (IgE) from patients’ sera. This paper presents the identification of high‐molecular‐weight material in the extensively hydrolyzed casein formula, Nutramigen. The material was concentrated by simple centrifugation. The proteins in the pellet were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), and protein‐containing bands were analyzed by protein sequencing after electroblotting. The proteins were identified as maize zeins, which are water‐insoluble proteins of apparent Mr 20 000 and 23 000, presumably originating from the maize starch in Nutramigen. Rabbits immunized with this formula developed antibodies against zeins but not against milk proteins. The maize zeins are probably identical to the recently reported components in Nutramigen ( 1 ), detected by binding of IgE from milk allergic patients, but not correlated to clinical allergic reactivity. The clinical relevance of maize proteins in Nutramigen remains to be established.

The Sensitising Capacity of Intact β-Lactoglobulin Is Reduced by Co-Administration with Digested β-Lactoglobulin

Background: It is generally believed that protein hydrolysis in the gastrointestinal tract decreases the allergenicity of food allergens. However, it remains unknown if specific properties of digestion products determine whether a sensitisation or tolerogenic immune response will develop. We sought to examine the sensitising capacity of the cow’s milk allergen β-lactoglobulin (BLG) and digestion products thereof in a Brown Norway (BN) rat model. Methods: Intact BLG was digested in an in vitro model simulating the gastro-duodenal digestion process and subsequently fractionated by gel permeation chromatography. BN rats were dosed with either PBS, 200 µg of intact BLG, 30 µg of intact BLG, 200 µg of partially digested BLG, 200 µg of digested BLG, or with 200 µg of a fraction of large complexes or a fraction of small complexes. Sera from BN rats were analysed for specific antibodies and avidity was measured. Results: BLG partly resisted the digestion process. However, the BLG molecules that did not survive the digestion process were rapidly broken down to peptides of sizes less than Mr 4,500. Specific antibody responses revealed that both 200 and 30 µg of intact BLG had immunogenic as well as sensitising capacity, while digested BLG could not induce any specific antibodies. Most importantly, while intact BLG showed a significant sensitising capacity when administered alone, this sensitising capacity was significantly reduced when co-administered with digested BLG. Conclusions: Co-immunisation of intact BLG with digested BLG reduces the sensitising capacity of intact BLG, which could result from tolerogenic mechanisms induced by the digestion products.

Lipopolysaccharide Contamination of β-Lactoglobulin Affects the Immune Response against Intraperitoneally and Orally Administered Antigen

Background: Microbial components in the environment are potent activators of the immune system with capacity to shift the active immune response towards priming of Th1 and/or Th2 cells. Lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria, is extensively present in food products like cow’s milk. It is not well established, however, how this presence of LPS affects oral tolerance induction. Methods: We studied the effect of LPS contamination in a commercial preparation of the cow milk protein β-lactoglobulin (β-LG) on antigen-specific immune responses. IgG1/IgG2a production upon intraperitoneal immunization without adjuvant was measured, and oral tolerance induction against β-LG after administration of either an aqueous solution or water-in-oil (w/o) emulsion of β-LG was evaluated. Results: LPS contamination of β-LG provoked a β-LG-specific IgG2a response, as well as an enhanced β-LG-specific IgG1 response upon intraperitoneal immunization. Oral tolerance induction to β-LG was induced by aqueous solutions of β-LG with and without LPS administration. Conversely, oral administration of w/o-emulsified β-LG prevented oral tolerance to β-LG only when the β-LG was contaminated with LPS. Conclusions: LPS contamination of an aqueous protein solution does not affect oral tolerance induction, whereas LPS present in emulsion prevents oral tolerance induction towards the food protein.