Tomas Bolumar

Quality considerations with high pressure processing of fresh and value added meat products

Pressure can be applied by high hydrostatic pressure, better known as high pressure processing (HPP), or by hydrodynamic pressure (HDP) in the form of shockwaves to alter quality parameters, such as shelf-life and texture of meat and meat products. The aim of this review is to give an overview of the use of pressure in the meat industry and to highlight its usage as a method to inactivate microorganisms but also a novel strategy to alter the structure and the quality parameters of meat and meat products. Benefits and possibilities of the technologies are presented, as well as how to overcome undesired product changes caused by HPP. The use of hydrodynamic shockwaves is briefly described and a promising newly developed industrial prototype for the generation of shockwaves by underwater explosion is presented.

Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

ABSTRACT A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg2+, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The Km for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

Hydrolysis of pork muscle sarcoplasmic proteins by Debaryomyces hansenii.

Strains of Debaryomyces hansenii originally isolated from sausages were screened for proteinase and aminopeptidase activity towards synthetic substrates. On the basis of these results, D. hansenii CT12487 was selected for further assays. The activities of the whole cells (WC), cell-free extracts (CFE) and a combination of both from the selected strain on pork muscle sarcoplasmic protein extracts were determined by protein, peptide and free amino acid analyses. There was a pronounced hydrolysis of protein bands of 110 kDa and 27-64 kDa regardless the incorporation of WC, CFE or a combination of both. The proteolytic activity also resulted in the generation of polar and non-polar peptides showing noticeable differences depending on the addition of WC or CFE. Whole cells generated greater amounts of free amino acids than the cell-free extracts.

Purification and properties of an arginyl aminopeptidase from Debaryomyces hansenii

A metallo arginyl aminopeptidase (EC 3.4.11.6) activated by Co(2+) was isolated from Debaryomyces hansenii CECT 12487. The enzyme was purified after precipitation with protamine sulphate, followed by a weak anion exchange chromatography, gel filtration chromatography and a strong anion exchange chromatography. The arginyl aminopeptidase (AAP) was purified 337 folds, with a 18% recovery. The AAP appeared to be a dimer with a molecular mass of 101 kDa. The enzyme was active in the pH range from 6 to 9. The optimal activity was detected at pH 7.0 and at 37 degrees C. AAP activity was inhibited by typical aminopeptidase inhibitors (puromycin and bestatin), reducing agents (DTT), chelating agents (EDTA, EGTA and phenantroline) and sulphydryl groups reagents (iodoacetate). Ca(2+), Mn(2+) and Co(2+) activated the enzyme, while Cu(2+), Cd(2+), Hg(2+) and Mg(2+) inhibited it. The K(m) values calculated for Arg-AMC (7-amido-4-methylcoumarin) and Leu-AMC were 0.071 and 0.094 mM, respectively. The enzyme showed maximum specificity for basic amino acids (Arg and Lys), but was also able to hydrolyze non-charged amino acids (Leu, Met and Ala) and, at a minor rate, aromatic amino acids (Phe and Tyr). AAP showed higher activity when an acid residue was located at the C-terminal position of dipeptides. The described purification of an arginyl aminopeptidase from the yeast D. hansenii can contribute to the lack of knowledge about the exopeptidase activity in one of the yeasts more frequently isolated in sausage and to understand its role during the ripening of a fermented sausage.

Sensory improvement of dry-fermented sausages by the addition of cell-free extracts from Debaryomyces hansenii and Lactobacillus sakei

The effects of the addition of a combined cell-free extract from Lactobacillus sakei and Debaryomyces hansenii (D+L) or just a D. hansenii cell-free extract (D) to the initial formulation of a dry-fermented sausage were evaluated. The differences found among batches in the main microbial populations, pH, moisture content and global proteolytic and lipolytic indexes (total free amino acids, non protein nitrogen, acidity and tiobarbituric acid index) were not significant. Only, the acidity value of batch D was significantly higher (p<0.05) than that of batch D+L. Thus, cell-free extract from D. hansenii accelerated the lipolysis. Moreover, there were some significant differences (p<0.05) in the amino acid profile and, especially, in the aroma profile. The combination D+L and D promoted the generation of volatile compounds derived from lipid oxidation and carbohydrate fermentation. In batch D, the production of volatile compounds derived from amino acid catabolism and microbial fermentation was also enhanced. The overall quality was improved by both treatments (D+L, D) and also the aroma by addition of the combination of extracts (D+L). It is concluded that the addition of cell-free extracts from D. hansenii and, particularly, D. hansenii plus L. sakei could be useful to improve the final quality of fermented sausages.

Purification and characterisation of Proteases A and D from Debaryomyces hansenii

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.

Effect of electrohydraulic shockwave treatment on tenderness, muscle cathepsin and peptidase activities and microstructure of beef loin steaks from Holstein young bulls

Hydrodynamic pressure processing (HDP) or shockwave treatment improved tenderness (18% reduction in Warner-Bratzler shear force (WBSF) of beef loin steaks. Endogenous muscle proteolyic activities (cathepsins and peptidases) and protein fragmentation of sarcoplasmic and myofibrillar proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were not influenced by HDP. However, microstructure changes were clearly detected using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Specifically a disruption of the structure at the muscle fiber bundles and an increased endomysium space were observed. The present paper supports the evidence of physical disruption of the muscle fibers as a cause behind the tenderness improvement. The paper discusses the possible mechanisms responsible for the meat tenderisation induced by HDP treatment.

High pressure processing (HPP) of foods and its combination with electron beam processing

High pressure processing (HPP) is non-thermal processing which has been applied in the food industry mainly as a post-packaging pasteurization method in order to ensure food safety with minimal effects on nutritional and sensorial profiles and thus to obtain enhanced “freshness” of preserved foods. The present chapter is a brief comprehensive review of the thermodynamic aspects under high pressure conditions and the HPP effect on microbial inactivation and food matrix. Up-to-date industrial HPP equipment and commercial HPP food products are presented. The combination of HPP and eBeam technology is discussed as a method of hurdles addition for an optimized preservation.

Structural Changes in Foods Caused by High-Pressure Processing

High-pressure processing (HPP) has been mainly applied in the food industry as a post-packaging pasteurization method in order to ensure food safety. In more recent years, HPP has also been extensively considered in relation to the structural changes that HPP treatments induce in food systems. These structural changes are based on the effect of high pressure on the cell structure and on the biopolymers present in food. These changes can lead to diverse food applications such as creation of novel textures, improvement of the water binding, or mediation of gelation processes. This research area has been intensified in the last 15 years and is currently evolving at a rapid pace. Different applications have already described in the literature for different foodstuffs such as processed fruits and vegetables, meat, and dairy products. The present chapter explains the main mechanisms underlying these modifications and summarizes the research carried out in this novel field. HPP provides an ideal tool for structure modifications by means of physical nonthermal processing and can play an important role in future product development and in the production of food ingredients with enhanced functionality.

Ultrasonic velocity measurements in the ternary mixtures water-lactose-lactate, for the purpose of monitoring the lactic acid fermentation of lactose

A preliminary study of the lactic acid fermentation of lactose using an ultrasonic velocity technique is presented. During this fermentation, lactose (milk sugar) is transformed into lactate (lactic acid) by the action of bacteria. It is shown that the changes occurring during the course of the process can be monitored on-line by measuring the changes experienced by ultrasonic waves propagating through the fermenting media. Measurements of density and ultrasonic velocity in the ternary mixtures water-lactose-lactate and during the fermentation of a lactose solution were carried out. The ultrasonic propagation velocity has been correlated with the mass concentration of the mixture components using a semi-empirical model. These relations may be used to achieve an on-line concentration control for microbial lactic acid production.