Effect of varying pH on solution interactions of soluble meat proteins with different plant proteins.

The exchange of animal-based for plant-based proteins is becoming more and more popular due to an increasing demand for alternative and more sustainable protein sources. In this study, solubilized water- (ws) or salt-and-water (sws) meat proteins were evaluated in their pH-dependent interactions with soluble protein fractions from wheat, pumpkin, sunflower, rapeseed, or potato proteins. For this purpose, 1 : 1 (v/v) mixtures of 1.0 wt% meat (ws or sws) and plant proteins were prepared at a sodium chloride concentration of 1.8 wt% (ionic strength: 0.31 mol L-1) and adjusted to different pH-values in between 4.5-7.0. While only slight differences were found upon comparison of interactions of ws and sws batches (p > 0.05), interactions among these animal-based and soluble plant proteins took place. First, optical observations, light microscopy, and SDS-PAGE revealed increasing protein solubility with increasing pH. Second, particle size distributions (PSDs) revealed a shift towards slightly larger particle sizes e.g. at pH 5.3 and 7.0 with d4,3 of 43.2 and 21.3 µm (sws) to 45.4 and 23.9 µm (sws + potato), respectively. Furthermore, heat-induced gel formation was improved at pH > 6.0, in particular in mixtures of meat and wheat or rapeseed proteins that formed a homogenous gel structure. Based on the obtained results, protein-protein complexations mainly by electrostatic forces are suggested which occur due to various pI of meat and plant proteins e.g. pH 7.5 (wheat), 7.2 (potato), and 6.6 (rapeseed) in comparison to 5.1 (ws) and 5.6 (sws). The filamentous microstructure of some gels (soluble fraction of rapeseed, potato and wheat proteins) led to the assumption that meat proteins, mainly at pH values greater than 5.8 (optimally ≥6.5), had a structuring effect on plant proteins.

Influence of protein and solid fat content on mechanical properties and comminution behavior of structured plant-based lipids.

Recently an approach has been developed to structure plant-based lipids with the intention to mimic animal fat tissue in processed meat products or analogues. This study investigated the comminution behavior in a bowl chopper of such structured lipids with varying mechanical properties. For products like salami-type sausages these systems need to withstand comminution to yield particles for inclusion in product matrices. Therefore, samples were prepared from protein suspensions with 6%, 8%, 10%, and 12% soy protein isolate (SPI) and 70% (w/w) total fat with varying solid fat contents (0-30%, w/w). The hardness of samples prepared with 6% and 8% SPI varied between 4.5 and 35.9 N. When comminuted in a bowl chopper, these structures had insufficient mechanical strengths to facilitate the formation of small particles and yielded a coarse paste. Higher concentrations of protein increased hardness (15.9-76.2 N and 15.6-96.1 N, for 10% and 12% SPI, respectively). These samples retained their structural integrity upon comminution yielding individual intact particles. The size of these particles increased with sample firmness, i.e. with increasing amount of protein. The shape of the particles was more elongated the higher the solid fat content as indicated by a higher aspect ratio. Taken together, results show that structural characteristics of the gelled emulsions can be tuned to yield desired fat particles after comminution.

Influence of phosphate chelators on white efflorescence formation in dry fermented sausages with co-extruded alginate casings.

The aim of the study was to investigate the effect of different condensed phosphates on the white efflorescence formation on dry fermented sausages with calcium alginate casings. The efflorescence formation is induced by the complexation of the divalent cations magnesium and calcium with lactate on the surface of the product. Phosphates are known to complex divalent cations like magnesium and calcium, which are responsible for the efflorescence formation with lactate. To treat the surface of the raw fermented sausages a mixture of di- and polyphosphates and a polyphosphate were used. The sausages were dipped in 5% solutions of the phosphates. The mixture of di- and polyphosphate showed the best results in reducing the white efflorescence formation, the amount of efflorescences on the surface was 18.12% after 8 weeks of storage. The diffusion of magnesium was significantly reduced by ~40% after 8 weeks of storage compared to the control for both phosphates. However, the crystals formed during storage had a different structure and size compared to the control. Isothermal titration calorimetry measurements showed that the divalent calcium ions and the polyphosphates from aggregates caused the crystal formation on the surface, whereas this effect was not seen in combination with magnesium. The surface treatment with the phosphates did not inhibit the white efflorescence formation. However, the extent was reduced but different kind of crystals were formed on the surface during the storage.

Effects of combined measures to minimize white efflorescence formation on dry fermented sausages co-extruded with alginate casings.

On dry fermented sausages, especially co-extruded with calcium alginate casings, the formation of white crystals on the surface of the product is a common problem. In this study a hurdle system of combinations of different metal complexing chelators (citric acid and polyphosphate) was investigated. The control and the sample produced with 1.1% citric acid in the alginate gel showed strong white efflorescence formation during the storage (∼60% of the surface was covered), whereas the samples with added 0.3% polyphosphate showed no efflorescence formation. The efflorescence-causing substances such as lactate, magnesium, and calcium increased significantly in the samples, which showed the strongest white efflorescence formation. In the control, lactate increased by 45.0%, calcium by 23.9%, and magnesium by 150.8%, whereas in the samples without white efflorescence, the magnesium and lactate content increased only slightly, and the calcium content even decreased during the storage of 8 weeks. The best results were observed on the addition of 0.3% polyphosphates and the citric acid surface treatment directly after the co-extrusion. Moreover, the strong complex formation could be due to the complexation of magnesium and calcium by lactate, whereby they are removed from the diffusion equilibrium leading to an increased diffusion of the efflorescence-causing substances. PRACTICAL APPLICATION: White efflorescence formation on dry fermented sausages is a major problem on dry fermented sausages, which causes food waste and financial loss for the producer. This study investigated different combinations of promising methods and it was possible to inhibit the white efflorescence formation during the storage. The methods are easy to implement during the manufacturing of the products and are therefore quite interesting for the industrial applications.

Influence of calcium on white efflorescence formation on dry fermented sausages with co-extruded alginate casings.

The effect of the concentration of calcium in the crosslinking solution during co-extrusion of dry fermented sausages with calcium alginate casing on the white efflorescence was investigated. With the co-extrusion technology, a continuous sausage production is possible and, furthermore, snack products with very small calibers can be produced. Therefore, crosslinking solutions with different concentrations of CaCl2 (15, 20, 25, and 30%) were used during the production. High concentrations of calcium led to a very rapid and intensive white efflorescence formation; the efflorescences covered ~90% of the surface of the samples produced with 20-30% of CaCl2 after 8 weeks of storage. However, the batch produced with the lowest amount of CaCl2 showed a slow efflorescence formation and a significantly decreased area covered by it (~70% after 8 weeks). The differences in the formation were attributed to the excess of calcium on the surface of the sausages (saturated calcium alginate film), therefore leading to rapid complexation of lactate to mostly calcium lactate. Whereas with 15% of CaCl2 in the solution, only small amounts of calcium are not bound by the alginate film, and the formation of white efflorescence is due to the time-delayed formation with magnesium, which diffuses from the core to the surface.

Influence of homopolysaccharide-producing lactic acid bacteria on the spreadability of raw fermented sausages (onion mettwurst).

The purpose of the study was to investigate the effect of a reduced pH value (5.1 instead of 5.5 to 5.6) on the properties of highly perishable, spreadable raw fermented sausages (onion mettwurst) with or without the addition of homopolysaccharide (HoPS)-producing lactic acid bacteria (LAB). Hence, sausages with HoPS-producing LAB and a pH value of 5.1 were produced and compared to sausages (pH 5.1) produced with a non-exopolysaccharide (EPS)-forming strain (Lactobacillus sakei TMW 1.2037). Microbial growth and pH values were monitored during processing (24 °C for 48 hr, 10 °C for 24 hr) and storage (14 days at 0 to 2 °C). Furthermore, fat (Weibull-Stoldt) and EPS contents were determined in the final products. Sausages were characterized using texture profile and sensory analysis. The fat contents ranged from 16% to 19% and the determined EPS concentrations ranged from 0.17 to 0.59 g/kg for L. sakei TMW 1.411 and Lactobacillus curvatus TMW 1.1928 and from 0.67 to 1.58 g/kg for L. curvatus TMW 1.51. The strains L. sakei TMW 1.411 and L. curvatus TMW 1.51 reduced the hardness of the samples significantly (P < 0.05) compared to the control samples. Regarding spreadability and mouthfeel, sausages containing an EPS-forming culture were rated slightly better than the control samples and the taste was not negatively influenced. PRACTICAL APPLICATION: This study clearly demonstrated that it is promising to apply HoPS-producing LAB to maintain the spreadability of pH-reduced (pH 5.1) spreadable raw fermented onion mettwurst, which may prospectively give the opportunity to increase the safety of this highly perishable product.

Application of exopolysaccharide-forming lactic acid bacteria in cooked ham model systems.

The meat industry often applies hydrocolloids (not label-free) to improve quality attributes of meat products including reconstructed cooked ham. A new approach to improve product quality could be the usage of in-situ Exopolysaccharide (EPS)- forming lactic acid bacteria (LAB) provided that these strains are able to produce EPS in meat matrices under typical processing conditions (here: cooked ham). Two homopolysaccharide- (L. curvatus TMW 1.624 and L. sakei TMW 1.411; 106CFUmL) and heteropolysaccharide-forming LABs (L. plantarum TMW 1.1478 and TMW 1.25; 106CFUmL) were hence examined for EPS formation in a cooked ham model system consisting of minced pork topside (<2% fat) and 16.67% brine containing either 0.5% sucrose or dextrose. Samples were stored for 48 h at either 2 °C to simulate typical tumbling conditions, or at 15 °C to examine in-situ EPS production under reduced stress conditions. Microbial growth behavior and pH development (48 h) were monitored and EPS qualitatively as well as semi-quantitatively analyzed using both confocal laser scanning microscopy and MATLAB enabling a better comparison of the investigated strains. All LAB were able to tolerate the suboptimal growth conditions in the cooked ham model systems (2 °C, 1.92% nitrite curing salt) and were found to already produce EPS within 10 h of storage at 2° and 15 °C. EPS amounts detected after 24 h of incubation were significantly (p < 0.05) higher than those determined after sample preparation. EPS were found to be predominately located at the outer edge of meat proteins. All investigated strains seem to be promising for prospective studies in cooked ham.

Impact of food structure on the compatibility of heated WPI-pectin-complexes in meat dispersions.

Process-stable complexes composed of whey protein isolate (WPI) and sugar beet pectin have great potential as structuring agents or fat replacers in foods. The current study investigates the compatibility of heated WPI : pectin complexes in different meat matrices. Spreadable raw-fermented sausages and sliceable emulsion-type sausages were therefore manufactured containing biopolymer complexes with various WPI : pectin ratios r (2 : 1, 8 : 1). Macro- and microstructural properties of the meat dispersions were analyzed in terms of colour, texture, rheometry, sensory, and confocal laser scanning microscopy (CLSM) measurements. Textural and sensorial results demonstrated that the meat products became increasingly soft and yellowish as the biopolymer ratio r was increased regardless of the meat matrix, whereas pH and water activity values were not affected. CLSM images revealed that the meat protein network became disrupted and loose in the presence of pectin, which was attributed to a thermodynamic incompatibility effect. The results obtained from this study highlight that biopolymer complexes might be suitable fat mimetics, particularly for spreadable meat products.

Optimum hexametaphosphate concentration to inhibit efflorescence formation in dry fermented sausages.

The occurrence of efflorescences on the surface of dry fermented sausages represents a current issue for the meat processing industry. Preventing the efflorescence formation by the addition of sodium hexametaphosphate (SHMP) was shown to be promising in a previous study. The optimum SHMP addition was studied by adding SHMP (0.0, 1.0, 3.0, and 5.0g/kg) directly to the sausage batter. Visual and chemical analyses were conducted during 8weeks of storage under modified atmosphere. Visual analyses revealed significant lower amounts of efflorescences on the sausage surface after 8weeks when 1.0 (27.1%), 3.0 (9.0%), and 5.0g/kg SHMP (3.4%) were added, compared to the control with 38.0% efflorescences. SHMP significantly affected the occurrence (8weeks) of magnesium on the surface: +85.5%, +23.7%, +3.5%, and -28.2% for 0.0, 1.0, 3.0, and 5.0g/kg, respectively. The addition of 4.785g/kg was calculated to fully inhibit the formation of efflorescences by complexing magnesium ions.

Inhibitory effect of phosphates on magnesium lactate efflorescence formation in dry-fermented sausages.

This study aimed to prevent the phenomena of efflorescence formation on the surface of dry fermented sausages due to the complexation of efflorescence forming cations with phosphates. Efflorescence formation is a critical issue constituting a major quality defect, especially of dry fermented sausages. Different phosphates (di- and hexametaphosphate) were added (3.0g/kg) to the sausage batter. As a hypothesis, these additives should complex with one of the main efflorescence-causing substances such as magnesium. The formation of efflorescences was determined for dry fermented sausages without phosphate addition, with diphosphate, or hexametaphosphate addition during 8weeks of storage under modified atmosphere. The visual analyses of the sausage surface revealed high amounts of efflorescences for the control (42.2%) and for the sausages with added diphosphate (40.9%), whereas the sausages containing hexametaphosphate had significantly reduced amounts of efflorescence formation, showing only 11.9% efflorescences after 8weeks of storage. This inhibition was a result of strong complexation of hexametaphosphate with magnesium ions, thus preventing the diffusion of magnesium towards the sausage surface. This can be explained by the magnesium content on the sausage surface that increased by 163.9, 127.8, and 52.8% for the sausages without phosphate, diphosphate, and hexametaphosphate addition, respectively. The mass transport of lactate and creatine was not affected by phosphate addition. Isothermal titration calorimetry confirmed that, theoretically, 4.5g/kg of diphosphate or 2.8g/kg hexametaphosphate are required to complex 0.2g/kg magnesium ions naturally occurring in dry fermented sausages and, thus, the chosen overall phosphate concentration of 3.0g/kg was enough when adding hexametaphosphate, but not for diphosphate, to inhibit the efflorescence formation.

Chemical and optical characterization of white efflorescences on dry fermented sausages under modified atmosphere packaging.

BACKGROUND: Dry fermented sausages that are packed under modified atmosphere are often affected by the formation of white crystals on the surface. These so called efflorescences are rejected by consumers and lead to high financial losses for the meat processing industry. In this study, the distribution of efflorescence-causing components was investigated over the sausage profile during 8 weeks of storage under modified atmosphere at 4 °C. In addition, two visual methods (image and sensory analyses) were compared regarding the ability to quantify the efflorescence content. RESULTS: The initial formation of efflorescences was observed after 2 weeks (7%). After 4 weeks of storage, 23.4% of the sausage surface was covered with efflorescences, and the amount of efflorescences did not change significantly by the end of storage. Furthermore, chemical analyses revealed that magnesium (increased by 98.1%), lactate (increased by 54.2%) and creatine (increased by 51.8%) are enriched on the sausage surface during storage. CONCLUSION: Sensory and image analyses lead to comparable results (r = 0.992) and therefore both are suitable to quantify the amount of efflorescences. The moisture gradient in the interior of the sausages which is built upon drying is supposed to be the driving force for the movement of efflorescence-causing compounds. © 2017 Society of Chemical Industry.

Mixing behaviour of WPI-pectin-complexes in meat dispersions: impact of biopolymer ratios.

Particulated complexes composed of oppositely charged biopolymers were incorporated into highly concentrated protein matrices as potential fat replacers and structuring agents. A multistep procedure was therefore utilized to generate process-stable complexes, which were subsequently embedded into emulsion-type sausages, whereas macro- and microstructural properties were then assessed. Firstly, stock WPI and sugar beet pectin solutions were mixed under neutral conditions (pH 7) at various biopolymer ratios r (2 : 1, 5 : 1, 8 : 1). Secondly, the pH of the biopolymer mixture was decreased to 3.5 to promote associative complexation. Thirdly, electrostatically attracted biopolymer particles were subjected to a heat treatment (ϑ = 85 °C, 20 min) to enhance their stability against superimposed stresses. Finally, fat-reduced emulsion-type sausages were fabricated containing stable WPI-pectin complexes. The results revealed that the heat treatment increased the pH-stability of the biopolymer complexes. In addition, textural and sensorial analysis demonstrated that the meat products became increasingly soft as the biopolymer ratio r increased. This effect was attributed to thermodynamic incompatibility between meat proteins and beet pectin. The results obtained from this study might have important implications for the fabrication of processed meat products with reduced fat levels.

Meat batter production in an extended vane pump-grinder injecting curing salt solutions to reduce energy requirements: variation of curing salt amount injected with the solution.

BACKGROUND: The integration of a nozzle in an extended vane pump-grinder system may enable the continuous injection of curing salt solutions during meat batter production. The purpose of this work was to examine the influence of the curing salt amount injected with the solution (0-100%) on protein solubilisation, water-binding, structure, colour and texture of emulsion-type sausages. RESULTS: The amount of myofibrillar protein solubilised during homogenisation varied slightly from 33 to 36 g kg-1 . Reddening was not noticeably impacted by the later addition of nitrite. L* ranged from 66.9 ± 0.3 to 67.8 ± 0.3, a* from 10.9 ± 0.1 to 11.2 ± 0.1 and b* from 7.7 ± 0.1 to 8.0 ± 0.1. Although softer sausages were produced when only water was injected, firmness increased with increasing curing salt amount injected and was similar to the control when the full amount of salt was used. The substitution of two-thirds of ice with a liquid brine may enable energy savings due to reduced power consumptions of the extended vane pump-grinder system by up to 23%. CONCLUSION: The injection of curing salt solutions is feasible without affecting structure and colour negatively. This constitutes a first step towards of an 'ice-free' meat batter production allowing for substantial energy savings due to lower comminution work. © 2016 Society of Chemical Industry.

Extension of the Vane Pump-Grinder Technology to Manufacture Finely Dispersed Meat Batters.

A vane pump-grinder system was extended to enable the manufacture of finely dispersed emulsion-type sausages by constructing and attaching a high-shear homogenizer at the outlet. We hypothesized that the dispersing capabilities of the extended system may be improved to the point of facilitating meat-fat emulsification due to an overall increased volumetric energy input EV . Coarsely ground raw material mixtures were processed to yield meat batters at varying volume flow rates (10 to 60 L/min) and rotational rotor speeds of the homogenizer nrotor (1000 to 3400 rpm). The normalized torques acting on pump, grinder, and homogenizer motors were recorded and unit power consumptions were calculated. The structure of the manufactured meat batters and sausages were analyzed via image analysis. Key physicochemical properties of unheated and heated batters, that is, texture, water-binding, color, and solubilized protein were determined. The mean diameter d10 of the visible lean meat particles varied between 352 and 406 µm whereas the mean volume-surface diameter d32 varied between 603 and 796 µm. The lightness L* ranged from 66.2 to 70.7 and correlated with the volumetric energy input and product structure. By contrast, varying process parameters did not impact color values a* (approximately 11) and b* (approximately 8). Interestingly, water-binding and protein solubilization were not affected. An exponential process-structure relationship was identified allowing manufacturers to predict product properties as a function of applied process parameters. Raw material mixtures can be continuously comminuted, emulsified, and subsequently filled into casings using an extended vane pump-grinder.

Oil-in-water emulsions as a delivery system for n-3 fatty acids in meat products.

The oxidative and physical stabilities of oil-in-water emulsions containing n-3 fatty acids (25 wt.% oil, 2.5 wt.% whey protein, pH 3.0 or pH 6.0), and their subsequent incorporation into meat products were investigated. The physical stability of fish oil emulsions was excellent and neither coalescence nor aggregation occurred during storage. Oxidative stability was better at pH 6.0 compared to pH 3.0 likely due to antioxidative continuous phase proteins. Incorporation of fish oil emulsions into pork sausages led to an increase in oxidation compared to sausages without the added fish oil emulsion. Confocal microscopy of pork sausages with fish oil emulsions revealed that droplets had coalesced in the meat matrix over time which may have contributed to the decreased oxidative stability. Results demonstrate that although interfacial engineering of n-3 fatty acids containing oil-in-water emulsions provides physical and oxidative stability of the base-emulsion, their incorporation into complex meat matrices is a non-trivial undertaking and products may incur changes in quality over time.

Impact of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) on functional characteristics of emulsified sausages.

Inclusion of fibers, such as carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), at the expense of fat or protein in meat batters could be used to produce healthier sausages while lowering production costs. To study the impact of CMC/MCC on structural/functional characteristics of emulsified sausages, standard-fat Lyoner-style sausages were formulated with CMC/MCC at concentrations of 0.3-2.0%. Methods of analysis included rheology, water binding capacity (WBC), texture measurements, and Confocal Laser Scanning Microscopy (CLSM). WBC, texture measurements, and rheology all indicated that addition of CMC (>0.7%) led to destabilization of the batter, which upon heating could no longer be converted into a coherent protein network, a fact that was also revealed in CLSM images. In contrast, MCC was highly compatible with the matrix and improved firmness (1405-1651N/100g) with increasing concentration compared to control (1381N/100g) while keeping WBC (4.6-5.9%) with <2% MCC at the level of the control (4.8%). Results were discussed in terms of molecular interactions of meat proteins with celluloses.

Differential in vivo zymography: a method for observing matrix metalloproteinase activity in the zebrafish embryo.

Investigations into the molecular mechanisms of, and cellular signaling pathways modulating ECM remodeling are especially challenging due to the complex post-translational regulation of the primary effectors of ECM catabolism - the matrix metalloproteinases (MMPs). Recently a variety of approaches to the detection of MMP activity have been developed, and the prospect of visualizing ECM remodeling activity in living tissues is now opening exciting avenues of research for matrix biologists. In particular the use of FRET-quenched MMP substrates, which generate a fluorescent signal upon hydrolysis, is becoming increasingly popular, especially because linkers with defined and/or restricted proteolytic sensitivity can be used to bind fluorophore-quencher pairs, making these probes useful in characterizing the activity of specific proteases. We have taken advantage of the transparency and amenability to reverse genetics of the zebrafish embryo, in combination with these fluorogenic MMP substrates, to develop a multiplex in vivo assay for MMP activity that we dub "differential in vivo zymography."