Impact of raw ham quality and tumbling time on the technological properties of polyphosphate-free cooked ham

The effect of tumbling time (5 h30, 19 h and 26 h) and raw ham quality (superior, inferior or mixed quality) on the quality of polyphosphate-free cooked ham was investigated. The water holding capacity and total yield of the polyphosphate-free tumbled hams were dependent on both tumbling time and ham quality. Higher values of both parameters were obtained with an increase in tumbling time from 5 h30 to 19 h and with superior hams. The exudate after 19 h and 26 h tumbling showed a higher gel forming ability compared to 5 h30, which, in case of polyphosphate-free cooked hams produced with mixed and inferior meat quality, resulted in a better sliceability (less holes). However, tumbling time did not affect hardness, which was only influenced by ham quality, resulting in a softer polyphosphate-free cooked ham produced with inferior ham quality compared to the other quality classes

N-nitrosamines in Dry Fermented Sausages: Occurrence and Formation of N-nitrosopiperidine

The occurrence of carcinogenic N-nitrosamines in food cannot be ignored as food safety issue. Since the intake of N-nitrosamine contaminated food may induce all kinds of cancer tumors, the presence of these carcinogens must be reduced to the lowest possible concentrations (below the limit of detection). Also in meat products N-Nitrosamines can regularly be detected, although mostly in low concentrations. Generally, it is assumed that N-nitrosamines are formed by the nitrosation of a secondary amine with a nitrosating agent. In dry fermented sausages, the nitrosating agent mainly originates from sodium nitrite, which is added to the meat as preservative and colouring agent. In addition, during the fermentation and the subsequent ripening period, microorganisms can decarboxylate amino acid to biogenic amines. These biogenic amines, some of which can cause food poisoning themselves, may be transformed to the secondary amines, which are the direct precursors of N-nitrosamines. Therefore, biogenic amines are considered to be a risk for the formation of N-nitrosamines in dry fermented sausages. However, this hypothesis has not been confirmed experimentally yet. Therefore, the objective of this work was to gain additional insight in the occurrence and formation of N-nitrosamines in relation to nitrite and the accumulation of biogenic amines in dry fermented sausages. More specifically, the study was focused on the mechanism of N-nitrosopiperidine (NPIP) formation in a dry fermented sausage model.Firstly, a method was optimised for the determination of biogenic amines in dry fermented sausages. To increase the sensitivity, a derivatisation was necessary. Therefore the commonly used dansylation was compared to an alternative dabsylation procedure. The derivatisation with dabsyl chloride was preferred since it was realized in 25 min at 70 °C instead of 45 min at 40 °C, which is a substantial time gain. The interferences in the chromatogram, which originated from the complex protein-fat matrix, were removed by a solid phase extraction (SPE). As a result, a reliable and sensitive method was developed to determine the biogenic amines, i.e., tryptamine (TRYP), phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), serotonin (SER), tyramine (TYR), and the natural polyamines spermidine (SPD) en spermine (SPM), in dry fermented sausages.Next, the concentrations of N-nitrosamines, biogenic amines and residual nitrite were determined in 101 commercial dry fermented sausages, which were available on the Belgian market. In this way, the food safety of the commercial products in relation to the occurrence of these compounds were assessed. In general, the product could be considered safe since the concentrations of biogenic amines and N-nitrosamines remained low. Traces of N-nitrosomorpholine (NMOR) and NPIP were found in 22% and 28% of the samples, respectively. In some cases (3%), the NPIP concentrations were measured in quantifiable concentrations (above the method quantification limit (MQL) of 2,5 µg/kg). In addition, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used in to search for patterns in the occurrence of biogenic amines and N-nitrosamines in the commercial meat products, and their relation with physical and chemical characteristics. However, no correlation could be observed between the N-nitrosamine contamination and the amounts of biogenic amines or the physical and chemical characteristics. Moreover, the contamination with N-nitrosamines could not be linked to a specific type of dry fermented sausage.To study the N-nitrosamine formation, the use of a dry fermented sausage model, produced under strictly controlled conditions (Good Manufactruing Practices, GMP) was preferred. In the technicum of the Research Group of Technology and Quality of Animal Products, a North European type dry fermented sausage was developed. The physical (pH = 5.1 and aw = 0.93) and chemical (salt, moisture, fat and protein content were 3.5, 42.9, 32.0, and 19.0 g/100g, respectively) characteristics were comparable with results found for commercial products. In addition, no extreme biogenic amine accumulation and no N-nitrosamine formation was observed during the production of the dry fermented sausage model. Therefore, this model was proven to be suitable for the study of the N-nitrosamine formation.In the second part of this work, the NPIP formation was studied in detail since the commercial samples were mainly contaminated with NPIP.By means of the dry fermented sausage model, the role of the biogenic amine CAD and the direct precursor piperidine (PIP) on the formation of NPIP was studied during the production of dry fermented sausages. In this study, the influence of pH (4.9 and 5.3), sodium nitrite (0 and 150 mg/kg) and sodium ascorbate (0 and 500 mg/kg) was investigated. When the meat batter was enriched with CAD (500 mg/kg cadaverine dihydrochloride, CAD.2HCl), no increased NPIP concentrations were observed. In contrast, the enrichment with PIP (10 and 100 mg/kg) resulted in an increased NPIP formation. On the one hand, no effect on the NPIP formation was seen by the small difference in pH. On the other hand, the NPIP formation was significantly higher when the sausages were prepared with sodium nitrite and when sodium ascorbate was omitted. In the case that PIP was present in excessive amounts, nitrite could be identified as source of the nitrosating agent. Also the role of ascorbate as inhibitor of N-nitrosamine formation was confirmed. However, this effect could only be observed in the beginning of the production since NPIP degraded during the subsequent production phases. In the next part, the influence of pH and aw on the NPIP formation was investigated, using two protein-based liquid systems. In the first system (NaCl-system), the NaCl concentration (0 30%) was varied to reduce the aw (between 0.99 and 0.79) at two pH levels (4.0 and 5.0). At both pH levels, the reduction of aw resulted in a decreased amount of NPIP. However, the applied NaCl concentrations, necessary for the reduction of aw, were much higher than the NaCl content in dry fermented sausages (ca. 3%). As a consequence, it was unclear to what extent this effect could be attributed to the higher ionic strength or the lower aw. In a second system (PEG-system), poly ethylene glycol (PEG) was used to reduce the aw. Response surface methodology (RSM) was applied to evaluate the combined effect of pH (3.0 7.0), aw (0.80 0.99) and the incubation time (1.3 98.7 h) on the NPIP formation. It was observed that the NPIP concentrations increased when the incubation time was longer, the aw was higher and the pH was lower. The results obtained in the liquid systems contributed to a better understanding of the inhibition of the NPIP formation during the production of dry fermented sausages. Hereby, the possible NPIP formation, partially promoted by a slight acidification during the fermentation, is inhibited by the reduction of aw.Finally, it was investigated if the presence of NPIP in dry fermented sausages can emanate from the use of NPIP contaminated spices. First, analytical methods were developed to determine the precursors, namely piperine and PIP, in the spices. Piperine was determined by HPLC-DAD (λ = 343 nm) after accelerated solvent extraction (ASE) with dichloromethane (DCM). To determine PIP, a hydroextraction by means of ASE followed by HPLC-ELSD was applied. Commonly used spices in dry fermented sausages, i.e., paprika (Capsicum annuum), chilli (Capsicum frutescens), allspice (Pimenta dioica), and nutmeg (Myristica fragrans), contained only traces of both precursors. Only in samples of black and white pepper high concentrations of piperine (max. 21.12 mg/g) and PIP (max. 11.42 mg/g) were measured. However, the addition of piperine and PIP containing spices in nitrite curing salt mixtures, did not always result in the formation of NPIP. Only in the mixture containing white pepper, a small amount of NPIP (9.8 ng/g) was detected after a two month storage period. However, this amount was not sufficient to explain the sporadic occurrence of quantifiable concentrations of NPIP in commercial dry fermented sausages. It should be noticed that the storage conditions were optimal and the storage time was relatively short. Future studies are needed to reveal if changed storage conditions can result in increased amounts of NPIP in the spice mixtures. The results obtained in this work demonstrated that in general the risk of N-nitrosamine contamination in dry fermented sausages is low. On the one hand, it was proven that the accumulation of biogenic amines, especially with regard to CAD, will not result in the formation of NPIP during the production of dry fermented sausages. On the other hand, NPIP can be formed from PIP, but only when extreme concentrations of PIP are present. Common amounts of PIP, which can be introduced in the sausage by the additionof PIP containing spices, are not a risk for the formation of NPIP. The sporadic occurrence of quantifiable concentrations in commercial dry fermented sausages will probably be attributed to the use of highly NPIP contaminated spice mixtures.status: publishe

Smart living biomaterials : development of a mycelium-bacteria cocultivation platform to engineer material functionalities

During the last decade, there was a drastic transition towards a more circular and sustainable bio-economy. Nevertheless, evolving and innovative technology keeps raising expectations for developing all kinds of smart materials. This project addresses a development gap in biological materials to keep up with this trend and aims to exploit nature’s strategies that have been developing and optimising for millions of years to cope and interact with its environment, yielding a smart biological material. To this end, special interest grew in mycelium materials because of their robustness, versatility and rapid growth. The major advances in bacterial synthetic biology engineering will be exploited to use bacterial strains as a chassis for sensor-containing genetic circuits that render advanced functionalities to the mycelium material through cocultivation of both partners. This creates an opportunity to meet the expectations of both the biobased economy and innovative technology by creating an engineered living material (ELM), consisting of only biological compounds and being able to interact with its environment. Synthetic biology will be implemented to introduce bioswitches in the bacterial hosts that are activated by environmental cues of light, temperature or chemical compounds and render an advanced functionality to the material, either by direct activity (metabolite production) or indirect effects (influencing growth or morphology of the mycelium material). Various engineered living material (ELM) products will be developed, composed of a synthetic cocultivation consortium of a filamentous fungus and a bacterial strain, ranging from consumer goods to applications in the environmental or construction sector

Volatile N-nitrosamines in meat products: Potential precursors, influence of processing and mitigation strategies

Meat products can be contaminated with carcinogenic N-nitrosamines, which is ascribed to the reaction between a nitrosating agent, originating from nitrite or smoke, and a secondary amine, derived from protein and lipid degradation. Although in model systems it is demonstrated that many amine containing compounds can be converted to N-nitrosamines, the yield is dependent of reaction conditions (e.g., low pH and high temperature). In this article, the influence of the composition of the meat products (e.g., pH, aw, spices) and processing (e.g., ageing, ripening, fermentation, smoking, heat treatment and storage) on the presence and availability of the amine precursors and the N-nitrosamine formation mechanism is discussed. In addition, this article explores the current N-nitrosamine mitigation strategies in order to obtain healthier and more natural meat products.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=bfsn20status: publishe

High-throughput in vivo screening of novel prokaryotic, metabolite-responsive transcription factors for biosensor development

In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority is not characterized to date, which hampers both the understanding of fundamental processes and the development of biosensor applications. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility and specificity. A set of 30 prokaryotic TFs belonging to the Leucine-responsive Regulatory Protein (= Lrp) family, with respective promoters of interest, were selected for analysis as metabolite inducible systems. A reporter system was designed to use for cloning and expression of the TFs and promoters in Escherichia coli. By using an automized platform, fluorescence measurements were performed with strains containing each TF-promoter pair. While inducing the TF’s expression at different levels, the functionality of the heterologous promoters in E. coli was determined, as well as the TF’s regulation mechanism. Furthermore, experiments were performed in the presence of possible effector molecules, to learn more about the ligand specificity and sensitivity of each TF. The selection of TF-promoter pairs seemed to be a good representation of the Lrp family, since different regulatory mechanisms could be found, both ligand dependent and independent, and since some pairs were characterized by one specific amino acid as effector molecule, whereas others were sensitive to a whole range of amino acids. To conclude, this screening led to the initial characterization of numerous TFs and contributes to the general knowledge currently available about the Lrp family and the development of well-functioning biosensors

In vivo screening for the identification and characterization of prokaryotic, metabolite-responsive transcription factors

In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority is not characterized to date, which hampers both the understanding of fundamental processes and the development of biosensor applications. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility and specificity. A set of 30 prokaryotic TFs belonging to the Leucine-responsive Regulatory Protein (= Lrp) family, with respective promoters of interest, were selected for analysis as metabolite inducible systems. A reporter system was designed to use for cloning and expression of the TFs and promoters in Escherichia coli. By using an automized platform, fluorescence measurements were performed with strains containing each TF-promoter pair. While inducing the TF’s expression at different levels, the functionality of the heterologous promoters in E. coli was determined, as well as the TF’s regulation mechanism. Furthermore, experiments were performed in the presence of possible effector molecules, to learn more about the ligand specificity and sensitivity of each TF. The selection of TF-promoter pairs seemed to be a good representation of the Lrp family, since different regulatory mechanisms could be found, both ligand dependent and independent, and since some pairs were characterized by one specific amino acid as effector molecule, whereas others were sensitive to a whole range of amino acids. To conclude, this screening led to the initial characterization of numerous TFs and contributes to the general knowledge currently available about the Lrp family and the development of well-functioning biosensors