First evaluation of alkylpyrazine application as a novel method to decrease microbial contaminations in processed meat products.

Every year about 20% of the globally produced meat gets lost due to microbial spoilage. Nevertheless, the demand for processed meat is constantly rising and producers are searching for novel strategies to reduce microbial contaminations in their products. In the present study, we evaluated the applicability of alkylpyrazines as antimicrobial agents. These fragrant molecules naturally occur in different vegetables, fruits, roasted nut and meat. Several pyrazine derivatives are readily added to processed products for flavoring purposes in the food industry. To evaluate their potential for application, two derivatives were tested for their antimicrobial activity against meat-associated bacterial contaminants and chicken meat as a whole. Isolates assigned to Carnobacteriaceae, Enterobacteriaceae, Listeriaceae, and Moraxellaceae were substantially inhibited in the pilot tests. Moreover, treatments of pyrazine-susceptible isolates resulted in 4-log reductions in bacterial cell counts. The effect was more pronounced when the model contaminants were exposed to higher concentrations of 5-isobutyl-2,3-dimethylpyrazine. In a first small-scale application with processed chicken meat, it was demonstrated that the antimicrobial effects of 2-isobutyl-3-methylpyrazine can be improved by additionally lowering the water activity on the meat surface when maltodextrin is used as a carrier substance. At low pyrazine dosages, the number of viable bacteria was decreased up to 95% in comparison to the corresponding controls. A complementary imaging method that was developed to assess the efficacy on the product, reinforced the applicability of this two-component system.

Replacing conventional decontamination of hatching eggs with a natural defense strategy based on antimicrobial, volatile pyrazines.

The treatment of hatching eggs relies on classic yet environmentally harmful decontamination methods such as formaldehyde fumigation. We evaluated bacteria-derived volatiles as a replacement within a fundamentally novel approach based on volatile organic compounds (VOCs), which are naturally involved in microbial communication and antagonism due to their high antimicrobial efficiency. Pyrazine (5-isobutyl-2,3-dimethylpyrazine) was applied passively and actively in prototypes of a pre-industry-scale utilization. Altogether, pyrazine decontamination rates of up to 99.6% were observed, which is comparable to formaldehyde fumigation. While active evaporation was highly efficient in all experiments, passive treatment showed reducing effects in two of four tested groups only. These results were confirmed by visualization using LIVE/DEAD staining microscopy. The natural egg shell microbiome was characterized by an unexpected bacterial diversity of Pseudomonadales, Enterobacteriales, Sphingomonadales, Streptophyta, Burkholderiales, Actinomycetales, Xanthomonadales, Rhizobiales, Bacillales, Clostridiales, Lactobacillales, and Flavobacteriales members. Interestingly, we found that especially low pyrazine concentrations lead to a microbiome shift, which can be explained by varying antimicrobial effects on different microorganisms. Micrococcus spp., which are linked to embryonic death and reduced hatchability, was found to be highly sensitive to pyrazines. Taken together, pyrazine application was shown to be a promising, environmentally friendly alternative for fumigation treatments of hatchery eggs.

New model substrates for enzymes hydrolysing polyethyleneterephthalate and polyamide fibres.

Recently the potential of enzymes for surface hydrophilisation and/or functionalisation of polyethyleneterephthalate (PET) and polyamide (PA) has been discovered. However, there was no correlation between enzyme class/activity (e.g. esterase, lipase, cutinase) and surface hydrolysis of these polymers and consequently no simple assay to estimate this capability. Enzymes active on the model substrates bis (benzoyloxyethyl) terephthalate and adipic acid bishexyl-amide, were also capable of increasing the hydrophilicity of PET and PA. When dosed at the identical activity on 4-nitrophenyl butyrate, only enzymes from Thermobifida fusca, Aspergillus sp., Beauveria sp. and commercial enzymes (TEXAZYME PES sp5 and Lipase PS) increased the hydrophilicity of PET fibres while other esterases and lipases did not show any effect. Activity on PET correlated with the activity on the model substrate. Hydrophilicity of fibres was greatly improved based on increases in rising height of up to 4.3 cm and the relative decrease of water absorption time between control and sample of the water was up to 76%. Similarly, enzymes increasing the hydrophilicity of PA fibres such as from Nocardia sp., Beauveria sp. and F. solani hydrolysed the model substrate; however, there was no common enzyme activity (e.g. protease, esterase, amidase) which could be attributed to all these enzymes.

A novel assay for the detection of bioactive volatiles evaluated by screening of lichen-associated bacteria.

Volatile organic compounds (VOCs) produced by microorganisms are known both for their effect on pathogens and their role as mediators in various interactions and communications. Previous studies have demonstrated the importance of VOCs for ecosystem functioning as well as their biotechnological potential, but screening for bioactive volatiles remained difficult. We have developed an efficient testing assay that is based on two multi-well plates, separated by a sealing silicone membrane, two tightening clamps, and variable growth media, or indicators. The experiment design as presented here is a novel and robust technique to identify positive as well as negative VOC effects on the growth of a target organism and to test for specific substances e.g., hydrogen cyanide which can be detected with a suitable indicator. While the first pre-screening assay is primarily based on indicator color change and visible growth diameter reduction, we also introduce an advanced and quantitatively precise experiment design. This adaptation involves qPCR-based quantification of viable target cells after concluding the treatment with VOCs. Therefore, we chose preselected active isolates and compared the partial 16S rRNA gene copy number of headspace-exposed E. coli with non-treated controls. Separately obtained headspace SPME and GC/MS-based profiles of selected bacterial isolates revealed the presence of specific and unique signatures which suggests divergent modes of action. The assay was evaluated by screening 100 isolates of lung lichen-associated bacteria. Approximately one quarter of the isolates showed VOC-based antibacterial and/or antifungal activity; mainly Pseudomonas and Stenotrophomonas species were identified as producers of bioactive volatiles.

Oxidation of glycerol by 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) in the presence of laccase.

Glycerol has the potential of being a low-cost and extremely versatile building block. However, current transformation strategies such based on noble-metal-catalysts show several disadvantages including catalyst deactivation or negative environmental impacts. In this study glycerol was oxidized by 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) in the presence of laccase from Trametes hirsuta. Analysis of the reaction production indicated sequential oxidation to glyceraldehyde, glyceric acid and tartronic acid, finally resulting in mesoxalic acid. The number and nature of oxidation products was depended on the concentration of TEMPO used. At lower TEMPO concentrations (<6mM) the major initial reaction product was glyceraldehyde while at higher concentration in addition considerable amounts of glyceric acid were formed. Glycerol oxidation was also shown with laccase immobilised on alumina pellets which increased laccase stability.