Sodium reduction in foods: Challenges and strategies for technical solutions.

In many parts of the world, sodium consumption is higher than recommended levels, representing one of the most important food-related health challenges and leading to considerable economical costs for society. Therefore, there is a need to find technical solutions for sodium reduction that can be implemented by food producers and within food services. The aims of this review are to discuss the barriers related to sodium reduction and to highlight a variety of technical solutions. The barriers relate to consumer perception, microbiology, processing, and physicochemistry. Existing technical solutions include inhomogeneous salt distribution, coated salt particles, changing particle sizes and forms, surface coating, multisensory combinations, sodium replacements, double emulsions, adapted serum release by microstructure design, and adapted brittleness by microstructure design. These solutions, their implementation and the associated challenges, and applicable product categories are described. Some of these solutions are ready for use or are in their early development stages. Many solutions are promising, but in most cases, some form of adaptation or optimization is needed before application in specific products, and care must always be taken to ensure food safety. For instance, further research and innovation are required in the dynamic evolution of saltiness perception, consumer acceptance, the binding and migration of sodium, juiciness, microbiological safety, and the timing of salt addition during processing. Once implemented, these solutions will undoubtedly support food producers and food services in reducing sodium content and extend the application of the solutions to different foods.

Putative antibiotic resistance genes present in extant Bacillus licheniformis and Bacillus paralicheniformis strains are probably intrinsic and part of the ancient resistome.

Whole-genome sequencing and phenotypic testing of 104 strains of Bacillus licheniformis and Bacillus paralicheniformis from a variety of sources and time periods was used to characterize the genetic background and evolution of (putative) antimicrobial resistance mechanisms. Core proteins were identified in draft genomes and a phylogenetic analysis based on single amino acid polymorphisms allowed the species to be separated into two phylogenetically distinct clades with one outlier. Putative antimicrobial resistance genes were identified and mapped. A chromosomal ermD gene was found at the same location in all B. paralichenformis and in 27% of B. licheniformis genomes. Erythromycin resistance correlated very well with the presence of ermD. The putative streptomycin resistance genes, aph and aadK, were found in the chromosome of all strains as adjacent loci. Variations in amino acid sequence did not correlate with streptomycin susceptibility although the species were less susceptible than other Bacillus species. A putative chloramphenicol resistance gene (cat), encoding a novel chloramphenicol acetyltransferase protein was also found in the chromosome of all strains. Strains encoding a truncated CAT protein were sensitive to chloramphenicol. For all four resistance genes, the diversity and genetic context followed the overall phylogenetic relationship. No potentially mobile genetic elements were detected in their vicinity. Moreover, the genes were only distantly related to previously-described cat, aph, aad and erm genes present on mobile genetic elements or in other species. Thus, these genes are suggested to be intrinsic to B. licheniformis and B. paralicheniformis and part of their ancient resistomes. Since there is no evidence supporting horizontal transmission, these genes are not expected to add to the pool of antibiotic resistance elements considered to pose a risk to human or animal health. Whole-genome based phylogenetic and sequence analysis, combined with phenotypic testing, is proposed to be suitable for determining intrinsic resistance and evolutionary relationships.

Antimicrobial Susceptibility Testing and Tentative Epidemiological Cutoff Values for Five Bacillus Species Relevant for Use as Animal Feed Additives or for Plant Protection.

Bacillus megaterium (n = 29), Bacillus velezensis (n = 26), Bacillus amyloliquefaciens (n = 6), Bacillus paralicheniformis (n = 28), and Bacillus licheniformis (n = 35) strains from different sources, origins, and time periods were tested for the MICs for nine antimicrobial agents by the CLSI-recommended method (Mueller-Hinton broth, 35°C, for 18 to 20 h), as well as with a modified CLSI method (Iso-Sensitest [IST] broth, 37°C [35°C for B. megaterium], 24 h). This allows a proposal of species-specific epidemiological cutoff values (ECOFFs) for the interpretation of antimicrobial resistance in these species. MICs determined by the modified CLSI method were 2- to 16-fold higher than with the CLSI-recommended method for several antimicrobials. The MIC distributions differed between species for five of the nine antimicrobials. Consequently, use of the modified CLSI method and interpretation of resistance by use of species-specific ECOFFs is recommended. The genome sequences of all strains were determined and used for screening for resistance genes against the ResFinder database and for multilocus sequence typing. A putative chloramphenicol acetyltransferase (cat) gene was found in one B. megaterium strain with an elevated chloramphenicol MIC compared to the other B. megaterium strains. In B. velezensis and B. amyloliquefaciens, a putative tetracycline efflux gene, tet(L), was found in all strains (n = 27) with reduced tetracycline susceptibility but was absent in susceptible strains. All B. paralicheniformis and 23% of B. licheniformis strains had elevated MICs for erythromycin and harbored ermD The presence of these resistance genes follows taxonomy suggesting they may be intrinsic rather than horizontally acquired. Reduced susceptibility to chloramphenicol, streptomycin, and clindamycin could not be explained in all species.IMPORTANCE When commercializing bacterial strains, like Bacillus spp., for feed applications or plant bioprotection, it is required that the strains are free of acquired antimicrobial resistance genes that could potentially spread to pathogenic bacteria, thereby adding to the pool of resistance genes that may cause treatment failures in humans or animals. Conversely, if antimicrobial resistance is intrinsic to a bacterial species, the risk of spreading horizontally to other bacteria is considered very low. Reliable susceptibility test methods and interpretation criteria at the species level are needed to accurately assess antimicrobial resistance levels. In the present study, tentative ECOFFs for five Bacillus species were determined, and the results showed that the variation in MICs followed the respective species. Moreover, putative resistance genes, which were detected by whole-genome sequencing and suggested to be intrinsic rather that acquired, could explain the resistance phenotypes in most cases.

Development of Bacillus subtilis mutants to produce tryptophan in pigs.

OBJECTIVES: To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. RESULTS: A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l. Genome sequence analysis revealed that M1 had three single nuclear polymorphisms (SNPs) and M2 had two SNPs compared to the wild type strains. In both mutants SNPs were found in genes regulating tryptophan synthesis. Reverse transcription PCR confirmed up-regulation of the tryptophan synthesis genes in both mutants, the expression was up to 3 times higher in M2 than in M1. CONCLUSIONS: Tryptophan-excreting B. subtilis strains were obtained with UV-mutagenesis and analogue selection and can be used in animal feed applications.