Sam Crauwels

Microbial community assessment of mealworm larvae (Tenebrio molitor) and grasshoppers (Locusta migratoria migratorioides) sold for human consumption.

In Western countries, the popularity of edible insects as an alternative animal protein source is increasing. Nevertheless, there is a lack of profound insight into the microbial safety and shelf life of living insects sold for human consumption. The purpose of this study was to characterise the microflora of fresh edible mealworm larvae and grasshoppers in a quantitative and qualitative way. Therefore, culture-dependent analyses (the total viable aerobic count, Enterobacteriaceae, lactic acid bacteria, yeasts and moulds, and bacterial endospores) and next-generation sequencing (454amplicon pyrosequencing) were performed. High microbial counts were obtained for both insect species. Different insect batches resulted in quite similar microbial numbers, except for bacterial endospores. However, the bacterial community composition differed between both insect species. The most abundant operational taxonomic unit in mealworm larvae was Propionibacterium. Also members of the genera Haemophilus, Staphylococcus and Clostridium were found. Grasshoppers were mainly dominated by Weissella, Lactococcus and Yersinia/Rahnella. Overall, a variety of potential spoilage bacteria and food pathogens were characterised. The results of this study suggest that a processing step with a microbiocidal effect is required to avoid or minimize risks involved with the consumption of edible insects.

Assessing genetic diversity among Brettanomyces yeasts by DNA fingerprinting and whole-genome sequencing.

ABSTRACT Brettanomyces yeasts, with the species Brettanomyces (Dekkera) bruxellensis being the most important one, are generally reported to be spoilage yeasts in the beer and wine industry due to the production of phenolic off flavors. However, B. bruxellensis is also known to be a beneficial contributor in certain fermentation processes, such as the production of certain specialty beers. Nevertheless, despite its economic importance, Brettanomyces yeasts remain poorly understood at the genetic and genomic levels. In this study, the genetic relationship between more than 50 Brettanomyces strains from all presently known species and from several sources was studied using a combination of DNA fingerprinting techniques. This revealed an intriguing correlation between the B. bruxellensis fingerprints and the respective isolation source. To further explore this relationship, we sequenced a (beneficial) beer isolate of B. bruxellensis (VIB X9085; ST05.12/22) and compared its genome sequence with the genome sequences of two wine spoilage strains (AWRI 1499 and CBS 2499). ST05.12/22 was found to be substantially different from both wine strains, especially at the level of single nucleotide polymorphisms (SNPs). In addition, there were major differences in the genome structures between the strains investigated, including the presence of large duplications and deletions. Gene content analysis revealed the presence of 20 genes which were present in both wine strains but absent in the beer strain, including many genes involved in carbon and nitrogen metabolism, and vice versa, no genes that were missing in both AWRI 1499 and CBS 2499 were found in ST05.12/22. Together, this study provides tools to discriminate Brettanomyces strains and provides a first glimpse at the genetic diversity and genome plasticity of B. bruxellensis.

Does Virulence Assessment of Vibrio anguillarum Using Sea Bass (Dicentrarchus labrax) Larvae Correspond with Genotypic and Phenotypic Characterization

Background Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays. Methodology/Principal Findings We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog’s Phenotype MicroArray™ technology and some virulence factor assays. Conclusions/Significance Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum.

Bacterial community dynamics during industrial malting, with an emphasis on lactic acid bacteria.

Characterization of the microflora during malting is an essential step towards process management and optimization. Up till now, however, microbial characterization in the malting process has mostly been done using culture-dependent methods, probably leading to biased estimates of microbial diversity. The aim of this study was to characterize the bacterial communities using two culture-independent methods, including Terminal Restriction Fragment Length Polymorphism (T-RFLP) and 454 pyrosequencing, targeting the 16S rRNA gene. Studied samples originated from two harvest years and two malting houses malting the same batch of barley. Besides targeting the entire bacterial community (T-RFLP), emphasis was put on lactic acid bacteria (LAB) (T-RFLP and 454 pyrosequencing). The overall bacterial community richness was limited, but the community structure changed during the process. Zooming in on the LAB community using 454 pyrosequencing revealed a total of 47 species-level operational taxonomic units (OTUs). LAB diversity appeared relatively limited since 88% of the sequences were covered by the same five OTUs (representing members of Weissella, Lactobacillus and Leuconostoc) present in all samples investigated. Fluctuations in the relative abundances of the dominant LAB were observed with the process conditions. In addition, both the year of harvest and malting house influenced the LAB community structure.

Microbial counts of mealworm larvae (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from different rearing companies and different production batches

The rising interest in insects for human consumption and the changing regulations in Europe require a profound insight into the food safety of insects reared and sold in Western society. The microbial quality of edible insects has only been studied occasionally. This study aimed at generating an overview of intrinsic parameters (pH, water activity and moisture content) and microbial quality of fresh mealworm larvae and crickets for several rearing companies and for several batches per rearer. In total, 21 batches obtained from 7 rearing companies were subjected to analysis of intrinsic parameters, a range of plate counts and presence-absence tests for Salmonella spp. and Listeria monocytogenes. The microbial counts of the fresh insects were generally high. Different rearing batches from a single rearing company showed differences in microbial counts which could not be explained by variations in intrinsic properties. The largest variations were found in numbers of bacterial endospores, psychrotrophs and fungi. Salmonella spp. and L. monocytogenes were not detected in any of the samples. Altogether, our study shows that large variations were found between batches from individual rearers. As a consequence, no overall differences between rearers could be observed.

Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains

Recent studies have suggested a correlation between genotype groups of Brettanomyces bruxellensis and their source of isolation. To further explore this relationship, the objective of this study was to assess metabolic differences in carbon and nitrogen assimilation between different B. bruxellensis strains from three beverages, including beer, wine, and soft drink, using Biolog Phenotype Microarrays. While some similarities of physiology were noted, many traits were variable among strains. Interestingly, some phenotypes were found that could be linked to strain origin, especially for the assimilation of particular α- and β-glycosides as well as α- and β-substituted monosaccharides. Based upon gene presence or absence, an α-glucosidase and β-glucosidase were found explaining the observed phenotypes. Further, using a PCR screen on a large number of isolates, we have been able to specifically link a genomic deletion to the beer strains, suggesting that this region may have a fitness cost for B. bruxellensis in certain fermentation systems such as brewing. More specifically, none of the beer strains were found to contain a β-glucosidase, which may have direct impacts on the ability for these strains to compete with other microbes or on flavor production.

Metagenetic analysis of the bacterial communities of edible insects from diverse production cycles at industrial rearing companies

Despite the continuing development of new insect-derived food products, microbial research on edible insects and insect-based foods is still very limited. The goal of this study was to increase the knowledge on the microbial quality of edible insects by comparing the bacterial community composition of mealworms (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from several production cycles and rearing companies. Remarkable differences in the bacterial community composition were found between different mealworm rearing companies and mealworm production cycles from the same company. In comparison with mealworms, the bacterial community composition of the investigated crickets was more similar among different companies, and was highly similar between both cricket species investigated. Mealworm communities were dominated by Spiroplasma and Erwinia species, while crickets were abundantly colonised by (Para)bacteroides species. With respect to food safety, only a few operational taxonomic units could be associated with potential human pathogens such as Cronobacter or spoilage bacteria such as Pseudomonas. In summary, our results implicate that at least for cricket rearing, production cycles of constant and good quality in terms of bacterial composition can be obtained by different rearing companies. For mealworms however, more variation in terms of microbial quality occurs between companies.

Assessing the xylanolytic bacterial diversity during the malting process

The presence of microorganisms producing cell wall hydrolyzing enzymes such as xylanases during malting can improve mash filtration behavior and consequently have potential for more efficient wort production. In this study, the xylanolytic bacterial community during malting was assessed by isolation and cultivation on growth media containing arabinoxylan, and identification by 16S rRNA gene sequencing. A total of 33 species-level operational taxonomic units (OTUs) were found, taking into account a 3% sequence dissimilarity cut-off, belonging to four phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) and 25 genera. Predominant OTUs represented xylanolytic bacteria identified as Sphingobacterium multivorum, Stenotrophomonas maltophilia, Aeromonas hydrophila and Pseudomonas fulva. DNA fingerprinting of all xylanolytic isolates belonging to S. multivorum obtained in this study revealed shifts in S. multivorum populations during the process. Xylanase activity was determined for a selection of isolates, with Cellulomonas flavigena showing the highest activity. The xylanase of this species was isolated and purified 23.2-fold by ultrafiltration, 40% ammonium sulfate precipitation and DEAE-FF ion-exchange chromatography and appeared relatively thermostable. This study will enhance our understanding of the role of microorganisms in the barley germination process. In addition, this study may provide a basis for microflora management during malting.

Microbial dynamics during production of lesser mealworms (Alphitobius diaperinus) for human consumption at industrial scale

In this study, the microbial dynamics during an industrial production cyle of lesser mealworms (Alphitobius diaperinus), sold for human consumption, were characterised. The microbial numbers as well as the microbial diversity were generally higher for the substrate, existing of remaining feed, faeces and exuviae, than for the larvae. Most of the species-level operational taxonomic units, identified using Illumina MiSeq sequencing, that were present in the feed were also detected in the larvae and vice versa. However, bacterial diversity decreased in the larvae during rearing. These results suggested that the feed is an important determinant of the insect bacterial community, but that some bacterial species show a competitive advantage inside the insect gut and become dominant. A blanching treatment of the larvae after harvest reduced most microbial counts, but the number of aerobic endospores remained at 4.0 log cfu/g. Whereas food pathogens Salmonella spp., Listeria monocytogenes, Bacillus cereus or coagulase-positive staphylococci were not detected in our study, fungal isolates corresponding to the genera Aspergillus and Fusarium were recovered. Therefore, it cannot be excluded that mycotoxins were present. The results of this study contribute to a better understanding of the microbial dynamics and food safety aspects during the production of edible insects.

Fermentation assays reveal differences in sugar and (off-) flavor metabolism across different Brettanomyces bruxellensis strains

Abstract Brettanomyces (Dekkera) bruxellensis is an ascomycetous yeast of major importance in the food, beverage and biofuel industry. It has been isolated from various man‐made ecological niches that are typically characterized by harsh environmental conditions such as wine, beer, soft drink, etc. Recent comparative genomics studies revealed an immense intraspecific diversity, but it is still unclear whether this genetic diversity also leads to systematic differences in fermentation performance and (off‐)flavor production, and to what extent strains have evolved to match their ecological niche. Here, we present an evaluation of the fermentation properties of eight genetically diverse B. bruxellensis strains originating from beer, wine and soft drinks. We show that sugar consumption and aroma production during fermentation are determined by both the yeast strain and composition of the medium. Furthermore, our results indicate a strong niche adaptation of B. bruxellensis, most clearly for wine strains. For example, only strains originally isolated from wine were able to thrive well and produce the typical Brettanomyces‐related phenolic off‐flavors 4‐ethylguaiacol and 4‐ethylphenol when inoculated in red wine. Sulfite tolerance was found as a key factor explaining the observed differences in fermentation performance and off‐flavor production. Sequence analysis of genes related to phenolic off‐flavor production, however, revealed only marginal differences between the isolates tested, especially at the amino acid level. Altogether, our study provides novel insights in the Brettanomyces metabolism of flavor production, and is highly relevant for both the wine and beer industry.