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Aims This study evaluated pH reduction and microbial growth during fermentation of maize stover (MS) mixed with banana pseudostem (BPS) under South Ethiopian conditions. Materials and Results The MS and BPS were chopped and mixed into six treatments (T): 80% BPS plus 20% DMS (T1), 70% BPS plus 30% DMS (T2), 40% BPS plus 60% FMS (fresh maize stover) (T3), 20% BPS plus 80% FMS (T4), 100% FMS (T5), and 95% BPS plus 5% molasses (T6). At 0, 7, 14, 30, 60 and 90 days, pH and dry matter were determined. Microbiological quality was assessed using plate counts and Illumina MiSeq sequencing. On day 60 and 90, aerobic stability was investigated. The results showed a significant reduction in pH in all mixtures, except in T1 and T2. Lactic acid bacteria counts reached a maximum in all treatments within 14 days. Sequencing showed marked changes in dominant bacteria, such as Buttiauxella and Acinetobacter to Lactobacillus and Bifidobacterium. Conclusions The fresh maize stover and banana pseudostem mixtures and fresh maize showed significant pH reduction and dominance of desirable microbial groups. Significance and Impact of the study The study enables year-round livestock feed supplementation to boost milk and meat production in South Ethiopia. ispartof: Journal Of Applied Microbiology vol:132 issue:1 pages:1-14 ispartof: location:England status: published

Enset (Ensete ventricosum) is a drought tolerant food crop consumed in Ethiopia after fermentation into ‘kocho’. In most enset growing regions of Ethiopia, traditional starters are prepared in local households and added to the scraped enset mash to aid the fermentation. The preparation of the starters differs among ethnic groups. The aim of this study was twofold. The first objective was to obtain an overview of the preparation methods and the specific usage of the starters. To this end, a survey was conducted in eighteen districts (covering seven zones or ethnic groups) in southern Ethiopia. The second objective was to collect starter samples from these districts and characterize their microbial community based on sequencing of the V4 region of the 16S rRNA gene. The survey results showed that the main ingredients used to prepare the starters were generally the same within a zone but varied between zones. The collected starters had a pH ranging from 3.68 ± 0.16 to 5.62 ± 0.33. Plate counts showed that lactic acid bacteria were present in high numbers in samples collected from Amaro (9.07 ± 0.00 log cfu/g), Tocha (8.70 ± 0.50 log cfu/g) and Mareka (8.86 ± 0.60 log cfu/g) districts. Moreover, Enterobacteriaceae were identified in samples collected from Kochere (7.16 ± 0.30), Dale (7.08 ± 0.30), Shembedino (6.93 ± 0.60) and Wondeo Genet (6.36 ± 0.70) districts. Overall, Lactobacillus, Lactococcus and Leuconostoc were the dominant LAB genera identified in the starters. These isolates will be further considered for the development of a commercial enset starter culture.

Currently, there is a strong interest in barrel ageing of finished, conventionally fermented beers, as a novel way to produce sour beers with a rich and complex flavour profile. The production process, however, remains largely a process of trial and error, often resulting in profit losses and inconsistency in quality. To improve product quality and consistency, a better understanding of the interactions between microorganisms, wood and maturing beer is needed. The aim of this study was to describe the temporal dynamics in microbial community composition, beer chemistry and sensory characteristics during barrel ageing of three conventionally fermented beers that differed in parameters like alcohol content and bitterness. Beers were matured for 38 weeks in new (two types of wood) and used (one type of wood) oak barrels. Beer samples were taken at the start of the maturation and after 2, 12 and 38 weeks. Microbial community composition, determined using amplicon sequencing of the V4 region of the bacterial 16S rRNA gene and the fungal ITS1 region, beer chemistry and sensory characteristics substantially changed throughout the maturation process. Likewise, total bacterial and fungal population densities generally increased during maturation. PerMANOVA revealed significant differences in the bacterial and fungal community composition of the three beers and across time points, but not between the different wood types. By contrast, significant differences in beer chemistry were found across the different beers, wood types and sampling points. Results also indicated that the outcome of the maturation process likely depends on the initial beer properties. Specifically, results suggested that beer bitterness may restrain the bacterial community composition, thereby having an impact on beer souring. While the bacterial community composition of moderately-hopped beers shifted to a dominance of lactic acid bacteria, the bacterial community of the high-bitterness beer remained fairly constant, with low population densities. Bacterial community composition of the moderate-bitterness beers also resembled those of traditional sours like lambic beers, hosting typical lambic brewing species like Pediococcus damnosus, Lactobacillus brevis and Acetobacter sp. Furthermore, results suggested that alcohol level may have affected the fungal community composition and extraction of wood compounds. More specifically, the concentration of wood compounds like cis-3-methyl-4-octanolide, trans-3-methyl-4-octanolide, eugenol and total polyphenols was higher in beers with a high alcohol content. Altogether, our results provide novel insights into the barrel ageing process of beer, and may pave the way for a new generation of sour beers. ispartof: International Journal Of Food Microbiology vol:339 ispartof: location:Netherlands status: Published online

Specific processing steps after industrial rearing of insects for food and feed, being starvation and rinsing, are assumed to have an impact on their microbial quality. The aim of this study was to assess the effect on the microbiota of starvation (24 or 48h, 10 or 30°C) and rinsing (1min using tap water) at the end of the rearing period of yellow mealworm larvae (Tenebrio molitor). Microbial numbers were determined using plate counts and the microbial community composition using metagenetic analyses. Total viable counts ranged from 7.7 to 8.4logcfu/g for all treatments. Starvation did not evoke prominent shifts in the bacterial community, which was predominated by Proteobacteria and Firmicutes. No bacterial food pathogens were detected using metagenetics. Our data suggest that the processing steps under study do not contribute to a better microbial quality of fresh mealworm larvae.As insects and insect-based foods are receiving more attention and are already being marketed in some European countries, more insects farms are being established. Rearing companies often optimise their practices by trial and error and no general hygiene codes are available. According to the Netherlands Food and Consumer Product Safety Authority (2014), mealworm larvae are generally starved and rinsed after rearing to empty their gut, but the impact of these practices has not been investigated so far. Hence, the necessity for rearers to incorporate these steps in their rearing procedures has not been demonstrated. The Belgian SHC (Superior Health Council) and FASFC (Federal Agency for the Safety of the Food Chain) have recommended in their advice (2014) to investigate these steps. In addition, as edible insects will be defined as novel foods as from 1 January 2018 according to the European Novel Food Regulation (EU) No. 2015/2283, more information is needed on their safety, which is related to production hygiene. ispartof: Innovative Food Science & Emerging Technologies vol:42 pages:8-15 status: published

The purpose of this study was to investigate the effect of the production process as well as the storage conditions (air, 60% CO 2 /40% N 2 ) on the microbial counts and the bacterial community composition of a minced meat-like product from yellow mealworm larvae (YM) and from lesser mealworm larvae (LM). It was necessary to design a different production process for each larva type in order to obtain a minced meat-like product. Both production methods had an effect on the microbiota of the finished products, and that effect was extended during storage. Immediately after production, YM and LM showed aerobic counts between 1.4 and 2.3 log cfu/g and between 2.0 and 3.6 log cfu/g, respectively. The bacterial community composition differed between both products. The use of modified atmosphere during storage reduced bacterial growth compared to storage in air. In conclusion, the study points out that for the two insect types considered, it is possible to obtain a minced meat-like product with low microbial numbers and a potential shelf life that is attractive to retailers and consumers. Industrial relevance The consumption of traditional meat sources is under pressure due to its high environmental impact. Entomophagy (the consumption of insects by humans) can be one of the solutions to the globally increasing protein demand. However, in Western countries, some aversion for edible insects still exists. In those markets, people prefer the inclusion of insects in an invisible way into familiar food products. In order to stimulate the implementation of edible insects in the Western market, new insect-based products were developed that closely resemble a very popular meat product, being minced meat. Recent studies showed that fresh edible insects contain a high number of spoilage organisms and potential food pathogens. Therefore, monitoring the dynamics of the microbiota during preparation and storage of the new insect-based products is of utmost importance. We were able to produce end products with low microbial numbers that could be kept low during storage, especially by the use of modified atmosphere packaging.

In this study, lactic acid bacteria strains were screened for the development of (a) starter culture(s) specific for enset (Ensete ventricosum) fermentation. To this end, 158 isolates grown on de Man, Rogosa and Sharpe agar were obtained from fermenting enset and traditional starter material. They were screened based on their biochemical characteristics. Three promising strains, two Lactobacillus plantarum and one Leuconostoc mesenteroides, were selected and evaluated under field conditions. Aliquots of pulverized enset (without adding a liquid) were inoculated with one of the isolates at 3 log cfu/g and one uninoculated portion was used as control. Fermentation was performed in sauerkraut jars. Samples were taken on days 0, 7, 15, 30 and 75. On day 7 and onwards, the inoculated samples showed a larger pH reduction compared to the control. Enterobacteriaceae counts were reduced below the detectable level on day 7 and onwards. Clostridium spore counts were significantly lower for the inoculated than for the uninoculated samples on day 75. Inoculated enset showed a faster colonization of lactic acid bacteria than the uninoculated biomass. The three starters appeared to be suitable for enset fermentation. Further research is necessary to elucidate the proximate composition of enset fermented with these starters. ispartof: LWT-FOOD SCIENCE AND TECHNOLOGY vol:115 status: Published online

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. publisher: Elsevier articletitle: Microbial counts of mealworm larvae (Tenebrio molitor) and crickets (Acheta domesticus and Gryllodes sigillatus) from different rearing companies and different production batches journaltitle: International Journal of Food Microbiology articlelink: http://dx.doi.org/10.1016/j.ijfoodmicro.2016.11.007 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved. ispartof: International Journal of Food Microbiology vol:242 pages:13-18 ispartof: location:Netherlands status: published

AIMS: The present study was conducted to assess the effect of three different fermentation systems on fermentation of enset into kocho. METHODS AND RESULTS: Nine enset plants were processed, mixed and fermented in either a pit, a bamboo basket or a sauerkraut jar. Samples were taken on days 1, 7, 15, 31, 60 and 90. Moisture content and pH generally decreased and titratable acidity increased during fermentation. Total viable aerobic counts were generally high for all samples and Enterobacteriaceae counts were reduced to below the detectable level after day 1 for the pits and jars and after day 7 for the baskets. Illumina MiSeq sequencing of 16S ribosomal RNA genes revealed that Leuconostoc and Lactococcus spp. were the most abundant lactic acid bacteria in the initial phases of the fermentation. Later on, Lactobacillus, Weissella and Bifidobacterium dominated. CONCLUSIONS: The type of fermentation system used had an effect on the microbial dynamics and the effect increased towards the end of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Millions of people in Ethiopia daily consume kocho prepared in either a pit or a basket. These systems show practical problems, but this study shows that fermentation is also possible in a sauerkraut jar. ispartof: JOURNAL OF APPLIED MICROBIOLOGY vol:126 issue:3 pages:842-853 ispartof: location:England status: published

© 2018 Elsevier Ltd The attractiveness of fresh edible insects might be enhanced by increasing their shelf life and taste. Two processes that have never been documented in the scientific literature for mealworms as such but that may achieve these goals are marination and fermentation. The objective of this study was to apply both processes on larvae of the yellow mealworm (Tenebrio molitor) and to monitor pH and microbial numbers during and after (for marination) the treatment. Blanched larvae were marinated for 6 days in either red wine or soy sauce. The total aerobic and spore counts increased with approximately 1 log cfu/g during marination, but during subsequent chilled storage for 17 days there was no microbial growth. Total viable counts were maximally 3.8 ± 0.1 log cfu/g for all samples. In contrast, a rapid increase of the total count to >7.0 log cfu/g was observed after 10–12 days for larvae that were only blanched. Therefore, marination can extend the shelf life for (at least) 7 days. Fermentation of crushed larvae with the starter Bactoferm F-LC and supplemented with 2.8% NaCl (w/w), 0.75% D(+)-glucose (w/w) and 0.015% NaNO2 (w/w) resulted in fast acidification and was effective inhibiting bacterial endospores and sulphite reducing clostridia. ispartof: Food Control vol:92 pages:47-52 status: published

In this study, the microbiota during industrial rearing, processing, and storage of the edible tropical house cricket, Gryllodes sigillatus , was investigated. To this end, we analyzed samples from the cricket feed, obtained before feeding as well as from the cages, and from the crickets during rearing, after harvest, and after processing into frozen, oven-dried, and smoked and oven-dried (smoked/dried) end products. Although the feed contained lower microbial numbers than the crickets, both were dominated by the same species-level operational taxonomic units, as determined by Illumina MiSeq sequencing. They corresponded, among others, to members of Porphyromonadaceae , Fusobacterium , Parabacteroides , and Erwinia . The harvested crickets contained high microbial numbers, but none of the investigated food pathogens Salmonella spp., Listeria monocytogenes , Bacillus cereus , or coagulase-positive staphylococci. However, some possible mycotoxin-producing fungi were isolated from the crickets. A postharvest heat treatment, shortly boiling the crickets, reduced microbial numbers, but an endospore load of 2.4 log CFU/g remained. After processing, an increase in microbial counts was observed for the dried and smoked/dried crickets. Additionally, in the smoked/dried crickets, a high abundance of a Bacillus sp. was observed. Considering the possible occurrence of food-pathogenic species from this genus, it is advised to apply a heat treatment which is sufficient to eliminate spores. Nevertheless, the microbial numbers remained constant over a 6-month storage period, whether frozen (frozen end product) or at ambient temperature (oven-dried and smoked/dried end products). IMPORTANCE The need for sustainable protein sources has led to the emergence of a new food sector, producing and processing edible insects into foods. However, insight into the microbial quality of this new food and into the microbial dynamics during rearing, processing, and storage of edible insects is still limited. Samples monitored for their microbiota were obtained in this study from an industrial rearing and processing cycle. The results lead first to the identification of process steps which are critical for microbial food safety. Second, they can be used in the construction of a Hazard Analysis and Critical Control Points (HACCP) plan and of a Novel Food dossier, which is required in Europe for edible insects. Finally, they confirm the shelf-life period which was determined by the rearer.

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.

Comparative analyses of bacterial and archaeal community structures and dynamics in three biogas digesters during start-up and subsequent operation using microwaved, ultrasonicated or untreated waste activated sludge were performed based on 454 pyrosequencing datasets of part of 16S ribosomal RNA sequences and quantitative PCR. The pre-treatment increased the solubility, and thus the availability of the substrate for microbial degradation and significantly affected the succession of the anaerobic community structure over the course of the digestion. Bacteroidetes, Proteobacteria and Firmicutes were the dominant phyla in all digesters throughout operation. Proteobacteria decreased in relative abundance from 23-26 % to 11-13 % in association with enhanced substrate availability. Negative correlations between relative abundance of Alpha-, Beta- and Gammaproteobacteria and the substrate availability and/or biogas production were disclosed in statistical analyses. Clostridiales was the dominant order in Firmicutes, and Clostridiales, Clostridia and Firmicutes relative abundance and richness were shown to positively correlate with substrate availability and biogas generation. Methanogenic communities had a fairly restricted structure, highly dominated by Methanosaeta and Methanobrevibacter phylotypes. A gradual decline in Methanobrevibacter and increased representation of Methanosaeta concilii over time were particularly apparent in the digester receiving untreated waste activated sludge, whereas more diversified archaeal communities were maintained in the pre-treatment digesters. The quantitative PCR analyses revealed a methanogenic community distribution that coincided with the 454 pyrosequencing data.

Enset (Ensete ventricosum) provides staple food for 15 million people in Ethiopia after fermentation into kocho. The fermentation process has hardly been investigated and is prone to optimization. The aim of this study was to investigate the physicochemical and microbial dynamics of fermentation practices in the Gamo highlands. These practices show local variation, but two steps were omnipresent: scraping of the pseudostem and fermenting it in a pit or a bamboo basket. Enset plants were fragmented and fermented for two months in order to investigate the physicochemical (temperature, moisture content, pH and titratable acidity) and microbial dynamics (total viable aerobic counts, counts of Enterobacteriaceae, lactic acid bacteria, yeasts and moulds and Clostridium spores counts, and Illumina Miseq sequencing). Samples were taken on days 1, 7, 15, 17, 31 and 60. The pH decreased, whereas the titratable acidity increased during fermentation. Of all counts those of lactic acid bacteria and Clostridium spores increased during fermentation. Leuconostoc mesenteroides initiated the fermentation. Later on, Prevotella paludivivens, Lactobacillus sp. and Bifidobacterium minimum dominated. These three species are potential candidates for the development of a starter culture. ispartof: FOOD MICROBIOLOGY vol:73 pages:342-350 ispartof: location:England status: published

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.

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. ispartof: International Journal of Food Microbiology vol:261 pages:11-18 ispartof: location:Netherlands status: published

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 .

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.

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.