Determination of mycobiota and aflatoxin contamination in commercial bee pollen from eight provinces and one autonomous region of China.

The co-occurrence of fungi and mycotoxins in various foods has been frequently reported in many countries, posing a serious threat to the health and safety of consumers. In this study, the mycobiota in five types of commercial bee pollen samples from China were first revealed by DNA metabarcoding. Meanwhile, the content of total aflatoxins in each sample was investigated by high-performance liquid chromatography with fluorescence detection. The results demonstrated that Cladosporium (0.16 %-89.29 %) was the most prevalent genus in bee pollen, followed by Metschnikowia (0-81.12 %), unclassified genus in the phylum Ascomycota (0-81.13 %), Kodamaea (0-73.57 %), and Penicillium (0-36.13 %). Meanwhile, none of the assayed aflatoxins were determined in the 18 batches of bee pollen samples. In addition, the fungal diversity, community composition, and trophic mode varied significantly among five groups. This study provides comprehensive information for better understanding the fungal communities and aflatoxin residues in bee pollen from different floral origins in China.

Wild bee and pollen microbiomes across an urban-rural divide.

Wild pollinators and their microbiota are sensitive to land use changes from anthropogenic activities that disrupt landscape and environmental features. As urbanization and agriculture affect bee habitats, human-led disturbances are driving changes in bee microbiomes, potentially leading to dysbiosis detrimental to bee fitness. This study examines the bacterial, fungal, and plant compositions of the small carpenter bee, Ceratina calcarata, and its pollen provisions across an urban-rural divide. We performed metabarcoding of C. calcarata and provisions in Toronto by targeting the 16S rRNA, ITS, and rbcL regions. Despite similar plant composition and diversity across bees and their provisions, there was a greater microbial diversity in pollen provisions than in bees. By characterizing the differences in land use, climate, and pesticide residues that differentiate urban and rural landscapes, we find that urban areas support elevated levels of microbial diversity and more complex networks between microbes and plants than rural areas. However, urban areas may lead to lower relative abundances of known beneficial symbionts and increased levels of pathogens, such as Ascosphaera and Alternaria fungi. Further, rural pollen provisions indicate elevated pesticide residues that may dysregulate symbiosis. As anthropogenic activities continue to alter land use, ever changing environments threaten microbiota crucial in maintaining bee health.

Individual Dietary Specialization in a Generalist Bee Varies across Populations but Has No Effect on the Richness of Associated Microbial Communities.

AbstractDespite the increasingly documented occurrence of individual specialization, the relationship between individual consumer interactions and diet-related microbial communities in wild populations is still unclear. Using data from nests of Ceratina australensis from three different wild bee populations, we combine metabarcoding and network approaches to explore the existence of individual variation in resource use within and across populations and whether dietary specialization affects the richness of pollen-associated microbes. We reveal the existence of marked dietary specialization. In the most specialized population, we also show that individuals' diet breadth was positively related to the richness of fungi but not bacteria. Overall, individual specialization appeared to have a weak or negligible effect on the microbial richness of nests, suggesting that different mechanisms beyond environmental transmission may be at play regarding microbial acquisition in wild bees.

Do amino and fatty acid profiles of pollen provisions correlate with bacterial microbiomes in the mason bee Osmia bicornis?

Bee performance and well-being strongly depend on access to sufficient and appropriate resources, in particular pollen and nectar of flowers, which constitute the major basis of bee nutrition. Pollen-derived microbes appear to play an important but still little explored role in the plant pollen-bee interaction dynamics, e.g. through affecting quantities and ratios of important nutrients. To better understand how microbes in pollen collected by bees may affect larval health through nutrition, we investigated correlations between the floral, bacterial and nutritional composition of larval provisions and the gut bacterial communities of the solitary megachilid bee Osmia bicornis. Our study reveals correlations between the nutritional quality of pollen provisions and the complete bacterial community as well as individual members of both pollen provisions and bee guts. In particular pollen fatty acid profiles appear to interact with specific members of the pollen bacterial community, indicating that pollen-derived bacteria may play an important role in fatty acid provisioning. As increasing evidence suggests a strong effect of dietary fatty acids on bee performance, future work should address how the observed interactions between specific fatty acids and the bacterial community in larval provisions relate to health in O. bicornis. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Microbial communities associated with honey bees in Brazil and in the United States.

Honey bee colony losses worldwide call for a more in-depth understanding of the pathogenic and mutualistic components of the honey bee microbiota and their relation with the environment. In this descriptive study, we characterized the yeast and bacterial communities that arise from six substrates associated with honey bees: corbicular pollen, beebread, hive debris, intestinal contents, body surface of nurses and forager bees, comparing two different landscapes, Minas Gerais, Brazil and Maryland, United States. The sampling of five hives in Brazil and four in the USA yielded 217 yeast and 284 bacterial isolates. Whereas the yeast community, accounted for 47 species from 29 genera, was dominated in Brazil by Aureobasidium sp. and Candida orthopsilosis, the major yeast recovered from the USA was Debaryomyces hansenii. The bacterial community was more diverse, encompassing 65 species distributed across 31 genera. Overall, most isolates belonged to Firmicutes, genus Bacillus. Among LAB, species from Lactobacillus were the most prevalent. Cluster analysis evidenced high structuration of the microbial communities, with two distinguished microbial groups between Brazil and the United States. In general, the higher difference among sites and substrates were dependents on the turnover effect (~ 93% of the beta diversity), with a more pronounced effect of nestedness (~ 28%) observed from Brazil microbiota change. The relative abundance of yeasts and bacteria also showed the dissimilarity of the microbial communities between both environments. These results provide a comprehensive view of microorganisms associated with A. mellifera, highlighting the importance of the environment in the establishment of the microbiota associated with honey bees.

Transmitting silks of maize have a complex and dynamic microbiome.

In corn/maize, silks emerging from cobs capture pollen, and transmit resident sperm nuclei to eggs. There are > 20 million silks per U.S. maize acre. Fungal pathogens invade developing grain using silk channels, including Fusarium graminearum (Fg, temperate environments) and devastating carcinogen-producers (Africa/tropics). Fg contaminates cereal grains with mycotoxins, in particular Deoxynivalenol (DON), known for adverse health effects on humans and livestock. Fitness selection should promote defensive/healthy silks. Here, we report that maize silks, known as styles in other plants, possess complex and dynamic microbiomes at the critical pollen-fungal transmission interval (henceforth: transmitting style microbiome, TSM). Diverse maize genotypes were field-grown in two trial years. MiSeq 16S rRNA gene sequencing of 328 open-pollinated silk samples (healthy/Fg-infected) revealed that the TSM contains > 5000 taxa spanning the prokaryotic tree of life (47 phyla/1300 genera), including nitrogen-fixers. The TSM of silk tip tissue displayed seasonal responsiveness, but possessed a reproducible core of 7-11 MiSeq-amplicon sequence variants (ASVs) dominated by a single Pantoea MiSeq-taxon (15-26% of sequence-counts). Fg-infection collapsed TSM diversity and disturbed predicted metabolic functionality, but doubled overall microbiome size/counts, primarily by elevating 7-25 MiSeq-ASVs, suggestive of a selective microbiome response against infection. This study establishes the maize silk as a model for fundamental/applied research of plant reproductive microbiomes.

Composition and acquisition of the microbiome in solitary, ground-nesting alkali bees.

Increasing evidence suggests the microbiome plays an important role in bee ecology and health. However, the relationship between bees and their bacterial symbionts has only been explored in a handful of species. We characterized the microbiome across the life cycle of solitary, ground-nesting alkali bees (Nomia melanderi). We find that feeding status is a major determinant of microbiome composition. The microbiome of feeding larvae was similar to that of pollen provisions, but the microbiome of post-feeding larvae (pre-pupae) was similar to that of the brood cell walls and newly-emerged females. Feeding larvae and pollen provisions had the lowest beta diversity, suggesting the composition of larval diet is highly uniform. Comparisons between lab-reared, newly-emerged, and nesting adult females suggest that the hindgut bacterial community is largely shaped by the external environment. However, we also identified taxa that are likely acquired in the nest or which increase or decrease in relative abundance with age. Although Lactobacillus micheneri was highly prevalent in pollen provisions, it was only detected in one lab-reared female, suggesting it is primarily acquired from environmental sources. These results provide the foundation for future research on metagenomic function and development of probiotics for these native pollinators.

Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.

Deciphering the Impact of a Bacterial Infection on Meiotic Recombination in Arabidopsis with Fluorescence Tagged Lines.

Plants are under strong evolutionary pressure to maintain surveillance against pathogens. One major disease resistance mechanism is based on NB-LRR (NLR) proteins that specifically recognize pathogen effectors. The cluster organization of the NLR gene family could favor sequence exchange between NLR genes via recombination, favoring their evolutionary dynamics. Increasing data, based on progeny analysis, suggest the existence of a link between the perception of biotic stress and the production of genetic diversity in the offspring. This could be driven by an increased rate of meiotic recombination in infected plants, but this has never been strictly demonstrated. In order to test if pathogen infection can increase DNA recombination in pollen meiotic cells, we infected Arabidopsis Fluorescent Tagged Lines (FTL) with the virulent bacteria Pseudomonas syringae. We measured the meiotic recombination rate in two regions of chromosome 5, containing or not an NLR gene cluster. In all tested intervals, no significant difference in genetic recombination frequency between infected and control plants was observed. Although it has been reported that pathogen exposure can sometimes increase the frequency of recombinant progeny in plants, our findings suggest that meiotic recombination rate in Arabidopsis may be resilient to at least some pathogen attack. Alternative mechanisms are discussed.

Kluyveromyces osmophilus is not a synonym of Zygosaccharomyces mellis; reinstatement as Zygosaccharomyces osmophilus comb. nov.

Kluyveromyces osmophilus, a single-strain species isolated from Mozambique sugar, has been treated a synonym of Zygosaccharomyces mellis. Analyses of D1/D2 LSU rRNA gene sequences confirmed that the species belongs to the genus Zygosaccharomyces but showed it to be distinct from strains of Z. mellis. During studies of yeasts associated with stingless bees in Brazil, nine additional isolates of the species were obtained from unripe and ripe honey and pollen of Scaptotrigona cfr. bipunctata, as well as ripe honey of Tetragonisca angustula. The D1/D2 sequences of the Brazilian isolates were identical to those of the type strain of K. osmophilus CBS 5499 (=ATCC 22027), indicating that they represent the same species. Phylogenomic analyses using 4038 orthologous genes support the reinstatement of K. osmophilus as a member of the genus Zygosaccharomyces. We, therefore, propose the name Zygosaccharomyces osmophilus comb. nov. (lectotype ATCC 22027; MycoBank no. MB 833739).

Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing.

Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%-99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%-85.51%, 0.20%-29.11%, and 0.11%-32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.

Genomic analyses of Pseudomonas syringae pv. actinidiae isolated in Korea suggest the transfer of the bacterial pathogen via kiwifruit pollen.

Introduction. The bacterial pathogen, Pseudomonas syringae pv. actinidiae (Psa), has emerged as a major threat to kiwifruit cultivation throughout the world. One pandemic strain (from the Psa3 group) has occurred in various geographical regions. It is important to understand how this pathogen is being transmitted.Aim. Although Psa has been found in Korea since 1992, the isolates were until recently of a distinct type (Psa2). Recently, the more virulent Psa3 type has been detected. The purpose of this study was to describe the variety of Psa3 now found in Korea.Methodology. Strains were isolated from kiwifruit plants in Korea and from pollen imported into Korea from New Zealand. The genomes of 10 isolates were sequenced using the Illumina platform and compared to the completely assembled genomes of pandemic Psa3 strains from New Zealand and China. Comparisons were also made with pandemic strains from Chile and non-pandemic Psa3 isolates from China.Results. Six of the 10 Psa3 isolates from Korea show a clear relationship with New Zealand isolates. Two isolates show a distinct relationship to isolates from Chile; one further isolate has a sequence that is highly similar to that of M228, a strain previously isolated in China; and the last isolate belongs to the Psa3 group, but is not a member of the pandemic lineage.Conclusion. This analysis establishes that there have been multiple routes of transmission of the Psa3 pandemic strain into Korea. One route has involved the importation of pollen from New Zealand. A second route probably involves importation from Chile.

Selection of yeasts from bee products for alcoholic beverage production.

The use of appropriate yeast strains allows to better control the fermentation during beverage production. Bee products, especially of stingless bees, are poorly explored as sources of fermenting microorganisms. In this work, yeasts were isolated from honey and pollen from Tetragonisca angustula (Jataí), Nannotrigona testaceicornis (Iraí), Frieseomelitta varia (Marmelada), and honey of Apis mellifera bees and screened according to morphology, growth, and alcohol production. Bee products showed to be potential sources of fermenting microorganisms. From 55 isolates, one was identified as Papiliotrema flavescens, two Rhodotorula mucilaginosa, five Saccharomyces cerevisiae, and nine Starmerella meliponinorum. The S. cerevisiae strains were able to produce ethanol and glycerol at pH 4.0-8.0 and temperature of 10-30 °C, with low or none production of undesirable compounds, such as acetic acid and methanol. These strains are suitable for the production of bioethanol and alcoholic beverages due to their high ethanol production, similar or superior to the commercial strain, and in a broad range of conditions like as 50% (m/v) glucose, 10% (v/v) ethanol, or 500 mg L-1 of sodium metabisulfite.

Structural diversity and functional variability of gut microbial communities associated with honey bees.

Microbial consortia accompanied to all eukaryotes can be inherited from ancestors, environment, and/or from various food source. Gut microbiota study is an emerging discipline of biological sciences that expands our understanding of the ecological and functional dynamics of gut environments. Microorganisms associated with honey bees play an important role in food digestion, colony performance, immunity, pollination, antagonistic effect against different pathogens, amelioration of food and many more. Although, many repots about honey bee gut microbiota are well documented, microbiome with other key components of honey bees such as larvae, adults, their food (pollen, beebread, and honey), honey combs, and floral nectar are poorly understood. Mutual interactions and extent of the roles of microbial communities associated with honey bees are still unclear and demand for more research on the nutritional physiology and health benefits of this ecologically and economically important group. Here in this study, we highlighted all the honey bee microbiome that harbored from different life stages and other relevant components. The anatomical parts of honey bee (larvae, adults), food source (pollen, beebread, and honey), honey combs, and floral nectar were highly flourished by numerous microorganisms like bacteria (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Actinomycetes, Bacilli, Bacteroidetes, Cocci, Clostridia, Coliforms, Firmicutes, Flavobacteriia, Mollicutes) and fungi (Dothideomycetes, Eurotiomycetes, Mucormycotina, Saccharomycetes, Zygomycetes, Yeasts, Molds). Some distinctive microbial communities of a taxonomically constrained species have coevolved with social bees. This contribution is to enhance the understanding of honey bee gut microbiota, to accelerate bee microbiota and microbiome research in general and to aid design of future experiments in this growing field.

Omnivory in Bees: Elevated Trophic Positions among All Major Bee Families.

As pollen and nectar foragers, bees have long been considered strictly herbivorous. Their pollen provisions, however, are host to abundant microbial communities, which feed on the pollen before and/or while it is consumed by bee larvae. In the process, microbes convert pollen into a complex of plant and microbial components. Since microbes are analogous to metazoan consumers within trophic hierarchies, the pollen-eating microbes are, functionally, herbivores. When bee larvae consume a microbe-rich pollen complex, they ingest proteins from plant and microbial sources and thus should register as omnivores on the trophic "ladder." We tested this hypothesis by examining the isotopic compositions of amino acids extracted from native bees collected in North America over multiple years. We measured bee trophic position across the six major bee families. Our findings indicate that bee trophic identity was consistently and significantly higher than that of strict herbivores, providing the first evidence that omnivory is ubiquitous among bee fauna. Such omnivory suggests that pollen-borne microbes represent an important protein source for larval bees, which introduces new questions as to the link between floral fungicide residues and bee development.

Pollen-borne microbes shape bee fitness.

Teeming within pollen provisions are diverse communities of symbiotic microbes, which provide a variety of benefits to bees. Microbes themselves may represent a major dietary resource for developing bee larvae. Despite their apparent importance in sustaining bee health, evidence linking pollen-borne microbes to larval health is currently lacking. We examined the effects of microbe-deficient diets on the fitness of larval mason bees. In a series of diet manipulations, microbe-rich maternally collected pollen provisions were replaced with increasing fractions of sterilized, microbe-deficient pollen provisions before being fed to developing larvae. Convergent findings from amino acid and fatty acid trophic biomarker analyses revealed that larvae derived a substantial amount of nutrition from microbial prey and occupied a significantly higher trophic position than that of strict herbivores. Larvae feeding on increasingly sterile diets experienced significant adverse effects on growth rates, biomass and survivorship. When completely deprived of pollen-borne microbes, larvae consistently exhibited marked decline in fitness. We conclude that microbes associated with aged pollen provisions are central to bee health, not only as nutritional mutualists, but also as a major dietary component. In an era of global bee decline, the conservation of such bee-microbe interactions may represent an important facet of pollinator protection strategies.

Five-Year Data on Pollen Monitoring, Distribution and Health Impact of Allergenic Plants in Bucharest and the Southeastern Region of Romania.

Background and objectives: Respiratory allergies induced by allergenic pollen represent an important public health problem with increasing prevalence and severity in Europe. Romania has no aerobiology network and pollen measurements have been done for about ten years in the west region only. Materials and Methods: We established the first pollen monitoring center in the capital of Bucharest in 2013, based on collaboration with the Réseau National de Surveillance Aérobiologique (RNSA) from France. The aim of our paper is to present results from five years of pollen monitoring in the city center of Bucharest and preliminary data on distribution and health impact of some allergenic plants, mainly Ambrosia artemisiifolia, which is considered a real danger for the public health. Results: Our data show a significant atmospheric amount and a longer season than previously considered of grass (Gramineae) pollen and short period with a high level of Ambrosia pollen, while tree pollen looks less important in this area. The plant distribution data provided by specialists and information from affected persons showed the wide and increasing spread of Ambrosia in Bucharest and other cities from the south region. Preliminary health data from allergists confirmed that the number of patients with allergies to Ambrosia pollen is increasing from one year to another and almost all patients describe a high urban exposure from their living or working place. Conclusions: We consider that the recently implemented Law 62/2018 against Ambrosia may help reduce weed distribution and the atmospheric pollen load, but a more complex and coordinated strategy for controlling urban vegetation and reducing biologic pollution is needed.

Determination of microbiological contamination, antibacterial and antioxidant activities of natural plant hazelnut (Corylus avellana L.) pollen.

The aim of our study is to determine microbial contamination, antibacterial and antioxidant activities of 14 pollen samples of Corylus avellana collected from different locations in Slovakia. Microbiological analysis was carried out in two steps: microbiological assays and studies of antibacterial activity of pollen extracts. The antimicrobial properties of pollen extracts were carried out with the disc-diffusion method. Methanol (70%), ethanol (70%) and distilled water were used for pollen extracts. Five strains of bacteria such as gram-negative (Salmonella enterica subsp. enterica CCM 3807, Escherichia coli CCM 2024, and Yersinia enterocolitica CCM 5671) and gram-positive (Staphylococcus aureus CCM 2461 and Bacillus thuringiensis CCM 19T) were tested. Antioxidant activity of pollen extracts was determined by the DPPH method. Bacterial analysis includes the determination of the total bacterial count ranged from 4.08 to 4.61 log CFU g-1, mesophilic aerobic bacteria ranged from 3.40 to 4.89 log CFU g-1, mesophilic anaerobic bacteria ranged from 3.20 to 4.52 log CFU g-1, coliform bacteria ranged from 3.30 to 4.55 log CFU g-1, yeasts and filamentous fungi ranged from 3.00 to 3.56 log CFU g-1. Microscopic filamentous fungi Aspergillus spp., Alternaria spp., Penicillium spp., Cladosporium spp., Rhizopus spp., and Paecylomyces spp. were isolated from hazelnut pollen. Yersinia enterocolitica was the most sensitive strain among ethanolic and methanolic pollen hazelnut extracts. Staphylococcus aureus was the most sensitive strain against aqueous hazelnut pollen extracts. We determined the following sensitivity against ethanol pollen extracts respectively: Yersinia enterocolitica > Salmonella enterica > Staphylococcus aureus > Bacillus thuringiensis > Escherichia coli. Methanol pollen extracts had shown following sensitivity: Yersinia enterocolitica > Salmonella enterica > Escherichia coli > Staphylococcus aureus > Bacillus thuringiensis. Aqueous extracts had shown the following sensitivity: Staphylococcus aureus > Salmonella enterica > Escherichia coli > Bacillus thuringiensis > Yersinia enterocolitica. Hazelnut pollen extracts have over 82% antioxidant capacity in samples from non-urban zones. An elevated level of antioxidant potential in the pollen is determined by its biological properties conditioned by biologically active substances. DPPH method allowed characterizing pollen as a source of antioxidants.

Production and characterization of collagenase from a new Amazonian Bacillus cereus strain.

A new collagenase producing a strain of Bacillus cereus, isolated from the pollen of a bee of Amazon Region (Brazil), had its enzyme characterized and the production medium composition and culture conditions enhanced. A two-level design on three factors, namely initial medium pH, the substrate (gelatin) concentration and agitation intensity, allowed identifying the first two variables as the most significant ones, while a central composite design (CCD) was subsequently used to identify their optimal levels. Statistics highlighted maximized collagenolytic activity when substrate concentration and initial medium pH were selected at their highest levels (positive effects), whereas agitation intensity at the lowest (negative effect). Triplicate runs performed under predicted optimal conditions (pH 7.8 and 1.7% gelatin concentration) yielded a collagenolytic activity (305.39 ± 5.15 U) 4.6- to 15-fold those obtained with the preliminary design. The enzyme displayed optimum activity at 45 °C and pH 7.2, was stable over wide ranges of pH values and temperatures (7.2-11.0 and 25-50 °C, respectively) and was strongly inhibited by 10 mM phenylmethylsulphonyl fluoride. The zymogram showed two prominent bands at 50 and 76 kDa. These results are a first attempt to elucidate the features of this new collagenase, its production conditions, and possible scale-up.

Novel solid-state fermentation of bee-collected pollen emulating the natural fermentation process of bee bread.

Structure of lactic acid bacteria biota in ivy flowers, fresh bee-collected pollen (BCP), hive-stored bee bread, and honeybee gastrointestinal tract was investigated. Although a large microbial diversity characterized flowers and fresh BCP, most of lactic acid bacteria species disappeared throughout the bee bread maturation, giving way to Lactobacillus kunkeei and Fructobacillus fructosus to dominate long stored bee bread and honeybee crop. Adaptation of lactic acid bacteria was mainly related to species-specific, and, more in deep, to strain-specific features. Bee bread preservation seemed related to bacteria metabolites, produced especially by some L. kunkeei strains, which likely gave to lactic acid bacteria the capacity to outcompete other microbial groups. A protocol to ferment BCP was successfully set up, which included the mixed inoculum of selected L. kunkeei strains and Hanseniaspora uvarum AN8Y27B, almost emulating the spontaneous fermentation of bee bread. The strict relationship between lactic acid bacteria and yeasts during bee bread maturation was highlighted. The use of the selected starters increased the digestibility and bioavailability of nutrients and bioactive compounds naturally occurring in BCP. Our biotechnological protocol ensured a product microbiologically stable and safe. Conversely, raw BCP was more exposed to the uncontrolled growth of yeasts, moulds, and other bacterial groups.