Liquid sourdough from stone-ground soft wheat (Triticum aestivum) flour: Development and exploitation in the breadmaking process.

The aim of the study was to set up a liquid sourdough obtained using stone-ground soft wheat (Triticum aestivum) flour to be exploited in breadmaking. Therefore, a Type II sourdough (dough yield = 350) was developed from a stable stone-ground wheat Type I sourdough (dough yield = 156) used as inoculum. Both sourdoughs were analyzed for lactic acid bacteria (LAB) viable counts, pH and total titratable acidity (TTA), LAB biodiversity by a combined culture-dependent and -independent approach (PCR-DGGE) and they were tested for their breadmaking ability. In addition, the chemical and rheological features and volatile organic compounds of the stone-ground soft wheat flour used in the experiment were investigated. The flour had a high protein content, good bakery properties and it also presented a rich aroma pattern characterized not only by the prevalence of green grass, flowery, and sweet aromas but also nutty, roasted and popcorn aromas. The sourdoughs I and II used in the trial were characterized by viable LAB counts, pH and TTA values typical of mature sourdoughs, i.e., approximately 9 log cfu gr-1 and mL, pH 3.9 and 10 mL 0.1 N NaOH. In addition, Levilactobacillus brevis and Companilactobacillus paralimentarius species represented the LAB stable microbiota of both sourdoughs. Both sourdoughs efficiently produce acceptable experimental breads characterized by different volatile profiles thus indicating that the type of sourdough fermentation significantly influenced the features of the final products. Overall, for the first time in the present study stone-ground wheat flour and bread have been characterized for their volatile aroma profile and sensory properties.

Effect of inoculated azotobacteria and Phanerochaete chrysosporium on the composting of olive pomace: Microbial community dynamics and phenols evolution.

The effect of inoculated azotobacteria and basidiomycetes white-rot fungi on the population dynamics of bacteria and eumycetes during the co-composting of olive mill pomace and wheat straw was evaluated by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis combined with sequencing of rRNA gene amplicons from selected DGGE bands. The evolution of pH, temperature, phytotoxicity and water-soluble phenol content during co-composting was also monitored. In general, a similar evolution of microbial biodiversity was seen in both the inoculated and uninoculated (control) piles, which was in keeping with a similar evolution of phytotoxicity and water-soluble phenol content. Overall, under the conditions applied, data suggest a marginal influence of the inoculated starters on the physical, chemical and microbiological properties of compost piles, with the resident microbiota playing a major role.

Profiling white wine seed vinegar bacterial diversity through viable counting, metagenomic sequencing and PCR-DGGE.

The production of traditional vinegar is usually carried out using the so-called "seed vinegar" or "mother of vinegar" that is composed of an undefined and complex pool of microorganisms deriving from a previous vinegar production. To date, there have been relatively few studies on the microbiota of seed vinegars. The present study was carried out to discover the bacterial biota of seed vinegar samples used in the homemade production of local vinegars obtained from the acetic fermentation of white wine. The seed vinegar samples were subjected to viable counting and advanced molecular analyses, namely, Illumina sequencing and PCR-DGGE. The adopted polyphasic approach allowed the bacterial diversity of the analyzed samples to be profiled, thus revealing the presence of acetic acid bacteria ascribed to the genera Acetobacter, Gluconacetobacter, Gluconobacter and Komagataeibacter. Moreover, other microbial genera as Pseudomonas, Bacillus and Clostridium were abundantly found in almost all the samples, together with other minority genera. The results of viable counting confirmed the well-acknowledged limitations inherent with acetic acid bacteria recovery on plate growth media. The overall results confirmed that seed vinegars have a complex and heterogeneous biodiversity, thus encouraging their exploitation for the isolation and future technological characterization of cultures to be selected for the manufacture of mixed starter cultures.

Microbial dynamics of model Fabriano-like fermented sausages as affected by starter cultures, nitrates and nitrites.

The present study promotes the valorization of Fabriano-like fermented sausages, which are central-Italy salami with an origin that dates to the early 17th century, for the possible future selection of autochthonous starter cultures to be used with respect to local traditions. To the best of the authors' knowledge, this study represents the first attempt to define the microbial dynamics in Fabriano-like fermented sausage and the effect of nitrates/nitrites and starter cultures on its natural bacterial biota. Culture and RNA-based techniques (RT-PCR-DGGE and Illumina sequencing) were used to assess the microbial ecology of model Fabriano-like fermented sausages together with the impact of starter cultures and different nitrate and nitrite concentrations. The meat batter was used to produce two batches of fermented sausages that were prepared as follows: i) without commercial starters or ii) with the use of starter cultures composed of Pediococcus pentosaceus and Staphylococcus xylosus. Each batch was further divided into three different batches with the addition of 0/0 mg kg-1 nitrate/nitrite, 75/60 mg kg-1 nitrate/nitrite and 150/125 mg kg-1 nitrate/nitrite to the first, second and third batch, respectively. The samples, which were produced in triplicate, were analyzed on the day of production and after 7, 21, and 42 days of ripening. Enterobacteriaceae counts were always higher in model Fabriano-like sausages produced without the use of starter cultures at all of the sampling times irrespective of the tested nitrate/nitrite concentrations. Lactobacilli counts were positively influenced by the starters, although this influence was not evident over time; moreover, the effect of nitrates and nitrites on the counts of lactobacilli differed over time. As a general trend, coagulase-negative cocci counts were apparently not influenced by the tested nitrate/nitrite concentrations. Regarding the effect of nitrates/nitrites on the microbial diversity revealed by RT-PCR-DGGE, the higher the concentration, the lower the presence of some genera/species such as Pseudomonas spp., Serratia liquefaciens and Staphylococcus spp. However, Illumina sequencing detected Pseudomonas spp. as a minority species after 7, 21 and 42 days of ripening irrespective of the nitrate/nitrite concentration. The presence of Staphylococcus species was highlighted by both RT-PCR-DGGE and Illumina sequencing at all of the stages of ripening, although its presence was massively detected in fermented sausages produced without the use of nitrates/nitrites at the end of ripening. Overall, the data collected clearly highlighted the dominance of Lactobacillus sakei in all of the fermented sausages during ripening (from day 7 to day 42) and irrespective of the nitrate/nitrite concentration and added starter cultures. Moreover, Pediococcus spp. was principally detected in model Fabriano-like fermented sausage with added starter cultures irrespective of the nitrate/nitrite concentration.

Impact of thistle rennet from Carlina acanthifolia All. subsp. acanthifolia on bacterial diversity and dynamics of a specialty Italian raw ewes' milk cheese.

Caciofiore della Sibilla is an Italian specialty soft cheese manufactured with Sopravissana raw ewes' milk and thistle rennet prepared with young fresh leaves and stems of Carlina acanthifolia All. subsp. acanthifolia, according to an ancient tradition deeply rooted in the territory of origin (mountainous hinterland of the Marche region, Central Italy). In this study, the impact of thistle rennet on the bacterial dynamics and diversity of Caciofiore della Sibilla cheese was investigated by applying a polyphasic approach based on culture and DNA-based techniques (Illumina sequencing and PCR-DGGE). A control cheese manufactured with the same batch of ewes' raw milk and commercial animal rennet was analyzed in parallel. Overall, a large number of bacterial taxa were identified, including spoilage, environmental and pro-technological bacteria, primarily ascribed to Lactobacillales. Thistle rennet was observed clearly to affect the early bacterial dynamics of Caciofiore della Sibilla cheese with Lactobacillus alimentarius/paralimentarius and Lactobacillus plantarum/paraplantarum/pentosus being detected in the phyllosphere of C. acanthifolia All., thistle rennet and curd obtained with thistle rennet. Other bacterial taxa, hypothetically originating from the vegetable coagulant (Enterococcus faecium, Lactobacillus brevis, Lactobacillus delbrueckii, Leuconostoc mesenteroides/pseudomesenteroides), were exclusively found in Caciofiore della Sibilla cheese by PCR-DGGE. At the end of the maturation period, Illumina sequencing demonstrated that both cheeses were dominated by Lactobacillales; however curd and cheese produced with thistle rennet were co-dominated by Lactobacillus and Leuconostoc, whereas Lactoccous prevailed in curd and cheese produced with commercial animal rennet followed by Lactobacillus. Differences in the bacterial composition between the two cheeses at the end of their maturation period were confirmed by PCR-DGGE analysis.

Insight into the bacterial diversity of fermentation woad dye vats as revealed by PCR-DGGE and pyrosequencing.

The bacterial diversity in fermenting dye vats with woad (Isatis tinctoria L.) prepared and maintained in a functional state for approximately 12 months was examined using a combination of culture-dependent and -independent PCR-DGGE analyses and next-generation sequencing of 16S rRNA amplicons. An extremely complex ecosystem including taxa potentially contributing to both indigo reduction and formation, as well as indigo degradation was found. PCR-DGGE analyses revealed the presence of Paenibacillus lactis, Sporosarcina koreensis, Bacillus licheniformis, and Bacillus thermoamylovorans, while Bacillus thermolactis, Bacillus pumilus and Bacillus megaterium were also identified but with sequence identities lower than 97%. Dominant operational taxonomic units (OTUs) identified by pyrosequencing included Clostridium ultunense, Tissierella spp., Alcaligenes faecalis, Erysipelothrix spp., Enterococcus spp., Virgibacillus spp. and Virgibacillus panthothenicus, while sub-dominant OTUs included clostridia, alkaliphiles, halophiles, bacilli, moderately thermophilic bacteria, lactic acid bacteria, Enterobacteriaceae, aerobes, and even photosynthetic bacteria. Based on the current knowledge of indigo-reducing bacteria, it is considered that indigo-reducing bacteria constituted only a small fraction in the unique microcosm detected in the natural indigo dye vats.

Transferable Antibiotic Resistances in Marketed Edible Grasshoppers (Locusta migratoria migratorioides).

Grasshoppers are the most commonly eaten insects by humans worldwide, as they are rich in proteins and micronutrients. This study aimed to assess the occurrence of transferable antibiotic resistance genes in commercialized edible grasshoppers. To this end, the prevalence of 12 selected genes [aac(6')-Ie aph(2″)-Ia, blaZ, erm(A), erm(B), erm(C), mecA, tet(M), tet(O), tet(S), tet(K), vanA, vanB] coding for resistance to antibiotics conventionally used in clinical practice was determined. The majority of samples were positive for tet(M) (70.0%), tet(K) (83.3%) and blaZ (83.3%). A low percentage of samples were positive for erm(B) (16.7%), erm(C) (26.7%), and aac(6')-Ie aph(2″)-Ia (13.3%), whereas no samples were positive for erm(A), vanA, vanB, tet(O), and mecA. Cluster analysis identified 4 main clusters, allowing a separation of samples on the basis of their country of origin.

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing.

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota.

The occurrence of spoilage yeasts in cream-filled bakery products.

BACKGROUND: Filling creams can provide an adequate substrate for spoilage yeasts because some yeasts can tolerate the high osmotic stress in these products. To discover the source of spoilage of a cream-filled baked product, end products, raw materials, indoor air and work surfaces were subjected to microbiological and molecular analyses. The efficacy of disinfectants against spoilage yeasts was also assessed. RESULTS: The analyses on end products revealed the presence of the closest relatives to Zygosaccharomyces bailii with counts ranging from 1.40 to 4.72 log cfu g-1 . No spoilage yeasts were found in the indoor air and work surfaces. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis, carried out directly on filling creams collected from unopened cans, showed the presence of bands ascribed to the closest relatives to Z. bailii sensu lato, although with counts < 1 log cfu g-1 . Susceptibility testing of yeast isolates to disinfectants showed a significantly lower effect of 10% alkyl dimethyl benzyl ammonium chloride. Different responses of isolates to the tested disinfectants were seen. CONCLUSION: To guarantee the quality of end products, reliable and sensitive methods must be used. Moreover, hygiene and the application of good manufacturing practices represent the most efficient way for the prevention and minimization of cross-contamination. © 2016 Society of Chemical Industry.

Microbial Diversity of Type I Sourdoughs Prepared and Back-Slopped with Wholemeal and Refined Soft (Triticum aestivum) Wheat Flours.

The fermentation of type I sourdough was studied for 20 d with daily back-slopping under laboratory and artisan bakery conditions using 1 wholemeal and 2 refined soft wheat (Triticum aestivum) flours. The sourdough bacterial and yeast diversity and dynamics were investigated by plate counting and a combination of culture-dependent and culture-independent PCR-DGGE approach. The pH, total titrable acidity, and concentration of key organic acids (phytic, lactic, and acetic) were measured. Three flours differed for both chemical and rheological properties. A microbial succession was observed, with the atypical sourdough species detected at day 0 (i.e. Lactococcus lactis and Leuconostoc holzapfelii/citreum group for bacteria and Candida silvae and Wickerhamomyces anomalus for yeasts) being progressively replaced by taxa more adapted to the sourdough ecosystem (Lactobacillus brevis, Lactobacillus alimentarius/paralimentarius, Saccharomyces cerevisiae). In mature sourdoughs, a notably different species composition was observed. As sourdoughs propagated with the same flour at laboratory and artisan bakery level were compared, the influence of both the substrate and the propagation environment on microbial diversity was assumed.

Getting insight into the prevalence of antibiotic resistance genes in specimens of marketed edible insects.

This study was aimed at investigating the occurrence of 11 transferable antibiotic resistance (AR) genes [erm(A), erm(B), erm(C), vanA, vanB, tet(M), tet(O), tet(S), tet(K), mecA, blaZ] in 11 species of marketed edible insects (small crickets powder, small crickets, locusts, mealworm larvae, giant waterbugs, black ants, winged termite alates, rhino beetles, mole crickets, silkworm pupae, and black scorpions) in order to provide a first baseline for risk assessment. Among the AR genes under study, tet(K) occurred with the highest frequency, followed by erm(B), tet(S) and blaZ. A high variability was seen among the samples, in terms of occurrence of different AR determinants. Cluster Analysis and Principal Coordinates Analysis allowed the 11 samples to be grouped in two main clusters, one including all but one samples produced in Thailand and the other including those produced in the Netherlands.

Yeast and mould dynamics in Caciofiore della Sibilla cheese coagulated with an aqueous extract of Carlina acanthifolia All.

Caciofiore della Sibilla is a speciality ewes' milk cheese traditionally manufactured in a foothill area of the Marche region (Central Italy) with a crude extract of fresh young leaves of Carlina acanthifolia All. subsp. acanthifolia as a coagulating agent. The fungal dynamics and diversity of this speciality cheese were investigated throughout the manufacturing and 20-day ripening process, using a combined PCR-DGGE approach. The fungal biota of a control ewes' milk cheese, manufactured with the same batch of milk coagulated with a commercial animal rennet, was also monitored by PCR-DGGE, in order to investigate the contribution of the peculiar vegetable coagulant to the fungal diversity and dynamics of the cheese. Based on the overall results collected, the raw milk and the dairy environment represented the main sources of fungal contamination, with a marginal or null contribution of thistle rennet to the fungal diversity and dynamics of Caciofiore della Sibilla cheese. Copyright © 2016 John Wiley & Sons, Ltd.

The Occurrence of Beer Spoilage Lactic Acid Bacteria in Craft Beer Production.

Beer is one of the world's most ancient and widely consumed fermented alcoholic beverages produced with water, malted cereal grains (generally barley and wheat), hops, and yeast. Beer is considered an unfavorable substrate of growth for many microorganisms, however, there are a limited number of bacteria and yeasts, which are capable of growth and may spoil beer especially if it is not pasteurized or sterile-filtered as craft beer. The aim of this research study was to track beer spoilage lactic acid bacteria (LAB) inside a brewery and during the craft beer production process. To that end, indoor air and work surface samples, collected in the brewery under study, together with commercial active dry yeasts, exhausted yeasts, yeast pellet (obtained after mature beer centrifugation), and spoiled beers were analyzed through culture-dependent methods and PCR-DGGE in order to identify the contaminant LAB species and the source of contamination. Lactobacillus brevis was detected in a spoiled beer and in a commercial active dry yeast. Other LAB species and bacteria ascribed to Staphylococcus sp., Enterobaceriaceae, and Acetobacter sp. were found in the brewery. In conclusion, the PCR-DGGE technique coupled with the culture-dependent method was found to be a useful tool for identifying the beer spoilage bacteria and the source of contamination. The analyses carried out on raw materials, by-products, final products, and the brewery were useful for implementing a sanitization plan to be adopted in the production plant.

Screening of yeasts for growth on crude glycerol and optimization of biomass production.

Out of 113 yeast strains tested, 45 grew on pure glycerol with growth rates ranging from 0.11 to 0.37h(-1). Twenty-three strains showed specific growth rates (h(-1)), biomass production and biomass yields higher or comparable to those on glucose which suggests that crude glycerol can be utilized as carbon source in yeast cultivation for biomass production. Response surface methodology was applied to optimize crude glycerol concentration and temperature for biomass production and yield by Yarrowia lipolytica (DiSVA C 12.1), Metschnikowia sp. (DiSVA 50), Debaryomyces sp. (DiSVA 45/9), and Rhodotorula mucilaginosa (DiSVA C 7.1). A biomass concentration of 25.7g/l and a biomass yield of 0.92g/g (Y/Xglyc) was obtained with Y. lipolytica DiSVA C 12.1 and with R. mucilaginosa DiSVA C 7.1, respectively. These results demonstrate the potential use of crude glycerol as carbon source in yeast cultivation and the yeast ability to convert low-value crude glycerol to added-value products.

Use of Pichia fermentans and Candida sp. strains for the biological treatment of stored olive mill wastewater.

Of 105 isolates screened for growth on plates containing olive mill wastewater (OMW), five were selected and identified as Pichia fermentans (Y1, Y4) and Candida sp. (Y2, Y11, and Y18). On the basis of their ability to use phenol at 716 mg l(-1), strains Y2 (15% reduction) and Y4 (18% reduction) were then used to detoxify stored OMW under various operational conditions. Yeast treatment of OMW increased the pH and, in the best conditions (aeration and no glucose addition), the COD decreased (47%) and phytotoxicity was also decreased (56%) probably due to the changes in the composition of phenolic compounds.

Fungicides degradation in an organic biomixture: impact on microbial diversity.

Biological systems are being developed all over EU countries to protect water-bodies from pesticide contamination at farm level. A laboratory experiment was carried out to test the efficiency of a mixture of compost and straw in bio-degrading different mixtures of fungicides usually applied in vineyards. At the same time the effects of fungicide applications on microbial community of biomixture were also evaluated. Results showed that the biomixture had a good capability of degrading pesticides. Indeed, at the end of the experiment (112 days), the concentration of most of the pesticides was close to complete degradation. Denaturing gradient gel electrophoresis (DGGE) analysis showed an evident modification of microbial diversity after the addition of fungicides. However, at the end of degradation process, no significant changes in the composition of microbial community were seen. In this specific substrate used in the biomixture, yeast flora and ascomycete filamentous fungi seemed to be involved in the degradation activity.

Yeast diversity during tapping and fermentation of palm wine from Cameroon.

In the present study, we have investigated the occurrence of yeast flora during tapping and fermentation of palm wine from Cameroon. The yeast diversity was investigated using both traditional culture-dependent and culture-independent methods. Moreover, to characterize the isolates of the predominant yeast species (Saccharomyces cerevisiae) at the strain level, primers specific for delta sequences and minisatellites of genes encoding the cell wall were used. The results confirm the broad quantitative presence of yeast, lactic acid bacteria and acetic acid bacteria during the palm wine tapping process, and highlight a reduced diversity of yeast species using both dependent and independent methods. Together with the predominant species S. cerevisiae, during the tapping of the palm wine the other species found were Saccharomycodes ludwigii and Zygosaccharomyces bailii. In addition, denaturing gradient gel electrophoresis (DGGE) analysis detected Hanseniaspora uvarum, Candida parapsilopsis, Candida fermentati and Pichia fermentans. In contrast to the progressive simplification of yeast diversity at the species level, the molecular characterization of the S. cerevisiae isolates at the strain level showed a wide intraspecies biodiversity during the different steps of the tapping process. Indeed, 15 different biotypes were detected using a combination of three primer pairs, which were well distributed in all of the samples collected during the tapping process, indicating that a multistarter fermentation takes place in this particular natural, semi-continuous fermentation process.

Yeast diversity in crop-growing environments in Cameroon.

In the present study, we have investigated the occurrence of yeast flora on several agricultural products coming from crop-growing environments in Cameroon, to provide better knowledge of the biodiversity of yeast flora, and to thus define the impact of this biodiversity on food products. The yeast biodiversity was investigated using traditional culture-dependent methods, along with culture-independent methods. The culture-dependent approach was carried out using both direct and enrichment procedures, to detect the broadest possible presence of yeast species. A total of 151 strains belonging to 26 different yeast species were isolated and identified using restriction pattern analysis of the internal transcribed spacer region 5.8S-ITS and sequence analysis of D1/D2 domain of 26S rRNA gene. The enrichment isolation procedures carried out in high-sugar media allowed the recognition of fermentative species such as Saccharomyces cerevisiae and Torulaspora delbrueckii, which have previously not been detected using direct isolation methodology. The results of culture-independent method using DGGE patterns and sequencing of the DNA bands revealed a lower number of yeast species when compared with the culture-dependent methodology even if the identification of several yeast species not detected by traditional microbiological procedures such as Candida tropicalis and Hanseniaspora uvarum is allowed. Thus, these multiphasic approaches to study yeast biodiversity (culture-dependent and -independent methods) have allowed us to get a more complete picture of the microbial diversity in these natural environments.