Evaluation of the inhibitory activity of essential oils against spoilage yeasts and their potential application in yogurt.

Yeasts are the leading cause of spoilage in yogurt. Considering the high demand from consumers to use natural products as an alternative to additives, essential oils (EOs) could be a promising solution to guarantee high microbiological standards. The present study highlighted the in vitro antifungal potential of cinnamon, ginger, lemongrass, mandarin, orange, lemon and lime EOs against spoilage yeasts isolated from yogurts prepared with pasteurized buffalo milk. A total of 74 isolates represented by 14 different species of Candida, Rhodotorula, Debaryomyces, Kluyveromyces and Yarrowia genera were subjected to a disc diffusion assay, showing lemongrass EO to have the highest antifungal activity (40.97 ± 9.86 mm), followed by cinnamon (38.46 ± 6.59 mm) and orange (12.00 ± 4.52 mm) EOs. Yarrowia lipolytica was less susceptible to lemongrass EO than Candida sake and Yarrowia deformans isolates. Ginger EO exhibited the lowest efficacy. A minimum inhibitory concentration (MIC) assay showed the ability of lemongrass and cinnamon EOs to inhibit the growth of all selected isolates at concentrations between ≤0.31 and 1.25 µL/mL. Therefore, for the first time, the two best-performing EOs (lemongrass and cinnamon) based on in vitro assays were assessed for their potential roles as preservatives in an in vivo yogurt model prepared at the laboratory scale. Since some limitations, such as the inhibition of lactic acid bacteria by cinnamon EO, consequently leading to fermentation failure as well as species-specific antifungal activity of lemongrass EO, were observed, further studies are needed to explore the possibility of using a slightly higher concentration of lemongrass EO and/or combinations of different EOs and/or their components. Finally, since yogurt spoilage could also be prevented by correct sanitation procedures of the production environment, the sanitizers commonly used in the food industry were tested against all isolates, showing the high efficiency of alcohol-based sanitizers and the ineffectiveness of chlorine-based sanitizers.

Study of kefir drinks produced by backslopping method using kefir grains from Bosnia and Herzegovina: Microbial dynamics and volatilome profile.

Kefir is a well-known health-promoting beverage that can be produced by using kefir grains (traditional method) or by using natural starter cultures from kefir (backslopping method). The aim of this study was to elucidate the microbial dynamics and volatilome profile occurring during kefir production through traditional and backslopping methods by using five kefir grains that were collected in Bosnia and Herzegovina. The results from conventional pour plating techniques and amplicon-based sequencing were combined. The kefir drinks have also been characterized in terms of their physico-chemical and colorimetric parameters. A bacterial shift from Lactobacillus kefiranofaciens to Acetobacter syzygii, Lactococcus lactis and Leuconostoc pseudomesenteroides from kefir grains in traditional kefir to backslopped kefir was generally observed. Despite some differences within samples, the dominant mycobiota of backslopped kefir samples remained quite similar to that of the kefir grain samples. However, unlike the lactic acid and acetic acid bacteria, the yeast counts decreased progressively from the grains to the backslopped kefir. The backslopped kefir samples showed higher protein, lactose and ash content and lower ethanol content compared to traditional kefir samples, coupled with optimal pH values that contribute to a pleasant sensory profile. Concerning the volatilome, backslopped kefir samples were correlated with cheesy, buttery, floral and fermented odors, whereas the traditional kefir samples were correlated with alcoholic, fruity, fatty and acid odors. Overall, the data obtained in the present study provided evidence that different kefir production methods (traditional vs backslopping) affect the quality characteristics of the final product. Hence, the functional traits of backslopped kefir should be further investigated in order to verify the suitability of a potential scale-up methodology for backslopping.

Lesser mealworm (Alphitobius diaperinus) powder as a novel baking ingredient for manufacturing high-protein, mineral-dense snacks.

Increasing interest in consuming foods that are high in protein, vitamin, amino acid, and mineral contents is steering growth in the market for fortified snacks. The aim of the present study was to evaluate the use of lesser mealworm (Alphitobius diaperinus) powder (LP) (at 10 or 30% substitution for wheat flour) for the protein and mineral fortification of crunchy snacks (rusks). Hence, the technological, microbiological, nutritional, and sensory characteristics of the fortified rusks were evaluated. The protein content was enriched up to 99.3% in rusks with 30% substitution; moreover, a notable increase in the essential amino acids content was observed, with histidine fortification reaching up to 129.1% in rusks with 30% substitution. The incorporation of LP has led to an enrichment of almost all the minerals considered here, and especially Fe, P and Zn, with Zn showing fortification percentages of up to 300% in rusks with 30% substitution for LP. The experimental rusks showed pleasant sensory traits and low aw values. In view of the potential industrial manufacturing of insect-based rusks, the proposed product can be assigned to level 4 (validation in a laboratory environment) of the Technology Readiness Level (TRL) scale, and it is thus ready to be tested in a simulated production environment.

A Glimpse into the Microbiota of Marketed Ready-to-Eat Crickets (Acheta domesticus).

The present study was aimed to get an insight into the bacterial biota of ready-to-eat small crickets (Acheta domesticus) already marketed in the European Union. 16S rRNA gene of the DNAs extracted from thirty-two samples of ready-to-eat crickets commercialized by 4 European Union producers located in Austria, Belgium, France and the Netherlands (2 batches per producer) was analyzed by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The species belonging to the genera Hespellia, Ruminococcus and Clostridium were detected in samples from Austria, while those from genera Lysobacter, Staphylococcus and Clostridium were detected in samples from Belgium. Moreover, samples from France were characterized by Staphylococcus, Pseudomonas, and Hydrogenophilus genera. Finally, the genera Staphylococcus, Hydrogenophilus, Clostridium and Ruminococcus were identified in the samples produced in the Netherlands. When insects are intended for commercialization, rearing, processing and handling could affect the presence of the occurring microbial species. Hence, to assure a safe product, the need for a full standardization of production technologies, including feed supply as well as rearing and processing practices, is recommended.

Investigating Antibiotic Resistance Genes in Marketed Ready-to-Eat Small Crickets (Acheta domesticus).

The present investigation was aimed at evaluating the occurrence of transferable genes conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B (MLSB ), vancomycin, beta-lactams, and aminoglycosides in 32 samples from eight batches of ready-to-eat crickets (Acheta domesticus) commercialized by four European Union producers (two batches per producer). Bacterial DNA extracted directly from the insects was subjected to optimized polymerase chain reaction (PCR) and nested-PCR assays for the qualitative detection of 12 selected antibiotic resistance (AR) genes. Microbial enumeration demonstrated high counts of spore-forming bacteria and total mesophilic aerobes. Statistical analyses revealed significant differences between different producers and insect batches. Regarding AR genes, a high prevalence of genes conferring resistance to tetracycline [tet(M), tet(O), tet(K), tet(S)] was observed, together with the presence of genes conferring resistance to erythromycin [erm(B), erm(C)], beta-lactams (blaZ and mecA), and aminoglycosides [aac(6')-Ie aph(2")-Ia]. We performed a principal component analysis based on the AR gene frequencies that differentiated samples of batch 1 from those of batch 2. This analysis provided evidence for a difference between the producer from France and all the other producers among the batch 1 samples. PRACTICAL APPLICATION: Overall, an intrabatch variation was seen in the transferable resistances among different producers. This evidence, coupled with the observed differences in the viable counts, suggests a low standardization of the production processes. Hence, a prudent use of antimicrobials during the rearing of insects destined for human consumption is strongly recommended, as well as a need for a full standardization of production technologies.

Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish.

Hákarl is produced by curing of the Greenland shark (Somniosus microcephalus) flesh, which before fermentation is toxic due to the high content of trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Despite its long history of consumption, little knowledge is available on the microbial consortia involved in the fermentation of this fish. In the present study, a polyphasic approach based on both culturing and DNA-based techniques was adopted to gain insight into the microbial species present in ready-to-eat hákarl. To this aim, samples of ready-to-eat hákarl were subjected to viable counting on different selective growth media. The DNA directly extracted from the samples was further subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and 16S amplicon-based sequencing. Moreover, the presence of Shiga toxin-producing Escherichia coli (STEC) and Pseudomonas aeruginosa was assessed via qualitative real-time PCR assays. pH values measured in the analyzed samples ranged from between 8.07 ±â€¯0.06 and 8.76 ±â€¯0.00. Viable counts revealed the presence of total mesophilic aerobes, lactic acid bacteria and Pseudomonadaceae. Regarding bacteria, PCR-DGGE analysis highlighted the dominance of close relatives of Tissierella creatinophila. For amplicon sequencing, the main operational taxonomic units (OTUs) shared among the data set were Tissierella, Pseudomonas, Oceanobacillus, Abyssivirga and Lactococcus. The presence of Pseudomonas in the analyzed samples supports the hypothesis of a possible role of this microorganism on the detoxification of shark meat from TMAO or TMA during fermentation. Several minor OTUs (<1%) were also detected, including Alkalibacterium, Staphylococcus, Proteiniclasticum, Acinetobacter, Erysipelothrix, Anaerobacillus, Ochrobactrum, Listeria and Photobacterium. Analysis of the yeast and filamentous fungi community composition by PCR-DGGE revealed the presence of close relatives of Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Saccharomyces cerevisiae, Debaryomyces, Torulaspora, Yamadazyma, Sporobolomyces, Alternaria, Cladosporium tenuissimum, Moristroma quercinum and Phoma/Epicoccum, and some of these species probably play key roles in the development of the sensory qualities of the end product. Finally, qualitative real-time PCR assays revealed the absence of STEC and Pseudomonas aeruginosa in all of the analyzed samples.

Protein fortification with mealworm (Tenebrio molitor L.) powder: Effect on textural, microbiological, nutritional and sensory features of bread.

In the present study, inclusion of mealworm (Tenebrio molitor L.) powder into bread doughs at 5 and 10% substitution level of soft wheat (Triticum aestivum L.) flour was tested to produce protein fortified breads. The addition of mealworm powder (MP) did not negatively affect the technological features of either doughs or breads. All the tested doughs showed the same leavening ability, whereas breads containing 5% MP showed the highest specific volume and the lowest firmness. An enrichment in protein content was observed in experimental breads where the highest values for this parameter were recorded in breads containing 10% MP. Breads fortified with 10% MP also exhibited a significant increase in the content of free amino acids, and especially in the following essential amino acids: tyrosine, methionine, isoleucine, and leucine. By contrast, no differences in nutritional quality of lipids were seen between fortified and control breads. Results of sensory analyses revealed that protein fortification of bread with MP significantly affected bread texture and overall liking, as well as crust colour, depending on the substitution level. Overall, proof of concept was provided for the inclusion of MP into bread doughs started with different leavening agents (sourdough and/or baker's yeast), at 5 or 10% substitution level of soft wheat flour. Based on the Technology Readiness Level (TRL) scale, the proposed bread making technology can be situated at level 4 (validation in laboratory environment), thus suggesting that the production of breads with MP might easily be scaled up at industrial level. However, potential spoilage and safety issues that need to be further considered were highlighted.

Brettanomyces Spoilage in Albanian Wines Assessed by Culture-Dependent and Culture-Independent Methods.

In the Albanian winemaking industry, there is little awareness of the potential detrimental effect of Brettanomyces in wines. The aim of this study was to detect and quantify Brettanomyces cells in 22 Albanian bottled wines, representing all the viticultural areas of Albania. A combined approach, including culture-dependent (viable plate counting) and culture-independent (qPCR) methods, was applied. Spoilage indicators (ethylphenols and total and volatile acidity), as well as the primary factors known to influence the growth of Brettanomyces in wine (pH, SO2 , and ethanol concentration), were also investigated. Brettanomyces was detected in only five (one Merlot, four Sheshi i Zi) out of 22 samples analyzed using viable counting, with loads ranging from 1.30 ± 0.03 log CFU/mL to 3.99 ± 0.00 log CFU/mL, whereas it was never detected in the Kallmet samples. When qPCR was applied, Brettanomyces cells were detected and quantified in all of the samples with a generally low load ranging from 0.47 ± 0.13 to 3.99 ± 0.01 log cells/mL. As a general trend, the loads of spoilage by this yeast were low (≤1.92 log cells/mL), with the exception of five samples that were also positive by plate counting. A positive correlation between the growth of this spoilage yeast on Dekkera/Brettanomyces differential media and its detection at high levels by qPCR was observed. A significant positive correlation between Brettanomyces and the concentration of ethylphenols and volatile acidity was also found. In summary, the results of this study demonstrated the low incidence of Brettanomyces spoilage yeasts in Albanian red wines. PRACTICAL APPLICATION: The awareness of Brettanomyces spoilage in the Albanian winemaking industry is very low. This study represents the first contribution to understand the extent of this spoilage yeast in Albanian autochthonous cultivars, which tend to have high economic value, to ensure product quality and safety. qPCR is confirmed to be a very sensitive method to rapidly detect Brettanomyces spoilage in wine samples.

Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source.