Comparisons among barley-pea mixed crop combinations in a replacement design as related to N fertilization and soil variation.

Two field trials (2017 and 2018) evaluated the performance of barley-pea mixed cropping by comparing different sowing densities (replacement design) and tailoring N fertilization on barley sowing density (split-plot design). High and Low N inputs were applied to whole plots whereas barley and pea, as pure and in mixed crops, were applied to subplots. The 2017 trial suggested the occurrence of an interaction between soil physical properties and N fertilization. Therefore, in 2018 a pedological survey allowed the soil effect to be included in the ANOVA model applied to evaluate crop performance parameters, showing that N fertilization positively affected barley performance only in the soil unit located downslope. A significantly lower presence of weeds was observed in mixed crops rather than in pea pure crops. Overall, increasing pea density and reducing barley density in mixed crops, and tailoring N fertilization were effective approaches to obtain a more balanced mixed grain at harvest. The combination of crop performance evaluation and assessments of soil conditions suggested that more sustainable agricultural systems, based on mixed cropping and a significant reduction of N fertilizers and herbicides, can be achieved with barley-pea mixed cropping as an alternative to pure cropping systems.

A Comprehensive Approach to Evaluate Durum Wheat-Faba Bean Mixed Crop Performance.

Plant breeding for intercropping is lagging because most varieties currently available in the market are selected for sole cropping systems. The present study analyzed the response of durum wheat (12 varieties) and faba bean (3 varieties) in pure and mixed cropping. Field trials were conducted in 2019 and 2020. The performance of each variety in mixed and pure cropping was evaluated using both univariate and multivariate analyses of the grain yield and land equivalent ratio (LER). For durum wheat, grain protein content was also evaluated. Durum wheat varieties were characterized by good performance in both years, whereas faba bean varieties were more affected by the growing season, suggesting that much breeding effort is warranted to improve the latter as a pure and mixed crop. Moreover, the relative performance of all varieties was affected by their combination in mixed cropping, as evaluated based on the ratio (LERratio) between LER for wheat (LERw) and LER for faba bean (LERfb). To further evaluate the overall performance of wheat and faba bean in mixed cropping, total yield, LERtotal (LERw + LERfb), and ln(LERratio) were subjected to principal component and cluster analyses. The first principal component combined the total yield and LERtotal in a single index of the overall performance of each mixed crop combination. The second principal component, based on ln(LERratio), highlighted the relative performance of varieties in each mixed crop combination. The proposed multivariate approach can be applied in the breeding programs for intercropping to identify variety combinations based on crop performance and the relative importance of the proportion of cereal and legume grains in the total harvest.

Effect of Myostatin Gene Mutation on Slaughtering Performance and Meat Quality in Marchigiana Bulls.

The myostatin gene also called Growth Differentiation Factor 8 gene (GDF8) is one of the most investigated loci that can be responsible for several quantitative and qualitative carcass and meat traits in double-muscled beef cattle. The objective of the study was to bring to light the effect of the myostatin polymorphism on slaughtering performance and meat quality in Marchigiana beef cattle. The experiment was carried out on 78 bulls reared according to the "cow-calf" extensive managing system. At the end of the fattening period, in vivo and carcass data were recorded. From each carcass, a steak of Longissimus thoracis was taken and used to determine the meat's analytical composition and colorimetric properties. Finally, from each steak a sample of Longissimus thoracis was collected, then used for DNA extraction and genotyping at the myostatin locus. The heterozygous bulls showed slight superiority in the carcass data (e.g., hot carcass weight: 426.09 kg-heterozygotes vs. 405.32 kg-normal) and meat quality parameters, although not always with statistical significance. Only fat and ashes content were significantly affected by the myostatin genotype (heterozygotes: 2.01%, 1.26%; normal: 3.04%, 1.15%). The greater muscularity of heterozygous animals compared to normal ones could be a starting point to improving productive efficiency in Marchigiana beef cattle.

Occurrence of Antibiotic Resistance Genes in Hermetia illucens Larvae Fed Coffee Silverskin Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens larvae are among the most promising insects for use as food or feed ingredients due to their ability to convert organic waste into biomass with high-quality proteins. In this novel food or feed source, the absence of antibiotic-resistant bacteria and their antibiotic resistance (AR) genes, which could be horizontally transferred to animal or human pathogens through the food chain, must be guaranteed. This study was conducted to enhance the extremely scarce knowledge on the occurrence of AR genes conferring resistance to the main classes of antibiotics in a rearing chain of H. illucens larvae and how they were affected by rearing substrates based on coffee silverskin supplemented with increasing percentages of Schizochytrium limacinum or Isochrysis galbana microalgae. Overall, the PCR and nested PCR assays showed a high prevalence of tetracycline resistance genes. No significant effect of rearing substrates on the distribution of the AR genes in the H. illucens larvae was observed. In contrast, the frass samples were characterized by a significant accumulation of AR genes, and this phenomenon was particularly evident for the samples collected after rearing H. illucens larvae on substrates supplemented with high percentages (>20%) of I. galbana. The latter finding indicates potential safety concerns in reusing frass in agriculture.

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.

Distribution of Antibiotic Resistance Genes in the Saliva of Healthy Omnivores, Ovo-Lacto-Vegetarians, and Vegans.

Food consumption allows the entrance of bacteria and their antibiotic resistance (AR) genes into the human oral cavity. To date, very few studies have examined the influence of diet on the composition of the salivary microbiota, and even fewer investigations have specifically aimed to assess the impact of different long-term diets on the salivary resistome. In this study, the saliva of 144 healthy omnivores, ovo-lacto-vegetarians, and vegans were screened by nested PCR for the occurrence of 12 genes conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B, vancomycin, and ß-lactams. The tet(W), tet(M), and erm(B) genes occurred with the highest frequencies. Overall, no effect of diet on AR gene distribution was seen. Some differences emerged at the recruiting site level, such as the higher frequency of erm(C) in the saliva of the ovo-lacto-vegetarians and omnivores from Bologna and Turin, respectively, and the higher occurrence of tet(K) in the saliva of the omnivores from Bologna. A correlation of the intake of milk and cheese with the abundance of tet(K) and erm(C) genes was seen. Finally, when the occurrence of the 12 AR genes was evaluated along with geographical location, age, and sex as sources of variability, high similarity among the 144 volunteers was seen.

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.

Trends in the quality and hygiene parameters of bulk Italian Mediterranean buffalo (Bubalus bubalis) milk: A three year study.

Buffalo milk represents an indispensable source of nourishment in many parts of the world and it is the second most consumed milk worldwide. Buffalo milk is actually used for the production of many dairy products such as pasteurized or concentrated milk, butter, yogurt, ice-cream, dehydrated milk products and cheeses. Due to its high nutritional value and the presence of natural bioactive substances, buffalo milk can also provide health benefits to consumers. In Italy, buffalo milk is used mainly for cheese making, mozzarella PDO (Protected Designation of Origin), which is a highly valued dairy product. This 3-year study, carried out between 2011 and 2013, was aimed at evaluating the quality of bulk Italian Mediterranean buffalo milk by monitoring physico-chemical parameters, somatic cell and total bacterial counts. A total of 51 samples of bulk milk were collected from one herd throughout the monitored period. Analysis of variance, carried out to test month, season and year main effects, highlighted remarkable seasonal effects for fat, protein and lactose content, as well as for predicted mozzarella cheese yield, and somatic cell counts. The calculation of simple correlations allowed the identification of positive correlations between estimated cheese yield and fat and protein content.

Investigation of the Dominant Microbiota in Ready-to-Eat Grasshoppers and Mealworms and Quantification of Carbapenem Resistance Genes by qPCR.

In this study, 30 samples of processed edible mealworms (Tenebrio molitor L.) and 30 samples of grasshoppers (Locusta migratoria migratorioides) were obtained from producers located in Europe (Belgium and the Netherlands) and Asia (Thailand) and subjected to PCR-DGGE analyses. The PCR-DGGE analyses showed that species in the genus Staphylococcus were predominant in the samples of mealworms from Belgium and grasshoppers from the Netherlands; species in the genus Bacillus were detected in the samples of mealworms and grasshoppers from Thailand. Moreover, Weissella cibaria/confusa/spp. was found in grasshoppers from Belgium. Since data concerning the role of novel foods such as edible insects in the dissemination of carbapenem resistance are currently lacking, the quantification of five carbapenemase encoding genes (bla NDM-1, bla VIM, bla GES, bla OXA-48, and bla KPC) by qPCR was also carried out in all the samples under study. The genes coding for GES and KPC were not detected in the analyzed samples. A very low frequency of bla OXA-48 (3%) and bla NDM-1 (10%) genes was detected among mealworms. In contrast, grasshoppers were characterized by a high incidence of the genes for OXA-48 and NDM-1, accounting for 57 and 27% of the overall grasshopper samples, respectively. The bla VIM gene was detected exclusively in two grasshopper samples from Thailand, showing only 7% positivity. The analysis of variance showed that all the effects (producers, species, and producers × species) were statistically significant for bla NDM-1, whereas for bla OXA-48 and bla VIM, no significant effects were detected for the same source of variation. Further studies are necessary to assess the possible role of edible insects as reservoirs for the resistance to carbapenems and to understand the correlation with the insect microbiota. Furthermore, an intensified surveillance plan examining the occurrence of carbapenemase encoding genes in the food chain and in environmental compartments is needed for a proper risk assessment. In such a context, the appropriate use of antimicrobials represents the main preventive action that should always be applied.

Occurrence of transferable antibiotic resistances in commercialized ready-to-eat mealworms (Tenebrio molitor L.).

The present study aimed to assess the occurrence of transferable determinants conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B, vancomycin, beta-lactams, and aminoglycosides in 40 samples of commercialized edible mealworms (Tenebrio molitor L.) purchased from European Union (EU) and non-EU producers. A high prevalence of tet(K) was observed in all of the samples assayed, with percentages of PCR-based positivity that ranged from 80% (samples from Thailand) to 100% (samples from the Netherlands, Belgium and France). For macrolides, erm(B) prevailed, being detected in 57.5% of the samples assayed, whereas erm(A) and erm(C) were detected with lower frequencies. Genes for resistance to vancomycin were only detected in samples produced in France and Belgium, with 90% and 10% of the samples being positive for vanA, respectively. Beta-lactamase genes were found with low occurrence, whereas the gene aac-aph, conferring high resistance to aminoglycosides, was found in 40% of the samples produced in the Netherlands and Belgium and 20% of the samples produced in Thailand. The results of Principal Coordinate Analysis and Principal Component Analysis depicted a clean separation of the samples collected from the four producers based on the distribution of the 12 AR determinants considered. Given the growing interest on the use of mealworms as a novel protein source, AR detection frequencies found in the present study suggest further investigation into the use of antibiotics during rearing of this insect species and more extensive studies focused on the factors that can affect the diffusion of transferable ARs in the production chain. Until such studies are completed, prudent use of antibiotics during rearing of edible insects is recommended.

Occurrence of antibiotic resistance genes in the fecal DNA of healthy omnivores, ovo-lacto vegetarians and vegans.

SCOPE: The effects of long-term omnivore, ovo-lacto vegetarian and vegan diets on the occurrence of 12 antibiotic resistance (AR) genes in the human gut were studied. METHODS AND RESULTS: The feces of 144 healthy volunteers recruited from Turin, Bari, Bologna, and Parma were screened for the occurrence of genes conferring resistance to tetracyclines, macrolide-lincosamide-streptogramin B, vancomycin, and ß-lactams. Overall, erm(B), tet(W) and tet(M) were detected at the highest frequency. A low effect from the diet on the AR gene distribution emerged, with tet(K) and vanB occurring at a lower and higher frequency in vegans and omnivores, respectively. A correlation of the intake of eggs, milk from animal sources and cheese with an increased occurrence of tet(K) was observed, together with a higher incidence of vanB in consumers of eggs, poultry meat, fish and seafood. When the detection frequencies of AR genes in volunteers from Bari and the other sites were comparatively evaluated, a north-to-south gradient was observed, whereas no effect of sex or age was highlighted. Except for tet(K), a negligible three-factor interaction was seen. CONCLUSION: A high impact of the geographical location on AR gene distribution was seen in the cohort of subjects analyzed, irrespective of their dietary habits.

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.

Bioluminescence ATP monitoring for the routine assessment of food contact surface cleanliness in a university canteen.

ATP bioluminescence monitoring and traditional microbiological analyses (viable counting of total mesophilic aerobes, coliforms and Escherichia coli) were used to evaluate the effectiveness of Sanitation Standard Operating Procedures (SSOP) at a university canteen which uses a HACCP-based approach. To that end, 10 cleaning control points (CPs), including food contact surfaces at risk of contamination from product residues or microbial growth, were analysed during an 8-month monitoring period. Arbitrary acceptability limits were set for both microbial loads and ATP bioluminescence readings. A highly significant correlation (r = 0.99) between the means of ATP bioluminescence readings and the viable counts of total mesophilic aerobes was seen, thus revealing a strong association of these parameters with the level of surface contamination. Among CPs, the raw meat and multi-purpose chopping boards showed the highest criticalities. Although ATP bioluminescence technology cannot substitute traditional microbiological analyses for the determination of microbial load on food contact surfaces, it has proved to be a powerful tool for the real time monitoring of surface cleanliness at mass catering plants, for verify the correct application of SSOP, and hence for their implementation/revision in the case of poor hygiene.

Evaluation of the HACCP system in a university canteen: microbiological monitoring and internal auditing as verification tools.

Food safety is essential in mass catering. In Europe, Regulation (EC) No. 852/2004 requires food business operators to put in place, implement and maintain permanent procedures based on Hazard Analysis and Critical Control Point (HACCP) principles. Each HACCP plan is specifically implemented for the processing plant and processing methods and requires a systematic collection of data on the incidence, elimination, prevention, and reduction of risks. In this five-year-study, the effectiveness of the HACCP plan of a University canteen was verified through periodic internal auditing and microbiological monitoring of meals, small equipment, cooking tools, working surfaces, as well as hands and white coats of the canteen staff. The data obtained revealed no safety risks for the consumers, since Escherichia coli, Salmonella spp. and Listeria monocytogenes were never detected; however, a quite discontinuous microbiological quality of meals was revealed. The fluctuations in the microbial loads of mesophilic aerobes, coliforms, Staphylococcus aureus, Bacillus cereus, and sulphite-reducing clostridia were mainly ascribed to inadequate handling or processing procedures, thus suggesting the need for an enhancement of staff training activities and for a reorganization of tasks. Due to the wide variety of the fields covered by internal auditing, the full conformance to all the requirements was never achieved, though high scores, determined by assigning one point to each answer which matched with the requirements, were achieved in all the years.

An eight-year report on the implementation of HACCP in a university canteen: impact on the microbiological quality of meals.

An investigation aimed at assessing the microbiological quality of meals consumed at a university canteen after implementation of the HACCP system and personnel training was carried out. Cooked and warm-served products (74 samples), cooked and cold-served products (92 samples) and cold gastronomy products (63 samples) sampled from 2000 to 2007 underwent microbiological analyses. All the samples were tested for: Samonella spp., Listeria monocytogenes, total mesophilic aerobes, coliforms, Escherichia coli, Staphylococcus aureus, Bacillus cereus, and sulphite-reducing clostridia. The microbiological contamination of work surfaces (tables, tablewares, cutters, ladles, slicing machines, wash-basins, etc.), hands and white coats of members of the canteen staff was also assessed. The microbiological results clearly demonstrated the success of the HACCP plan implementation, through a general improvement of the hygiene conditions of both meals and work surfaces.

Agronomic, qualitative (ß-ODAP) and molecular variability in grasspea populations of the Marche region (central Italy).

Grasspea is an interesting crop for rational crop rotations in inner areas of the Marche region. However, breeding efforts are needed to improve its agronomic performance and to lower as much as possible the neuro-active amino acid ß-ODAP in the seeds. Since the year 2000, a germplasm collection and evaluation program has been carried out. Accessions were evaluated by field trials for morphological, physiological and agronomic traits and for seed ß-ODAP content. Significant variability was detected. In particular, populations collected in local farms showed a higher seed production compared with unadapted accessions. ß-ODAP content varied between 0.26% and 0.53%. Moreover, AFLP molecular markers were applied to investigate genetic variation characterizing the populations included in the collection. In the year 2008 a breeding plan was started using two household populations, one with the lowest average ß-ODAP content and one with the highest agronomic performance. Based on these results, a within population analysis for this trait will be started using the accession with the lowest ß-ODAP content. Interesting information was also gathered using AFLP molecular markers, since a wide range of genetic variation supported the possibility of starting a mapping project aimed at the identification of genes of the biochemical pathway for ß-ODAP.