Biodiversity and antibiotic resistance profile provide new evidence for a different origin of enterococci in bovine raw milk and feces.

Enterococci are widely distributed in dairy sector. They are commensals of the gastrointestinal tract of animals, thus, via fecal contamination, could reach raw milk and dairy products. The aims of this study were: 1) to investigate the enterococcal diversity in cow feces and milk samples and 2) to evaluate the antibiotic resistance (AR) of dairy-related enterococci and their ability to transfer resistance genes. E. faecalis (59.9%), E. faecium (18.6%) and E. lactis (12.4%) were prevalent in milk, while E. faecium (84.2%) and E. hirae (15.0%) were dominant in bovine feces. RAPD-PCR highlighted a high number of Enterococcus biotypes (45 from milk and 37 from feces) and none of the milk strains exhibited genetic profiles similar to those of feces biotypes. A high percentage of enterococci isolated from milk (71%) were identified as multidrug resistant and resistance against streptomycin and tetracycline were widespread among milk strains while enterococci from feces were commonly resistant to linezolid and quinupristin/dalfopristin. Only E. faecalis strains were able to transfer horizontally the tetM gene to Lb. delbrueckii subsp. lactis. Our results indicated that Enterococcus biotypes from milk and bovine feces belong to different community and the ability of these microorganisms to transfer AR genes is strain-dependent.

Surface layer proteins from Lactobacillus helveticus ATCC® 15009™ affect the gut barrier morphology and function.

Paraprobiotics and postbiotics represent a valid alternative to probiotic strains for ameliorating and preserving a healthy intestinal epithelial barrier (IEB). The present study investigated the effects of surface layer proteins (S-layer) of the dairy strain Lactobacillus helveticus ATCC® 15009™ (Lb ATCC® 15009™), as paraprobiotic, on the morpho-functional modulation of IEB in comparison to live or heat-inactivated Lb ATCC® 15009™ in an in vitro co-culture of Caco-2/HT-29 70/30 cells. Live or heat-inactivated Lb ATCC® 15009™ negatively affected transepithelial electrical resistance (TEER) and paracellular permeability, and impaired the distribution of Claudin-1, a tight junction (TJ) transmembrane protein, as detected by immunofluorescence (IF). Conversely, the addition of the S-layer improved TEER and decreased permeability in physiological conditions in co-cultures with basal TEER lower than 50 ohmcm2, indicative of a more permeable physiological IEB known as leaky gut. Transmission electron microscopy (TEM) and IF analyses suggested that the S-layer induces a structural TJ rearrangement and desmosomes' formation. S-layer also restored TEER and permeability in the presence of LPS, but not of a mixture of pro-inflammatory cytokines (TNF-α plus IFN-γ). IF analyses showed an increase in Claudin-1 staining when LPS and S-layer were co-administered with respect to LPS alone; in addition, the S-layer counteracted the reduction of alkaline phosphatase detoxification activity and the enhancement of pro-inflammatory interleukin-8 release both induced by LPS. Altogether, these data corroborate a paraprobiotic role of S-layer from Lb ATCC® 15009™ as a possible candidate for therapeutic and prophylactic uses in conditions related to gastrointestinal health and correlated with extra-intestinal disorders.

NMR-Based Metabolomic Study on Phaseolus vulgaris Flour Fermented by Lactic Acid Bacteria and Yeasts.

In recent years, fermented foods have attracted increasing attention due to their important role in the human diet, since they supply beneficial health effects, providing important sources of nutrients. In this respect, a comprehensive characterization of the metabolite content in fermented foods is required to achieve a complete vision of physiological, microbiological, and functional traits. In the present preliminary study, the NMR-based metabolomic approach combined with chemometrics has been applied, for the first time, to investigate the metabolite content of Phaseolus vulgaris flour fermented by different lactic acid bacteria (LAB) and yeasts. A differentiation of microorganisms (LAB and yeasts), LAB metabolism (homo- and heterofermentative hexose fermentation), LAB genus (Lactobacillus, Leuconostoc, and Pediococcus), and novel genera (Lacticaseibacillus, Lactiplantibacillus, and Lentilactobacillus) was achieved. Moreover, our findings showed an increase of free amino acids and bioactive molecules, such as GABA, and a degradation of anti-nutritional compounds, such as raffinose and stachyose, confirming the beneficial effects of fermentation processes and the potential use of fermented flours in the production of healthy baking foods. Finally, among all microorganisms considered, the Lactiplantibacillus plantarum species was found to be the most effective in fermenting bean flour, as a larger amount of free amino acids were assessed in their analysis, denoting more intensive proteolytic activity.

Fatty acids composition and lipolysis of Parmigiano Reggiano PDO cheese: effect of the milk cooling temperature at the farm.

OBJECTIVE: The aim was to study the influence of cooling milk at 9°C at the farm versus keeping it at 20°C on Parmigiano Reggiano cheese lipolysis. METHODS: A total of six cheesemaking trials (3 in winter and 3 in summer) were performed. In each trial, milk was divided continuously into two identical aliquots, one of which was kept at 9°C (MC9) and the other at 20°C (MC20). For each trial and milk temperature, vat milk (V-milk) and the resulting 21 month ripened cheese were analysed. RESULTS: Fat and dry matter and fat/casein ratio were lower in MC9 V-milk (p≤0.05) than in MC20. Total bacteria, mesophilic lactic acid and psychrotrophic and lipolytic bacteria showed significant differences (p≤0.05) between the two V-milks. Regarding cheese, fat content resulted lower and crude protein higher (p≤0.05) both in outer (OZ) and in inner zone (IZ) of the MC9 cheese wheels. Concerning total fatty acids, the MC9 OZ had a lower concentration of butyric, capric (p≤0.05) and medium chain fatty acids (p≤0.05), while the MC9 IZ had lower content of butyric (p≤0.05), caproic (p≤0.01) and short chain fatty acids (p≤0.05). The levels of short chain and medium chain free fatty acids (p≤0.05) were lower and that of long chain fatty acids (p≤0.05) was higher in MC9 OZ cheese. The principal component analysis of total and free fatty acids resulted in a clear separation among samples by seasons, whereas slight differences were observed between the two different milk temperatures. CONCLUSION: Storing milk at 9°C at the herd affects the chemical composition of Parmigiano Reggiano, with repercussion on lipolysis. However, the changes are not very relevant, and since the cheese can present a high variability among the different cheese factories, such changes should be considered within the "normal variations" of Parmigiano Reggiano chemical characteristics.

Antifungal activity of lactic acid bacteria and their application in food biopreservation.

Lactic acid bacteria (LAB) are ubiquitous bacteria associated with spontaneous lactic fermentation of vegetables, dairy and meat products. They are generally recognized as safe (GRAS), and they are involved in transformation of probiotic lacto-fermented foods, highly desired for their nutraceutical properties. The antifungal activity is one of the exciting properties of LAB, because of its possible application in food bio-preservation, as alternative to chemical preservatives. Many recent research works have been developed on antifungal activity of LAB, and they demonstrate their capacity to produce various antifungal compounds, (i.e. organic acids, PLA, proteinaceous compounds, peptides, cyclic dipeptides, fatty acids, and other compounds), of different properties (hydrophilic, hydrophobic and amphiphilic). The effectiveness of LAB in controlling spoilage and pathogenic fungi, demonstrated in different agricultural and food products, can be due to the synergistic effect between their antifungal compounds of different properties; where the amphiphilic-compounds allow the contact between the target microbial cell (hydrophilic compartment) and antifungal hydrophobic-compounds. Further studies on the interaction between compounds of these three properties are to de be developed, in order to highlight more their mechanism of action, and make LAB more profitable in improving shelf life and nutraceutical properties of foods.

Air Quality, Management Practices and Calf Health in Italian Dairy Cattle Farms.

Among factors that can affect calf health, microbial quality of the pen air is poorly studied. In 25 Italian dairy farms, data concerning air quality in the calf pens, hygiene of pens and equipment, microclimatic conditions, calf health and management were collected during the winter season (January-March 2020 and December-March 2021). The average air Standard Plate Count (SPC) of 85 pens was 4.51 (SD = 0.52) log10 cfu/m3 whereas the average air ammonia concentration was 0.66 (SD = 0.53) ppm. Positive correlations were found between average Temperature Humidity Index (THI) in the pen and air SPC, night maximum THI and air SPC and between SPC and yeast, mould and ammonia concentration in the pen air. The concentrations of E. coli, Enterobacteriaceae and yeasts in the pen air were higher and calf cough increased as the renewal interval of bedding material became longer. High bedding dry matter and low THI were associated with low air SPC, good calf health scores and low mortality. Maintaining low bedding humidity and controlling microclimatic conditions can contribute to enhancing air microbiological quality in the pen and reduce calf diseases and mortality.

Characterization of antimicrobial compounds obtained from the potential probiotic Lactiplantibacillus plantarum S61 and their application as a biopreservative agent.

This work aimed to characterize the antimicrobial compounds obtained from the potential probiotic Lactiplantibacillus plantarum S61, isolated from traditional fermented green olive, involved in their activity against fungi and bacteria responsible for food spoilage and poisonings. Their application as a biopreservative agent was also investigated. The culture of L. plantarum S61 showed substantial antifungal and antibacterial activity against yeasts (Rhodotorula glutinis and Candida pelliculosa), molds (Penicillium digitatum, Aspergillus niger, Fusarium oxysporum, and Rhizopus oryzae), and pathogenic bacteria (Listeria monocytogenes ATCC 19,117, Salmonella enterica subsp. enterica ATCC 14,028, Staphylococcus aureus subsp. aureus ATCC 6538, Pseudomonas aeruginosa ATCC 49,189), with inhibition zones > 10 mm. Likewise, the cell-free supernatant (CFS) of L. plantarum S61 showed an essential inhibitory effect against fungi and bacteria, with inhibition diameters of 12.25-22.05 mm and 16.95-17.25 mm, respectively. The CFS inhibited molds' biomass and mycelium growth, with inhibition ranges of 63.18-83.64% and 22.57-38.93%, respectively. The antifungal activity of the CFS was stable during 4 weeks of storage at 25 °C, while it gradually decreased during storage at 4 °C. Several antimicrobial compounds were evidenced in the CFS of L. plantarum S61, including organic acids, ethanol, hydrogen peroxide, diacetyl, proteins, and fatty acids. The protein fraction, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), demonstrated important antifungal activity, in relation to the fraction with molecular weight between 2 and 6 kDa. L. plantarum S61 and its CFS, tested in apple and orange fruit biopreservation, demonstrated their protective effect against P. digitatum spoilage. The CFS exhibited effectiveness in reducing Salmonella enterica subsp. enterica ATCC 14,028 in apple juice. L. plantarum S61 and/or its bioactive compounds CFS represent a promising strategy for biocontrol against pathogens and spoilage microorganisms in the agro-industry.

Antibiotic resistance in wild and commercial non-enterococcal Lactic Acid Bacteria and Bifidobacteria strains of dairy origin: An update.

Antibiotic Resistance is a growing concern for public health and global economy. Lactic acid bacteria (LAB) involved in the production of dairy products and commonly present in the agro-zootechnical environment can act as reservoirs of antibiotic resistance genes, acquiring or transferring them to other microorganisms. The review focuses on LAB group of dairy origin (Lactobacillus, Lactococcus, Streptococcus, Leuconostoc, Pediococcus and Weissella) and Bifidobacterium genus, considering its large use in dairy industry. We have analyzed data in the last 25 years, highlighting atypical resistance, genetic traits correlated to antibiotic resistance and their ability to be transmitted to other microorganisms; comparative analysis of resistomes was also considered. Differences were observed among wild strains isolated from different regions because of authorized antibiotic use. Commercial strains belonging to Lactobacillus, Streptococcus and Bifidobacterium currently used for industrial dairy products are frequently resistant to gentamycin, kanamycin, chloramphenicol together with tetracycline. The presence of resistant wild LAB in raw milk products has been significantly reduced as a result of worldwide restrictions on the use of antibiotics in animal husbandry. Transmissible resistances are still present in industrial cultures, despite the great effort of starter industries in the process control and the safety screening of commercial cultures.

Recent Advances in the Mechanisms and Regulation of QS in Dairy Spoilage by Pseudomonas spp.

Food spoilage is a serious issue dramatically impacting the worldwide need to counteract food insecurity. Despite the very expensive application of low temperatures, the proper conservation of fresh dairy products is continuously threatened at different stages of production and commercialization by psychrotrophic populations mainly belonging to the Pseudomonas genus. These bacteria cause discolouration, loss of structure, and off-flavours, with fatal implications on the quality and shelf-life of products. While the effects of pseudomonad decay have been widely reported, the mechanisms responsible for the activation and regulation of spoilage pathways are still poorly explored. Recently, molecule signals and regulators involved in quorum sensing (QS), such as homoserine lactones, the luxR/luxI system, hdtS, and psoR, have been detected in spoiled products and bacterial spoiler species; this evidence suggests the role of bacterial cross talk in dairy spoilage and paves the way towards the search for novel preservation strategies based on QS inhibition. The aim of this review was to investigate the advancements achieved by the application of omic approaches in deciphering the molecular mechanisms controlled by QS systems in pseudomonads, by focusing on the regulators and metabolic pathways responsible for spoilage of fresh dairy products. In addition, due the ability of pseudomonads to quickly spread in the environment as biofilm communities, which may also include pathogenic and multidrug-resistant (MDR) species, the risk derived from the gaps in clearly defined and regulated sanitization actions is underlined.

Bacterial Communities in the Embryo of Maize Landraces: Relation with Susceptibility to Fusarium Ear Rot.

Locally adapted maize accessions (landraces) represent an untapped resource of nutritional and resistance traits for breeding, including the shaping of distinct microbiota. Our study focused on five different maize landraces and a reference commercial hybrid, showing different susceptibility to fusarium ear rot, and whether this trait could be related to particular compositions of the bacterial microbiota in the embryo, using different approaches. Our cultivation-independent approach utilized the metabarcoding of a portion of the 16S rRNA gene to study bacterial populations in these samples. Multivariate statistical analyses indicated that the microbiota of the embryos of the accessions grouped in two different clusters: one comprising three landraces and the hybrid, one including the remaining two landraces, which showed a lower susceptibility to fusarium ear rot in field. The main discriminant between these clusters was the frequency of Firmicutes, higher in the second cluster, and this abundance was confirmed by quantification through digital PCR. The cultivation-dependent approach allowed the isolation of 70 bacterial strains, mostly Firmicutes. In vivo assays allowed the identification of five candidate biocontrol strains against fusarium ear rot. Our data revealed novel insights into the role of the maize embryo microbiota and set the stage for further studies aimed at integrating this knowledge into plant breeding programs.

Effects of the Cooling Temperature at the Farm on Milk Maturation and Cheesemaking Process in the Manufacture of Parmigiano Reggiano PDO Cheese.

Parmigiano Reggiano is a hard PDO cheese made from bovine raw milk, whose microbiological characteristics have important repercussions on cheese quality. According to the EU official production protocol, milk temperature at the farm must not drop below 18 °C. The present research aimed to study the effect of cooling milk at the farm at 9 °C on the characteristics of milk and on the cheesemaking process and losses during manufacture. Six cheesemaking trials were performed in two different dairies. In each of them, two cheesemakings were made in parallel: one with milk kept at 9 °C (TM9) and the other with milk kept at 20 °C (TM20). TM9 milk, in comparison with TM20, showed after the creaming process a significant reduction not only of total bacterial count but also of psychrotrophic and lipolytic bacteria. At the same time, TM9 milk showed a higher creaming capacity and, consequently, a lower fat content than TM20. TM9 vat milk had worst coagulation properties than TM20, which caused slightly higher loss of fat and curd fines into the whey. Nevertheless, these changes were too small to influence the efficiency of the cheesemaking process; conversely, maintaining milk at the farm at 9 °C led to a reduction of the number of spoilage bacteria.

High biodiversity in a limited mountain area revealed in the traditional production of Historic Rebel cheese by an integrated microbiota-lipidomic approach.

Historic Rebel (HR) cheese is an Italian heritage cheese, produced from raw milk during the summer grazing period in the Alps. The aim of this work was (i) to characterize the cheese microbiota, by 16S rRNA gene amplicons sequencing, and the volatile and non-volatile lipophilic fraction, by Gas Chromatography and Dynamic Headspace Extraction-Gas Chromatography-Mass Spectrometry, and (ii) to evaluate their respective associations. HR cheese was dominated by Firmicutes phylum (99% of the entire abundance). The core microbiota was formed by Streptococcus, Lactobacillus, Lactococcus, Leuconostoc and Pediococcus genera together representing 87.2-99.6% of the total abundance. The polyunsaturated fatty acids composition showed a high PUFA n-3, PUFA n-6 and CLA content, two fold higher than typical plain cheeses, positively correlated with pasture altitude. A complex volatilome was detected, dominated in terms of abundance by ketones, fatty acids and alcohols. Total terpene levels increased at higher altitudes, being the main terpenes compounds α-pinene, camphene and ß-pinene. The HR cheese showed a great diversity of bacterial taxa and lipophilic fractions among producers, despite belonging to a small alpine area, revealing a scarce cheese standardization and a chemical fingerprint of a typical mountain cheese produced during the grazing period. A deeper knowledge of the variability of HR cheese due to its composition in microbial community and volatile compounds will be appreciated, in particular, by elite consumers looking for niche products, adding economic value to farming in these alpine areas.

Proteolytic Traits of Psychrotrophic Bacteria Potentially Causative of Sterilized Milk Instability: Genotypic, Phenotypic and Peptidomic Insight.

The proteolytic traits of the psychrotrophic strains Pseudomonas poae LP5, Pseudomonas fluorescens LPF3, Chryseobacterium joostei LPR1, Pseudomonas fulva PS1, Citrobacter freundii PS37, Hafnia alvei PS46, and Serratia marcescens PS92 were initially investigated by phenotypic and genotypic approaches. Six strains elicited extracellular proteolytic activity, and five expressed the thermostable AprX or (likely) Ser1 enzymes. Then, the strains were inoculated (104 CFU/mL) in microfiltered pasteurized milk and kept at 4 °C for five days. All of the strains reached 108 CFU/mL at the end of storage and five produced thermostable extracellular proteolytic enzymes. The freshly inoculated samples and the corresponding samples at 108 CFU/mL were batch-sterilized (131 °C, 30 s) and kept at 45 °C up to 100 days. The former samples did not gel until the end of incubation, whereas the latter, containing P. poae, P. fluorescens, C. joostei, C. freundii, and S. marcescens, gelled within a few days of incubation. The thermostable proteolytic activity of strains affected the peptidomic profile, and specific proteolyzed zones of ß-CN were recognized in the gelled samples. Overall, the results confirm some proteolytic traits of psychrotrophic Pseudomonas spp. strains and provide additional insights on the proteolytic activity of psychrotrophic bacteria potentially responsible for sterilized milk destabilization.

Paraprobiotics: A New Perspective for Functional Foods and Nutraceuticals.

Probiotics are live microorganisms that confer health benefits on the host. However, in recent years, several concerns on their use have been raised. In particular, industrial processing and storage of probiotic products are still technological challenges as these could severely impair cell viability. On the other hand, safety of live microorganisms should be taken into account, especially when administered to vulnerable people, such as the elderly and immunodeficient individuals. These drawbacks have enhanced the interest toward new products based on non-viable probiotics such as paraprobiotics and postbiotics. In particular, paraprobiotics, defined as "inactivated microbial cells (non-viable) that confer a health benefit to the consumer," hold the ability to regulate the adaptive and innate immune systems, exhibit anti-inflammatory, antiproliferative and antioxidant properties and exert antagonistic effect against pathogens. Moreover, paraprobiotics can exhibit enhanced safety, assure technological and practical benefits and can also be used in products suitable for people with weak immunity and the elderly. These features offer an important opportunity to prompt the market with novel functional foods or nutraceuticals that are safer and more stable. This review provides an overview of central issues on paraprobiotics and highlights the urgent need for further studies aimed at assessing safety and efficacy of these products and their mechanisms of action in order to support decisions of regulatory authorities. Finally, a definition is proposed that unambiguously distinguishes paraprobiotics from postbiotics.

Assessment of Possible Application of an Atmospheric Pressure Plasma Jet for Shelf Life Extension of Fresh-Cut Salad.

Ready-to-eat salads are very perishable with quality losses within 6-7 days, and the extension of their shelf life is still a challenge. In this work, an atmospheric pressure plasma jet (APPJ) was applied for the surface decontamination of fresh-cut lettuce baby leaves. The APPJ antimicrobial efficiency on the natural microbiota and its impact on some physicochemical attributes of lettuce were evaluated as a function of the treatment duration (0-30 s). Then, the influence of plasma treatment on the salad shelf life was studied, following the growth of aerobic mesophilic bacteria in both untreated and plasma-treated samples during 9 days of storage at 4 °C, together with the plasma-induced changes in physicochemical parameters of lettuce leaves. The APPJ induced a fast (15 s) microbial decontamination (1.3 log10 CFU/g) of the salad surface. Exposure time and salad-plasma plume distance were the parameters that substantially affected the microbial inactivation. APPJ treatment retarded bacterial growth during the refrigerated storage, as plasma-treated samples were noticeably less contaminated than the non-treated ones in the first 3-4 days. No significant effect were observed on electrolyte leakage, pH, and dry matter content in both the set up phase and the shelf life study.

Effect of Different Farming Practices on Lactic Acid Bacteria Content in Cow Milk.

The natural load of lactic acid bacteria (LAB) in milk is the basis of the production of raw milk cheeses, such as Grana Padano PDO. In the last decades, improvements in livestock hygiene management resulted in bulk cow milk with less than 20,000 colony forming units (CFU) of bacterial count, unable to ensure a sufficient supply of LAB, with a negative impact on cheese quality. This study investigated the relations between farm management practices and prevalence of different groups of bacteria in cow milk. Sixty-two intensive dairy farms located in Lombardy (Italy) where involved, most of them destined as milk for the production of Grana Padano. Season had no significant effect on the content of most of the bacterial groups, except for coliforms. A strong relation among standard plate count (SPC) and other bacterial groups was evidenced. Cluster analysis showed that the most productive farms applied a complete milking routine and produced milk with the lowest value of SPC, the lowest count of the other bacteria, including LAB, but the highest LAB/SPC. The study suggests that complexity of farming practices can affect the microbial population of milk.

The prebiotics (Fructo-oligosaccharides and Xylo-oligosaccharides) modulate the probiotic properties of Lactiplantibacillus and Levilactobacillus strains isolated from traditional fermented olive.

This study aimed to examine the influence of two prebiotics, fructo-oligosaccharides (FOS) and xylo-oligosaccharides (XOS), on probiotic properties (resistance to low pH and bile salt, hydrophobicity and auto-aggregation), metabolites production, and antimicrobial activity of probiotic Lactiplantibacillus (L. pentosus S42 and L. plantarum S61) and Levilactobacillus (L. brevis S27) strains isolated from fermented olive. The results demonstrated the ability of strains to ferment XOS more than FOS as a sole carbon source, resulting in pH reduction. The prebiotics (FOS and XOS) significantly increased (p < 0.05) their survival in gastro-intestinal conditions (low pH and 0.3% of bile salts), as well as their hydrophobicity, auto-aggregation and production of proteins, compared to glucose (control). The major organic acids produced by Lactiplantibacillus and Levilactobacillus strains, were oxalic, malic and lactic acids from FOS, XOS and glucose, respectively. No antimicrobial activity was observed from cell-free supernatant (CFS) of Lactiplantibacillus and Levilactobacillus strains obtained from FOS. In the presence of XOS the organic acids, produced by Lactiplantibacillus and Levilactobacillus strains, were more diverse with high contents, and exhibited higher antifungal and antibacterial activities, more than that of FOS and glucose. The combination of L. plantarum S61 and XOS demonstrated the highest inhibition zones ranges of 20.7-22.2 mm against pathogenic bacteria and 29.2-30 mm against yeasts. This combination can be used in production of antifungal preservatives and pharmaceuticals, against pathogenic and spoilage yeasts.

Raw Milk Microbiota Modifications as Affected by Chlorine Usage for Cleaning Procedures: The Trentingrana PDO Case.

Milk microbiota represents a key point in raw milk cheese production and contributes to the development of typical flavor and texture for each type of cheese. The aim of the present study was to evaluate the influence of chlorine products usage for cleaning and sanitizing the milking equipment on (i) raw milk microbiota; (ii) the deriving whey-starter microbiota; and (iii) Trentingrana Protected Designation of Origin (PDO) cheese microbiota and volatilome. Milk samples from three farms affiliated to a Trentingrana PDO cheese factory were collected three times per week during a 6-weeks period in which a sodium hypochlorite detergent (period C) was used and during a subsequent 6-weeks period of non-chlorine detergent usage (period NC). Samples were subjected to microbiological [Standard Plate Count; coliforms; coagulase-positive staphylococci; and lactic acid bacteria (LAB)] and metagenomic analysis (amplification of V3-V4 regions of 16S rRNA gene performed on Illumina MiSeq platform). In addition, cheese volatilome was determined by SPME-GC-MS. In the transition from period C to period NC, higher SPC and LAB counts in milk were recorded. Milk metagenomic analysis showed a peculiar distinctive microbiota composition for the three farms during the whole experimental period. Moreover, differences were highlighted comparing C and NC periods in each farm. A difference in microbial population related to chlorine usage in bulk milk and vat samples was evidenced. Moreover, chlorine utilization at farm level was found to affect the whey-starter population: the usually predominant Lactobacillus helveticus was significantly reduced during NC period, whereas Lactobacillus delbrueckii had the exact opposite trend. Alpha- and beta-diversity revealed a separation between the two treatment periods with a higher presence of L. helveticus, L. delbrueckii, and Streptococcus thermophilus in cheese samples after NC detergent period. Cheese volatilome analysis showed a slight decrease in lipolysis during C period in the inner part of the cheese wheel. Although preliminary, these results suggest a profound influence on milk and cheese microbiota, as well as on raw milk cheese production and quality, due to the use of chlorine. However, further studies will be needed to better understand the complex relationship between chlorine and microbiota along all the cheese production steps.

Effect of Different Percentage of Camelina sativa Cake in Laying Hens Diet: Performance, Welfare, and Eggshell Quality.

Although camelina [Camelina sativa (L.) Crantz] is a good source of protein, antioxidants, and polyunsaturated fatty acids, its antinutritional compounds limit its use in animal feeding. The aim of this study was to verify the effect of feeding laying hens with up to 20% of camelina cake from a breeding line containing a low level of glucosinolates on performance, welfare, and eggshell quality. Two hundred and forty Hy-Line® hens from 18 to 51 weeks of age were divided into three treatments: control (C), camelina cake 10% (CAM10), and camelina cake 20% (CAM20). Egg number was recorded daily, while egg weight, feed consumption, and mortality were recorded weekly. At 24 and 43 weeks of hen age, shell resistance to fracture was measured. Our results demonstrate no detrimental effects for CAM10 and CAM20 diets on feed intake, growth performance, and welfare. No difference in egg production was detected among the diets. The significant (p < 0.05) interaction of diet and age factors suggest that the addition of camelina cake, up to 20%, likely protects the eggshell of older hens. Our findings confirm that camelina cake might be an alternative and sustainable protein source for hens.