Disclosing Lactobacillus delbrueckii subsp. bulgaricus intraspecific diversity in exopolysaccharides production.

Exopolysaccharides production by 3 ropy strains of Lactobacillus delbrueckii subsp. bulgaricus of dairy origin was evaluated in synthetic medium by combining different approaches: impedometric measurements, fluorescent microscopy and flow cytometry analyses. The evaluation of ΔE by impedometric measurement (E%max-E%40h) allowed the detection of EPS production in synthetic medium, but the differences in EPS production kinetic was highlighted by flow cytometry analysis and fluorescent microcopy. This approach enabled us to unravel the diversity in EPS synthesis and release into the laboratory medium during the growth of the strains. Our results showed that the maximum EPS production occurred after 8 h of incubation, when cells were in late exponential growth phase. Furthermore, flow cytometry analysis revealed that only part of the cell population could be identified as EPS producer or as EPS-bounded cell. Therefore, the combined approach used, allowed us to define at the same time the kinetics of EPS production and release by three strains belonging to the same species and, highlight that the production of EPS depends also on the number of EPS-producing cells within the same population. This approach could be useful for the selection of strains to be used as starter cultures in dairy products where EPS production is considered an important feature.

Probiotics and Covid-19.

Coronavirus disease 2019 (COVID-19) has become pandemic very rapidly at the beginning of 2020. In the rush to possible therapeutic options, probiotics administration has been proposed mainly based on indirect observation. Some evidence of COVID-19 effects on intestinal microbiota dysbiosis has been shown and probiotics have been considered for their efficacy in the management of respiratory tract viral infections. These observations could be reinforced by the more and more evident existence of a lung-gut axis, suggesting the modulation of gut microbiota among the approaches to the COVID-19 prevention and treatment. As different possible roles of probiotics in this extremely severe illness have been contemplated, the aim of this work is to collect all the currently available information related to this topic, providing a starting point for future studies focussing on it.

Safety and technological application of autochthonous Streptococcus thermophilus cultures in the buffalo Mozzarella cheese.

Thermophilic and mesophilic lactic acid bacteria (LAB), such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, and Lactococcus lactis, play a crucial role in the technological and sensory quality of Mozzarella cheese. In this study, the safety (genes encoding virulence factors and antibiotic resistance) and acidifying activity of autochthonous S. thermophilus cultures were evaluated in order to choose the most suitable strain for industrial application. The safe and good acidifying culture was tested in two buffalo Mozzarella cheese batches: Mozzarella cheeses produced with autochthonous culture (SJRP107) and commercial culture (STM5). The cultivable LAB was evaluated by culture-dependent method (plate counting) and the quantification of S. thermophilus cultures (commercial and autochthonous) were evaluated by culture-independent method RealT-qPCR (real-time quantitative polymerase chain reaction). The texture, physicochemical and proteolytic properties of the Mozzarella cheeses were similar for both batches. The nonstarter LAB count was higher during manufacture than in the storage, and the RealT-qPCR indicated the presence of S. thermophilus culture until the end of storage. S. thermophilus SJRP107 presented high potential for safety application in the production of Mozzarella cheese. Furthermore, considering the culture characteristics and their relationship with product quality, further studies could be helpful to determine their effect on the sensory characteristics of the cheese.

Potential prebiotic effect of a long-chain dextran produced by Weissella cibaria: an in vitro evaluation.

Dextrans are homopolysaccharides of D-glucose units produced by lactic acid bacteria. They have several technological applications and potential utilisation in positively modulating gut microbiota is attracting increasing attention. Whereas the prebiotic activity of low polymerisation degree (DP) dextrans has been established, high DP dextrans still deserve deeper investigation. In the present study, a long linear chain dextran produced by Weissella cibaria was compared to inulin with regards to the growth of specific health-related taxa and to the production of organic acids in pH-controlled batch cultures of intestinal microbiota. qPCR quantification of Lactobacillus, Bifidobacterium, Prevotella, Bacteroides fragilis, and Faecalibacterium prausnitzii revealed differences in their relative abundance, depending on the carbon source, that reflected the pattern of fermentation products determined by HPLC. Dextran mainly enhanced the relative amount of Prevotella and Bacteroides, consistently with a favourable acetate-propionate ratio suggesting a promising utilisation as functional ingredient in the food industry.

Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products.

Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in "Food Genomics;" a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or "end-point" PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.

Effective identification of Lactobacillus casei group species: genome-based selection of the gene mutL as the target of a novel multiplex PCR assay.

Lactobacillus casei,Lactobacillus paracasei and Lactobacillusrhamnosus form a closely related taxonomic group (the L. casei group) within the facultatively heterofermentative lactobacilli. Strains of these species have been used for a long time as probiotics in a wide range of products, and they represent the dominant species of nonstarter lactic acid bacteria in ripened cheeses, where they contribute to flavour development. The close genetic relationship among those species, as well as the similarity of biochemical properties of the strains, hinders the development of an adequate selective method to identify these bacteria. Despite this being a hot topic, as demonstrated by the large amount of literature about it, the results of different proposed identification methods are often ambiguous and unsatisfactory. The aim of this study was to develop a more robust species-specific identification assay for differentiating the species of the L. casei group. A taxonomy-driven comparative genomic analysis was carried out to select the potential target genes whose similarity could better reflect genome-wide diversity. The gene mutL appeared to be the most promising one and, therefore, a novel species-specific multiplex PCR assay was developed to rapidly and effectively distinguish L. casei, L. paracasei and L. rhamnosus strains. The analysis of a collection of 76 wild dairy isolates, previously identified as members of the L. casei group combining the results of multiple approaches, revealed that the novel designed primers, especially in combination with already existing ones, were able to improve the discrimination power at the species level and reveal previously undiscovered intraspecific biodiversity.

Invited review: Microbial evolution in raw-milk, long-ripened cheeses produced using undefined natural whey starters.

The robustness of the starter culture during cheese fermentation is enhanced by the presence of a rich consortium of microbes. Natural starters are consortia of microbes undoubtedly richer than selected starters. Among natural starters, natural whey starters (NWS) are the most common cultures currently used to produce different varieties of cheeses. Undefined NWS are typically used for Italian cooked, long-ripened, extra-hard, raw milk cheeses, such as Parmigiano Reggiano and Grana Padano. Together with raw milk microbiota, NWS are responsible for most cheese characteristics. The microbial ecology of these 2 cheese varieties is based on a complex interaction among starter lactic acid bacteria (SLAB) and nonstarter lactic acid bacteria (NSLAB), which are characterized by their different abilities to grow in a changing substrate. This review aims to summarize the latest findings on Parmigiano Reggiano and Grana Padano to better understand the dynamics of SLAB, which mainly arise from NWS, and NSLAB, which mainly arise from raw milk, and their possible role in determining the characteristics of these cheeses. The review is presented in 4 main sections. The first summarizes the main microbiological and chemical properties of the ripened cheese as determined by cheese-making process variables, as these variables may affect microbial growth. The second describes the microbiota of raw milk as affected by specific milk treatments, from milking to the filling of the cheese milk vat. The third describes the microbiota of NWS, and the fourth reviews the knowledge available on microbial dynamics from curd to ripened cheese. As the dynamics and functionality of complex undefined NWS is one of the most important areas of focus in current food microbiology research, this review may serve as a good starting point for implementing future studies on microbial diversity and functionality of undefined cheese starter cultures.

The role of airways microbiota on local and systemic diseases: a rationale for probiotics delivery to the respiratory tract.

INTRODUCTION: Recent discoveries in the field of lung microbiota have enabled the investigation of new therapeutic interventions involving the use of inhaled probiotics. AREAS COVERED: This review provides an overview of what is known about the correlation between airway dysbiosis and the development of local and systemic diseases, and how this knowledge can be exploited for therapeutic interventions. In particular, the review focused on attempts to formulate probiotics that can be deposited directly on the airways. EXPERT OPINION: Despite considerable progress since the emergence of respiratory microbiota restoration as a new research field, numerous clinical implications and benefits remain to be determined. In the case of local diseases, once the pathophysiology is understood, manipulating the lung microbiota through probiotic administration is an approach that can be exploited. In contrast, the effect of pulmonary dysbiosis on systemic diseases remains to be clarified; however, this approach could represent a turning point in their treatment.

Development of inhalation powders containing lactic acid bacteria with antimicrobial activity against Pseudomonas aeruginosa.

OBJECTIVES: The aim of the project was to develop and characterise powders containing a probiotic (Lactiplantibacillus plantarum [Lpb. plantarum], Lacticaseibacillus rhamnosus, or Lactobacillus acidophilus) to be administered to the lung for the containment of pathogen growth in patients with lung infections. METHODS: The optimised spray drying process for the powder manufacturing was able to preserve viability of the bacteria, which decreased of only one log unit and was maintained up to 30 days. RESULTS: Probiotic powders showed a high respirability (42%-50% of particles had a size < 5 µm) suitable for lung deposition and were proven safe on A549 and Calu-3 cells up to a concentration of 107 colony-forming units/mL. The Lpb. plantarum adhesion to both cell lines tested was at least 10%. Surprisingly, Lpb. plantarum powder was bactericidal at a concentration of 106 colony-forming units/mL on P. aeruginosa, whereas the other two strains were bacteriostatic. CONCLUSION: This work represents a promising starting point to consider a probiotic inhalation powder a value in keeping the growth of pathogenic microflora in check during the antibiotic inhalation therapy suspension in cystic fibrosis treatment regimen. This approach could also be advantageous for interfering competitively with pathogenic bacteria and promoting the restoration of the healthy microbiota.

Diversity and dynamic of lactic acid bacteria strains during aging of a long ripened hard cheese produced from raw milk and undefined natural starter.

The aim of this study was to explore diversity and dynamic of indigenous LAB strains associated with a long ripened hard cheese produced from raw milk and undefined natural starter such as PDO Grana Padano cheese. Samples of milk, curd, natural whey culture and cheeses (2nd, 6th, 9th and 13th months of ripening) were collected from 6 cheese factories in northern Italy. DNA was extracted from each sample and from 194 LAB isolates. tRNA(Ala)-23S rDNA-RFLP was applied to identify isolates. Strain diversity was assessed by (GTG)5 rep-PCR and RAPD(P1)-PCR. Finally, culture-independent LH-PCR (V1-V2 16S-rDNA), was considered to explore structure and dynamic of the microbiota. Grana Padano LAB were represented mainly by Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus helveticus and Pediococcus acidilactici, while the structure and dynamic of microbiota at different localities was specific. The strength of this work is to have focused the study on isolates coming from more than one cheese factories rather than a high number of isolates from one unique production. We provided a valuable insight into inter and intraspecies diversity of typical LAB strains during ripening of traditional PDO Grana Padano, contributing to the understanding of specific microbial ecosystem of this cheese.

Feeding Lactic Acid Bacteria with Different Sugars: Effect on Exopolysaccharides (EPS) Production and Their Molecular Characteristics.

Exopolysaccharides (EPS) are complex molecules produced by some microorganisms and used in foods as texturizers and stabilizers, their properties depending on their chemical structure. In this work, three different lactic acid bacteria (LAB), were tested for their ability to produce EPS, by using five different mono- and disaccharides as their sole carbon source. The growth and acidifying ability were analysed, the EPSs were quantified by the official method AOAC 991.43, and their chemical structure was investigated. The amount of EPS varied from 0.71 g/L to 2.38 g/L, and maltose was the best sugar for EPS production by Lacticaseibacillus paracasei 2333. Lacticaseibacillus rhamnosus 1019 produced the highest amount when fed with lactose, whereas the EPS amount of Lactobacillus bulgaricus 1932 was not significantly different depending on the sugar type. The EPS chains consisted of fructose, galactose, glucose, mannose, ribose, glucosamine, galactosamine, and in some cases rhamnose in different proportions, depending on the strain and carbon source. The molecular weight of EPS ranged from <10 KDa to >500 KDa and was again highly dependent on the strain and the sugar used, suggesting the possibility of growing different strains under different conditions to obtain EPS with different potential applications in the food system.

Lactobacillus delbrueckii subsp. bulgaricus derivatives for 3D printed alginate/hyaluronic acid self-crosslinking hydrogels: Manufacturing and wound healing potential.

Derivatives [i.e. proteins and exopolysaccharides (EPS)] from Lactobacillus delbrueckii subsp. bulgaricus (LB) were extracted, characterized, and for the first time used in the production of novel self-crosslinking 3D printed alginate/hyaluronic acid (ALG/HA) hydrogels, as high-value functional biomaterials with therapeutic potentials in regenerative medicine applications. Derivatives coming from two different LB strains, LB1865 and LB1932, were tested in-vitro and compared for their cytotoxicity and effect on proliferation and migration on human fibroblast. EPS received particular attention as showing relevant dose-dependent cytocompatibility against the human fibroblast. The derivatives showed an ability to increase cell proliferation and migration, quantifiable between 10 and 20 % if compared to controls, with higher values for the derivatives obtained from the LB1932 strain. These were explained by liquid chromatography-mass spectrometry targeted protein biomarker analysis as a decrease in matrix-degrading and proapoptotic proteins, associated with an increase in collagen and antiapoptotic proteins production. LB1932 enriched hydrogel was found to be of benefit compared to control dressings, giving the more promising results as potential for in vivo skin wound healing tests.

Lacticaseibacillus Strains Isolated from Raw Milk: Screening Strategy for Their Qualification as Adjunct Culture in Cheesemaking.

The microbial ecology fundamentals of raw milk and long-ripened cheeses consist of a complex interaction between starter lactic acid bacteria (SLAB) and non-starter LAB (NSLAB). Although NSLAB aromatic properties are paramount, other phenotypic traits need to be considered for their use as adjunct cultures, such as the capability to endure technological parameters encountered during cheesemaking. The present study focused on the isolation and characterization of NSLAB from spontaneously fermented raw cow's milk coming from 20 dairies that produce Grana Padano PDO cheese. From 122 isolates, the screening process selected the 10 most diverse strains belonging to Lacticaseibacillus spp. to be phenotypically characterized. The strains were tested for their growth performance in milk in combination with the application of technological stresses, for their ability to produce volatile compounds after their growth in milk, and for their ability to use different nutrient sources and resist chemicals. The complex characterization qualified the strains 5959_Lbparacasei and 5296_Lbparacasei as the best candidates to be used as adjunct strains in the production of raw milk and long-ripened cheeses, provided that antibiotic resistance is measured before their employment. Other strains with interesting aromatic capabilities but lower heat resistance were 5293_Lbparacasei, 5649_Lbparacasei and 5780_Lbparacasei, which could be candidates as adjunct strains for uncooked cheese production.

Lactic acid bacteria in cow raw milk for cheese production: Which and how many?

Lactic Acid Bacteria (LAB) exert a fundamental activity in cheese production, as starter LAB in curd acidification, or non-starter LAB (NSLAB) during ripening, in particular in flavor formation. NSLAB originate from the farm and dairy environment, becoming natural contaminants of raw milk where they are present in very low concentrations. Afterward, throughout the different cheesemaking processes, they withstand chemical and physical stresses becoming dominant in ripened cheeses. However, despite a great body of knowledge is available in the literature about NSLAB effect on cheese ripening, the investigations regarding their presence and abundance in raw milk are still poor. With the aim to answer the initial question: "which and how many LAB are present in cow raw milk used for cheese production?," this review has been divided in two main parts. The first one gives an overview of LAB presence in the complex microbiota of raw milk through the meta-analysis of recent taxonomic studies. In the second part, we present a collection of data about LAB quantification in raw milk by culture-dependent analysis, retrieved through a systematic review. Essentially, the revision of data obtained by plate counts on selective agar media showed an average higher concentration of coccoid LAB than lactobacilli, which was found to be consistent with meta-taxonomic analysis. The advantages of the impedometric technique applied to the quantification of LAB in raw milk were also briefly discussed with a focus on the statistical significance of the obtainable data. Furthermore, this approach was also found to be more accurate in highlighting that microorganisms other than LAB are the major component of raw milk. Nevertheless, the variability of the results observed in the studies based on the same counting methodology, highlights that different sampling methods, as well as the "history" of milk before analysis, are variables of great importance that need to be considered in raw milk analysis.

Peptide profile of Parmigiano Reggiano cheese after simulated gastrointestinal digestion: From quality drivers to functional compounds.

Time of ripening has a strong impact on shaping the valuable and recognizable characteristics of long-ripened types of cheese such as Parmigiano Reggiano (PR) due to the interrelationship between microbiota and proteolysis that occurs during ripening. The derived peptide profile is linked to cheese quality and represents the canvas for enzymes upon digestion, which could be responsible for the release of potentially bioactive peptides (BPs). In this study, we aimed at investigating the presence of BP in 72 PR cheese samples of different ripening times, from curd to 24 months of ripening, produced in six different dairies, and following their fate after simulated gastrointestinal digestion. A small number of peptide sequences sharing 100% similarity with known antimicrobial, antioxidant, and ACE-inhibitor sequences were found in PR cheeses, while a higher number of potential BPs were found after their simulated gastrointestinal digestion, in different amounts according to ripening time. Taking advantage of the complex organization of the sampling plan, we were able to follow the fate of peptides considered quality drivers during cheese ripening to their release as functional compounds upon digestion.

"Re-engineering of a food oven for thermal sanitization of Personal Protective Equipment against Sars-CoV-2 virus".

One of the main issues addressed by the recent COVID-19 pandemic which affected the whole world is the availability of Personal Protective Equipment (PPE) (e.g., face masks, white coats, or disposable gloves). This issue impacts on sustainability from different perspectives, such as more generated waste or environmental pollution, both for manufacturing and disposal, or more inequalities deriving from who can afford and access PPE and who cannot, since many shortages were recorded during the pandemic as well as fluctuating unit prices. Moreover, quite often PPE intended for single use are improperly used more times, thus generating a biological risk of infection. In an attempt to propose an innovative solution to face this problem, in this paper the re-design of an oven originally intended for food purposes is presented, with the aim of operating a thermal sanitization of PPE. The machinery and its components are detailed, together with physical and microbiological tests performed on non-woven PPE to assess the effect of treatment on mechanical properties and viral load. The pilot machinery turned out to be effective in destroying a bovine coronavirus at 95 °C and thus reducing contaminating risk in one hour without compromising the main properties of PPE, opening perspectives for the commercialization of the solution in the near future.

Arthrospira platensis Extract: A Non-Invasive Strategy to Obtain Adjunct Attenuated Cultures.

This study aims at proposing the use of Arthrospira platensis, commonly known as Spirulina, extract as a non-invasive method to attenuate the growth rate of non-starter adjunct cultures, thus preventing the over-acidification that may occur during cheese manufacturing. A preliminary screening using four different concentrations (0.20%, 0.30%, 0.50%, and 0.70%) of A. platensis extract and four starter and three non-starter lactic acid bacteria strains was performed by impedometric analysis. This allowed us to select one starter and one non-starter strain to be used in the in vitro simulation of a co-culture in milk with the best antimicrobial concentration (0.3%). The growth dynamics of the two selected strains, starter Lactococcus lactis 1426 and non-sarter Lacticaseibacillus rhamnosus 1473, co-cultured for 120 h was monitored by three different approaches: (i) plate counting on M17, for the enumeration of lactococci, and MRS for lactobacilli; (ii) fluorescence microscopic counting of viable and non-viable coccoid Lactococcus lactis 1426 and rod-shaped Lacticaseibacillus rhamnosus 1473 cells; (iii) the overall estimation of co-culture growth behavior by impedometric parameters Lag, Rate, and yEnd. All the data obtained from the in vitro simulation were in agreement, revealing that a slowdown of non-starter growth occurred, while the starter strain was not affected, or slightly stimulated, from the antimicrobial presence. In particular, the growth of Lb. rhamnosus 1473 was delayed without adversely compromise the cells' integrity, connected with metabolic functions, showing a great potential for use in cheese production.

Eating Fermented: Health Benefits of LAB-Fermented Foods.

Lactic acid bacteria (LAB) are involved in producing a considerable number of fermented products consumed worldwide. Many of those LAB fermented foods are recognized as beneficial for human health due to probiotic LAB or their metabolites produced during food fermentation or after food digestion. In this review, we aim to gather and discuss available information on the health-related effects of LAB-fermented foods. In particular, we focused on the most widely consumed LAB-fermented foods such as yoghurt, kefir, cheese, and plant-based products such as sauerkrauts and kimchi.

Inactivation of indicator microorganisms and biological hazards by standard and/or alternative processing methods in Category 2 and 3 animal by-products and derived products to be used as organic fertilisers and/or soil improvers.

The European Commission requested EFSA to assess if different thermal processes achieve a 5 log10 reduction in Enterococcus faecalis or Salmonella Senftenberg (775W) and (if relevant) a 3 log10 reduction in thermoresistant viruses (e.g. Parvovirus) as well as if different chemical processes achieve a 3 log10 reduction of eggs of Ascaris sp., in eight groups of Category 2 and 3 derived products and animal by-products (ABP). These included (1) ash derived from incineration, co-incineration and combustion; (2) glycerine derived from the production of biodiesel and renewable fuels; (3) other materials derived from the production of biodiesel and renewable fuels; (4) hides and skins; (5) wool and hair; (6) feathers and down; (7) pig bristles; and (8) horns, horn products, hooves and hoof products. Data on the presence of viral hazards and on thermal and chemical inactivation of the targeted indicator microorganisms and biological hazards under relevant processing conditions were extracted via extensive literature searches. The evidence was assessed via expert knowledge elicitation. The certainty that the required log10 reductions in the most resistant indicator microorganisms or biological hazards will be achieved for each of the eight groups of materials mentioned above by the thermal and/or chemical processes was (1) 99-100% for the two processes assessed; (2) 98-100% in Category 2 ABP, at least 90-99% in Category 3 ABP; (3) 90-99% in Category 2 ABP; at least 66-90% in Category 3 ABP; (4) 10-66% and 33-66%; (5) 1-33% and 10-50%; (6) 66-90%; (7) 33-66% and 50-95%; (8) 66-95%, respectively. Data generation on the occurrence and reduction of biological hazards by thermal and/or chemical methods in these materials and on the characterisation of the usage pathways of ABP as organic fertilisers/soil improvers is recommended.

Wild Lactobacillus casei Group Strains: Potentiality to Ferment Plant Derived Juices.

Plant derived beverages have recently gained consumers' interest, particularly due to their intrinsic functional properties. They can also act as non-dairy carriers for probiotics and prebiotics, meeting the needs of lactose allergic/intolerant people and vegans. Direct fermentation of fruit and vegetables juices by probiotic lactic acid bacteria could be a tool to increase safety, shelf-life, nutrients bioavailability and to improve sensorial features of plant derived juices. This study aims to screen wild Lactobacillus casei-group strains isolated from dairy matrices for probiotic features, such as acid and bile salts resistance, and test them for the potentiality to ferment celery and orange juices. Strains' ability to produce exopolysaccharides (EPS) in situ is also checked. These evaluations were performed for the first time in fruit and vegetables matrices by means of an impedometric analysis, recently shown to be a suitable and rapid method to measure microorganisms' growth, acidification performances and EPS production. This study allowed the selection of three potentially probiotic L. casei-group wild strains able to ferment fruit and vegetable juices and also producing EPS. These strains with three-in-one abilities could be used to produce new functional fermented plant derived juices.