Data_Sheet_2_Lactic acid bacteria in cow raw milk for cheese production: Which and how many?.docx

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.</p

Data_Sheet_1_Lactic acid bacteria in cow raw milk for cheese production: Which and how many?.xlsx

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.</p

Abundance (%) of the <i>lacS</i> sequence types identified in this study.

<p>The abundance values are averaged for the three cheeses and only sequence types with an occurrence of at least 0.5% are showed in the color legend.</p

Nucleotide sequence alignment of the 28 <i>lacS</i> gene sequence types identified in this study.

<p>Sequences are aligned to the reference sequence of strain A147 (accession no. M23009). The ribosome binding site (RBS), the −35 and −10 sequences and the start codon are boxed. The putative <i>cre</i> site is underlined and aligned with the consensus sequence. The primer sequences are underlined.</p

Total number and average relative abundance (%) of the prevalent <i>lacS</i> sequence types identified in NWC and curd samples from Grana Padano (GP), Parmigiano Reggiano (PR) and Mozzarella manufactures from Caserta (MC) and Salerno (MS) area of production.

<p>Only types occurring at >0.5% abundance were included.</p

Pseudo-heatmap depicting distribution (%) of bacterial genera and species in NWC and curd samples from Grana Padano (GP), Parmigiano Reggiano (PR) and Mozzarella manufactures from Caserta (MC) and Salerno (MS) area of production.

<p>Only OTUs (except those reported in Fig. 1) occurring at >0.01% abundance in at least 2 samples were included.</p

Simplified illustration of possible cheese - microbe networks.

<p>Network nodes are color coded by cheese type. Only OTUs with abundance >0.1% were considered.</p

Data_Sheet_1_Selective enrichment of the raw milk microbiota in cheese production: Concept of a natural adjunct milk culture.docx

In cheese production, microorganisms are usually added at the beginning of the process as primary starters to drive curd acidification, while secondary microorganisms, with other pro-technological features important for cheese ripening, are added as selected cultures. This research aimed to investigate the possibilities of influencing and selecting the raw milk microbiota using artisanal traditional methods, providing a simple method to produce a natural supplementary culture. We investigated the production of an enriched raw milk whey culture (eRWC), a natural adjunct microbial culture produced from mixing an enriched raw milk (eRM) with a natural whey culture (NWC). The raw milk was enriched by spontaneous fermentation for 21 d at 10°C. Three milk enrichment protocols were tested: heat treatment before incubation, heat treatment plus salt addition, and no treatment. The eRMs were then co-fermented with NWC (ratio of 1:10) at 38°C for 6 h (young eRWC) and 22 h (old eRWC). Microbial diversity during cultures’ preparation was evaluated through the determination of colony forming units on selective growth media, and next-generation sequencing (16S rRNA gene amplicon sequencing). The enrichment step increased the streptococci and lactobacilli but reduced microbial richness and diversity of the eRMs. Although the lactic acid bacteria viable count was not significantly different between the eRWCs, they harbored higher microbial richness and diversity than NWC. Natural adjunct cultures were then tested in cheese making trials, following the microbial development, and assessing the chemical quality of the 120 d ripened cheeses. The use of eRWCs slowed the curd acidification in the first hours of cheese making but the pH 24 h after production settled to equal values for all the cheeses. Although the use of diverse eRWCs contributed to having a richer and more diverse microbiota in the early stages of cheese making, their effect decreased over time during ripening, showing an inferior effect to the raw milk microbiota. Even if more research is needed, the optimization of such a tool could be an alternative to the practice of isolating, geno-pheno-typing, and formulating mixed-defined-strain adjunct cultures that require knowledge and facilities not always available for artisanal cheese makers.</p

Minimalist Hybrid Ligand/Receptor-Based Pharmacophore Model for CXCR4 Applied to a Small-Library of Marine Natural Products Led to the Identification of Phidianidine A as a New CXCR4 Ligand Exhibiting Antagonist Activity

Here, we present a minimal hybrid ligand/receptor-based pharmacophore model (PM) for CXCR4, a chemokine receptor deeply involved in several pathologies, such as HIV infection, rheumatoid arthritis, cancer development/progression, and metastasization. This model, considerably simpler than those thus far proposed for this receptor, has been used to search for new CXCR4 inhibitors in a small marine natural product library available at ICB-CNR Institute (Pozzuoli, NA, Italy), since natural products, with their naturally selected chemical and functional diversity, represent a rich source of bioactive scaffolds; computational approaches allow searching for new scaffolds with a minimal waste of possibly precious natural product samples; and our “stripped-down” model substantially increases the probabilities of identifying potential hits even in small-sized libraries. This search, also validated by a systematic virtual screening of the same library, has led to the identification of a new CXCR4 ligand, phidianidine A (PHIA). Docking studies supported PHIA activity and suggested its possible binding modes to CXCR4. Using the CXCR4-expressing/CXCR7-negative GH4C1 cell line we show that PHIA inhibits CXCL12-induced DNA synthesis, cell migration, and ERK1/2 activation. The specificity of these effects was confirmed by the lack of PHIA activity in GH4C1 cells, in which siRNA highly reduces CXCR4 expression and the lack of cytoxicity of PHIA was also verified. Thus, PHIA represents a promising lead for a new family of CXCR4 modulators with wide margins of improvement in potency and specificity offered by the small and very simple underlying PM

<i>R</i>. <i>graveolens</i> a.e. induces apoptosis in A1 cells.

<p>(A) Cell cycle was analyzed by means of Tali image-based cytometry on proliferating A1 cells in control conditions (dark grey) and 48h after 1mg/ml <i>R</i>. <i>graveolens</i> a.e. treatment (light grey) *p<0.01 <i>vs</i> controls (B) Number of apoptotic nuclei/100 cells treated (R48) or not (CTRL) with 1 mg/ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours. *p<0.01 <i>vs</i> controls. (C-E) Caspase 3 activity expressed as absorbance at 400 nm in A1 cells (C), U87MG cells (D) and C6 cells (E) treated with vehicle (CTRL), 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 24 (R24) or 48 (R48) hours, 10μM PD98059 in combination with ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours (PD+R48), 1μM wortmannin in combination with ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours (W+R48) or the combination of the two inhibitors (PD+W+R48) for 48 hours. *p<0.01 <i>vs</i> control conditions.</p