Natural Whey Starter Research Articles

Beneficial effect of GABA-producing Lactiplantibacillus strain LPB145 isolated from cheese starters evaluated in anxiety- and depression-like behaviours in rats.

In a previous study, we reported the in vitro potential probiotic and gamma-aminobutyric acid (GABA) production, of several strains from a collection of Lactiplantibacillus (Lpb) strains within the community of natural whey starters from the artisanal cheese industry. GABA is a non-protein amino acid widely distributed in nature and produced in animals, plants, and microorganisms. However, the best known role of GABA is its function as the major inhibitory neurotransmitter of the central nervous system. Preclinical and clinical evidence suggests that the GABAergic system has a relevant role in mental health disorders, such as anxiety and major depression. The modulation of the GABAergic system has been suggested as a potential strategy for treatment, one such mechanism of modulation is the influence of the microbiota-gut-brain axis through probiotic treatments. The present study was designed to investigate the in vivo probiotic potential of LPB145, a Lactiplantibacillus strain previously characterised as a GABA-producing potentially probiotic strain. Therefore, we evaluated the behavioural effects of chronic oral administration of LPB145 on rats' anxiety- and depression-like behaviours, using the elevated plus maze, open field, and the forced swimming test. The impact of LPB145 strain treatment on the gut microbiota structure and diversity was assessed to discern a possible mechanism of action of the LPB145 treatment through the microbiota-gut-brain axis. Our results showed that LPB145 administration induced an antidepressive-like behaviour without changes in locomotor activity. In contrast, the treatment did not modify the experimental anxiety. The structure and diversity of the intestinal microbiota remained unaffected by the treatment when compared to the control. However, specific clades that could be implicated in the behavioural changes did show differences in their relative abundance. These findings provide evidence regarding the potential of probiotic strains isolated from alimentary sources, to modulate the microbiota-gut-brain axis and positively impact mental health.

Dynamic microbial and metabolic changes during Apulian Caciocavallo cheesemaking and ripening produced according to a standardized protocol

The microbiota of a cheese play a critical role in influencing its sensory and physicochemical properties. In this study, traditional Apulian Caciocavallo cheeses coming from 4 different dairies in the same area and produced following standardized procedures were examined, as well as the different bulk milks and natural whey starter (NWS) cultures used. Moreover, considering the cheese wheels as the blocks of Caciocavallo cheeses as whole, these were characterized at different layers (i.e., core, under-rind, and rind) of the block using a multi-omics approach. In addition to physical-chemical characterization, culturomics, quantitative PCR, metagenomics, and metabolomics analysis were carried out after salting and throughout the ripening time (2 mo) to investigate major shifts in the succession of the microbiota and flavor development. Culture-dependent and 16S rRNA metataxonomics results clearly clustered samples based on microbiota biodiversity related to the production dairy plant as a result of the use of different NWS or the intrinsic conditions of each production site. At the beginning of the ripening, cheeses were dominated by Lactobacillus, and in 2 dairies (Art and SdC), Streptococcus genera were associated with the NWS. The analysis allowed us to show that although the diversity of identified genera did not change significantly between the rind, under-rind, and core fractions of the same samples, there was an evolution in the relative abundance and absolute quantification, modifying and differentiating profiles during ripening. The real-time PCR, also known as quantitative or qPCR, mainly differentiated the temporal adaptation of those species originating from bulk milks and those provided by NWS. The primary starters detected in NWS and cheeses contributed to the high relative concentration of 1-butanol, 2-butanol, 2-heptanol, 2-butanone, acetoin, delta-dodecalactone, hexanoic acid ethyl ester, octanoic acid ethyl ester, and volatile free fatty acids during ripening, whereas cheeses displaying low abundances of Streptococcus and Lactococcus (dairy Del) had a lower total concentration of acetoin compared with Art and SdC. However, the subdominant strains and nonstarter lactic acid bacteria present in cheeses are responsible for the production of secondary metabolites belonging to the chemical classes of ketones, alcohols, and organic acids, reaffirming the importance and relevance of autochthonous strains of each dairy plant although only considering a delimited production area.

The Microbial Community of Natural Whey Starter: Why Is It a Driver for the Production of the Most Famous Italian Long-Ripened Cheeses?

The remarkable global diversity in long-ripened cheese production can be attributed to the adaptability of the cheese microbiota. Most cheese types involve intricate microbial ecosystems, primarily represented by lactic acid bacteria (LAB). The present study aims to review the microbial community’s diversity in dairy fermentation processes, focusing on two famous Italian cheeses, Grana Padano and Parmigiano Reggiano, produced using natural whey starter (NWS). NWS, created by retaining whey from the previous day’s cheese batches, forms a microbiological connection between daily cheese productions. Through this technique, a dynamic microbiota colonizes the curd and influences cheese ripening. The back-slopping method in NWS preparation ensures the survival of diverse biotypes, providing a complex microbial community in which interactions among microorganisms are critical to ensuring its technological functionality. As highlighted in this review, the presence of microbial cells alone does not guarantee technological relevance. Critical microorganisms can grow and colonize the curd and cheese. This complexity enables NWS to adapt to artisanal production technologies while considering variations in raw milk microbiota, inhibitory compounds, and manufacturing conditions. This critical review aims to discuss NWS as a key factor in cheese making, considering microbial communities’ ability to evolve under different selective pressures and biotic and abiotic stresses.

Probiotic Properties of Lactococcus lactis Strains Isolated from Natural Whey Starter Cultures.

Lactococcus lactis is a lactic acid bacterium (LAB), generally recognized as safe, and has been widely used in the food industry, especially in fermented dairy products. Numerous studies have evaluated the technological and probiotic properties of lactococci; however, few studies have reported the probiotic characteristics of L. lactis strains isolated from dairy products. In this work, probiotic potential, including survival in simulated gastric juice, tolerance to bile salts, hydrophobicity, and auto- and co-aggregation, was evaluated in L. lactis strains from natural whey starter cultures. The results highlighted the potential probiotic properties of some strains under study, which showed high values of hydrophobicity and auto-aggregation and low values of co-aggregation with the tested pathogenic strains. In addition, studies of safety parameters, such as antibiotic susceptibility and haemolytic activity, confirmed the safety status of all strains under study. Finally, the four most promising strains were investigated for their ability to inhibit the enteroinvasive Escherichia coli (EIEC) and Salmonella Typhimurium adhesion to epithelial cells, using a model of co-cultured epithelial cells. The results demonstrated that L. lactis strains A3-A5-I4-I7 showed the ability to compete with pathogens as well as the ability to exert a protective effect on cells previously infected with E. coli or S. Typhimurium. The identification of new probiotic LAB strains from dairy products aims to produce novel functional foods.

Microbial composition and viability of natural whey starters used in PDO Comté cheese-making

Natural whey starters (NWS) are cultures with undefined multiple-strains species commonly used to speed up the fermentation process of cheeses. The aim of this study was to explore the diversity and the viability of Comté cheese NWS microbiota. Culture-dependent methods, i.e. plate counting and genotypic characterization, and culture-independent methods, i.e. qPCR, viability-qPCR, fluorescence microscopy and DNA metabarcoding, were combined to analyze thirty-six NWS collected in six Comté cheese factories at two seasons. Our results highlighted that NWS were dominated by Streptococcus thermophilus (ST) and thermophilic lactobacilli. These species showed a diversity of strains based on Rep-PCR. The dominance of Lactobacillus helveticus (LH) over Lactobacillus delbrueckii (LD) varied depending on the factory and the season. This highlighted two types of NWS: the type-ST/LD (LD > LH) and the type-ST/LH (LD < LH). The microbial composition varied depending on cheese factory. One factory was distinguished by its level of culturable microbial groups (ST, enterococci and yeast) and its fungi diversity. The approaches used to estimate the viability showed that most NWS cells were viable. Further investigations are needed to understand the microbial diversity of these NWS.

Cultivable microbial diversity, peptide profiles, and bio-functional properties in Parmigiano Reggiano cheese.

Lactic acid bacteria (LAB) communities shape the sensorial and functional properties of artisanal hard-cooked and long-ripened cheeses made with raw bovine milk like Parmigiano Reggiano (PR) cheese. While patterns of microbial evolution have been well studied in PR cheese, there is a lack of information about how this microbial diversity affects the metabolic and functional properties of PR cheese. To fill this information gap, we characterized the cultivable fraction of natural whey starter (NWS) and PR cheeses at different ripening times, both at the species and strain level, and investigated the possible correlation between microbial composition and the evolution of peptide profiles over cheese ripening. The results showed that NWS was a complex community of several biotypes belonging to a few species, namely, Streptococcus thermophilus, Lactobacillus helveticus, and Lactobacillus delbrueckii subsp. lactis. A new species-specific PCR assay was successful in discriminating the cheese-associated species Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, and Lacticaseibacillus zeae. Based on the resolved patterns of species and biotype distribution, Lcb. paracasei and Lcb. zeae were most frequently isolated after 24 and 30 months of ripening, while the number of biotypes was inversely related to the ripening time. Peptidomics analysis revealed more than 520 peptides in cheese samples. To the best of our knowledge, this is the most comprehensive survey of peptides in PR cheese. Most of them were from β-caseins, which represent the best substrate for LAB cell-envelope proteases. The abundance of peptides from β-casein 38-88 region continuously increased during ripening. Remarkably, this region contains precursors for the anti-hypertensive lactotripeptides VPP and IPP, as well as for β-casomorphins. We found that the ripening time strongly affects bioactive peptide profiles and that the occurrence of Lcb. zeae species is positively linked to the incidence of eight anti-hypertensive peptides. This result highlighted how the presence of specific LAB species is likely a pivotal factor in determining PR functional properties.

New insights into nonenzymatic browning of non‐PDO Italian hard cheese through a metabolomics approach

Nonenzymatic browning poses a severe problem to the dairy industry, causing economic losses. In this work, an untargeted metabolomics approach based on ultra‐high‐performance liquid chromatography coupled with Orbitrap mass spectrometry was used to investigate the discoloration found on the inner parts of nonprotected designation of origin Italian hard cheese. Alkyl‐pyrazines, imidazo‐quinoxalines and β‐carbolines were the best markers, suggesting the involvement of microbial methylglyoxal in modulating the browning defect. A better understanding of the microbial composition of natural whey starter could be of interest in future studies to better elucidate the biochemical mechanisms involved and to help generate discriminant metabolites.

Proteomics Parameters for Assessing Authenticity of Grated Grana Padano PDO Cheese: Results from a Three-Year Survey.

Assessing the authenticity of PDO cheeses is an important task because it allows consumer expectations to be fulfilled and guarantees fair competition for manufacturers. A 3-year survey was carried out, analyzing 271 samples of grated Grana Padano (GP) PDO cheese collected on the European market. Previously developed analytical methods based on proteomics approaches were adopted to evaluate the compliance of market samples with selected legal requirements provided by the specification for this cheese. Proteolysis follows highly repeatable pathways in GP cheese due to the usage of raw milk, natural whey starter, and consistent manufacturing and ripening conditions. From selected casein breakdown products, it is possible to calculate the actual cheese age (should be >9 months) and detect the presence of excess rind (should be <18%). Furthermore, due to the characteristic pattern of free amino acids established for GP, distinguishing it from closely related cheese varieties is feasible. Cheese age ranged from 9 to 25 months and was correctly claimed on the label. Based on the amino acid pattern, three samples probably contained defective cheese and there was only one imitation cheese. Few samples (9%) were proven to contain some excess rind. Overall, this survey highlighted that the adopted control parameters can assure the quality of grated GP.

Untargeted metabolomics provide new insights into the implication of Lactobacillus helveticus strains isolated from natural whey starter in methylglyoxal-mediated browning

Hard cheeses may occasionally show a brown discolouration during ripening due to multifactorial phenomena that involve bacteria and give rise to pyrazines arising from methylglyoxal. The present work aimed at developing a novel approach to investigate the role of natural starters in browning. To this object, 11 strains of L. helveticus were incubated in a medium containing 10 % rennet casein dissolved in whey, and then growth was monitored by measuring pH and number of genomes/mL. Browning was assessed through CIELab analysis, methylglyoxal production was determined by targeted mass spectrometry, and untargeted metabolomics was used to extrapolate marker compounds associated with browning discoloration. The medium allowed the growth of all the strains tested and differences in colour were observed, especially for strain A7 (ΔE* value 15.92 ± 0.27). Noteworthy, this strain was also the higher producer of methylglyoxal (2.44 µg/mL). Metabolomics highlighted pyrazines and β-carboline compounds as markers of browning at 42 °C and 16 °C, respectively. Moreover, multivariate statistics pointed out differences in free amino acids and oligopeptides linked to proteolysis, while 1,2-propanediol and S-Lactoylglutathione suggested specific detoxification route in methylglyoxal-producing strains. Our model allowed detecting differences in browning amid strains, paving the way towards the study of individual L. helveticus strains to identify the variables leading to discoloration or to study the interaction between different strains in natural whey starters.

Microbial Community Profiling from Natural Whey Starter to Mozzarella among Different Artisanal Dairy Factories in Apulia Region (Italy)

Mozzarella is one of the most popular unripened Apulian cheeses. Knowledge about microbial composition and variability of artisanal mozzarella and its production chain is increasingly growing. In this study, microbial communities from natural whey starters to end products from four renowned Apulian artisanal dairy factories have been explored by means of 16S metagenomics. The chemical properties of mozzarella samples were also detected and analyzed. Lactobacillus is the core acidifying component of the used starters, while some psychrophilic or contaminants bacteria appear in site-specific products. Biodiversity was found to be quite similar between the whey and mozzarella sample pools, while a significant variability among production sites (factories) has been detected. Furthermore, mozzarella microbial diversity seems to be in positive correlation with its lactic acid content. Targeted metagenomics would then be a powerful and relatively quick technique to characterize the microbiological variability of traditional milk-based foods.

The microbiota of Mozzarella di Bufala Campana PDO cheese: a study across the manufacturing process.

Mozzarella di Bufala Campana PDO cheese (MBC) is a globally esteemed Italian cheese. The traditional cheesemaking process of MBC relies on natural whey starter culture, water buffalo's milk, and the local agroecosystem. In this study, the microbial ecology of intermediate samples of MBC production, coming from two dairies with slightly different cheesemaking technology (dairy M large producer, and dairy C medium-small), was investigated using 16S rRNA amplicon sequencing. This research aimed to provide insights into the dynamics of microbial consortia involved in various cheesemaking steps. All samples, except for raw buffalo milk, exhibited a core microbiome predominantly composed of Streptococcus spp. and Lactobacillus spp., albeit with different ratios between the two genera across the two MBC producers. Notably, the microbiota of the brine from both dairies, analyzed using 16S amplicon sequencing for the first time, was dominated by the Lactobacillus and Streptococcus genera, while only dairy C showed the presence of minor genera such as Pediococcus and Lentilactobacillus. Intriguingly, the final mozzarella samples from both producers displayed an inversion in the dominance of Lactobacillus spp. over Streptococcus spp. in the microbiota compared to curd samples, possibly attributable to the alleviation of thermal stress following the curd stretching step. In conclusion, the different samples from the two production facilities did not exhibit significant differences in terms of the species involved in MBC cheesemaking. This finding confirms that the key role in the MBC cheesemaking process lies with a small-sized microbiome primarily composed of Streptococcus and Lactobacillus spp.

Application of flow cytometry for rapid bacterial enumeration and cells physiological state detection to predict acidification capacity of natural whey starters

Natural whey starter cultures are undefined microbial communities mainly consisting of thermophilic lactic acid bacteria (LAB). The technological pressure that shapes the natural whey starter community before and during the back-slopping procedure can impact the amount and viability of the different thermophilic LAB. Traditional culture-dependent analytical methods are useful for evaluating natural whey cultures based on plate enumeration with various culture media and are commonly used as self-control procedures in dairy items. These methods have high variability and require days to obtain results. As the dairy industry has been searching for a solution to this problem for a long time, researchers must explore alternative methods for the technological evaluation of natural whey and assessment of the health status of the thermophilic acidifying bacteria community in the cheesemaking process. The flow cytometry approach has been considered an alternative to classical methods in this work sector. This study compared bacterial enumeration by plate counting and flow cytometry on natural whey samples. Flow cytometry results showed positive agreement with a tendency to overestimate, linearity, and correlation with plate counting. Other parameters have also been introduced for evaluating a natural whey starter, measuring the physiological state of the cells. Specifically, cell-wall damage and metabolic activity were also evaluated, allowing us to quantify the number of cells even in sub-optimal physiological conditions.

Impact of Spontaneous Fermentation and Inoculum with Natural Whey Starter on Peptidomic Profile and Biological Activities of Cheese Whey: A Comparative Study

Fermentation is a promising solution to valorize cheese whey, the main by-product of the dairy industry. In Parmigiano Reggiano cheese production, natural whey starter (NWS), an undefined community of thermophilic lactic acid bacteria, is obtained from the previous day residual whey through incubation at gradually decreasing temperature after curd cooking. The aim of this study was to investigate the effect of fermentation regime (spontaneous (S) and NWS-inoculated (I-NWS)) on biofunctionalities and release of bioactive peptides during whey fermentation. In S and I-NWS trials proteolysis reached a peak after 24 h, which corresponded to the drop out in pH and the maximum increase in lactic acid. Biological activities increased as a function of fermentation time. NWS inoculum positively affected antioxidant activity, whilst S overcame I-NWS in angiotensin-converting enzyme (ACE) and DPP-IV (dipeptidyl peptidase IV) inhibitory activities. Peptidomics revealed more than 400 peptides, mainly derived from β-casein, κ-casein, and α-lactalbumin. Among them, 49 were bioactive and 21 were ACE-inhibitors. Semi-quantitative analysis strongly correlated ACE-inhibitory activity with the sum of the peptide abundance of ACE-inhibitory peptides. In both samples, lactotripeptide isoleucine-proline-proline (IPP) was higher than valine-proline-proline (VPP), with the highest content in S after 24 h of fermentation. In conclusion, we demonstrated the ability of whey endogenous microbiota and NWS to extensively hydrolyze whey proteins, promoting the release of bioactive peptides and improving protein digestibility.

Distinct Bacterial Communities in São Jorge Cheese with Protected Designation of Origin (PDO)

São Jorge cheese is an iconic product of the Azores, produced from raw cow's milk and natural whey starter (NWS). Although it is produced according to Protected Designation of Origin (PDO) specifications, the granting of the PDO label depends crucially on sensory evaluation by trained tasters. The aim of this work was to characterize the bacterial diversity of this cheese using next-generation sequencing (NGS) and to identify the specific microbiota that contributes most to its uniqueness as a PDO by distinguishing the bacterial communities of PDO and non-PDO cheeses. The NWS and curd microbiota was dominated by Streptococcus and Lactococcus, whereas Lactobacillus and Leuconostoc were also present in the core microbiota of the cheese along with these genera. Significant differences (p < 0.05) in bacterial community composition were found between PDO cheese and non-certified cheese; Leuconostoc was found to play the chief role in this regard. Certified cheeses were richer in Leuconostoc, Lactobacillus and Enterococcus, but had fewer Streptococcus (p < 0.05). A negative correlation was found between contaminating bacteria, e.g., Staphylococcus and Acinetobacter, and the development of PDO-associated bacteria such as Leuconostoc, Lactobacillus and Enterococcus. A reduction in contaminating bacteria was found to be crucial for the development of a bacterial community rich in Leuconostoc and Lactobacillus, thus justifying the PDO seal of quality. This study has helped to clearly distinguish between cheeses with and without PDO based on the composition of the bacterial community. The characterization of the NWS and the cheese microbiota can contribute to a better understanding of the microbial dynamics of this traditional PDO cheese and can help producers interested in maintaining the identity and quality of São Jorge PDO cheese.

Insights on the bacterial composition of Parmigiano Reggiano Natural Whey Starter by a culture-dependent and 16S rRNA metabarcoding portrait

Natural whey starters (NWS) are undefined bacterial communities produced daily from whey of the previous cheese-making round, by application of high temperature. As a result, in any dairy plant, NWS are continuously evolving, undefined mixtures of several strains and/or species of lactic acid bacteria, whose composition and performance strongly depend on the selective pressure acting during incubation. While NWS is critical to assure consistency to cheese-making process, little is known about the composition, functional features, and plant-to-plant fluctuations. Here, we integrated 16S rRNA metabarcoding and culture-dependent methods to profile bacterial communities of 10 NWS sampled in the production area of Parmigiano Reggiano cheese. 16S rRNA metabarcoding analysis revealed two main NWS community types, namely NWS type-H and NWS type-D. Lactobacillus helveticus was more abundant in NWS type-H, whilst Lactobacillus delbrueckii/St. thermophilus in NWS type-D, respectively. Based on the prediction of metagenome functions, NWS type-H samples were enriched in functional pathways related to galactose catabolism and purine metabolism, while NWS type-D in pathways related to aromatic and branched chain amino acid biosynthesis, which are flavor compound precursors. Culture-dependent approaches revealed low cultivability of individual colonies as axenic cultures and high genetic diversity in the pool of cultivable survivors. Co-culturing experiments showed that fermentative performance decreases by reducing the bacterial complexity of inoculum, suggesting that biotic interactions and cross-feeding relationships could take place in NWS communities, assuring phenotypic robustness. Even though our data cannot directly predict these ecological interactions, this study provides the basis for experiments targeted at understanding how selective regime affects composition, bacterial interaction, and fermentative performance in NWS.

Probiotic potential of GABA-producing lactobacilli isolated from Uruguayan artisanal cheese starter cultures.

In this study, we sought to identify and characterize a collection of 101 lactobacilli strains isolated from natural whey starters used in Uruguayan artisan cheese production, based on their capacity to produce gamma-aminobutyric acid (GABA) and their probiotic potential. The probiotic potential was assessed using low pH and bile salt resistance assays; bacterial adhesion to intestinal mucus was also evaluated. Selected strains were then identified by 16S sequencing, and their GABA-producing potential was confirmed and quantified using a UHPLC-MS system. Twenty-five strains were identified and characterized as GABA-producing lactobacilli belonging to the phylogenetical groups Lactiplantibacillus (n= 19) and Lacticaseibacillus (n= 6). Fifteen strains of the Lactiplantibacillus group showed a significantly higher GABA production than the rest. They showed the predicted ability to survive the passage through the gastrointestinal tract, according to the in vitro assays. A set of promising candidate strains was identified as potential probiotics with action on the gut-brain axis. Further studies are needed to assess their possible effects on behaviour using in vivo assay. This study shows the potential of strains isolated from local natural whey starters as probiotics and for biotechnological use in functional GABA-enriched foods formulation.

Fermentation of whey protein concentrate by Streptococcus thermophilus strains releases peptides with biological activities

Cheese whey is the principal by-product of cheese making and is perceived as an environmental pollutant. Different technological procedures have been applied to convert whey into value-added products including whey protein concentrate (WPC). In the present study, 2 Lactobacillus helveticus strains and 5 Streptococcus thermophilus strains, isolated from natural whey starter used in Parmigiano Reggiano production, have been evaluated for their capacity to ferment WPC and to release bioactive peptides. WPC fermented with S. thermophilus RBC06 showed the highest antioxidant activity as well as angiotensin-converting enzyme- and dipeptidyl-peptidase-IV-inhibitory activities followed by WPC fermented by S. thermophilus RBC20 and RBN16. An untargeted peptidomics approach was applied to profile the peptides released after WPC fermentation. Several bioactive peptides, mainly angiotensin-converting enzyme inhibitors, were identified and the anti-hypertensive lactotripeptides valine-proline-proline (VPP) and isoleucine-proline-proline (IPP) were quantified. S. thermophilus RBC06 produced the highest amount of VPP (12.80 ± 0.45 mg/L) whereas the highest quantity of IPP (0.56 ± 0.02 mg/L) was detected in WPC fermented with S. thermophilus RBN16. The present study paved the way for the further exploitation of S. thermophilus RBC06 as new bioactive-peptide producer and for the application of WPC hydrolysates as additives for designing whey-based beverages with improved healthy properties.

Effect of using mycotoxin-detoxifying agents in dairy cattle feed on natural whey starter biodiversity

Natural whey cultures (NWC) are undefined multiple-strain bacterial starter communities that can be affected by even small changes along the entire dairy chain. We applied a multidisciplinary approach to investigate how the addition of 2 mycotoxin-detoxifying agents [sodium smectite and lignocellulose-based material (B1); leonardite and betaine (B2)] to cow diets modified the microbiota of the NWC in manufacture of a Grana-like cheese. Microbiological and flow cytometry analyses showed that the content and viability of lactic acid bacteria (LAB) and the total whey microbiota were not affected by the detoxifying agents, and Streptococcus thermophilus, Lactobacillus helveticus, and Limosilactobacillus fermentum were the dominant taxa. Random amplified polymorphic DNA-PCR fingerprinting and metagenomic analysis highlighted differences in the bacterial community of the NWC and in the relative abundance of Bacteroidetes that increased when B1 and B2 were included in the diet. Two of 6 St. thermophilus biotypes were detected only in control samples; conversely, none of the Lb. helveticus biotypes found in control samples were isolated from B1 and B2. In vitro tests showed that the 2 binders did not significantly affect the development of St. thermophilus, but they stimulated the growth of Lb. helveticus strains recovered only from B1 and B2 NWC. The addition of binders in cow feed can affect the LAB biotypes present in NWC.

Investigating the influence of Food Safety Management Systems (FSMS) on microbial diversity of Canastra cheeses and their processing environments

Canastra Cheese is one of the most commercialized artisanal cheeses in Brazil and intrinsic characteristics of its production, such as the use of raw milk and natural whey starter cultures as well short ripening time on wooden shelves, offer risk of contamination by a plethora of microorganisms. Here, we used 16 S rRNA gene amplicon sequencing approach to characterize the bacterial communities from Canastra cheese processing environments and final products, accessing cheesemaking facilities with distinct profiles of Food Safety Management Systems (FSMS), in order to estimate whether differences in microbial composition and diversity could also be observed between the two sampled groups of facilities. Our results revealed that the diversity of bacterial communities in the processing environments was much higher than that observed for cheeses, with greater discrepancy for facilities with inadequate FSMS. Additionally, in facilities with inadequate FSMS the bacterial communities from environments, especially hand surfaces and ripening wooden shelves, were similar to those during processing and finished cheese. These evidences highlight the importance of implementing and maintaining FSMS in the facilities, in order to assure quality and safety of Canastra cheese, but also the stability and economic viability of the Canastra cheese production chain.

Distinctive Traits of Four Apulian Traditional Agri-Food Product (TAP) Cheeses Manufactured at the Same Dairy Plant.

This study aimed to highlight the distinctive features of four Traditional Agri-food Products (TAP), namely, Caprino, Pecorino, Vaccino, and Cacioricotta cheeses produced at the same dairy plant to reveal any possible relationships between their microbiological and biochemical characteristics. Two distinct natural whey starter (NWS) cultures were used during Caprino and Vaccino cheesemaking, whereas no starter was used for the other cheeses. Cacioricotta retained the highest concentrations of salt and residual carbohydrates. Lactic acid bacteria dominated the microbiota of the cheeses. Furthermore, staphylococci represented an additional dominant microbial population in Cacioricotta. Although culture-dependent analysis showed that the use of NWS cultures only slightly affected the microbial community of cheeses, 16S metagenetic analysis showed that Lactobacillus helveticus dominated both the NWS cultures and the corresponding Caprino and Vaccino cheeses. This analysis indicated that Staphylococcus equorum and Streptococcus thermophilus dominated Cacioricotta and Pecorino cheeses, respectively. The highest peptidase activities were found in either Caprino or Vaccino. Enzymes involved in the catabolism of free amino acids and esterase showed the highest activity in Pecorino cheese. Each cheese showed a distinct profile of volatile organic compounds, with Pecorino being the richest cheese in carboxylic acids, ketones, and esters, related to lipolysis. The results of this study contribute to valorizing and safeguarding these TAP cheeses, sustaining local farming.