Effectiveness of modified atmosphere and vacuum packaging in preserving the volatilome of Stelvio PDO cheese over time.

We aimed to assay the effectiveness of vacuum or modified atmosphere packaging in preserving the organoleptic characteristics of already ripened slices of Stelvio Protected Designation of Origin cheese during 3 months of storage. A multi-omics panel, including metagenomic and metabolomic analyses, was implemented together with physicochemical and sensory analyses. Among the 177 volatiles identified, 30 out of the 50 potent odorants were found to be prevalent, regardless of packaging. Isovaleric acid showed the highest relative intensity in all samples. Caproic and caprylic acids always increased during storage, while metabolites such as dodecane and 2,3-butanediol always decreased. Slow proteolysis occurred during storage, but did not differentiate cheese samples. The type of packaging differentiated the microbiota and volatile profile, with modified atmosphere packaging keeping the volatilome more stable. Out of the 50 potent odorants, 9 were relevant to sample discrimination, with 8-nonen-2-one, 2-nonanone, and caproic acid being more abundant in stored samples.

Comparative analysis of microbial succession and proteolysis focusing on amino acid pathways in Asiago-PDO cheese from two dairies.

In this study, a comprehensive and comparative analysis was conducted on Italian Asiago-PDO cheese obtained from two different dairies named Dairy I and Dairy II using industrial and natural fermented milk, respectively. The analysis encompassed the evaluation of chemical composition, the succession of the microbiota during manufacture and ripening, and proteolysis mainly focusing on free individual amino acid (FAA) profiles. A metagenomic approach was used to investigate the cheese microbiome functionality. Differences in gross chemical composition were more evident during ripening, with Dairy II showing higher variability within batches. The microbiota varied significantly between the two dairies and ripening stages. The choice of starter culture shaped the microbiota during production and affected the microbial diversity of non-starter lactic acid bacteria (NSLAB) originated from the raw milk during ripening. Peptide chromatographic profiles and FAA concentrations increased as ripening progressed, with Dairy I showing higher production of FAA. Functional analysis of the metagenomes linked species to specific amino acid metabolism/catabolism pathways. The amino acid metabolism pathways, particularly those related to aromatic amino acids, lysine, and branched-chain amino acids, were affected by the presence of specific NSLAB species, which differed between the two dairies. The results obtained in this study reveal the impact of starter culture on peculiar cheese microbiota assemblies, which selectively targets amino acid pathways, providing insights into the potential flavor and aroma characteristics of Asiago-PDO cheese.

Milk metagenomics and cheese-making properties as affected by indoor farming and summer highland grazing.

The study of the complex relationships between milk metagenomics and milk composition and cheese-making efficiency as affected by indoor farming and summer highland grazing was the aim of the present work. The experimental design considered monthly sampling (over 5 mo) of the milk produced by 12 Brown Swiss cows divided into 2 groups: the first remained on a lowland indoor farm from June to October, and the second was moved to highland pastures in July and then returned to the lowland farm in September. The resulting 60 milk samples (2 kg each) were used to analyze milk composition, milk coagulation, curd firming, and syneresis processes, and to make individual model cheeses to measure cheese yields and nutrient recoveries in the cheese. After DNA extraction and Illumina Miseq sequencing, milk microbiota amplicons were also processed by means of an open-source pipeline called Quantitative Insights Into Microbial Ecology (Qiime2, version 2018.2; https://qiime2.org). Out of a total of 44 taxa analyzed, 13 bacterial taxa were considered important for the dairy industry (lactic acid bacteria, LAB, 5 taxa; and spoilage bacteria, 4) and for human (other probiotics, 2) and animal health (pathogenic bacteria, 2). The results revealed the transhumant group of cows transferred to summer highland pastures showed an increase in almost all the LAB taxa, bifidobacteria, and propionibacteria, and a reduction in spoilage taxa. All the metagenomic changes disappeared when the transhumant cows were moved back to the permanent indoor farm. The relationships between 17 microbial traits and 30 compositional and technological milk traits were investigated through analysis of correlation and latent explanatory factor analysis. Eight latent factors were identified, explaining 75.3% of the total variance, 2 of which were mainly based on microbial traits: pro-dairy bacteria (14% of total variance, improving during summer pasturing) and pathogenic bacteria (6.0% of total variance). Some bacterial traits contributed to other compositional-technological latent factors (gelation, udder health, and caseins).

Sourdough performances of the golden cereal Tritordeum: Dynamics of microbial ecology, biochemical and nutritional features.

The novel cereal 'Tritordeum' was employed in sourdough fermentation for bread making using a traditional backslopping procedure over 10 days. Culture-dependent and culture-independent approaches were used to characterize microbial ecology during sourdough preparation and propagation. Sourdough reached the highest microbial diversity after three days of propagation. Microbial diversity decreased as sourdough reached maturity (day 5). Microbiota dominance shifted from Weissella to Lactiplantibacillus genera after 5 days of propagation. Lactic acid bacteria (LAB) showed a constant increase throughout the propagations starting from 3.9 ± 0.24 log CFU g-1 on day 0 up to 8.0 ± 0.39 log CFU g-1 on day 5. Weissella confusa/cibaria and Weissella paramesenteroides were the most prevalent LAB species until day 5 of propagation, while Lactiplantibacillus plantarum was the most prevalent thereafter. Yeasts were present in low cell density (2.0 ± 0.11 log CFU g-1) until the fourth backslopping (day 4) and then gradually increased until day 10 (5.0 ± 0.29 log CFU g-1), with Saccharomyces cerevisiae being the most prevalent and dominant species. Lactic and acetic acid concentrations increased throughout Tritordeum sourdough propagations, indicative of a proportional decrease of fermentation quotient (lactic acid/acetic acid) from 13.54 ± 1.29 to 4.08 ± 0.15. Utilization of glucose, fructose and sucrose was observed, followed a progressive increase in mannitol concentrations beginning from day 4. The nutritional potential (total phenol content, antioxidant activity, dietary fiber content and total free amino acids) remained elevated during sourdough propagations. Antinutritional factors (phytic acid and raffinose) were reduced to minimal concentrations by day 10. Finally, texture analysis of Tritordeum sourdough bread was demonstrated to have better cohesiveness, resilience and firmness compared to baker's yeast bread, confirming its potential to improve functionality and use in sourdough biotechnology.

Commercial Organic Versus Conventional Whole Rye and Wheat Flours for Making Sourdough Bread: Safety, Nutritional, and Sensory Implications.

Organic farming is gaining a broad recognition as sustainable system, and consumer demand for organic products has increased dramatically in the recent past. Whether organic agriculture delivers overall advantages over conventional agriculture is, however, contentious. Here, the safety, nutritional, and sensory implications of using commercial organic rye, soft, and durum wheat flours rather than conventional-made sourdough bread have been investigated. Culture-dependent and culture-independent approaches were used to explore the microbial architecture of flours and to study their dynamics during sourdough propagation. Besides biochemical features, the main nutritional (amino acid content, asparagine level, and antioxidant activity) characteristics of sourdoughs were investigated, and their effect on the structural, nutritional, and sensory profiles of breads assessed. Overall, the organic farming system led to flours characterized by lower content of asparagine and cell density of Enterobacteriaceae while showing higher concentration of total free amino acids. Differences of the flours mirrored those of sourdoughs and breads. The use of sourdough fermentation guaranteed a further improvement of the flour characteristics; however, a microbial and sensory profile simplification as well as a slight decrease of the biochemical parameters was observed between breads with sourdough after one-cycle fermentation and 10 days of propagation.

Massive Survey on Bacterial-Bacteriophages Biodiversity and Quality of Natural Whey Starter Cultures in Trentingrana Cheese Production.

This study focused on the microbial and bacteriophages identification and characterization in cheese-production facilities that use natural whey starter (NWS) cultures for Trentingrana production. Bacterial and phage screening was carried out on cooked not acidified whey and NWS samples isolated from six dairy factories, for 4 consecutive days in four different months. By means of a combined approach, using plate counts, bacterial isolation, and metataxonomic analysis Lactobacillus helveticus was found occurring as the dominant species in NWS cultures and Levilactobacillus brevis as codominant in the cheese factories where the temperature of NWS production was mainly lower than 40°C, suggesting that the variability in the parameters of the NWS culture preparation could differently modulate the bacterial species in NWS cultures. Using turbidity test approach on 303 bacterial isolates from the NWS cultures, 120 distinct phages were identified. L. helveticus phage contamination of NWS cultures was revealed in most of the analyzed samples, but despite the great recovery of bacteriophage contamination cases, the microbial quality of NWS cultures was high. Our results support the presence of natural bacteriophage resistance mechanisms in L. helveticus. The use of NWS cultures probably creates an ideal environment for the proliferation of different L. helveticus strains balanced with their phages without a clear dominance. It is evident, from this study, that the presence of a high biodiversity of NWS bacterial strains is relevant to avoid phages dominance in NWS cultures and consequently to keep a good acidification ability.

Modulating Wine Aromatic Amino Acid Catabolites by Using Torulaspora delbrueckii in Sequentially Inoculated Fermentations or Saccharomyces cerevisiae Alone.

Yeasts are the key microorganisms that transform grape juice into wine, and nitrogen is an essential nutrient able to affect yeast cell growth, fermentation kinetics and wine quality. In this work, we focused on the intra- and extracellular metabolomic changes of three aromatic amino acids (tryptophan, tyrosine, and phenylalanine) during alcoholic fermentation of two grape musts by two Saccharomyces cerevisiae strains and the sequential inoculation of Torulaspora delbrueckii with Saccharomyces cerevisiae. An UPLC-MS/MS method was used to monitor 33 metabolites, and 26 of them were detected in the extracellular samples and 8 were detected in the intracellular ones. The results indicate that the most intensive metabolomic changes occurred during the logarithm cellular growth phase and that pure S. cerevisiae fermentations produced higher amounts of N-acetyl derivatives of tryptophan and tyrosine and the off-odour molecule 2-aminoacetophenone. The sequentially inoculated fermentations showed a slower evolution and a higher production of metabolites linked to the well-known plant hormone indole acetic acid (auxin). Finally, the production of sulfonated tryptophol during must fermentation was confirmed, which also may explain the bitter taste of wines produced by Torulaspora delbrueckii co-fermentations, while sulfonated indole carboxylic acid was detected for the first time in such an experimental design.

Shift in the cow milk microbiota during alpine pasture as analyzed by culture dependent and high-throughput sequencing techniques.

In the present study, two groups of cows from a permanent lowland farm (PF) were divided during summer and reared in the PF or in a temporary alpine farm (ALP), respectively. Microbiological analyses were performed with the objective to investigate the microbial evolution of milk before, during, and after summer transhumance comparing, in particular, the two groups of cows to determine whether the alpine pasture could directly influence the milk microbiota. A significant increase of all microbial groups was registered in milk samples collected in the ALP. Interestingly, many strains belonging to species with well reported technological and probiotic activities were isolated from Alpine milk (20% Lactococcus lactis subsp. lactis/cremoris, 18% Lactobacillus paracasei, 14% Bifidobacterium crudilactis and 18% Propionibacterium sp.), whereas only 16% of strains isolated from the permanent farm milk belonged to the species Lactococcus lactis subsp. lactis/cremoris, 6% to Lactobacillus paracasei, 2% to Bifidobacterium crudilactis and 5% to Propionibacterium sp. The MiSeq Illumina data showed that Alpine milk presented a significant reduction of Pseudomonas and an increase of Lactococcus, Bifidobacterium and Lactobacillus genera. These data confirmed the practice of Alpine pasture as one of the main drivers affecting the milk microbiota. All the microbial changes disappeared when cows were delivered back from Alpine pasture to the indoor farm.

Metformin and Dipeptidyl Peptidase-4 Inhibitor Differentially Modulate the Intestinal Microbiota and Plasma Metabolome of Metabolically Dysfunctional Mice.

OBJECTIVES: Recent evidence indicates that gut microbiota is altered considerably by a variety of commonly prescribed medications. This study assessed the impact of 2 antidiabetic therapeutics on gut microbiota and markers of cardiometabolic disease in metabolically dysfunctional mice. METHODS: C57BL/6 mice were fed a high-fat diet for 24 weeks while receiving 1 of 2 antidiabetic therapeutics-metformin or dipeptidyl peptidase-4 (DPP-4) inhibitor, PKF-275-055-for the final 12 weeks. Mice were assessed for weight gain, glucose and cholesterol metabolism, and adiposity. In addition, cecal microbiota was analyzed by 16S compositional sequencing, and plasma metabolome was analyzed by liquid chromatography with tandem mass spectrometry. RESULTS: Both therapeutics had similar metabolic effects, attenuating mesenteric adiposity and improving cholesterol metabolism and insulin sensitivity. However, multivariate analyses of microbiota and metabolomics data revealed clear divergence of the therapeutic groups. Although both metformin and PKF-275-055 mice displayed significantly decreased Firmicutes/Bacteroidetes ratios, only metformin harboured metabolic health-associated Akkermansia, Parabacteroides and Christensenella. Paradoxically, metformin also reduced α diversity, a metric frequently associated with host metabolic fitness. PKF-275-055 mice displayed elevated levels of butyrate-producing Ruminococcus and acetogen Dorea, with reduced levels of certain plasma sphingomyelin, phosphatidylcholine and lysophosphatidylcholine entities. In turn, metformin reduced levels of acylcarnitines, a functional group associated with systemic metabolic dysfunction. Finally, several associations were identified between metabolites and altered taxa. CONCLUSIONS: This study represents the first direct comparison of the microbiota-modifying effects of metformin and a DPP-4 inhibitor, and proposes several putative microbial targets both in terms of novel therapeutic development and adverse effect prevention.

Effects of Summer Transhumance of Dairy Cows to Alpine Pastures on Body Condition, Milk Yield and Composition, and Cheese Making Efficiency.

Summer transhumance to alpine pastures (ALP) is widespread in dairy systems of alpine regions. This study aimed to investigate the effects of transhumance of Brown Swiss cows to ALP on the yield, composition, and coagulation properties of milk (MCP), and on cheese yield (CY). The study involved 12 multiparous cows kept at a mountain lowland permanent farm (PF), which were divided into two equal groups: One remained at the PF, the other was moved to the ALP (1860 m above sea level) from July to September. Every month (June to October), daily milk yield (MY) and body condition score (BCS) were recorded, and individual milk samples (n = 60, 2000 mL each) were collected to assess milk composition, MCP, and CY. Compared with PF, ALP cows had a reduced MY and BCS, which was maintained on return to the PF, greater fat and lower protein contents of milk. Neither MCP nor CY were affected by summer transhumance. In conclusion, summer transhumance did not affect the cheese making efficiency of milk but depressed MY and consequently daily cheese yield, which was nearly 2 kg/d lower for the ALP than the PF cows and was only partially recovered after returning to the PF in autumn.

Production of Naturally γ-Aminobutyric Acid-Enriched Cheese Using the Dairy Strains Streptococcus thermophilus 84C and Lactobacillus brevis DSM 32386.

The cheese-derived strains Streptococcus thermophilus 84C isolated from Nostrano cheese, and Lactobacillus brevis DSM 32386 isolated from Traditional Mountain Malga cheese have been previously reported as γ-aminobutyric acid (GABA)-producers in vitro. In the present study, the ability of these strains to produce GABA was studied in experimental raw milk cheeses, with the aim to investigate the effect of the culture and the ripening time on the GABA concentration. The cultures used consisted on S. thermophilus 84C alone (84C) or in combination with L. brevis DSM 32386 (84C-DSM). The control culture was a commercial S. thermophilus strain, which was tested alone (CTRL) or in combination with the L. brevis DSM 32386 (CTRL-DSM). The pH evolution, microbiological counts, MiSeq Illumina and UHPLC-HQOMS analysis on milk and cheese samples were performed after 2, 9, and 20 days ripening. During the whole ripening, the pH was always under 5.5 in all batches. The concentration of GABA increased during ripening, with the highest content in 84C after 9 days ripening (84 ± 37 mg/kg), in 84C-DSM and CTRL-DSM after 20 days ripening (91 ± 28 and 88 ± 24 mg/kg, respectively). The data obtained support the hypothesis that S. thermophilus 84C and L. brevis DSM 32386 could be exploited as functional cultures, improving the in situ bio-synthesis of GABA during cheese ripening.

Digestion and Colonic Fermentation of Raw and Cooked Opuntia ficus-indica Cladodes Impacts Bioaccessibility and Bioactivity.

The bioactivity of (poly)phenols from a food is an interplay between the cooking methods applied and the interaction of the food with the gastrointestinal tract. The (poly)phenolic profile and biological activity of raw and cooked cactus ( Opuntia ficus-indica Mill.) cladodes following in vitro digestion and colonic fermentation were evaluated. Twenty-seven (poly)phenols were identified and quantified by HPLC-ESI-MS, with piscidic acid being the most abundant. Throughout the colonic fermentation, flavonoids showed more degradation than phenolic acids, and eucomic acid remained the most relevant after 24 h. The catabolite 3-(4-hydroxyphenyl)propionic acid was generated after 24 h of fermentation. Cytotoxicity, genotoxicity, and cell cycle analyses were performed in HT29 cells. Cactus colonic fermentates showed higher cell viability (≥80%) in comparison to the control fermentation with no cactus and significantly ( p < 0.05) reduced H2O2-induced DNA damage in HT29 cells. Results suggest that, although phenolic compounds were degraded during the colonic fermentation, the biological activity is retained in colon cells.

Evaluation of autochthonous lactic acid bacteria as starter and non-starter cultures for the production of Traditional Mountain cheese.

Lactococcus lactis subsp. lactis 68, Streptococcus thermophilus 93 and Lactobacillus rhamnosus BT68, previously isolated from Traditional Mountain (TM) cheese, were tested for the production of four experimental mountain cheeses, with the aim to assess their effectiveness in leading the TM-cheese-making process. Lactococcus lactis subsp. lactis 68 and Streptococcus thermophilus 93 were used as starter cultures, whereas Lactobacillus rhamnosus BT68 was used as non-starter culture. Three control (CTRL) cheeses were manufactured without adding any starter, according to the traditional cheese-making process; nine, cheeses were produced inoculating the vat milk with the starters (ST), starter and low concentration of non-starter culture (STLC), starter and high concentration of non-starter culture (STHC). Samples of vat milk, cheese after 24 h and 7 months ripening were processed for microbiological counts. Mesophilic cocci were dominant in all 24 h-cheese samples, while a dominance of both cocci and lactobacilli was observed after 7 months ripening. The total genomic DNA was extracted, and a fragment of V1-V3 region was amplified and pyrosequenced. Lactococci and streptococci were the most abundant species, and Lc. lactis ssp. lactis 68 affected the proliferation of milk-resident Lc. lactis ssp. cremoris, during the early fermentation. Lb. rhamnosus BT68 showed to be responsible in reducing the abundance of other Lactobacillus species. Moreover, it likely competed against Sc. thermophilus 93 for the same energetic sources, when added in concentration higher than 5 × 103 CFU/mL milk. The sensorial and fatty acid (FA) composition analysis were performed on cheese samples at the end of ripening, demonstrating that the inoculated cheeses had better sensorial characteristics (aspect, smell, taste, texture) than CTRL cheeses, and that Lb. rhamnosus BT68 at high concentration is related to the increase of short chain fatty acids and conjugated linoleic acid in cheese after 7 months ripening.

Exploring the microbiota of the red-brown defect in smear-ripened cheese by 454-pyrosequencing and its prevention using different cleaning systems.

Red-brown pigmentation can occasionally form in smeared-ripened cheese such as Fontina during the ripening process. This reaction is due to over-development of the typical microbiota present on the rind. Previous studies have demonstrated the relationship between red-brown pigmentation and the traditional utilization of wooden shelves during cheese ripening. The first part of the paper focuses on the characterisation of yeast and bacterial microbiota: plate counts and 454-pyrosequencing were performed in spoiled (n = 6) and non-spoiled cheeses (n = 6) and on the wooden shelves used during ripening. The second part shows different systems tested for cleaning the wooden shelves and avoiding the development of the red-brown defect in cheese: washing with hot water and ozone treatment. Actinobacteria, dominated on the wooden shelves, suggesting to be responsible for the red-brown pigmentation; they were also found in traces in the defected cheese samples. Galactomyces and Debaryomyces were the main species characterizing the yeast population, with Debaryomyces being the most dominant species on the shelves used during ripening of the red-brown defective cheese. Hot water treatment reduced the microbial contamination of shelves, whereas only the ozone treatment ensured complete elimination of both yeast and bacteria, resulting in the cheese rind not having the red-brown defect.

Microbial evolution of traditional mountain cheese and characterization of early fermentation cocci for selection of autochtonous dairy starter strains.

The microbial population of Traditional Mountain (TM) cheese was investigated and characterized for the selection of cocci suitable for developing new starter cultures. Samples of milk, curd and cheese at different ripening times were enumerated in selective culture media and 640 colonies were isolated from curd and cheese after 24 h of ripening. The Lactic Acid Bacteria (LAB) isolated from M17 were clustered into 231 biotypes by RAPD-PCR analysis and identified as Lactococcus lactis, Streptococcus thermophilus and Enterococcus faecalis. Forty percent of enterococci showed the in vitro ability to inhibit raw milk resident coliforms, but they were excluded as possible starters due to the presence of associated risk factors. All lactococci and streptococci were tested for their technological properties; 4 Lc. lactis subsp. lactis and 2 Sc. thermophilus which were fast acidifiers and did not produce unpleasant flavours were subjected to the freeze-drying stability test. Lc. lactis subsp. lactis biotype 68 and Sc. thermophilus biotype 93 showed the best technological properties and may be appropriate for cheese production. This work gave evidence of the high biodiversity of TM-cheese autochthonous biotypes which could be used as starter cultures for the improvement of TM-cheese technology.

Biodiversity and γ-aminobutyric acid production by lactic acid bacteria isolated from traditional alpine raw cow's milk cheeses.

"Nostrano-cheeses" are traditional alpine cheeses made from raw cow's milk in Trentino-Alto Adige, Italy. This study identified lactic acid bacteria (LAB) developing during maturation of "Nostrano-cheeses" and evaluated their potential to produce γ-aminobutyric acid (GABA), an immunologically active compound and neurotransmitter. Cheese samples were collected on six cheese-making days, in three dairy factories located in different areas of Trentino and at different stages of cheese ripening (24 h, 15 days, and 1, 2, 3, 6, and 8 months). A total of 1,059 LAB isolates were screened using Random Amplified Polymorphic DNA-PCR (RAPD-PCR) and differentiated into 583 clusters. LAB strains from dominant clusters (n = 97) were genetically identified to species level by partial 16S rRNA gene sequencing. LAB species most frequently isolated were Lactobacillus paracasei, Streptococcus thermophilus, and Leuconostoc mesenteroides. The 97 dominant clusters were also characterized for their ability in producing GABA by high-performance liquid chromatography (HPLC). About 71% of the dominant bacteria clusters evolving during cheeses ripening were able to produce GABA. Most GABA producers were Lactobacillus paracasei but other GABA producing species included Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Pediococcus pentosaceus, and Streptococcus thermophilus. No Enterococcus faecalis or Sc. macedonicus isolates produced GABA. The isolate producing the highest amount of GABA (80.0±2.7 mg/kg) was a Sc. thermophilus.

Identification and characterization of wild lactobacilli and pediococci from spontaneously fermented Mountain cheese.

The Traditional Mountain Malga (TMM) cheese is made from raw cow's milk by spontaneously fermentation in small farms called "Malga" located in Trentino region. This study was designed to characterize the lactic acid bacteria (LAB) growing on MRS medium, of TMM-cheese at the end of the ripening. Ninety-five LAB were isolated and genotypically characterized by Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR) with two primers, species-specific PCR and partial sequencing of 16S rRNA gene. The 95 LAB clustered in 70 biotypes. Pediococcus pentosaceus and Lactobacillus paracasei were the dominant species. Isolates were tested for their growth properties, carbohydrate metabolism, acidifying ability, proteolytic and lipolytic activities, acetoin production, amino-peptidase (AP) activity, biogenic amines production, bile salts hydrolysis, conjugated linoleic acid and γ-aminobutyric acid production. Lb. paracasei isolates resulted to be well adapted to Malga environment and to show the best AP activity and acetoin production. TMM-cheese related LAB showed also interesting health promoting properties and produced bioactive substances. In particular, one Lb. brevis biotype produced a GABA mean value of 129 mg/L that is considered a high concentration. The results confirmed that TMM-cheese resident LAB could be exploited for dairy production.