Cheese Ripening Research Articles

Peptidomics and biological activity analysis of in vitro digested Parmigiano Reggiano cheese at different ripening stages

The relationships among Parmigiano Reggiano (PR) cheese ripening, in vitro digestion, and biological activity have been investigated. PR cheese samples at different ripening time (from 12 to 30 months) were collected from two dairies and in vitro digested. No effect of ripening was found when the total peptide profiles were considered. Nevertheless, ripening affected the biological activities and the bioactive peptide profiles of in vitro digested PR. The long-ripened PR samples showed higher ACE-inhibitory and DPP-IV-inhibitory activities after in vitro digestion. The ACE-inhibitory activity of in vitro digested PR samples was positively correlated with the relative amount of potent ACE-inhibitory peptides whereas DPP-IV-inhibitory activity with protein hydrolysis. Some bioactive peptides that, according to the literature, exhibit proven in vivo bioactivity and bioavailable in humans were detected, suggesting their potential role in the protective effects on human health resulting from cheese consumption. PR samples at different ripening time may have potentially different effects on human health.

Advantages and disadvantages of autochthonous enterococci strains for their potential use in cheese ripening: a preliminary study

SummaryThe enzymatic activity exhibited by native enterococci could, in turn, result in a reduction in the cheese ripening time. However, they may also carry virulence factors or resistance to antibiotics that prevent their use in food. A total of 69 enterococci isolates were obtained from a Spanish traditional raw goat's milk cheese and identified by Maldi‐Tof/MS. Different enzymatic activities of technological interest were evaluated and the safety of those with greater technological suitability was studied. The strains were identified as E. malodoratus, E. italicus, E. gilvus, E. faecium, E. durans, E. casseliflavus and E. faecalis. Some strains had very high acidifying and proteolytic activities. Most strains had medium or low amino and carboxypeptidase activities and high dipeptidase activities, while esterase activities were medium. A total of 10 strains were selected based on their enzymatic activities. Of these, five showed β‐haemolysin activity, eight showed gelatinase activity and eight produced tyramine. These activities represent virulence factors that could potentially affect food safety. Finally, all selected strains were susceptible to the antibiotics tested.

Dynamics of the viral community on the surface of a French smear-ripened cheese during maturation and persistence across production years.

The surface of smear-ripened cheeses constitutes a dynamic microbial ecosystem resulting from the successive development of different microbial groups such as lactic acid bacteria, fungi, and ripening bacteria. Recent studies indicate that a viral community, mainly composed of bacteriophages, also represents a common and substantial part of the cheese microbiome. However, the composition of this community, its temporal variations, and associations between bacteriophages and their hosts remain poorly characterized. Here, we studied a French smear-ripened cheese by both viral metagenomics and 16S metabarcoding approaches to assess both the succession of phages and bacterial communities on the cheese surface during cheese ripening and their temporal variations in ready-to-eat cheeses over the years of production. We observed a clear transition of the phage community structure during ripening with a decreased relative abundance of viral species (vOTUs) associated with Lactococcus phages, which were replaced by vOTUs associated with phages infecting ripening bacteria such as Brevibacterium, Glutamicibacter, Pseudoalteromonas, and Vibrio. The dynamics of the phage community was strongly associated with bacterial successions observed on the cheese surface. Finally, while some variations in the distribution of phages were observed in ready-to-eat cheeses produced at different dates spanning more than 4 years of production, the most abundant phages were detected throughout. This result revealed the long-term persistence of the dominant phages in the cheese production environment. Together, these findings offer novel perspectives on the ecology of bacteriophages in smear-ripened cheese and emphasize the significance of incorporating bacteriophages in the microbial ecology studies of fermented foods.IMPORTANCEThe succession of diverse microbial populations is critical for ensuring the production of high-quality cheese. We observed a temporal succession of phages on the surface of a smear-ripened cheese, with new phage communities showing up when ripening bacteria start covering this surface. Interestingly, the final phage community of this cheese is also consistent over large periods of time, as the same bacteriophages were found in cheese products from the same manufacturer made over 4 years. This research highlights the importance of considering these bacteriophages when studying the microbial life of fermented foods like cheese.

Quality and healthy aspects of Lider cheese at different stages of ripening

Lider cheese is a commercial full- fat semi-hard cheese produced from pasteurized goat`s milk in mountain area of Nikšić, Montenegro. The aim of this paper is to characterize the change of protein and fatty acid profiles, mineral content and antioxidant potential during the period of one month of its ripening. Proteolysis was monitored using electrophoresis of total extracts and water-soluble fractions under reducing conditions. Fatty acid profiles were determined by gas chromatography whereas the mineral content of cheese at different stage of ripening was determined by Inductively coupled plasma - optical emission spectrometry (ICP-OES) for macroelements and Inductively coupled plasma mass spectrometry (ICP-MS) for microelements, respectively. One month of ripening of Lider cheese is characterized by a relatively slow proteolysis which can be attributed to intensive lose of moisture, by the increase in desirable fatty acid content and consequently by the improvement of fatty acid healthy indices. After 30 days of ripening, mainly due to the degradation of αs-CN, the WSN/ TN and TCA/TN contents increased from 4.69 % to 6.06 % and 1.15 % to 1.90 %, respectively. One- month-old cheese had improved indexes of lipid quality as well as improved reducing power and ability to scavenge free radicals. Ripening did not affect mineral content of cheese if it is expressed on dry matter basis. According to obtained results, the optimal ripening period of Lider cheese is 15 days.

BENEFITS AND RISKS OF USING ENTEROCOCCUS SPECIES IN FOOD OR AS PROBIOTICS

Background: Enterococci are ubiquitous and can be found in water, soil, plants and several food products. They are also considered part of the normal beneficial gastrointestinal flora of both humans and animals. The aim of this review was to provide comprehensive information regarding the benefits and risks of Enterococcus speciesuse in food or as probiotics. Review Results: The presence of enterococci in foodstuffs has long been considered a sign of poor sanitation. Contrariwise, due to their enzymatic and proteolytic activities, they are used as starters in food fermentation and play an important role in the development of the organoleptic properties of fermented foods. Enterococci can spoil processed meats but, in contrast, are important for the maturation and flavor development of some traditional cheeses and sausages. Because of the ability to produce bacteriocins possessing antimicrobial action, enterococci are used as protective cultures in food biopreservation. Additionally, they showed probiotic properties with various beneficial characteristics. However, in the last few decades, enterococci have emerged as one of the most important bacteria worldwide, major opportunistic agents causing a wide range of nosocomial infections. They are resistant or tolerant to many antimicrobials, and multidrug resistant enterococci belong to the so-called "therapeutic problematic microorganisms". According to WHO, vancomycin-resistant representatives are high-priority pathogens on the list of microorganisms for the treatment of which the development of new antibiotics is urgently needed. In addition, they possess multiple virulence factors. Conclusion: The role of enterococci in the pathogenesis of numerous and severe infections requires serious comments and discussion about the benefits and risks regarding their presence in food and their use as probiotics.

Adjunct cultures of autochthonous Lactobacillus strains to diversify and improve cheese maturation

This study evaluated the influence of 11 autochthonous mesophilic lactobacilli strains on maturation profile of a miniature semihard cheese. Five strains of Lactiplantibacillus plantarum, three Lacticaseibacillus rhamnosus, two Lacticaseibacillus casei and one Lacticaseibacillus paracasei, were evaluated individually as adjunct cultures. Three groups of strains were distinguished regarding their influence: One group was characterised by consumption of citrate and production of several volatile compounds, another group by its acidification and peptidolytic activity, while two strains had no influence. The combined use of strains with different properties as adjunct cultures probably allows further improvement and diversification of cheese flavour.

Investigation of Freezing and Freeze-Drying for Preserving and Re-Using a Whole Microbial Cheese Community.

Preserving microbial ecosystems obtained from traditional cheese-making processes is crucial to safeguarding the biodiversity of microbial cheese communities and thus ensuring that the high flavor quality of traditional cheeses is maintained. Few protocols have been proposed for the long-term storage of microbial consortia. This work aimed to develop preservation methods to stabilize the entire microbial community in smear-ripened cheese without multiplication or isolation. A simplified microbial community, capable of reproducing the metabolic pattern of cheese maturation, was used in three independent cheese productions. Cheese samples were taken before and after the ripening step, mixed with maltodextrin or saline solution, and subjected to different stabilization conditions including freezing and freeze-drying, followed by 1 month of storage. Microbial survival was quantified using the colony-forming unit assay. Differential scanning calorimetry was used to relate the physical events occurring within the samples to the microbial storage stability. Freezing at -80 °C resulted in the lowest loss of culturability (<0.8 log unit), followed by freezing at -20 °C and freeze-drying. The ripening bacteria appeared as the most sensitive microorganisms within the community. Moreover, a successful cheese production using the best-stabilized community showed the possibility of preserving and re-using an entire microbial community of interest.

Probiotic Potential and Application of Indigenous Non-Starter Lactic Acid Bacteria in Ripened Short-Aged Cheese.

There are massive sources of lactic acid bacteria (LAB) in traditional dairy products. Some of these indigenous strains could be novel probiotics with applications in human health and supply the growing needs of the probiotic industry. In this work, were analyzed the probiotic and technological properties of three Lactobacilli strains isolated from traditional Brazilian cheeses. In vitro tests showed that the three strains are safe and have probiotic features. They presented antimicrobial activity against pathogenic bacteria, auto-aggregation values around 60%, high biofilm formation properties, and a survivor of more than 65% to simulated acid conditions and more than 100% to bile salts. The three strains were used as adjunct cultures separately in a pilot-scale production of Prato cheese. After 45days of ripening, the lactobacilli counts in the cheeses were close to 8 Log CFU/g, and was observed a reduction in the lactococci counts (around -3 Log CFU/g) in a strain-dependent manner. Cheese primary and secondary proteolysis were unaffected by the probiotic candidates during the ripening, and the strains showed no lipolytic effect, as no changes in the fatty acid profile of cheeses were observed. Thus, our findings suggest that the three strains evaluated have probiotic properties and have potential as adjunct non-starter lactic acid bacteria (NSLAB) to improve the quality and functionality of short-aged cheeses.

Physicochemical and microbiological characterisation of a typical Italian raw ewe's milk cheese: Pecorino bagnolese

Pecorino Bagnolese is a typical Italian cheese made from raw ewe's milk in province of Avellino (southern Italy). The aim of this study was to evaluate the physicochemical and microbiological parameters during cheese-making and ripening (30, 90 and 180 d). Results showed that the cheese-making and ripening influenced the pH, water activity, moisture, fat content and levels of Enterobacteriaceae. Changes in the fatty acids profile of ripened cheeses compared to milk were observed, suggesting the influence of ripening on the fatty acids profile. Nutritional indices demonstrated positive health attributes. The concentration of malondialdehyde remained consistently low, while free fatty acids showed an increasing trend during ripening. Coagulase-positive staphylococci complied with the legislative threshold value, but colonies of Staphylococcus aureus were found. Pathogens were not detected. In conclusion, the data collected provided a better characterisation of this traditional cheese and can be used for broadening knowledge of raw ewe's milk cheeses.

The addition of Citrus aurantium leaves and tomato pomace in the diet of Awassi ewes and the impact on milk composition and matured cheese

The purpose of this study was to use concentrated feed alternatives with other less expensive feed alternatives in feeding the Awassi ewes and the impact on milk composition and matured cheese. A total of twenty Awassi ewes were used, they were selected in the middle of lactation and divided into two homogeneous groups with weights (47±1.7 kg) and aged 3-4 years, given two different servings of feed (1.5 kg/sheep/day) Milk composition was measured weekly, to make cheese, raw milk control (CON) and Citrus aurantium leaves and tomato pomace (CALTP) is kept separately and chilled. Results indicated that fat and total solid content were high in milk and cheese manufactured in the diet that contained CALTP; the proportion of medium-chain fatty acids (c10-c16) and free fatty acids was lower compared to the diet (CON). While the proportion of unsaturated fatty acids (UFA) in the diet increased CALTP. Therefore, the inclusion of CALTP in the diet is helpful because it has a positive impact on the composition of milk and mature cheese, reduces the cost of the diet and helps recycle byproducts.

Short communication: Comparing the microbiota diversity from the core, middle part and rind of six Spanish commercial goat cheeses

The cheese core has a lower oxygen saturation and salinity and a higher acidity than the rind, but there is controversy about the incidence of such factors on the magnitude of microbial diversity. The goal of the current work was to investigate the existence of differences in α-diversity between the core, middle part, and rind of six Spanish commercial cheeses through a sequencing approach. To this end, we have collected rind, middle part, and core samples from fresh (H and M), soft semi-ripened (C and P), hard semi-ripened (B) and semi-hard aged (G) goat cheeses. After purifying deoxyribonucleic acid from these 18 samples, the V3-V4 ultravariable region of the 16S rRNA gene was sequenced. The analysis of microbial composition revealed that lactic acid bacteria from the genera Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc are predominant in all six goat cheeses. Furthermore, we identified several psychrophilic taxa often associated with the post-pasteurization contamination of refrigerated milk. Comparison of three α-diversity estimators (Chao1, Shannon and Faith's phylogenetic diversity indices) of microbiota in the core, middle part, and rind of all six goat cheeses did not reveal substantial differences, being only significant (at the nominal level) the comparison of rind vs middle part for the Shannon index (P-value = 0.031). Moreover, the construction of a dendrogram based on Aitchison distances revealed that cheese samples cluster according to their manufacturing characteristics, with a clear distinction between fresh vs semi-ripened or aged cheeses. We conclude that the magnitude of microbial α-diversity in the cheese core is similar to that in the rind despite their different physicochemical attributes. This result could be because physicochemical differences between cheese compartments are often attenuated during cheese ripening.

Biofunctional, chemical and sensory profile of mature cheese produced with artichoke flower coagulant (Cynara cardunculus L. var scolymus)

The objective was to analyze how the use of artichoke flower coagulant (Cynara cardunculus L. var. scolymus) affects the chemical, biofunctional and sensorial characteristics of cheeses during the ripening process. The cheeses were produced with bovine milk and extracts of artichoke flowers (TA), thistle flowers (TT) and microbial chymosin (TC), analyzed 1 d after production and during maturation for 10, 20 and 30 d. The reactions that occurred in the TA during maturation mainly impacted the proteolysis and texture of the cheeses. All cheeses had antioxidant and antibacterial properties. Sensorially, the TA cheese was similar to the TT, mainly in the bitter taste. The presence of hydrophobic peptides was greater in TA and TT cheeses when compared to TT, suggesting that proteolysis occurred in a similar way in both types of cheese, indicating the effectiveness of using the artichoke flower coagulant in the production of cheeses with a pasty texture.

Effect of Lactiplantibacillus plantarum CNPC003 and milk pasteurization on artisanal goat coalho cheese characteristics

This study evaluated the effect of adding Lactiplantibacillus plantarum CNPC003 (LP) and applying milk pasteurization on the microbiological, technological, and physicochemical characteristics and protein profile of artisanal goat coalho cheese during 60 days of ripening. Four types of cheese were produced, including cheeses made with raw and pasteurized goat milk and with and without LP (RC, PC, RCLP, and PCLP). All cheeses were safe for consumption and maintained high lactic acid bacteria counts (above 8 log CFU mL−1) at the end of the ripening period. RC and RCLP showed higher hardness and lower chewiness than cheeses made with pasteurized milk (PC and PCLP) after 60 days of ripening. As expected, there was a reduction in lactose content with a simultaneous increase in acidity and syneresis for all cheeses over the 60 days of ripening, with no significant change in the other evaluated physicochemical parameters. Proteolysis rates increased in all cheeses up to 60 days of ripening, aligning with elevated levels of soluble protein and free amino acids. After 60 days of ripening, PC showed a higher extent of proteolysis index, higher amount of high molecular weight peptides, higher number of identified proteins, and lower content of free amino acids. RCLP showed a higher depth of proteolysis index, higher free amino acids amount (1583 ± 3.95 mg 100 g−1), and fewer identified proteins. Among the peptides identified, it was possible to define 30 potential bioactive peptides according to the bioactivity score (>0.5), where both cheeses added with LP had 18 of these peptides, mostly potentially ACE inhibition sequences. The addition of LP and the pasteurization of milk positively affected the microbiological safety of ripened coalho goat cheeses, primarily contributing to the differentiation of their texture and protein and bioactivity profile.

The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits

BackgroundArtisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese.ResultsBoth the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and β-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product.ConclusionOverall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese.A8MAe52mDCf21YyaVzbpABVideo

Selection of adjunct cultures for the ripening of plant cheese analogues

Fermentation contributes to the taste and odor of plant cheeses. The selection of functional cultures for the fermentation of plant cheeses, however, is in its infancy. This study aimed to select lactic acid bacteria for ripening of soy and lupin cheese analogues. Bacillus velezensis and B. amyloliquefaciens were used for germination of seeds to produce proteolytic enzymes; Lactococcus lactis and Lactiplantibacillus plantarum served as primary acidifying cultures. Levilactobacillus hammesii, Furfurilactobacillus milii, or Lentilactobacillus buchneri were assessed as adjunct cultures for the ripening of plant cheese. Growth of bacilli was inhibited at low pH. Both Lc. lactis and Lp. plantarum were inactived during plant cheese ripening. Cell counts of Lv. hammesii remained stable over 45 d of ripening while Ff. milii and Lt. buchneri grew slowly. Sequencing of full length 16S rRNA genes confirmed that the inocula the plant cheeses accounted for more than 98% of the bacterial communities. HPLC analysis revealed that Lt. buchneri metabolized lactate to acetate and 1,2-propanediol during ripening. Bacilli enhanced proteolysis as measured by quantification of free amino nitrogen, and the release of glutamate. LC-MS/MS analysis quantified kokumi-active dipeptides. The concentrations of γ-Glu-Leu, γ-Glu-Ile, and γ-Glu-Ala, γ-Glu-Cys in unripened cheeses were increased by seed germination but γ-Glu-Phe was degraded. Lt. buchneri but not Lv. hammesii or Ff. milii accumulated γ-Glu-Val, γ-Glu-Ile or γ-Glu-Leu during ripening, indicating strain-specific differences. In conclusion, a consortium of bacilli, acidification cultures and adjunct cultures accumulates taste- and kokumi-active compounds during ripening of plant cheeses.

Cocoa byproduct inclusion in dairy sheep diet: Effects on sensory, volatile, and antioxidant properties of cheese

The possibility of inclusion of agro-industrial byproducts in the diet of small ruminants represents both an economic and an environmental strategy for reducing waste management by industries and costs of feeding as well as the impact of livestock farming. Large amounts of wastes from the cocoa industry are produced annually, with a considerable part represented by cocoa bean shells, considered a suitable ingredient to be included in the diet of ruminants within the limits established by European legislation. The aim of this study was to assess the effect of including cocoa bean shells in the diet of dairy sheep on the sensory, volatile, and antioxidant properties of cheese. To this purpose, 20 lactating Comisana ewes were randomly assigned to 2 experimental groups: control (CTRL) and cocoa bean shells (CBS), and received alfalfa hay ad libitum and 800 g of conventional (CTRL) or experimental (CBS) concentrate containing 11.7% CBS to partially replace corn and barley of the CTRL concentrate. Bulk milk collected from each group was used to produce a total of 15 cheeses per group, obtained in 5 different days of cheesemaking (3 cheeses a day per group). After 60 d of aging, each cheese of each experimental group was sampled for the analyses. The results on chemical composition revealed a greater content of monounsaturated fatty acids and an increase in the nutritional indices, suggesting a favorable role of cocoa bean shell dietary inclusion on the nutritive value of cheese. The cheese sensory profile was affected by the cocoa bean shell inclusion, with more pronounced appearance, odor, aroma, and taste attributes in the product. The volatile profile showed only a few significant differences, mainly related to the cheese ripening process, and no differences were found in α-tocopherol contents in cheese fat between the 2 groups. Therefore, the inclusion of coca bean shells in the diet of dairy sheep allowed us to obtain a good-quality cheese, without altering the characteristics associated with the typical profiles of sheep cheese. Furthermore, the use of this byproduct could contribute to decreasing feed costs and waste management, representing a good practice for increasing the sustainability of dairy products.

Phenotypic and genotypic characterization of antimicrobial resistances reveals the effect of the production chain in reducing resistant lactic acid bacteria in an artisanal raw ewe milk PDO cheese

Antimicrobial resistance (AMR) is a significant public health threat, with the food production chain, and, specifically, fermented products, as a potential vehicle for dissemination. However, information about dairy products, especially raw ewe milk cheeses, is limited. The present study analysed, for the first time, the occurrence of AMRs related to lactic acid bacteria (LAB) along a raw ewe milk cheese production chain for the most common antimicrobial agents used on farms (dihydrostreptomycin, benzylpenicillin, amoxicillin and polymyxin B). More than 200 LAB isolates were obtained and identified by Sanger sequencing (V1-V3 16S rRNA regions); these isolates included 8 LAB genera and 21 species. Significant differences in LAB composition were observed throughout the production chain (P ≤ 0.001), with Enterococcus (e.g., E. hirae and E. faecalis) and Bacillus (e.g., B. thuringiensis and B. cereus) predominating in ovine faeces and raw ewe milk, respectively, along with Lactococcus (L. lactis) in whey and fresh cheeses, while Lactobacillus and Lacticaseibacillus species (e.g., Lactobacillus sp. and L. paracasei) prevailed in ripened cheeses. Phenotypically, by broth microdilution, Lactococcus, Enterococcus and Bacillus species presented the greatest resistance rates (on average, 78.2 %, 56.8 % and 53.4 %, respectively), specifically against polymyxin B, and were more susceptible to dihydrostreptomycin. Conversely, Lacticaseibacillus and Lactobacillus were more susceptible to all antimicrobials tested (31.4 % and 39.1 %, respectively). Thus, resistance patterns and multidrug resistance were reduced along the production chain (P ≤ 0.05). Genotypically, through HT-qPCR, 31 antimicrobial resistance genes (ARGs) and 6 mobile genetic elements (MGEs) were detected, predominating Str, StrB and aadA-01, related to aminoglycoside resistance, and the transposons tnpA-02 and tnpA-01. In general, a significant reduction in ARGs and MGEs abundances was also observed throughout the production chain (P ≤ 0.001). The current findings indicate that LAB dynamics throughout the raw ewe milk cheese production chain facilitated a reduction in AMRs, which has not been reported to date.

Autochthonous cultures to improve the quality of PGI Castellano cheese: Impact on proteolysis, microstructure and texture during ripening

The aim of this research was to find out the effect of different combinations of starter and non-starter cultures on the proteolysis of Castellano cheese during ripening. Four cheese batches were prepared, each containing autochthonous lactobacilli and or Leuconostoc, and were compared with each other and with a control batch, that used only a commercial starter. To achieve this, nitrogen fractions (pH 4.4-soluble nitrogen and 12 % trichloroacetic acid soluble nitrogen, polypeptide nitrogen and casein nitrogen), levels of free amino acids and biogenic amines were assessed. Texture and microstructure of cheeses were also evaluated. Significant differences in nitrogen fractions were observed between batches at different stages of ripening. The free amino acid content increased throughout the cheese ripening process, with a more significant increase occurring after the first 30 days. Cheeses containing non-starter lactic acid bacteria exhibited the highest values at the end of the ripening period. Among the main amino acids, GABA was particularly abundant, especially in three of the cheese batches at the end of ripening. The autochthonous lactic acid bacteria were previously selected as non-producers of biogenic amines and this resulted in the absence of these compounds in the cheeses. Analysis of the microstructure of the cheese reflected the impact of proteolysis. Additionally, the texture profile analysis demonstrated that the cheese's hardness intensified as the ripening period progressed. The inclusion of autochthonous non-starter lactic acid bacteria in Castellano cheese production accelerated the proteolysis process, increasing significantly the free amino acids levels and improving the sensory quality of the cheeses.

A Metabolomics Study by 1H HRMAS NMR: From Sheep Milk to a Pressed-Curd Cheese: A Proof of Concept

For the first time, High-Resolution Magic Angle Nuclear Magnetic Resonance spectroscopy (NMR-HRMAS) was applied to directly identify specific metabolites from a Spanish raw ewe’s milk and enzymatic coagulation pressed-curd cheese (Protected Geographical Indication: Castellano) manufactured by two procedures (traditional/artisanal vs. industrial) and including the ewe’s raw milk. The NMR parameters were optimized to study the complex matrixes of this type of cheese. In addition, conventional overcrowded 1H-NMR-HRMAS spectra were selectively simplified by a Carr–Purcell–Meiboom–Gill (CPMG) sequence or a stimulated echo pulse sequence by bipolar gradients (DIFF), thus modulating spin–spin relaxation times and diffusion of molecular components, respectively. 1H-NMR-HRMAS spectroscopy displayed important information about cheese metabolites, which can be associated with different manufacturing processes (industrial vs. traditional) and ripening times (from 2 to 90 days). These results support that this spectroscopy is a useful technique to monitor the ripening process, from raw milk to commercial ripened cheese, using a minimum intact sample, implying the absence of time-consuming sample pretreatments.

Brazilian indigenous nonstarter lactic acid bacteria enhance the diversification of volatile compounds in short-aged cheese.

There is growing interest in using autochthonous lactic acid bacteria (LAB) that provide unique sensory characteristics to dairy products without affecting their safety and quality. This work studied the capacity of three Brazilian indigenous nonstarter LABs (NSLAB) to produce biogenic amines (BAs) and evaluated their effect on the volatile organic compounds (VOCs), microbial LAB communities, and physicochemical profile of short-aged cheese. Initially, the strain's potential for biosynthesis of BAs was assessed by PCR and in vitro assays. Then, a pilot-scale cheese was produced, including the NSLAB, and the microbial and VOC profiles were analyzed after 25 and 45 days of ripening. As a results, the strains did not present genes related to relevant BAs and did not produce them in vitro. During cheese ripening, the Lactococci counts were reduced, probably in the production of alcohols and acid compounds by the NSLAB. Each strain produces a unique VOC profile that changes over the ripening time without the main VOCs related to rancid or old cheese. Particularly, the use of the strain Lacticaseibacillus. paracasei ItalPN16 resulted in production of ester compounds with fruity notes. Thus, indigenous NSLAB could be a valuable tool for the enhancement and diversification of flavor in short-aged cheese.