Omics Approaches to Assess Flavor Development in Cheese

Cheese is characterized by a rich and complex microbiota that plays a vital role during both production and ripening, contributing significantly to the safety, quality, and sensory characteristics of the final product. In this context, it is vital to explore the microbiota composition and understand its dynamics and evolution during cheese manufacturing and ripening. Application of high-throughput DNA sequencing technologies have facilitated the more accurate identification of the cheese microbiome, detailed study of its potential functionality, and its contribution to the development of specific organoleptic properties. These technologies include amplicon sequencing, whole-metagenome shotgun sequencing, metatranscriptomics, and, most recently, metabolomics. In recent years, however, the application of multiple meta-omics approaches along with data integration analysis, which was enabled by advanced computational and bioinformatics tools, paved the way to better comprehension of the cheese ripening process, revealing significant associations between the cheese microbiota and metabolites, as well as their impact on cheese flavor and quality.

Manganese modulates metabolic activity and redox homeostasis in translationally-blocked Lactococcus cremoris, impacting metabolic persistence, cell-culturability, and flavor formation.

Manganese (Mn) is an essential trace element that is supplemented in microbial media with varying benefits across species and growth conditions. We found that growth of Lactococcus cremoris was unaffected by manganese omission from the growth medium. The main proteome adaptation to manganese omission involved increased manganese transporter production (up to 2000-fold), while the remaining 10 significant proteome changes were between 1.4 and 4 fold. Further investigation in translationally-blocked (TB), non-growing cells showed that Mn supplementation (20 µM) led to approximately 1.5X faster acidification compared to Mn-free conditions. However, this faster acidification stagnated within 24 hours, likely due to draining of intracellular NADH that coincides with substantial loss of culturability. Conversely, without manganese, non-growing cells persisted to acidify for weeks, albeit at a reduced rate, but maintaining redox balance and culturability. Strikingly, despite being unculturable, α-keto acid-derived aldehydes continued to accumulate in cells incubated in the presence of manganese, whereas without manganese cells predominantly formed the corresponding alcohols. This is most likely reflecting NADH availability for the alcohol dehydrogenase-catalyzed conversion. Overall, manganese influences the lactococcal acidification rate, and flavor formation capacity in a redox dependent manner. These are important industrial traits especially during cheese ripening, where cells are in a non-growing, often unculturable state.

Developments in effective use of volatile organic compound analysis to assess flavour formation during cheese ripening

In the burgeoning demand for optimization of cheese production, ascertaining cheese flavour formation during the cheese making process has been the focal point of determining cheese quality. In this research reflection, we have highlighted how valuable volatile organic compound (VOC) analysis has been in assessing contingent cheese flavour compounds arising from non-starter lactic acid bacteria (NSLAB) along with starter lactic acid bacteria (SLAB), and whether VOC analysis associated with other high-throughput data might help provide a better understanding the cheese flavour formation during cheese process. It is widely known that there is a keen interest to merge all omics data to find specific biomarkers and/or to assess aroma formation of cheese. Towards that end, results of VOC analysis have provided valuable insights into the cheese flavour profile. In this review, we are pinpointing the effective use of flavour compound analysis to perceive flavour-forming ability of microbial strains that are convenient for dairy production, intertwining microbiome and metabolome to unveil potential biomarkers that occur during cheese ripening. In doing so, we summarised the functionality and integration of aromatic compound analysis in cheese making and gave reflections on reconsidering what the role of flavour-based analysis might have in the future.

Development of a new Brazilian semi-hard (Coalho) Buffalo cheese made with the inclusion of cow milk and functional potential / Desenvolvimento de um novo queijo semiduro brasileiro (Coalho) de búfalo feito com a inclusão de leite de vaca e potencial funcional

This study aimed to produce a new Brazilian Coalho buffalo cheese with 45% cow milk and potential functional through inulin and Lactobacillus plantarum addition. The Four treatments were made: the control (CTL) without functional ingredients, prebiotic (PRE) with Inulin, probiotic (PRO) with L. plantarum, and symbiotic (SYM) with both Inulin and L. plantarum. The starter culture (Lactococcus spp.) and L. plantarum viability and pH were analyzed during cheese ripening (0, 4, 8, and 10 days). While moisture, fat, and fat in the dry matter parameters were evaluated after ten days of storage ripening. During the ripening, the Lactococcus spp. growth was significant in the first eight days. The starter culture stabilized in counts above 7.1 log UFC.g-1. In the same period, L. plantarum was no growth ( 10) neither in PRO nor SYM treatments. The higher Lactococcus spp. counts (up to 10.6 Log UFC.g-1) in PRE and PRO cheeses leads to the most intense pH decrease (5.4) during the ripening. All treatments pointed to a moisture content between 38.09% to 40.49% and classified as Brazilian Coalho cheeses. The PRE had a higher FDM content (42.66%), while the lowest values were from SYM (35.53%). Thus, inulin addition does not negatively affect parameters tested, indicating a good fiber source for prebiotic semi-cooked and semi-hard cheeses. The L. plantarum viability was compromised during the process. Future studies about temperature adaptation, previous activation of strains or even encapsulation techniques associated with starter function should be evaluated to draw the best inoculation strategies for this probiotic for Coalho buffalo cheese.

FT-MIR Analysis of Water-Soluble Extracts during the Ripening of Sheep Milk Cheese with Different Phospholipid Content

The purpose of this work was to study the suitability of the water-soluble extracts (WSE) of semi-hard sheep milk cheese for analysis by diffuse reflectance Fourier transform mid-infrared spectroscopy (FT-MIR) and the development of classification models using discriminant analysis and based on cheese age or phospholipid content. WSE was extracted from three types of sheep milk cheeses (full-fat, reduced-fat and reduced-fat fortified with lyophilized sweet sheep buttermilk) at various stages of ripening from six to 168 days and lyophilized. The first model used 1854–1381 and 1192–760 cm−1 regions of the first-derivative spectra and successfully differentiated samples of different age, based on changes in the water-soluble products of ripening biochemical events. The second model used the phospholipid absorbance spectral regions (3012–2851, 1854–1611 and 1192–909 cm−1) to successfully discriminate cheeses of markedly different phospholipid content. Cheese WSE was found suitable for FT-MIR analysis. According to the results, a fast and simple method to monitor cheese ripening based on water-soluble substances has been developed. Additionally, the results indicated that a considerable amount of phospholipids migrates to the cheese WSE and that FT-MIR can be a useful tool for their assessment.

An in-Depth Investigation of the Safety of Wooden Shelves Used for Traditional Cheese Ripening

The main goal of this research was to characterize the bacterial diversity of wooden boards used for aging traditional Sicilian cheeses and to evaluate whether pathogenic bacteria are associated with these surfaces. Eighteen cheese dairy factories producing three traditional cheese typologies (PDO Pecorino Siciliano, PDO Piacentinu Ennese and Caciocavallo Palermitano) were selected within Sicily region. The wooden shelf surfaces were sampled by a destructive method to detach wood splinters as well as by a non-destructive brushing to collect microbial cells. Scanning electron microscopy showed the presence of almost continuous bacterial formations on the majority of the shelves analysed. Yeasts and fungal hyphae were also visualized, indicating a complexity of the plank communities. The amplicon library of the 16S rRNA gene V3-V4 region was pair-end sequenced using the Illumina MiSeq system allowing the identification of 14 phyla, 32 classes, 52 orders, 93 families and 137 genera. Staphylococcus equorum was identified from all wooden surfaces with a maximum abundance of 64.75%. Among cheese surface ripening bacteria, Brevibacterium and Corynebacterium were detected in almost all samples. Several halophilic ( Halomonas , Tetragenococcus halophilus , Chromohalobacter, Salimicrobium , Marinococcus , Salegentibacter , Haererehalobacter , Marinobacter and Idiomarinaceae) and moderately halophilic ( Salinicoccus , Psychrobacter and Salinisphaera ) bacteria were frequently identified. Lactic acid bacteria (LAB) were present at low percentages with the genera Leuconostoc , Lactococcus , Lactobacillus , Pediococcus and Streptococcus . The levels of wooden shelf viable microorganisms ranged between 2.4 and 7.8 log CFU/cm 2 . In some cases, LAB were counted at very high levels (8.2 log CFU/cm 2 ). Members of Enterobacteriaceae family were detected in a viable state only for six samples. Coagulase positive staphylococci, Salmonella spp. and Listeria monocytogenes were not detected. Seventy-five strains belonged to Leuconostoc , Lactococcus , Pediococcus , Enterococcus , Lactobacillus and Weissella genera. IMPORTANCE This study provides evidence for the lack of pathogenic bacteria on the wooden shelves used to ripen internal bacterially ripened semi-hard and hard cheeses produced in Sicily. These three cheeses are non-inoculated on their surfaces and surface ripening is not considered to occur or, at least, not at the same extent of surface inoculated smear cheeses. Several bacterial groups identified from the wooden shelves are typically associated with smear cheeses strongly suggesting that PDO Pecorino Siciliano, PDO Piacentinu Ennese and Caciocavallo Palermitano cheese rind contributes to their final organoleptic profiles.

Adaptive laboratory evolution as a means to generate Lactococcus lactis strains with industrially relevant traits – Characterization of isolates with improved thermotolerance and ability to autolyze

Lactococcus lactis subsp. lactis ( L. lactis ) is a model lactic acid bacterium and one of the main constituents of the mesophilic cheese starter used for producing soft or semi-hard cheeses. Most dairy L. lactis strains grow optimally at around 30°C and are not particularly well-adapted to the elevated temperatures (37-39°C), which they are often exposed to during cheese production. To overcome this challenge, we used Adaptive Laboratory Evolution (ALE) in milk, using a setup where the temperature was gradually increased over time, and isolated two evolved strains (RD01 and RD07) better able to tolerate high growth temperatures. One of these, strain RD07, was isolated after one and a half years of evolution (400 generations) and efficiently acidified milk at 41°C, which has not been reported for industrial L. lactis strains until now. Moreover, RD07 appeared to autolyze 2-3 times faster than its parent strain, which is another highly desired property of dairy lactococci and rarely observed in the lactis subspecies used in this study. Model cheese trials indicated that RD07 could potentially accelerate cheese ripening. Transcriptomics analysis revealed the potential underlying causes responsible for the enhanced growth at high temperatures for the mutants. These included downregulation of the pleiotropic transcription factor CodY and overexpression of genes, which most likely lowered the guanidine nucleotide pool. Cheese trials at ARLA Foods using RD01 blended with the commercial Flora Danica starter culture, including a 39.5°C cooking step, revealed better acidification and flavor formation than the pure starter culture. Importance In commercial mesophilic starter cultures, L. lactis is generally more thermotolerant than L. cremoris , whereas L. cremoris is more prone to autolysis, which is the key to flavor and aroma formation. In this study, we find that adaptation to higher thermotolerance can improve autolysis. Using whole-genome sequencing and RNA-sequencing, we attempt to determine the underlying reason for the observed behavior. In terms of dairy applications, there are obvious advantages associated with using L. lactis strains with high thermotolerance, as these are less affected by curd cooking, which generally hampers the performance of the mesophilic starter. Cheese ripening, the most costly part of cheese manufacturing, can be reduced using autolytic strains. Thus, the solution presented here could simplify starter cultures, make the cheese manufacturing process more efficient, and enable novel types of harder cheese variants.

Preliminary Study of Extended Ripening Effects on Peptides Evolution and DPPH Radical Scavenging Activity in Mexican Goat Cheese

Cheese ripening causes intense proteolysis, particularly when the cheese contains starter cultures. Several studies have shown the presence of bioactive peptides in goat’s milk cheeses with antioxidant activity. Mexican goat cheeses’ peptide fractions were evaluated at different ripening stages. Additionally, they were correlated with their antioxidant activity. Proteolysis was measured in the acid-soluble nitrogen and non-protein nitrogen fractions using reverse-phase high-performance liquid chromatography. While the antioxidant activity in both nitrogenous fractions was determined using a 2,2-diphenyl-1-picrylhydrazyl solution. Analyzed cheeses showed peptides fraction in the retention time of 2.05, 18.36, and 50.11 min for acid-soluble fraction and non-protein protein nitrogen, and showed antioxidant activity from the first day of ripening to 73% discoloration in the DPPH solution at 55 ripening days. Obtained results suggested that ripened Mexican goat cheese had a DPPH radical scavenging activity related to peptides present originally in the milk or released by starter culture action during cheese ripening.

Identification of Red Pigments Produced by Cheese-Ripening Bacterial Strains of Glutamicibacter arilaitensis Using HPLC

Glutamicibacter arilaitensis is one of the predominant bacterial species involved in the coloration of cheese rinds, especially smear-ripened cheeses. Besides well-known yellow-pigmented carotenoids, this species exhibits an ability to produce red pigments, as the occurrence of pink/red formation was previously found when co-cultured with a fungal strain. In this work, the red pigments synthesized by G. arilaitensis strains grown on cheese-based (curd) solid medium deacidified using Debaryomyces hansenii were identified. The analyses using HPLC equipped with both fluorescence and diode array detectors were performed to characterize the pigments extracted from a dry matter of the medium inoculated with either G. arilaitensis Re117, Po102, or Stp101. Based on the UV–vis absorption spectra, the elution order, and fluorescent property, compared to those of the porphyrin standards, eight metal-free porphyrins, including UPI, UPIII, 7PI, 6PI, 5PI, CPI, CPIII, and MPIX, were indicated as components of the red pigments produced by these G. arilaitensis strains. However, following the chromatographic profiles, the degree of porphyrins formed by each strain was apparently different. Regardless of precise quantitative measurement, the type strains Re117 and Po102 manifested a potential to produce a high amount of CPIII, whereas MPIX was formed by the strains Po102 and Stp101, but exceptionally high by the strain Stp101. The variation in both yield and form of the red pigments synthesized by the cheese-related bacterial G. arilaitensis has not previously been reported; therefore, our results provide the first information on these aspects.