Cheese Ripening Research Articles

Proteomic methods for separation and identification of milk proteins

The review presents the most common proteomics methods based on separation and identification of proteins, with examples of their use for studying the protein system of milk and dairy products. The essence of electrophoretic and chromatographic methods for separating proteins and peptides and their identification using Western blotting and mass spectrometry is described. The main types of electrophoresis methods (native, two-dimensional, in polyacrylamide gel with urea, immunoelectrophoresis, capillary) and chromatography methods (gas, liquid, ion exchange, gel filtration, affinity) are given. It is noted that the main direction of research using these methods, which has both scientific and applied significance, is the detection of falsifications in the composition of milk and dairy products. Such falsifications are associated with the deliberate addition of dry milk and cheese whey, as well as with the replacement of raw milk by milk from other animals, which is unacceptable in the production of cheeses with a protected designation of origin (PDO) or a protected geographical indication (PGI). Another area is research into the proteolytic processes that occur during the maturation of cheeses, on the basis of which their degree of maturity and species can be determined depending on the manufacturing technology. Given the wide range of cheeses from different countries, such research is clearly insufficient.

Acceleration RAS Cheese Ripening Using Extracts of Some Plants

Acceleration RAS Cheese Ripening Using Extracts of Some Plants

Properties of processed cheese made from cagliata cheese: Influence of fat content and ripening of cagliata cheese and homogenization of cheese milk

Properties of processed cheese made from cagliata cheese: Influence of fat content and ripening of cagliata cheese and homogenization of cheese milk

Mycobiota composition through the ripening of artisanal soft cheeses ‘Torta del Casar’ and ‘Queso de la Serena’ monitored by high-throughput sequencing

Mycobiota composition through the ripening of artisanal soft cheeses ‘Torta del Casar’ and ‘Queso de la Serena’ monitored by high-throughput sequencing

Biochemical Processes During Cheese Ripening

Cheese ripening entails specific biochemical changes that occur under certain conditions during storage. These changes allow different cheese varieties to develop their unique characteristics. The ripening process is influenced by both primary and secondary biochemical events. These are driven by coagulating enzymes, milk's natural enzymes, and the enzymes of both starter and non-starter microflora. The main biochemical events during cheese ripening include proteolysis, lipolysis, and the metabolism of citrate and lactate. Secondary biochemical reactions then process the primary metabolic products, such as lactic acid, fatty acids, and amino acids. This leads to the formation of volatile compounds like alcohols, aldehydes, ketones, acids, lactones, phenols, esters, and sulfur compounds, which play a crucial role in determining the cheese's quality. These processes give each cheese type its distinctive features, like aroma, taste, color, texture, and pore structure, influencing consumer preferences. This review provides insights into the biochemical events that occur during the cheese ripening period.

Bacteriocin-Producing Enterococci Modulate Cheese Microbial Diversity

Cheese production involves various lactic acid bacteria (LAB) that break down lactose, milk proteins, and fats, producing key nutrients and influencing the cheese’s flavor. They form communities that play a crucial role in determining the cheese’s organoleptic properties. The composition of cheeses’ microbial communities is shaped by physicochemical factors (e.g., temperature, pH, and salinity) and biological factors (i.e. microbial interactions). While starter cultures are introduced to control these communities, non-starter LAB represent a significant portion of the final microbial assemblage, but their interactions remain unclear. LAB often produce bacteriocins, antimicrobial peptides that antagonize other bacteria, but their role within LAB communities is not fully understood. This study aimed to assess the impact of bacteriocin production on LAB diversity in cheese, using Enterococcus as a model organism, a common bacteriocin producer. We analyzed enterocin production of enterococcal isolates by antimicrobial assays and microbial diversity differences in raw milk cheeses by two approaches: 16S RNA gene amplicon metagenomic sequencing for the whole microbial community and multi-locus sequence analysis (MLSA) for the enterococcal diversity. Our results revealed that LAB communities were dominated by lactococci, lactobacilli, and streptococci, with enterococci present in lower numbers. However, cheeses containing bacteriocin-producing enterococci exhibited higher microbial diversity. Interestingly, the highest diversity occurred at low levels of bacteriocin producers, but this effect was not observed within enterococcal populations. These findings suggest that bacteriocin production plays a key role in shaping LAB communities during cheese ripening, although further research is needed to understand its broader implications in other microbial ecosystems.

A Global Review of Cheese Colour: Microbial Discolouration and Innovation Opportunities

Cheese is a biologically active food product, characterised by its colour, texture, and taste. Due to its rich matrix of fats and proteins, as well as the fact that the cheese’s surface acts as its own packaging, the cheese becomes more susceptible to contamination by microorganisms during the ripening process, particularly bacteria and fungi. The ripening of cheese involves several biochemical reactions, with the proteolytic activity of the cheese microbiota being particularly significant. Proteolysis results in the presence of free amino acids, which are precursors to various metabolic mechanisms that can cause discolouration (blue, pink, and brown) on the cheese rind. Surface defects in cheese have been documented in the literature for many years. Sporadic inconsistencies in cheese appearance can lead to product degradation and economic losses for producers. Over the past few decades, various defects have been reported in different types of cheese worldwide. This issue also presents opportunities for innovation and development in edible and bioactive coatings to prevent the appearance of colour defects. Therefore, this review provides a comprehensive analysis of cheese colour globally, identifying defects caused by microorganisms. It also explores strategies and innovation opportunities in the cheese industry to enhance the value of the final product.

FibroScan's evolution: a critical 20-year review.

FibroScan, initially designed for assessing cheese maturity, has evolved into a crucial medical tool for liver fibrosis diagnosis. This systematicreview explores its development history, functionality, and pros and cons compared to traditional liver biopsy. Precision in various clinical settings is scrutinised, emphasising FibroScan's accuracy in conditions like NAFLD and viral-induced liver disease. The article also delves into its potential in paediatrics, its relevance in monitoring COVID-19-related liver complications, and its role in predicting hepatocellular carcinoma risk, Technical aspects, including transducers, imaging integration, and portability, are examined. Various methods for evaluating liver fibrosis are discussed, highlighting FibroScan's suitability for advanced stages, contrasting with the gold standard of liver biopsy for early stages. The impact of FibroScan on long-term liver conditions is emphasised, focusing on early detection, progression monitoring, reduced invasive biopsies, and hepatocellular carcinoma risk prediction. This systematicreview underscores FibroScan's transformative potential in liver disease treatment and predicts ongoing research to enhance early detection, disease monitoring, and explore new clinical applications. Anticipated advances include FibroScan-guided liver biopsy, artificial intelligence data analysis, and point-of-care device development, promising a further revolution in liver disease management. The article concludes with optimistic prospects for FibroScan's future.

Iron fortification modifies the microbial community structure and metabolome of a model surface-ripened cheese

Iron is a vital micronutrient for nearly all microorganisms, serving as a co-factor in critical metabolic pathways. However, cheese is an iron-restricted environment. Furthermore, it has been demonstrated that iron represents a growth-limiting factor for many microorganisms involved in cheese ripening and that this element is central to many microbial interactions occurring in this ecosystem. This study explores the impact of iron fortification on the growth and activity of a reduced microbial community composed of nine strains representative of the microbial community of surface-ripened cheeses. Three different iron compounds (ferrous sulfate, ferric chloride, ferric citrate) were used at three different concentrations, i.e., 18, 36, and 72 μM, to fortify cheese curd after inoculation with the consortium. This treatment significantly enhanced the growth of certain cheese-ripening bacteria in curd, resulting in substantial changes in the volatilome and metabolome profiles. These observations were dose-dependent, with more pronounced effects detected with higher iron concentrations. No statistically significant difference was observed in the microbial composition based on the iron compounds used for fortification, but this factor had an impact on the volatilome and amino acids profile. These findings highlight the importance of iron availability for the behavior of cheese microbial communities. They also open novel perspectives on cheesemakers' use of iron fortification to control microbial growth and improve cheese quality.

How to Keep Lactose Avoiders Healthy

A large portion of the world’s population has lactose intolerance. Fundamentally, this condition occurs when the small intestine does not produce enough of the lactase enzyme, which digests the disaccharide lactose in milk. Lactose avoiders might unconsciously decide to limit or exclude milk and dairy products from their diets. This group includes people with lactose intolerance, people with an allergy to milk protein, vegans, and those expressing personal preferences. Lactose avoiders are often self-reported as being milk intolerant. In this review, specific amounts of lactose in different types of milk and milk products are presented. The amounts of micro- and macronutrients in them are compared with the daily requirements established by accepted sources. Foods are suggested that can play vital roles in permanently avoiding lactose-containing dairy products, for example, brussels sprouts, as a good source of vitamin B1; kale, as a source of vitamin K; and cereals at breakfast for vitamin B6. Attention is paid to mature cheeses as they are extremely beneficial for health due to their rich vitamin and elemental compositions, and they are also suitable for people with lactose intolerance due to their low lactose content. This information is rarely provided on packaging. In addition, the current state of labeling for the presence of lactose in food and pharmaceutical products is discussed. The term “hidden lactose” is introduced to include added lactose in unexpected foods, drinks, and even medicines.

Antioxidant, Antihypertensive, and Anti-Inflammatory Activities of Long-Term Ripened Cheddar Cheese Water-Soluble Extract.

During cheese ripening, the proteins in the cheese are broken down, and various functional peptides are produced. This research aimed to investigate the changes in peptides and their physiological activities during the long-term maturation of Cheddar cheese. Young (YC), medium (MC), and 3-year-aged extra-sharp (EC) Cheddar cheeses were extracted with water, and bioactive peptides were identified using ultra performance liquid chromatography-high resolution mass spectrometer. Peptides reported to have antioxidant, angiotensin-converting enzyme (ACE)-inhibitory, and anti-inflammatory effects were identified and evaluated in the extracts. MC and EC showed stronger antioxidant activity than YC. The 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid inhibition rates of MC and EC were similar, but EC exhibited a higher 2,2-diphenyl-1-picrylhydrazyl inhibition rate. The antihypertensive effect was found to increase owing to the appearance of peptides with ACE-inhibitory activity in MC and EC. Quantitative real time polymerase chain reaction and immunoblot were run to evaluate the anti-inflammatory effects. YC did not show anti-inflammatory activity, but MC and EC were shown to effectively inhibit inflammatory mRNA expression. The immunoblot results showed that EC did not inhibit IκBα phosphorylation, but had an inhibitory effect at the mRNA expression level. Overall, the peptides contained in aged Cheddar cheese were shown to have excellent antioxidant, anti-inflammatory, and antihypertensive activities, and long-term ripening appeared to have a positive effect on these activities. This is presumed to have affected not only the already identified peptides but also unknown peptides; therefore, it is expected that the discovery of bioactive peptides will be possible through additional research.

Evaluation of Physiological Activity of Long-Term Ripened Gouda Water Extract.

This study investigated peptide changes and their bioactive functions through the long-term ripening of Gouda. Young Gouda (YG), medium Gouda (MG), and extra-sharp Gouda (EG) water extracts were prepared and functional peptides were recognized using liquid chromatography-high-resolution mass spectrometry. Two peptides with ACE-inhibitory effects (IQP and LQP) were identified in YG, while in MG and EG were identified eight (EL, IVP, VP, LPP, VIP, IPP, VPP, and VVPP) and six (EL, YL, VP, IR, YPEL, and DKIHPF) functional peptides, respectively. MG (70.26%) and EG (46.81%) showed stronger antioxidant activity than YG (25.99%) in ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid) inhibition, though the DPPH (2,2-diphenyl-1-picrylhydrazyl) inhibition rate decreased with ripening. The antihypertensive effect increased in MG (79.76%) and EG (94.50%) due to ACE-inhibitory peptides. Measurements of inflammatory mRNA expression levels and immunoblotting were conducted to assess the anti-inflammatory properties. MG and EG suppressed the transcription of IL-1β and IL-6 mRNA. Immunoblotting indicated that EG suppressed IκBα phosphorylation to 57%. The enhancement of bioactive function in the water-soluble part of long-term ripened Gouda cheese may have affected identified peptides as well as unknown peptides. Further studies are expected to aid in discovering these novel bioactive peptides.

Protein fractionation by microfiltration of goat milk: Influence on proteolysis, metabolites and sensory characteristics of cheeses produced throughout lactation

This study addresses seasonal variations in goat milk by examining the effects of microfiltration (MF) and micro- and diafiltration (MDF) in standardising goat milk composition for cheese production. The research focuses on MF and MDF treatments at two lactation stages, analysing their impact on the quality of a hard goat milk cheese. Key parameters such as total solids, pH, organic acids, free amino acids, protein profiles, and sensory properties during cheese ripening were evaluated. Sensory attributes were assessed using Quantitative Descriptive Analysis (QDA), revealing different profiles between treatments. The MF cheeses showed a balanced profile, while MDF cheeses varied more in sourness, acidity, and texture, also influenced by lactation stage.

Advances in Spectroscopic Methods for Predicting Cheddar Cheese Maturity: A Review of FT-IR, NIR, and NMR Techniques

The quest for reliable techniques to predict Cheddar cheese maturity has gained momentum to ensure quality and consistency in large-scale production. Given the complexity of cheese ripening and the industry’s need for fast and reliable evaluation methods, this review addresses the challenge by scrutinising the application of spectroscopic techniques such as Fourier transform infrared (FT-IR), near-infrared (NIR), and nuclear magnetic resonance (NMR). These methods are evaluated for their noninvasive and rapid on-site analysis capabilities, which are essential for ensuring quality in cheese production. This review synthesises current research findings, discusses the potential and limitations of each technique, and highlights future research directions. Overall, NIR spectroscopy emerges as the most promising, offering quick, nondestructive assessments and reasonably accurate compositional predictions, crucial for real-time maturation monitoring. It provides rapid results within minutes, making it significantly faster than FT-IR and NMR. While FT-IR also offers high accuracy, it typically requires longer analysis times due to extensive calibration and can be sensitive to sample conditions, while NMR, although highly accurate, involves complex and time-consuming procedures. Nonetheless, further studies are necessary to refine these spectroscopic techniques, enhance their predictive accuracy, and deepen the understanding of the correlations between chemical attributes and sensory qualities in Cheddar cheese.

Antioxidant Peptides Derived from Cheese Products via Single and Mixed Lactobacillus Strain Fermentation.

Probiotics are used in cheese fermentation to endow the product with unique functional properties, such as enhanced flavor and aroma development through proteolysis and lipolysis. In this study, two probiotic Lactobacillus strains, Lactobacillus plantarum A3 and Lactobacillus reuteri WQY-1, were selected to develop new probiotic cheeses in the form of single- and mixed-strain starters. The results demonstrated that the L. plantarum A3 single-strain group and the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group exhibited superior product performance, particularly the release of functional hydrolysates during cheese ripening. Furthermore, Label-free quantitative proteomic analysis revealed 26 unique antioxidant peptides in the L. plantarum A3 single-strain group and 53 in the L. plantarum A3/L. reuteri WQY-1 mixed fermentation group. Among these, CMENSAEPEQSLACQCL (β-lactoglobulin), CMENSAEPEQSLVCQCL (β-lactoglobulin), and IQYVLSR (κ-casein) have been found to possess potential antioxidant properties both in vitro and in vivo. This confirmed that milk-derived protein peptides in cheese products exhibit potential antioxidant functions through the hydrolysis of probiotic strains.

Microbiological Evaluation of Two Mexican Artisanal Cheeses: Analysis of Foodborne Pathogenic Bacteria in Cotija Cheese and Bola de Ocosingo Cheese by qPCR.

Cotija and Bola de Ocosingo are artisanal ripened cheeses produced in Mexico. Both are made with raw bovine milk from free-grazing cows and with no starter cultures. Unlike culture-based techniques, molecular methods for pathogen detection in food allow a shorter turnaround time, higher detection specificity, and represent a lower microbiological risk for the analyst. In the present investigation, we analyzed 111 cheese samples (95 Cotija and 16 Bola de Ocosingo) by qPCR (TaqMan®) after an enrichment-culture step specific to each foodborne bacterium. The results showed that 100% of the samples were free of DNA from Listeria monocytogenes, Brucella spp., Escherichia coli enterotoxigenic (ETEC), and O157:H7; 9% amplified Salmonella spp. DNA; and 11.7%, Staphylococcus aureus DNA. However, the threshold cycle (Ct) values of the amplified targets ranged between 23 and 30, indicating DNA from non-viable microorganisms. Plate counts supported this assumption. In conclusion, 100% of the cheeses analyzed were safe to consume, and the enrichment step before DNA extraction proved essential to discern between viable and non-viable microorganisms. Hygienic milking, milk handling, cheese manufacturing, and ripening are crucial to achieve an adequate microbiological quality of cheeses made with raw milk.

Comprehensive analysis of Moringa oleifera leaves’ antioxidant properties in ovine cheese

This study aimed to enhance “Pecorino” type ovine cheese by adding Moringa oleifera leaves powder (MOLP). Cheese-making trials, conducted at industrial level, used raw ewes' milk and two selected Lactococcus lactis strains. The experimental plan included a control production (CTR), and two experimental productions with 1% or 2% MOLP addition (1-MOLP and 2-MOLP, respectively). MOLP did not hinder starters development, which reached about 8.0 Log CFU/g in 2-month ripened cheeses. Illumina results highlighted lactococci dominance in all trials [45.98%–62.48% of relative abundance (RA)]. Physicochemical analysis showed that MOLP-enriched cheeses had higher protein content and lower secondary lipid oxidation. The addition of MOLP increased total phenolic compounds in cheese, reaching 3.64 mg GAE/g in the 2-MOLP sample. MOLP-enriched cheeses showed significantly higher radical scavenging activity than CTR production (p < 0.0001). Ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) revealed increased levels of chlorogenic acid, protocatechuic acid, caffeic acid, and ferulic acid due to MOLP enrichment. In the presence of MOLP, cheese volatile organic compounds were affected by compounds like 2-octanone, 3-hexen-2-one, heptane, nonanol, and linalool. 1-MOLP cheese was comparable to CTR production in overall satisfaction (sensory evaluation). Including MOLP in cheese production offers exciting opportunities for functional Sicilian ewes’ milk products.

An Overview: Specificities and Novelties of the Cheeses of the Eastern Mediterranean

The aim of this study is to provide an overview of the specificities (milk characteristics, production process, ripening biochemistry, composition, and sensory properties) and novelties of the world-famous traditional cheeses of the Eastern Mediterranean (EM). The EM area is remarkably heterogeneous (11 countries—Egypt, Israel, Lebanon, Syria, Türkiye, Cyprus, Greece, Albania, Montenegro, Bosnia and Herzegovina, and Croatia) in terms of cheese production, but there are some common features that can be associated with EM which are connected to the difficult geoclimaticconditions (hilly terrain and hot summers). Cheesemakers resort to some preservation methods, such as high salt content (in white-brined cheeses), high total solids content (in hard cheeses), the use of hot water in the treatment of the curd (in pasta filata cheeses), the addition of some local herbs with antimicrobial properties, and the use of animal skin sacks for cheese ripening. Due to the high proportion of whey as a by-product, whey is traditionally used in EM for the production of whey cheeses. Preserving the production of traditional EM cheeses is critical to maintaining their cultural significance and meeting the demand of consumers interested in the provenance, craftsmanship, and nutritional value of these unique products.

Microbiome mapping in dairy industry reveals new species and genes for probiotic and bioprotective activities

The resident microbiome in food industries may impact on food quality and safety. In particular, microbes residing on surfaces in dairy industries may actively participate in cheese fermentation and ripening and contribute to the typical flavor and texture. In this work, we carried out an extensive microbiome mapping in 73 cheese-making industries producing different types of cheeses (fresh, medium and long ripened) and located in 4 European countries. We sequenced and analyzed metagenomes from cheese samples, raw materials and environmental swabs collected from both food contact and non-food contact surfaces, as well as operators’ hands and aprons. Dairy plants were shown to harbor a very complex microbiome, characterized by high prevalence of genes potentially involved in flavor development, probiotic activities, and resistance to gastro-intestinal transit, suggesting that these microbes may potentially be transferred to the human gut microbiome. More than 6100 high-quality Metagenome Assembled Genomes (MAGs) were reconstructed, including MAGs from several Lactic Acid Bacteria species and putative new species. Although microbial pathogens were not prevalent, we found several MAGs harboring genes related to antibiotic resistance, highlighting that dairy industry surfaces represent a potential hotspot for antimicrobial resistance (AR) spreading along the food chain. Finally, we identified facility-specific strains that can represent clear microbial signatures of different cheesemaking facilities, suggesting an interesting potential of microbiome tracking for the traceability of cheese origin.

Exploring a Cheese Ripening Process That Hinders Ochratoxin A Production by Penicillium nordicum and Penicillium verrucosum.

A lack of control of the technological abiotic parameters apparent during cheese manufacture, including temperature and relative humidity, results in this dairy product being prone to mold contamination. Sometimes, inoculant molds are used to obtain the characteristic sensory properties of this type of product. However, during the maturation process, some unwanted molds can colonize the ripening cheese and produce mycotoxins. Mycotoxigenic molds such as Penicillium nordicum and Penicillium verrucosum can colonize ripened cheeses, contaminating them with ochratoxin A (OTA), a nephrotoxic 2B toxin. Thus, the presence of OTA in cheeses could represent a hazard to consumers' health. This study has evaluated the growth and OTA production of P. nordicum and P. verrucosum on a cheese analogue under simulated ripening conditions of 10 and 15 °C and 0.96 water activity (aw). Ecophysiological, molecular, and analytical tools assessed the mold growth, gene expression, and OTA production under these environmental conditions. Both species were able to effectively colonize the cheese under these ripening conditions. However, neither species expressed the otapks and otanps biosynthetic genes or produced phenotypic OTA. Therefore, these results suggest a relatively low risk of exposure to OTA for consumers of this type of cheese product. The conditions used were thus appropriate for cheese ripening to minimize the potential for contamination with such mycotoxins. An appropriate adjustment of the technological ripening parameters during such cheese manufacture could contribute to OTA-free cheeses.