Cheese Surface Research Articles

Redefining the effect of salt on thermophilic starter cell viability, culturability and metabolic activity in cheese

This study investigated the differential effect of salt concentration in the outside and inside layers of brine salted cheeses on viability, culturability and enzyme activity of starter bacteria. The high-salt environment of the outside layer caused a sharp decrease in L. helveticus viability as measured by traditional plate counts. Remarkably, this was associated with lower release of intracellular enzymes (LDH), reduced levels of proteolysis and larger membrane integrity as measured by flow cytometry (FC) following classical Live/Dead staining. FC analysis of light scattering properties highlighted a significant reduction in size and granularity of the microbiota located in the cheese surface, suggestive of cell shrinkage and condensation of internal macromolecules probably due to hyperosmotic stress. The microbiota of the cheese surface were found to experience greater oxidative stress, as measured by FC analysis of the total levels of reactive oxygen species, compared to that of the interior layer. These results lead us to postulate that the physiology and health status of the microbiota were significantly different in the outer and inner layers of the cheese. The hyperosmotic environment of the outer layer resulted in reduced cell lysis, as measurable by assays based upon membrane integrity, but rather triggered cell death via mechanisms involving cell shrinkage and ROS-mediated damage of vital intracellular components. This study challenges the current thinking on how salt controls microbial activity in ripening cheese, especially in cheeses which are brine salted as local variations in biochemical ripening indices can differ significantly from the outside to the inside of a ripening cheese.

α, β-citral from Cymbopogon citratus on cellulosic film: Release potential and quality of coalho cheese

Cellulose acetate films (CAF) with Cymbopogon citratus were tested on coalho cheese to investigate the migration of α, β-Citral and the quality cheese. The film was evaluated for mechanical properties, water vapor permeability, thickness and solubility and the cheese packed was evaluated every 25 days to texture, color, and presence of the citral by volatile analyzes, using gas chromatography with FID detector. The film thickness varied, mechanical analysis of the film showed good condition, demonstrating a film with rigidity and flexibility. The cheese's texture had no changes and the yellow color was enhanced during the 25 days. In the first two hours, cheese packed control percentage areas of the neral and geranial on the surface of the cheese were around 1.2 × 105, indicating that the volatiles passed to the cheese surface quickly and reached their maximum levels in ten days. From day 15, citral levels in the cheese decreased with this tendency until the 25th day. Dispersion of the citral on the surface to the inner part of the coalho cheese was observed, reaching great migration from the 20th day. These results suggest that the film used with the essential oil was active and could guarantee a safer food.

Utilization of Edible Films and Coatings as Packaging Materials for Preservation of Cheeses

The diversity of cheese varieties, dynamic microbiological and biochemical changes which undergo during cheese manufacture, ripening and marketing need special care to meet the desired consumer acceptability and satisfaction. In this area packaging plays an important role in cheese industry. Application of films or coatings to cover cheese surfaces has long been recognized as an important treatment for the protection of the quality and safety of the product. The growing environmental concern about the use of un-biodegradable materials in cheese packaging encouraged research to develop edible materials for coating and packaging of cheese. Several polysaccharides and proteins with or without plasticizers, antimicrobial agents and nanoparticles have been used as basis for the development of edible cheese coating. The present manuscript gives an overview on the development and use of edible cheese coating in relation to the cheese quality and safety.

Genome Sequence of Staphylococcus saprophyticus DPC5671, a Strain Isolated from Cheddar Cheese

ABSTRACTThe draft genome sequence of Staphylococcus saprophyticus DPC5671, isolated from cheddar cheese, was determined. S. saprophyticus is a common Gram-positive bacterium detected on the surface of smear-ripened cheese and other fermented foods.

Pulsed light and antimicrobial combination treatments for surface decontamination of cheese: Favorable and antagonistic effects

Postprocessing cross-contamination of cheese can lead to both food safety issues and significant losses due to spoilage. Pulsed light (PL) treatment, consisting of short, high-energy, broad-spectrum light pulses, has been proven effective in reducing the microbial load on cheese surface. As PL treatment effectiveness is limited by light-cheese interactions, the possibility to improve its effectiveness by combining it with the antimicrobial nisin was explored. The effect of natamycin, which is added to cheeses as an antifungal agent, on PL effectiveness was also investigated. Pseudomonas fluorescens, Escherichia coli ATCC 25922, and Listeria innocua were used as challenge microorganisms. Bacterial cultures in stationary growth phase were diluted to initial inoculum levels of 5 or 7 log cfu per cheese slice. Slices of sharp white Cheddar cheese and white American singles were cut in rectangles of 2.5 × 5 cm. For cheese slices receiving antimicrobial treatment before PL, slices were dipped in natamycin or nisin, spot inoculated with 100 μL of bacterial suspension, and then treated with PL. Cheese slices receiving PL treatment before antimicrobials were spot inoculated, treated with PL, and then treated with antimicrobials. The PL fluence levels from 1.02 to 12.29 J/cm2 were used. Survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. All treatments were performed in triplicate, and the data were analyzed using a general linear model. Treatment with nisin or natamycin before PL decreased the effectiveness of PL for all bacteria tested. For instance, PL reduced P. fluorescens on Cheddar cheese by 2.19 ± 0.27 log after 6.14 J/cm2, whereas combination treatments at the same PL fluence yielded barely 1 log reduction. Inactivation of L. innocua on Cheddar was only 0.78 ± 0.01 log when using PL after nisin, compared with a 1.30 ± 0.76 log reduction by nisin alone. This was attributed to the absorption of UV light by the 2 antimicrobials, which diminished the UV fluence received by the bacteria. Increased inactivation was obtained when antimicrobials were applied after PL. On process cheese, a maximum reduction of 3.73 ± 0.96 log of L. innocua was obtained at 9.22 J/cm2 for PL followed by nisin, compared with 3.01 ± 0.48 by PL alone. This study demonstrates that antimicrobials may increase the antimicrobial effectiveness of PL on cheese surface, but the order of treatments is critical.

Minerals In Food: Crystal Structures of Ikaite and Struvite From Bacterial Smears On Washed-Rind Cheese

Abstract Food contains inorganic elements and compounds that are important for human nutrition and human health. Although these substances have been investigated and characterized in many foods for their nutritive value, little is known about the crystal phases that form in foods. In this study, we investigated crystals that form in the bacterial smears on the surface of washed-rind cheese. Washed-rind cheeses have been consumed for centuries, but the crystals that often contribute detectable grittiness to the surface of these cheeses have never been identified. The crystals were characterized with petrographic microscopy and identified with single crystal X-ray diffractometry as ikaite (CaCO 3 ·6H 2 O), a rare metastable phase that has only been observed in freezing marine and lacustrine environments, and struvite (NH 4 MgPO 4 ·6H 2 O), a mineral that is often associated with bacterial activity. These crystals are important to cheesemakers because they affect cheese texture and sensory characteristics. The potential importance of the bacterial smear in the nucleation of these phases is discussed, and the possibility of using cheese as a model system to investigate geological biomineralization phenomena is explored.

Application of a 222-nm krypton-chlorine excilamp to control foodborne pathogens on sliced cheese surfaces and characterization of the bactericidal mechanisms

This study was conducted to investigate the basic spectral properties of a 222-nm krypton-chlorine (KrCl) excilamp and its inactivation efficacy against major foodborne pathogens on solid media, as well as on sliced cheese compared to a conventional 254-nm low-pressure mercury (LP Hg) lamp. Selective media and sliced cheese inoculated with Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes were irradiated with a KrCl excilamp and a LP Hg lamp at the same dose. The KrCl excilamp showed full radiant intensity from the outset at a wide range of working temperatures, especially at low temperatures of around 0 to 10°C. Irradiation with 222nm UV-C showed significantly (P<0.05) higher inactivation capacity against all three pathogens than 254-nm radiation on both media and sliced cheese surfaces without generating many sublethally injured cells which potentially could recover. The underlying inactivation mechanisms of 222-nm KrCl excilamp treatment were evaluated by fluorescent staining methods and damage to cellular membranes and intracellular enzyme inactivation were the primary factors contributing to the enhanced bactericidal effect. The results of this study suggest that a 222-nm UV-C surface disinfecting system can be applied as an alternative to conventional LP Hg lamp treatment by the dairy industry.

Effect of chestnut tannin extract (Castanea sativa Miller) on the proliferation of Cladosporium cladosporioides on sheep cheese rind during the ripening

Strains belonging to the genus Cladosporium can cause black spots on the surface of sheep cheese, making it impossible to sell. Two water solutions of chestnut tannin extract (i) 200 g L−1 (CHE200) and (ii) 400 g L−1 (CHE400), and the chestnut tannin extract powder (CHEP) were tested in a cheese making trial in which 60 cheese units were allotted to 5 experimental groups (each of 12 cheeses: C1, control 1 without any treatment; C2, control 2 treated with a silver ion solution; and cheeses LCHE200, LCHE400, and LCHEP, treated with CHE200, CHE400, and CHEP, respectively). The cheeses were ripened in a room polluted with Cladosporium cladosporioides with the aim to create conditions for the proliferation of this fungus on the cheeses. The results indicated that chestnut tannin extract at a concentration of 200 g L−1 is capable of completely inhibiting C. cladosporioides proliferation, avoiding spoilage of the sheep cheese.

Evaluation of antimicrobial effectiveness of pimaricin-loaded thermosensitive nanohydrogel coating on Arzúa-Ulloa DOP cheeses

Pimaricin is a fungicide commonly used on the cheese surface. In this work poly(N-isopropylacrylamide) nanohydrogels copolymerized with acrylic acid (PNIPA-20AA(5)) were proposed to encapsulate pimaricin and release it in a controlled manner to prevent a real food product, Arzúa-Ulloa DOP cheese, from food spoilage. The results reflect that the nanohydrogel coating neither impart any adverse effects (compared to the control) nor influence the natural ripening process of Arzúa-Ulloa DOP cheese. In the bioassay with artificial contamination, the total fungal counts in cheeses with the pimaricin-loaded nanohydrogel coating (NP) were lower than those of all the other treatments, during all the assay (30 days). Despite significant (p < 0.05) differences were not maintained for a long time, probably due to the very high initial microbial load, the antimicrobial effect of the NP-coating evidenced its protective effect in cheeses. A new qPCR method that allows the total fungal counts to be quantified directly from the cheese samples was also developed to avoid the inconveniences associated with the traditional methods based on Standard Plate Count (SPC). The efficiency of the reaction in the cheese matrix was good, and the qPCR assay was highly linear. A good correlation between both analysis (SPC and qPCR) was observed (around 100% of accuracy). The results obtained in this work reveal the usefulness of thermosensitive nanohydrogels for encapsulating and releasing pimaricin to reduce the amount of free pimaricin in the foods bulk but getting sufficient inhibition of fungal growth.

Biodegradable films obtained from triticale (x Triticosecale Wittmack) flour activated with natamycin for cheese packaging

Abstract The objective of this study was to investigate the properties of films made from triticale flour activated with natamycin for cheese packaging. Films control and with different concentration of natamycin were prepared. Moisture content, water solubility, color, puncture test, water vapor permeability and microstructure of films and diffusion coefficients of natamycin were determined. The addition of natamycin reduced solubility values and water permeability, increases L*, b* and ΔE, whereas it did not significantly affect the moisture content of the films or puncture force. The analysis of the microstructure indicated that natamycin was incorporated homogenously onto the films. The diffusional exponents were between 0.5 and 1 (non-fickian diffusion). The antifungal activity of the active triticale flour films against Candida albicans and Aspergillus niger were demonstrated by agar diffusion test. In the case of C. albicans the zone of inhibition was greater at higher concentrations, while against A. niger there were no significant differences. In addition, we studied the performance of active films through testing soft cheese, covered partly with control and activated films and stored 14 days at 4 °C and at room temperature. Visual inspection showed growth of mold on cheese surfaces that were not covered and on the coated with film control, while those covered with activated films did not show mold growth. In conclusion, the addition of natamycin to triticale flour films allows obtaining a material suitable for use in food packaging as active container of cheese to inhibit deterioration.

Growth inhibition of Listeria monocytogenes by bacteriocin-producing Staphylococcus equorum SE3 in cheese models

The anti-listerial activity of Staphylococcus equorum SE3 isolated from cheese brine was tested in two different model cheese systems to ascertain its potential for use as a protective culture for smear cheese ripening. Co-cultivation of Listeria monocytogenes L129 and the antilisterial S. equorum SE3 was performed on “milk agar” or “modified milk agar” (model cheese surface systems). S. equorum inoculated at concentrations of 106 cfu/cm2 completely inhibited growth of L. monocytogenes inoculated at 10–500 cfu/cm2 on modified milk agar within 24 h of incubation, in the negative controls L129 grew to >107 cfu/cm2. At a higher inoculation level, growth inhibition was still more than 7 log units after 24 h. L. monocytogenes strains of different serotypes were also inhibited. Co-cultivation of S. equorum SE3 with other smear bacteria or yeasts, however, showed no growth inhibition of these important ripening microorganisms. The antilisterial effect was not diminished on the modified milk agar when co-cultivation was performed with the added smear cheese microbiota. However, on milk agar with no adjuncts (“green cheese model”), only a slight (<1 log unit) growth inhibition of L. monocytogenes was observed. Addition of peptides or amino acids to milk agar could restore growth inhibition of listeriae at different levels.

Application of non-starter lactic acid bacteria as biopreservative agents to control fungal spoilage of fresh cheese

The effects of six non-starter lactic acid bacteria (NSLAB) as biopreservative agents against mycotoxigenic moulds in fresh cheeses were investigated. The potential of NSLAB to inhibit the growth of contaminating fungi was evaluated using end point (antifungal overlay assay and mycelium inhibition growth) and descriptive (antifungal assay on cell-free cheese extract) methods. The application of all NSLAB except Lactobacillus plantarum KU13 as the sole starter culture of fresh cheese resulted in significant delay of mycelial growth of Aspergillus flavus and Aspergillus parasiticus on the cheese surface. Compared with other NSLAB strains, Lb. plantarum PIN exhibited the most antifungal activity and delayed the mycelia growth of A. flavus and A. parasiticus on the cheese surface by 19 and 22 days, respectively (at 4 °C). The present study indicated that NSLAB could be used as a natural biopreservatives, for controlling the problems caused by mould growth in cheese.

Stress relaxation/creep compliance behaviour of kashar cheese: Scanning electron microscopy observations

In this study, uniaxial and creep analyses were used to characterise stress relaxation and creep compliance behaviour of kashar cheese as a function of different compression (1.5, 3.0, 4.5, 6.0 and 7.5 mm) and stress (12.25, 24.50, 36.75, 49.00 and 61.25 kPa) levels. For this purpose, mechanical simulation models, namely generalised Maxwell, Nussinovitch, Peleg and Burger models were used to simulate the viscoelastic behaviour of kashar cheese as a function of different compression and stress levels. The results revealed that the compression levels could remarkably change internal structure and deformation properties of kashar cheese. Stress relaxation test demonstrated that kashar cheese showed more elastic behaviour, namely higher resistance to deformation at higher compression levels. Creep tests showed that rigidity of kashar cheese decreased as the stress level applied increased. Scanning electron microscopy (SEM) images showed that compression resulted in a less homogeneous protein matrix, an interrupted casein/fat network, and destroyed fat globules by forcing them to exude from the cheese surface. These results should be useful for dairy industry.

Starch-halloysite nanocomposites containing nisin: Characterization and inhibition of Listeria monocytogenes in soft cheese

Abstract Starch/halloysite/nisin nanocomposite films were developed as active antimicrobial packaging. Nanocomposites without nisin and control films (containing starch and glycerol only) were also prepared. Scanning electron microscopy revealed that all samples were homogeneous, halloysite nanotubes (HNT) were dispersed in starch matrix, and film surfaces denoted aggregates as higher amount of nisin was added. X-ray diffraction spectra displayed alterations in typical starch peaks after HNT and nisin incorporation, indicating a decrease in polymer crystallization. Mechanical properties were improved when HNT was incorporated, but inferior values of Young modulus and tensile strength were obtained with nisin addition. Thermal stability decreased for bionanocomposites containing nisin, which showed T max values about 20 °C lower than films without nisin. The antimicrobial activity was tested against Listeria monocytogenes, Clostridium perfringens and Staphylococcus aureus in skimmed milk agar and all microorganisms were inhibited by nanocomposites containing nisin. These active films were applied on Minas Frescal cheese surface previously inoculated with L. monocytogenes . After 4 days, antimicrobial nanocomposite films with 2 g/100 g nisin significantly reduced the initial counts of the bacterium and those with 6 g/100 g nisin completely inhibited L. monocytogenes . Results showed that nisin supported in starch/halloysite films can be an active and useful barrier to control food contamination.

Effects of controlling ripening conditions on the dynamics of the native microbial population of Mexican artisanal Cotija cheese assessed by PCR-DGGE

Diversity and dynamics of autochthonous microbiota of Cotija, a traditional Mexican raw-milk cheese that resembles Parmesan, were compared in cheeses during 90 days of ripening under traditional and controlled conditions (temperature and relative humidity). Molecular assessments by PCR-DGGE 26S and 16S rRNA encoding regions were performed on various sections of the cheese at different ripening times. A complex microbial profile was found differentially distributed in the cheese. Several spices of lactic-acid-bacteria (LAB) from the genera Leuconostoc, Lactobacillus, Weissella, Aerococcus, Enterococcus, Carnobacterium and the yeasts Candida etchellsii, Pichia kudriavzevii and Moniliella suaveolens were found in the cheese matrix, with Leuconostoc mesenteroides being dominant. The cheese surface was populated by some of these microorganisms before the characteristic crust was formed, but Kluyveromyces lactis, Kodamaea ohmeri together with non-lactic-acid-bacteria (nLAB) Psychrobacter celer and Corynebacter variabile became distinctive in this part of the cheese. The microbial spatial/temporal variations showed similar patterns in samples from both ripening conditions, which suggested a robust ecosystem that is likely responsible for the safety, sensorial richness, and potential bio-functionality of Cotija. This knowledge could open the possibility for standardizing the ripening operation to avoid defects and improve the marketing of the product.

Microbial diversity and dynamics throughout manufacturing and ripening of surface ripened semi-hard Danish Danbo cheeses investigated by culture-independent techniques

Microbial successions on the surface and in the interior of surface ripened semi-hard Danish Danbo cheeses were investigated by culture-dependent and -independent techniques. Culture-independent detection of microorganisms was obtained by denaturing gradient gel electrophoresis (DGGE) and pyrosequencing, using amplicons of 16S and 26S rRNA genes for prokaryotes and eukaryotes, respectively. With minor exceptions, the results from the culture-independent analyses correlated to the culture-dependent plating results. Even though the predominant microorganisms detected with the two culture-independent techniques correlated, a higher number of genera were detected by pyrosequencing compared to DGGE. Additionally, minor parts of the microbiota, i.e. comprising <10.0% of the operational taxonomic units (OTUs), were detected by pyrosequencing, resulting in more detailed information on the microbial succession. As expected, microbial profiles of the surface and the interior of the cheeses diverged. During cheese production pyrosequencing determined Lactococcus as the dominating genus on cheese surfaces, representing on average 94.7%±2.1% of the OTUs. At day 6 Lactococcus spp. declined to 10.0% of the OTUs, whereas Staphylococcus spp. went from 0.0% during cheese production to 75.5% of the OTUs at smearing. During ripening, i.e. from 4 to 18weeks, Corynebacterium was the dominant genus on the cheese surface (55.1%±9.8% of the OTUs), with Staphylococcus (17.9%±11.2% of the OTUs) and Brevibacterium (10.4%±8.3% of the OTUs) being the second and third most abundant genera. Other detected bacterial genera included Clostridiisalibacter (5.0%±4.0% of the OTUs), as well as Pseudoclavibacter, Alkalibacterium and Marinilactibacillus, which represented <2% of the OTUs. At smearing, yeast counts were low with Debaryomyces being the dominant genus accounting for 46.5% of the OTUs. During ripening the yeast counts increased significantly with Debaryomyces being the predominant genus, on average accounting for 96.7%±4.1% of the OTUs. The interior of the cheeses was dominated by Lactococcus spp. comprising on average 93.9%±7.8% of the OTUs throughout the cheese processing. The microbial dynamics described at genus level in this study add to a comprehensive understanding of the complex microbiota existing especially on surface ripened semi-hard cheeses.

QUALITY IMPROVEMENT OF DAMIETTA CHEESE USING SOME SPICES EXTRACT

The experiment was carried out to investigate the effect of some spices extract on the quality of Damietta cheese. The sensory evaluation of cheese supplemented with cumin oil only gave slightly pronounced flavor, compared with control one. The curcumin treated cheese showed faint-yellow color of slight good appearance.The microbiological quality revealed that cumin and N.sativa oils have strong antimicrobial effect against microbial groups than their aqueous extracts. Curcumin was found to be the most effective additives among the aqueous spices extract used. According to the effect of storage periods, there was a significant reduction of microbial groups in treated cheeses. The multiplication rates of microbial groups in control cheeses of 5 % salt were rapid compared with control one of 10 % salt during the storage periods. On contrary, the reduction rates of microbial groups were rapid in treated cheeses of 10 % salt compared with 5 % salt. Also, there was a high significant difference between treated cheeses and control one. Concerning salt concentration, there was a significant difference between both types of cheese (5 & 10 % salt) against all microbial groups, except S.aureus. Also, there was a significant difference for all microbial groups between cheeses of 5 % and 10 % salt in all cheese groups, except S.aureus. Fungal growth was observed at 21 days of storage over cheese surface and whey of control one of 5 % salt only.

Pulsed-light inactivation of pathogenic and spoilage bacteria on cheese surface

Cheese products are susceptible to postprocessing cross-contamination by bacterial surface contamination during slicing, handling, or packaging, which can lead to food safety issues and significant losses due to spoilage. This study examined the effectiveness of pulsed-light (PL) treatment on the inactivation of the spoilage microorganism Pseudomonas fluorescens, the nonenterohemorrhagic Escherichia coli ATCC 25922 (nonpathogenic surrogate of Escherichia coli O157:H7), and Listeria innocua (nonpathogenic surrogate of Listeria monocytogenes) on cheese surface. The effects of inoculum level and cheese surface topography and the presence of clear polyethylene packaging were evaluated in a full factorial experimental design. The challenge microorganisms were grown to early stationary phase and subsequently diluted to reach initial inoculum levels of either 5 or 7 log cfu/slice. White Cheddar and process cheeses were cut into 2.5×5 cm slices, which were spot-inoculated with 100 µL of bacterial suspension. Inoculated cheese samples were exposed to PL doses of 1.02 to 12.29 J/cm2. Recovered survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. The PL treatments were performed in triplicate and data were analyzed using a general linear model. Listeria innocua was the least sensitive to PL treatment, with a maximum inactivation level of 3.37±0.2 log, followed by P. fluorescens, with a maximum inactivation of 3.74±0.8 log. Escherichia coli was the most sensitive to PL, with a maximum reduction of 5.41±0.1 log. All PL inactivation curves were nonlinear, and inactivation reached a plateau after 3 pulses (3.07 J/cm2). The PL treatments through UV-transparent packaging and without packaging consistently resulted in similar inactivation levels. This study demonstrates that PL has strong potential for decontamination of the cheese surface.

Starch edible film supporting natamycin and nisin for improving microbiological stability of refrigerated argentinian Port Salut cheese

The effectiveness of natamycin and nisin supported in edible films (NANI) was evaluated, at 7 ± 1 °C, in relation to the improvement of the microbiological stability of Port Salut cheese. This film inhibited the growth of yeasts and moulds and controlled the growth of psychrotrophic bacteria originally present in the Port Salut cheese stored at refrigeration temperature. It also inhibited the development of a mixed culture (Saccharomyces cerevisiae and Listeria innocua) present in the cheese due to a superficial contamination, throughout the entire storage. With respect to a postprocessing contamination affecting the film present in a covered cheese, control film, commercial film and film containing nisin and natamycin (NANI), acted as barriers precluding the mixed culture growth on the cheese surface. But only NANI inhibited the growth of that culture on the film. These results are demonstrating that the film NANI is an extremely effective method to control the population of microorganisms present in both the cheese and the film, enabling to offer the consumer a safer product.

Crystal fingerprinting: elucidating the crystals of Cheddar, Parmigiano-Reggiano, Gouda, and soft washed-rind cheeses using powder x-ray diffractometry.

Crystals in cheese may be considered defects or positive features, depending on the variety and mode of production (industrial, artisanal). Powder x-ray diffractometry (PXRD) offers a simple means to identify and resolve complex combinations of crystals that contribute to cheese characteristics. The objective of the present research was to demonstrate the application of PXRD to study crystals from a range of different cheese types, specifically Cheddar, Parmigiano-Reggiano, Gouda, and soft washed-rind (smear ripened) cheeses. In studies of Parmigiano-Reggiano and long-aged Gouda, PXRD has confirmed that hard (crunchy) crystals that form abundantly within these cheeses consist of tyrosine. Furthermore, PXRD has tentatively identified the presence of an unusual form of crystalline leucine in large (up to 6 mm in diameter) spherical entities, or “pearls”, that occur abundantly in 2-year-old Parmigiano Reggiano and long-aged Gouda cheeses, and on the surface of rindless hard Italian-type cheese. Ongoing investigations into the nature of these “pearls” are providing new insight into the roles that crystals play in the visual appearance and texture of long-aged cheeses. Crystals also sometimes develop profusely in the eyes of long-aged Gouda, which have been shown by PXRD to consist of tyrosine and the aforementioned presumptive form of crystalline leucine. Finally, crystals have been shown by PXRD to form in the smears of soft washed-rind cheeses. These crystals may be associated in some cheeses with gritty mouth feel and with zonal body softening that occurs during ripening. Heightened interest in artisanal cheeses highlights the need to better understand crystals and their contributions to cheese characteristics.