Effect of lactose standardization of milk using low-concentration factor ultrafiltration: Effect of reducing the lactose-to-casein ratio on the properties of milled-curd Cheddar cheese.

The pH of cheese is determined by the amount of lactose fermented and the buffering capacity of the cheese. The buffering capacity of cheese is largely determined by the protein contents of milk and cheese and the amount of insoluble calcium phosphate in the curd, which is related to the rate of acidification. The objective of this study was to standardize both the lactose and casein contents of milk to better control final pH and prevent the development of excessive acidity in Cheddar cheese. This approach involved the use of low-concentration factor ultrafiltration of milk to increase the casein content (∼5%), followed by the addition of water, ultrafiltration permeate, or both to the retentate to adjust the lactose content. We evaluated milks with 4 different lactose-to-casein ratios (L:CN): 1.8 (control milk), 1.4, 1.1, and 0.9. All cheesemilks had similar total casein (2.3%) and fat (3.4%) contents. These milks were used to make milled-curd Cheddar cheese, and we evaluated cheese composition, texture, functionality, and sensory properties over 9 mo of ripening. Cheeses made from milks with varying levels of L:CN had similar moisture, protein, fat, and salt contents, due to slight modifications during manufacture (i.e., cutting the gel at a smaller size than control) as well as control of acid development at critical steps (i.e., cutting the gel, whey drainage, salting). As expected, decreasing the L:CN led to cheeses with lower lactic acid, residual lactose, and insoluble Ca contents, as well as a substantial pH increase during cheese ripening in cheeses. The L:CN ratio had no significant effect on the levels of primary and secondary proteolysis. Texture profile analysis showed no significant differences in hardness values during ripening. Maximum loss tangent, an index of cheese meltability, was lower until 45 d for the L:CN 1.4 and 0.9 treatments, but after 45 d, all reduced L:CN cheeses had higher maximum loss tangent values than the control cheese (L:CN 1.8). Sensory analyses showed that cheeses made from milks with reduced L:CN contents had lower acidity, sourness, sulfury notes, and chewdown cohesiveness. Standardization of milk to a specific L:CN ratio, while maintaining a constant casein level in the milk, would allow Cheddar cheese manufacturers to have tighter control of pH and acidity.

Detection of Volatile Compounds of Cheese and Their Contribution to the Flavor Profile of Surface-Ripened Cheese.

The volatiles responsible for the typical aroma of cheese are produced mainly by lipolytic and proteolytic pathways and by the metabolism of lactose, lactate, and citrate. The volatile profile of cheese is determined by gas chromatography (GC), which includes the extraction, separation, and detection of volatiles. A wide range of extraction techniques is available, and technological improvements have been developed in GC separation and detection that enhance our understanding of the role of individual key volatiles to cheese flavor. To date, for surface-ripened cheese, the main volatiles detected that contribute to flavor include acids, ketones, alcohols, and sulfur compounds. However, based on the limited number of studies undertaken and the approaches used, it appears that a significant degree of bias possibly exists that may have over- or underestimated the impact of specific chemical classes involved in the flavor of these types of cheese.

Compromised Lactobacillus helveticus starter activity in the presence of facultative heterofermentative Lactobacillus casei DPC6987 results in atypical eye formation in Swiss-type cheese.

Nonstarter lactic acid bacteria are commonly implicated in undesirable gas formation in several varieties, including Cheddar, Dutch-, and Swiss-type cheeses, primarily due to their ability to ferment a wide variety of substrates. This effect can be magnified due to factors that detrimentally affect the composition or activity of starter bacteria, resulting in the presence of greater than normal amounts of fermentable carbohydrates and citrate. The objective of this study was to determine the potential for a facultatively heterofermentative Lactobacillus (Lactobacillus casei DPC6987) isolated from a cheese plant environment to promote gas defects in the event of compromised starter activity. A Swiss-type cheese was manufactured, at pilot scale and in triplicate, containing a typical starter culture (Streptococcus thermophilus and Lactobacillus helveticus) together with propionic acid bacteria. Lactobacillus helveticus populations were omitted in certain vats to mimic starter failure. Lactobacillus casei DPC6987 was added to each experimental vat at 4 log cfu/g. Cheese compositional analysis and X-ray computed tomography revealed that the failure of starter bacteria, in this case L. helveticus, coupled with the presence of a faculatively heterofermentative Lactobacillus (L. casei) led to excessive eye formation during ripening. The availability of excess amounts of lactose, galactose, and citrate during the initial ripening stages likely provided the heterofermentative L. casei with sufficient substrates for gas formation. The accrual of these fermentable substrates was notable in cheeses lacking the L. helveticus starter population. The results of this study are commercially relevant, as they demonstrate the importance of viability of starter populations and the control of specific nonstarter lactic acid bacteria to ensure appropriate eye formation in Swiss-type cheese.

Control of oxidation-reduction potential during Cheddar cheese ripening and its effect on the production of volatile flavour compounds.

In cheese, a negative oxidation-reduction (redox) potential is required for the stability of aroma, especially that associated with volatile sulphur compounds. To control the redox potential during ripening, redox agents were added to the salted curd of Cheddar cheese before pressing. The control cheese contained only salt, while different oxidising or reducing agents were added with the NaCl to the experimental cheeses. KIO3 (at 0·05, 0·1 and 1%, w/w) was used as the oxidising agent while cysteine (at 2%, w/w) and Na2S2O4 (at 0·05 and 0·1%, w/w) were used as reducing agents. During ripening the redox potential of the cheeses made with the reducing agents did not differ significantly from the control cheese (E h ≈ -120 mV) while the cheeses made with 0·1 and 0·05% KIO3 had a significantly higher and positive redox potential in the first month of ripening. Cheese made with 1% KIO3 had positive values of redox potential throughout ripening but no starter lactic acid bacteria survived in this cheese; however, numbers of starter organisms in all other cheeses were similar. Principal component analysis (PCA) of the volatile compounds clearly separated the cheeses made with the reducing agents from cheeses made with the oxidising agents at 2 month of ripening. Cheeses with reducing agents were characterized by the presence of sulphur compounds whereas cheeses made with KIO3 were characterized mainly by aldehydes. At 6 month of ripening, separation by PCA was less evident. These findings support the hypothesis that redox potential could be controlled during ripening and that this parameter has an influence on the development of cheese flavour.

Physicochemical and nutritional qualities of grape pomace powder-fortified semi-hard cheeses.

Powders obtained from three grape pomaces (Barbera, Chardonnay before distillation, Chardonnay after distillation) were added at two concentration levels (0.8 and 1.6 % w/w) into semi-hard and hard cheeses (Italian Toma-like and Cheddar, respectively) to increase their polyphenol content. Effects on physicochemical characteristics, total phenolic content (TPC), radical scavenging activity (RSA), proteolysis, organic acids content, starter and non-starter bacteria were evaluated during ripening. The amount and the type of powder used did not significantly affect the physicochemical parameters of cheese with the exception of pH their values. Italian Toma-like and Cheddar cheeses fortified with Chardonnay after distillation powder showed at the end of ripening (30 days and 120 days respectively) the highest TPC and RSA values. Proteolysis and microbial counts did not show statistically significant differences between fortified and control cheeses. This study demonstrated that grape pomace powder can be a functional ingredient to increase TPC and RSA in consumers' diets and the addition of this by-product to cheese is an environmentally friendly way to manage winemaking wastes.

Temporal and spatial differences in microbial composition during the manufacture of a continental-type cheese.

We sought to determine if the time, within a production day, that a cheese is manufactured has an influence on the microbial community present within that cheese. To facilitate this, 16S rRNA amplicon sequencing was used to elucidate the microbial community dynamics of brine-salted continental-type cheese in cheeses produced early and late in the production day. Differences in the microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that cheeses produced late in the day had a more diverse microbial population than their early equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were initially found to have a more diverse microbial population but thereafter the opposite was the case. Interestingly, the genera Thermus, Pseudoalteromonas, and Bifidobacterium, not routinely associated with a continental-type cheese produced from pasteurized milk, were detected. The significance, if any, of the presence of these genera will require further attention. Ultimately, the use of high-throughput sequencing has facilitated a novel and detailed analysis of the temporal and spatial distribution of microbes in this complex cheese system and established that the period during a production cycle at which a cheese is manufactured can influence its microbial composition.

High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses.

BACKGROUND: The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. RESULTS: Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. CONCLUSIONS: In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.

Effect of curd washing on the properties of reduced-calcium and standard-calcium Cheddar cheese.

Washed (W) and nonwashed (NW) variants of standard (SCa) and reduced-calcium (RCa) Cheddar cheeses were made in triplicate, ripened for a 270-d period, and analyzed for composition and changes during maturation. Curd washing was applied to cheeses to give a target level of lactose plus lactic acid in cheese moisture of 3.9 g/100 g in the W cheese, compared with a value of 5.3 g/100 g of lactose plus lactic acid in cheese moisture in the control NW cheeses. The 4 cheese types were denoted standard calcium nonwashed (SCaNW), standard calcium washed (SCaW), reduced-calcium nonwashed (RCaNW), and reduced-calcium washed (RCaW). The mean calcium level was 760 mg/100 g in the SCaNW and SCaW and 660 mg/100 g in the RCaNW and RCaW cheeses. Otherwise the gross composition of all cheeses was similar, each with protein, fat, and moisture levels of ~26, 32, and 36 g/100 g, respectively. Curd washing significantly reduced the mean level of lactic acid in the SCaW cheese and residual lactose in both SCaW and RCaW cheeses. The mean pH of the standard-calcium cheese over the 270-d ripening period increased significantly with curd washing and ripening time, in contrast to the reduced-calcium cheese, which was not affected by the latter parameters. Otherwise curd washing had little effect on changes in populations of starter bacteria or nonstarter lactic acid bacteria, proteolysis, rheology, or color of the cheese during ripening. Descriptive sensory analysis at 270 d indicated that the SCaW cheese had a nuttier, sweeter, less fruity, and less rancid taste than the corresponding SCaNW cheese. In contrast, curd washing was not as effective in discriminating between the RCaW and RCaNW cheeses. The RCaW cheese had a more buttery, caramel odor and flavor, and a more bitter, less sweet, and nutty taste than the SCaW cheese, whereas the RCaNW had a more pungent and less fruity flavor, a less fruity odor, a saltier, more-bitter, and less acidic taste, and a more astringent mouthfeel than SCaNW. Washing of curd during manufacture provides a means of reducing the contents of lactic acid and residual lactose, increasing pH, and altering the sensory properties of Cheddar cheese, with the level of these effects being significantly less pronounced as the calcium content was reduced.

The influence of alkaline earth metal equilibria on the rheological, melting and textural properties of Cheddar cheese.

The total calcium content of cheese, along with changes in the equilibrium between soluble and casein (CN)-bound calcium during ripening can have a major impact on its rheological, functional and textural properties; however, little is known about the effect of other alkaline earth metals. NaCl was partially substituted with MgCl2 or SrCl2 (8·7 and 11·4 g/kg curd, respectively) at the salting stage of cheesemaking to study their effects on cheese. Three cheeses were produced: Mg supplemented (+Mg), Sr supplemented (+Sr) and a control Cheddar cheese. Ca, Mg and Sr contents of cheese and expressible serum obtained therefrom were determined by atomic absorption spectroscopy. Addition of Mg2+ or Sr2+ had no effect on % moisture, protein, fat and extent of proteolysis. A proportion of the added Mg2+ and Sr2+ became CN-bound. The level of CN-bound Mg was higher in the +Mg cheese than the control throughout ripening. The level of CN-bound Ca and Mg decreased during ripening in all cheeses, as did % CN-bound Sr in the +Sr cheese. The presence of Sr2+ increased % CN-bound Ca and Mg at a number of ripening times. Adding Mg2+ had no effect on % CN-bound Ca. The +Sr cheese exhibited a higher G' at 70 °C and a lower LTmax than the control and +Mg cheeses throughout ripening. The +Sr cheese had significantly lower meltability compared with the control and +Mg cheeses after 2 months of ripening. Hardness values of the +Sr cheese were higher at week 2 than the +Mg and control cheeses. Addition of Mg2+ did not influence the physical properties of cheese. Supplementing cheese with Sr appeared to have effects analogous to those previously reported for increasing Ca content. Sr2+ may form and/or modify nanocluster crosslinks causing an increase in the strength of the para-casein matrix.

Comparison of the carotenoid profiles of commonly consumed smear-ripened cheeses.

The objective of this study was to identify the carotenoids imparting the orange colour to the rind, and pale yellow color to the core, of selected smear-ripened cheeses. The cheeses investigated were Charloe, Ashbrook, Taleggio, and Limburger, and were sourced from artisanal markets. Samples of the rind and core were extracted using non-polar solvents, followed by saponification to hydrolyze triglycerides to remove fatty acids, and to release carotenoid esters. Extracts were tested using ultra-high pressure liquid chromatograph-diode array detector-high resolution mass spectrometry (UHPLC-DAD-MS and -MS/MS), and identities of α- and ß-carotene, lycopene, and ß-cryptoxanthin confirmed with authentic standards. ß-Carotene was the predominant species in both the rind and core, absorbing ~70% of the signal at 450 nm in all cheese extracts tested, as well as minor quantities of ß-cryptoxanthin and α-carotene. Carotenoids unique to the rind included lycopene as well as the rare bacterial carotenoids previously identified in bacterial isolates of cheeses (i.e. decaprenoxanthin, sarcinaxanthin, and echinenone). This is the first detailed characterisation of carotenoids extracted directly from smear-ripened cheeses, and reveals that smear-ripened cheese can contribute both provitamin A carotenoids as well as C50 carotenoids to the human diet.

Proteolysis in Irish farmhouse Camembert cheese during ripening.

Proteolysis in an Irish farmhouse Camembert cheese was studied during 10 weeks of ripening. Urea-polyacrylamide gel electrophoresis of pH 4.6-insoluble fractions of cheese showed the degradation of caseins, initially due to the action of chymosin and plasmin and later due to Penicillium camemberti proteinases. Proteolytic specificities of Penicillium camemberti proteinases on the caseins in milk hydrolysates were determined and 64, 6, 28, and 2 cleavage sites were identified in αs1 -, αs2 -, ß-, and κ-casein, respectively. Proteolysis in cheese was studied and peptides produced were determined and compared to the cleavage specificities of Penicillium camemberti proteinases. Regions most susceptible to proteolysis were 1-40, 79-114, and 168-199 in αs1 -casein; 42-79 and 97-116 in αs2 -casein; 40-57, 101-125, 143-189, and 165-209 in ß-casein; and 31-81 and 124-137 in κ-casein. The present study describes in detail the proteolytic action of proteinases from Penicillium camemberti in Camembert cheese during ripening. PRACTICAL APPLICATIONS: Camembert cheese is a major international cheese variety, made in many countries around the world. The ripening of the cheese involves many biochemical changes and this study provides new information on peptides produced during ripening. Penicillium camemberti is an important mold used in the production of this type of cheese and detailed information is provided on the action of its enzymes on the caseins. Data reported in this study furthers the understanding of the ripening of Camembert cheese.

Comparison of the level of residual coagulant activity in different cheese varieties.

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify residual coagulant in 9 cheese varieties by measuring its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) assayed using reversed-phase HPLC. The level of residual coagulant activity was highest in Camembert cheese, probably due to its low pH at whey drainage and the high moisture content of the cheese, followed in order by Feta=Port du Salut=Cheddar>Gouda>Emmental=Parmigiano Reggiano=low-moisture part-skim Mozzarella=Mozzarella di Bufala Campana. The high cooking temperature (50-54 degrees C) used during the manufacture of Emmental and Parmigiano Reggiano cheeses and the cooking and stretching step in hot water during the manufacture of Mozzarella cheese may be the reasons for the lowest residual coagulant activity in these cheeses. The level of residual coagulant activity was higher in Feta cheese made from milk concentrated by ultrafiltration than in conventional Feta.

Sequencing of the Cheese Microbiome and Its Relevance to Industry.

The microbiota of cheese plays a key role in determining its organoleptic and other physico-chemical properties. It is essential to understand the various contributions, positive or negative, of these microbial components in order to promote the growth of desirable taxa and, thus, characteristics. The recent application of high throughput DNA sequencing (HTS) facilitates an even more accurate identification of these microbes, and their functional properties, and has the potential to reveal those microbes, and associated pathways, responsible for favorable or unfavorable characteristics. This technology also facilitates a detailed analysis of the composition and functional potential of the microbiota of milk, curd, whey, mixed starters, processing environments, and how these contribute to the final cheese microbiota, and associated characteristics. Ultimately, this information can be harnessed by producers to optimize the quality, safety, and commercial value of their products. In this review we highlight a number of key studies in which HTS was employed to study the cheese microbiota, and pay particular attention to those of greatest relevance to industry.

A casein hydrolysate increases GLP-1 secretion and reduces food intake.

In an effort to control weight gain, much attention has focused on the identification of bioactive peptides from food sources that induce satiety hormone secretion and increase the feeling of fullness. In this study, a screening platform identified a sodium caseinate hydrolysate, LFC25, that significantly increased calcium signalling in the enteroendocrine cell line, STC-1, and as a result increased secretion of the satiety hormone, GLP-1, in a dose-dependent manner. Administration of this hydrolysate to mice reduced the cumulative food intake over an eight hour period. To determine the feasibility of LFC25 as a food ingredient, production was scaled up to 10 L and spray-dried or freeze-dried without loss of bioactivity.

Aggregation of rennet-altered casein micelles at low temperatures.

The rennet-induced coagulation of bovine milk at 10 degrees C was investigated. The rate of change of absorbance at 600 nm was higher in milk renneted at 30 degrees C than that at 10 degrees C. The amount of casein sedimented on centrifuging skim milk at 5000g for 1 h at 10 degrees C increased with time after renneting. The viscosity of milk at 10 degrees C at low shear rates did not change significantly until 10 h after rennet addition, but it increased markedly after 20 h. Smaller particles in milk at 10 degrees C disappeared slowly over 36 h after rennet addition and aggregated into larger particles. These results suggested that casein micelles in milk aggregate at low temperatures. Reasons for the slow aggregation of milk renneted at 10 degrees C were investigated by inhibiting chymosin activity by pepstatin A. It is likely that beta-casein, or its hydrolysis, plays a role in aggregation of rennet-altered casein micelles at low temperatures.

Factors affecting the retention of rennet in cheese curd.

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the retention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle size in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged.

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

The 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.

Proteolysis in model sourdough fermentations.

Model wheat doughs started with six different lactic acid bacteria (LAB), with or without a commercial baker's yeast culture, were used to study proteolysis in sourdough fermentations. Cell counts, pH, and free amino acid concentration were measured. Sequential extraction of dough samples was performed to separate wheat proteins. The salt-soluble protein fraction (albumins and globulins) was analyzed by RP-HPLC and SDS-PAGE, whereas propanol-soluble (gliadins) and insoluble (glutenins) protein fractions were analyzed by SDS-PAGE only. Multivariate statistical methods were used for the analysis of results. The presence of yeasts and LAB affected RP-HPLC and SDS-PAGE patterns of the salt-soluble fraction in a complex way. The only changes in the gluten proteins that could be related to the presence of LAB were the appearance of new protein fragments (20 and 27 kDa) from gliadins and the degradation of high molecular weight glutenin subunits.

Manufacture and Incorporation of Liposome-Entrapped Ethylenediaminetetraacetic Acid into Model Miniature Gouda-Type Cheese and Subsequent Effect on Starter Viability, pH, and Moisture Content.

Liposome-encapsulated ethylenediaminetetraacetic acid (EDTA) was incorporated into a model miniature Gouda-type cheese (20 g) in order to assess its effect on rennet gelation, starter viability, pH, and moisture content. EDTA was encapsulated within 2 different food-grade proliposome preparations, Pro-Lipo Duo and Pro-Lipo C (50% and 40% unsaturated soybean phospholipids and 50% and 60% aqueous medium, respectively), using the following high-shear technologies: Ultra-Turrax (5000 rpm), 2-stage homogenization (345 bar), or microfluidization (690 bar). Liposome size distribution was affected by the high-shear technology employed with the proportion of large vesicles (>100 nm) decreasing in the order microfluidization < 2-stage homogenization < Ultra-Turrax. All EDTA-containing liposomes were stable during 28 d refrigerated storage, with no significant (P ≤ 0.05) change in size distribution or EDTA entrapment efficiency (%EE). Liposome composition affected the entrapment of EDTA, with Pro-Lipo C having a significantly greater %EE than Pro-Lipo Duo, 63% and 54%, respectively. For this reason, Pro-Lipo C EDTA liposomes, with and without EDTA, were incorporated into model miniature Gouda-type cheese. Addition of liposome-encapsulated EDTA to milk during cheese making did not impact pH or rennet gel formation. No differences in composition or pH were evident in liposome-treated cheeses. The results of this study show that the incorporation of liposome-encapsulated EDTA into milk during cheese manufacture did not affect milk fermentation, moisture content, or pH, suggesting that this approach may be suitable for studying the effects of calcium equilibrium on the texture of brine-salted cheeses.

Thermus and the Pink Discoloration Defect in Cheese.

A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch's postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.