Thermal inactivation of chymosin during cheese manufacture

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.     

Recombinant lamb chymosin as an alternative coagulating enzyme in cheese production

Recombinant lamb chymosin (RLC) was prepared and tested for its potential use in cheese production. The milk clotting activity and proteolytic activity of RLC were evaluated in comparison with commercial recombinant calf chymosin (RCC), cow rennet (CR), and microbial coagulant (MC). RLC, RCC, and MC showed similar responses to pH, with a sharp increase of the coagulation time at pH 6.6 to 6.8 and decrease of curd firmness at the pH 6.5 to 6.6. In the case of CR, we observed two clear increases in the coagulation time and decreases in the curd firmness, at pH 6.4 to 6.5 and 6.6 to 6.8. Optimal clotting activity was obtained for RLC at 40 degrees C, for both CR and RCC at 45 degrees C, and for MC at 60 degrees C. The temperature instability of RLC at temperatures above 45 degrees C could constitute a benefit in making hard cheese varieties. The additon of CaCl2 to milk resulted in enhanced clotting activity of all coagulants, most prominently for CR. The proteolytic activity of RLC was significantly lower from that of CR but not significantly different from the activity of RCC. The lower proteolytic activity in the cheese made with RLC did not have negative effect on organoleptic properties. The overall quality of the cheese made with RLC was at least comparable to that of the cheese made with RCC, and both cheeses were better scored than the cheese made with CR.     

Interaction between sodium chloride and texture in semi-hard Danish cheese as affected by brining time,dl-starter culture,chymosin type and cheese ripening

Reduced NaCl in semi-hard cheeses greatly affects textural and sensory properties. The interaction between cheese NaCl concentration and texture was affected by brining time (0–28 h), dl-starter cultures (C1, C2, and C3), chymosin type (bovine or camel), and ripening time (1–12 weeks). Cheese NaCl levels ranged from <0.15 to 1.90% (w/w). NaCl distribution changed during ripening; migration from cheese edge to core led to a more homogeneous NaCl distribution after 12 weeks. As ripening time increased, cheese firmness decreased. Cheeses with reduced NaCl were less firm and more compressible. Cheeses produced with C2 were significantly firmer than those produced with C1; cheeses produced with C3 had higher firmness and compressibility. In NaCl reduced cheese, use of camel chymosin as coagulant resulted in significantly higher firmness than that given using bovine chymosin. Overall, cheese NaCl content is reducible without significant textural impact using well-defined starter cultures and camel chymosin.     

Effects of blends of camel and calf chymosin on proteolysis,residual coagulant activity,microstructure, and sensory characteristics of Beyaz peynir

Beyaz peynir, a white brined cheese, was manufactured using different blends of camel chymosin (100, 75, 50, 25, and 0%) with calf chymosin and ripened for 90 d. The purpose of this study was to determine the best mixture of coagulant for Beyaz peynir, in terms of proteolysis, texture, and melting characteristics. The cheeses were evaluated in terms of chemical composition, levels of proteolysis, total free amino acids, texture, meltability, residual coagulant activity, microstructure, and sensory properties during 90 d of ripening. Differences in the gross chemical composition were statistically significant for all types of cheeses. Levels of proteolysis were highly dependent on the blends of the coagulants. Higher proteolysis was observed in cheeses that used a higher ratio of calf chymosin. Differences in urea-PAGE and peptide profiles of each cheese were observed as well. Meltability values proportionally increased with the higher increasing levels of calf chymosin in the blend formula. These coagulants had a slight effect on the microstructure of cheeses. The cheese made with camel chymosin had a harder texture than calf chymosin cheese, and hardness values of all cheese samples decreased during ripening. The cheeses with a high ratio of calf chymosin had higher residual enzyme activity than those made with camel chymosin. No significant difference in sensory properties was observed among the cheeses. In conclusion, cheeses made with a high level of calf chymosin had a higher level of proteolysis, residual coagulant activity, and meltability. The cheeses also had a softer texture than cheeses made with a high content of camel chymosin. Camel chymosin may be used as a coagulant alone if low or limited levels of proteolysis are desired in cheese.     

Suitability of recombinant camel (Camelus dromedarius) chymosin as a coagulant for Cheddar cheese

Cheddar-type cheeses were manufactured using fermentation-produced camel or calf chymosin. There were no significant differences in the composition and pH between the cheeses made with either coagulant. The extent of primary proteolysis was significantly lower in cheeses made with camel chymosin than in cheeses made with calf chymosin. There were large quantitative differences between the peptide profiles of cheeses; however, the levels of amino acids were similar except for isoleucine, histidine and lysine. The cheeses made with camel chymosin were characterized by lower intensities of sulphur and brothy flavours and showed less bitter taste; however, the cheeses made with calf chymosin had greater breakdown of texture, higher smoothness and mouthcoating and were more cohesive and adhesive. The results of this study suggest that camel chymosin appears to be suitable for making Cheddar cheese with lower levels of proteolysis but with good flavour.     

Impact of milk preacidification with CO2 on the aging and proteolysis of cheddar cheese

To determine the influence of milk preacidification with CO(2) on Cheddar cheese aging and proteolysis, cheese was manufactured from milk with and without added CO(2). The experiment was replicated 3 times. Carbon dioxide (approximately 1600 ppm) was added to the cold milk, resulting in a milk pH of 5.9 at 31 degrees C in the cheese vat. The starter and coagulant usage rates were equal for the control and CO(2) treatment cheeses. The calcium content of the CO(2) treatment cheese was lower, but no difference in moisture content was detected. The higher CO(2) content of the treatment cheeses (337 vs. 124 ppm) was maintained throughout 6 mo of aging. In spite of having almost one and a half times the salt-in-moisture, proteolysis as measured by pH 4.6 and 12% trichloroacetic acid soluble nitrogen expressed as percentages of total nitrogen, was higher in the CO(2) treatment cheeses throughout aging. The ratio of alpha(s)-casein (CN) to para-kappa-CN decreased faster in the CO(2) treatment cheeses than in the control cheeses, especially before refrigerated storage. No difference was detected in the ratio of beta-CN to para-kappa-CN between the control and CO(2) treatment cheeses. Intact alpha(s)- and beta-CN were found in the expressible serum (ES) from the CO(2) treatment cheese as well as alpha(s1)-I-CN, but they were not detected in the ES from the control cheese. No CN was detected in the ES from the curd before the salting of either the control or CO(2) treatment cheese. Higher proteolysis in the cheese made from milk preacidified with CO(2) may have been due to increased substrate availability in the water phase or increased chymosin activity or retention in the cheese.     

Effect of camel chymosin on the texture,functionality, and sensory properties of low-moisture,part-skim Mozzarella cheese

The objective of this study was to compare the effect of coagulant (bovine calf chymosin, BCC, or camel chymosin, CC), on the functional and sensory properties and performance shelf-life of low-moisture, part-skim (LMPS) Mozzarella. Both chymosins were used at 2 levels [0.05 and 0.037 international milk clotting units (IMCU)/mL], and clotting temperature was varied to achieve similar gelation times for each treatment (as this also affects cheese properties). Functionality was assessed at various cheese ages using dynamic low-amplitude oscillatory rheology and performance of baked cheese on pizza. Cheese composition was not significantly different between treatments. The level of total calcium or insoluble (INSOL) calcium did not differ significantly among the cheeses initially or during ripening. Proteolysis in cheese made with BCC was higher than in cheeses made with CC. At 84 d of ripening, maximum loss tangent values were not significantly different in the cheeses, suggesting that these cheeses had similar melt characteristics. After 14 d of cheese ripening, the crossover temperature (loss tangent = 1 or melting temperature) was higher when CC was used as coagulant. This was due to lower proteolysis in the CC cheeses compared with those made with BCC because the pH and INSOL calcium levels were similar in all cheeses. Cheeses made with CC maintained higher hardness values over 84 d of ripening compared with BCC and maintained higher sensory firmness values and adhesiveness of mass scores during ripening. When melted on pizzas, cheese made with CC had lower blister quantity and the cheeses were firmer and chewier. Because the 2 types of cheeses had similar moisture contents, pH values, and INSOL Ca levels, differences in proteolysis were responsible for the firmer and chewier texture of CC cheeses. When cheese performance on baked pizza was analyzed, properties such as blister quantity, strand thickness, hardness, and chewiness were maintained for a longer ripening time than cheeses made with BCC, indicating that use of CC could help to extend the performance shelf-life of LMPS Mozzarella.     

Factors influencing the gelation and rennetability of camel milk using camel chymosin

The effects of temperature, pH, concentration of camel chymosin and addition of CaCl₂ on the hydrolysis of κ-casein (κ−CN) and the coagulation kinetics of camel milk were investigated. The rate of κ−CN hydrolysis was higher at 40 °C than at 30 °C and with increasing addition of chymosin and decreasing pH. For all samples gelation was initiated at levels of camel milk κ−CN hydrolysis >95%. The gelation time (T_g) of camel milk was significantly reduced (from 717 to 526 s) at 30 °C when the concentration of chymosin was increased, but was independent of chymosin concentration at 40 °C. Reducing pH also reduced T_g. The gel firmness increased at 40 °C (58 Pa) compared with 30 °C (44 Pa) and effect of CaCl₂ addition on the gelation properties of camel milk was found to be dependent on pH; a significant improvement was only found at pH 6.3.     

Short communication: A comparative analysis of recombinant chymosins

The first step in cheesemaking is the milk clotting process, in which κ-caseinolytic enzymes contribute to micelle precipitation. The best enzyme for this purpose is chymosin because of its high degree of specificity toward κ-casein. Although recombinant bovine chymosin is the most frequently used chymosin in the industry, new sources of recombinant chymosin, such as goat, camel, or buffalo, are now available. The present work represents a comparative study of 4 different recombinant chymosins (goat and buffalo chymosins expressed in Pichia pastoris, and bovine and camel chymosin expressed in Aspergillus niger). Recombinant goat chymosin exhibited the best catalytic efficiency compared with the buffalo, bovine, or camel recombinant enzymes. Moreover, recombinant goat chymosin exhibited the best specific proteolytic activity, a wider pH range of action, and a lower glycosylation degree than the other 3 enzymes. In conclusion, we propose that recombinant goat chymosin represents a serious alternative to recombinant bovine chymosin for use in the cheesemaking industry.     

A method for the large-scale isolation of β-casein

A method for the large-scale isolation of β-casein from renneted skim milk was developed. The curd from renneted skim milk was dispersed in hot (?70 °C) water to inactivate residual chymosin. The heated curd was subsequently recovered by centrifugation, resuspended in water and incubated at 5 °C, during which β-casein dissociated from the curd; the suspension was centrifuged and the aqueous phase lyophilised. The isolated protein consisted mainly of β-casein, containing a minor amount of γ-caseins and traces of other caseins. Unless chymosin was fully inactivated by heating, some β-casein was hydrolysed at the Leu₁₉₂–Tyr₁₉₃ bond. The yield of β-casein increased with incubation time, up to ∼20% of the β-casein present in the milk after 24 h at 5 °C. Reducing milk pH to 5.5 or 6.0, prior to renneting, caused a high level of contamination with α_s-caseins. This isolation procedure can be easily scaled-up to an industrial process and the β-casein-depleted curd may be used for the manufacture of rennet casein or processed cheese.     

Influence of proteolytic Lactococcus lactis subsp. cremoris on ripening of reduced-fat Cheddar cheese made with camel chymosin

This study investigated proteolysis in reduced-fat Cheddar cheese produced with camel chymosin and Lactococcus lactis subsp. cremoris with the ability to cleave the N-terminus of α_S1-casein. The aim was to match the activity of bovine chymosin, which leads to softer cheese structure than camel chymosin. Cheeses were analysed for gross composition, casein and peptide breakdown, release of free amino acids, structure parameters and sensory characteristics. Selected Lc. lactis subsp. cremoris increased the amount of peptides and, to a limited extent, the total amount of free amino acids in the cheeses. One group of experimental cheeses was found to have a significantly firmer structure, higher stress at fracture and modulus of deformability than the reference cheeses. The addition of the selected proteolytic dairy strains of Lc. lactis subsp. cremoris to the cheeses did not result in extended breakdown of α_S1-casein or a softer cheese structure.     

The effect of concentration of chymosin on the yield and sensory properties of camel cheese and on its microbiological quality

The transformation of camel milk into soft cheese by using chymosin and yoghurt starter culture (Streptococcus thermophilus and Lactobacillus bulgaricus) was investigated. The cheese yield and sensory properties were related to the concentration of chymosin. A yield of 16.74 g/100 mL of milk was obtained with a chymosin concentration of 1.7 mL/L of milk. The cheeses obtained with concentrations ranging between 1.0 mL and 2.9 mL of chymosin/L of milk scored highly regarding their sensory properties and had an acceptable microbiological quality. This study demonstrated that cheesemaking from camel milk can be made successfully providing that the appropriate chymosin concentration is used; and that 1.7 mL of chymosin/L of milk was optimal.     

微小毛霉与米黑毛霉凝乳酶的制备及其凝乳质构特性研究

将经乙醇分步沉淀所制得的微小毛霉和米黑毛霉凝乳酶以小牛皱胃酶为对照在不同温度、不同时间和不同pH值下凝乳,以下压过程的平均力为衡量标准,对其物性进行了比较,得出在相同条件下,米黑毛霉凝乳酶的凝乳要好于微小毛霉凝乳酶,并在高温和酸性环境要比微小毛霉凝乳酶稳定。     

Effects of indirect indicators of udder health on nutrient recovery and cheese yield traits in goat milk

In dairy goats, very little is known about the effect of the 2 most important indirect indicators of udder health [somatic cell count (SCC) and total bacterial count (TBC)] on milk composition and cheese yield, and no information is available regarding the effects of lactose levels, pH, and NaCl content on the recovery of nutrients in the curd, cheese yield traits, and daily cheese yields. Because large differences exist among dairy species, conclusions from the most studied species (i.e., bovine) cannot be drawn for all types of dairy-producing animals. The aims of this study were to quantify, using milk samples from 560 dairy goats, the contemporary effects of a pool of udder health indirect indicators (lactose level, pH, SCC, TBC, and NaCl content) on the recovery of nutrients in the curd (%REC), cheese yield (%CY), and daily cheese yields (dCY). Cheese-making traits were analyzed using a mixed model, with parity, days in milk (DIM), lactose level, pH, SCC, TBC, and NaCl content as fixed effects, and farm, breed, glass tube, and animal as random effects. Results indicated that high levels of milk lactose were associated with reduced total solids recovery in the curd and lower cheese yields, because of the lower milk fat and protein contents in samples rich in lactose. Higher pH correlated with higher recovery of nutrients in the curd and higher cheese yield traits. These results may be explained by the positive correlation between pH and milk fat, protein, and casein in goat milk. High SCC were associated with higher recovery of solids and energy in the curd but lower recovery of protein. The higher cheese yield obtained from milk with high SCC was due to both increased recovery of lactose in the curd and water retention. Bacterial count proved to be the least important factor affecting cheese-making traits, but it decreased daily cheese yields, suggesting that, even if below the legal limits, TBC should be considered in order to monitor flock management and avoid economic losses. The effect of NaCl content on milk composition was linked with lower recovery of all nutrients in the curd during cheese-making. In addition, high milk NaCl content led to reductions in fresh cheese yield and cheese solids. The indirect indicators of the present study significantly affected the cheese-making process. Such information should be considered, to adjust the milk-to-cheese economic value and the milk payment system.     

Glycolysis and related reactions during cheese manufacture and ripening

Fermentation of lactose to lactic acid by lactic acid bacteria is an essential primary reaction in the manufacture of all cheese varieties. The reduced pH of cheese curd, which reaches 4.5 to 5.2, depending on the variety, affects at least the following characteristics of curd and cheese: syneresis (and hence cheese composition), retention of calcium (which affects cheese texture), retention and activity of coagulant (which influences the extent and type of proteolysis during ripening), the growth of contaminating bacteria. Most (98%) of the lactose in milk is removed in the whey during cheesemaking, either as lactose or lactic acid. The residual lactose in cheese curd is metabolized during the early stages of ripening. During ripening lactic acid is also altered, mainly through the action of nonstarter bacteria. The principal changes are (1) conversion of L-lactate to D-lactate such that a racemic mixture exists in most cheeses at the end of ripening; (2) in Swiss-type cheeses, L-lactate is metabolized to propionate, acetate, and CO2, which are responsible for eye formation and contribute to typical flavor; (3) in surface mold, and probably in surface bacterially ripened cheese, lactate is metabolized to CO2 and H2O, which contributes to the increase in pH characteristic of such cheeses and that is responsible for textural changes, (4) in Cheddar and Dutch-type cheeses, some lactate may be oxidized to acetate by Pediococci. Cheese contains a low level of citrate, metabolism of which by Streptococcus diacetylactis leads to the production of diacetyl, which contributes to the flavor and is responsible for the limited eye formation characteristic of such cheeses.     

Microbiological,chemical, textural and sensory characteristics of Hispánico cheese manufactured using frozen ovine milk curds scalded at different temperatures

Hispánico cheese is a semi-hard variety, manufactured in Spain from a mixture of pasteurized bovine and ovine milk. To study one strategy for overcoming a seasonal shortage of ovine milk in summer and autumn, curds made from ovine milk, scalded at 32, 35 or 38 °C, were pressed for 30 min and frozen at ?24 °C for 4 months. After thawing, they were added to fresh bovine milk curd for the manufacture of experimental Hispánico cheeses. Control cheese was made from a mixture of pasteurized bovine and ovine milk in the same (80:20) proportion. No significant effect of the addition of frozen ovine milk curd or scalding temperature was found for lactic acid bacteria counts, dry matter content, hydrophilic and hydrophobic peptides, 45 out of 65 volatile compounds, texture, and sensory characteristics throughout a 60-day ripening period. Differences between cheeses, of low magnitude and little practical significance, were found for pH value, aminopeptidase activity, proteolysis, free amino acids, free fatty acids, and the remaining 20 volatile compounds. Thus, the addition of frozen ovine milk curd to fresh bovine milk curd does not alter the main physicochemical and sensory characteristics of Hispánico cheese.     

Low-fat akawi cheese made from bovine-camel milk blends: Rheological properties and microstructural characteristics

Camel milk (CM) can be used as an ingredient to produce various dairy products but it forms weak rennet-induced and acid-induced gels compared with bovine milk (BM). Therefore, in this study, we aimed to investigate the effect of blending bovine milk with camel milk on the physicochemical, rheological (amplitude sweep and frequency sweep), and microstructural properties of low-fat akawi (LFA) cheese. The cheeses were made of BM only or BM blended with 15% (CM15%) or 30% (CM30%) camel milk and stored at 4°C for 28 d. The viscoelastic properties as a function of temperature were assessed. The LFA cheeses made from blended milks had higher moisture, total Ca, and soluble Ca contents, and had higher pH 4.6–water-soluble nitrogen compared with those made from BM. Analysis by scanning electron microscopy demonstrated that the microstructures formed in BM cheese were rough with granular surfaces, whereas those in blended milk cheeses had smooth surfaces. Hardness was lower for LFA cheeses made from blended milk than for those made from BM only. The LFA cheeses demonstrated viscoelastic behavior in a linear viscoelastic range from 0.1 to 1.0% strain. The storage modulus (G′) was lower in LFA cheese made from BM over a range of frequencies. Adding CM reduced the resistance of LFA cheeses to flow as temperature increased. Blended cheeses exhibited lower complex viscosity values than BM cheeses during temperature increases. Thus, the addition of camel milk improved the rheological properties of LFA cheese.     

Interactions of Calcium,pH, Temperature,and Chymosin During Milk Coagulation,

Holstein milk samples with good and poor chymosin-coagulation characteristics were coagulated in the Formagraph using different combinations of five levels of chymosin, three pH, and three temperatures in the presence and absence of .02% added calcium chloride.All the main factors significantly altered both coagulation time and curd firmness. Multiple comparisons of mean coagulation times showed that lower concentrations of chymosin (.01, .02, and .03 rennin units/ml milk) were significantly different in altering coagulation time and were different from higher concentrations (.04 and .05 rennin units/ml milk). The three pH produced significantly different mean coagulation times. Addition of more than .02 rennin units/ml milk was not necessary for adequate curd firmness in 30 min after chymosin addition where the pH of the milk was 6.4 or lower. Addition of .02% calcium chloride to milk was not necessary for adequate curd firmness 30 min after chymosin addition if other milk coagulation factors were adequately adjusted (pH ≤ 6.4; chymosin concentration = .02 rennin unit/ml milk; temperature = 37°C).     

Variation in Coagulation Properties of Milk from Individual Cows,

Milk samples from 50 Holstein cows were tested monthly for 10 mo for total protein, casein, fat, somatic cells, and pH. A Formagraph was used to measure chymosin coagulation properties. Significant variations in coagulation time and curd firmness were observed in relation to period of lactation, individual cows, and milk pH. A high negative correlation coefficient (?.86) was observed between coagulation time and curd firmness measured 30 min after addition of chymosin. The mean coagulation time generally increased as lactation progressed and milk yield decreased. Curd firmness was generally greatest in midlactation samples.Milk from 38% of the cows did not coagulate in 30 min 1 mo prior to their dry periods. The frequency of failure to coagulate was 68% in winter and 32% in fall. Milk pH was the most significant factor that affected coagulation time and curd firmness. Simulated cheese making procedures were utilized to estimate recovery of fat and protein in curd. Curd yield calculated from the recovery data ranged from 5.4 to 14% with a mean of 9.2%.     

Pathway-based genome-wide association analysis of milk coagulation properties,curd firmness,cheese yield,and curd nutrient recovery in dairy cattle

It is becoming common to complement genome-wide association studies (GWAS) with gene-set enrichment analysis to deepen the understanding of the biological pathways affecting quantitative traits. Our objective was to conduct a gene ontology and pathway-based analysis to identify possible biological mechanisms involved in the regulation of bovine milk technological traits: coagulation properties, curd firmness modeling, individual cheese yield (CY), and milk nutrient recovery into the curd (REC) or whey loss traits. Results from 2 previous GWAS studies using 1,011 cows genotyped for 50k single nucleotide polymorphisms were used. Overall, the phenotypes analyzed consisted of 3 traditional milk coagulation property measures [RCT: rennet coagulation time defined as the time (min) from addition of enzyme to the beginning of coagulation; k₂₀: the interval (min) from RCT to the time at which a curd firmness of 20 mm is attained; a₃₀: a measure of the extent of curd firmness (mm) 30 min after coagulant addition], 6 curd firmness modeling traits [RCT_eq: RCT estimated through the CF equation (min); CF_P: potential asymptotic curd firmness (mm); k_CF: curd-firming rate constant (% × min^?1); k_SR: syneresis rate constant (% × min^?1); CF_max: maximum curd firmness (mm); and t_max: time to CF_max (min)], 3 individual CY-related traits expressing the weight of fresh curd (%CY_CURD), curd solids (%CY_SOLIDS), and curd moisture (%CY_WATER) as a percentage of weight of milk processed and 4 milk nutrient and energy recoveries in the curd (REC_FAT, REC_PROTEIN, REC_SOLIDS, and REC_ENERGY calculated as the % ratio between the nutrient in curd and the corresponding nutrient in processed milk), milk pH, and protein percentage. Each trait was analyzed separately. In total, 13,269 annotated genes were used in the analysis. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway databases were queried for enrichment analyses. Overall, 21 Gene Ontology and 17 Kyoto Encyclopedia of Genes and Genomes categories were significantly associated (false discovery rate at 5%) with 7 traits (RCT, RCT_eq, k_CF, %CY_SOLIDS, REC_FAT, REC_SOLIDS, and REC_ENERGY), with some being in common between traits. The significantly enriched categories included calcium signaling pathway, salivary secretion, metabolic pathways, carbohydrate digestion and absorption, the tight junction and the phosphatidylinositol pathways, as well as pathways related to the bovine mammary gland health status, and contained a total of 150 genes spanning all chromosomes but 9, 20, and 27. This study provided new insights into the regulation of bovine milk coagulation and cheese ability that were not captured by the GWAS.