Improvement of camel milk-clotting: Usefulness of crude extract from green pods of carob (Ceratonia siliqua. L) as a substitute for commercial rennet.

Camel milk transformation into cheese remains an objective to be improved today. This study aimed to improve camel milk clotting using a crude extract from green pods of carob as a substitute for commercial rennet. The composition of the crude carob extract was determined for dry matter and protein content. Milk clotting conditions were studied at different temperatures, pH and CaCl2 concentrations. Milk clotting properties were assessed by milk clotting activity, specific activity and proteolytic activity. Enzymatic hydrolysis of camel milk caseins by crude carob extract and its inhibition were demonstrated by SDS-polyacrylamide gel electrophoresis. Crude carob extract analysis showed a protein and dry matter content of 23.26±0.5 mg/ml and 30.66±0.5 g/l, respectively. Optimal milk clotting activity was observed at 53.6 °C, pH 4.5, and 0.09 M CaCl2. The crude carob extract showed a high milk clotting activity (4.97 U/ml) and a low proteolytic activity (0.04U/ml) with camel milk. The cheese yield of curd produced from camel milk using crude carob extract was the highest (23.95%) compared with that of Camel chymosin (20.5%). The high ratio of milk-clotting to proteolytic activity shows the potential of this extract as a substitute for commercial rennet in the dairy industry.

Milk coagulation properties are moderately heritable in dairy cows: a meta-analysis using the random-effects model.

This study aimed to conduct a meta-analysis using the random-effects model to merge published genetic parameter estimates for milk coagulation properties (MCP: comprising rennet coagulation time (RCT), curd-firming time (k20), curd firmness 30 min after rennet addition (a30), titrable acidity (TA) and milk acidity or pH) in dairy cows. Overall, 80 heritability estimates and 157 genetic correlations from 23 papers published between 1999 and 2020 were used. The heritability estimates for RCT, a30, k20, TA, and pH were 0.273, 0.303, 0.278, 0.189 and 0.276, respectively. The genetic correlation estimates between RCT-a30, RCT-pH, and RCT-TA were 0.842, 0.549 and -0.565, respectively. Genetic correlation estimates between RCT and production traits were generally low and ranged from -0.142 (between RCT and casein content) to 0.094 (between RCT and somatic cell score). Moderate and significant genetic correlations were observed between a30-pH (-0.396) and a30-TA (0.662). Also, the genetic correlation estimates between a30 and production traits were low to moderate and varied from -0.165 (between a30 and milk yield) to 0.481 (between a30 and casein content). Genetic correlation estimates between pH and production traits were low and varied from -0.190 (between pH and milk protein percentage) to 0.254 (between pH and somatic cell score). The results of this meta-analysis indicated the existence of additive genetic variation for MCP that could be used in genetic selection programs for dairy cows. Because of the moderate heritability of MCP and small genetic correlations with production traits, it could be possible to improve MCP with negligible correlated effects on production traits.

Saccharomyces cerevisiae improves rennet-induced gelation of ultra-high temperature milk.

Heating milk at high temperatures impairs its renneting properties, but rennet-induced curds can be formed from ultra-high temperature (UHT) milk inoculated with Saccharomyces cerevisiae. Herein, we measured physicochemical indices of UHT milk inoculated with S. cerevisiae before rennet addition, monitored the kinetics of gel formation, and investigated the physicochemical properties and microstructure of rennet-induced curds to explore the mechanisms by which S. cerevisiae influenced rennet-induced gelation of UHT milk. Compared with untreated pasteurized cow milk and UHT milk, the ethanol content was increased, the pH was decreased, the particle size and ζ-potential were increased, the time points at which the elasticity index began to increase were advanced, and the maximum elasticity index was increased for UHT milk inoculated with S. cerevisiae. The number of S. cerevisiae cells affected the structure of rennet-induced curds; with few cells added, the protein network of curds was continuous and tight, the mean square displacement curves showed an asymptotic behavior, and the water retention capacity and curd yield were high; with more cells added, the loosely entangled proteins aggregated, the continuity of the network was destroyed, and the curd yield decreased. In summary, a low number of S. cerevisiae cells (<1.0 × 107 cfu/mL) can increase particle size, ζ-potential, and ethanol content, and decrease pH of S. cerevisiae-inoculated UHT milk, thereby accelerating the aggregation reactions after enzymatic reaction and improving the renneting properties.

Phenotypic and genetic characterization of the occurrence of noncoagulating milk in dairy sheep.

Milk coagulation ability is of central importance for the sheep dairy industry because almost all sheep milk is destined for cheese processing. The occurrence of milk with impaired coagulation properties is an obstacle to cheese processing and, in turn, to the profitability of the dairy companies. In this work, we investigated the causes of noncoagulation of sheep milk; specifically, we studied the effect of milk physicochemical properties on milk coagulation status [coagulating and noncoagulating (NC) milk samples, which do or do not coagulate within 30 min, respectively], and whether mid-infrared spectroscopy (MIR) could be used to assess variability in coagulation status. We also investigated the genetic background of milk coagulation ability. Individual milk samples were collected from 996 Sarda ewes farmed in 47 flocks located in Sardinia (Italy). Considered traits were daily milk yield, milk composition traits, and milk coagulation properties (rennet coagulation time, curd firming time, and curd firmness), and MIR spectra were acquired. About 9% of samples did not coagulate within 30 min. A logistic regression approach was used to test the effect of milk-related traits on milk coagulation status. A principal component (PC) analysis was carried out on the milk MIR spectra, and PC scores were then used as covariates in a logistic regression model to assess their relationship with milk coagulation status. Results of the present work demonstrated that the probability of having NC samples increases as milk contents of proteins and chlorides and somatic cell score increase. The analysis of PC extracted from milk spectra that influenced coagulation status highlighted key regions associated with lactose and protein concentrations, and others not associated with routinely collected milk composition traits. These results suggest that the occurrence of NC is mostly related to damage of the epithelium secretory mammary cells, which occurs with the advancement of a lactation or due to unhealthy mammary gland status. Genetic analysis of milk coagulation status and of the extracted PC confirmed the genetic background of the milk coagulability of sheep milk.

Liddle syndrome misdiagnosed as primary aldosteronism is caused by inaccurate aldosterone-rennin detection while a novel SCNN1G mutation is discovered.

PURPOSE: Through describing the confusing misdiagnosis process of Liddle syndrome, we try to reveal the importance of accurate aldosterone-renin detection and a genetic test for Liddle syndrome. METHODS: We found a family of hypertension and hypokalaemia with the proband of a 21-year-old female who had been misdiagnosed as primary aldosteronism (PA). She presented with high aldosterone and low renin levels. Aldosterone is not suppressed in the saline infusion test and captopril challenge test. However, treatment with a standard dose of spironolactone has no blood pressure improvement effect. A heterozygous variant of SCNN1G was found with whole exome sequencing and Liddle syndrome is indicated. Treatment with amiloride was effective. We rechecked aldosterone-renin levels with two different aldosterone and renin test kits. Clinical features and the mutant gene SCNN1G of each family member were determined by the Sanger method. RESULTS: The two kits had nearly opposite results. Among those Liddle syndrome patients confirmed by a genetic test, for Test kit A all ARR were screened positive while for test kit B negative. It seems Test kit B is consistent with the diagnosis while test kit A misleads the diagnosis. A novel SCNN1G mutation, c.1729 C > T, was found in this family, which introduce a premature stop codon in the γ subunit in the epithelial Na+ channel (ENaC) and resulted in a deletion of 72 amino acids at the carboxyl end. CONCLUSION: inaccurate ARR detection might misdiagnose Liddle syndrome. A Gene test is an important method for the diagnosis of Liddle syndrome. A novel SCNN1G missense mutation, c.1729 C > T, is found in a Chinese family.

Co-evolution of drug resistance and broadened substrate recognition in HIV protease variants isolated from an Escherichia coli genetic selection system.

A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.

Characterization of the Altai Maral Chymosin Gene, Production of a Chymosin Recombinant Analog in the Prokaryotic Expression System, and Analysis of Its Several Biochemical Properties.

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichia coli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis-Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s-1 and 2.27± 0.10 µm M-1·s-1, respectively.

Effect of fermentation time and acid casein concentration as nitrogen source on microbial rennet production.

We evaluated the effects of fermentation time and acid casein content on the microbial rennet obtained by solid-state fermentation using wheat bran as the carbon source. The experiments used two fermentation times (72 and 96 h), while acid casein content was 1.5, 2.0, 2.5, and 3.0 g. Rennet strength from eight enzymatic extracts was measured using pasteurized whole milk. Rennet strength of samples from 72 h of fermentation showed an increase when acid casein content increased. The rennet strength increased at 96 h of fermentation with increasing amount of casein (up to 2.5 g), and then decreased with the largest addition (3.0 g) of casein. Coagulation time for the sample with highest rennet strength was 420 s.

Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.

Diafiltration affects the gelation properties of concentrated casein micelle suspensions obtained by filtration.

Using membrane filtration it is possible to selectively concentrate proteins and, in the case of microfiltration, concentrate casein micelles. During filtration, water is often added and this practice, called diafiltration, causes further release of permeable components and maintains filtration efficiency. Filtration causes changes in composition of the protein as well as the soluble phase, including soluble calcium, which is a critical factor controlling the gelation properties of the casein micelles in milk. It was hypothesized that concentrates obtained using membrane filtration with or without diafiltration would have different gelation behavior. To test this hypothesis, two concentrates of similar casein micelle volume fraction were prepared, using spiral wound polymeric microfiltration membranes with a 800 kDa molecular weight cutoff, with or without diafiltration. The concentrates showed a gelation behavior comparable to that of skim milk, with a similar gelation time and with a higher firmness, due to the higher number of protein linkages in the network. In contrast, the hydrolysis of κ-casein by chymosin and casein aggregation were inhibited in diafiltered casein micelle suspensions. When the concentrates were recombined with the original skim milk to a final concentration of 5% protein, which re-established a similar soluble phase composition, differences in gelation behavior were no longer observed: both treatments showed similar gelation time and gel firmness. These results confirmed that membrane filtration can result in concentrates with different functionality, and that ionic environmental conditions are critical to the aggregation behavior of casein micelles. This is of particular significance in industrial settings where these fractions are used as a way to standardize proteins in cheese making.

Effects of animal versus vegetal rennet on milk coagulation traits in Mediterranean buffalo bulk milk.

Given consumer interest in Mozzarella di latte di Bufala and other cheeses, and the growing interest of the cheese industry in offering products adequate for lactovegetarian consumers, this study aimed to compare clotting capacity of vegetal and animal rennet in buffalo milk. Milk coagulation properties of 1,261 buffalo bulk milk samples collected during milk quality testing were assessed by lactodynamography using commercial animal (75% chymosin and 25% bovine pepsin) and vegetal (Cynara cardunculus) rennets. Chemical composition of milk samples was predicted by MilkoScan (Foss Analytics, Hillerød, Denmark) calibrated with specific buffalo standards. Rennet effect (animal versus vegetal) was statistically analyzed with a paired t-test. Fat, protein, and lactose contents of milk samples were 7.94%, 4.52%, and 4.80%, respectively. A similar variability of milk coagulation properties was observed with both rennets, with the exception of greater variability of curd firmness at 30 min after the addition of vegetal rennet compared with animal rennet (73 and 26%, respectively). On average, when using plant rennet, milk started to coagulate and reached the 20-mm coagulum 12 ± 0.22 min and 1.9 ± 0.20 min, respectively, later than with animal rennet. Thirty minutes after rennet addition, curds were almost twice as firm in animal as in vegetal rennet (difference of 23.92 ± 0.66 mm). However, curd firmness at 60 min was only 1.21 ± 0.39 mm thicker with vegetal than with animal rennet. Moreover, when using animal rennet, 99.52% of samples started coagulating within the first 30 min of analysis, whereas only 70.42% did so when using vegetal rennet. We conclude that vegetal rennet has the capacity to coagulate buffalo milk, achieving a similar curd firmness to that of animal rennet at 60 min. Further studies are needed to evaluate the sensory characteristics and consumer acceptability of Mozzarella di latte di Bufala processed with vegetal rennet.

Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii.

Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.

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.

Goat cheese yield and recovery of fat, protein, and total solids in curd are affected by milk coagulation properties.

The aims of the present research were to quantify the effects of each coagulation trait, traditional milk coagulation properties [MCP: rennet coagulation time (RCT), curd-firming time (k20), and curd firmness at 30 min (a30)], and modeled curd-firming over time (CFt) parameters [estimated rennet coagulation time (RCTeq), curd-firming instant rate constant (kCF), and potential curd firmness (CFP)] directly on the following: (1) recovery of 3 milk components in the curd (%REC), (2) 3 measures of cheese yield (%CY), and (3) 3 daily cheese yield traits (dCY) from goat milk. Cheese-making traits were analyzed using 2 mixed different models, the first to test MCP and the second to test CFt parameters. Pearson correlations were also calculated. Significant and favorable relationships (negative for time intervals and positive for CF measures) were found between the traditional MCP and the CFt parameters and %REC and %CY traits. The effects of milk fat and protein contents were particularly important on all cheese-making traits, with the only exception being the effect of fat content on water retention in cheese (%CYWATER). We found an optimum value of milk k20, associated with the highest recovery of components and cheese yield in solids (%CYSOLIDS). In addition, a lower level of curd water retention and an increased fresh curd yield (%CYCURD) were associated with greater recovery of fat. The collection of all available information during the process of milk coagulation and curd-firming allowed us to discover the effect of RCTeq on %REC traits and %CYSOLIDS, which had not previously been revealed for traditional RCT. Moreover, higher kCF values were associated with increased %CYCURD and %CYSOLIDS. Given that CFt parameters showed a high level of independence from one another, these can also be easily used and characterized in future applications at the industry level. Information provided by traditional and modeled coagulation properties could efficiently support the goat dairy industry and lay the foundations for a quality payment scheme for goat milk.

Biotechnological potential of a cysteine protease (CpCP3) from Calotropis procera latex for cheesemaking.

This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos' morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin.

High-Level Expression and Substrate-Binding Region Modification of a Novel BL312 Milk-Clotting Enzyme To Enhance the Ratio of Milk-Clotting Activity to Proteolytic Activity.

A novel BL312 milk-clotting enzyme (MCE) exhibited high-level expression and remarkable milk-clotting activity (MCA) (865 ± 20 SU/mL) that was 3.3-fold higher than the control by optimizing induction conditions in recombinant Escherichia. coli harboring pET24a-proMCE. Through substrate-binding region analyses and modification, MCE-G165A was identified from nine mutants and showed a proteolytic activity of 49.4 ± 2.4 U/mL and an MCA/PA ratio of 18.2, which were respectively 1.9-fold lower and 2.0-fold higher than those of the control. The purified MCE-G165A (28 kDa) exhibited weak αs-casein, ß-casein, and strong κ-casein (κ-CN) hydrolysis levels as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase high-performance liquid chromatography. The milk-clotting mechanism for MCE-G165A was the primary hydrolysis of Met106-Ala107 and Asn123-Thr124 bonds in κ-CN, as determined by mass spectrometry. MCE-G165A showed different hydrolysis sites in casein, leading to various functional peptides. Feasible methods for obtaining MCEs suitable as calf rennet substitutes are presented.

Microstructure and fracture properties of semi-hard cheese: Differentiating the effects of primary proteolysis and calcium solubilization.

The individual roles of hydrolysis of αS1- and ß-caseins, and calcium solubilization on the fracture properties of semi-hard cheeses, such as Maasdam and other eye-type cheeses, remain unclear. In this study, the hydrolysis patterns of casein were selectively altered by adding a chymosin inhibitor to the curd/whey mixture during cheese manufacture, by substituting fermentation-produced bovine chymosin (FPBC) with fermentation-produced camel chymosin (FPCC), or by modulating ripening temperature. Moreover, the level of insoluble calcium during ripening was quantified in all cheeses. Addition of a chymosin inhibitor, substitution of FPBC with FPCC, or ripening of cheeses at a consistent low temperature (8 °C) decreased the hydrolysis of αS1-casein by ~95%, ~45%, or ~30%, respectively, after 90 d of ripening, whereas ~35% of ß-casein was hydrolysed in that time for all cheeses, except for those ripened at a lower temperature (~17%). The proportion of insoluble calcium as a percentage of total calcium decreased significantly from ~75% to ~60% between 1 and 90 d. The rigidity or strength of the cheese matrix was found to be higher (as indicated by higher fracture stress) in cheeses with lower levels of proteolysis or higher levels of intact caseins, primarily αS1-casein. However, contrary to the expectation that shortness of cheese texture is associated with αS1-casein hydrolysis, fracture strain was significantly positively correlated with the level of intact ß-casein and insoluble calcium content, indicating that the cheeses with low levels of intact ß-casein or insoluble calcium content were more likely to be shorter in texture (i.e., lower fracture strain). Overall, this study suggests that the fracture properties of cheese can be modified by selective hydrolysis of caseins, altering the level of insoluble calcium or both. Such approaches could be applied to design cheese with specific properties.

Association between the GHR, GHRHR and IGF1 gene polymorphisms and milk coagulation properties in Sarda sheep.

We investigated whether variation of the sheep Growth Hormone Receptor (GHR), Growth Hormone Releasing Hormone Receptor (GHRHR) and Insulin-Like Growth Factor 1 (IGF1) genes were associated with milk coagulation properties (MCP) in sheep. The GHR, GHRHR and IGF1 genes are part of the GH system, which is known to modulate metabolism, growth and reproduction as well as mammogenesis and galactopoiesis in dairy species. A total of 380 dairy Sarda sheep were genotyped for 36 SNPs mapping to these three genes. Traditional MCP were measured as rennet coagulation time (RCT), curd-firming time (k20) and curd firmness at 30 m (a30). Modeling of curd firming over time (CFt) was based on a 60 m lactodynamographic test, generating a total of 240 records of curd firmness (mm) for each milk sample. The model parameters obtained included: the rennet coagulation time as a result of modeling all data available (RCTeq, min); the asymptotic potential value of curd firmness (CFP, mm) at an infinite time; the CF instant rate constant (kCF, %/min); the syneresis instant rate constant (kSR, %/min); the maximum value of CF (CFmax, mm) and the time at achievement of CFmax (tmax, min). Statistical analysis revealed that variation of the GHR gene was significantly associated with RCT, kSR and CFP (P < 0.05). No other significant associations were detected. These findings may be useful for the dairy industry, as well as for selection programs.

Interactive effects of casein micelle size and calcium and citrate content on rennet-induced coagulation in bovine milk.

Interactive effects of casein micelle size and milk calcium and citrate content on rennet-induced coagulation were investigated. Milk samples containing small (SM) and large (LM) micelles, obtained from individual Holstein cows, were modified by addition of calcium and/or citrate and milk coagulation properties were evaluated in a full factorial design. The results showed that LM milk had a higher relative proportion of casein, coagulated faster, and resulted in a stronger gel than SM milk. Addition of calcium slightly decreased casein micelle size, while addition of citrate slightly increased micelle size. Calcium addition resulted in a shorter coagulation time and the strongest gels, while citrate addition increased the coagulation time and resulted in the weakest gels. Addition of calcium and citrate in combination resulted in intermediate coagulation properties. The interactive effect of micelle size and citrate was significant for gel strength. Microstructural differences between the milk gels were consistent with the rheological properties, for example, the micrographs revealed that a more homogeneous network was formed when calcium was added, resulting in a stronger gel. A more inhomogeneous network structure was formed when citrate was added, resulting in a weaker gel. Thus, variations in casein micelle size and in calcium and citrate content influence rennet-induced coagulation in bovine milk. The calcium and citrate contents in Swedish milk have changed over time, whereby calcium content has increased and citrate content has decreased. In practical cheese making, calcium is added to cheese milk, most likely altering the role of inherent citrate and possibly influencing casein micelle size. The observed interaction effect between casein micelle size and citrate in this study, suggests that larger micelles with moderate citrate level will result in firmer gels, whereas a higher citrate content reduced gel strength more in case of large than SM. Since firmer gels are likely to retain more protein and fat than less firmer gels, this interaction effect could have implications in practical cheese production.

Genetic relationships of lactose and freezing point with minerals and coagulation traits predicted from milk mid-infrared spectra in Holstein cows.

The aim of the present study was to assess the relationships of lactose percentage (LP), lactose yield (LY), and freezing point (FRP) with minerals and coagulation properties predicted from mid-infrared spectra in bovine milk. To achieve this purpose, we analyzed 54,263 test-day records of 4,297 Holstein cows to compute (co)variance components with a linear repeatability animal model. Parity, stage of lactation, season of calving, and herd-test-date were included as fixed effects in the model, and additive genetic animal, within- and across-lactation permanent environment, and residual were included as random effects. Lactose percentage was more heritable (0.405 ± 0.027) than LY (0.121 ± 0.021) and FRP (0.132 ± 0.014). Heritabilities (± standard error) of predicted milk minerals varied from 0.375 ± 0.027 for Na to 0.531 ± 0.028 for P, and those of milk coagulation properties ranged from 0.348 ± 0.052 for rennet coagulation time to 0.430 ± 0.026 for curd firming time. Lactose percentage showed favorable (negative) genetic correlations with milk somatic cell score (SCS) and FRP, and it was almost uncorrelated with casein-related minerals (Ca and P) and coagulation properties. Moreover, LP was strongly correlated with Na (-0.783 ± 0.022), a mineral known to increase in the presence of intramammary infection (IMI) and high somatic cell count. Indeed, Na is the main osmotic replacer of lactose in mastitic milk when the blood-milk barrier is altered during IMI. Being strongly associated with milk yield, LY did not favorably correlate with coagulation properties, likely because of the negative correlation of this trait with protein and casein percentages. Milk FRP presented moderate and null genetic associations with Na and SCS, respectively. Results of the present study suggest that the moderate heritability of LP and its genetic correlations with IMI-related traits (Na and SCS) could be exploited for genetic selection against mastitis. Moreover, selection for LP would not impair milk coagulation characteristics or Ca and P content, which are important for cheesemaking.