Functionality of process cheese made from Cheddar cheese with various rennet levels and high-pressure processing treatments.

Due to its versatility and shelf stability, process cheese is gaining interest in many developing countries. The main structural component (base) of most processed cheese formulations is young Cheddar cheese that has high levels of intact casein. Exporting natural Cheddar cheese base from the United States to distant overseas markets would require the aging process to be slowed or reduced. As Cheddar cheese ripens, the original structure is broken down by proteolysis and solubilization of insoluble calcium phosphate. We explored the effect of varying rennet levels (we also used a less proteolytic rennet) and application of high-pressure processing (HPP) to Cheddar cheese, as we hoped these treatments might limit proteolysis and concomitant loss of intact casein. To try to retain high levels of insoluble Ca, all experimental cheeses were made with a high-draining pH and from concentrated milk. To compare our intact casein results with current practices, we manufactured a Cheddar cheese that was prepared according to typical industry methods (i.e., use of unconcentrated milk, calf chymosin [higher levels], and low draining pH value [∼6.2]). All experimental cheeses were made from ultrafiltered milk with protein and casein contents of ∼5.15% and 4.30%, respectively. Three (low) rennet levels were used: control (38 international milk clotting units/mL of rennet per 250 kg of milk), and 25% and 50% reduced from this level. All experimental cheeses had similar moisture contents (∼37%) and total Ca levels. Four days after cheese was made, half of the experimental samples from each vat underwent HPP at 600 MPa for 3 min. Cheddar cheese functionality was monitored during aging for 240 d at 4°C. Cheddar cheese base was used to prepare process cheese after aging for 14, 60, 120, 180, and 240 d. Loss tangent (LT) values of cheese during heating were measured by small strain oscillatory rheology. Intact casein levels were measured using the Kjeldahl method. Acid or base titrations were used to determine the buffering capacity and insoluble Ca levels as a percentage of total Ca. The LTmax values (an index of meltability) in process cheese increased with aging for all the cheese bases; the HPP treatment significantly decreased LTmax values of both base (natural) and process cheeses. All experimental cheeses had much higher levels of intact casein compared with typical industry-make samples. Process cheese made from the experimental treatments had visually higher stretching properties than process cheese made from Cheddar with the typical industry-make procedure. Residual rennet activity was not affected by rennet level, but the rate of proteolysis was slightly slower with lower rennet levels. The HPP treatment of Cheddar cheese reduced residual rennet activity and decreased the reduction of intact casein levels. The HPP treatment of Cheddar cheese resulted in process cheeses that had slightly higher hardness values, lower LTmax values, and retained higher storage modulus values at 70°C. We also observed that the other make procedures we used in all experimental treatments (i.e., using a less proteolytic chymosin, using a concentrated cheese milk, and maintaining a high draining pH value) had a major effect on retaining high levels of intact casein.

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.

Effect of high hydrostatic pressure processing on the rennet coagulation kinetics and physicochemical properties of sheep milk rennet-induced gels.

The effects of high hydrostatic pressure on the constituents and coagulation ability and their effect on cheese production of sheep milk have not been studied in detail. The objective of this work was to evaluate the effect of high hydrostatic pressure processing on the coagulation kinetics and physicochemical properties of sheep milk and to explore how such treatment could improve the cheesemaking process. Five batches of milk were tested: 1 untreated control batch and 4 batches each subjected to a different pressure (150, 300, 450, or 600 MPa) for 5 min at 10°C. As treatment pressure increased, values of electrical conductivity and oxidation-reduction potential were found to decrease. However, no significant reduction in pH was recorded. Treatment pressures >300 MPa produced milk with lower lightness (luminosity) and a more yellow and green hue. Pressures >150 MPa resulted in micellar fragmentation, as well as significant increases in particle size, viscosity, and water-holding capacity as a consequence of the denaturing of soluble proteins. High-pressure treatments increased the solubility of colloidal calcium phosphate, leading to a considerable increase in the concentration of minerals in the serum phase. The highest concentrations of calcium and phosphorus in the rennet whey of milk were reached at 300 MPa. Curd coagulation time was reduced by 28% at pressures >300 MPa, and an increase in the curd firming rate was observed. As treatment pressure increased to 450 MPa, the firmness, elasticity, and the percentage creep recovery of gels increased, whereas values of compliance and fracture strain were reduced. Thus, we can conclude that 300 MPa is the optimum treatment pressure for milk intended for cheesemaking by enzymatic coagulation. This pressure produced milk with optimal coagulation kinetics and water-holding properties with the least loss of fat and protein to the whey.

Acid and rennet gelation properties of sheep, goat, and cow milks: Effects of processing and seasonal variation.

Gelation is an important functional property of milk that enables the manufacture of various dairy products. This study investigated the acid (with glucono-δ-lactone) and rennet gelation properties of differently processed sheep, goat, and cow milks using small-amplitude oscillatory rheological tests. The impacts of ruminant species, milk processing (homogenization and heat treatments), seasonality, and their interactions were studied. Acid gelation properties were improved (higher gelation pH, shorter gelation time, and higher storage modulus (G') by intense heat treatment (95°C for 5 min) to comparable extents for sheep and cow milks, both better than those for goat milk. Goat milk produced weak acid gels with low G' (<100 Pa) despite improvements induced by heat treatments. Seasonality had a marked impact on the acid gelation properties of sheep milk. The acid gels of late-season sheep milk had a lower gelation pH, no maximum in tan δ following gel formation, and 70% lower G' values than those from other seasons. We propose the potential key role of a critical acid gelation pH that induces structural rearrangements in determining the viscoelastic properties of the final gels. For rennet-induced gelation, compared with cow milk, the processing treatments of the goat and sheep milks had much smaller impacts on their gelation properties. Intense heat treatment (95°C for 5 min) prolonged the rennet gelation time of homogenized cow milk by 8.6 min (74% increase) and reduced the G' of the rennet gels by 81 Pa (85% decrease). For sheep and goat milks, the same treatment altered the rennet gelation time by only less than 3 min and the G' of the rennet gels by less than 14 Pa. This difference may have been caused by the different physicochemical properties of the milks, such as differences in their colloidal stability, proportion of serum-phase caseins, and ionic calcium concentration. The seasonal variations in the gelation properties (both acid and rennet induced) of goat milk could be explained by the minor variation in its protein and fat contents. This study provides new perspectives and understandings of milk gelation by demonstrating the interactive effects among ruminant species, processing, and seasonality.

Carbocyanine-Based Optical Sensor Array for the Discrimination of Proteins and Rennet Samples Using Hypochlorite Oxidation.

Optical sensor arrays are widely used in obtaining fingerprints of samples, allowing for solutions of recognition and identification problems. An approach to extending the functionality of the sensor arrays is using a kinetic factor by conducting indicator reactions that proceed at measurable rates. In this study, we propose a method for the discrimination of proteins based on their oxidation by sodium hypochlorite with the formation of the products, which, in turn, feature oxidation properties. As reducing agents to visualize these products, carbocyanine dyes IR-783 and Cy5.5-COOH are added to the reaction mixture at pH 5.3, and different spectral characteristics are registered every several minutes (absorbance in the visible region and fluorescence under excitation by UV (254 and 365 nm) and red light). The intensities of the photographic images of the 96-well plate are processed by principal component analysis (PCA) and linear discriminant analysis (LDA). Six model proteins (bovine and human serum albumins, γ-globulin, lysozyme, pepsin, and proteinase K) and 10 rennet samples (mixtures of chymosin and pepsin from different manufacturers) are recognized by the proposed method. The method is rapid and simple and uses only commercially available reagents.

The introduction of an N-glycosylation site into prochymosin greatly enhances its production and secretion by Pichia pastoris.

BACKGROUND: N-glycosylation is one of the most important post-translational modifications. Many studies have shown that N-glycosylation has a significant effect on the secretion level of heterologous glycoproteins in yeast cells. However, there have been few studies reporting a clear and unified explanation for the intracellular mechanism that N-glycosylation affect the secretion of heterologous glycoproteins so far. Pichia pastoris is an important microbial cell factory producing heterologous protein. It is of great significance to study the effect of N-glycosylation on the secretion level of heterologous protein. Camel chymosin is a glycoprotein with higher application potential in cheese manufacturing industry. We have expressed camel prochymosin in P. pastoris GS115, but the lower secretion level limits its industrial application. This study attempts to increase the secretion level of prochymosin through N-glycosylation, and explore the molecular mechanism of N-glycosylation affecting secretion. RESULTS: Adding an N-glycosylation site at the 34th amino acid of the propeptide of prochymosin significantly increased its secretion in P. pastoris. N-glycosylation improved the thermostability of prochymosin without affecting the enzymatic activity. Immunoprecipitation coupled to mass spectrometry (IP-MS) analysis showed that compared with the wild prochymosin (chy), the number of proteins interacting with N-glycosylated mutant (chy34) decreased, and all differential interacting proteins (DIPs) were down-regulated in chy34-GS115 cell. The DIPs in endoplasmic reticulum were mainly concentrated in the misfolded protein pathway. Among the five DIPs in this pathway, overexpression of BiP significantly increased the secretion of chy. The knockout of the possible misfolded protein recognition elements, UDP-glycose:glycoprotein glucosyltransferase 1 and 2 (UGGT1/2) had no effect on the growth of yeast cells and the secretion of prochymosin. CONCLUSIONS: In conclusion, N-glycosylation increased the secretion of prochymosin in P. pastoris trough the adjustment of intracellular interacted proteins. The results of our study may help to elucidate the molecular mechanism of N-glycosylation affecting secretion and provide a new research method to improve the secretion of heterologous glycoprotein in P. pastoris.

Effect of mild thermal and pH changes on the sol-gel transition in skim milk.

The present work aimed to improve acid and rennet milk gelation properties with mild thermal and pH changes to skim milk, with emphasis on heating temperatures below the denaturation temperature of whey proteins. We hypothesized the heat-induced, pH-dependent micellar changes, namely the shifts in casein and calcium equilibria between the micellar (or colloidal) and serum phases, result in firmer acid and rennet milk gels and reduced gelation time. Homogenized, pasteurized skim milk was adjusted to pH values in the range of 6.4 to 7.3, heated at temperatures in the range of 50 to 80°C, cooled to refrigeration temperature, and restored to native pH (pH 6.7). Then, acid and rennet gels were made by the addition of glucono-δ-lactone and chymosin, respectively. We monitored the storage modulus (G', Pa) during gel formation with small-amplitude oscillatory shear and the gelation time and maximum G' (G'max, Pa) of acid and rennet gels, were measured at 3 and 2 h, respectively. When skim milk was heated at 50°C for 15 min, there was a 58 and 163% increase in the G'max of acid and rennet gels, respectively, as the pH at heating was raised from pH 6.7 to 7.3. Increases in gel strength were greater for skim milk heated at 60°C for 15 min. There was a positive correlation between G'max of acid gels and the heat-induced casein protein exchanges between the micellar and serum phases on heating milk at pH in the range from 6.4 to 7.3 (r = 0.78). We also found positive correlations between the variation in G'max of rennet gels with the heat-induced, pH-dependent migration of casein (r = 0.83) and calcium (r = 0.80) from the micelle into the serum phase, as determined by PAGE and atomic emission spectroscopy. Under these mild heating temperatures (50 and 60°C), rennet coagulation time was significantly reduced from 45 ± 5 to 27 ± 3 min when the pH at heating was raised from pH 6.7 to 7.3. The ability to enhance milk gelation properties with a scalable pretreatment allows for the expression of novel functionality of casein.

Recombinant expression and characterization of Oryctolagus cuniculus chymosin in Komagataella phaffii (Pichia pastoris).

This study was conducted for investigating expression and enzymatic characteristics of recombinant Oryctolagus cuniculus chymosin (ROCC) expressed in Pichia pastoris. SDS-PAGE of partially purified supernatant displayed two distinct molecular bands approximately at the sizes of 40 kDa and 45 kDa corresponding to chymosin and partially glycosylated chymosin, respectively. Proteolysis assay demonstrated that rabbit chymosin was more specific compared to bovine and camel chymosins when it comes to hydrolyzing α, ß, and κ-casein. Rabbit chymosin kept its stability in a wide pH range (3.0-6.0) at 37 °C for 8 h. Active chymosin exhibited maximum enzymatic activity at 40 °C and pH 4.0 with the addition of 75 mM CaCl2. The ROCC clotting activity on donkey, cow, goat, lamb, camel milk was determined as 40, 10, 5.7, 3.07, and 2.66 IMCU/mL, respectively. These results revealed that ROCC might possess a potential for incorporation into cheese manufacture technology as a milk-clotting enzyme.

Study on the Correlation between the Protein Profile of Lupin Milk and Its Cheese Production Compared with Cow's Milk.

Australian sweet lupin, the largest legume crop grown in Western Australia, is receiving global attention from the producers of new foods. To understand the effect of protein on cheese yield, lupin milk proteins were separated from the first, second, and third filtrations by cheesecloths. However, proteins from the first and second were analyzed using two-dimensional polyacrylamide gel electrophoresis; then, the isolated proteins associated with cheese production were identified. The research also focused on identifying the optimal method of cheese production based on the coagulation process, temperature, yield, and sensory evaluation. Lupin curds from the two cultivars, Mandelup and PBA Jurien, were produced using vinegar, lemon juice, starter culture, vegetable rennet enzyme as coagulant, as well as curd generated using starter culture and vegetable rennet enzyme. Cow's milk was used as a control. The results indicated that first-time filtration produced better extraction and higher yield of lupin proteins and cheese than the second filtration. A sensory analysis indicated that lupin cheese produced from PBA Jurien lupin milk using vinegar, 7.80% expressed as acetic acid, and ground in 45 °C water, was the most acceptable. The cheeses were examined for their protein, carbohydrates, fat, ash, and moisture contents. The concentration of protein was approximately 27.3% and 20.6%, respectively, in the cheese from PBA Jurien and Mandelup. These results suggest that lupin milk can adequately supply the proteins needed in human diets and, thus, could be used in the production of many existing products that require animal milk as an input.

The influence of yak casein micelle size on rennet-induced coagulation properties.

BACKGROUND: Yak milk formed stronger rennet-induced gels if the milk contained smalled casein micelles and a higher concentration of calcium. Also casein gels could formed after a shorter incubation time if the milk contained smalled casein micelles. The objective of this study was to estimate the importance of yak casein micelle size on rennet-induced coagulation properties. RESULTS: Three fractions of different-sized, undamaged casein micelles (Ф112.17 ± 0.83 nm, Ф207.13 ± 0.59 nm and Ф269.37 ± 2.89 nm) were obtained by ultracentrifugation. The smallest casein micelles had the highest concentrations of calcium (803.21 ± 8.49 mM), phosphate (445.52 ± 10.66 mM), and κ-casein/total casein (19.45%). Rheological analyses determined the optimal gelation times of small, medium, and large casein micelles to be 9.5 ± 0.5, 10.8 ± 0.5, and 13.3 ± 0.2 min, respectively. Higher κ-casein concentration in the small casein micelles appeared to facilitate their shorter incubation time. Both the faster caseinomacropeptide (CMP) release rate and rennet-induced aggregation rate of small casein micelles contributed to a faster change in turbidity. Furthermore, small casein micelles had the highest elastic modulus (G', 73.21 ± 4.5 Pa) 60 min after the addition of rennet. This was consistent with micro-photographs, which showed that small casein micelles could form a more homogeneous gel, which had smaller pore sizes. Trial cheese manufacture verified that yak cheese containing small casein micelles, formed curd faster and the cheese had higher texture profile analysis (TPA) values for hardness, cohesiveness, and springiness. CONCLUSION: This is important information for the optimization of yak cheese industrial production. © 2020 Society of Chemical Industry.

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.

Minor acidification of diafiltration water using various acidification agents affects the composition and rennet coagulation properties of the resulting microfiltration casein concentrate.

Cheese made from microfiltration (MF) retentate may suffer from textural defects due to a high Ca concentration. The reduction of colloidal minerals by the acidification of milk before MF at pH below 6.0 has been well documented in the literature. This process, however, creates less valuable side streams to the MF process and induces changes in the casein micelles that negatively affect their coagulation properties. The objective of this study was to determine whether a minor reduction in pH by using different acidifiers in the diafiltration (DF) water could induce changes in composition and renneting properties of the MF retentate. A 2-stage filtration process was used, with the first designed to increase the casein concentration to 8% and the second to slightly reduce the casein concentrate by 0.1 pH unit by DF, without influencing the total protein concentration. Four acidifying agents were tested during DF: lactic acid, hydrochloric acid, citric acid, and carbon dioxide. Diafiltration with water was used as a reference. At the start of DF, the retentates of acid DF had a slightly reduced pH, with an average of 0.09, whereas the pH of the reference retentate increased by an average of 0.07 unit. The reference retentate regained its starting pH by the end of DF. The carbonated retentate gradually increased in pH during processing, whereas the pH of the lactic, hydrochloric, and citric acid retentates remained constant. The permeate from the lactic acid and carbonated treatments had a reduced whey protein content compared with the reference. The total P and inorganic phosphate were lowered in the retentate by using carbonation. The total amount of Mg and Na were lowered in the retentate by using citric acid. The ionic Ca content in the retentate increased with use of lactic or hydrochloric acid. The type of acidifier used reduced the rennet clotting time. Combined acidified diafiltration with a slight reduction affects the permeate composition and improves the retentate clotting time despite the minimal mineral modification.

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.

The combined effects of different fat replacers and rennet casein on the properties of reduced-fat processed cheese.

Reduced-fat food products can help to prevent obesity and other diet-related diseases. However, the removal of fat often impairs the sensory and textural properties of foods, leading to low consumer acceptance. In this study, we tested various concentrations of fat replacers (inulin, corn dextrin, polydextrose, and microparticulated whey protein) combined with rennet casein to investigate their effects on the melting behavior, dynamic rheological properties, and hardness of reduced-fat processed cheese. We found that increasing concentrations of inulin and corn dextrin reduced the flowability of cheese in the melting test and can thus be used to inhibit flow during heating. Microparticulated whey protein did not affect flowability but caused an increase in the storage and loss moduli as well as the temperature at gel-sol transition. A similar effect was also shown for rennet casein, whereas inulin and polydextrose had little or no effect on these rheological parameters. Corn dextrin had no effect on the storage and loss moduli, but affected the gel-sol transition temperature. No changes in hardness were detected for any concentration of the fat replacers, but increasing the rennet casein content also increased the hardness of the samples, regardless of the fat replacer used. Our results indicate the different concentrations and combinations of fat replacers and rennet casein that can be included in reduced-fat processed cheese to develop products with specific rheological properties, thus meeting future demand for reduced-fat products with attractive sensory attributes.

Bovine colostrum whey: Postpartum changes of particle size distribution and immunoglobulin G concentration at different filtration pore sizes.

Bovine colostrum, as vital as it is for calves, is also a valuable source of functional components with rich health benefits for humans. Bovine colostrum whey consists of a large number of bioactive proteins and peptides. The most abundant of these is IgG. Particle size distribution (PSD) is an important feature of many of the processes in the dairy food industries. Despite this, scientific literature on PSD of colostrum whey is scarce. The goal of this research was to describe bovine colostrum whey PSD with an emphasis on postpartum milking time, filtration (pore size 450, 100, and 20 nm), IgG concentration, and lactation number. For this purpose, 4 postpartum milking colostrum samples were sequentially milked from 46 Holstein cows at 12 ± 1 h intervals. Colostrum whey was prepared by renneting and diluted (1:200) for PSD analyses by a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK). Immunoglobulin G concentration of these diluted colostrum whey samples were analyzed by an Octet K2 (Molecular Devices LLC, San Jose, CA) system. Linear mixed model analysis revealed significant effects of filter pore size, postpartum milking, and lactation on colostrum whey IgG concentrations. The percentage of particles in the size interval 5 to 15 nm (the hydrodynamic diameter of IgG is around 10 nm) had an intermediate positive correlation (r = 0.50) with IgG concentration. Furthermore, we showed that PSD was associated with IgG concentration, postpartum milking time, and lactation number. The PSD measurement results showed the mean hydrodynamic diameter of 100 nm pore size filtered colostrum whey to be around 10 nm. This, with the IgG concentration results, suggests that even though the size of IgG is around 10 nm, a 100 nm pore size is adequate for membrane-involved IgG separations. In terms of energy efficiency of the filtration process, the use of a larger filter pore size can make a remarkable difference, for example, in pressurizing and cooling costs. Our work contributes to the development of sustainable and widely available colostrum-derived food and feed supplements.

Effect of thermoresistant protease of Pseudomonas fluorescens on rennet coagulation properties and proteolysis of milk.

This study aimed to investigate the effect of different activity levels of a thermoresistant protease, produced by Pseudomonas fluorescens (ATCC 17556), on the cheesemaking properties of milk and proteolysis levels. Sterilized reconstituted skim milk powder was inoculated with the bacteria, and after incubation, centrifuged to obtain a supernatant-containing protease. Raw milk was collected and inoculated to obtain a protease activity of 0.15, 0.60, and 1.5 U/L of milk (treatments P1, P4, and P10, respectively). One sample was not inoculated (control) and noninoculated supernatant was added to a fifth sample to be used as a negative control. Samples were stored at 4°C for 72 h. After 0, 48, and 72 h, the rennet coagulation properties and proteolysis levels were assessed. The protease produced was thermoresistant, as no significant differences were observed in the activity in the pasteurized (72°C for 15 s) and nonpasteurized supernatants. The chromatograms and electrophoretograms indicated that the protease preferably hydrolyzed κ-casein and ß-casein, and levels of proteolysis increased with added protease activity over storage time. The hydrolysis of αS-caseins and major whey proteins increased considerably in P10 milk samples. At 0 h, the increase in the level of protease activity decreased the rennet coagulation time (RCT, min) of the samples, possibly due to synergistic proteolysis of κ-casein into para-κ-casein. However, over prolonged storage, hydrolysis of ß-casein and αS-casein increased in P4 and P10 samples. The RCT of P4 samples increased over time and the coagulum became softer, whereas P10 samples did not coagulate after 48 h of storage. In contrast, the RCT of P1 samples decreased over time and a firmer coagulum was obtained, possibly due to a lower rate of hydrolysis of ß-casein and αS-casein. Increased levels of protease could result in further hydrolysis of caseins, affecting the processability of milk over storage time.

Effects of milk proteins and posttranslational modifications on noncoagulating milk from Swedish Red dairy cattle.

Milk that does not coagulate after rennet addition, also called noncoagulating (NC) milk, is unwanted in cheese production due to prolonged processing time. Amounts of whey and casein proteins, genetic variants, as well as posttranslational modifications (PTM) of proteins are all contributing factors in rennet-induced coagulation of milk. In this study, we conducted a wide-ranging investigation of milk proteins in milk samples from 616 Swedish Red dairy cattle using liquid chromatography-high resolution mass spectrometry. Relative concentration of proteins, genetic variants, and PTM were compared between NC milk and coagulating milk. The PTM investigated were phosphorylation of caseins and glycosylation of κ-casein. Several genetic variants and PTM were found, including rare phosphorylation variants of the αS-caseins. Genetic variants were found to effect the expressed amount of different proteins. Further, the effect of protein amounts and PTM on a binary NC milk trait was modeled using a generalized linear model. The model showed that NC milk significantly correlated with higher relative concentrations of α-lactalbumin and ß-casein and lower relative concentrations of ß-lactoglobulin and κ-casein. Regarding PTM of caseins, an effect on NC milk from a lower relative concentration of αS1-casein with 8 phosphate groups were found, even though an effect from total relative concentration of αS1-casein was not found. This study has provided insights into protein variants and PTM important for NC milk to improve this undesirable property.

Genetic parameters for noncoagulating milk, milk coagulation properties, and detailed milk composition in Swedish Red Dairy Cattle.

The rennet-induced coagulation ability of milk is important in cheese production. For Swedish Red Dairy Cattle (RDC), this ability is reduced because of a high prevalence of noncoagulating (NC) milk. In this study, we simultaneously combined genetic parameters for NC milk, milk coagulation properties, milk composition, physical traits, and milk protein composition. Our aim was to estimate heritability and genetic and phenotypic correlations for NC milk and 24 traits (milk coagulation properties, milk composition, physical traits, and milk protein composition). Phenotypes and ∼7,000 SNP genotypes were available for all 600 Swedish RDC. The genotypes were imputed from ∼7,000 SNP to 50,000 SNP. Variance components and genetic parameters were estimated with an animal model. In Swedish RDC, a moderate heritability estimate of 0.28 was found for NC milk. For the other 24 traits, heritability estimates ranged from 0.12 to 0.77 (standard errors from 0.08 to 0.18). A total of 300 phenotypic and genetic correlations were estimated. For phenotypic and genetic correlations, 172 and 95 were significant, respectively. In general, most traits showing significant genetic correlations also showed significant phenotypic correlations. In this study, phenotypic and genetic correlations with NC milk suggest that many correlations between traits exist, making it difficult to predict the real consequences on the composition of milk, if selective breeding is applied on NC milk. We speculate that some of these consequences may lead to changes in the composition of milk, most likely affecting its physical and organoleptic properties. However, our results suggest that κ-casein could be used as an indicator trait to predict the occurrence of NC milk at the herd level.

Large-scale production of yak (Bos grunniens) chymosin A in Pichia pastoris.

Milk-clotting enzymes used in the dairy industry can be obtained from different sources such as plants, animals, and microorganisms. Recombinant chymosin is the best alternative for the dairy industry due to the differences in physicochemical properties of coagulating enzymes and scarcity of chymosin from animal sources. In this study, glycosylated and non-glycosylated forms of yak chymosin were extracellularly produced in a methylotrophic yeast, Komagataella phaffii (Pichia pastoris). Synthetic yak prochymosin genes were cloned into the pPICZαA vector, expressed in P. pastoris GS115 (PDI) strain. Active chymosin expression was achieved into supernatant with Saccharomyces cerevisiae α-mating factor under the control of methanol-inducible AOXI promoter. The glycosylation of yak chymosin did not have a significant effect on yield and activity at shake flask level. In a 5L fermentor, production of native yak-chymosin was achieved and the enzyme activity was found as 214 IMCU/ml. pH of 6-7 and temperature of 40 °C values were optimum for the enzyme. The laboratory scale white cheese production yield with recombinant yak chymosin was very similar to a commercial bovine chymosin. These results indicate that P. pastoris expression system is very suitable for recombinant yak chymosin production to meet the needs of the cheese industry.

Effect of pH adjustment on the composition and rennet-gelation properties of milk concentrates made from ultrafiltration and reverse osmosis.

The objective of this work was to investigate the effect of pH adjustment (initial pH vs. pH 6.50) on the rennet-gelation properties of concentrates made by ultrafiltration (UF) and reverse osmosis (RO). Rennet-gelation kinetics were followed by dynamic rheology and κ-casein hydrolysis by reverse-phase HPLC. At initial pH, RO concentrates had better rennet-coagulation behavior than UF concentrates and skim milk, whereas adjusting the pH to 6.50 produced the opposite results. The kinetics of κ-casein hydrolysis were similar in skim milk, and both concentrates and were not affected by pH adjustment. Differences in rennet coagulation were then related to the extent of hydrolysis required to trigger casein micelle aggregation. Small pH adjustments (<0.2 pH unit) enabled the use of RO concentrate with similar rennet-gelation behavior to UF concentrate, despite major compositional differences. This study shows that pH adjustment of RO concentrates can be a simple approach to improve their coagulation properties; however, the mechanisms behind these improvements remain to be elucidated.