Microsyringe-assisted visual volume detection based on phase separation: the case of chymosin milk-clotting activity study.

The constantly diverse demand scenarios for rapid on-site analysis have put forward high requirements for developing low-cost and user-friendly visual detection methods. Therefore, developing a visual detection method with simple operation and intuitive results has important practical value in the field of analysis and detection, but it is also challenging. In this work, we propose a microsyringe-assisted visual volume detection method based on phase separation, and apply it to analyze the milk-clotting activity of chymosin. Chymosin can cause phase separation of milk with whey in the mobile phase and curd in the gel state. The network structures of casein in curd can trap water molecules, resulting in separation of whey from curd gradually. Therefore, the analysis of chymosin milk-clotting activity can be realized according to the volume of whey measured using a portable microsyringe. This method shows a good linear correlation when the concentration of chymosin ranges from 1.02 U L-1 to 1020 U L-1 and the limit of detection of this method for chymosin is calculated to be 0.03 U mL-1. This work successfully realizes the visual analysis of chymosin milk-clotting activity based on the enzyme-triggered phase separation. It also shows great promise to be applied in other phase separation-based detection systems with the advantages of high accuracy, great portability and user-friendliness.

Isolation and Characterization of Milk Exosomes for Use in Advanced Therapies.

Exosomes are cell-derived extracellular vesicles (EVs) with diameters between 30 and 120 nm. In recent years, several studies have evaluated the therapeutic potential of exosomes derived from different fluids due to their low immunogenicity and high biocompatibility. However, producing exosomes on a large scale is still challenging. One of the fluids from which they could be isolated in large quantities is milk. Moreover, regeneration is a well-known property of milk. The present work seeks to optimize a method for isolating exosomes from bovine and human milk, comparing different storage conditions and different extraction protocols. We found differences in the yield extraction associated with pre-storage milk conditions and observed some differences according to the processing agent. When we removed milk fat globules and added rennet before freezing, we obtained a cleaner final fraction. In summary, we attempted to optimize a rennet-based new milk-exosome isolation method and concluded that pre-treatment, followed by freezing of samples, yielded the best exosome population.

Effect of jujube pulp on acid- and rennet-induced coagulation properties of milk.

Milk coagulation is an important step in the production of fermented dairy products such as yogurt and cheese. Jujube is gaining popularity and acceptance as a food ingredient. In China, jujube yogurt is popular among consumers. However, there is limited information on the effect of jujube on acid- and rennet-induced coagulation properties of milk. The objective of this study was to evaluate the effects of jujube pulp at different concentrations on acid- and rennet-induced coagulation kinetics of milk and the microstructure of acid- and rennet-induced gels. During acid-induced coagulation, with increasing jujube pulp concentration, the initial pH value decreased; however, the final pH value increased. The initial elasticity index (EI) value increased, and the time point at which the mean square displacement curves lost the linear trend advanced. The sample with 10% jujube pulp had the densest structure and highest EI value. During rennet-induced coagulation, with increasing jujube pulp concentration, the production rate and amount of caseinomacropeptide decreased, and the final EI value increased. Protein aggregates in rennet-induced gels became rough, and the sample with 20% jujube pulp had the highest EI value. This study provides a new perspective and understanding of the application of jujube in fermented dairy products.

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.

Analysis of Some Biochemical Properties of Recombinant Siberian Roe Deer (Capreolus pygargus) Chymosin Obtained in the Mammalian Cell Culture (CHO-K1).

Structure of the chymosin gene of Siberian roe deer (Capreolus pygargus) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene of C. pygargus was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1' + S3' specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to express recombinant roe deer prochymosin gene in CHO-K1 cells. CHO-K1-CYM-Cap pool cells were obtained, allowing synthesis and secretion of recombinant prochymosin into the culture fluid. As a result of zymogen activation, a recombinant roe deer chymosin was obtained and its total milk-clotting activity was estimated to be 468.4 ± 11.1 IMCU/ml. Yield of the recombinant roe deer chymosin was 500 mg/liter or ≈468,000 IMCU/liter, which exceeds the yields of genetically engineered chymosins in most of the expression systems used. Basic biochemical properties of the obtained enzyme were compared with the commercial preparations of recombinant chymosins from one-humped camel (Camelus dromedarius) and cow (Bos taurus). Specific milk-clotting activity of the recombinant chymosin of C. pygargus was 938 ± 22 IMCU/mg, which was comparable to that of the reference enzymes. Non-specific proteolytic activity of the recombinant roe deer chymosin was 1.4-4.5 times higher than that of the cow and camel enzymes. In terms of coagulation specificity, recombinant chymosin of C. pygargus occupied an intermediate position between the genetically engineered analogs of B. taurus and C. dromedarius chymosins. Thermostability threshold of the recombinant roe deer chymosin was 55°C. At 60°C, the enzyme retained <1% of its initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.

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.

Optimization of caseinolytic and coagulating activities of Solanum tuberosum rennets for cheese making.

BACKGROUND: In recent years, the rising global demand for cheese, the high cost and limited supply of calf rennet, and consumer choices have increased research into new alternatives to animal or recombinant chymosins for cheese making. Plant proteases with caseinolytic activity (CA) and milk-clotting activity (MCA) have been proposed as alternatives for milk clotting to obtain artisanal cheeses with new organoleptic properties. They have been named vegetable rennets (vrennets). The aim of this study was to evaluate the performance of two Solanum tuberosum aspartic proteases (StAP1 and StAP3) as vrennets for cheese making and to obtain a statistical model that could predict and optimize their enzymatic activity. RESULTS: To optimize the CA and MCA activities, a response surface methodology was used. Maximum values of CA and MCA for both enzymes were found at pH 5.0 and 30-35 °C. Analysis of the degradation of casein subunits showed that it is possible to tune the specificity of both enzymes by changing the pH. At pH 6.5, the αS - and ß- subunit degradation is reduced while conserving a significant MCA. CONCLUSION: The statistical models obtained in this work showed that StAP1 and StAP3 exert CA and MCA under pH and temperature conditions compatible with those used for cheese making. The casein subunit degradation percentages obtained also allowed us to select the best conditions for the degradation of the κ-casein subunit by StAPs. These results suggest that StAP1 and StAP3 are good candidates as vrennets for artisan cheese making. © 2023 Society of Chemical Industry.

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.

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.

Chymosin pretreatment accelerated papain catalysed hydrolysis for decreasing casein antigenicity by exposing the cleavage site at tyrosine residues.

Cow milk protein allergy (CMPA) induced by casein poses major health challenges that hinders the consumption of milk-based formulas. In this study, a novel sequential enzymatic hydrolysis catalysed by chymosin and papain was proposed to reduce casein antigenicity. Its effects on reducing casein antigenicity, structural properties and peptide profiles were evaluated by ELISA, multispectral techniques and peptidome analysis. It was revealed that the sequential enzymatic hydrolysis obtained a similarly residual antigenicity level in a shorter time (60 min) compared to papain-hydrolysis for 360 min. The hydrolysis-site at Tyr residues accessibility of papain was increased to 36.84 % by the chymosin pretreatment and it was significantly higher than 26.93 % obtained by only papain for 60 min. Moreover, the sequential enzymatic hydrolysis led to decrease in the large fragment peptides from αs1 casein. These findings suggested that the proposed sequential enzymatic hydrolysis can be exploited in the development of CMPA-free formulas.

Use of Calotropis procera cysteine peptidases (CpCPs) immobilized on glyoxyl-agarose for cheesemaking.

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture.

Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk-clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk-clotting activity under a solid-state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal-sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost-effective production, milk-clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese-making industry. PRACTICAL APPLICATION: The milk-clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.

Application of Calcium Citrate in the Manufacture of Acid Rennet Cheese Produced from High-Heat-Treated Goat's Milk from Spring and Autumn Season.

The stability of milk proteins is affected by changes in the pH value of milk, the heating temperature, and the addition of calcium compounds or chelating agents, which can cause alterations in calcium distribution. The purpose of this study was to determine the potential of the use of calcium citrate to manufacture fresh acid rennet cheese from high-temperature-pasteurized goat's milk (90 °C, 15 s) from the spring and autumn season and the effect of the calcium dose used on the physicochemical and organoleptic properties of the cheese. Autumn milk was found to be a richer source of total solids, confirming the effect of the production season on milk quality. The applied doses of calcium did not cause the denaturation of goat milk proteins and allowed pasteurization to take place at 90 °C for 15 s. The addition of calcium citrate resulted in a significant increase in the pH value of milk and cheese compared to the control sample. Adding 15 and 20 mg of Ca 100 g-1 to milk as citrate had the most beneficial effect on increasing protein retention in cheese in both seasons, showing a rise from 1.33% to 2.40%. The production season significantly influenced the cheese yield. The control goat cheese from the autumn season showed a 6.85% higher yield compared to the spring cheese. An increase in cheese yield was also observed as the calcium dose of milk increased. The content of micro- and microelements in cheese was affected by the production season. The addition of calcium citrate to milk resulted in a significant increase in the calcium content of cheese-from 120.83 to 147.45 mg 100 g-1 in the spring season and from 130.66 to 151.21 mg 100 g-1 in the autumn season. Increasing the dose of calcium increased the hardness of cheese samples by 1.37 N in the spring and 0.90 N in the autumn. The organoleptic evaluation showed that adding calcium to milk did not significantly affect the organoleptic characteristics of goat cheese.

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.

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.

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.

Transfer of antibiotics from goat's milk to rennet curd and whey fractions during cheese-making.

The transfer of 35 antibiotics from milk to curd and whey was evaluated. Cheeses were produced at laboratory scale, from antibiotic-free goat's milk spiked with different antibiotic concentrations between 0.25 and 4 times the Maximum Residue Limits established in milk. Drug concentrations in milk, curd and whey were analysed by UHPLC-HRMS. Results indicated that most antibiotics were mainly transferred from milk to whey (up to 85.9%), with retention percentages in the curd lower than 50%, except for ceftiofur (59.7%) and dicloxacillin (52.8%). In most cases, drug distribution was unaffected by the antibiotic concentration in milk and correlated significantly to the drug lipophilicity (Log P) for ß-lactams (R2 = 0.54) and sulfonamides (R2 = 0.62). When drug ionization was considered (Log D), improved correlation coefficients were obtained for macrolides (R2 = 0.98). However, other factors besides the drug solubility should be considered to explain and predict the partitioning of antibiotics during cheese-making.