Characterization of "lettucine", a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting

In this work we focused on the characterization of a novel plant rennet purified from lettuce leaves (Lactuca sativa L. cv Romana). The lettuce protease, lettucine, showed trypsin-like, SV8-like, and caseinolytic activities. Although the enzyme did not recognize peptides having hydrophobic amino acid residues in the P(1) position of the target bond, it did show milk-clotting activity, suggesting that different bonds rather than the Phe(105)-Met(106) of the kappa-casein might be cleaved, still inducing milk-clotting. The enzyme exhibited proteolytic activity toward alpha-casein, beta-casein, kappa-casein, and milks with different fat contents, with the highest activity observed with partially skimmed milk, total casein, and alpha- and kappa-casein. SDS-PAGE studies showed that lettucine cleaved alpha-casein, beta-casein, and kappa-casein. In particular, we showed that alpha-casein breakdown occurred even though total casein or milks were supplied, suggesting that the lettuce enzyme is able to operate a significant disorganization of the casein's micellar structure. Moreover, the proteolytic activity of the enzyme analyzed under various technological parameters, such as temperature and pH, indicated that the lettuce enzyme is highly consistent with the milk-clotting process.     

Comparison of the hydrolysis of bovine κ-casein by camel and bovine chymosin: a kinetic and specificity study

Bovine chymosin constitutes a traditional ingredient for enzymatic milk coagulation in cheese making, providing a strong clotting capacity and low general proteolytic activity. Recently, these properties were surpassed by camel chymosin, but the mechanistic difference behind their action is not yet clear. We used capillary electrophoresis and reversed-phase liquid chromatography-mass spectrometry to compare the first site of hydrolysis of camel and bovine chymosin on bovine κ-casein (CN) and to determine the kinetic parameters of this reaction (pH 6.5; 32 °C). The enzymes showed identical specificities, cleaving the Phe105-Met106 bond of κ-CN to produce para-κ-CN and caseinomacropeptide. Initial formation rates of both products validated Michaelis-Menten modeling of the kinetic properties of both enzymes. Camel chymosin bound κ-CN with ～30% lower affinity (K(M)) and exhibited a 60% higher turnover rate (k(cat)), resulting in ～15% higher catalytic efficiency (k(cat)/K(M)) as compared to bovine chymosin. A local, less dense negatively charged cluster on the surface of camel chymosin may weaken electrostatic binding to the His-Pro cluster of κ-CN to simultaneously impart reduced substrate affinity and accelerated enzyme-substrate dissociation as compared to bovine chymosin.     

Heat-induced covalent complex between casein micelles and beta-lactoglobulin from goat's milk: identification of an involved disulfide bond.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.     

Role of kappa-casein in the association of denatured whey proteins with casein micelles in heated reconstituted skim milk

Reconstituted skim milk at pH from 6.5 to 7.1 was unheated, preheated (68 degrees C/20 min), or heated at 90 degrees C for 20-30 min. On preheating, the size of the casein micelles decreased by about 5-20 nm, with a greater effect at higher pH. The casein micelle size of the heated milk at pH 6.5 increased by about 30 nm when compared to that of the unheated or preheated milk. As the pH was increased before heating, the particle size gradually decreased so that, at pH 7.1, the size was markedly smaller than that for the unheated milk and slightly smaller than that for the preheated milk. High levels (about 85%) of denatured whey protein associated with the casein micelles at pH 6.5, and this level decreased as the pH increased so that, at pH 7.1, low levels (about 15%) were associated with the micelles. Low levels of alphaS-casein and beta-casein were found in the serum regardless of the heat treatment or the pH of the milk. At pH 6.5, low levels (about 10%) of kappa-casein were also found in the milk serum. In the unheated milk, the level of serum kappa-casein increased slightly with increasing pH; in the heated samples, the level of serum kappa-casein increased markedly and linearly with increasing pH so that, at pH 7.1, about 70% of the kappa-casein was in the serum phase. The results of this study indicate that the pH dependence of the levels of serum phase kappa-casein may be responsible for the change in distribution of the whey proteins between the colloidal and serum phases. This is the first report to demonstrate significant levels of dissociation of kappa-casein from the micelles at pH between 6.5 and 6.7, although this dissociation phenomenon is well known on heating milk at high temperatures at pH above 6.7.     

Controlled proteolysis and the properties of milk gels

Milk gels induced by partial proteolysis of the kappa-casein followed by acidification were studied, and their gelation behavior was compared to that of milk gels induced by simultaneous acidification and renneting, using dynamic rheology. There were generally two stages (at pH values below and above 5.0) in the gelation of the milk whose kappa-casein had been partially proteolyzed and acidified. The onset of gelation was at higher pH as the degree of kappa-casein proteolysis increased. The development of G' immediately after the onset of gelation was faster in the milk gels induced by simultaneous acidification and renneting, because of the continuing kappa-casein proteolysis. Preheat treatment caused the onset of gelation to occur at higher pH than for unheated milk. However, the maximum tan delta during gelation always occurred at the same pH (for a given concentration of acidulant), and its value and position were independent of the extent of renneting and whether the milks had been heat treated. The results are discussed in terms of the interactions between casein micelles occurring during gelation.     

Limited enzymatic treatment of skim milk using chymosin affects the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates

The effects of heat treatment and limited kappa-casein hydrolysis on the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates were investigated as a possible explanation for the gelation properties of combined rennet and acid gels. Reconstituted skim milk was submitted to combinations of 0-67% hydrolysis of the kappa-casein at 5 degrees C and heat treatment at 90 degrees C for 10 min. The protein composition of the ultracentrifugal fractions was obtained by reverse-phase high-performance liquid chromatography (RP-HPLC). The aggregates contained in each phase were isolated by size-exclusion chromatography and analyzed by RP-HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon heating only, 20-30% of the total kappa-casein dissociated, while 20-30% of the total whey protein attached to the micelles. When heated milk was renneted, little changes were observed in the distribution and composition of the aggregates. Conversely, the heat treatment of partially renneted milk induced the formation of essentially micelle-bound aggregates. The results were discussed in terms of the preferred interaction between hydrophobic para-kappa-casein and denatured whey proteins.     

Plasmin system and microbial proteases in milk: characteristics,roles, and relationship

Proteolysis of milk proteins can be attributed to both native proteases and the proteases produced by psychrotrophic bacteria during storage of fresh raw milk. These proteases cause beneficial or detrimental changes, depending on the specific milk product. Plasmin, the major native protease in milk, is important for cheese ripening. Milk storage and cheese-making conditions can affect the level of plasmin in the casein and whey fractions of milk. A microbial protease from a psychrotrophic microorganism can indirectly increase plasmin levels in the casein curd. This relationship between the plasmin system and microbial proteases in milk provides a means to control levels of plasmin to benefit the quality of dairy products. This paper is a short review of both the plasmin system and microbial proteases, focusing on their characteristics and relationship and how the quality of dairy products is affected by their proteolysis of milk proteins.     

Heat-induced redistribution of disulfide bonds in milk proteins. 2. Disulfide bonding patterns between bovine beta-lactoglobulin and kappa-casein

Heat treatment of milk causes the heat-denaturable whey proteins to aggregate with kappa-casein (kappa-CN) via thiol-disulfide bond interchange reactions. The particular disulfide bonds that are important in the aggregates are uncertain, although Cys(121) of beta-lactoglobulin (beta-LG) has been implicated. The reaction at 60 degrees C between beta-LG A and an activated kappa-CN formed small disulfide-bonded aggregates. The tryptic peptides from this model system included a peptide with a disulfide bond between a Cys residue in the triple-Cys peptide [beta-LG(102-124)] and kappa-CN Cys(88) and others between kappa-CN Cys(88) or kappa-CN Cys(11) and beta-LG Cys(160). Only the latter two novel disulfide bonds were identified in heated (90 degrees C/20 min) milk. Application of computational search tools, notably MS2Assign and SearchXLinks, to the mass spectrometry (MS) and collision-induced dissociation (CID)-MS data was very valuable for identifying possible disulfide-bonded peptides. In two instances, peptides with measured masses of 4275.07 and 2312.07 were tentatively assigned to beta-LG(102-135):kappa-CN(11-13) and beta-LG A(61-69):kappa-CN(87-97), respectively. However, sequencing using the CID-MS data demonstrated that they were, in fact, beta-LG(1-40) and beta-LG(41-60), respectively. This study supports the notion that reversible intramolecular disulfide-bond interchange precedes the intermolecular interchange reactions.     

Human IgE binding to the glycosidic moiety of bovine kappa-casein

IgE ability for recognizing milk proteins was assayed in the serum of an adult atopic patient who outgrew cow milk allergy in early childhood. A number of protein species included in casein from bovine milk were detected by human IgE in immunoblotting experiments. Comparing these results with those obtained from an analysis using antibody preparations specifically directed toward the different casein fractions, IgE-reactive bands were identified as isoforms of kappa-casein. IgE-reactive protein was not present in neither bovine cheese, regardless of cheese-making technology and time ripening, nor milk from any other dairy animal, such as ewe, goat, and water buffalo. Chemical deglycosylation of protein bands immobilized onto nitrocellulose proved that the glycosidic moiety of bovine kappa-casein was principally involved in IgE recognition.     

Specificity of disulfide bond formation during thermal aggregation in solutions of beta-lactoglobulin B and kappa-casein A

Heat-induced (90 degrees C, 10 min, pH 6.7) intermolecular disulfide bond formation in 1:1 mixtures of beta-lactoglobulin B (beta-Lg) and kappa-casein A (kappa-CN) was studied by enzymatic digestion with trypsin or glu-C, reverse-phase HPLC, and MALDI-TOF-MS. Observed masses were compared to theoretically calculated masses of disulfide-bonded peptide dimers and trimers, and the number of different masses matching peptide combinations involving each bond was used as a measure of confidence of identification. The beta-Lg cysteine residues 121 or 119 were involved in bonds with both cysteines of kappa-CN and all cysteines of beta-Lg. This agrees with the supposed initiatory role of beta-C121 in heat-induced SH/SS interchange. The largest numbers of matches corresponded to bonds linking beta-C119/C121 with kappa-C11 or with beta-C66. Multiple matches were recorded for beta-C119/C121 bonding with beta-C119/C121, with beta-C160, or with kappa-C88. However, beta-C106 was observed only in bonds with beta-C119/C121 and did not appear to bond to kappa-CN, suggesting it remains buried in the core of the protein.     

Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels

Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.     

Effect of the pH of heating on the qualitative and quantitative compositions of the sera of reconstituted skim milks and on the mechanisms of formation of soluble aggregates

The effect of the pH of heating (6.3-7.3) on the composition of sera in reconstituted skimmed milks was investigated. A combination of SDS-PAGE analysis and size exclusion chromatography (SEC) combined with an original approach to the analysis of the SEC profiles was performed. The composition of the sera varied greatly when the pH of heating was adjusted below and above the natural pH of milk. The formation, composition, and concentration of heat-induced soluble complexes depended on the combination of the effect of adjusting the pH of the milk and the heat treatment. Two types of mechanism for the formation of soluble aggregates appeared to exist, depending on the pH of the milk. The first type results from the formation of WP/kappa-casein aggregates at the surface of the micelle, and these were detached partially into the serum in larger amount as the pH increased up to 6.7, where it reaches a maximum. The second type of complexes, whose amount increased as the pH of heating increased from 6.7 to 7.3, may be formed between caseins (kappa- but also perhaps some alpha(s)-casein) and aggregated WP resulting in complexes that are smaller in size and with a higher kappa-casein/whey protein ratio than the first type.     

Changes in structures of milk proteins upon photo-oxidation

Changes in protein structures as a result of riboflavin-induced photo-oxidation were studied for six milk proteins: alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin. The milk proteins showed significant variability in sensitivity to photo-oxidation. After photo-oxidation, an increase in carbonyl content because of oxidation of tryptophan, histidine, and methionine, as well as formation of dityrosine, was observed for all proteins studied, although at very different levels. Generally, the increment was highest for alpha- and beta-casein and was lowest for lactoferrin. Loss of tryptophan because of photo-oxidation was well-correlated with the formation of the tryptophan oxidation products, N-formylkynurenine and kynurenine. Changes at the tertiary protein structure level were observed after photo-oxidation of the globular proteins, where tryptophan fluorescence emission indicated unfolding of alpha-lactalbumin and beta-lactoglobulin, whereas lactoferrin achieved a more compact tertiary structure. Changes in secondary structure were observed for alpha-lactalbumin and beta-lactoglobulin, whereas the secondary structure of lactoferrin did not change. Polymerization of alpha- and beta-casein and of lactoferrin was observed, whereas kappa-casein, alpha-lactalbumin, and beta-lactoglobulin showed little tendency to polymerize after photo-oxidation. Lability toward photo-oxidation is discussed according to the structural stabilities of the globular proteins.     

Functionality of beta-casein peptides: importance of amphipathicity for emulsion-stabilizing properties.

To investigate structure-function relationships with regard to emulsion-stabilizing properties, peptides from bovine beta-casein (betaCN), obtained by plasmin hydrolysis and fractionation of the hydrolysate, were isolated and identified on the basis of their masses determined by electrospray ionization mass spectrometry, the primary structure of the intact protein, and the known specificity of the enzyme. An amphipathic peptide fraction was fractionated further by ion-exchange chromatography and subsequent hydrophobic interaction chromatography resulting in the components betaCN[f 1-105/107] and betaCN[f 29-105/107]. The latter peptides had poor emulsion-stabilizing properties compared to the former ones, and the stability of an emulsion formed with betaCN[f 29-105/107] was also more sensitive to hydrophobic impurities than that of an emulsion formed with betaCN[f 1-105/107]. The highly charged N-terminal part appeared to be important for the emulsion-stabilizing properties of these peptides. A hypothesis for the structure-function relationship is given.     

Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 degrees C). The pasteurizing heat treatment (72 degrees C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 degrees C for 120 s, 120 degrees C for 120 s, and 140 degrees C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and kappa-casein (kappa-CN) and a proportion of the alpha(s2)-CN. Pressure treatment of milk at 200 MPa caused beta-lactoglobulin (beta-LG) to form disulfide-bonded dimers and incorporated beta-LG into aggregates, probably disulfide-bonded to kappa-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated beta-LG and most of the minor whey proteins, as well as kappa-CN and much of the alpha(s2)-CN, into aggregates. The accessibility of alpha(s2)-CN and formation of complexes involving alpha(s2)-CN, kappa-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the alpha-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities.     

Casein proteins as molecular chaperones

Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.     

影响原料乳品质的纤溶酶活性的因素分析

为了充分了解原料乳中纤溶酶活性,以便从源头上提高原料乳品质,该文针对中国原料乳的生产现状,分析了奶牛品种、胎次、养殖模式及挤奶时间对原料乳中纤溶酶活性的影响,并对纤溶酶的热稳定性进行了研究。结果表明,养殖模式、奶牛品种及胎次对原料乳中纤溶酶活性影响显著（P＜0.05）,牧场养殖、1、4胎和娟珊牛原料乳中纤溶酶活性显著低于养殖小区、2、3胎及荷斯坦牛乳中的纤溶酶活性;挤奶时间对原料乳中纤溶酶活性影响不显著（P＞0.05）;原料乳中纤溶酶活性随热处理温度的升高而逐渐下降,巴氏杀菌（75℃、15 s）仅可使原料乳中纤溶酶活性下降25%;半胱氨酸对乳中纤溶酶活性具有一定的抑制作用。研究结果对根据纤溶酶活性对原料乳进行品质评价及分级具有一定借鉴作用。     

Simple and rapid liquid chromatography-tandem mass spectrometry confirmatory assay for determining amoxicillin and ampicillin in bovine tissues and milk

A simple specific and rapid confirmatory method for determining the two amphoteric penicillins, that is, amoxicillin and ampicillin, in bovine muscle, liver, kidney, and milk is presented. This method is based on the matrix solid-phase dispersion technique with hot water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry. With this instrumentation, the selected reaction monitoring acquisition mode with two fragmentation reactions for each analyte was adopted. After acidification and filtration of the aqueous extracts, 25 microL of the tissue final extracts and 50 microL of the milk final extract were injected into the LC apparatus. Absolute recovery of the two analytes in any biological matrix at the 50 ppb level in tissues and the 4 ppb level in milk was 74-95% with relative standard deviations (RSDs) of no larger than 9%. When penicillin V was used as surrogate internal standard, relative recovery of the targeted compounds present in bovine tissues and milk at, respectively, 25 and 2 ppb levels ranged between 100 and 106% with RSDs of no larger than 11%. When fractionation of analytes by using a short chromatographic run was attempted, remarkable signal weakening for the two analytes was experienced. This effect was traced to polar endogenous coextractives eluted in the first part of the chromatographic run that interfered with the gas-phase ion formation of the two penicillins. Slowing the chromatographic run eliminated this unwelcome effect. Limits of quantification of the two analytes in bovine milk were estimated to be <1 ppb, whereas amoxicillin and ampicillin could be quantified in bovine tissues down to 3.1 and 0.8 ppb levels, respectively.     

Effects of added sodium caseinate on the formation of particles in heated milk

The effects of heat at temperatures in the range of 80-90 degrees C on mixtures of reconstituted skim milk powder (RSMP) and sodium caseinate have been determined. In the absence of caseinate, the action of heat on RSMP produces soluble complexes of whey proteins and kappa-casein, as well as complexes of whey protein with the casein micelles. When sodium caseinate was added to RSMP at levels of 0.5 and 1.0%, the denaturation of the whey protein and the production of the soluble complexes in the serum were hardly affected, either in rate or in amount. However, during the heating, the caseinate disappeared from the serum. Further studies on model mixtures of the different components showed that it was probable that the bulk of the caseinate associated with the casein micelles during heating, probably by binding inside the surface layer of kappa-casein, because no increase in the diameters of the casein micelles could be observed.