Peptone Fraction Research Articles

PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation

The milk protein proteose peptone component 3 (PP3), also called lactophorin, is a small phosphoglycoprotein that is expressed exclusively in lactating mammary tissue. The C-terminal part of the protein contains an amphipathic helix, which, upon proteolytic liberation, shows antibacterial activity. Previous studies indicate that PP3 forms multimeric structures and inhibits lipolysis in milk. PP3 is the principal component of the proteose peptone fraction of milk. This fraction is obtained by heating and acidifying skimmed milk, and in the dairy industry milk products are also typically exposed to treatments such as pasteurization, which potentially could result in irreversible denaturation and inactivation of bioactive components. We show here, by the use of CD, that PP3 undergoes reversible thermal denaturation and that the α-helical structure of PP3 remains stable even at gastric pH levels. This suggests that the secondary structure survives treatment during the purification and possibly some of the industrial processing of milk. Finally, asymmetric flow field-flow fractionation and multi-angle light scattering reveal that PP3 forms a rather stable tetrameric complex, which dissociates and unfolds in guanidinium chloride. The cooperative unfolding of PP3 was completely removed by the surfactant n-dodecyl-β-d-maltoside and by oleic acid. We interpret this to mean that the PP3 monomers associate through hydrophobic interactions via the hydrophobic surface of the amphipathic helix. These observations suggest that PP3 tetramers act as reservoirs of PP3 molecules, which in the monomeric state may stabilize the milk fat globule.

HPLC profile and dynamic surface properties of the proteose–peptone fraction from bovine milk and from whey protein concentrate

Extraction of proteose–peptones (PPs) was carried out from fresh milk, from milk after prolonged incubation (12 days at 37 °C) and from two different whey protein concentrates (WPCs). The high performance liquid chromatography profiles of these extracts were compared. PPs eluted as three chromatographic peaks, which increased during milk incubation. The PP extracts from WPCs showed a number of peaks that are attributable to small peptides belonging to the proteose–peptone fraction and probably derive from proteolysis during storage of WPC. The dynamic properties at the air–water interface of the PP extracts were investigated by the drop-volume method. The PPs extracted from fresh milk showed the lowest values and a more rapid reduction in surface tension. PPs from WPCs were found to be effective as surfactants, even though a less marked reduction in surface tension compared with PPs from milk samples was shown.

β-CN-5P and β-CN-4P components of bovine milk proteose–peptone: Large scale preparation and influence on the growth of cariogenic microorganisms

Indigenous proteolytic activity in milk, mostly due to plasmin, gives rise to many casein-derived peptides that subsequently are found in the proteose–peptone fraction of milk where they comprise 10% or more of the total whey protein. Prominent amongst proteose–peptone components are β-CN-5P (β-casein residues, 1–105/107) and β-CN-4P (β-casein residues 1–28). Many peptides have potentially valuable functional or biological properties that differ from those of the parent proteins, and this paper describes simple, rapid and cost-effective preparation of these two milk peptide components in a high degree of purity, and in gramme quantities, for evaluation of such properties. The purification process was more efficient if β-casein was used as starting material. In this work, we prepared 46 g of β-casein from sodium caseinate in a simple rapid DEAE-cellulose ion-exchange chromatography stage. This was followed by in vitro hydrolysis with plasmin and precipitation and gel filtration steps to yield 4.8 g of highly purified β-CN-5P and 1.2 g of β-CN-4P. Utilising either unfractionated sodium caseinate, or milk itself, as starting material was satisfactory but gave less purified material containing other peptide impurities. Peptides similar to these proteose–peptone components have been implicated in the protective effects of milk and dairy products against dental caries in teeth. The mechanism(s) by which this protection occurs is unclear, but some antibiotics are peptides. However, we have found that, even at peptide concentrations as high as 0.5 mg/ml, neither β-CN-5P nor β-CN-4P had any effect on the in vitro growth of cariogenic Streptococcus mutans bacteria, ruling out a simple antibiotic mechanism.

Characterization and Proteolytic Origins of Specific Peptides Appearing During Lipopolysaccharide Experimental Mastitis

Based on the compositional change of the proteose peptone fraction, proteolysis was studied over time following lipopolysaccharide-induced experimental mastitis. Electrophoresis of the proteose peptone fraction revealed many degradation products. Five peptides were identified by amino-terminal sequencing as internal fragments of β-, κ-, αs1-, and αs2-casein that were generated by somatic cell proteases. Although κ-casein is considered particularly resistant to endogenous proteolysis, a κ-casein peptide was electrophoretically isolated in association with a β-casein fragment. The in vitro kinetic studies of caseinate hydrolysis by elastase, one of the main polymorphonuclear neutrophil (PMN) proteases, suggested that the β-casein peptide might be generated by elastase. In addition, elastase activity in milk PMN was higher during the inflammation of the mammary gland than prior to infusion.

Proteolysis and consistency changes of Gouda and Eidamský blok cheeses during ripening

Composition and rheological and sensory properties of Gouda cheese with different time of ripening (4 weeks, 4, 10, and 14 months) was evaluated. The level of proteolysis was measured by both the determination of nitrogenous compound fractions and the electrophoretic determination of casein proteolysis products. These properties were compared to those determined in Eidamský blok cheese with ripening time of 4 weeks. According to rheological evaluation, cheese hardness increased with ripening time. Sensory evaluation showed that hardness between fingers and in mouth increased and, on the other hand, the elasticity between fingers and in mouth as well as cohesiveness decreased with ripening time because of proteolysis. Based on the intensity sensory evaluations, the salt taste appears more intensive whereas the sour and bitter tastes were less intensive in Gouda cheese. Gouda cheese was evaluated as more pleasant in all three tastes in the hedonic sensory evaluation. The width and depth of ripening increased quite fast in Gouda cheese up to 10 months of maturation. In a four-week old Eidamský blok cheese the slightly lower values of ripening width and depth were obtained. The electrophoretic evaluation showed that the number of identifiable zones rose with ripening time. The growing content of g1-casein, proteose peptone fraction 5, and g2/g3-caseins, which are the products of b-caseins hydrolysis, can be observed as the increase of their zone intensity.

Component PP3 from bovine milk is a substrate for transglutaminase. Sequence location of putative crosslinking sites.

As the demands for food products of high quality increase, it will become more important to be able to modify the properties of food proteins. One possibility is to use enzymic modifications to improve functional properties and nutritional values. A highly efficient class of enzymic crosslinkers are transglutaminases (TG, EC 2.3.2.13), calcium-dependent enzymes that catalyse an acyl transfer reaction between protein-bound glutaminyl residues and primary amines (for review, see Aeschlimann & Paulsson, 1994). When protein-bound lysyl residues act as acyl acceptors, the reaction leads to the formation of intramolecular and/or intermolecular isopeptide bonds. Although a wide variety of amines such as putrescine, cadaverine or lysyl residues may act as amine donors in this reaction, only a limited number of glutamine residues in certain proteins will act as amine acceptors (Gorman & Folk, 1980).Ikura et al. (1981, 1985) reported that TG can be used to introduce methionine into casein and soyabean proteins and lysine into wheat gluten, thereby showing that TG can also be utilized to improve the nutritional value of food proteins. There have been a number of reports concerning the TG-mediated polymerization of food proteins such as α-lactalbumin and β-lactoglobulin (Aboumahmoud & Savello, 1990; Færgemand et al. 1997), soyabean proteins and casein components (Ikura et al. 1980a, b) and pea legumin (Larré et al. 1993). Recently, improved protein foaming capacity and stability have been demonstrated in TG-catalysed polymers of soyabean protein and whey protein isolate (Yildirim et al. 1996).Bovine PP3 (for review, see Girardet & Linden, 1996) is a phosphorylated glycoprotein isolated from the proteose peptone fraction of milk (Sørensen & Petersen, 1993a). The primary structure of PP3 has been determined (Sørensen & Petersen, 1993b) and comprises a polypeptide backbone of 135 amino acid residues containing five phosphorylated serines, two threonine-linked O-glycosylations, and one N-glycosylation. Immunological studies have shown that PP3 is present in the milk fat globule membrane, and that PP3 forms multimeric aggregates in bovine milk (Sørensen et al. 1997). Several functions have been suggested and investigated for bovine PP3 and fractions enriched with PP3, including emulsification (Shimizu et al. 1989), inhibition of lipolysis (Girardet et al. 1993) and mitogenesis (Mati et al. 1993).Previously, we have localized the potential TG-reactive glutamines in the four bovine caseins (Christensen et al. 1996) and in milk osteopontin (Sørensen et al. 1994). In the present study we have shown that component PP3 is a substrate for guinea-pig liver TG containing both reactive glutamine and lysine residues. In addition, we have localized the glutamine residues that act as amine acceptors.

Aqueous two-phase systems: a novel approach for the separation of proteose peptones

Poly(ethylene glycol) and dextran aqueous two-phase systems (ATPS) were developed to facilitate the separation of components of the proteose peptone fraction of bovine milk, which are mostly large casein derived peptides or glycoproteins. These have proved difficult to purify using conventional chromatographic procedures. ATPS exploit differences in hydrophobicity, size and ionic properties of the proteose peptones with a view to developing methods for future large scale preparations of the individual components of this whey protein fraction.

Structure of the O-glycopeptides isolated from bovine milk component PP3.

The heat-stable acid-soluble phosphoglycoprotein component PP3 was isolated from the bovine milk proteose peptone fraction by concanavalin A affinity chromatography. Glycopeptides from the ConA-bound fraction corresponding to the component PP3 were obtained by Pronase digestion and were separated by gel filtration into high and low-molecular-mass glycopeptides. In a previous work, we have investigated the structure of the N-glycans from the high-molecular-mass glycopeptides [Girardet et al. (1995) Eur J Biochem 234: 939-46]. Here, we describe the structure of the O-glycans from the low-molecular-mass glycopeptides. By combining methylation analysis, mass spectrometry, 400 MHz 1H-NMR spectroscopy and peptide sequence analysis, we show that the low-molecular-mass fraction contains several neutral glycopeptides. A mixture of the following three glycan structures linked to the Thr86 has been identified: GalNac alpha1-O-Thr, Gal(beta1-3)GalNAc alpha1-O-Thr and Gal(beta1-4)GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc alpha1-O-Thr.

Structure of glycopeptides isolated from bovine milk component PP3.

The heat-stable acid-soluble phosphoglycoprotein component PP3 was isolated from the bovine milk proteose peptone fraction by concanavalin A affinity chromatography. Glycopeptides were released by pronase digestion of the milk component PP3 and were subsequently separated by high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of the glycan and peptide moieties of eight N-glycopeptides have been established by combining methylation analysis, mass spectrometry, 400-MHz 1H-NMR spectroscopy, and peptide sequence analysis. All the analyzed fractions contained biantennary N-acetyllactosamine-type carbohydrate chains, some of them with a GalNAc(beta 1-4)GlcNAc or a NeuAc(alpha 2-6)GalNAc(beta 1-4)GlcNAc group. This particular sequence did or did not replace the Gal(beta 1-4)GlcNAc group usually found in most N-linked glycans. Moreover, the sialylated Gal and GalNAc residues were only found on the Man(alpha 1-3) antenna.

Phosphorylation, glycosylation and amino acid sequence of component PP3 from the proteose peptone fraction of bovine milk

Component PP3 is a phosphorylated glycoprotein with an apparent molecular mass of 28 kDa isolated from the proteose peptone fraction of bovine milk. The function of the protein is not known. The primary structure has been determined and shown to contain 135 amino acid residues (EMBL accession no. P80195). It was phosphorylated at Ser29, Ser34, Ser38, Ser40 and Ser46. Two O-linked carbohydrate groups were found at Thr16 and Thr86, while one N-linked carbohydrate group was present at Asn77. Thr16 was only approximately 50% glycosylated. The amino sugar detected by the amino acid analyser at Thr86 was mainly galactosamine but a small amount of glucosamine was also present. The amino sugars found in the carbohydrate group linked to Asn77 were both glucosamine and galactosamine. A fragment of PP3 has been isolated from milk and shown to correspond to residues 54-135. This fragment was probably generated by plasmin hydrolysing the Arg53-Ser54 bond.

Heat Inactivation and Reactivation of Alpha Toxin from Clostridium perfringens

Crude and purified preparations of commercial alpha toxin from Clostridium peifringens were heated in physiological saline and in fractions separated from proteose peptone (Difco, 0120). Heating temperatures ranged from 55–95 C for 6–18 min. Alpha toxin remained active at temperatures ⩽ 85 C when heated in the presence of a fraction of proteose peptone having a MW ⩾ 50,000. At 95 C, some destruction was observed with a D-value of 444 min. In saline or a proteose peptone fraction having a MW < 50,000, heat inactivation of alpha toxin occurred at 65 C. Gel filtration revealed that heat protection was associated with complex formation of the alpha toxin with proteose peptone. Alpha toxin heat-treated in saline was reactivated when proteose peptone was added after heat treatment and the toxin was subsequently incubated in hemolysin indicator plates at 37 C for 24 h. Reactivation of alpha toxin was not observed when saline was added after the toxin was heat-treated and then incubated in hemolysin indicator plates at 37 C for up to 48 h.

Studies on a serum substitute for mammalian cells in culture

Amino acid analyses of two biologically efficacious fractions from a peptone dialysate (PD) revealed high concentrations of glycine, presumably present in PD as glycine-rich peptides. Before undertaking further analyses of PD the growth-promoting abilities of several commercially available glycine peptides were tested with strain L (NCTC 2071) cells continuously subcultured in chemically defined medium NCTC 135, and with RH-PD cells which require peptone or peptone fractions for continued survival and proliferation. NCTC 2071 populations 5 to 10% greater than control cultures were obtained with 0.18 to 0.72mm peptide-supplemented medium NCTC 135; slightly better results were obtained with peptides containing an even number of glycine residues. No amplification of this response was observed after 28 to 29 weekly culture transfers in 0.72mm supplemented NCTC 135, and evidence suggested that the peptides were poorly hydrolyzed to monomeric glycine. Addition of the glycine peptides may permit use of a reduced minimum inoculum for NCTC 2071. Marked growth enhancement of peptone-dependent RH-PD cells was obtained only when glycine peptides were tested in the presence of 0.5 mg of PD per ml.

12. The use of peptone fractions as cryoprotective agents: J. D. Davies (Department of Pathology, University of Cambridge, Cambridge, England)

12. The use of peptone fractions as cryoprotective agents: J. D. Davies (Department of Pathology, University of Cambridge, Cambridge, England)

Long-term cultivation of human cells (Chang) in chemically defined medium and effect of added peptone fractions.

Long-term cultivation of human cells (Chang) in chemically defined medium and effect of added peptone fractions.

168. Bitterness and thinning in canned cream

1. Three organisms have been isolated from defective commercially-canned cream which, on inoculation into normal cream, cause bitterness and thinning.2. These organisms are described and are shown to be strains ofB. subtilis.Their spores are capable of withstanding temperatures up to 120°C. for as long as 40 min.3. The rate of development of bitterness and thinning in experimentally inoculated cans is shown to vary with the culture used and with the temperature of incubation.4. Both bitterness and thinning are associated with the breakdown of protein, as indicated by determinations of the non-protein nitrogen and of the peptone and subpeptone fractions.5. The alterations in viscosity do not appear to be due to any fundamental change in the structure of the cream, nor to differences in the degree of clumping of the fat globules.6. The applications of the results in commercial practice are discussed.

The Effect of Lecithin and Horse Serum on the Hemolytic Action of Certain Peptones

Abstract In a preceding communication (1) it was shown that certain peptone fractions cause hemolysis and agglutination of different species of red blood cells. The peptones used in the tests were the individual fractions obtained by separation of Witte's peptone and those arising from the peptic digestion of casein. As stated there, corresponding peptone fractions from the two sources, possess similar properties, although in certain instances the reactions obtained differ in degree. The hemolysis produced by the peptones is as follows: Peptone B (Witte) hemolyzes ten species of red blood cells: Human beef, horse, goat, rabbit, guinea-pig, cat, dog, mouse and pig. Peptone B (Casein) hemolyzes ten species of red blood cells: Human, beef, horse, goat, rabbit, guinea-pig, cat, mouse, goose and pig. Peptone A (Witte) hemolyzes ten species of red blood cells: Human beef, horse, goat, sheep, rabbit, guinea-pig, dog, goose and pig. Deuteroalbumose B (Witte) hemolyzes nine species of red blood cells: Human, beef, horse, goat, sheep, rabbit, guinea-pig, cat and goose.

IMMUNOCHEMICAL STUDIES WITH PEPTONES

The experiments here described indicate that the peptone fractions can alter the different blood elements in such a manner as to affect their immunological reactions. This power is not possessed by all the peptones equally, either qualitatively or quantitatively. If we regard the phenomenon of hemolysis by immune serum in the light of a biochemical process, depending upon properties inherent in certain constituents of the serum and the red blood cells, the experiments described reveal a number of facts concerning the immunochemical action of albumoses and peptones. These substances, which depend for their separation and identification upon purely physical means, show differences in their behavior toward the elements concerned in the production of hemolysis. Inagaki (7) has shown that the albumoses can combine with nucleohiston. It is not improbable, therefore, that some of the reactions are chemical as well as physicochemical in character. Further studies are necessary to determine whether the reactions elicited can aid us in the differentiation of the peptone bodies, for which at present we possess but few tests. The results, however, are suggestive, and may aid in recognizing the presence of these substances in blood serum.