Tryptic Hydrolysis Research Articles

Implementing visible 473 nm photodissociation in a Q-Exactive mass spectrometer: towards specific detection of cysteine-containing peptides.

Improvement of the fragmentation specificity may streamline data processing of bottom-up proteomic experiments by drastically reducing either the amount of MS/MS data to process in the discovery phase or the detection of interfering signals in targeted quantification. Photodissociation at appropriate wavelengths is a promising alternative technique to the non-discriminating conventional activation mode by collision. Here, we describe the implementation of visible LID at 473 nm in a Q-Exactive-Orbitrap mass spectrometer for the specific detection of cysteine-containing peptides tagged with a Dabcyl group. HCD cell DC offset and irradiation time were optimized to obtain high fragmentation yield and spectra free of contaminating CID product ions, while keeping the irradiation time scale compatible with chromatographic separation. With this optimized experimental set-up, the selective detection of cysteine-containing peptides in a whole tryptic hydrolysate of three combined proteins is demonstrated by comparing all ion fragmentation (AIF) spectra recorded online with and without laser irradiation.

Antioxidant activities of two metallothionein-like proteins from sweet potato (Ipomoea batatas [L.] Lam. `Tainong 57') storage roots and their synthesized peptides.

BackgroundMetallothionein (MT) characterized by their low molecular weight and high cysteine content.ResultsTwo recombinant proteins of MT-I and MT-II overproduced in E. coli (M15) was purified by Ni2+-chelated affinity chromatography. The molecular mass of MT-I and MT-II are ca. 6,600 and 8,000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Total antioxidant status, DPPH radical scavenging activity, reducing power method, Fe2+-chelating ability, ferric thiocyanate (FTC) method, and protecting calf thymus DNA against hydroxyl radical-induced damage were studied. The MT-I and MT-II proteins with a concentration of 100 μg/mL exhibited the highest activity (expressed respectively as 61.72 ± 0.13 and 74.28 ± 1.15 μM Trolox equivalent antioxidative capacity, TEAC) in total antioxidant status test. Like total antioxidant status, DPPH radical scavenging activity, reducing power, Fe2+-chelating ability, FTC activity, and protecting calf thymus DNA against hydroxyl radical-induced damage all showed that MT-1 and MT-II proteins have antioxidant activities. In this study, we also found that antioxidant activities of MT-I and MT-II increased from 17% and 16% (0 h) to about 26% and 28% (24 h) after 24 h hydrolysis by trypsin. Smaller peptides increased the antioxidant activities. Four and three peptides, respectively, from MT-I and MT-II protein sequences for testing antioxidative activity were synthesized according to tryptic hydrolysis simulation. The obtained MSSGCK, CGSDCK, LTLEGSSEK, ATEGGHACK, CGNGCGGCK, and CDPCNCK showed IC50 values of 309.87, 1423.37, 3925.54, 561.32, 300.76, and 610.12 μM, respectively, when scavenging activity of DPPH radicals (%) was measured.ConclusionsThese findings mean that a cysteine residue is most important in antiradical activities. It was suggested that MT-I and MT-II might contribute their antioxidant activities against hydroxyl and peroxyl radicals.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0064-4) contains supplementary material, which is available to authorized users.

Caseinophosphopeptides released after tryptic hydrolysis versus simulated gastrointestinal digestion of a casein-derived by-product

The production of caseinophosphopeptides from a casein-derived by-product generated during the manufacture of a functional ingredient based on antihypertensive peptides was attempted. The casein by-product was submitted to tryptic hydrolysis for 30, 60 and 120min and further precipitated with calcium chloride and ethanol at pH 4.0, 6.0 and 8.0. Identification and semi quantification of the derived products by tandem mass spectrometry revealed some qualitative and quantitative changes in the released caseinophosphopeptides over time at the different precipitation pHs. The by-product was also subjected to simulated gastrointestinal digestion. Comparison of the resulting peptides showed large sequence homology in the phosphopeptides released by tryptic hydrolysis and simulated gastrointestinal digestion. Some regions, specifically αS1-CN 43-59, αS1-CN 60-74, β-CN 1-25 and β-CN 30-50 showed resistance to both tryptic hydrolysis and simulated digestion. The results of the present study suggest that this casein-derived by-product can be used as a source of CPPs.

Extraction And Characterization of Bioactive Peptides Derived from the Hydrolysates of Total Soluble Proteins of Pistachio Nuts (Pistacia vera L.)

This study explored the potential of pistachio nut (Pistacia vera L.) total soluble proteins to release bioactive peptides exhibiting angiotensin-converting enzyme (ACE) inhibition, antioxidative, and antibacterial activities. The total soluble proteins were extracted from the ground, defatted nut using 0.010 M phosphate buffered saline, pH 6.8. The extracted proteins were hydrolyzed at different time intervals using trypsin and chymotrypsin. Hydrolysates of the total soluble proteins at 24 h digestion time exhibited the highest ACE-inhibition activity of 76.67 ± 0.10% and 70.83 ± 0.00% for chymotrypsin and trypsin digestion, respectively. The 24 h enzymatic hydrolysates were further fractionated in RP-HPLC using a C18 Vydac column. The C1 fraction from the 24 h chymotryptic hydrolysates and T2 from the 24 h tryptic hydrolysates exhibited the highest ACE-inhibition activities with an IC50 value of 147.7 ± 0.8 and 148.7 ± 0.6 μg/mL, respectively. The 24 h chymotryptic and tryptic hydrolysates also exhibited a DPPH radical scavenging activity of 83.7 ± 1.1% (EC50 = 356.5 ± 1.0 μg/mL) and 80.4 ± 0.2% (EC50 = 402.7 ± 1.1 μg/mL), respectively. The hydroxyl radical scavenging activities of the 24 h chymotryptic and tryptic hydrolysates were found to be 22.8 ± 1.0 and 16.6 ± 3.7%, respectively. However, the 24 h tryptic and chymotryptic hydrolysates did not exhibit any antibacterial activity against the gram-negative Escherichia coli and gram-positive Staphylococcus aureus. Therefore, the total soluble proteins of pistachio nuts were found to contain peptides exhibiting ACE-inhibition and antioxidative activities upon hydrolysis with trypsin and chymotrypsin.

Steady-state kinetics of tryptic hydrolysis of β-lactoglobulin after dynamic high-pressure microfluidization treatment in relation to antigenicity

Our previous research revealed that dynamic high-pressure microfluidization (DHPM) increased the antigenicity of β-lactoglobulin (β-Lg) below 80 MPa, which was related to the unfolding of protein. To test the hypothesis that the unfolding of protein may change proteolytic susceptibility of β-Lg and modulate its antigenicity during the digestion, we developed that the steady-state kinetics of tryptic hydrolysis of β-Lg subjected to DHPM (0.1–80 MPa) have been investigated in relation to the antigenicity in this study. According to the steady-state kinetics analysis, the improved digestion of β-Lg was accompanied with the obvious decrease of antigenicity during the hydrolysis with pressure increasing, reflected by the increase of k c , the decrease of K m, the increase of overall catalytic efficiency (k c/K m), and the increase of the binding volume. It was indicated that although DHPM can increase the antigenicity of β-Lg, the enhanced digestibility of β-Lg at elevated pressure contributed to a decrease of antigenicity during the hydrolysis.

Identification of a NFκB inhibitory peptide from tryptic β-casein hydrolysate

Several bioactive peptides are encrypted within the sequence of major milk proteins, requiring enzymatic proteolysis for release and activation. The present study aimed at the identification of potential anti-inflammatory activities in tryptic hydrolysates of bovine β-casein. Inflammatory processes involve in most cases an activation of Nuclear factor Kappa-light-chain enhancer of activated B cells (NFκB), which is a pro-inflammatory transcription factor of several genes. Hence, a NFκB reporter cell line was established, and TNF-α mediated activation of NFκB was used as a measurement. Bovine β-casein (β-CN) was hydrolysed by trypsin and fractionated by ultrafiltration. Total proteolysate as well as the fraction containing peptides between 1 and 5kDa showed an inhibitory effect in the cell-based assay, while the fraction containing molecules smaller than 1kDa did not. This anti-inflammatory effect was ascribed to a group of large, hydrophobic peptides, which were identified using LC–MS. The main peptide was synthesised and showed a significant anti-inflammatory effect in HEKnfkb-RE-cells. Thus, for the first time, a casein-derived peptide having an anti-inflammatory effect in vitro has been identified.

Vasorelaxant effects of camel and bovine casein hydrolysates in rat thoracic aorta and mesenteric artery

We evaluated the vasorelaxant effect of camel and bovine casein tryptic hydrolysates (CTH and BTH, respectively) and their mechanism of action in aorta and mesenteric artery of Wistar Kyoto rats. CTH produced a more potent relaxation than that induced by BTH. This relaxation was endothelium-dependent and was mediated through the activation of the nitric oxide (NO)/cyclic guanosine 3,5-monophosphate (cGMP) pathway. The intracellular level of cGMP was increased by CTH. Moreover, CTH-induced relaxation was inhibited by HOE 140, a B2 bradykinin receptors antagonist, indicating that B2 bradykinin receptors play a role in the CTH effect. In endothelium-intact mesenteric artery, CTH and BTH produced a similar concentration-dependent relaxation with a lesser maximal effect than in aortic rings. Moreover, this relaxation was inhibited by Nω nitro-l-arginine methyl ester hydrochloride, a NO synthase inhibitor, but the addition of indomethacin, a cyclooxygenase inhibitor, and tetraethylammonium, a K+ channels blocker did not potentiate the inhibition.

Selective enrichment of bioactive properties during ultrafiltration of a tryptic digest of β-lactoglobulin

Whey proteins are rich sources of bioactive peptides which may play a role in the dietary management of chronic diseases. Fractionation via ultrafiltration (UF) was investigated for the enrichment of antioxidant, dipeptidyl peptidase IV (DPP-IV) and angiotensin converting enzyme (ACE) inhibitory activity in a tryptic hydrolysate of β-lactoglobulin (β-Lg TH). UF processing selectively enhanced the biofunctional properties of β-Lg TH with the permeate obtained using a 1 kDa polyethersulfone (HFP-1) membrane having highest in vitro multifunctional bioactivity. Compared to β-Lg TH, the antioxidant activity was 1.7-fold higher (46,765 ± 2504 vs. 77,251 ± 5124 µmol Trolox equivalent/100 g dw; P < 0.05), DPP-IV half maximal inhibitory concentration (IC50) decreased threefold (1.6 ± 0.31 vs. 0.53 ± 0.05 mg/mL; P < 0.05) and there was a twofold reduction in ACE IC50 (0.131 ± 0.005 vs. 0.068 ± 0.018 mg/mL; P < 0.05). The multifunctional peptide VAGTWY, β-Lg f(15–20) was shown to have potent antioxidant activity (5.63 µmol TE/µmol peptide). Two new ACE inhibitory peptides, IIAEK and IPAVFK, having IC50 values of 63.7 ± 7.22 and 144.8 ± 25.3 μM, respectively, were also identified in β-Lg TH. This study demonstrates the multifunctional bioactive properties of a β-Lg TH and its associated UF fractions.

원양산 오징어(Illex argentinus) 간췌장 유래 Exopeptidase 분획물의 쓴맛개선 효과

Exopeptidase active fractions from the hepatopancreas of the Argentina shortfin squid Illex argentinus, were obtained with acetone (AC 30-40%), ammonium sulfate (AS 60-70% saturation), anion exchange chromatography (AE-II, 0.2 M NaCl) and gel filtration chromatography (GF-I, 30-50 kDa) fractionation methods. A bitter peptide solution that has a bitterness equivalent to that of 2% glycylphenylalanine and prepared by tryptic hydrolysis of milk casein, was treated with the exopeptidase active fractions. The GF-I fraction was the best based on aminopeptidase activity (35.3 U/mg), percentage of recovery (30.7%) and a sensory evaluation (1.7). The amount of released amino acids increased as incubation time increased, and the bitterness of the enzyme reaction mixtures decreased. Incubation with the GF-I fraction for 24 h resulted in the hydrolysis of several peptides as revealed by the reverse-phase high performance liguid chromatography profile, with three peaks (3, 5 and 6) decreasing in area (%) and three peaks (1, 2 and 4) increasing in area (%). Therefore, the GF-I fraction appeared to be ideally suited to reduce bitterness in protein hydrolysates by catalyzing the hydrolysis of bitter peptides.

Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography–tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard

A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3mg/100g and 1.0mg/100g, respectively. Bovine lactoferrin within the concentration range of 10–1000nmolL−1 showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD<6.5% and RSD<7.1%, respectively. Excellent repeatability (RSD<6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method was successfully validated and applied to determination of bovine lactoferrin in dairy products including infant formulas.

Fractionation of dairy based functional peptides using ion-exchange membrane adsorption chromatography and cross-flow electro membrane filtration

Two food grade fractionation processes using ion-exchange membrane adsorption chromatography (IE-MAC) and cross-flow electro membrane filtration (CFEMF) were successfully developed for the fractionation of complex peptide mixtures. Feed solutions for the fractionation steps were generated by tryptic hydrolysis of aqueous solutions of β-lactoglobulin (β-Lg) and micellar casein (MCN). The fractionation efficiency of both processes, to separate bioactive peptides was determined by analysing the yield of angiotensin-I-converting enzyme (ACE)-inhibitory peptides in the obtained fractions and by measuring their ACE-inhibitory activities in vitro. Both techniques were successfully applied for the fractionation of complex hydrolysates and the ACE-inhibitory peptides were enriched in individual fractions, resulting in yields of 52% for β-Lg(9–14) by IE-MAC and 25% for β-casein f(108–113) by CFEMF. Additionally, the ACE-inhibitory activity of the fractions was determined to be 3- to 6-fold higher compared with the hydrolysates.

Antioxidative effect of yak milk caseinates hydrolyzed with three differenct proteases

Aim: Yak milk is a type of milk that people are less familiar with due to its remote geographical location which may have significant effects on composition, microbiota and hydrolytic outcome. Present work was designed with the aim to evaluate the antioxidative effect of peptides derived from yak milk caseinate on hydrolysis with three different proteases. Materials and Methods: In this investigation Yak milk casein was hydrolyzed by three commercially available proteases (Trypsin, Pepsin and chymotrypsin). These hydrolysates collected at different hydrolysis times (30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, 270 min, 300 min, 330 min and 360 min) were assayed for their antioxidant activity with respect to the effect of incubation period. Results: Among all the enzyme hydrolysates, the tryptic hydrolysates showed highest antioxidant activity followed by chymotryptic hydrolysates. Further, the peptide samples showing highest activity were subjected to RP-HPLC for their partial characterization. Tryptic and peptic hydrolysates produced peaks mainly in the region of hydrophillic solvent indicating the presence of hydrophillic peptides/peptides. Conclusion: The results indicated that yak milk casein could be a resource to generate antioxidative peptides and be used as multifunctional active ingredients for many value-added functional foods as well as a traditional food protein.

Membrane fractionation of a β‐lactoglobulin tryptic digest: effect of the membrane characteristics

AbstractBACKGROUNDMembrane separation technologies seem to be one of the best available techniques to fractionate protein hydrolysates containing bioactive peptides and obtain peptide fractions with increased functionality. The influence of the molecular weight cut‐off (MWCO) of the membrane and its hydrophilic/hydrophobic characteristics on the fractionation of a β‐lactoglobulin tryptic hydrolysate were studied in this research work in order to obtain more purified peptidic fractions.RESULTSThe molecular weight of the solutes, their charge and, ultimately, their hydrophobicity influenced peptide transmission. As regards the composition of the active layer of the membrane, high permeate flux values were maintained throughout the pH working range (from pH 4.0 to pH 10.0) when an extremely hydrophilic membrane was employed, even in the presence of peptide aggregates at acidic pH values. In addition, the decrease in the MWCO of the membrane (from 5 kDa to 1 kDa) did not improve the selectivity of the fractionation process, however, it assumed a significant decrease of the permeate flux through the membrane.CONCLUSIONThe low efficiency of the process in terms of peptide recovery and the poor separation observed between bioactive and non‐bioactive peptides work against the use of extremely hydrophilic membranes to fractionate peptide hydrolysates. © 2013 Society of Chemical Industry

Effects of a peptide isolated from γ-casein hydrolysates on rat arterial blood pressure assay carried out under different anaesthetic drugs treatment

The action of the peptide YPVQPFTE, isolated from the tryptic hydrolysate of γ-casein, displays a bradykinin potentiating activity, characterized by the arterial blood pressure recording analysis. The intravenous injection of bradykinin (0.5μg) pre-treated with this peptide (30μg) was performed by chronic assay in conscious spontaneously hypertensive rats (SHR) and also by acute assay in normotensive Wistar rats, anaesthetized by three currently used anaesthetic drugs: sodium pentobarbitone (68mg/kg, i.p.); ketamine hydrochloride (25mg/kg, i.p.) associated with xylazine hydrochloride (10mg/kg, i.p.) and atropine (0,05mg/Kg, i.p.); and chloral hydrate (160mg/kg, i.p.). The peptide displayed bradykinin potentiating activity, by means that was not related to angiotensin converting enzyme (ACE) inhibition. The hypotensive areas were compared by two parameters, namely magnitude decrease (mmHg) and time course (min) to recover the pressure baseline values. In the first parameter, conscious SHR presented a greater decrease of the arterial blood pressure, followed by chloral hydrate and sodium pentobarbitone, ketamine–xylazine, which presented a small decrease. For the time parameter, the two depressants of the CNS presented the higher time to recover from the hypotension and ketamine–xylazine association resulted similar to conscious SHR group. The results demonstrated that the magnitude of the hypotension (mmHg) caused by the action of the vasoactive compounds was not directly altered by anaesthetic drugs even with compounds not related to ACE but the time to recover from this hypotension was significantly different depending on pharmacological mechanism proprieties of the anaesthetic agent used, suggesting that the main effects observed in this case is the failure to recover basal Mean Arterial Pressure (MAP) in the presence of depressant of the central nervous system.

Inhibition of Listeria monocytogenes growth in Cheddar cheese by an anionic peptides-enriched extract from whey proteins

The efficacy of an anionic peptides-enriched extract (APEE), produced by nanofiltration of a tryptic hydrolysate from whey proteins, to inhibit the growth of Listeria innocua and Listeria monocytogenes in reconstituted Cheddar cheese was studied. The antimicrobial activity of APEE in reconstituted cheese was greater against L. monocytogenes than L. innocua and was higher in storage at 30 °C than at 4 °C. The combination of 20 mg g−1 of APEE and 1.75% salt/moisture (S/M) in cheeses incubated for 7 days at 30 °C was the most efficient condition to inhibit the growth of Listeria. Using these conditions, L. monocytogenes counts were significantly reduced by 1.1 and 1.5 log cfu g−1, compared with cheeses without APEE and prepared with lactococci at 1.75 and 3.5% S/M, respectively. These results suggest that antimicrobial anionic peptides from whey proteins can contribute to control pathogen in reduced-salt Cheddar cheeses.

Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease involved in the degradation and inactivation of incretin hormones that act by stimulating glucose-dependent insulin secretion after meal ingestion. DPP-IV inhibitors have emerged as new and promising oral agents for the treatment of type 2 diabetes. The purpose of this study was to investigate the potential of β-lactoglobulin as natural source of DPP-IV inhibitory peptides. A whey protein concentrate rich in β-lactoglobulin was hydrolysed with trypsin and fractionated using a chromatographic separation at semipreparative scale. Two of the six collected fractions showed notable DPP-IV inhibitory activity. These fractions were analysed by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) to identify peptides responsible for the observed activity. The most potent fragment (IPAVF) corresponded to β-lactoglobulin f(78–82) which IC50 value was 44.7μM. The results suggest that peptides derived from β-lactoglobulin would be beneficial ingredients of foods against type 2 diabetes.

Antioxidant activity of bioactive peptides derived from bovine casein hydrolysate fractions

Bovine casein hydrolysates were prepared by enzymatic hydrolysis using trypsin alone and with a combination of trypsin and pepsin at different time intervals. Tryptic hydrolysate prepared at 4 h showed the highest antioxidant activity (13.7 %). Crude casein hydrolysate (CH) was further fractionated into 2 main types by using ultrafiltration with two different molecular weight cut-off at 10 kDa and 1 kDa. The crude hydrolysate was then compared with the collected permeates. Crude hydrolysates and the permeates, using trypsin showed radical scavenging effect by 13.7, 17.3 and 24.4 %, respectively. Furthermore, a reduced radical scavenging effect of 13.1, 15.5 and 19.6 % was observed by the combine action of pepsin and trypsin. Ultrafiltration fractions and crude hydrolysate showed the radical scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) by 1 kDa > 10 kDa > crude hydrolysate. The combined effect of trypsin and pepsin did not prove to be effective in radical scavenging. However, fractionation by ultrafiltration proved to be effective in radical scavenging. Bioactive peptides < 1 kDa possess the potential to be used in physiologically functional foods or pharmaceutical industries.

Effects of Enteric-coated Lactoferrin Tablets Containing Lactobacillus brevis subsp. coagulans on Fecal Properties, Defecation Frequency and Intestinal Microbiota of Japanese Women with a Tendency for Constipation: a Randomized Placebo-controlled Crossover Study.

The effects of oral administration of enteric-coated tablets containing lactoferrin (LF; 100 mg/tablet) and heat-killed Lactobacillus brevis subsp. coagulans FREM BP-4693 (LB; 6×109 bacteria/tablet) on fecal properties were examined in 32 Japanese women (20–60 years of age) with a tendency for constipation (defecation frequency at equal to or less than 10 times/2 weeks) by a double-blind placebo-controlled crossover design. A significant increase in defecation days per week was obserbed in the subjects who ingested the tablets containing LF and LB compared with the placebo group. The number of bifidobacteria in feces also significantly increased compared with the placebo group. In an in vitro study, LF and tryptic hydrolysate of LF, but not peptic hydrolysate of LF, upregulated the growth of Bifidobacterium longum ATCC15707 when added to the culture. These results demonstrate the capability of the enteric-coated tablets containing LF and LB in improving intestinal function and suggest that they have a growth promoting function for bifidobacteria.

Antihypertensive Property of Yak Milk Caseinates Hydrolyzed with Different Proteases

Yak milk is a type of milk that people are less familiar with due to its remote geographical location. Among the bioactive peptides derived from milk proteins, those with blood pressure-lowering effects are receiving special attention due to the prevalence and importance of hypertension in the world population. In the present work antihypertensive activity of yak caseinate studied after treatment with different enzymes and the effect of incubation period on this activity has been further studied. Degree of hydrolysis goes on increasing with incubation period. Maximum hydrolysis obtained with enzyme pepsin followed by chymotrypsin and trypsin. Tryptic hydrolysates showed highest antihypertensive activity followed by peptic and chymotryptic hydrolysates. Activity increases with the increase in time of incubation in case of all the enzyme hydrolysates. Thus, it could be concluded that bioactive peptides from the milk of yak possesses good antihypertensive activity which has useful implications in food science & nutrition.

Ultrafiltration of whey buttermilk to obtain a phospholipid concentrate

Concentrates of milk phospholipids (PLs) were extracted from whey buttermilk. The whey buttermilk was centrifuged and deproteinised before ultrafiltration (UF) using peptic or tryptic hydrolysis. UF was carried out using membranes with molecular mass cut-off limits of 30, 50, 100, and 300 kDa. A degree of hydrolysis of >2% was sufficient to separate the whey proteins from the lipids by means of UF. Protein hydrolysis resulted in an increase in PL concentration in the dried powder of about 2.5%. The purity of the PL was enhanced with increasing molecular mass cut-off limits of the UF membranes and with increasing filtration temperatures up to 40 °C. A transmembrane pressure of 1.5–2.0 bar was optimal for both the purity of PLs and the permeation flux. After a four-step diafiltration the final powder contained up to 14.0% PL and the total recovery of PLs after UF and DF was 95–99%.