Developments in downstream processing of whey for new products. Prof. Dr.-Ing. Ulrich Kulozik

Transcription

1 Developments in downstream processing of whey for new products Prof. Dr.-Ing. Ulrich Kulozik Technische Universität München Institute for and Dairy Technology Freising-Weihenstephan, Germany

2 Protein composition of milk Milk protein 100 % Casein ca. 80 % Whey protein ca. 20 % α-casein ca. 40 % β-casein ca. 30 % κ-casein ca. 10 % α-lactalbumin ca. 4 % β-lactoglobulin ca. 10 % immunoglobulins serum albumin proteose peptone ca. 6 % para-κ-casein ca. 6 % CMP ca. 4 % Motivation peptides Physiological effects Therapeutic effects Technological functions

3 Release of CMP during renneting k-casein Pyg 1 Phe 105 -Met 106 Val 169 Chymosin Milk: 3,3 4,5 g/l κ-casein 1,2 2,5 g/l CMP CMP Pyg 1 Phe 105 para-k-casein + Met 106 Val 169 CMP Chymosin Casein micelle CMP Whey "para-casein micelle" 15-20% of the total protein content is CMP, the other 85 % are whey proteins Aggregation of the casein micelles via Ca 2+ -bridges Green Cheese

4 Amino acid sequence of Caseinmacropeptide [CMP var. A (var. B)] Met - Ala - Ile - Pro - Pro - Lys - Lys - Asn - Gln - Asp - Lys - Glu - Ile - Pro Ile - Asn - Ile - Ala - Ser - Gly - Glu - Pro - Ser - Pro - Thr (Ile) -Glu - Ala - Val - 35 Glu - Ser - Val - Ala - 40 Leu - Glu - Asp (Ala) -Ser- 45 Pro Glu - Val - Ile - Glu - Ser - Pro - Pro - Glu - Ile - Asn - Val - Gln - Val - 64 Ser - Ala - Val. genetic var. A and B essential amino acid branched-chain amino acid - High amount of essential amino acids - High amount of branched-chain amino acids - Absence of aromatic amino acids (Phe, Tyr, Trp) acceptable protein source for PKU patients

5 Glycosylation sites and types of CMP var. A (var. B) NeuNAc 1 Met - Ala - Ile - Ile - Asn- Pro - 5 Pro - 20 Ile - Lys - NeuNAc Gal Ala - Lys - Ser - Asn - Gly - Gln - Glu - 10 Asp - Gal Lys - GalNAc 25 Pro - Ser - Glu - Ile - NeuNAc Pro - 15 Gal Pro - GalNAc 30 Thr (Ile) -Glu - Ala - NeuNAc GalNAc 35 Val - Glu - Ser - Val - Ala - 40 Leu - Glu - GalNAc Asp (Ala) Gal -Ser- 45 Pro - Glu - Val - Ile - Glu Ser - GalNAc Pro - Pro - Gal Glu - Ile - 55 Asn - Val - Gln - Val - 60 Ser - Ala - Val. Gal NeuNAc GalNAc 50 % of CMP unglycosylated 50 % of CMP glycosylated (GMP) Heatstablein comparisonto wheyproteins Galactose Sialic acid N-Acetylgalactosamine Site of saccharide attachment variations in: point of attachment type degree of glycosyltn.

6 Osteopontin 3-10 mg/l Milk protein 100 % Laktokinine Albutensin A Casein 80 % α-lactorphin β-lactorphin Serorphin Immunoglobulin D (IgD) α S1 -Casein g/l Casecidin Isradicin α S1 -Casein Exorphin α-casein g/l α S2 -Casein 3-4 g/l Phosphopeptide CPP β-cn-4p α S1 -CN-5P Casokinine Casocidin-I Angiotensin-1-Converting Enzyme Inhibitor-Peptide (ACEI) Oxidoreduktasen Xanthin Oxidase XO Lactoperoxidase LP 30 mg/l β 2 -Microglobulin (Lactollin) 9,5 mg/l Glutathion Peroxidase GPX Superoxid Dismutase SOD Catalase Enzyme β-casein 9-11 g/l Transferasen Hydrolasen (Alkalische Phosphatase) Lyasen Isomerasen Ligasen β-casomorphine β-cm-7 β-cm-9 β-cm-13 β-cm-21 β-neocasomorphin -6 Morphiceptin Proteose Pepton 5 β-cn-5p Proteose Pepton 8f β-cn-4p Proteose Pepton 8s β-cn-1p Aglyco-CMP (acmp) ca. 50 % Gonadotropinreleasing P Casoxin A,B,C Casecidin κ-casein 2-4 g/l Proteose Pepton 3 (Lactophorin) HFPP (300 mg/l) Hydrophobic fraction of proteose peptones Gesamtanteil: 1,2 g/l Gastrinreleasing P. Somatostatin Insulin EGF ~ 2 ng/ml Caseinomakropeptid (CMP) ~ 1,5 g/l Casoplatelin Casopiastrin para-κ-casein ~ 2 g/l Hormone Wachstumsfaktoren Lysozym 0,3 mg/l α-lactalbumin 0,6-1,7 g/l Caseinomacropeptide ca. 4 % of the total milk protein ca. 4 % des Gesamtproteins von Milch IGF-I 30 ng/ml IGF-II 75 ng/ml Pareathyroid- Hormon Motilin TGF-α TGF-β NGF β-lactoglobulin 2-4 g/l Bradykinin Serumalbumin 0,4 g/l Hochmolekulares Kin Kininogen Immunoglobuline ~ 0,7 g/l Niedermolekulares Kin Metall bindende Proteine Wheyproteins Transferrin Lactoferrin GMP 20 % (soluble or at phglyco-cmp 4.6) (gcmp) ~ 100 mg/l ca. 50 % Lactoferricin B Lactoferroxine Ceruloplasmin (Ferroxidase) Angiogenine Immunoglobulin E (IgE) Immunoglobulin M (IgM) 90 mg/l Immunoglobulin A (IgA) 10 mg/l Immunoglobulin G2 (IgG2) 50 mg/l Immunoglobulin G1 (IgG1) mg/l Angiogenin mg/l Angiogenin 2 PAS-1 Sekretkomponente (SC) mg/l PAS-3 Glycolactin PAS-4 Minore Glykoproteine Vitamin bindende Proteine Butyrophilin Milk fatglobule membrane = Source/Group BRCA-1 = Protein = Peptid = Glykoprotein MUC-1 CD-36 Lactogenin M-1 Orcosomucoid (α 1 -Säure GP) ~ 20 mg/l Betacellulin Prosaposin 6 mg/l restl. Mucine Riboflavin bp Folat bp 6-10 mg/l Vitamin D bp 16 mg/l Vitamin B 12 bp (Transcobalamin ) 0,1-0,2 mg/l by Markus Kreuß & Nadja Siegert 2006

7 Methods to obtain CMP Skim milk MF-Diafiltration Casein retentate Labfällung Renneting (Chymosin) Casein coagulum Permeate CMP-freie Molke CMP-containing whey UF Water MF Casein particles Conventional cheese making Wheyproteins UF UF-Permeat Rennet whey MF Ion exchange Chromatography CMPconcentrate UF- Diafiltration Glyco-CMP Aglyco-CMP Ionen- Chromatographie Fractionation by IEC

8 Isoelectric points of whey proteins and CMP Glyco-CMP CMP α-la BSA β-lg + Net-charge protein ph - Isoelectric properties of whey proteins (Source: Swiss Protein Database / European Protein Database)

9 Schematic of the experimental pilot plant set-up Circulation loop loading Buffer 2 Buffer 1 CMPsolution Eluate

10 Membrane Adsorber (MAC) IEX Column in comparison 0,0 0,5 1,0 1,5 2, TIme (min) MAC 2000 Adsorber UV Absorption 218 nm IEX-column Time (min) IEX column Variation Phase

11 MAC process based on CMP solution Permeate (Salt) Glyco Glyko-CMP CMP 10 kda UF-Diafiltration Direct Direct-Capture Capture MAC Membranadsorber Wasser Pure Reine Fractions Fraktionen (Concentrate) (Konzentrat) Rennet whey Erhitzung Heat treatment Aglyco CMP Aglyko-CMP + whey Molkenproteine proteins Erhitzung Heat treatment 10 kda UF-Diafiltration Whey proteins aggregated Whey proteins aggregated Permeate(Salt) Key data Purity eluate: 91 % Flow rate: 8 L/min Capacity: 7,5 g glyco-cmp/cycle = 0,21 mg/cm² Capacity loss from cycle to cycle

12 Whey proteins and their thermal reactivity β-lg ΔT SH SHSH unfolding S H SH SH SH Extraction of native α- La/denaturated ß-Lg α-la aggregation Extraction of native ß- Lg/denaturated α-la Some influencing factors Protein content ph Lactose content Temperature Calcium content Treatment time

13 Heat induced aggregation of α-lactalbumin and β-lactoglobulin α-lactalbumin, n = 1 β-lactoglobulin, n = 1, Geschwindigkeitskons tante [s ] C -1 1-n Ges chwindig keitsko nstante [(gl ) s ] C ,5 1,5 Lactosegehalt [%] 2,5 3,5 4,5 5,5 0,5 1,5 2,5 3,5 4,5 Proteingehalt [%] 5,5 0 1 Lactosegehalt [%] Proteingeha lt [%]

14 Co-aggregates: Effect of temperature on the particulates serum binding capacity

15 Ice cream: Assessment of the melt behaviour t = 0 min Ice cream samples with different whey protein supplements: Melt down characteristics at ambient temperature initial state t = 60 min t = 60 min Addition of whey powder Addition of whey protein aggregates (Koxholt 2000)

16 Manufacture of pure, native α-la in conjunction with ß-Lg aggregates Whey UF/Diafiltration 3,5% Protein Heat treatment 93 C/20 s; ph 7,5 5 0 native heated α-la β-lg B β-lg A ph adjustment min MF 0.1 µm Retentate denatured β-lg Permeate native α-la

17 Manufacture of pure, native ß-Lg in conjunction with α-la aggregates Wheyor WPC UF-Diafiltration 6 % ß-Lg, citrate Heat treatment 56 C/40 min; ph 3,6 native heated Microfiltration 0.1 µm Retentate denatured α-la Permeate native ß-Lg RO of UFpermeate Diafiltration (UF)

18 Process to obtain CMP depleted rennet whey WPC Treatment with transglutaminase TG-ase Gln-C-NH-Lys O= UF permeate MF, 0.14 µm UF, 25 kda MF permeate Crosslinked CMP and remaining casein curd MF retentate WPC w/o CMP UF retentate Fractionation of whey proteins

19 Process to obtain CMP depleted rennet whey by means of enzymatic crosslinking (Transglutaminase) Remaining CMP [%] WPC c pr = 6 % [TG]: 3 U/g protein ϑ =40 C Incubation time [min] Glycosylated CMP CMP A untreated 10 min 120 min CMP B

20 Microcapsulation of sensitive components using casein or whey protein as matrix material Caseinate Proteins Probiotic Cells Transglutaminase Formation of a water-in-oil- Emulsion Water Mix Oil Gelation by means of cross-linking Emulsifying at 40 C Separation of the microcapsules via centrifugation Developement of isopeptide bonds

21 Microcapsulation of sensitive components using whey proteins as matrix material Protein (Hydrocolloids) Fruit extract Water insoluble microcapsules Size impact on sensory and protective effects Heat induced gel network

22 Novel whey protein based nanotubes made from a-lactalbumin (Left) Schematic presentation of the self-assembly of partially hydrolysed a-lactalbumin into nanotubes in presence of Ca 2+. (Right) Transmission electron micrograph of negatively stained a-lactalbumin nanotubes (negative staining was performed with 3% uranyl acetate for 1 min) J.F. Graveland-Bikker, C.G. de Kruif Trends in Food Science Technology 17 (2006)

23 Approach for production of bioactive peptides Precursor protein selection -Single vs. mixed -Pre-treated or native -Glyco-conjugated Production Purification/ Fractionation Enzyme selection: -single or mixed enzymes -enzyme cascades Desalting, MAC, HPLC, GPC, UF, NF membranes Analysis Elucidate structure-function relationships, use of quantitative structureactivity relationships (QSAR) Techno-functional/ sensory properties Interfacial activity, Network formation Fat-holding Bitterness Bioactivity In vitro or In vivo Cell cultures Toxicological tests Human nutrition group Structure analysis MALDI-TOF-MS, ESI Peptide synthesis, etc. Structure-Function-Relationships (?)

24 Contact: TUM campus at Freising-Weihenstephan

25 Bioactive components in Milk - Target compounds Proteins -Whey proteins + α-lactalbumin, ß-Lactoglobulin + Peptides -Casein fractions + Peptides (Hormone activity, mineral binding capacity, immune system modulation, etc.) + Glycomacropeptide Fat globule membrane components -Phospholipoproteins -Sphingomyelins Minor Components -Minerals (Calcium) -Lactoferrin -Prolactin -Lactulose -Oligosaccharides Motivation Physiological effects Therapeutic effects Technological functions Functional Products

26 Ways to produce bioactive peptides Protein Hydrolysis by digestive enzymes Fermentation bymeans of proteolytic microbial cultures Lactic acid bacteria (L. lactis; L. helveticus; L.delbrueckii) Release of peptides as fragments of the protein Proteolysis by means of isolated microbial or plant derived enzymes Peptide mix Fractionation and concentration

27 Solid phase alternatives Conventional gels Membrane Adsorption Chromatography (Lab scale units) Polymer beads Diffusion is rate limiting High backpressure Cellulosic membrane Convective flow of target component to adsorber surface Low backpressure

28 MAC pilot plant unit Adsorber hold-up volume: 1000 ml Flow rate: 8 L/min

29 MAC process based on CMP solution Permeate (Salt) CMP CMP-Konzentrat solution Glyco Glyko-CMP CMP 10 kda UF-Diafiltration Direct Direct-Capture Capture MAC Membranadsorber Water Pure Reine Fractions Fraktionen (Concentrate) (Konzentrat) Aglyco CMP Aglyko-CMP + whey Molkenproteine proteins Erhitzung Heat treatment 10 kda UF-Diafiltration Key data Purity eluate: 97 % Flow rate: 8 L/min Whey proteins aggregated Permeate(Salt) Capacity: 10,2 g glyco-cmp/cycle = 0,28 mg/cm² No capacity loss after 20 cycles observed

30 IEC Analytical method development Selection of solid phase -Strong, weak exchangers (Q, DEAE) -conventional bead gels, membrane adsorbers Selection of liquid buffer -Citrate, Acetate, Methyl-Piperazin -Molarity of buffer (0,01 M to 1 M) ph -Variation depending on buffer between ph 3 and ph 6 Dual buffer gradient Flow rate Protein concentration

31 Wheyprotein micro-aggregate size under various thermal and compositional conditions 100 Aggregate size d 50,3 [µm] UF-whey concentrate phase separation 10% total protein; ph 6.7 SSHE: 1200 min -1 DD : > 0.95 β-lg lactose content: 1.5% 13.5% Temperature [ C] sandy very mealy slightly mealy smooth

32 Milk protein fractionation by means of microfiltration (linear scale) Skim milk MF-permeate MF/Diafiltration Casein micelles UFpermeate Ultrafiltration Whey proteins

33 Milk protein fractionation by means of microfiltration (log scale) Amount of whey protein to be washed out Diafiltration step [-]

34 Influence of ph on size and permeation of ß-Lg Permeation ß-Lg [%] ,9 nm 3,6 nm 6,9 nm 10 3,6 nm ph

35 Critical filtration rate as a function of particle size forming membrane deposits w = w + krit Diff w hydro 10 4 Filtration rate [l/m²h] Sum Hydrodynamics Diffusion Casein micelles Particle size [m] Grein; MVT Kaiserslautern 2006

36 Deckschichtbildung: Grundlagen Überströmgeschwindigkeit Diffusion Liftkraft F L F L F y 1,5 ~ τ w ~ v ( TMP) y Schleppkraft F Y =0 Filtratstrom Altmann 2000