Controlling the aggregation and gelation of ß-lactoglobulin by the addition of its peptides
Kosters, H.A. - \ 2012
Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Peter Wierenga. - S.l. : s.n. - ISBN 9789461732040 - 171
bèta-lactoglobuline - aggregatie - gelering - peptiden - eiwithydrolysaten - beta-lactoglobulin - aggregation - gelation - peptides - protein hydrolysates
In this thesis the effects of peptides, or protein hydrolysates on the heat-induced aggregation and gelation of (concentrated) protein systems were studied. First, it was investigated if specific peptides could influence the heat-induced denaturation and aggregation of intact proteins solutions, and which peptide properties dominated the different interactions. Next, the effects of the peptides on the heat-induced gelation of intact proteins as a model for a potential high protein food system were studied.
It was found that certain peptides in the hydrolysate show binding to native proteins, and some additional peptides bind to unfolded proteins. Since the same peptides were shown to bind to not only β-lactoglobulin, but also other to proteins, it is concluded that the binding does not depend on specific molecular details of the protein. The hydrophobicity and charge were found to be important in determining the binding and the effect on aggregation. With the hydrolysates, as well as with two synthesized peptides (modelled on those found in the hydrolysate) it was confirmed that the addition of these binding peptides has significant effects on the heat-induced aggregation. In the gelation experiments performed in this study a dominant effect was found for peptides containing free SH groups. While it is expected that the changes in aggregation behaviour, induced by the binding of non-cysteine-containing peptides also affects the gel properties, this was not found with the techniques used. Finally, disulfide-containing peptides were found to reduce the presence of sulfurous volatiles formed after heating of β-lactoglobulin, WPI and lysozyme.
Since only certain peptides exhibit binding to intact proteins, it is expected that control over the hydrolysis process and, thereby the concentrations of such specific peptides, can be used to produce hydrolysates with specific functionalities in this respect.
Review of the potential health impact of ß-casomorphins and related peptides: Report of the DATEX Working Group on ß-casomorphins
Noni, I. De; FitzGerald, R.J. ; Korhonen, H.J.T. ; Roux, Y. Le; Livesey, C.T. ; Thorsdottir, I. ; Tomé, D. ; Witkamp, R.F. - \ 2009
Brussel : European Food Safety Authority (EFSA Scientific report / European Food Safety Authority 231) - 107
caseïnehydrolysaat - peptiden - nadelige gevolgen - casein hydrolysate - peptides - adverse effects
Characterization of genes coding for small hypervariable peptides in Globodera rostochiensis
Bers, N.E.M. van - \ 2008
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - [S.l.] : S.n. - ISBN 9789085049579 - 229
globodera rostochiensis - plantenparasitaire nematoden - peptiden - genen - genexpressie - solanum tuberosum - arabidopsis - nicotiana - gensplitsing - gastheer-pathogeen interacties - moleculaire interacties - globodera rostochiensis - plant parasitic nematodes - peptides - genes - gene expression - solanum tuberosum - arabidopsis - nicotiana - gene splicing - host pathogen interactions - molecular interactions
Plant parasitic nematodes secrete a cocktail of effector molecules, which are involved
in several aspects of the interaction with the host, eg. in host defense suppression, in
migration and in feeding cell formation. In this thesis, we performed the first study on
10 novel peptide genes, believed to be important for parasitism of the potato cyst
nematode, Globodera rostochiensis. Nine of the peptide genes described here belong
to the SECPEP gene family. The SECPEP genes are all expressed in the dorsal
esophageal gland, which is one of the main sites for the production of effector
molecules. This, together with the predominant expression in preparasitic and early
parasitic juvenile nematodes, makes it very likely that the SECPEPs code for effector
peptides essential for succesful infection and feeding site formation.
In chapter 2, we show that diversifying selection is a likely driver of the molecular
evolution of the SECPEPs. The sequences of the mature peptides appear to be highly
diverse, while the non)coding 3’UTR and intronic regions as well as the region coding
for the signal peptide for secretion are relatively conserved. In fact, a pairwise
comparison of the SECPEPs reveals no significant sequence similarity between family
members at all. In chapter 5 we further speculate on a possible role for RNA)editing as
a mechanism to yield hypervariability in the SECPEPs, because the sequence diversity
at the transcript level significantly exceeds that of the genomic locus. Chapter 5 further
elaborates on the analysis of trans)splicing in SECPEP1 transcripts. We show that
SECPEP1 transcripts are trans)spliced to a surprising diversity of novel spliced)leader
sequences. The first approach to unravel the role of the members of the SECPEP family
in plant parasitism, is described in chapter 4. We generated transgenic potato and
Arabidopsis plants expressing SECPEP3 while using the CaMV 35S promotor. The
phenotype associated with SECPEP3 in both potato and Arabidopsis plants includes a
reduction of root growth and an alteration of the leaf morphology. The SECPEP3
peptide harbors several sequence motifs first found in the cyclin)dependent kinase
inhibitors ICK1/KRP1, SIM and Smr1. We, therefore, suggest a role for SECPEP3 in cell
cycle alteration in nematode feeding site formation. Although the SECPEP genes show
only a low level of primary sequence similarity, all code for positively charged,
hydrophilic peptides with a C)x)G γ)core motif (chapter 2). These are characteristics
typical for host defense peptides, and in chapter 6 we investigate whether these
characteristics are also found for other peptides involved in plant)parasite interactions.
We show that a considerable number of these effector peptides share a positive
charge, hydrophilicity and C)x)G γ)core motif with the SECPEPs, and we speculate on a
role for the positive charge in peptide)ligand interaction.
In chapter 3 we describe the NEMPEP peptide, secreted by G. rostochiensis. NEMPEP
is also a positively charged, hydrophilic peptide with a C)x)G γ)core motif, although it is
genetically unrelated to the SECPEP gene family. During the life cycle of G.
rostochiensis, the expression pattern of NEMPEP reveals a striking regulation. NEMPEP
is highly expressed in preparasitic juveniles and in the parasitic life stages after initial
feeding cell formation. However, NEMPEP expression was hardly detectable in the
juveniles just after entering the plant root. Several disease resistance genes condition
nematode resistance at the onset of parasitism. The downregulation of NEMPEP at
exactly this timepoint could be a strategy to avoid recognition by the host’s immune
system. In planta expression of NEMPEP, as a fusion to GFP, shows that NEMPEP
accumulates in the nucleolus of tobacco cells. Potato plants transformed with
35S::NEMPEP were slow at forming roots and the internodes between the leaflets were
shortened. This, together with a reduced transformation efficiency, led us to
hypothesize a role for NEMPEP in cytokinin signaling (Chapter 3).
Currently, there are two models regarding the functional role of the SECPEPs and
NEMPEP. The first one concerns a role as an antimicrobial peptide, which could protect
the host plant against secondary infections by opportunistic microbes. As a competing
hypothesis, the high hydrophilicity of the peptides may point to a role as peptide
hormone. As such, they may be involved in redirecting cell cycle or hormonal regulation
upon feeding cell formation.
Peptides as inhibitors of lipoxygenase and tyrosinase
Minderhout-Schurink, M. - \ 2007
Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Willem van Berkel; Carmen Boeriu. - [S.l.] : S.n. - ISBN 9789085048459 - 144
lipoxygenase - tyrosine 3-monooxygenase - enzymremmers - peptiden - voedselkwaliteit - oxidatie - lipoxygenase - tyrosine 3-monooxygenase - enzyme inhibitors - peptides - food quality - oxidation
Oxidation reactions catalyzed by enzymes such as lipoxygenase (LOX) and tyrosinase (TYR) initiate food quality decay. Besides their physiological role in the human body, LOX and TYR are involved in certain types of cancer, neurodegenerative diseases and processes of aging. Most common antioxidants able to retard these oxidations, function by scavenging free radicals or by reducing oxidation products formed by these enzymes. The enzyme activity, which is the cause of the formation of these oxidized compounds, remains unaffected. In addition, most of the conventional oxidative enzyme inhibitors are synthetic, unstable, difficult to obtain and cannot be used in food products, cosmetics or medicines from viewpoints of safety and economics. Hydrolyzed proteins from animal and plant sources have been found to possess antioxidant activity. They have a potential to be used as alternative, natural antioxidants. In the research described in this thesis a novel approach was chosen in order to identify protein-based inhibitors for soybean LOX and mushroom TYR. SPOT synthesis was used to synthesize cellulose-bound peptide libraries containing overlapping peptides derived from proteins originating from different industrial sources such as milk, egg, soy, or wheat. Screening of these libraries with a fluorescent labeled LOX or TYR resulted in a set of peptides that specifically bind to these enzymes. The presence of positively charged residues within the peptide sequence appears to be important for interaction with LOX. Preparative synthesis of some binding peptides and subsequent inhibition assays confirmed a true, noncompetitive inhibition of LOX by the octapeptide RINKKIEK from the milk protein b‑casein. A substitutional analysis showed that replacement of the glutamic acid residue in RINKKIEK significantly improves binding and inhibition of LOX. The molecular determinants for TYR-inhibiting peptides are different from LOX-inhibiting peptides. Strong TYR-binding peptides always contain one or more arginine residues, often in combination with phenylalanine. Furthermore, the presence of valine, alanine and/or leucine contributes to TYR inhibition. An example of a good TYR-binding and -inhibiting peptide is LFRVASMA from egg ovalbumin, which comprises a combination of these amino acid residues. In conclusion, several inhibitory peptides were identified with potencies comparable to nonpeptidic inhibitors. However, further experiments are required in order to assess the applicability of these antioxidant peptides.
Aggregation of peptides in soy protein isolate hydrolysates : the individual contributions of glycinin- and ß-conglycinin-derived peptides
Kuipers, B.J.H. - \ 2007
Wageningen University. Promotor(en): Harry Gruppen. - [S.l.] : S.n. - ISBN 9789085046097 - 160
sojaeiwit - gelering - peptiden - eiwithydrolysaten - soya protein - gelation - peptides - protein hydrolysates
Keywords: Soy proteins, glycinin, β-conglycinin, enzymatic hydrolysis, peptides, aggregation, gelation, identification, mass-spectrometry, mappingThe aim of the work presented in this thesis is to understand howlimited enzymatic hydrolysis can influence the aggregation behavior of soy protein material. This is performed by uncoupling the enzymatic degradation and the aggregation process. Subsequently, the regions in soy proteins from which the aggregating peptides originate were identified.Hydrolysates of soy protein isolates (SPI) were prepared using the protease subtilisin Carlsberg. The enzyme was inhibited when the desired degree of hydrolysis (DH) was reached. It was shown that with increasing DH, the pH at which a gel could be formed was increasing as well. The aggregation behavior of SPI, glycinin- and β-conglycinin-derived hydrolysates showed that glycinin-derived peptides are responsible for the aggregation of SPI-derived peptides. This was also found when glycinin was hydrolyzed with other enzymes (bromelain, papain, and chymotrypsin) using SPI, β-conglycinin, and bovine whey proteins as reference materials. Only hydrolysis of glycinin by trypsin did not result in strong aggregating peptides.Subsequently, a new method was developed, denoted accumulative-quantitative-peptide-mapping. This method reveals those regions in the parental protein from which the aggregating peptides originate. This method comprises a second hydrolysis of the aggregating peptides, followed by separation, identification, and quantification of individual peptides obtained. Quantification was performed based on absorbance at 214 nm, corrected for the calculated molar extinction coefficient based on the amino acid composition. This novel method revealed that mainly the basic polypeptide and that part of the acidic polypeptide close to the location of the disulfide bridge connecting the basic to the acidicpolypeptide,are the predominant regions of glycinin yielding aggregating peptides. These regions have a relative high hydrophobicity compared to that of the total protein. Upon hydrolysis the net hydrophobicity of the remaining glycinin is increasing, eventually resulting in aggregation and subsequent gelation.
Enzyme-induced aggregation of whey proteins with Bacillus licheniformis protease
Creusot, N.P. - \ 2006
Wageningen University. Promotor(en): Harry Gruppen. - [S.l.] : S.n. - ISBN 9789085045014 - 128
wei-eiwit - aggregatie - proteïnasen - peptiden - interacties - bacillus licheniformis - whey protein - aggregation - proteinases - peptides - interactions - bacillus licheniformis
Whey proteins are commonly used as ingredient in food. In relation with the gelation properties of whey proteins, this thesis deals with understanding the mechanism of peptide-induced aggregation of whey protein hydrolysates made with Bacillus licheniformis protease (BLP). The results show that BLP breaks down hydrophilic segments in the substrate and, therefore, preserves hydrophobic segments that aggregate once exposed to the solvent. Aggregation during hydrolysis prevented further degradation of the substrate, explaining that aggregating peptides are larger than the non-aggregating ones. Solubility experiments performed on fractionated aggregating peptides showed that peptide co-aggregation is an important factor in the aggregation process. Results also showed that the hydrolysates are able to aggregate added parental protein. The aggregating peptides could form a network in which the presence of both insoluble and partly insoluble peptides was required for the aggregation of intact protein.
Limiting factors for the enzymatic accessibility of soybean protein
Fischer, M. - \ 2006
Wageningen University. Promotor(en): Harry Gruppen; Fons Voragen. - [S.l.] : S.n. - ISBN 9789085044963 - 139
sojaeiwit - hydrolyse - aggregatie - koolhydraten - eiwitextractie - eiwitvertering - eiwitverteerbaarheid - peptiden - soya protein - hydrolysis - aggregation - carbohydrates - protein extraction - protein digestion - protein digestibility - peptides
Soy is a commonly used ingredient is food and animal feed. With particular focus on the in-soluble fractions, this thesis deals with the effects of proteases and carbohydrate degrading enzymes on different soybean meals subjected to different extent of heating. The primary aim is to improve the understanding of enzymatic hydrolysis of SBM with emphasis on proteins and to identify barriers limiting the efficiency of the process. The results show that aggregation behavior of peptides during enzymatic processing of soy proteins is potentially a limiting factor for efficacy of protein extraction. Surprisingly, it is also demonstrated that aggregation is not limited to in vitro incubations, but is also occurring in vivo in the digestive system of pigs.
Biochemical and functional characterisation of casein and whey protein hydrolysates : a study on the correlations between biochemical and functional properties using multivariate data analysis
Ven, C. van der - \ 2002
Wageningen University. Promotor(en): A.G.J. Voragen; H. Gruppen; D.B.A. de Bont. - S.l. : S.n. - ISBN 9789058086532 - 155
melkeiwitten - caseïnehydrolysaat - wei-eiwit - eiwithydrolysaten - peptiden - multivariate analyse - vloeistofchromatografie met omgekeerde fase - gelfiltratiechromatografie - infraroodspectroscopie - molecuulgewicht - emulsies - schuim - oplosbaarheid - bitterheid - milk proteins - casein hydrolysate - whey protein - protein hydrolysates - peptides - multivariate analysis - reverse phase liquid chromatography - gel filtration chromatography - infrared spectroscopy - molecular weight - emulsions - foams - solubility - bitterness
Whey protein and sodium caseinate were hydrolysed with commercially available enzyme preparations. The resulting hydrolysates were characterised using several analytical characterisation methods and by determination of several functional properties. Subsequently, correlations between the biochemical characteristics themselves and between biochemical and functional properties were studied using multivariate regression analysis.
Biochemical characteristics of hydrolysates were determined using unifactorial methods like the degree of hydrolysis, and by multifactorial methods, i.e . reversed phase (RPC) and size exclusion chromatography (SEC), and Fourier transform infrared (FTIR) spectroscopy. FTIR spectroscopy appeared to discriminate most effectively between hydrolysates made from different protein sources and classes of proteolytic enzymes, followed by RPC and SEC.
Emulsion and foam properties of hydrolysates were similar or inferior to those of the parental proteins. Casein hydrolysates generally showed better emulsion and foam forming ability than whey protein hydrolysates. Foam forming ability of whey protein hydrolysates was correlated to the molecular weight distribution (MWD) of the peptides, showing that especially peptides with MW of 3-5 kDa contributed to foam forming ability.
Concerning prevention of emulsion instability due to coalescence it was shown that peptides with a molecular weight larger than 2 kDa are needed. Foam stabilising ability of casein hydrolysates also depended on the MWD of hydrolysates, but higher molecular weight peptides, i.e. larger than 7 kDa, were needed to obtain good foam stability.
The ability of the three multifactorial characterisation methods (SEC, RPC, FTIR spectroscopy) to predict functional properties was investigated. It appeared that SEC profiles were able to predict emulsion and foam stability of all hydrolysates, as well as foam forming ability, Angiotensin Converting Enzyme (ACE) inhibiting ability and bitterness of whey hydrolysates. RPC profiles were also able to predict these properties and additionally predicted solubility and bitterness of casein hydrolysates. FTIR spectra were best suited to predict a variety of hydrolysate properties, since apart from the before-mentioned properties, the spectra can also be used to predict emulsion forming ability and to improve prediction of bitterness of hydrolysates.
Finally, the influence of hydrolysis process conditions on ACE inhibiting ability of whey hydrolysates was investigated, showing that ACE inhibiting activity could be optimised by using process optimisation techniques like experimental design and response surface optimisation.
Cell to cell communication by autoinducing in gram-positive bacteria
Sturme, M.H.J. ; Kleerebezem, M. ; Nakayama, J. ; Akkermans, A.D.L. ; Vaughan, E.E. ; Vos, W.M. de - \ 2002
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 81 (2002)1-4. - ISSN 0003-6072 - p. 233 - 243.
grampositieve bacteriën - peptiden - nisine - aftasten - gram positive bacteria - peptides - nisin - sensing
While intercellular communication systems in Gram-negative bacteria are often based on homoserine lactones as signalling molecules, it has been shown that autoinducing peptides are involved in intercellular communication in Gram-positive bacteria. Many of these peptides are exported by dedicated systems, posttranslationally modified in various ways, and finally sensed by other cells via membrane-located receptors that are part of two-component regulatory systems. In this way the expression of a variety of functions including virulence, genetic competence and the production of antimicrobial compounds can be modulated in a co-ordinated and cell density- and growth phase-dependent manner. Occasionally the autoinducing peptide has a dual function, such as in the case of nisin that is both a signalling pheromone involved in quorum sensing and an antimicrobial peptide. Moreover, biochemical, genetic and genomic studies have shown that bacteria may contain multiple quorum sensing systems, underlining the importance of intercellular communication. Finally, in some cases different peptides may be recognised by the same receptor, while also hybrid receptors have been constructed that respond to new peptides or show novel responses. This paper provides an overview of the characteristics of autoinducing peptide-based quorum sensing systems, their application in various gram-positive bacteria, and the discovery of new systems in natural and engineered ecosystems
Formation and stability of emulsions made with proteins and peptides
Smulders, P.E.A. - \ 2000
Agricultural University. Promotor(en): P. Walstra. - S.l. : S.n. - ISBN 9789058083135 - 143
emulsies - formatie - stabiliteit - caseïne - lactalbumine - lysozym - ovalbumine - peptiden - emulsions - formation - stability - casein - lactalbumin - lysozyme - ovalbumin - peptides
The formation and stabilization of oil-in-water emulsions using well-defined and well-characterized proteins and peptides was studied in order to elucidate the relation between their molecular and functional properties. The emulsions were formed with a high-pressure homogenizer. To study the effect of the homogenizer scale on the emulsion properties, emulsions were prepared with a laboratory and a small industrial homogenizer. The flow in the industrial homogenizer was shown to be turbulent. In the laboratory homogenizer, droplet break-up was found to occur in a bounded laminar type of flow, resulting in a poor operating efficiency. The effect of the flow type on the emulsion properties, however, appeared to be small, if the number of passes through the laboratory homogenizer was sufficiently high.
Proteins appeared to have good emulsion forming properties as long as protein aggregation was absent. In those cases, the recoalescence rate during homogenization was found to be similar and only small differences in the droplet size of emulsions could be determined. The surface excess of the emulsion droplets appeared to be governed by the conformational stability and the aggregated state of the proteins. Globular proteins with a high conformational stability yielded relatively low surface excesses, while a flexible random coil protein, likeβ-casein, yielded a relatively high surface excess. Protein aggregation may be due to physicochemical conditions and surface or heat denaturation. If protein aggregates were present, the emulsion droplets were also often aggregated. The droplet size, surface excess, and rate of recoalescence of these aggregated emulsions were usually found to be relatively high.
The emulsion forming properties ofβ-casein peptides appeared to be comparable or superior to those of intact proteins. Amphiphilic peptides without the hydrophobic C-terminal domain ofβ-casein yielded a relatively low surface excess, likely due to strong electrostatic interactions between the highly charged groups of the N-terminal end. The surface excess of emulsions made with hydrophobic peptides with a removed N-terminal domain was comparable to those of emulsions made with intactβ-casein. The peptides were due to their relatively small molar mass more readily desorbed from the oil/water interface than intact proteins.
The coalescence stability of emulsions made with proteins was high even at low protein concentrations and appeared to be mainly determined by the surface excess of the droplets. The emulsion stabilizing properties ofβ-casein peptides were inferior to those of intact proteins probably due to their relatively low molar mass. Comparison of the stability of emulsions made with amphiphilic peptides with an intact or partially removed N-terminal domain showed that this domain was of great importance for providing stability against coalescence. The coalescence stability of emulsions made with hydrophobic peptides was relatively high, which was attributed to the high surface excess of the droplets. The electrostatic and steric interactions appeared to be of great importance for stabilizing emulsions made with peptides against coalescence as was indicated by the effect of changes in pH and ionic strength on the stability.
Keywords: emulsions, formation, stability, molecular properties,β-casein,β-lactoglobulin,α-lactalbumin, lysozyme, ovalbumin, peptides.
|Proceedings First International Symposium on Enzymatic Protein Processing (ISEPP-1), Noordwijkerhout, The Netherlands, 1998
Gruppen, H. ; Hartingsveldt, W. van - \ 2000
Zeist : TNO Nutrition and Food Research Institute - ISBN 9789067436960 - 234
eiwitten - peptiden - enzymen - proteolyse - eiwithydrolysaten - proteins - peptides - enzymes - proteolysis - protein hydrolysates
|Immunochemistry at interfaces. Peptides as antigen mimics in ELISA
Loomans, E.E.M.G. - \ 1997
Loomans - ISBN 9789090109114 - 143
immunochemie - peptiden - elisa - immunochemistry - peptides - elisa
|Formation and stability of foam made from aqueous protein solutions.
Prins, A. - \ 1997
Industrial Proteins 4 (1997)2. - ISSN 1381-0022 - p. 3 - 5.
eiwitten - peptiden - structuur - schuim - schuimen - reologie - fysica - vloeistofmechanica - reologische eigenschappen - moleculaire fysica - proteins - peptides - structure - foams - foaming - rheology - physics - fluid mechanics - rheological properties - molecular physics
Onderzoek naar de relatie tussen moleculaire structuur van eiwitten en het schuimgedrag van de oplossing
|Peptiden als emulgator en schuimvormer.
Caessens, P.W.J.R. ; Kalsbeek, H.K.A.I. van; Smulders, P.E.A. - \ 1997
Voedingsmiddelentechnologie 30 (1997)25. - ISSN 0042-7934 - p. 11 - 14.
eiwitten - emulsies - schuim - functionele reacties - peptiden - proteins - emulsions - foams - functional responses - peptides