Conformation and association of κ-carrageenan in the presence of locust bean gum in mixed NaI/CsI solutions from rheology and cryo-TEM

Mixtures of locust bean gum (LBG) with κ-carrageenan (KC) in 0.1 M aqueous solutions of the mixed salts NaI/CsI were investigated by cryo-transmission electron microscopy (cryo-TEM) and dynamic viscoelastic measurements. Previous studies have shown that as the cesium content is increased in such mixed salt solutions, a transition occurs from molecularly dispersed helices to ‘superhelical rods’ of KC. We now found that LBG stabilises the superhelical rods, shifting the transition to a lower content of Cs for the mixtures than for KC alone. The formation of superhelical rods was evidenced both by cryo-TEM images and by an onset of thermal hysteresis in the coil–helix transition of KC. In the mixtures, the transition temperatures on cooling and heating were insensitive to the proportions of LBG and KC present at all cesium contents. Under conditions where no helix aggregation occurred (no hysteresis) the mixtures showed high tan δ values and low storage moduli. Under aggregated conditions, gels formed, and gels with added LBG had enhanced moduli compared to gels with KC alone. On the basis of these results we propose that LBG associates to the super-helical rods of KC.     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part I: gelation of individual components

As a prelude to studies of co-gelation with galatin, the gelation behaviour of Paselli maltodextrins SA-6 and SA-2 (DE ≈ 6 and 2, respectively) was mapped out over the experimentally-accessible range of temperature (T) and concentration (c), using a simple visual method to determine the time required for formation of a self-supporting network (t_g). For both samples, log t_g decreased linearly with log c and increased linearly with T. At equivalent temperatures and concentrations, SA-2 gelled between 20 and 60 times faster than SA-6.

Selected samples were monitored more rigorously by mechanical spectroscopy, taking t_g as the time at which elastic response (G′) became greater than viscous response (G″). In all cases the values of t_g obtained by this procedure were lower than those from visual inspection, by a constant factor of about 3·4.

The concentration-dependence of gel moduli (G′) for SA-2 and for gelatin (second-extract limed ossein; LO-2) fitted accurately to the form anticipated from cascade theory for normal polymer networks. For SA-6, by contrast, log G′ varied linearly with log c over the entire range at which measurements could be made, indicating a different mechanism of structure-formation (such as the agglomeration of short, aggregated helices).     

Gelation of konjac glucomannan crosslinked by organic borate

A series of thermoreversible konjac glucomannan gels crosslinked by organic borate were prepared. The gel network was formed through the crosslinking reaction between borate ions dissociated from organic borate and the cis-diol hydroxyl groups on the mannose units of polysaccharide chains. The rheological properties of the complex gels were studied by dynamic viscoelastic measurement. The gelation kinetics of the gels were studied and the critical gelation points of the gels were exactly determined by the Winter–Chambon criterion. The effects of temperature and composite ratio on the shear storage modulus (G^′), the loss modulus (G^″), and the sol-gel transition points were investigated. The critical gel-sol temperatures of the complex gels were successfully elucidated by Winter–Chambon criterion. The effect of crosslinking density on the critical gelation temperature and the elasticity of the gels were discussed.     

Effect of water-soluble and insoluble non-starch polysaccharides isolated from wheat flour on the rheological properties of wheat starch gel

Water-soluble (WSP) and insoluble non-starch polysaccharides (WIP) were isolated from wheat flour to evaluate the effects of WSP and WIP on starch gel properties. Isolated WSP and WIP were added to two types of isolated wheat starch with different amylose content at a concentration of 3% based on the dry weight of starch. 30% starch gels were prepared and stored at 5 °C for 1, 8, or 24 h. The dynamic viscoelasticity of 30% starch gels mixed with WSP and WIP was measured using parallel plate geometry, showing that WSP and WIP affected the elastic component of starch gels in opposite ways. Adding WIP increased the storage shear modulus (G′) of starch gels, while adding WSP decreased G′ and dramatically increased the loss tangent (tan δ=G″/G′).     

Phase equilibria and gelation in gelatin/maltodextrin systems — Part IV: composition-dependence of mixed-gel moduli

The storage moduli (G′) of phase-separated co-gels formed by quench-cooling mixed solutions of gelatin and potato maltodextrin (Paselli SA-2 and SA-6) have been related quantitatively to the experimentally-determined concentration-dependence of G′ for the constituent polymers. Distribution of water between the phases was examined explicitly by using polymer blending laws to derive calculated moduli for gelatin-continuous and maltodextrin-continuous networks over the entire range of solvent partition. Allowance was made for the direct contribution of polymer chains, and for density differences between the phases, in calculating relative phase-volumes. The effect of gel formation prior to phase-separation was calculated using classical theory for network de-swelling. Good agreement with observed moduli for more than 30 gelatin/maltodextrin combinations was achieved using a single adjustable parameter, p (the ratio of solvent to polymer in the gelatin phase divided by the same ratio for the maltodextrin phase), with an optimum value of p ≈ 1·8 for both SA-6 and SA-2.     

Correlation between sagging and shear elasticity in pectin, gelatin and polyacrylamide gels

The relationship between the height of gels determined by a sag test and their elastic shear modulus (G′) has been both investigated experimentally and simulated using a finite element analysis for the inhomogeneous deformation of gels due to gravity. It was assumed in the simulations that gels can be modeled as incompressible linear elastic materials. General relationships between the sag of gels and their elastic modulus were obtained from the simulations for slip and no-slip conditions. The relationships were tested experimentally on pectin, gelatin and polyacrylamide gels with a range of concentrations and rigidities. The good agreement between the predictions and the results shows that these gels can be modeled accurately as incompressible elastic materials. A standard 150° SAG pectin gel, which sags 23.5% in the SAG test, has G′ moduli of 429 and 379 Pa under slip and no-slip conditions, respectively.     

Rheological properties of mixtures of kappa-carrageenan from Hypnea musciformis and galactomannan from Cassia javanica

Mixed gels of κ-carrageenan (κ-car) from Hypnea musciformis and galactomannans (Gal) from Cassia javanica (CJ) and locust bean gum (LBG) were compared using dynamic viscoelastic measurements and compression tests. Mixed gels at 5 g/l of total polymer concentration in 0.1 M KCl showed a synergistic maximum in viscoelastic measurements for κ-car/CJ and κ-car/LBG at 2:1 and 4:1 ratios, respectively. The synergistic maximum obtained from compression tests carried out for mixed gels at 10 g/l of total polymer concentration in 0.25 M KCl was the same for both κ-car/CJ and κ-car/LBG gels. An enhancement in the storage modulus (G′) and the loss modulus (G″) was observed in the mechanical spectra for the mixtures in relation to κ-car. The proportionally higher increase in G″ compared with G′, as indicated by the values of the loss tangent (tan δ), suggests that the Gal adhere non-specifically to the κ-car network.     

Effect of polymeric cosolutes on calcium pectinate gelation. Part 2. Dextrans and inulin

In a continuation of the work reported in the preceding paper, the effect of chicory root inulin (M_r≈4.5 kD) and branched dextrans of M_r 67, 464 and 2000 kD on gelation of low methoxy pectin (DE 31; 2.0 wt%; pH≈2.9–3.0) on cooling from 90 to 5 °C in the presence of stoichiometric Ca²⁺ has been characterised by low amplitude oscillatory measurements of G′ and G″. As found for other polymeric cosolutes studied previously (oxidised starch, potato maltodextrin, guar gum and locust bean gum), increasing concentrations of dextran or inulin caused a progressive increase of G′ and G″ in the solution state at 90 °C (attributed to segregative interactions promoting formation of calcium-mediated ‘egg-box’ junctions between pectin chains) and a progressive reduction in final moduli at 5 °C (attributed to excessive association of calcium pectinate into large aggregated bundles), with cooling curves crossing those for calcium pectinate alone at 55 °C. For the dextran samples, the ability to promote initial association of pectin chains at high temperature decreased with increasing molecular weight (i.e. with the smallest molecules having the greatest effect, as was also found for guar gum), but the reduction in final modulus on completion of cooling to 5 °C became more pronounced. By the criterion of the decrease in the change in log G′ during cooling, the three dextran samples studied seem roughly comparable in their overall effectiveness in promoting self-association of pectin, with inulin having a substantially smaller effect. Both materials, however, are less effective than the partially depolymerised starches and galactomannans studied previously. It is tentatively suggested that this may be associated with the flexibility introduced by the presence of three single bonds in the predominant glycosidic linkages of both dextran and inulin.     

1,10-Phenanthroline-5,6-dione as a building block for the synthesis of homo- and heterometallic complexes

1,10-Phenanthroline-5,6-dione (C₁₂H₆N₂O₂ (1)) reacts with V(η⁶-mesitylene)₂ and Ti(η⁶-toluene)₂ affording coordination compounds of general formula M(O,O′---C₁₂H₆N₂O₂)₃ (M=Ti (2); M=V (3)) which further react with TiCl₄ or TiCp₂(CO)₂ yielding the tetrametallic species M(O,O′---C₁₂H₆N₂O₂---N,N′)₃(M′L_n)₃ (M=V, M′L_n=TiCl₄ (4); M=Ti, M′L_n=TiCp₂ (5); M=V, M′L_n=TiCp₂ (6)). The complex salt [Fe(N,N′---C₁₂H₆N₂O₂)₃][PF₆]₂ (7) has been obtained from iron(II) chloride tetrahydrate and 1 in the presence of NH₄PF₆. The reaction of 7 with TiCp₂(CO)₂ affords the tetrametallic derivative [Fe(N,N′---C₁₂H₆N₂O₂---O,O′)₃(TiCp₂)₃][PF₆]₂ (8). TiCl₂(THF)₂ reacts with MCp₂(O,O′---C₁₂H₆N₂O₂) to give MCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (M=Ti (9); M=V (10)). By reaction of TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (9) with C₁₂H₆N₂O₂, the bimetallic derivative TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂(O,O′---C₁₂H₆N₂O₂) (11) has been prepared, which readily adds to TiCl₄, to give the trimetallic titanium derivative TiCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₄ (12). VCp₂(O,O′---C₁₂H₆N₂O₂---N,N′)TiCl₂ (10) reacts with the tris-chelate iron(II) cation 7 affording the heptametallic cationic complex [Fe(N,N′---C₁₂H₆N₂O₂---O,O′)TiCl₂(N,N′---C₁₂H₆N₂O₂---O,O′)VCp₂]₃ ⁺² isolated as the hexafluorophosphate 13.     

“Melt-in-the-mouth” gels from mixtures of xanthan and konjac glucomannan under acidic conditions: A rheological and calorimetric study of the mechanism of synergistic gelation

The effect of acidification on a typical commercial xanthan and on pyruvate-free xanthan (PFX), alone and in gelling mixtures with konjac glucomannan (KGM), has been studied by differential scanning calorimetry (DSC) and small-deformation oscillatory measurements of storage modulus (G′) and loss modulus (G″). For both xanthan samples, progressive reduction in pH caused a progressive increase in temperature of the disorder–order transition in DSC, and a progressive reduction in gelation temperature with KGM. This inverse correlation is interpreted as showing that synergistic gelation involves disruption of the xanthan 5-fold helix, probably by attachment of KGM to the cellulosic backbone of the xanthan molecule (as proposed previously by a research group in the Institute of Food Research, Norwich, UK). Higher transition temperature accompanied by lower gelation temperature for PFX in comparison with commercial xanthan at neutral pH is explained in the same way. However, an additional postulate from the Norwich group, that attachment of KGM (or galactomannans) can occur only when the xanthan molecule is disordered, is inconsistent with the observation that gelation of acidified mixtures of KGM with PFX can occur at temperatures more than 60 °C below completion of conformational ordering of the PFX component (as characterised by DSC). Increase in G′ on cooling for mixtures of commercial xanthan with KGM at pH values of 4.5 and 4.25 occurred in two discrete steps, the first following the temperature-course observed for the same mixtures at neutral pH and the second occurring over the lower temperatures observed for mixtures of KGM with PFX at the same values of pH. These two “waves” of gel formation are attributed to interaction of KGM with, respectively, xanthan sequences that had retained a high content of pyruvate substituents, and sequences depleted in pyruvate by acid hydrolysis. At pH values of 4.0 and lower, gelation of mixtures of KGM with commercial xanthan followed essentially the same temperature-course as for mixtures with PFX, indicating extensive loss of pyruvate under these more strongly acidic conditions. Mixtures prepared at pH values in the range 4.0–3.5 gave comparable moduli at room temperature (20 °C) to those obtained at neutral pH, but showed substantial softening on heating to body temperature, suggesting possible applications in replacement of gelatin in products where “melt-in-the-mouth” characteristics are important for acceptability to the consumer.     

Non-equilibrium processes of interchain association induced by Cs+ ions in kappa-carrageenan aqueous solutions

We present preliminary results of the investigation of interchain association processes induced by Cs+ ions in kappa-carrageenan aqueous solution. The solutions contained variable amounts of NaI and CsI, under the condition that the total concentration of 1:1 electrolyte was 0.1M. The associative processes were observed by static light scattering under isothermal conditions (at T = 25 degrees C), after cooling molecularly dispersed solutions obtained at high temperature (80 degrees C). It was found that, under all the investigated conditions of polymer concentration (from 0.2 to 2 g l(-1)) and ionic composition, the onset of time-dependent association fails to lead to an equilibrium, but proceeds up to physical gelation of the associating system. Depending on the experimental variables, however, the gelation threshold may take up to several days to be achieved.     

Effect of polymeric cosolutes on calcium pectinate gelation. Part 3. Gum arabic and overview

Addition of gum arabic (average M_r≈450 kDa; 0.5–2.0 wt%) to solutions of low methoxy pectin (DE 31; 2.0 wt%; pH≈2.9–3.0) with stoichiometric Ca²⁺ caused massive increases in G′ and G″ in the pre-gel state at 90 °C (attributed to segregative interactions promoting formation of calcium-mediated ‘egg-box’ junctions between pectin chains) but had little effect on the gels formed on cooling to 5 °C. This is in marked contrast to the behaviour of other polymeric cosolutes studied in the investigations reported in the two preceding papers, which caused large reductions in gel moduli (attributed to excessive association of calcium pectinate into large aggregated bundles); the difference is tentatively ascribed to strengthening of the calcium pectinate network by divalent counterions to the uronate residues in gum arabic. When the complication of cation exchange was eliminated by extensive dialysis of gum arabic against 100 mM Na⁺ and use of the final dialysate in preparation of mixtures with calcium pectinate, massive increases in G′ and G″ at high temperature were again observed, but with accompanying reductions in moduli at low temperature, which, at gum arabic concentrations above 1.0 wt%, arose from collapse of the developing calcium pectinate network during cooling. The tentative conclusion from this work, and from the two preceding papers, is that enthalpically unfavourable (segregative) interactions between low methoxy pectin and polymeric cosolutes can be relieved in two ways: (i) Ca²⁺-mediated self-association of pectin into compact ordered assemblies which occupy less of the total volume, and (ii) conformational rearrangement of the cosolute molecules to minimise segmental interactions with pectin; conformational rearrangement is inhibited by chain stiffness and by branching; thus polymeric cosolute molecules of limited flexibility are more effective in promoting self-association of pectin than more flexible molecules of comparable size, and branched molecules are more effective than linear chains of comparable stiffness.     

Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer

Enantiomerically pure alkylphosphonate compounds RR′P(O)PNP (R=C_nH_2n+1, R′=OY with Y=C_n′H_2n′+1 with n=n′ or n≠n′; PNP=p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolecular films technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simultaneously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious correlation between the inhibitor molar fraction (₅₀) leading to half inhibition, and the chain length, R or Y was observed. (R₁₁Y₁₆)s were the best inhibitor of HPL and (R₁₀Y₁₁)s were the best inhibitors of HGL. We observed a highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a scalemic mixture. We also showed, for the first time, that this enantioselective recognition can occur either during the catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric inhibitors.     

Gelation of fractionated canine submaxillary mucin in a chaotropic solvent

Rheological measurements have been performed on three molecular weight fractions of purified canine submaxillary mucin (CSM) dissolved in the chaotropic solvent 6 M guanidine hydrochloride (GdnHCI). Solutions of the lower molecular weight fractions are viscoelastic sols, and their dynamic moduli can be scaled with respect to molecular weight and concentration according to linear viscoelasticity theory. In contrast, preparations of the highest molecular weight fraction form viscoelastic gels that exhibit an equilibrium shear modulus, G_e^′, which scales with mucin concentration as G_e^′ c³. Amino acid and carbohydrate analyses of all three fractions are similar; thus, the differences in rheological behavior are attributed to molecular weight differences, which affect the degree of coil overlap in solutions of a given concentration. These observations demonstrate conclusively that mucin glycoproteins of high molecular weight form gels under conditions in which the mucin chains physically interpenetrate, even when non-covalent intermolecular interactions are extensively disrupted. A comparison of these results with previous studies of purified submaxillary and tracheobronchial mucins indicates that the carbohydrate side-chain length, in addition to molecular weight, is an important determinant of the observed elastic response and the ability to form physical gels     

Rheology and gelation of water-insoluble dextran from Leuconostoc mesenteroides NRRL B-523

Dynamic oscillatory testing has been used to study the rheology of water-insoluble dextran. The rheological properties (storage and loss moduli) of dextran gel were measured and dextran was found to be neither a strong gel nor a weak gel, but an entanglement network at a concentration of 250 mg/ml. The extent of gelation, illustrated by the gel elastic modulus G′, is found to decrease with increasing concentration of calcium ions. This was confirmed by shift of crossover frequencies towards higher values on the dynamic spectra and lower yield stress τ values obtained from stress ramp experiments. Finally, a comparison between gelation of dextran and alginate (a similar biopolymer) was made for clear understanding of effect of calcium ions on the dextran gelation.     

Pumpkin pectin: gel formation at unusually low concentration

The gel properties of high-methoxy pectin from pumpkins have been investigated to assess the potential of this material as a hard-currency export from the former Soviet Union. Comparison was made with commercial slow-set, medium-set and rapid-set pectins from citrus peel. Gels were formed by cooling pectin solutions (pH 3·0; 60% (w/w) sucrose; 5% (w/w) corn syrup) from 95°C to 25°C, and the time-temperature course of network formation was monitored by small-deformation oscillatory measurements of storage modulus (G′). At concentrations above 1% (w/w) the pumpkin pectin gave weaker gels than the other three samples, but its minimum critical gelling concentration (c₀) was found to be much lower (by at least a factor of five). Compression testing gave similar results, with pumpkin pectin giving useful breaking-stress (‘hardness’) at concentrations down to 0·5% (w/w), about a factor of two lower than for the citrus samples. Its gelation was also less rapid, giving G′ values below those of the other three samples at temperatures down to 60°C, but then setting sharply; this behaviour could be useful in avoiding ‘pregelation’ in commercial processing. The commercial slow-set pectin showed typical ‘weak gel’ properties in the solution state at 95°C, with systematic reduction in gel-like character with increasing ester content in the other samples. The rigidity of the final gels also decreased systematically through the series: rapid-set < medium-set < slow-set. These observations are tentatively ascribed to stable association of unesterified galacturonate chain segments at low pH, where electrostatic repulsion is suppressed.     

Rheological and calorimetric study of the sol–gel transition of κ-carrageenan

Rheological and DSC techniques were used to study the effect of κ-carrageenan and KCl concentrations, 0–300 mM, on the sol–gel transition as well as on the linear viscoelasticity, at 25 °C, of the resulting gels. In heating and cooling DSC tests, the peak temperature was taken as the sol–gel transition point. In rheological tests, sol–gel transitions were determined from the variation of dynamic moduli with frequency and temperature, the independence of the phase angle on frequency and the evolution with temperature of dynamic moduli on cooling and heating at constant frequency and strain. Transition temperatures from DSC and rheology were in good agreement among them and with those previously reported. The three procedures yielded similar results, but the transition temperatures were more easily determined through the independence of the phase angle on frequency. Frequency sweeps showed gel behavior with stiffness increasing with polysaccharide and salt concentration. Below 100 mM KCl, G′ increased notably, whereas higher concentrations produced only marginal increases.     

Piezoelectric properties of poly-β-hydroxybutyrate and copolymers of β-hydroxybutyrate and β-hydroxyvalerate

The shear piezoelectricity was observed in oriented films of poly-β-hydroxybutyrate (PHB) and copolymers of β-hydroxybutyrate (HB) and β-hydroxyvalerate (HV). The piezoelectric stress constant 3₁₄ = e′₁₄ − ie″₁₄ (polarization/strain), the piezoelectric strain constant d₁₄ = d′₁₄ − id″₁₄ (polarization/stress), the elastic constant c = c′ + ic″ and the dielectric constant = ′ − i″ were determined at a frequency of 10 Hz over a temperature range from −150° to +150°C. Piezoelectric relaxations as well as elastic and dielectric relaxations were clearly observed at the glass transition temperature of about 15°C. In order to evaluate the piezoelectric constants (e₂ and d₂) for the piezoelectric phase which consists of the crystalline region and the oriented non-crystalline region, a spherical dispersion two phase model was utilized. Assuming the appropriate fixed values for the elastic and dielectric constants in the piezoelectric phase, d₂ and d₂ were calculated as a function of temperature. For a PHB and a copolymer (17 HV/83 HB), e₂ and d₂ showed relaxations, leading to a conclusion that the instantaneous piezoelectric constant in the crystalline phase is constant independent of temperature but the piezoelectric constant in the oriented non-crystalline phase is relaxational and has the opposite sign. For a copolymer (25 HV/75 HB) and a chloroform treated copolymer (17 HV/83 HB), e₂ and d₂ were constant independent of temperature, indicating that the oriented non-crystalline phase has disappeared owing to the increased molecular flexibility due to copolymerization or annealing in chloroform vapour.     

Conductance and relaxations of gelatin films in glassy and rubbery states

The dielectric constant, ′, and the dielectric loss, ″, for gelatin films were measured in the glassy and rubbery states over a frequency range from 20 Hz to 10 MHz; ′ and ″ were transformed into M* formalism (M*=1/(′−i″)=M′+iM″; i, the imaginary unit). The peak of ″ was masked probably due to dc conduction, but the peak of M″, e.g. the conductivity relaxation, for the gelatin used was observed. By fitting the M″ data to the Havriliak–Negami type equation, the relaxation time, τ_HN, was evaluated. The value of the activation energy, E_τ, evaluated from an Arrhenius plot of 1/τ_HN, agreed well with that of E_σ evaluated from the DC conductivity σ₀ both in the glassy and rubbery states, indicating that the conductivity relaxation observed for the gelatin films was ascribed to ionic conduction. The value of the activation energy in the glassy state was larger than that in the rubbery state.     

Impact of the structure of arabinoxylan gels on their rheological and protein transport properties

Water-extractable arabinoxylan (WEAX) gels exhibiting different structural, rheological and protein transport properties were obtained upon laccase treatment of WEAX solutions, by modifying (i) the initial ferulic acid (FA) content of WEAX from 2.3 to 1.6 μg/mg AX or (ii) the AX concentration of the WEAX solution from 0.5 to 2.0% (w/v). WEAX gels with di and tri-ferulic acid (di-FA, tri-FA) contents varying from 6.2 to 3.2 μg/ml gel and from 0.61 to 0.27 μg/ml gel, respectively, were obtained. In parallel, increases in gel mesh sizes from 201 to 331 nm and reduction of G′ of gels from 160 to 12 Pa were observed. The differences in structural and rheological characteristics of WEAX gels were reflected in their capacity to load and release proteins of M_w ranging from 43 to 669 kDa. The possibility of modulating protein release from WEAX gels makes these gels potential candidates for the controlled delivery of proteins.