De Graaf et al.: Requirements for non-food applications of pea proteins

Requirements for non-food applications of pea proteinsA Review

L. A. De Graaf, P. F. H. Harmsen,J. M. Vereijken and M. Mönikes

1 Introduction

Industrial proteins can be derived from plants, e.g. from pea,wheat and soy, or from the milk or skin and bones from ani-mals (e.g. casein and gelatin, respectively). Proteins have beenused in technical applications for over 3000 years already.Casein and keratin (derived from deer’s antlers) were used asadhesives already by the Romans. In the 1930s and 1940s, soyand casein plastics and fibers were developed [1], and the useof soy adhesive for plywood production increased rapidly.However, in the 1960s, the use of proteins declined, to a largeextent due to the relatively high price of proteins compared topetrochemical based polymers, but also due to differences inperformance.

Currently, still a number of large-scale industrial applica-tions is based on industrial proteins. Casein is still used inlabelling adhesives, and gelatin finds application in hot meltsin bookbinding and as a stabilizer/binder in photographicemulsions (with non-surpassed performance). Soy protein isstill applied as paper sizing agent and as plywood adhesive. Amore recent development is the use of (plant) proteins in sur-factants and cosmetics.

As casein and gelatin are expensive proteins, there is a mar-ket pull to replace these proteins by cheaper (mostly plant)proteins. Recent research makes clear that proteins haveunique properties that can be exploited for several technicalapplications [2–4]. Relevant protein properties are the varietyin sources, ease of modification, processability (in melt andaqueous solutions and dispersions), adhesion and film forma-tion, resistance toward oils and organic solvents, and gas bar-rier properties [2].

Pea is a comparatively recent commercial crop. Its cultiva-tion has shown a rapid expansion in the European Union since1980 [5]. However, the cultivation areas are still modest. Untilnow the applicability of peas is rather limited: they are mainlyused for animal feed. The development of new applications ofmajor components (such as protein) could enhance their culti-vation. However, limited research is performed on technicalapplications of pea proteins and no applications are yetreported. This can partly be contributed to the recent commer-cial introduction of pea proteins, and to the limited informationavailable for possible end-users [6].

Researchers have shown that casted films can be preparedfrom alkaline media, using glycols or polyols as plasticizers[7–9]. Film strength could be increased by use of crosslinkerssuch as formaldehyde [7, 9]. One paper describes pea proteinas a coating for extruded starch [10]. Pea proteins have goodemulsifying properties for preparing oil in water emulsions[11–13]. As pea protein contains a large amount of reactiveamino groups (lysine residue), chemical modification reactionsonto the amino group, such as acetylation or succinylation,could effectively be carried out. These reactions were powerfulto improve for instance emulsifying properties [6, 14]. Noreferences concerning the use of pea proteins in adhesives, as(extruded) plastics or as encapsulation medium were found.

The aim of this paper is first to discuss general requirementsfor non-food applications, and secondly to show the potentialof pea proteins in technical applications such as surfactants,films from neutral aqueous media and microspheres for encap-sulation of active ingredients.

2 Requirements for non-foodapplications

Opposite to food applications of proteins, in the case oftechnical applications there seem to be less (or no) restrictions

408 Nahrung/Food 45 (2001) No. 6, pp. 408 – 411 i WILEY-VCH Verlag GmbH, D-69451 Weinheim 2001 0027-769X/2001/0610-0408$17.50+.50/0

Agrotechnological Research Institute (ATO), Division of RenewableMaterials, P.O. Box 17, NL-6700 AA Wageningen, The Netherlands.Correspondence to:Dr. L. A. De Graaf (e-mail: l.a.degraaf@ato.wag-ur.nl).

So far, limited research is performed on technical applications of peaproteins and no applications have yet been reported. At ATO, three tech-nical applications were investigated: surfactants, films, and micro-spheres as encapsulation matrices.

Pea protein hydrolysates are surfactants with good emulsifying andfoaming properties. By variation of enzyme type and degree of hydroly-sis, the surfactant properties can be tailored toward specific applica-tions. Pea protein films could be prepared by casting from dispersions atpH 7 and 10, and by compression moulding at 140 8C. Opposite to many

other proteins, pea protein films combine strength (5–7.5 MPa) withhigh elongation at break (150%).

A protein isolate derived from peas was applied as matrix material forthe microencapsulation of b-carotene, intended for cosmetic applica-tions. Supercritical CO2 technology appeared to be a promising encap-sulation technique for b-carotene in porous pea protein microspheres.Advantages of this method are that no organic solvents are used, andthat encapsulation is achieved under mild conditions, thereby prevent-ing the sensitive b-carotene from degradation.

Legume Protein Modification and Non-foodApplication

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with respect to protein denaturation. On the contrary, for mostapplications, protein denaturation through processing or modi-fication seems to be a requirement.

In order to reach high quality technical products, a numberof functional and structural requirements have to be met [15].Examples are the good adhesion and bond strength for adhe-sives, resistance against water for coatings and strength forplastic materials. Adhesion is reached upon exposure of speci-fic groups, such as polar groups onto polar surfaces (wood,glass, metals, paper) and apolar groups onto apolar surfaces(most synthetic plastics). Molecules have to be entangled inorder to obtain a high cohesive strength in plastics, coatingsand (dried) adhesives. The cohesive strength and water resis-tance of protein polymers can be enhanced by crosslinking, forwhich reactivity is a prerequisite. Reactivity implies exposureof reactive (polar) groups such as carboxylic acid, amino,hydroxyl and sulphydryl groups. Exposure of specific groupsand the formation of entanglements (physically entangledchains) imply that the protein has to become less structured oreven denatured. In general, the decrease in water uptake ofproteins, and thereby the dependence of properties on the watercontent, is an important aspect of research on technical appli-cations of proteins.

3 Applications of pea protein

In this paragraph three applications of pea proteins will bediscussed in more detail. With respect to functional and struc-tural requirements of the proteins for these applications, forfilms/coatings the protein should be processable in aqueousdispersion, and it is important to reach high film strength andgood water resistance. The latter is also very important formicrospheres. The requirements were met by the film formingconditions (neutral pH, stirring, drying temperature) and byapplying a temperature treatment to the microspheres. Gener-ally, surface activity of proteins can be enhanced by reductionof the molecular weight, by the exposure of (internal) hydro-phobic groups of the protein, and by the introduction of addi-tional hydrophobic groups to the protein molecule (creatingamphiphilicity).

3.1 Surfactants

Surface active agents cover a range of molecules which canact as emulsifier and foaming agent, but also as pigment dis-perser, wetting agent and defoamer. Virtually all paint, coatingand adhesive formulations contain surfactants for the purposesmentioned before. To improve surface activity, pea protein wasenzymatically hydrolysed in order to reduce its molecularweight and to expose internal hydrophobic groups. No addi-tional modifications were carried out.

The solubility in water of pea protein (Propulse, DPS, TheNetherlands) shows a strong dependence on pH, with a mini-mum around pH 5.8. By hydrolysing the protein, the pHdependence disappears and the surface activity is improved, ascan be seen from Figure 1. Depending on the protease used, inthis case Alcalase or Protamex (Novo Nordisk), the surfaceactive behaviour varied with degree of hydrolysis (DH). Alca-lase hydrolysates show a broad maximum for foaming andemulsifying properties, while Protamex hydrolysates are moreDH specific. In conclusion, by variation of enzyme type andDH, the surfactant properties can be tailored toward specificapplications.

3.2 Films

So far, films based on pea protein have been prepared fromalkaline media [7–9]. However, it appeared to be well possibleto prepare films by casting a dispersion of pea protein (Pisane,Provital, Warcoing, Belgium) at pH 7, containing 20 wt.-%glycerol as plasticizer. Transparent, flexible and strong filmswere obtained, which were sligtly yellow coloured. Alterna-tively, pea protein powder containing 30 wt.-% glycerol and 30wt.-% water was compression moulded at 1408C (40 tons ofpressure, 5 min).

Table 1 lists the mechanical properties of films based on dif-ferent plant and animal proteins. All films were approximately300 lm thick and had a water content of 9–10% upon meas-urement. The table shows that, whereas most proteins showeither a high strength (soy, rapeseed) or a high elongation atbreak (wheat gluten), pea protein films combine the two prop-erties. This combination is rare. There was no difference inproperties of films casted at neutral pH or pH 10. Compressionmoulded films, which contain 30% of glycerol, show a lowerstrength and higher elongation. Despite the higher plasticizercontent, the material retains a good strength with high elonga-tion.

In conclusion, it can be stated that pea protein films can beprepared by casting from dispersions at pH 7 and 10, and bycompression moulding at 1408C. Opposite to many other pro-teins, pea protein films combine strength (5–7.5 MPa) withhigh elongation at break (150%).

Figure 1. Foam expansion (full lines) and emulsifying activity(dotted lines) of pea proteins hydrolysates, enzymatically modified byAlcalase (squares) and Protamex (triangles), as a function of degree ofhydrolysis. Emulsifying activity and foaming capacity were deter-mined as described by Sijtsma and Massoura [11, 16].

Table 1. Comparison of film properties of several plant proteins(20% glycerol w/w unless stated otherwise).

Protein Tensile strength[MPa]

Elongation atbreak [%]

Pea pH 7/10 7.4 140Pea compression moulded

at 140 8C (30% glycerol)5.2 170

Soy isolate 6.5 50Soy isolate compression

moulded at 140 8C7.0 75

Wheat gluten 1.9 230Rapeseed protein 8.2 30

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3.3 Microspheres

Cosmetic ingredients, such as b-carotene (the precursor ofvitamin A) are often sensitive to oxidation. In order to improvethe stability, but also to mask the deep-purple colour, b-caro-tene can be encapsulated in small particles like microspheres,and added to cosmetic formulations [17]. In this study, firstmicrospheres were prepared, followed by encapsulation of b-carotene in the microspheres via supercritical CO2 (scCO2)treatment.

Microspheres based on pea protein (Pisane, Provital, Warco-ing, Belgium) were prepared by a water-in-oil emulsion techni-que. Stable microspheres were obtained by denaturation of thepea protein by heat treatment in a microwave (1–4 min). Theparticles were isolated and dried to give a free flowing powder.b-Carotene (natural 30% carotene oleoresin, Alban MullerInternational) was encapsulated in the microspheres by scCO2

treatment at 30 MPa using CO2 and 32 8C during 15 min.The best results with respect to microsphere morphology

were obtained at pH 10. This is mainly due to the improvedsolubility of pea protein at high pH. A correlation wasobserved between particle porosity and temperature reachedduring heat treatment. Microspheres with a highly porousstructure were obtained at a relatively low temperature (i. e.908C), whereas a higher temperature (i. e. 1268C) resulted in aless porous structure (Figure 2).

When no b-carotene was present, scCO2 treatment did notaffect the microsphere morphology. However, when b-caro-tene was added by means of mixing or scCO2 treatment, theporous structure dispappeared, and became denser and moreuniform (Figure 3). Up to a load of 20% w/w b-carotene, afree-flowing powder was obtained with porous microspheresand a paste-like product with smooth ones. No morphologicaldifferences were observed between the mixed and scCO2 treat-ed samples. In both cases, the b-carotene can diffuse into thepores of the structure, thus “filling in the holes”. Additionally,applying CO2 treatment could plasticize the protein. Therebythe matrix is swollen, and the overall free volume is increased,allowing rapid diffusion of the b-carotene into the free volumeof the matrix. By releasing the CO2 pressure, the b-carotene istrapped in the protein matrix.

Release experiments showed that microspheres mixed withb-carotene showed an extremely quick release, with release ofessentially all material in 30 min. In contrast, the microspheresthat had undergone the scCO2 procedure showed a differentrelease profile. There was an initial rapid release of b-carotenein the first 15 min, followed by a more gradual release of theactive ingredient. No further release of b-carotene occurredafter approximately 200–300 min.

Though each sample showed burst release, this effect wasless pronounced for the scCO2 sample as shown by the releaserate of b-carotene (Figure 4). The release of b-carotene from

Figure 2. SEM micrograph of microspheres made at pH 10 at 90 8C(top) and 126 8C (bottom).

Figure 3. Cryo-SEM micrographs of microspheres with b-caroteneprepared by mixing (top) and by scCO2 treatment (bottom).

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the scCO2 microspheres was much more delayed compared tothe mixed sample. Based on these results, it was concludedthat b-carotene was better entrapped in the protein matrix bythe scCO2 treatment.

It can be concluded that microspheres based on pea proteincould be prepared at high pH and elevated temperatures usinga water-in-oil emulsion technique. Subsequent encapsulationof b-carotene into these microspheres using supercritical CO2

can give a free flowing material with a loading of 20% w/w b-carotene, depending on the porosity of the microspheres.Supercritical CO2 technology appears to be a promising encap-sulation technique for b-carotene in pea protein matrices, andmore in general for lipophilic materials in various matrices.

Conclusions

So far, pea protein has not been used in technical applica-tions. However, films based on pea protein combine goodstrength with high elongation at break, allowing for instancepackaging applications. Its surface activity can be tailored byvariation of molecular weight, and by modifying the hydropho-bicity. This opens the possiblilty for pea protein to be used assurfactants in coatings, paints and adhesives. Pea protein iso-late shows good potential as matrix material for the microen-capsulation of b-carotene, by means of supercritical CO2 tech-nology. Both protein and technology are not restricted to thisingredient only.

In order to introduce pea protein into technical applications,there are three requirements to be met for the end-products:acceptable price, processability on regular equipment, and aperformance which equals or exceeds the current products.More research and development is needed to reach all threerequirements, but a good start has been made.

AcknowledgementsThe European Community (Fair CT98-3527, INCO COPERNICUS

CIPA CT94-177) is acknowledged for financial support.

References[1] Bietz J., and G. L. Lookhart, Cereal Foods World 41 (1996)

376–382.[2] De Graaf, L. A., W. J. Mulder and P. Kolster, Proceedings 6th

Symposium on Renewable Resources for the Chemical Industry,pp. 120–128. Bonn 1999.

[3] De Graaf, L. A., Industrial Proteins 6 (1998) 9–11.[4] Fossen, M., and W. Mulder, Lebensmittel-Verfahrungs- und Ver-

packungstechnik 43 (1998) 108–111.[5] Schneider, A., and J. P. Lacampagne, Industrial Proteins 8–1

(2000) 3–6.[6] Gueguen, J., Industrial Proteins 8 (2000) 6–8.[7] Gueguen, J., G. Viroben, P. Noireaux and M. Subirade, Industrial

Crops and Products 7 (1998) 149–157.[8] Viroben, G., J. Barbot, Z. Moloungi and J. Gueguen, J. Agric.

Food Chem. 48 (2000) 1064–1069.[9] Rampon, V., Sciences des Aliments 18 (1998) 95–100.

[10] Juhel, F., Sciences des Aliments 19 (1999) 73–84.[11] Sijtsma, L. D. Tezera, J. Hustinx and J. M. Vereijken, Nahrung/

Food 42 (1998) 215–16.[12] Lu, B. Y., L. Quillien and Y. Popineau, J. Sci. Food Agric. 80

(2000), 1964–1972.[13] Franco, J. M., P. Partal et al., J. Am. Oil Chem. Soc. 77 (2000)

975–983.[14] Legrand, J., J. Gueguen, S. Berot, Y. Popineau and L. Nouri,

Biotechnol. Bioengin. 53 (1997) 409–414.[15] De Graaf, L. A., J. Biotechnol. 79 (2000) 299–306.[16] Massoura, E., J. M. Vereijken, P. Kolster and J. T. P. Derksen,

J. Am. Oil Chem. Soc. 75 (1998) 329.[17] Harmsen, P. F. H., M. H. Vingerhoeds et al., IFSSC magazine

4(1) (2001).

Received: 27 March 2001.Accepted: 06 April 2001.

Figure 4. Release rate of b-carotene (19% w/w) from the mixedsample (dotted line) and from the scCO2 sample (full line). The insertis a detail of the large Figure.