ELSEVIER

Field Crops Research 53 (1997) 31-45

F i e l d
Cr o p s
R e s e a r c h
Legume seeds: protein content and nutritional value
Ma r c e l l o Du r a n t i *, Cr i s t i na Gi u s
Dipartimento di Scienze Molecolari Agroalimentari and Centro lnteruniversitario per lo Studio delle Macromolecole Informazionali
(CISMI), Universith di Milano, via Celoria, 2, 1-20133 Milano, Italy
Abs t r ac t
Proteins are major components of legume seeds. Their nutritional and functional properties dramatically affect the overall
quality of the seed and its technological performance. In this review legume seed proteins are considered, taking into account
both their molecular and nutritional properties. Other compounds of non-protein origin affect the utilization of grain legumes
for food and feed. They have also been reviewed both for their involvement in the seed life cycle and for human nutrition.
The perspectives for improvement of legume traits, starting from the molecules considered and also including the
biotechnological approaches, are also discussed. 1997 Elsevier Science B.V.
1. I n t r o d u c t i o n
Grain l egumes are widely recogni zed as important
sources of food and feed proteins. In many regions
of the world, l egume seeds are the unique supply of
protein in the diet. Very often they represent a
necessary suppl ement to other protein sources. On
the other hand in devel oped countries, plant proteins
can now be regarded as versatile functional ingredi-
ents or as biologically active component s more than
as essential nutrients. This evolution towards health
and functionality is mai nl y driven by the demands of
consumers and health professionals (the partial re-
pl acement of animal foods with l egumes is cl ai med
to i mprove overall nutritional status (Guillon and
Champ, 1996)) and the needs of the food industry,
respectively.
* Corresponding author. Dipartimento di Scienze Molecolari
Agroalimentari, Universith di Milano, via Celoria, 2 1-20133
Milano, Italy. Tel.: +39-2-2663662; fax: + 39-2-70633062.
The world production of l egume seeds, ' t he poor
man' s meat ' as devel oped-count ry producers call
them, was about 58 million tons in 1994 (FAO
estimations). Of this, the maj or part, 40 million tons,
was produced by developing countries, especially
India and China with onl y 8.5% consumed outside
the country in which it was produced. Only Ar-
gentina, Mexico, the USA and China export signifi-
cant quantities of l egume grain, while Europe is the
mai n i mport i ng continent (Heiser, 1996). Proteins
are the maj or seed component in all grain legumes,
and are the reason for their relevant nutritional and
soci o-economi cal impact. New research approaches
which rely strongly on bi ot echnol ogy to i mprove
growth and utilization of grain legumes have had
significant impacts on the nutritional quality of
legumes.
In this revi ew we will focus on the mol ecul ar and
nutritional properties of l egume seed proteins. The
perspectives for the i mpr ovement of the protein qual-
ity traits of grain legumes will arise from these
considerations. The protein and non-protein com-
0378-4290/97/$17.00 1997 Elsevier Science B.V. All rights reserved.
PII S0378-4290(97)0002 I-X
32 M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45
pounds affecting the overall nutritional value of ma-
j or legume crops will also be considered.
2. The seed proteins
Legume seeds accumulate large amounts of pro-
teins during their development. Most are devoid of
catalytic activity and play no structural role in the
cotyledonary tissue. They are stored in membrane-
bound organelles (protein bodies) in the cotyle-
donary parenchyma cells, survive desiccation on seed
maturation and undergo hydrolysis at germination,
thus providing ammonia and carbon skeletons to the
developing seedlings. Seed proteins that behave in
this way are termed ' storage proteins' . However,
because of their insolubility in water and solubility
in salt solutions, the storage proteins are also named
globulins and the two terms are commonl y used
interchangeably (Derbyshire et al., 1976). Besides
storage proteins, legume seeds contain several com-
paratively minor proteins including trypsin in-
hibitors, lectins, lipoxygenase and urease, which are
relevant to the nutritional quality of the seed. Some
of them, like urease from jack-bean, also seem to
have adopted a storage role by virtue of their amount
in the seed (Casey et al., 1986).
The catalytic proteins, most of which belong to
the vast but less abundant class of the albumins, i.e.
the proteins soluble in water, are mainly represented
by the thousands of different enzymes necessary for
the cotyledonary cell metabolism and will only be
marginally considered in this review.
2.1. The globulins
The globulins are generally classified as 11S and
7S proteins according to their sedimentation coeffi-
cients. The well-studied 11S and 7S proteins of pea
are named legumin and vicilin respectively, so that
the corresponding proteins of other seeds are often
referred to as legumin- and vicilin-like globulins.
The 7S proteins are oligomeric proteins (usually
trimers) showing pH and ionic strength-dependent
association-dissociation equilibria. The 1 IS proteins
are also oligomers (usually hexamers), but are less
susceptible to dissociation, except at very low pH or
ionic strength. Moreover, larger aggregates of 15-
18S have been reported for soybean legumin-like
proteins (Koshiyama, 1983). In the presence of dena-
turing agents, such as urea or sodium dodecylsul-
phate, both the l l S and 7S proteins liberate their
constituent polypeptide chains. These polypeptides
are naturally heterogeneous: those which have been
purified to homogeneity will often appear to be
mixtures of different molecular species, if examined
by other methods (Pusztai and Stewart, 1980). Het-
erogeneity is evident at both size and charge levels
(Brown et al., 1981; Tucci et al., 1991; Horstmann et
al., 1993), and arises from a combination of two
factors, the multigene origin of each storage globulin
(Casey et al., 1986) and the post-translational modi-
fications of relatively few expression products
(Wright, 1986). The relative contribution of these
factors vary significantly intra- and inter-generically.
It appears that the rules for the assembly of native
l l S and 7S proteins from their heterogeneous sub-
units are not well defined and that random associa-
tion might occur in many instances (Gatehouse et al.,
1981; Akazawa and Hara-Nishimura, 1985), thus
exponentially increasing the number of molecular
species in each class. The situation is further compli-
cated by the fact that many of these proteins (espe-
cially 7S) are unevenly glycosylated.
Heterogeneity is the genetic and molecular start-
ing point for modem DNA-recombinant technologies
aimed to improve the overall performance of legume
proteins.
2.1.1. The l l S globulins
The widely accepted structural model of 11S pro-
teins is still that proposed for Vicia faba legumin
(Plietz et al., 1984). In this model six monomers are
arranged in a trigonal antiprism compact structure,
which is necessary to the dense packing of the
molecules in the water-poor medium of the protein
bodies. The monomeric unit of virtually all legumin-
like globulins consists of an acidic polypeptide chain
(ranging in size from 25,000 to 50,000 daltons (Casey
et al., 1986)) disulphide-bonded to a basic chain
(usually around 20,000), also named c~ and 13 sub-
units respectively; the c~ and /3 subunits are equimo-
lar, since, as we will see below, they arise from the
post-translational proteolytic cleavage of an c~ + [3
precursor polypeptide.
M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45 33
The a +/ 3 polypeptides of legumin-like proteins
are synthesized as pre-pro-polypeptides. The first
proteolytic event consists in the co-translational re-
moval of the highly hydrophobic signal peptide
(Blobel and Dobberstein, 1975; Miintz et al., 1985),
which produces a pro-polypeptide, where formation
of intramolecular disulphide bridge(s) takes place. A
further limited proteolytic degradation, occurring im-
mediately after the deposition of the legumin in the
protein bodies, gives rise to the - S- S- l i nked c~ and
/3 mature chains (Gatehouse et al., 1984; Miintz,
1989). Cleavage of the pro-polypeptide occurs be-
tween an As n- Gl y peptide bond in proximity of a
highly hydrophylic region (Nielsen, 1984). A ' con-
vertase' which has been isolated and characterized is
responsible for this specific cleavage (Scott et al.,
1992; Murumatsu and Fukazawa, 1993; Shimada et
al., 1994). The proteolytic modification of legumin
pro-polypeptide to mature a and /3 chains triggers
the conversion of the trimeric pro-polypeptide as-
sembly to the hexameric association of the mature
legumin (Nielsen et al., 1989; Duranti et al., 1992).
The molecular basis of this phenomenon will be
made clear once the crystals of both legumin forms
will be available for X-ray analysis.
A single point mutation of lupin 11S globulin has
realized a glycosylation site, by virtue of which lupin
1 IS globulin is the only known glycosylated protein
of this class (Duranti et al., 1995).
2.1.2. The 7S globulins
The 7S globulins or vicilin-like proteins are even
more heterogeneous than legumin-like proteins, due
to an uneven glycosylation of their subunits (Scholz
et al., 1983). As an example, phaseolin can exist in a
combination of differentially glycosylated poly-
peptides (Bollini et al., 1983).
The generally-accepted structure for vicilin is a
trimer of polypeptides of Mr 40, 000-75, 000 making
up a native protein of Mr about 150, 000-170, 000
(Plietz et al., 1983). The vicilins normally do not
contain cysteines and therefore have no disulphide
bonds (Casey et al., 1986).
Most vicilins undergo a more extensive prote-
olytic processing than the legumins, resulting in a
number of polypeptide fragments which, despite the
loss of covalent continuity, still keep together in an
associated structure (Gatehouse et al., 1983; Mon-
salve et al., 1990). The scheme for derivation of the
polypeptide fragments from the 50,000 pro-poly-
peptide in pea has been made possible by comparing
amino acids with cDNA sequences (Gatehouse et al.,
1983; Lycett et al., 1983; Spencer et al., 1983).
Biosynthetic studies have indicated that Pisum vi-
cilin precursor polypeptides, like those of the legu-
min precursors, are synthesized with a N-terminal
signal peptide (Higgins and Spencer, 1981), which is
co-translationally removed inside the endoplasmic
reticulum. One of the most studied vicilins is phase-
olin, the major seed storage protein from Phaseolus
vulgaris (Bollini and Chrispeels, 1978). Its three-di-
mensional structure has been determined (Lawrence
et al., 1990; Ko et al., 1993; Lawrence et al., 1994),
thus providing a powerful tool for the investigation
of the structure-function relationships and for the
manipulation of its nutritional and functional proper-
ties (Dyer et al., 1993). Phaseolin is an heteroge-
neous trimer of Mr between 140,000 and 160,000
(Sun et al., 1975; Bollini and Chrispeels, 1978). The
three size classes of phaseolin polypeptides are struc-
turally similar and appear to be encoded by a rela-
tively small and conserved multigene family (Talbot
et al., 1984). Phaseolin is a glycoprotein with two
potential glycosylation sites (Slightom et al., 1983).
Differential glycosylation can occur at either site
giving rise to further polypeptide heterogeneity (Lioi
and Bollini, 1983).
Despite the heterogeneity of storage proteins
among species and even within a single species
(Nielsen et al., 1989), l l S and 7S globulins have
also been identified in cereals (Wen and Luthe,
1985) and non-cereal seeds (Simon et al., 1985)
suggesting that the genes involved are ancient and
originated long before speciation of the ancestral
forms of the many diverse taxa (Casey et al., 1986).
Supposition of a common ancestral origin of 7S and
11S seed storage proteins is confirmed on a molecu-
lar and genetic basis (Borroto and Dure III, 1987;
Gibbs et al., 1989; Shutov et al., 1995); despite
extreme diversity of these families of seed globulins,
there seems to be high amino acid sequence conser-
vation and structural relationship among them (Argos
et al., 1985). From this point of view the globulins
can also be regarded as evolutionary markers of the
different legume species.
34 M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45
3. Nutritional value of legume seed proteins
All legume seed proteins are relatively low in
sulphur-containing amino acids and tryptophan, but
the amounts of another essential amino acid, lysine,
are much greater than in cereal grains (Rockland and
Radke, 1981; Ampe et al., 1986). Therefore, with
respect to lysine and sulphur amino acid contents,
cereal and legume proteins are nutritionally comple-
mentary. The degree of mutual supplementation may
also depend, however, on the contents of second
limiting amino acids, i.e. threonine in cereals and
tryptophan in legumes.
The essential and non-essential amino acid pattern
of eleven major legume seeds, including up to 3
species in each genus, is shown in Fig. 1.
However, amino acid composition represents the
potential quality of a protein food, their bioavailabil-
ity being critical for the supply of amino acids in the
diet. A number of experimental approaches, devised
to assess the bioavailability of amino acids in legume
foods, namely Protein Efficiency Ratio (PER), Net
Protein Ratio (NPR), Relative NPR (RNPR) (Sarwar
and McDonough, 1990), and True Protein Digestibil-
i t y-correct ed Ami no Aci d Score (AAS DTP )
( FAO/ WHO (1990)), concurrently demonstrate that
seed proteins have a lower overall nutritional quality
than animal proteins. This can be related to their low
content of sulphur-containing amino acids (Sarwar
and Peace, 1986), the compact proteolysis-resistant
structure of native seed proteins (Desphande and
Nielsen, 1987) and the presence of antinutritional
Arg I
P i l e i I i I
L . I
n e | ] i
Val I 5,
Thr i ~ i
Met ~ 4.0
I I
e~ ~ 4.o
~ I I
! I
~ I I I I
c ~ i 1~1
Ser I l
Asx I
t a x i
o |
m i
e i
EAA
8,0 12.0 16.0 24. 0
I I I I I
0. 0 12.0 16.0 24. 0
"l
NEAA
Fig. 1. Ami no acid content of selected legumes. The l owest (white bars) and the hi ghest (black bars) content of each amino acid among the
following l egume seeds is given: a: Cajanus cajan (Neme et al., 1975), b: Canavalia ensiformis (Mabesa, 1984), c: Cicer arietinum (Attia et
al., 1994), d: Glycine max (Wang and Chang, 1995), e: Lens culinaris (Hsu et al., 1980), f: Lupinus albus (Chango et al., 1995), g: Lupinus
angustifolius (Hove, 1974), b: Lupinus luteus (Chango et al., 1995), i: Pisum sativum (Leterme et al., 1990), l: Phaseolus lunatus (Alli et
al., 1994), m: Phaseolus mungo (Mabesa, 1984), n: Phaseolus vulgaris (Steel et al., 1995), o: Vicia faba (Hebblethwite, 1983), p: Vicia
angustifolia (Mabesa, 1984), q: Vigna unguiculata (Khan et al., 1980). Values are given as g / 1 0 0 g protein. EAA: essential amino acids.
NEAA: non-essential amino acids.
M. Duranti, 6". Gius / Field Crops Research 53 (1997) 31- 45 35
compounds, which may affect digestibility of pro-
teins themselves and other components (Liener, 1979;
Bressani et al., 1982; Aw and Swanson, 1985).
4. Legume seed ANtinutritional Compounds
(ANCs)
Legume seeds contain several antinutritional pro-
tein and non-protein compounds. The presence of
ANCs in crop plants is often the result of an evolu-
tionary adaptation which enables the plant to survive
and complete its life cycle under natural conditions,
regardless of the negative consequences on the qual-
ity and safety of the food products. Indeed, due to
their antinutritional or even toxic properties, various
potentially harmful compounds have been shown to
play a protective role against insects (Peumans and
van Damme, 1995), fungi (Jellis and Vassie, 1995),
predators and a number of stress conditions
(Chrispeels and Raikhel, 1991).
4.1. Protein ANCs
Seed hydrolase inhibitors are considered to be
important in determining the quality of legume seeds
and are classified as antinutritional compounds. Their
antinutritional effect in the irreversible inhibition of
various digestive enzymes is well documented
(Leterme et al., 1992).
However, their effect is usually manifest onl y i f
the seed or the flour are consumed uncooked, since
heat denaturation normally inactivates these proteins
(Vidal-Valverde et al., 1994). Once inactivated, the
protein inhibitors may even play a positive nutri-
tional role, due to their high content of sulphur-con-
taining amino acids relative to the majority of the
seed proteins (Ryan, 1990). The most characterized
protein inhibitors are Trypsin Inhibitors (TIs) of both
the Bowman- Bi r k type (Domoney et al., 1993) in
Pisum sativum and Kunitz type in Glycine max
(Horisberger and Tacchini-Vonlanthen, 1983) and
a-amyl ase inhibitors (Moreno et al., 1990). Recently
an in vitro digestion system has been used to evalu-
ate the importance of pea seed TI in protein diges-
tion (Domoney et al., 1993). A relatively low level
of inhibition is observed when purified pea TIs are
evaluated using this system, compared to experi-
ments where soybean TIs are used. Moreover, varia-
tion in pea protein digestibility has been observed
among a set of pea lines that are near-isogenic and
contain similar seed trypsin inhibitor activities, sug-
gesting that seed compounds, other than inhibitors,
may be of greater importance in affecting protein
digestibility (A1-Wesali et al., 1995).
TIs have been proven to act as protective agents
against insect attack (Hilder et al., 1990; Johnson et
al., 1989) and their involvement in wound responses
is currently receiving attention in a number of species
(Grosjean et al., 1993).
Seed lectins are sugar-binding proteins, as well as
hemagglutinins, which are able to agglutinate red
blood cells (Etzler, 1985). Many but not all, lectins
have hemagglutinating activity (Lis and Sharon,
1986). They are found in most plant products includ-
ing those that may be eaten without heat-treatment or
processing like Solanaceae (Sharon and Lis, 1990).
The toxicity of lectins is characterized by growth
inhibition in experimental animals, and by diarrhea,
nausea, bloating and vomiting when injected in hu-
mans. Some lectins can cause agglutination of the
red blood cells followed by hemolysis and, in ex-
treme cases, death (Liener et al., 1986). Heat pro-
cessing can reduce the toxicity of lectins, but low
temperature or insufficient cooking may not com-
pletely eliminate their toxicity (van Driessche, 1988).
Although the role of lectin in legume seeds is still
controversial, there is evidence that lectins are de-
fense proteins against potential plant enemies. Since
the majority of plant lectins exhibit a specificity
against carbohydrates of animal origin, one can rea-
sonably argue that plants use this type of protein as a
defense against phytophagous invertebrates and her-
bivorous animals. Feeding trials confirm the deleteri-
ous effect of various plant lectins on insects and rats.
However due to their limited toxicity, lectins proba-
bly did not evolve as protectants of individual plants,
but rather as resistance factors which are beneficial
for the survival of the species (Peumans and van
Damme, 1995).
Allergies to legume seeds are relatively uncom-
mon in humans due to the low allergenic capacity of
storage proteins (Lalles and Peltre, 1996), but they
could develop with increased consumption, as exem-
36 M. Duranti, C. Gius / FieM Crops Research 53 (1997) 31-45
plified by peanut and soybean, whose major aller-
gens are well-characterized proteins. Interesting ex-
periments on intestinal survival, absorption and anti-
genicity of soybean antinutrients in the rat (Gyongyi
ef al., 1995) showed that the elimination or substan-
tial reduction of this undesirable biological activity
would substantially increase the nutritive value of
soybean products. Moreover, studies of proteolytic
modification and methionine enrichment of soy pro-
tein antigens showed a reduction in allergenicity and
an overall improvement of the soybean meal value
(Hajos et al., 1993).
4.2. Non-protein ANCs
Legume seeds contain a number of non-protein
ANCs with significantly different structures and ef-
fects. The most important of them are listed below.
Alkaloids limit the acceptance of various legume
seeds, such as lupin, both for their strong bitter taste
and toxicity (Markievicz et al., 1988; Cuadrado et
al., 1992). The bitter seeds contain up to 2% alka-
loids, usually lupinine, cytisine, sparteine, lupanine
and anagyrine. Due to the water solubility of alka-
loids and their low size, it is possible to remove them
from the seeds by soaking and cooking in water
(Schoeneberger et al., 1982). Lupin varieties with
low alkaloids (0.04%), which are suitable to human
and ani mal consumpt i on wi t hout debi t t eri ng
(Cuadrado et al., 1992), have been selected (Harrison
and Williams, 1982). A probable role of alkaloids
can also be to provide the seeds pest protection. In
addition, alkaloids extracted from lupin seeds can be
used for pharmacological and other biomedical pur-
poses (De la Cuadra et al., 1992).
Together with alkaloids, other compounds may
contribute to the scarce palatability of some legume
seeds. This is the case of Vicia narbonensis seeds
where the presence of y-glutamyl-ethenyl-cysteine
causes the formation of sulphurous compounds on
enzymatic hydrolysis (Peter Eichinger, personal
communication).
Phytic acid, myoinositol 1,2,3,4,5,6 hexakis-dihy-
drogen phosphate, is present in legume seeds, mak-
ing up the major portion of the total phosphorous in
the seed. Phytic acid is responsible for the reduction
of the bioavailability of essential minerals, forming
insoluble complexes which are less available for
digestion and absorption in the small intestine (De-
sphande and Cheryan, 1984). Moreover, phytates
have also been shown to inhibit the activity of
several enzymes (Knuckles et al., 1989).
Phenolic compounds, such as tannins, can cross-
link with proteins by reacting with lysine or methion-
ine residues, making them unavailable during diges-
tion (Davis, 1981). Jaff6 (1950) had already sug-
gested that there may be an inverse relationship
between protein digestibility and seed-coat color in
Phaseolus vulgaris, which contain tannins and other
polyphenols.
Saponins are a diverse group of compounds com-
monly found in legumes (Hudson and E1-Difrawi,
1979). Their general structure consists of a steroid or
triterpene group linked to one or more sugar
molecules. The presence of both polar and non-polar
groups provide saponins with strong surface-active
properties which are responsible for their adverse
biological effects. A well known toxic effect of
saponins is their ability to lyse erythrocytes, as well
as other cells, such as those found in the intestinal
mucose, thus affecting nutrient absorption (Hudson,
1979).
Vicine and convicine are confined to Vicia and
are known to be responsible for a type of hemolytic
anemia, known as favism (Hudson, 1979).
Legumes are well known inducers of intestinal
gasses (flatulence), due to the presence of a-D-
galactopyranosyl residues bound to the glucose moi-
ety of sucrose. Animals and man are not able to
digest such oligosaccharides, because of the absence
of a-galactosidase in their intestinal mucose. Conse-
quently the a-galactosides pass into the colon and
are fermented by the intestinal bacteria with produc-
tion of gas (Fleming, 1981). It is clearly desirable to
decrease the oligosaccharide content of legumes, i f
they are to be more effectively exploited as relatively
inexpensive sources of protein (Gdala et al., 1995).
Many legumes have starchy seeds. Even i f most
of the starch is digested in the upper part of the
digestive tract, some resistant starch, usually encap-
sulated in the cell walls and accounting for up to
20% of the total starch (Noah et al., 1995), may
escape digestion by endogenous enzymes of the gas-
trointestinal tract, thus contributing to intestinal fer-
mentation.
M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45 37
5. Effect of processi ng and germi nat i on on the
nutri ti onal and technol ogi cal qual i ty of l egume
seeds
Removal of undesirable components is essential
to improve the nutritional quality of legumes and
effectively utilize their full potential. Reduction in
the amounts of most ANCs, including lectins (Reddy
and Pearson, 1994) and galacto-sugars (Vidal-
Valverde et al., 1992), either by technological and
house processing or by endogenous enzymatic catal-
ysis during seed germination have been reported.
Decrease of ANCs may occur either by physical
removal or by heat inactivation, especially with pro-
tein ANCs. Cooking generally inactivates heat-sensi-
tive factors such as protein inhibitors (Table 1). In
many instances, the use of only one method may not
completely remove a given ANC and a combination
of two or more methods is required (Vidal-Valverde
et al., 1992). Polyphenols are significantly reduced
by soaking and cooking. On the other hand, the
content of tannins and catechins in lentils and broad
beans seems to increase after these treatments
(Vidal-Valverde et al., 1994). This can be attributed
to a greater accessibility to analysis of tannins, which
are originally in the interior of the cell and often
linked to proteins and other macromolecules (Hager-
man, 1992).
Seed germination also has a documented effect in
the removal of antinutrients in legumes, since it
mobilizes those compounds which are thought to
function as reserve nutrients, e.g, phytates, oligo-
saccharides and, in some instances, protein ANCs
(Table 1). However due to the different legumes,
germination conditions and analytical methods used,
the results on the effect of germination on legume
ANCs, can hardly be compared.
Heat-treatment of legume seeds not only causes a
number of desirable modifications to their chemical
composition, nutritional and organoleptic properties
(Wu et al., 1995), but can also induce losses of
essential amino acids and vitamins. Heat denatura-
tion also has a mar ked i nfl uence on the
funct i onal / t echnol ogi cal properties of legume pro-
teins, which are strictly related to their modified
physico-chemical characteristics, namely dissociation
into constituent subunits, unfolding and surface ex-
posure of the hydrophobic side groups. Heat denatu-
ration is usually accompanied by a decrease of solu-
bility, which results from the aggregation of the
unfolded molecules. These changes affect a number
of functional properties, such as the ability of gelifi-
Tabl e 1
Effect of soaki ng and cooki ng (A) and germi nat i on (B) on selected l egume seed ANCs. Values
amount / act i vi t y after t reat ment
are gi ven as percent residual
Genus TI Phytic acid Pol yphenol s
A B A B A B
Cajanus cajan 0 a 65 b 85 a 35 b 60 a 30 b
Canavalia ensiformis 0 60 c 70 c 35 65 c 20 c
Cicer arietinum 0 d 30 c 75 d 40 e 40 d 20 e
Glycine max 0 f 60 f 0 f 35 e 25 f 30 f
Lens culinaris variabilis 0 g 70 h 65 g 55 h 70 g 50 h
Lens culinaris vulgaris 0 g 75 h 70 g 30 h 60 g 50 h
Phaseolus mungo 0 i 40 i 80 i 40 i 35 i 25 i
Phaseolus vulgaris (black) 10 1 60 m 60 1 35 m 50 1 35 m
Phaseolus vulgaris (red) 30 n 65 o 50 n 40 o 55 n 30 o
Pisum sativum 0 p 60 p 50 p 45 p 40 p 35 p
Psophocarpus tetragonobolus 0 q 70 r 50 q 40 r 80 q 30 r
Viciafaba 20 s 60 t 50 s 40 t 80 s 55 t
a Mul i mani and Paramj yot hi , 1993; b Weder and Link, 1993; c Babar et al., 1988; d Attia et al., 1994; e Savage and Thompson, 1993; f Liu
and Markakis, 1987; g Vi dal -Val verde et al., 1994; h Bat ra et al., 1986; i Reddy et al., 1978; i Desphande and Cheryan, 1984; m Trugo et
al., 1980; n Dhurandhar and Chang, 1990; o Ni el sen and Liener, 1988; p Gatel, 1994; q de Lumen and Sal amamat , 1980; r Ki ng and
Puwastien, 1987; s Al et or et al., 1994; t Rahma et al., 1987.
38 M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45
cation (Moil, 1988), rheological properties of the
gels, foaming and emulsification capacity, as often
shown in soybean protein systems (Yamagishi et al.,
1980; Iawabuchi et al., 1991a; and Iawabuchi et al.,
1991b). Gel formation is an important property for
the use of soybean proteins in food systems. Differ-
ent gels are formed with the major soybean storage
proteins 11S and 7S globulins: the gels formed by
the 7S globulin are transparent, contrary to the turbid
gels formed by the l l S globulin; these latter gels
being much harder (Nakamura et al., 1986). This has
been attributed to the formation of intermolecular
disulphide exchange reactions in the l l S globulin-
formed gels. In the gel formation of the mixed
system, the 7S and 11S globulins interact non-cova-
lently to form soluble aggregates (Nakamura et al.,
1986). The extent of this interaction is affected by
the proportions of the two globulins and their subunit
composition, leading to various gelation behaviours.
Since the composition of 11S hexamers varies among
soybean cultivars, it is possible to control the rate of
gelation and the gel properties of soybean proteins
through selection a nd/ or breeding of soybean culti-
vars by considering the contribution of each subunit
to the final properties of the gels (Nakamura et al.,
1985a and Nakamura et al., 1985b; Zarkadas et al.,
1994; Ko et al., 1995). The extreme variability in the
conditions of protein denaturation (pH, ionic strength,
presence of free sulphydryl or disulphyde groups,
temperature, heating time and rate of cooling) can
significantly alter the functionality of the protein and
consequently the technological performance of a de-
rived food.
From the practical viewpoint, the cooking time is
an important variable to consider for the utilization
of a legume seed, as it is evidenced by the well
known hard-to-cook problem in common beans (Re-
yes-Moreno and Paredes-Lbpez, 1993).
6. Positive effects of legume seeds in the diet
Legume seed components may play a positive
role in the diet of normal and even pathological
subjects. For example, it has been shown that an
indirect control of polyamine presence in the gut by
lectins can slow down intestinal turnout growth
(Bardocz et al., 1995). Other studies have shown that
inclusion of beans in the diet have some protective
effects in rats fed a hypercholesterolemic diet (De
Angelis et al., 1995).
Celiac patients, who show a severe intolerance
response to food containing gliadin and glutenin
(Allmann et al., 1992), can safely be fed with beany
foods which contain only albumins and globulins.
New processing technologies producing alternative
foods are being devised. For example, pea proteins
can not form the complex structure of the wheat
gluten (Adams et al., 1991; Bahnassey et al., 1986),
but the use of emulsifiers, which favour the interac-
tion of proteins with carbohydrates, have been used
to improve the structure of the dough (Schuster,
1984; Kovacs and Gero, 1995).
Dried legume seeds are known to elicit a low
glycemic response (Yokota et al., 1994), due to
various factors including a high proportion of amy-
lose in legume starch, which increases the probabil-
ity of their retrogradation, the possible interactions of
amylolytic enzymes with antinutritional compounds
and the content and composition of oligosaccharides.
' Sl ow carbohydrates' can be beneficial to health and
help to prevent diseases such as type II diabetes,
cardiovascular problems and obesity. The low insulin
requirement in the post-prandial phase of a meal rich
in ' slow carbohydrates' could explain these apparent
beneficial effects.
7. Developments and perspectives in the produc-
tion and use of seed proteins
Study of seed storage proteins and their genes
have played an important role in the establishment
and development of plant molecular biology tech-
niques, mainly due to the advantages provided by the
massive synthesis of few proteins in a specific organ
during seed development. A detailed understanding
of storage protein structure and diversity is an impor-
tant prerequisite for attempts to manipulate quality
because it indicates the extent to which the structure
of the protein can be altered without affecting seed
biology (Shewry et al., 1995).
Many of the storage protein genes of important
crop plants have been cloned and characterized and
M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45 39
their regulation investigated (Goldberg et al., 1989;
Shotwell and Larkins, 1989; Shirsat, 1991). These
studies have provided a molecular basis for the
introduction, apart from the traditional breeding and
selection approaches, of plant genetic engineering.
Generally speaking, three genetic approaches can be
considered to improve the quality of legume pro-
teins: (i) regulation of gene expression, (ii) introduc-
tion of foreign genes, and (iii) modification of genes
encoding a given storage protein (Tabe et al., 1993).
The possibility of expressing and accumulating
legume storage proteins in transgenic plants, such as
petunia (Petunia hybrida), tobacco (Nicotiana
tabacum L.) (Beachy et al., 1985; Bray et al., 1987;
Lawton et al., 1987), Arabidopsis and Brassica
napus (rape seed) (Altenbach et al., 1989; Clercq et
al., 1990a and Clercq et al., 1990b) have been ex-
ploited. This approach has often made available in-
formation on seed protein folding, assembly, trans-
port and deposition, and in many cases has allowed
to improve the content of S-containing amino acids,
as with the 2S storage proteins of Brazil nut
(Bertholletia excelsa) (18 methionine r esi dues/ 100
amino acids) (Altenbach et al., 1989; Altenbach et
al., 1992; Saalbach et al., 1995). Now that the trans-
formation of some legume plants, namely Glycine
max and Vicia narbonensis, has been established
(Saalbach et al., 1994; Saalbach et al., 1995), this
approach will become one of the most promising
methods for improving the nutritional quality of
legume proteins.
Gene transfer of seed proteins conferring insect
resistance into crop plants is an exciting goal in the
field of plant protection. Excellent candidates for
transfer and expression are serine proteinase in-
hibitors (An et al., 1989; Hilder et al., 1987), lectins
(Chrispeels and Raikhel, 1991) and a-amyl ase in-
hibitors (Huesing et al., 1991; Ishimoto and Kita-
mura, 1989; Schroeder et al., 1993).
From the viewpoint of legume protein functional-
ity, a challenging project is the domain-exchanging
gene technology, consisting in the construction of
new genes of a given protein, but containing selected
domain(s) of another subunit. Since the contribution
of each monomeric unit of a heterogeneous storage
protein to the functionality depends upon the proper-
ties of the monomer itself, the exchange of some
critical domain will allow the production of new
storage proteins with improved functional or nutri-
tional qualities. A soybean 11S globulin domain-ex-
changing gene can be easily expressed in a trans-
genic soybean plant as a stable monomer which can
self-assemble to a 11S globulin molecule (Utsumi,
1990).
The variable regions of the storage protein genes,
which have little function in forming and maintain-
ing the storage protein structure and may tolerate
modification (Nielsen, 1985), are the targets of choice
for these techniques. These regions are hydrophilic
and are thus usually located on the surface of the
protein molecule. Therefore, the deletion of a portion
or all of the variable regions causes an increase in
hydrophobicity of the monomer molecule as a whole.
This change affects the molecular stability of the
protein and, as a result, the emulsifying property, the
gel forming rate, and the gel hardness as shown for
the soybean 11S protein (Haque et al., 1982, Haque
and Kito, 1982; Nakamura et al., 1984; Kato and
Yutani, 1988; Ki m et al., 1990). These modern
approaches imply a preformed knowledge of protein
st ruct ure/ st abi l i t y relationships. When available, 3D
structures of storage proteins, unfortunately an un-
derdeveloped field (Suzuki et al., 1983; Lawrence et
al., 1990), can be used as a template to simulate
modifications aimed at improving the nutritional and
functional performance of a seed protein (simulated
mutagenesis) (Dyer et al., 1993).
8. Conclusions
Despite their ancient use for food and feed and
the many years of intense research, legume seeds and
their components still deserve further exploitation.
The direct benefits to human nutrition and health,
and the potentialities of the proteins and other com-
pounds for functionality purposes in food architec-
ture, as well as their relative inexpensiveness largely
compensate the, often easily removable, presence of
antinutritional compounds.
After the intensive use of biotechnological tech-
niques in the field during the last decade, many
scientists have recognized that a better utilization of
legume seeds may arise only from a highly inte-
grated approach, where the role of the various seed
components both in the seed and in our diets has to
40 M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45
be understood in detail and the desired modifications
developed accordingly.
Acknowl edgements
Research supported by National Research Council
of Italy, Special Project RAISA, Subproject No. 2.
References
Adams, W., Funke, A., G~litz, H. and Schuster, G., 1991. Activity
of emulsifiers in bakery goods natural or chemical. Getreide
Mehl und Brot, 45: 357-361.
Akazawa, T. and Hara-Nishimura, I., 1985. Topographic aspects
of biosynthesis, extracellular secretion and intracellular storage
of proteins in plant cells. Annu. Rev. Plant Physiol., 36:
441-472.
Aletor, V.A., Goodchild, A.V. and El moei m, A. M. A. , 1994.
Nutritional and antinutritional characteristics of selected Vicia
genotypes. Animal Feed Science and Technology, 47: 125-
139.
Alli, I., Gibbs, M. K. O. , Konishi, Y. and Dumas, F., 1994. Identi-
fication of phaseolin polypeptide subunits in a crystalline food
protein isolate from large lima beans (Phaseolus lunatus). J.
Agric. Food Chem. , 42: 2679-2683.
Allmann, M., Condrian, H. and Li]thy, J., 1992. PCR: a possi bl e
alternative to i mmunochemi cal met hods assuring safety and
quality food. Detection of wheat contamination in non wheat
products. Zeensm. Unteres. Forsch., 96: 248-251.
Altenbach, S.B., Kual, C.C., Staraci, L.C., Pearson, K.W., Wain-
wright, C., Georgescu, A. and Townsed, J., 1992. Accumula-
tion of a Brazil nut albumin in seeds of transgenic canola
results in enhanced levels of seed protein methionine. Plant
Mol. Biol., 18: 245-245.
Altenbach, S.B., Pearson, K.W., Meeker, G., Staraci, L.C. and
Sun, S.S.M., 1989. Enhancement of methionine content of
seed proteins by the expressi on of a chimeric gene encodi ng a
methionine-rich protein in transgenic plants. Plant Mol. Biol.,
13: 513-522.
A1-Wesali, M., Lambert, N., Welham, T. and Domoney, C., 1995.
The influence of pea seed trypsin inhibitors on the in vitro
digestibility of casein. J. Sci. Food Agric., 68: 431-437.
Ampe, C., Van Damme, J., de Castro, A., Sampaio, M.J., Van
Montagu, M. and Vanderkerckhove, J., 1986. The amino acid
sequence of the 2S sulphur-rich proteins from seeds of Brazil
nut (Bertholletia excelsa HBK). Eur. J. Biochem. , 159: 597-
604.
An, G., Johnsonn, R., Narvaez, J. and Ryan, C.A., 1989. Expres-
sion of proteinase inhibitors I and II in transgenic tobacco
plants, effects on natural defense against M. sexta larvae.
Proc. Natl. Acad. Sci. USA, 86: 9871-9875.
Argos, P., Narayana, S.V.L. and Nielsen, N.C., 1985. Structural
similarity bet ween legumin and vicilin, storage proteins from
legumes. EMBO J., 4: 1111-1117.
Atria, R.S., Shehata, A. M. E. , Aman, M.E. and Hamza, M. A. ,
1994. Effect of cooking and decorticarion on the physical
properties, the chemical-composition and nutritive-value of
chickpea (Cicer arietinum). Food Chem. , 50: 125-131.
Aw, T.L. and Swanson, B.J., 1985. Influence of tannin on Phase-
olus vulgaris protein digestibility and quality. J. Food Sci., 50:
67- 71.
Babar, V.S., Chavan, J.K. and Kadam, S.S., 1988. Effects of heat
treatments and germination on trypsin inhibitor activity and
polyphenols in j ack bean. Plant Foods Hum. Nutr., 38: 319-
324.
Bahnassey, Y., Khan, K. and Harrold, R., 1986. Fortification of
spaghetti wi t h edible legumes. I. Physicochemical, antinutri-
tional, amino acid and mineral composition. Cereal Chem., 63:
210-215.
Bardocz, S., Grant, G., Brown, D.S., Duguid, T.J., Pusztai, A. and
Pryme, I.F., 1995. Effect of PHA on intestinal cell prolifera-
tion and tumour growth: role of polyammines. Abstracts of the
16th International Lectin Meeting, p. 49.
Batra, V.I.P., Vasvshata, R. and Dhindsa, K.S., 1986. Effect of
heat and germination on trypsin inhibitor activity in lentil and
pigeon pea. J. Food Sci. Technol., 23: 260-263.
Beachy, R.N., Chen, Z.L., Horsch, R.B., Rogers, S.G., Hoffman,
N.J. and Fray, R.T., 1985. Accumulation and assembly of
soybean in seeds of transformed petunia plants. EMBO J., 4:
3047-3053.
Blobel, G. and Dobberstein, B., 1975. Transfer of protein across
membrane. Part 1: presence of proteolyrically processed and
unprocessed nascent immunoglobulin light chains on mem-
branes bound ri bosomes of murine myeloma. J. Cell Biol., 67:
835-851.
Bollini, R. and Chrispeels, M.J., 1978. Characterization and sub-
cellular localization of vicilin and phytohemagglutinin, the two
major proteins of Phaseolus vulgaris L. Planta, 142: 291-298.
Bollini, R., Vitale, A. and Chrispeels, M.J., 1983. In vivo and in
vitro processing of seed reserve protein in the endoplasmic
reticulum: evi dence for two glycosylation steps. J. Cell Biol.,
96: 999-1007.
Borroto, K. and Dure III, L., 1987. The globulin seed storage
proteins of flowering plants are derived from two ancestral
genes. Plant Mol. Biol., 8: 115-131.
Bray, E.A., Naito, S., Pan, N.S., Anderson, E., Dube, P. and
Beachy, R.N., 1987. Expressi on of the /3-subunit of /3-con-
glycinin in seeds of transgenic plants. Planta, 172: 364-370.
Bressani, R., Elias, L.G. and Braham, J.E., 1982. Reduction of
digestibility of legume proteins by tannins. J. Plant Food, 4:
43- 55.
Brown, J.W.S., Bliss, F.A. and Hall, T.C., 1981. Linkage relation-
ships bet ween genes controlling seed proteins in french bean.
Theor. Appl. Genet., 60: 251-259.
Casey, R., Domoney, C. and Ellis, N., 1986. Legumes storage
proteins and their genes. Oxford Surveys of Plant Molecular
and Cell Biology, 3: 1-95.
Chango, A., Villanme, C., Bau, H.M., Nicolas, J.P. and Mijean,
L., 1995. Fracrionation by thermal coagulation of lupin pro-
teins: Physi cochemi cal characteristics. Food Res. Int., 28:
91- 99.
M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45 41
Chrispeels, M.J. and Raikhel, N.V., 1991. Lectins, lectin genes
and their role in plant defence. Plant Cell, 3: 1-19.
Clercq, A.D., Vanderwiele, M., Damme, J.V., Guerche, P., Mon-
tagu, M. V. , Vanderkerckhove, J. and Krebbers, E., 1990b.
Stable accumulation of modi fi ed 2S albumin seed storage
proteins with hi gher met hi oni ne contents in transgenic plants.
Plant Physiol., 94: 970- 979.
Clercq, A.D., Vanderwiele, M., Rycke, R.D., Damme, J.V., Mon-
tagu, M. V. , Krebbers, E. and Vanderkerckhove, J., 1990a.
Expressi on and processing of an Arabidopsis 2S albumin in
transgenic tobacco. Mol. Genet., 221: 306-314.
Cuadrado, C., Burbano, C. and Muzquir, M., 1992. Analysis of
lupine alkaloids by hi gh performance liquid chromatography.
In: ed. J. Picard, Proc. 1st Eur. Conf. Grain Legumes, AEP,
Angers, 1992, pp. 421- 422.
Davis, K.R., 1981. Effect of processi ng on composi t i on of Te-
trahyrnena relative nutritive value on green and yellow peas,
lentils and whi t e pea beans. Cereal Chem. , 58: 454- 460.
De Angelis, R.C., Terra, I., Scialfa, J.H. and Klemps, I.F., 1995.
Beans (Phaseolus vulgaris) decrease bioavailability of some
nutrients but hypocholesterolemic. In: ed. H. Sorensen, Proc.
2nd Eur. Conf. Grain Legumes, AEP, Copenhagen, 1995, pp.
280-281.
De la Cuadra, C., Tello, J.C., Muzquiz, M. and Calvo, R., 1992.
Antifungal effect of quinolizidine alkaloids from Lupinus ssp.
In: ed. J. Picard, Proc. 1st Eur. Conf. Grain Legumes, AEP,
Angers, 1992, pp. 341-342.
de Lumen, B.O. and Salamamat, L.A., 1980. Trypsin inhibitor
activity in wi nged bean (Psophocarpus tetragonolobus) and
possi bl e role of polyphenol compound. J. Agric. Food Chem. ,
28: 533-537.
Derbyshire, E., Wright, D.J. and Boulter, D., 1976. Legumi n and
vicilin, storage proteins of legume seeds. Phytochem. , 15:
3- 24.
Desphande, S. and Cheryan, M., 1984. Effects of phytic acid,
divalent cations and their interactions on a-ami l ase activity. J.
Food Sci., 49: 516- 519.
Desphande, S.S. and Nielsen, S.S., 1987. In vitro enzymatic
hydrolysis of phaseolin, the maj or storage protein of Phaseo-
lus vulgaris L. J. Food Sci., 52: 1326-1329.
Dhurandhar, N.V. and Chang, K.C., 1990. Effect of cooking on
trypsin inhibitors, lectins and cys t i ne/ cys t ei ne content of navy
and red kidney beans (Phaseolus vulgaris). J. Food Sci., 55:
470-475.
Domoney, C., Wel ham, T. and Sidebottom, C., 1993. Purification
and characterization of Pisum seed trypsin inhibitors. J. Exp.
Bot., 44: 701- 709.
Duranti, M., Guerrieri, N., Cerletti, P. and Vecchi o, G., 1992. The
legumin precursor from white lupin seed. Eur. J. Biochem. ,
206: 941-947.
Duranti, M., Horstmann, C., Gilroy, J. and Croy, R.R.D., 1995.
The molecular basis for N-glycosylation in the 11S globulin
(legumin) of lupin seed. J. Prot. Chem. , 14: 107-110.
Dyer, J.M., Nelson, J.W. and Murai, M., 1993. Strategies for
selecting mutation sites for met hi oni ne enhancement in the
bean seed storage protein phaseolin. J. Prot. Chem. , 12: 545-
560.
Etzler, M.E., 1985. Plant lectins: molecular and biological aspects.
Annu. Rev. Plant Physiol., 36: 209-234.
FAO/ WHO, 1990. Protein quality evaluation report of a j oi nt
F AO/ WHO expert consultation. Food and Nutrition Paper,
51.
Fleming, S.E., 1981. A study of relationships bet ween flatus
potential and carbohydrate distribution in legume seeds. J.
Food Sci., 46: 794-798.
Gatehouse, J.A., Croy, R.D. and Boulter, D., 1984. The synthesis
and structure of pea storage proteins. CRC Crit. Rev. Plant
Sci., 1: 287-314.
Gatehouse, J.A., Croy, R.R.D., Morton, H., Tyler, M. and Boulter,
D., 1981. Characterization and subunit structure of the vicilin
storage proteins of pea (Pisum sativum L.). Eur. J. Biochem.,
118: 627-633.
Gatehouse, J.A., Lycett, G.W., Delauney, A.J., Croy, R.R.D. and
Boulter, D., 1983. Sequence specificity of the post-transla-
tional proteolytic cleavage of vicilin, a seed storage protein of
pea (Pisum sativum L.). Biochem. J., 212: 427- 432.
Gatel, F., 1994. Protein quality of l egume seeds for non-ruminant
animals. Animal Feed Sci. and Technol., 45: 317-348.
Gdala, J., Buraczewska, L. and Wasilewsko, J., 1995. Ileal di-
gestibility of lupin seed carbohydrates in young pi gs and the
effect of a-galactosidase supplementation. In: ed. H. Sorensen,
Proc. 2nd Eur. Conf. Grain Legumes, AEP, Copenhagen,
1995, p. 289.
Gibbs, P.E.M., Strongin, K.B. and McPherson, A., 1989. Evolu-
tion of legume seed storage proteins: a domain common to
legumins and vicilins is duplicated in vicilins. Mol. Biol.
Evol., 6: 614-623.
Goldberg, R.S., Barker, S.J. and Perez-Grau, L., 1989. Regulation
of gene expression during plant embryogenesis. Cell, 56:
149-160.
Grosjean, F., M&ayer, J.P., Peyronnet, C. and Carroure, B., 1993.
Variability in trypsin inhibitor activity of peas ( Pi sum sativum)
grown in France. In: eds. A.F.B. van der Poel, J. Heusman,
H.S. Saini, Recent Advances of Research in Antinutritional
Factors in Legume Seeds. Wageni ngen Press, Wageningen,
The Netherlands, pp. 411- 415.
Guillon, F. and Champ, M., 1996. Grain legumes and transit in
humans. Grain Legumes, AEP (ed.), 11: 18.
Gyongyi, H., Gelencser, E., Pusztai, A., Grant, G., Sakhiri, M.
and Bardocz, S., 1995. Biological effects and survival of
trypsin inhibitors and the agglutinin from soybean in the small
intestine of the rat. J. Agric. Food Chem. , 43: 165-170.
Hagerman, A.E., 1992. Phenolic compounds in food and their
effects on health. In: eds. C.T. Ho, C. Lee and M.T. Huang,
ACS Symposi um Series 506, Ameri can Chemical Society,
Washingthon, DC, 1: 236-247.
Hajos, G., Gelencser, E. and Polgar, M., 1993. Comparative study
on bioavailabilty of different soyprotein antigens. Ber. Bun-
desforshungsanst. Ernaehr., 1: 44- 52.
Haque, Z. and Kito, M., 1982. Lipophylization of soybean glycinin:
42 M. Duranti, C. Gius / FieM Crops Research 53 (1997) 31-45
covalent attachment to long chain fatty acids. Agric. Biol.
Chem. , 46: 597-599.
Haque, Z., Mataba, T. and Kito, M., 1982. Incorporation of fatty
acid into food proteins: palmitoyl soybean glycinin. J. Agric.
Food Chem. , 30: 481- 486.
Harrison, J.E.M. and Williams, W., 1982. Genetical control of
alkaloids in Lupinus albus. Euphytica, 31: 357-364.
Hebblethwite, P.D., 1983. The faba bean (Vicia faba L.). Butter-
worths Press, London, 600 pp.
Heiser, M., 1996. Trade and consumpt i on of l egume seeds. Grain
legumes, AEP, 11: 14-15.
Higgins, T.J.V. and Spencer, D., 1981. Precursor form of pea
vicilin subunits. Modification by microsomal membranes dur-
ing cell-free translation. Plant Physiol., 67: 209- 211.
Hilder, V.A., Gatehouse, A.M.R. and Boulter, D., 1990. Genetic
engineering of crops for insect resistance using genes of plant
origin. In: eds. D. Grierson, G. Lycett, Genetic Engineering of
Crop Plants. Butterworths, London, pp. 51- 56.
Hilder, V.A., Gatehouse, A.M.R., Sheerman, S.E., Barker, R.F.
and Boulter, D., 1987. A novel mechani sm of insect resistance
engi neered into tobacco. Nature, 330: 160-163.
Horisberger, M. and Tacchini-Vonlanthen, M., 1983. Ultrastruc-
tural localization of Kunitz inhibitor on thin sections of Glycine
max (soybean) CV. Maple Arrow by the gol d method. His-
tochem., 77: 37- 50.
Horstmann, C., Schlesier, B., Otto, A., Kostka, S. and Mtintz, K.,
1993. Pol ymorphi sm of legumin subunits from field bean
(Vicia faba L. var. minor) and its relation to the corresponding
multigene family. Theor. Appl. Genet., 86: 867-874.
Hove, E.L., 1974. Composi t i on and protein quality of sweet lupin
seed. J. Sci. Food Agile. , 25: 851-859.
Hsu, D., Leung, H.K., Finney, P.L. and Morad, M. M. , 1980.
Effect of germination on nutritive value and baking properties
of dry peas, lentils and faba beans. J. Food Sci., 45: 87-95.
Hudson, B.J.F., 1979. The nutritional quality of lupin seed. Qual.
P1. Foods Hum. Nutr., 29: 245-251.
Hudson, B.J.F. and E1-Difrawi, E.A., 1979. The sapogenins of the
seeds of four lupin species. J. Plant Foods, 3: 181-186.
Huesing, J.E., Shade, R.E., Chfispeels, M.J. and Murdock, L.L.,
1991. ot-amilase inhibitor, not phytohemagglutinin explains
the resistance of common bean seeds to cowpea weevil. Plant
Physiol., 96: 995-996.
Iawabuchi, S., Watanabe, H. and Yamauchi, F., 1991a. Thermal
denaturation of /3-conglycinin. Kinetic resolution of reaction
mechani sm. J. Agric. Food Chem., 39: 34- 40.
Iawabuchi, S., Watanabe, H. and Yamauchi, F., 1991b. Observa-
tion on the dissociation of / 3- congl yci ni n into subunits by heat
treatment. J. Agric. Food Chem., 39: 34-40.
Ishimoto, M. and Kitamura, K., 1989. Growth inhibitory effects of
an a-ami l ase inhibitor from kidney bean, Phaseolus vulgaris
(L.) on three species of bruchids (Coleoptera: Bruchidae).
Appl. Ent. Zool., 2: 281-286.
Jaff6, W.G., 1950. Protein digestibility and trypsin inhibitor activ-
ity of legume seeds. Proc. Soc. Exp. Biol. Med., 75: 219-221.
Jellis, G.J. and Vassie, V., 1995. Combi ni ng resistance to As-
cochyta blight with low anti-nutritional factors in faba bean.
In: ed. H. Sorensen, Proc. 2nd Eur. Conf. Grain Legumes,
AEP, Copenhagen, 1995, pp. 88-89.
Johnson, R., Narvaez, J., An, G. and Ryan, C.A., 1989. Expres-
sion of proteinase inhibitors I and II in transgenic tobacco
plants: effects on natural defence against Manduca sexta
larvae. Procl. Natl. Acad. Sci. USA, 86: 9871-9875.
Kato, A, and Yutani, K., 1988. Correlation of surface properties
with conformafional stabilities of wild-type six mutant trypto-
phan synthase ot-subunit substituted at the same position.
Protein Engin., 2: 153-156.
Khan, R.I., Gatehouse, J.A. and Boulter, D., 1980. The seed
proteins of cowpea (Vigna unguiculata L.). J. Exp. Bot., 31:
1599-1611.
Kim, C., Kamiy, S., Sato, T., Usami, S. and Kito, M., 1990.
Improvement of nutritional value and functional properties of
soybean glycinin by protein engineering. Prot. Engin., 3:
725-731.
King, R.D. and Puwastien, P., 1987. Effect of germination on the
proximate composition and nutritional quality of wi nged bean.
J. Food Sci., 52: 106-108.
Knuckles, B.E., Kuzmicky, D.D., Gumbmann, M.R. and Betschart,
A.A., 1989. Effect of myo-inositol phosphat e esters on in vitro
and in vivo digestion of proteins. J. Food Sci., 54: 1348-1350.
Ko, T.P., Ng, J.D. and McPherson, A., 1993. The three-dimen-
sional structure of canavalin from Jack bean (Canavalia ensi-
formis). Plant Physiol., 101: 729-744.
Ko, H., Yama, K., Murata, M., Tari, F., Sano, Y. and Doi, E.,
1995. Effects of protein composition on gelation of mixtures
containing soybean 7S and l l S globulins. Biosci. Biotech.
Biochem., 69: 240-245.
Koshiyama, I., 1983. Storage proteins of soybean. In: eds. W.
Gottschalk and H.P. Muller, Seed Proteins. Biochemistry,
Genetics, Nutritive Value. Martinus Ni j hof f / Dr . W. Junk
Publishers: The Hague, pp. 428-450.
Kovacs, E. and Gero, L., 1995. The effect of emulsifiers on the
quality of macaroni doughs from leguminous materials. In: ed.
H. Sorensen, Proc. 2nd Eur. Conf. Grain Legumes, AEP,
Copenhagen, 1995, p. 284.
Lalles, J.P. and Peltre, G., 1996. Some aspects of legume protein
allergens. Grain Legumes, AEP (ed.), 11: 20.
Lawrence, M.C., Izard, T., Beuchat, M., Blagrove, R.J. and
Colman, P.M., 1994. Structure of phaseolin at 2.2 A resolu-
tion. J. Mol. Biol., 238: 748-776.
Lawrence, M.C., Suzuki, E., Varghese, J.V., Davis, P.C., van
Donkelaar, A., Tulloch, P.A. and Colman, P.M., 1990. The
three-dimensional structure of the storage protein phaseolin at
3 A resolution. EMBO J., 9: 9- 15.
Lawton, M. A. , Tierney, M.A., Nakamura, I., Anderson, E., Ko-
mido, Y., Dube, P., Hoffman, N., Fraley, R.T. and Beachy,
R.N., 1987. Expression of a soybean fl-conglycinin gene
under the control of the cauliflower mosaic vires 35S and 19S
promotors in transformed petunia tissues. Plant Mol. Biol., 9:
315-324.
Leterme, P., Monmart, T. and Baudart, E., 1990. Ami no acid
composition of pea (Pisum sativum) proteins and proteins
profile of pea flour. J. Sci. Food Agric., 53: 107-110.
M. DurantL C. Gius / Field Crops Research 53 (1997) 31-45 43
Leterme, P., Monmart , T. and Thrwes, A., 1992. Varietal distribu-
tion of trypsin inhibitors in peas. In: ed. J. Picard, Proc. 1st
Eur. Conf. Grain Legumes, AEP, Angers, 1992, pp. 417-418.
Liener, I.E., 1979. Legume toxins in relation to protein digestibil-
ity. J. Food Sci., 41: 1076-1081.
Liener, I.E., Sharon, N. and Goldstein, I.J., 1986. The Lectins:
Properties, Functions and Applications in Biology and
Medicine. Academi c Press, Orlando, FL.
Lioi, L. and Bollini, R., 1983. Contribution of processing events
to the molecular heterogeneity of four banding types of phase-
olin, the maj or storage protein of Phaseolus vulgaris L. Plant
Mol. Biol., 3: 345-353.
Lis, H. and Sharon, N., 1986. Lectins as mol ecul es and as tools.
Annu. Rev. Biochem., 55: 35- 67.
Liu, K. and Markakis, P., 1987. Effect of maturity and processing
on the trypsin inhibitor and oligosaccharides of soybeans. J.
Food Sci., 52, 22- 225.
Lycett, G.W., Delauney, AJ . , Gatehouse, J.A., Gilroy, J., Croy,
R.R.D. and Boulter, D., 1983. The vicilin gene family of pea
(Pisum sativum L.): a compl et e cDNA coding sequence for
pre-pro-vicilin. Nucleic Aci d Res., 11: 2367-2380.
Mabesa, L., 1984. Protein quality of flours from germinated
legumes. J. Sci. Food Agric., 35: 212-214.
Marklevicz, M., Kolanowska, A. and Gulewics, K., 1988. The
chemi cal composi t i on of debittered lupine seeds and their
extract. Bull. Pol. Acad. Sci. Biol. Sci., 36: 1- 3.
Monsalve, R.I., Menendez, L.A., Lbpez-Ofin, C. and Rodriguez,
R., 1990. /3-turus as structural motifs for the proteolytic
processi ng of seed proteins. FEBS: 263: 209-212.
Moreno, J., Altabella, T. and Chrispeels, M.J., 1990. Characteriza-
tion of a-amyl ase inhibitor, a lectin-like protein in the seeds
of Phaseolus vulgaris. Plant Physiol., 92: 703-709.
Mori, T., 1988. Mechani sms of the gel formation of soybean
proteins. J. Agric. Chem. Soc. Jpn., 62: 882-885.
Mulimani, V.H. and Paramjyothi, S., 1993. Effect of heat and UV
on trypsin and chymot rypsi n inhibitor activities in red gram
(Cajanus cajan L.). J. Food Sci. Technol., 30: 62- 63.
Miintz, K., 1989. Intracellular protein sorting and the formation of
protein reserves in storage tissue cells of plant seeds. Biochem.
Physiol. Pflanzen, 185: 318-335.
Mtintz, K., Bassiiner, R., Lichtenfeld, C., Scholz, G. and Weber,
E., 1985. Proteolytic cleavage of storage proteins during em-
bryogenesi s and germination of seeds. Physiol. Veg., 23:
75- 94.
Murumatsu, M. and Fukazawa, C., 1993. A high order structure of
plant storage pro-protein allows its second conversion by
asparagine-specific cyst ei ne protease, a novel proteolytic en-
zyme. Eur. J. Biochem., 215: 123-132.
Nakamura, T., Utsumi, S., Kitamura, K., Harada, K. and Mori, T.,
1984. Cultivar difference in gelling characteristics of soybean
glycinin. J. Agric. Food Chem. , 32: 647-649.
Nakamura, T., Utsumi, S. and Mori, T., 1985a. Effects of temper-
ature on the different stages in thermal gelling of glycinin. J.
Agric. Food Chem. , 35: 1201-1203.
Nakamura, T., Utsumi, S. and Mori, T., 1985b. Formation of
pseudoglycinins from intermediary subunits of glycinin and
their gel properties and network structure. Agric. Biol. Chem. ,
49: 2733-2740.
Nakamura, T., Utsumi, S. and Mori, T., 1986. Interactions during
heat-induced gelation in a mi xed syst em of soybean 7S and
11S globulins. Agric. Biol. Chem. , 50: 2429-2435.
Neme, S.P., Vakil, V.K. and Srince-Vason, A., 1975. Effect of
gamma irradiation on red gram (Cajanus cajan) proteins. J.
Food Sci., 40: 815-819.
Nielsen, N.C., 1984. The chemi st ry of legume storage proteins.
Phyl. Trans. R. Soc. London, 304: 287-296.
Nielsen, N.C., 1985. The structure and complexity of the l l S
polypeptides in soybean. J. Am. Oil Chem. Soc., 62: 1680-
1686.
Nielsen, N.C., Dickinson, C.D., Cho, T.J., Thanh, V.H., Scallon,
B.J., Fisher, R.L., Sims, T.L., Drews, G.N. and Goldberg,
R.B., 1989. Characterization of the glycinin gene family in
soybean. Plant Cell, 1: 313-328.
Nielsen, S.S. and Liener, I.E., 1988. Effect on germination on
trypsin inhibitor and hemagglutinating activities in beans
(Phaseolus vulgaris). J. Food Sci., 53: 298-301.
Noah, L., Guillon, F., Mollis, C., Faisant, N., Galmiche, J.P. and
Champ, M., 1995. Digestion of carbohydrate component s of
dry beans (Phaseolus vulgaris) in healthy humans. In: ed. H.
Sorensen, Proc. 2nd Eur. Conf. Grain Legumes, AEP, Copen-
hagen, 1995, p. 276.
Peumans, W.J. and van Damme, E.J.M., 1995. The role of lectins
in plant defense. Histochem. J., 27: 253-271.
Plietz, P., Damaschun, G., Zirwer, D., Gast, K. and Schlesier, B.,
1983. Structure of 7S seed globulin from Phaseolus vulgaris
L. in solution. Int. J. Biol. Macromol. , 5: 356-360.
Plietz, P., Zirwer, D., Schlesier, B., Gast, K. and Damaschun, G.,
1984. Shape, symmetry, hydration and secondary structure of
the legumin from Vicia faba in solution. Biochem. Biophys.
Acta, 784: 140-146.
Pusztai, A. and Stewart, J.C., 1980. Molecular size, subunit
structure and microheterogeneity of glycoprotein II from the
seeds of ki dney bean (Phaseolus vulgaris L.). Biochem. Bio-
phys. Acta, 623: 418-428.
Rahma, E.M., E1-Bedawey, A. A. , E1-Adaw, T.A. and Goma,
M. A. , 1987. Changes in chemi cal and antinutritional factors
and functional properties of faba beans during germination.
Lebensm. Wiss. Technol., 20: 271-276.
Reddy, N.P., Balakrishnan, C.V. and Salunkhe, D.K., 1978. Phy-
tate, phosphorous and mineral changes during germination and
cooking of black gram (Phaseolus mungo) seeds. J. Food Sci.,
43: 540-543.
Reddy, N.R. and Pearson, M.D., 1994. Reduction in antinutri-
tional and toxic component s in plant foods (A) by fermenta-
tion. Food Res. Int., 27: 281-290.
Reyes-Moreno, C. and Paredes-Lbpez, O., 1993. Hard-to-cook
phenomenon in common beans. A review. CRC Crit. Rev.
Food Sci. Nutr., 33: 227-286.
Rockland, L.B. and Radke, T.M., 1981. Legume protein quality.
Food Technol., 28: 79- 82.
Ryan, C.A., 1990. Protease inhibitors in plants: genes for improv-
ing defenses against insects and pathogens. Annu. Rev. Phy-
topathol., 28: 425-449.
Saalbach, I., Pickard, T., Machmehl, F., Saalbach, G., Schieder,
O. and Miintz, K., 1994. A chimeric gene encodi ng the
methionine-rich 2S albumin of the Brazil nut Bertholletia
44 M. Duranti, C. Gius / Field Crops Research 53 (1997) 31- 45
excelsa HBK is stably expressed in inherited in transgenic
grain legumes. Mol. Gen. Genet., 242: 226-236.
Saalbach, I., Waddel, D., Pickard, T., Schieder, O. and Miintz, K.,
1995. Stable expressi on of the sulphur rich 2S albumin gene in
transgenic Vicia narbonensis increases the met hi oni ne content
of seeds. J. Plant Physiol., 145: 674-681.
Sarwar, G. and McDonough, F.E., 1990. Evolution of protein
digestibility-corrected amino acid score met hod for assessing
protein quality of foods. J. Assoc. Off. Anal. Chem. , 73:
347-356.
Sarwar, G. and Peace, R.W., 1986. Comparisons bet ween true
digestibility of a total nitrogen and limiting amino acids in
vegetable proteins fed to rats. J. Nutr., 116: 1172- 1181.
Savage, G.P. and Thompson, D.R., 1993. Effect of processing on
the trypsin inhibitor content and nutritive value of chick peas
(Cicer arietinum). Recent advances of research in antinutri-
tional factors in l egume seeds, Wageni ngen Press, The Nether-
lands, pp. 435-440.
Schoeneberger, H., Gross, R., Cremer, H.D. and Elmadfa, I.,
1982. The protein quality of water debittered lupines (Lupinus
mutabilis) in combination wi t h other protein sources. Nutr.
Rep. Intern., 25: 763-771.
Scholz, G., Manteuffel, R., Miintz, K. and Rudolph, A., 1983.
Low molecular-weight polypeptides of vicilin from Vicia f aba
L. are products of proteolytic breakdown. Eur. J. Biochem. ,
132: 103-107.
Schroeder, H.E., Scholtz, A.H., Wardley-Richardson, T., Spencer,
D. and Higgins, T.J.V., 1993. Transformation and regeneration
of two cultivars of pea (Pisum sativum L.). Plant Physiol.,
101: 751-757.
Schuster, G., 1984. Emulsifiers in bread and other baked goods,
direct and indirect effects. Zeitschrift Unt erschung und
Forschung, 179: 190-196.
Scott, M.P., Jung, R., Miintz, K. and Nielsen, N.C., 1992. A
protease responsible for post-translational cleavage of a con-
served As n- Gl y linkage in glycinin, the maj or seed storage
protein of soybean. Proc. Natl. Acad. Sci. USA, 89: 658-662.
Sharon, N. and Lis, H., 1990. Legume lectins a large family of
homol ogous proteins. FASEB J., 4: 3198-3208.
Shewry, P.R., Napier, J.A. and Tatham, A., 1995. Seed storage
proteins: structure and biosynthesis. Plant Cell, 7: 945-956.
Shimada, T., Hirawa, N., Nishimura, M. and Hara-Nishimura, I.,
1994. Vacuolar processing enzyme of soybean that converts
pro-proteins to the corresponding mature forms. Plant Cell
Physiol., 35: 1713-1718.
Shirsat, A.H., 1991. Control of gene expression in the developing
seed. In: ed. D. Grierson, Devel opment al Regulation of Plant
Gene Expression. Chapman and Hall, New York, pp. 153-181.
Shotwell, M.A. and Larkins, B.A., 1989. The biochemistry and
molecular biology of seed storage proteins. In: ed. A. Marcus,
The Biochemistry of Plants, Academi c Press, New York, pp.
287-345.
Shutov, A.D., Kakhovskaya, I.A., Braun, H., B~iumlein, H. and
Miintz, K., 1995. Legumin-like and vicilin-like seed storage
proteins: evi dence for a common single-domain ancestral gene.
J. Mol. Evol., 41: 1057-1069.
Simon, A.E., Tenbarge, K.M., Scofield, S.R., Finkelstein, R.R.
and Crauch, M.L., 1985, Nucleotide sequence of a cDNA
clone of Brassica napus 12S storage protein shows homol ogy
wi t h legumin from Pisum sativum. Plant Mol. Biol., 5: 191-
201.
Slightom, J.L., Sun, S.M. and Hall, T.C., 1983. Compl et e nu-
cleotide sequence of a french bean storage protein gene:
phaseolin. Proc. Natl. Acad. Sci. USA, 80: 1897-1901.
Spencer, D., Chandler, P.M., Higgins, T.J.V., Inglis, A.S. and
Rubira, M., 1983. Sequence inter-relationships of the subunits
of vicilin from pea seeds. Plant Mol. Biol., 2: 259-267.
Steel, J.C., Sgarbieri, V.C. and Jackx, M.H., 1995. Use of extru-
sion technology to overcome undesirable properties of hard to
cook dry beans ( Phaseol us vulgaris L.). J. Agric. Food Chem.,
43: 2487-2492.
Sun, S.M., Buchbinder, B.U. and Hal!, T.C., 1975. Cell free
synthesis of the major storage protein of the bean, Phaseolus
vulgaris L. Plant Physiol., 56: 780-785.
Suzuki, E., Vandonkelaar, A., Varghese, J.N., Lilley, G.G., Bla-
grove, R.J. and Colman, P.M., 1983. Crystallization of phase-
olin from Phaseolus vulgaris. J. Biol. Chem., 258: 2634-2636.
Tabe, L.M., Higgins, C.M., McNabb, W.C. and Higgins, T.J.V.,
1993. Genetic engineering of grain and pasture legumes for
i mproved nutritive value. Genetika, 90: 181-200.
Talbot, D.R., Adang, M.J., Slightom, J.L. and Hall, T.C., 1984.
Size and organization of a multigene family encoding phase-
olin, the maj or seed storage protein of Phaseolus vulgaris L.
Mol. Gen. Genet., 198: 42- 49.
Trugo, L.C., Ramos, L.A., Trugo, N.M.F. and Souza, M.C.P.,
1980. Oligosaccharide composition and trypsin inhibitor activ-
ity of Phaseolus vulgaris and the effect of germination on the
alpha-galactoside composition and fermentation in the human
colon. Food Chem., 36: 53-61.
Tucci, M., Capparelli, R., Costa, A. and Rao, R., 1991. Molecular
heterogeneity and genetics of Vicia f aba seed storage proteins.
Theor. Appl. Genet., 81: 50- 58.
Utsumi, S., 1990. Improvement of nutritional value and functional
properties of soybean proteins and protein engineering. New
Food Industry, 32: 71- 84.
van Driessche, E., 1988. Structure and function of leguminosae
lectins. In: ed. H. Franz, Advances in Lectin Research,
Springer, Berlin, 1: 73- 134.
Vidal-Valverde, C., Frias, J., Estrella, I., Gorospe, N.J., Ruiz, R.
and Bacon, J., 1994. Effect of processing on some antinutri-
tional factors of lentils. J. Agfic. Food Chem., 42: 2291-2295.
Vidal-Valverde, C., Frias, J. and Valverde, S., 1992. Effect of
processing on the soluble carbohydrate content of lentils. J.
Food Prot., 95: 301-304.
Wang, C.C.R. and Chang, S.K.C., 1995. Physicochemical proper-
ties and tofu quality of soybean cultivars Proto. J. Agric. Food
Chem. , 43: 3020-3034.
Weder, J.K.P. and Link, I., 1993. Effect of treatments on legume
inhibitor activity against human proteinases. In: Recent Ad-
vances of Research in Antinutrifional Factors in Legume
Seeds, Wageni ngen Press, The Netherlands, pp. 481-485.
Wen, T.N. and Luthe, D.S., 1985. Biochemical characterization of
rice glutelin. Plant Physiol., 78: 172-177.
Wright, D.J., 1986. The seed globulins. In: ed. B.J.F. Hudson,
M. Duranti, C. Gius / Field Crops Research 53 (1997) 31-45 45
Development in Food Proteins-5, Elsevier, Amsterdam, pp.
81-157.
Wu, W., Williams, W.P., Kunkel, M.E., Acton, J.C., Huang, Y.,
Wardlow, F.B. and Grimes, L.W., 1995. True protein di-
gestibility and digestibility-corrected amino acid score of red
kidney beans (Phaseolus vulgaris L.). J. Agric. Food Chem.,
43: 1295-1298.
Yamagishi, T., Yamauchi, F. and Shibasa, K., 1980. Isolation and
partial characterization of heat-induced products of soybean
11S globulin and their analysis by electrophoresis. Agric. Biol.
Chem., 44: 1575-1582.
Yokota, Y., Ishibashi, K., Kamada, K., Tanaka, S., Pondevida,
J.L., Dominguez, L.G., Lalusis, B.M., Pigao, C.G. and Pan-
lasigui, R.A., 1994. Preparation and performance of blow
release microcapsules containing nutrient by complex emul-
sion method. Philipp. J. Soc., 123: 121-133.
Zarkadas, C.G., Yu, Z., Voldeng, M.D., Hope, H.J., Minero-
Amador, A. and Rochemont, J.A., 1994. Comparison of the
protein-bound and free amino acid contents of two northem
adapted soybean cultivars. J. Agfic. Food Chem., 42: 21-33.