Food Biochemistry and Food Processing (2 edition)

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664 Part 5: Fruits, Vegetables, and Cereals

peptide bonds somewhere along the protein chain, whereas ex-

oproteases attack the ends of the protein chain and remove one

amino acid at a time. The latter are called carboxypeptidases

when acting from the carboxy terminus or aminopeptidases

when acting from the amino terminus. Based on the chemistry

of their catalytic mechanism, proteases have been classified into

four groups: serine (E.C. 3.4.21), metallo- (E.C. 3.4.24), aspartic

(E.C. 3.4.23), and cysteine (E.C. 3.4.22) proteases.

Nowak and Mierzwinska (1978) reported the presence of

endopeptidase, carboxypeptidase, and aminopeptidase activi-

ties in the embryo and endosperm of rye seeds. Endoprote-

olytic, exoproteolytic, carboxypeptidase, aminopeptidase, and

N-α-benzoylarginine-p-nitroanilide (BAPA) hydrolyzing activ-

ities have also been found in whole meal extracts from ungermi-

nated rye seeds by Brijs et al. (1999). During germination, pro-

teolytic activity increases during the first 3 days to then remain

constant (Brijs et al. 2002). Milling fractions of germinated rye

(Brijs et al. 2002) have much higher proteolytic activities than

those of ungerminated rye (Brijs et al. 1999). The enzymes are

mainly present in the bran and shorts fractions. Ungerminated

rye mainly contains two classes of proteases (serine and aspartic

proteases) (Brijs et al. 1999), whereas all four protease classes

are present during germination (Brijs et al. 2002). Germinated

rye contains mainly aspartic and cysteine protease activities;

serine and metalloproteases are less abundant.

Aspartic proteases have been purified from bran of unger-

minated rye (Brijs 2001). Two heterodimeric aspartic proteases

were found: a larger 48 k enzyme, consisting of 32 k and 16 k

subunits which are probably linked by disulphide bridges and a

smaller one of 40 k, consisting of two noncovalently bound 29 k

and 11 k subunits. Both proteins have isoelectric points close

to pI 4.6. Rye aspartic proteases show optimal activity around

pH 3.0 and 45◦C and can be completely inhibited by pepstatin

A (Brijs 2001). Amino acid sequence alignment showed that

the rye aspartic proteases resemble those from wheat (Bleukx

et al. 1998) and barley (Sarkkinen et al. 1992). Cysteine pro-

teases were isolated from the starchy endosperm of 3-day ger-

minated rye (Brijs 2001). SDS-PAGE revealed three protein

bands with apparent molecular weights of 67 k, 43 k, and 30 k,

although it was not established that all three protein bands are

cysteine proteases. The rye cysteine proteases are optimally ac-

tive around pH 4.0 and at 45◦C and can be totally inhibited

by l-transepoxysuccinyl-l leucylamido-(4-guanidino)butane

(or E-64).

Protease inhibitors can reduce the activity of proteases and

have also been found in rye. Boisen (1983) and Lyons et al.

(1987) isolated a trypsin inhibitor from rye endosperm and rye

seed, respectively. The inhibitor had a molecular weight of about

12 to 14 k, contained four disulphide bridges, and was resistant

to trypsin and elastase but was completely inactivated by chy-

motrypsin. The amino acid composition was very similar to that

of the barley and wheat trypsin inhibitors.

Mosolov and Shul’gin (1986) purified a subtilisin inhibitor

from rye that belongs to the Kunitz-type inhibitor family. The

inhibitor consisted of two forms with pI values of 6.8 and 7.1

and had a molecular weight of about 20 k. It inhibited subtilisin

(at a molar ration of 1:1) and fungal proteinases from the genus

Aspergillus, but was inactive against trypsin, chymotrypsin, and

pancreatic elastase. The amino acid composition of the rye sub-

tilisin inhibitor was similar to that from its wheat and barley

counterparts.

Hejgaard (2001) purified and characterized six serine protease

inhibitors, also called “serpins,” from rye grain. The serpins each

have different regulatory functions based on different reactive

center loop sequences, allowing for specific associations with the

substrate-binding subsites of the proteases. The reactive center

loops of the rye serpins were similar to those of wheat serpins.

Five of the six serpins contained one or two glutamine residues at

the reactive center bond, which were differently positioned than

in the wheat reactive center loops. The rye serpins have a molec-

ular weight of 43 k and inhibit proteases with chymotrypsin-like

specificity, but not pancreatic elastase or proteases with trypsin-

like specificity.

Protein Physicochemical Properties

The properties of rye proteins, for example, their foam-forming

and emulsifying capacities, have hitherto not been studied well.

Ludwig and Ludwig (1989) isolated a protein mixture from rye

flour that exhibited low foam stability but excellent emulsifying

properties at pH 3.5. Meuser et al. (2001) reported on a rye water-

soluble protein with foam-forming capacity. It had a molecular

weight of 12.3 k and an isoelectric point of 5.97. The optimum

pH value for the water-soluble rye protein to form stable foam

was pH 6.0. The protein did not appear to be located in spe-

cific morphological parts of the rye kernel. Wannerberger et al.

(1997) found that secalins are more surface-active than gliadins.

The lower molecular weight secalins spread more rapidly at the

air-water interface, resulting in a higher surface pressure, than

did the higher molecular weight gliadins. The surface pressure

developed by both secalin and gliadin decreased with decreasing

pH. Different rye milling fractions showed differences in surface

behavior in relation to their protein content. The milling fraction

with the highest protein content spread fastest and reached the

highest surface pressure value.

RYE PROCESSING

While rye processing has gained interest because of its perceived

positive health effects (Poutanen 1997), the functional properties

of the rye major constituents limit the processing performance

of rye in industrial processes (Weipert 1997). Milling and bread

making are the most common processes in rye grain processing

for human consumption.

Rye Milling

The objective of milling is to separate the bran and the germ from

the starchy endosperm and to reduce the starchy endosperm in

size, in order to obtain a refined product, that is, flour. Rye

milling differs somewhat from wheat milling. Rye kernels are

smaller than wheat kernels, and the separation of the endosperm

from the bran is poorer in rye than in wheat (Nyman et al. 1984).

The extraction rate of rye flour is lower than that of wheat flour,