Food Biochemistry 2B BIOCHEMICAL CONSTITUENTS OF PLANT FOODS

PROTEINS

Essential amino acids

The balancing of the pattern of amino acid supply against the needs for synthesis is a major
function of the liver.
C ® oxidized to produce energy and converted to fats for storage
N ® converted to urea for excretion or used to produce non-essential amino acids

The standard against which the nutritional value of protein is based is human milk. Animal
proteins do not differ significantly from human milk with regard to essential amino acid content but plant
protein foods present some problems.
Plant proteins are less efficient than animal proteins but the greater efficiency of agricultural
processes in providing plant protein sources must also be considered. For example, during storage or
processing, Lys, Met etc. undergo reactions that destroy their nutritional activity.

Free amino acids

In fruits, free amino acids are on average 50% of the soluble N compounds. The amino acid
pattern is typical of a fruit and can be utilized for the analytical characterization of a fruit product.

Peptides

Most peptides occur commonly and in variable levels in protein rich food as degradation products
of proteolytic processes. A number of peptides are of interest to food chemists.

glutathione (g-Lglutamyl-L-cysteinylglycine)
• adequate amounts are found in flour as the free form, oxidized dimer or protein
bound
• involved in many redox type reactions
• influences the rheological properties of wheat flour dough through thiol-disulfide
interchange with wheat gluten. High concentrations of reduced glutathione in flour
reduces protein disulfide bonds and causes a decrease in molecular weight of some
of the protein constituents of dough gluten.

Proteins

Osborne classification of proteins based on solubility:

1. albumins – soluble in water at pH 6.6 (e.g. serum albumin, ovalbumin)
2. globulins – soluble in dilute salt solution at pH 7 (e.g. lactoglobulin)
3. prolamins – soluble in 70% ethanol (e.g. zein, gliadins)
4. glutelins – insoluble in the above solvents but soluble in acid (pH 2 ) or alkali (pH 12)
(e.g. wheat glutenin)

Enzymes are present in the albumin and globulin fractions; prolamins and glutelins are mostly
storage proteins.
 Cereals: Protein distribution (%) in Osborne fractions

Fraction Wheat Rye Barley Oats Rice Corn

albumins 14.7 44.4 12.1 20.2 10.8 4.0
globulins 7.0 10.2 10.2 11.9 9.7 2.8
prolamins 32.6 20.9 20.9 14.0 2.2 47.9
glutelins 45.7 24.5 54.5 53.9 77.3 45.3

Cereal Proteins: Gluten

- in wheat, about 85% of endosperm proteins are storage proteins, insoluble in ordinary
aqueous media and responsible for dough formation
- the lack of solubility of these proteins can be explained by their amino acid compositions.
They have low proportions of charged amino acids compared with more typical proteins.
The contents of glutamine and proline are exceptionally high. Although hydrophilic, high
levels of Gln lead to the formation of H-bonds between polypeptide chains.
- gluten proteins can be separated on the basis of their solubilities. Gliadins can be
extracted with 70% ethanol; the remainder are the glutenins
- the structure of wheat glutenin includes a backbone of HMW subunits linked “nose to tail”
by S-S bridges, carrying short chains of LMW subunits
- HMW subunits have higher contents of Tyr and Thr and lower contents of Phe and Ile
than the LMW subunits
- Glutenin, consisting of HMW and LMW subunits polymerized by S-S bonds, may form an
insoluble but swellable skeleton in native gluten, the stability of which depends on the
type of subunits involved and on the degree of polymerization. Gliadins, added to the
glutenins soften the gluten formed. The gliadins act as plasticizers.

Essential features of the gluten proteins

% total gluten Molecular Distinctive features of

protein weight amino acid composition
(x 10-3)
gliadins S rich 51-64 32-42 Gln (32-42%)
Pro (15-24%)
Cys (2%)
Phe (7-9%)
S poor 7-13 55-79 Gln (42-53%)
Pro (20-31%)
Cys (0%)
glutenin Low MW 19-25 36-44 Gln (33-39%)
Gly (13-18%)
High MW 7-13 90-124 Cys (0.5-1%)

- the prolamin patterns of other cereals differ from that of wheat

 Legume Proteins

Legumes: Protein distribution (%) by Osborne fractions

Fraction Soybeans Peanuts Peas Mungbeans Broad beans

Albumin 10 15 21 4 20
Globulin 90 70 66 67 60
Glutelin 0 10 12 29 15

- the high content of globulins in seeds indicate that they function mostly as storage
proteins which are mobilized during the course of germination
- the globulin fraction can be separated into 2 major components: vicilin (7S) and legumin
(11S)
- the 7S globulins are glycosylated to different extents (5%); 11S globulins contain less
than 1% carbohydrate
- proteins exhibit marked gelling capacity and are suitable for production of foams and
emulsions

SOYBEAN PROTEIN

The protein fraction can be separated by ultracentrifugation or chromatography into 4 classes (identified by
the sedimentation coefficient): 2S, 7S, 11S, 15S. The major fractions are 11S representing about 40% of
total protein and containing one of two soybean globulins (glycinin) and
7S (35%) which contains the other globulin (conglycinin) in comparable amounts to the other biologically
active substances, namely the enzymes (lipoxygenase, b-amylase, cytochrome C) and the lectin,
hemagglutinin.
The two minor fractions (each 10-15%) contain trypsin inhibitors (2S) and, for 15S, heavy polymers of molar
mass 500,000 and over.

Trypsin inhibitors (2S) precipitate only imperfectly at pH 4.5; because of this, the protein concentrate only
includes about one-third of them. The soybean trypsin inhibitor makes up 6-8% of the total protein. These
are strongly resistant to heat, acids and the proteases. The most abundant (Kunitz’s inhibitor), has molar
mass of 21700 and contains two S-S linkages. The other inhibitor (Bowman-Birk) has molar mass 7900
and seven S-S linkages.

Glycinin (11S), the most insoluble globulin of soybean (molar mass 350000) is a polymer made up of 12
sub-units, 6 acidic (A) and 6 basic (B). In 6 sub-units, A and B are linked by disulfide bonds. The sub-units
may be dissociated at low ionic strength, in the presence of dissociating agents (urea), reducing agents or
in acid or alkali. Thermal denaturation starts at 70°C.

Conglycinin (7S) is a glycoprotein containing mannose and glucosamine. The molar mass of about
170000 corresponds to 3 sub-units, acidic in character; a,a’ and b. No cysteine residues are present (no
disulfide bonds) and the b sub-unit does not have Met either.

Soy proteins do not contain alcohol-soluble constituents and have no value in baking without some
technological contrivance.
Enzymes

Enzymes in Cereals

Some enzymes in cereal kernels which play a role in processing or are involved in reactions
which determine the quality of cereal product include:

1. amylases
- optimum activities of a and b-amylases are desirable in dough making in the presence of
yeast
- in mature kernels a-amylase activity is minimal, but increases abruptly during sprouting
or germination. Unfavorable harvest conditions (high moisture, high temperature) favor
premature germination, not visible externally. During this time a-amylase activity rises
resulting in extensive starch degradation during the baking process. Bread faults appear.
- The ratio of concentrations of the amylases depends on stage of development.

2. Lipases
- occur in various concentrations in all cereals
- a rise in free fatty acid observed during flour storage also involves lipases from
metabolism of microorganisms present in flour
- Oats contain a significant level of lipase. Its high activity is released once the oat kernel
is disintegrated, crushed or squeezed. Linoleic acid is released, converted into fatty
acids by lipoxygenase and hydroperoxide enzymes, giving rise to off-flavors.
- Phospholipase D (enzyme which transfers phosphatidyl residue from phospholipid to the
alcohol used to extract the lipid) activity is high in ripe cereals.
- Phopholipase B (enzyme in the reaction:
Lecithin ® acyl residues + phosphatidyl glycerol)
is found in germinating cereal, influences foam stability in beer

3. Phytase
- Phytin ® myo-inositol + 6H3PO4
- reaction occurs during dough-making

4. Lipoxygenases
- enzyme oxidizes fatty acids which contain a 1-cis-4-cis-pentadiene system
- linoleic and linolenic acids are preferred substrates
- unsaturated fatty acid ® monohydroperoxides
- in plants, 2 types exist:
type I – forms either 9- or 13-hydroperoxides from linoleic acid
type II – has lower specificity for linoleic acid;
produces 9- and 13-hydroperoxides in equal amounts and some other
products like ketodiene fatty acids;
also reacts with esterified substrate
- in cereals (except rye), enzyme oxidizes linoleic acid preferentially to 9-hydroperoxy
acids (i.e. type I)
- enzyme from wheat is type I, cooxidizes carotenoids at a slower rate but can still bring
about loss of yellow color in pasta products
 COOH

O2

(1) (2)

OOH
COOH COOH

OOH
13-S-hydroperoxy-cis-9, trans-11-octadecadienoic acid 9-S-hydroperoxy-trans-10, cis-12-octadecadienoic acid
(13-LOOH) (9-LOOH)

(1) lipoxygenase from soybean

(2) lipoxygenase from tomato
LOOH - linoleic acid hydroperoxide

5. Polyphenol oxidase
- have Cu2+/Cu+ system as prosthetic group
- cause enzymatic browning
- examples are tyrosinases, phenolases, catecholases, cresolases
- catalyze 2 reactions:
• hydroxylation of a monophenol to o-diphenol
• oxidation to o-quinone
- enzyme contains 2 Cu+ ions at its active site with 2 residues each in the ligand field
- enzyme first binds O2 and later, monophenol
- Cu ions change their valency: Cu+ ® Cu2+
 OH

H2O OH2 O OH2

N O O N O O
N N
Cu Cu Cu Cu
(II) (II) (II) (II)
N N N N
E E
O 2, H2O H2O

N N
Cu Cu
(I) (I)
N N O
N
E OH2
N O O
N 2 H+ Cu
O
N
(II) N
Cu Cu H2O O
(II) (II) Cu
N N (II)
E N
O O
E

Mechanism of polyphenol oxidase activity

Vegetable enzymes

1. Alliinase

H2O CH3
NH3
R S CH2 CH COOH
R S H + C O
O NH2 O COOH
alkyl cysteine sulfoxide alkenyl selfenic acid pyruvic acid

(a) Onion: 1-propenyl sulfenic acid spontaneously isomerizes to the lachrymatory

thiopropionaldehyde-S-oxide

CH3 CH CH SOH CH3 CH2 CH S O

(b) Garlic: Alkenyl sulfenic acid spontaneously dimerizes to allicin (diallyl thiosulfinate)

CH2 CH CH2 S OH CH2 CH CH2 S S CH2 CH CH2

2. Oxidoreductases (e.g. lipoxygenase, phenoloxidases,peroxidases)

3. Hydrolases (e.g. glycosidases, esterases, proteinases)
4. Transferases (e.g. transaminases)
5. Lyases (e.g. glutamic acid decarboxylase, alliinase)
6. Ligases (e.g. glutamine synthetase)

Fruit enzymes

The protein fraction in fruits varies with variety and ripeness. This fraction is primarily
enzymes

Examples:
1. Enzymes in carbohydrate metabolism (pectinolytic enzymes, cellulases, amylases,
phosphorylases, enzymes of pentose phosphate pathway)

2. Enzymes in lipid metabolism (lipases, lipoxygenases, enzymes of lipid biosynthesis, TCA

and glyoxylate cycles)

Legume enzymes

1. Lipoxygenase
- strongly affects legume aroma

2. Urease
- found in soybeans at relatively high concentration
H2NCONH2 + H2O ® CO2 + 2NH3

3. Proteinase inhibitors
- inhibitors of hydrolases, form stoichiometric inactive complexes with the enzyme
examples: inhibitors of trypsin and or chymotrypsin
- often found but not limited to seeds of plants; amount depends on variety, degree of
ripeness, storage time
- seeds of legumes, tubers of potatoes and cereal grains contain especially high
concentrations
 Occurrence and properties of various hydroperoxide-lyase

Occurrence Substrate Products of catalyses

Apple,tomato, cucumber, tea
leaf, soybeans, grape
Apple,tomato, cucumber, tea
leaf, soybeans, grape
Cucumber, pear
Cucumber, pear
Champignon
Champignon
13-hydroperoxy-9-cis, 11-trans-
octadecadienoic acid
(13-LOOH)
13-hydroperoxy-9-cis, 11-trans-
15 cis-octadecatrienoic acid (13-
LnOOH)
9-hydroperoxy-10-trans,
12-cis-octadecadienoic acid
(9-LOOH)
9-hydroperoxy-10-trans,
12-cis, 15-cis-octadecatrienoic
acid (9-LnOOH)
10-hydroperoxy-8-trans, 12-cis-
octadecadienoic acid
(10-LOOH)
10-hydroperoxy-8-trans, 12-cis-
15-cis-octadecatrienoic acid
(10-LnOOH)
Hexanal + 12-oxo-9-cis-
dodecenoic acid
3-cis-hexenal + 12-oxo-9-
cisdodecenoic acid
3-cis-nonenal + 9-oxo-
nonanoic acid
3-cis-6-cis-nonadienal + 9-
oxononanoic acid
1-octen-3-ol + 10-oxo-8-
trans-decenoic acid
1,5-cis-octadien-3-ol + 10-
oxo-8-trans-decenoic acid

Enzymatic degradation of hydroperoxides

(a) hydroperoxides with conjugated diene systems

- the bond between the C - atom bearing the HOO - group and the C - atom of the diene system
is cleaved

(b) hydroperoxides with isolated double bonds

- cleavage occurs in the opposite direction between the C - atom with the HOO - group and the C
- atom with the adjacent methylene group

H
R1

R2
O
OH

R3 OH
R1CHO
O
H
R4
HO R2
R3

OH
H H

O R2 O R4

(a) (b)