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Critical Reviews in Food Science and Nutrition

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Amylolytic enzymes and products derived from starch:

A review
a b c
Horacio Guzmán‐Maldonado , Octavio Paredes‐López & Dr. Costas G. Biliaderis
a
Instituto Nacional de Investigaciones Forestales y Agropecuarias (INIFAP‐CAEB), km. 6 Carr.
San Miguel Allende, Apdo. Postal 112, Celaya, Gto., México
b
Depto. de Biotecnología y Bioquímica, Centro de Investigatión y de Estudios Avanzados del
IPN, Unidad Irapuato, Apdo. Postal 629, Irapuato, Gto., 36500, México
c
Dept. of Food Science and Technology, School of Agriculture, Aristotle University,
Thessaloniki, Greece

Version of record first published: 29 Sep 2009

To cite this article: Horacio Guzmán‐Maldonado, Octavio Paredes‐López & Dr. Costas G. Biliaderis (1995): Amylolytic enzymes
and products derived from starch: A review, Critical Reviews in Food Science and Nutrition, 35:5, 373-403

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 Critical Reviews in Food Science and Nutrition, 35(5):373^M)3 (1995)

Amylolytic Enzymes and Products Derived from

Starch: A Review
Horacio Guzmán-Maldonadoa and Octavio Paredes-Lópezb
a
Instituto Nacional de Investigaciones Forestales y Agropecuarias (INIFAP-CAEB), km. 6 Carr. San
Miguel Allende, Apdo. Postal 112. Celaya, Gto., México; bCentro de Investigatión y de Estudios
Avanzados del IPN, Unidad Irapuato, Depto. de Biotecnología y Bioquímica, Apdo. Postal 629,
36500 Irapuato Gto., México

Referee: Dr. Costas G. Biliaderis, Dept. of Food Science and Technology, School of Agriculture, Aristotle University, Thessaloniki,
Greece

ABSTRACT: This review provides current information on starch and its molecular composition, common and
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potential sources, and manufacturing processes. It also deals with the five groups of enzymes involved in the
hydrolysis of starch: the endo- and exoamylases, which act primarily on the α-1,4 linkages; the debranching
enzymes, which act on the α-1,6 linkages; the isomerases, which convert glucose to fructose; and the cyclodextrin
glycosyltransferases, which degrade starch by catalyzing cyclization and disproportionation reactions.
This work mainly discusses the enzymatic processes for the manufacture of maltodextrins and corn syrup
solids, including the production, both batch and continuous, of glucose syrup, and the processes to obtain
sweeteners, such as maltose and 42, 55, and 90% high-fructose corn syrups. It highlights the novel production of
Schardinger's dextrins: the α-, β-, and γ-cyclodextrins, consisting of six, seven, and eight glucose monomers,
respectively.
New products are emerging on the market that can serve as fat and oil substitutes, moisture-retention
compounds, crystal-formation controllers, stabilizers for volatile materials like flavors and spices, or products for
the pharmaceutical industry. As a result, particular attention is given to functional properties and applications of
the above-cited compounds.

KEY WORDS: starch, amylolytic enzymes, maltodextrins, corn sweeteners, cyclodextrins.

I. INTRODUCTION food products and provide a source of numerous

After cellulose, starch is the principal carbo-
hydrate photosynthesized by plants by means of
solar energy. This polysaccharide is readily as-
similated in the human diet; in fact, a very high
proportion of the world's food energy intake is
starch. The most important sources of starch are
cereal grains (40 to 90% of their dry weight),
pulses (30 to 70%), and tubers (65 to 85%).66
Total world production is extracted mainly from
maize and potatoes, and the rest is derived from
wheat, sorghum, and sago. However, some ef-
forts have been made to study alternative sources
of starch such as triticale, wild rice, cassava, and
amaranth. A high percentage of starch produced
is intended for food purposes.64 Starches gener-
ally help to improve the functional properties of
oligo-, di-, and monosaccharides.
Historically, starch was hydrolyzed by min-
eral acid, but biotechnological application of
biocatalysts in chemical processes has led to the
use of enzymes as replacements for chemicals in
producing a variety of glucose and glucose-based
products. The use of enzymes on a large scale is
hardly new. Production processes and enzyme
applications, which may have seemed uninterest-
ing because of unacceptable economics, are now
on the brink of becoming a reality.
New enzymic products derived from starch
are emerging on the market, such as fat substi-
tutes, flavor stabilizers, and ice crystal growth
retarders. These have captured the interest of the
public and therefore the attention of the food-
processing industry. On the other hand, the most

1040-8398/95/$.50
© 1995 by CRC Press, Inc.
373
 commonly used sweetener has been sucrose, but
in the 1970s, a highly significant change took
place as a starch-derived sweetener, fructose syrup,
came into common use. Methods for chemical
separation of glucose and fructose have been de-
veloped and enrichment of the enzymatically pro-
duced syrup to 90% fructose is practiced by sev-
eral producers today.
This article reviews the amylolytic enzymes,
their types and action patterns, and the production
of novel derivatives of starch that are influencing
both the public and the food industry. In particu-
lar, emphasis is placed on the functional proper-
ties and food applications of these products.
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II. STARCH
A. Introduction
The cold water-insoluble starch granule forms
the major constituent of all cereal grains. It is a
reserve substance composed entirely of oc-D-glu-
cose for most higher plants and constitutes an
energy source essential for many organisms, es-
pecially humans.64 From a nutrition standpoint,
starch is the major component of the diet in all
human populations. However, in our culture, the
main purposes of starch utilization remain aes-
thetic rather than nutritional. This biopolymer
constitutes an excellent raw material for modify-
ing food structure and consistency.19
The commercial and technological uses of
starch are extremely numerous; this arises from
its unique character as it can be used directly as
intact granules, in the dispersed form, as a film
dried from a dispersion, or as an extrudate powder
after conversion to a mixture of oligosaccharides
or via hydrolysis and isomerization.66-91-177 As a
result, the academic aspects of the subject have
received much attention. The most important ad-
vance in the study of starch was the realization
that the starch granule was not chemically homo-
geneous,41-66-189 thus separation could be made into
the simpler component amylose, an essentially
linear polymer of glucopyranose units linked
through OC-D-1,4 linkages, and amylopectin, a
branched polymer containing chains with a short
degree of polymerization (DP = 20 to 25
glucopyranose residues) linked to the C-6
374
hydroxymethyl position of certain glucose moi-
eties via OC-D-1,6 linkages.19-64 Although it is now
accepted that amylose is not completely linear,74
its solution properties are typical of those of a
linear polymer.19
More than half of all starch production goes
to the manufacture of food and feed products,
either as such or in pregelatinized and chemically
modified form.66-119 However, modified starches
are used in the paper, textile, soap, laundry, cos-
metic, and pharmaceutical industries; in fireproof-
ing preparations; as explosive- and fuel-binding
agents; and as flocculating agents for water treat-
ments and in the building industry.21-66-91 These
industries, especially the paper and board indus-
try, consume a remarkable amount of starch,
modified as well as native.66-91
B. Common Sources
1. Maize
Of the common starches, regular corn, waxy
corn, and high amylose corn are by far the most
important sources. Nevertheless, only about 5%
of the annual world maize crop is used for the
manufacture of maize starch.20 About 70% of the
maize starch produced is converted into corn syrup,
high fructose corn syrup, and dextrose.20
Corn starch granules are medium sized and
round or polygonal in shape. Starch content of
corn ranges from 68 to 74%. The amylose content
ranges from about 80% in high amylose to about
1% in waxy starch.20-159
Maize starch has a wide variety of industrial
applications, with uses ranging from thickening
and gelling agents in puddings and fillings, to
molding for confections. Chemical modification
of maize starches can be used to extend the func-
tional characteristics to satisfy a wide range of
industrial requirements.21-159-168
2. Potato
About 3% of the world crop of potatoes is
used for the production of potato starch. Potato
starch granules are oval in shape, with pronounced
oyster shell-like striations around an acentrically
 placed hilum.20 Potato starch has the largest gran-
ules of any commercial starch, and its products
are used in the food, paper, textile, and adhesive
industries.96-183
3. Wheat
Wheat starch is produced in many countries
as a byproduct in the manufacture of wheat glu-
ten. Only 0.4% of the world crop of wheat is
processed to starch and gluten.20-135 The carbohy-
drate composition of wheat has been widely re-
ported.117'135 Starch is the principal carbohydrate
constituent of the endosperm (96%) and presents
two populations: the smaller spherical granules
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and the larger, lenticular granules.20
Wheat starch is used in the baking industry
and in the production of adhesives as well as
having applications in the confection and canning
industries.20
4. Rice
Most of the material for rice starch manufac-
turing consists of rice grains that are damaged or
broken in mills that produce rice for food.20 Rice
starch has the smallest granules of all commercial
starches; the granules tend to aggregate within the
amyloplast in spherical clusters and each amylo-
plast contains 20 to 60 small polyhedral gran-
ules.83 Rice starch is used as laundry starch, in
cosmetic dusting powders, and as a thickener in
puddings and ice cream.81
C. Manufacturing Processes
Some variations in procedure may be required
in isolating starches from different sources (Fig-
ure 1). It is necessary that starch be softened by
some type of process: cleaning by steeping with
S02 in maize,5-21'51 pulping in potato tubers,20 and
dough mixing in wheat.20 Also, it is necessary to
carry out the extraction process in the presence of
mercuric chloride (0.01 M) in order to inhibit
endogenous starch-degrading enzymes.20'21
Different steps of Figure 1 are repeated until
the material that passes through the sieves is free
from fiber, grits, pulp, or gluten. Basically, it is a
process that separates germ, oil, protein, and starch
using mainly screening, centrifugation, and plenty
of water without any concomitant degradation of
the starch. The starch suspension free of protein
and fine fiber material is dewatered, then dried,
and finally sifted.5-21-51
D. Other Potential Sources of Starch
Rye {Secale spp.), triticale (Triticale spp.),
wild rice (Zizania aquatica L.), cassava {Manihot
esculenta L.), and amaranth (Amaranthus
hypochondriacus L.) are common alternative
sources of starch. Rye has an approximately 52%
starch content, with 27% amylose.73'190 Triticale
has 54% starch, with an amylose content of ap-
proximately 23%.18'73 Wild rice has 60 to 65%
starch, with 16.8 to 23.9% amylose.82 Amaranth
has 48 to 69% starch, with an amylose content of
0 to 21.8%.134138'140'157'171'172 Some efforts have
been made to study the potential use of amy-
lolytic hydrolysis for these materials, especially
with amaranth14'61-69-70-139 and cassava16-43-44'61
starches.
III. AMYLOLYTIC ENZYMES
A. Introduction
The term enzyme, which means "in yeast,"
gives only a few clues as to what the sources of
enzymes are.40 Primitive man relied on plants and
certain organs of animals for their enzymatic roles
in brewing, cheesemaking, and other food pro-
cesses. Malting was done by the ancient Chinese
1000 years before Christ; it was used to manufac-
ture "Hung Fan," meaning "sweet from cereal."30
It is interesting to note that the first enzymes to be
recognized as specific biocatalysts were those
capable of hydrolyzing starch.51
The development of the starch/wet-milling
industry can be traced back to the French general
Napoleon in the eighteenth century. Because of
Napoleon's continental blockade, Europe found
itself without its traditional sources of sugar.
Therefore, rewards were offered for methods that
could result in a suitable substitute.51 While work-
375
 pre-«illin$ fine entrifuoinj
cleaning STARCH
•—• separation — —» nillUg — cyclone — —t.
steeping
uasMrig separation

SI ttf Fiber Gluten

I quor And
rS
Dil Cale
Grits

cleaning pulpin? centrifugift'

washing sieving cyclone STARCH
separation

Fulp Effluent
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"ft" STARCH

— —i
screening
dough gluten gluten centrifuging TSIATCH
FIOOR nixing dewelopnentl washing cuclone
separation

Solubles

FIGURE 1. Simplified diagrams of typical starch production processes

from maize, potato, and wheat. (Adapted from References 20, 21, 52.)

ing on this problem, the German chemist Kirchhoff
discovered that starch boiled with acid and then
neutralized could be converted into sugar.109-161
However, the nonspecific catalytic action of the
acid resulted in the formation of undesirable re-
version products and color. Furthermore, the syrup
had a relatively high salt content.142-166 Those
undesirable characteristics of the syrups were
caused by acid hydrolysis and enzymes are now
supplementing or replacing that process.51
In 1833, a Frenchman, Payen, succeeded in
isolating the active component in malt, which he
called diastase. Takamine, in 1894, developed the
idea of using an enzyme mixture from Aspergil-
lus oryzae for consumption as a digestive aid for
Asian populations who had difficulties in digest-
ing rice (starch).51-127 It was discovered that
A. oryzae produced a mixture of amylases. The
identification and use of these enzymes enabled
production of syrups not possible with acid.51
The real breakthrough in industrial terms came
in the mid-1960s, with the development of an
amyloglucosidase, which made it possible for
starch to be completely hydrolyzed to glucose.108
A full enzymatic starch hydrolyzate was not
achieved until the high-temperature stable bacte-
rial amylase from Bacillus licheniformis was in-
troduced in 1973. The use of this enzyme enabled
production of syrups containing up to 98% glu-
cose, which is an ideal substrate for glucose
isomerase to convert glucose to fructose.24-54-104-105
The worldwide market for industrial enzymes
in 1990 has an estimated value of $625 million,
with the U.S. as the largest sector. About 62% of

376
 the enzymes produced find their way into food
applications. In the food industry, the largest use
of enzymes is in starch processing followed by
cheese production, fruit and vegetable juice pro-
cessing, baking, and brewing.9-40
The screening of microorganisms and plants
has become standard procedure for yielding im-
proved forms of known enzymes. Recent advances
and creative approaches are demonstrating that
enzymes can catalyze reactions under conditions
previously thought to be too extreme. Rapid
progress is being made toward fine-tuning many
of the diverse techniques that are required for
successful gene cloning and protein engineering
of amylolytic enzymes.25-76'102-141-178'179-180
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B. Types of Enzymes and Action
Patterns
1. Introduction
There are essentially five groups of enzymes
involved in the hydrolysis of starch. The endo-
and exoamylases act primarily on the a-1,4 link-
ages,51-109 whereas the debranching enzymes act
exclusively on the a-1,6 linkages.130 A fourth
group, the isomerases, act on glucose syrups to
convert them to fructose syrups.51-66-68-109 Finally,
the cyclodextrin glycosyltransferases degrade
starch by catalyzing cyclization and dispropor-
tionation reactions.128-170-186 Table 1 classifies the
various enzymes involved in the primary produc-
tion of syrups, maltodextrins, and cyclodextrins
and gives the names of microbial species involved
in enzyme production. The action of these en-
zymes on starch is illustrated schematically in
Figure 2.
There is another group of starch enzymes, the
oligosaccharide-forming amylases, that produce
specific oligosaccharides, such as maltotriose,
maltotetraose, maltopentaose, and malto-
hexaose.133 Although the production of such en-
zymes has not been commercially exploited to a
large extent, the use of these specific exo-acting
amylases represents an interesting development
in the area of starch enzymology. There are two
other groups of enzymes that do not act on starch
but are involved in the starch hydrolysis industry.
The first group is comprised of the cellulases,
which are known as "filtration enzymes" that
positively affect the filterability of the syrups.93-95
The other group, named proteinases, is contained
in industrial preparations of starch-degrading en-
zymes. These release starch from protein-starch
complexes.55-60
2. Endo-enzymes
These enzymes cleave the a-1,4 glycosidic
bonds in amylose, amylopectin, and related
polysaccharides but not the a-1,6 bonds in amy-
lopectin.130 The products of hydrolysis are oli-
gosaccharides of varying chain lengths and have
the cc-configuration on the Cl of the reducing
glucose unit produced. They act on bonds in the
inner regions of the substrate and hence cause a
rapid decrease in the viscosity of the gelatinized
starches and a parallel decrease in iodine com-
plexation.51-108 Activity is usually defined in terms
of the amount of enzyme required to break down
starch under a given set of conditions to a fixed
degree of hydrolysis. This is usually determined
by reaction with iodine to give a blue color,
which is then compared with a standard
color.34-51-165
There are two varieties of endoamylases (EC
3.2.1.1): thermostable and thermolabile. The ther-
mostable oc-amylases are bacterial in origin and
are derived from the Bacillus species (Table 1).
Two types are of current commercial importance;
one is an amylase derived from B. subtilis (variant
amyloliquefaciens), which has been used for many
years.51 It has an optimum temperature, in the
presence of Ca2+, around 65 to 70°C but is active
even at 82°C.51-109 Because its stability is lower
than other a-amylases, it has not achieved much
commercial use.148 The other enzyme is derived
from B. licheniformis and was first introduced
around 1973. It is active and stable in starch so-
lutions at temperatures >90°C, relatively inde-
pendent of Ca2+ ions for stabilization, and is more
active over a wide pH range.113-150-166
Traditionally, starch solutions had to be cooled
to approximately 85°C before the enzyme could
be added, but with the introduction of cc-amylase
from B. licheniformis it was possible to add it at
temperatures >100°C. Additionally, the risk of
starch retrogradation (the event that occurs when
377
 « TABLE 1
3 Classification of Enzymes Involved in the Commercial Production of Starch Syrups, Maltodextrins and
Cyclodextrins
^•» *v w • • • VB p • •

Common Substrate
Type name Source specificity PH Temp. (°C)

Endoamylase Bacterial amylase Bacillus subtilis a-1,4-Glycosyl 6.0 65-70

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B. licheniformis a-1,4-Glycosyl 5.0-7.0 90

Fungal cc-amylase Aspergillus oryzae a-1,4-Glycosyl 4.5 50-60
Exoamylase Amyloglucosidase A. niger a-1,4-Glycosyl 4.0-5.0 60
a-1,6-Glycosyl
Bacterial p-amylase Bacillus sp. a-1,4-Glycosyl 5.0 55-60
Clostridium sp. a-1,4-Glycosyl 5.5-6.0 75-85
a-1,6-amylase Pullulanase Klebsiella aerogenes a-1,6-Maltotriosyl 5.0 60
Isoamylase Pseudomonas sp. a-1,6-Heptasacch. 4.0 50-55
Exo-a-amylases Exomaltotriohydrolase Streptomyces griseus a-1,4-Glycosyl —
B. subtilis a-1,4-Glycosyl —
Exomaltotetraohydrolase B. circulans a-1,4-Glycosyl —
Pseudomonas stutzeri a-1,4-Glycosyl —
Exomaltopentaohydrolase Pseudomonas sp. a-1,4-Glycosyl —
Exomaltohexaohydrolase B. subtilis a-1,4-Glycosyl —
B. circulans a-1,4-Glycosyl —
Isomerase Glucose isomerase B. circulans Aldo/keto pentose 8.2 65
Aldo/keto hexose
Glucanotransferase Cyclodextrinase B. macerans a-1,4-Glycosyl 5.0 50
Thermoanaerobacter sp. a-1,4-Glycosyl 4.5 90
Endo/Exocellulase Fungal cellulase Trichoderma reesei p-1,4-Glycosyl —
Endoprotease Bacterial protease B. licheniformis — 6.5 90
B. subtilis 6.5 65-70

Adapted from References 16, 43, 51, 65,133, and 136.

 B 6
II
o-o-o-o-o
/\
0 4-D
/ BG
A 0 0 II
"V
V \\ • •
0 0-0-0-0
G A n /
0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0-0

B tD D 0 1
J 0
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G 0 G A \
1
0 • \
o-o-o-o-o-o^ 0 \
B D
0
\ •

FIGURE 2. Action of amylolytic enzymes. A, a-amylase; B, f3-amylase; D, debranching

enzyme; G, amyloglucosidase; O, glucose unit; O-O, a-1,4 linkage; -»O, a-1,6 linkage; O,
reducing end.

starch molecules begin to reassociate into an or-
dered structure)12'118 is greatly reduced. Another
fact involves the requirements for Ca2+ ions for
full stabilization of the enzymes. B. licheniformis
needs only 5 ppm of Ca2+, whereas B. subtilis
needs 150 ppm for saturation level.166 A reduction
in the Ca2+ concentration required for enzyme
stabilization and its removal later in refining of-
fered economic advantages.165
Three other thermostable a-amylases have
been at least partially purified. One from
B. licheniformis MV10 is inhibited by calcium
ions and has an optimum temperature >80°C.97
Another from a mutant of B. licheniformis is ca-
pable of low pH (5.5) liquefaction.7 If a lower
operating pH can be achieved, then chemical ad-
dition and costs for ion exchange are significantly
reduced. The third enzyme, from B. licheniformis
M27, shows two peaks for pH optima at 6.5-7.0
and 8.5-9.0, a temperature optimum at 85 to 90°C,
and an activation energy 20.4 times lower than
that reported for the enzyme from another strain
of B. licheniformis.136-141 A higher reaction rate
due to lower activation energy is thus offered by
this enzyme.
The action pattern of B. licheniformis en-
zymes generates G5 (chain length expressed as
number of glucose units) and larger products ini-
tially, followed by later production of G3, G2, G^
and G4 in descending order. The hydrolysis rate
decreases sharply with decreasing chain length;
G2 and G3 are not attacked at all.148'165 The Michae-
lis-Menten model was found to be sufficient for
describing the kinetics at low starch concentra-
tions; however, a modified first-order model was
required at high concentrations.92 There is no

379
 evidence of substrate inhibition at high starch
concentration.166
The other group of endoamylases, thermo-
labile a-amylases, is derived from fungal sources,
usually A. oryzae (Table 1). In the starch industry,
their use is mostly confined to the saccharifica-
tion reaction. Products of this reaction are mainly
maltose and maltotriose, and thermolabile a-amy-
lases are hence referred to as maltogenic in action
(Figure 2).62'108'148 Units of activity are defined as
for thermostable a-amylases, but under different
conditions, especially pH because, unlike bacte-
rial enzymes, fungal enzymes are usually more
stable and active under acidic conditions.51
Properties of immobilized a-amylases can be
strongly influenced by diffusion limitations, which
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are aggravated by the high molecular weight of
the substrate and high solution viscosity. If an
immobilized a-amylase were packed in a col-
umn, usually the most efficient procedure, high
pressure drops at any reasonable flow rate would
also be expected.148
Genetic-engineering techniques have opened
new avenues for production of valuable enzymes.
One microorganism of interest, B. subtilis
TN106(pAT5), is a fast-growing bacterium, indi-
cating very efficient glucose utilization. Further-
more, batch culture experiments revealed that
a-amylase synthesis and secretion were initiated
in the early part of the growth phase.180
The a-amylase gene (amy) from Streptomy-
ces griseus IMRU 3570 was subcloned into
Brevibacterium lactofermentum. The amy gene
was expressed efficiently when the promoter of
the kanamycin resistance gene was inserted up-
stream of the promoterless amylase gene.25 Liu
et al.102 reported an overproduction of a-amylase
in recombinant Escherichia coli.
3. Exoamylases
These enzymes act on the same substrates as
endoamylases but in a different way. Some of
them are able to cleave a-1,6 linkages (Figure 2),
but the reaction rate is slow compared with their
hydrolysis of a-1,4 bonds. Exoamylases act ex-
ternally on substrate bonds from the nonreducing
end and produce low molecular weight products.
Currently two types of exoenzymes are important
380
in starch hydrolysis: the glucogenic and maltogenic
exoamylases.51
The glucogenic exoamylase, amyloglucosidase
or glucoamylase (EC 3.2.1.3) is fungal in origin
(Table 1). This enzyme attacks a-1,4 linkages from
the nonreducing end, releasing D-glucose molecules
in the P-configuration.109 Amyloglucosidase re-
moves D-glucose molecules up to the point of
branching in the amylopectin molecule; hydrolysis
may also proceed on the a-1,6 bond, but much
more slowly than on a-1,4 bonds.108 Activity is
usually stated in units per liter and is based on the
production of glucose (or reducing sugars) from a
defined substrate.51 This enzyme is one of the most
important industrial enzymes and has worldwide
applications.2-108'109 The most important use of
glucoamylase is in the production of high-glucose
syrups (96 to 98% glucose)2-16 and high-fructose
syrups (55% fructose).2'104
At high glucose concentrations, amylo-
glucosidase repolymerizes glucose molecules in a
reaction called reversion, forming maltose and
isomaltose.151 This phenomenon is more pro-
nounced at high substrate and high enzyme con-
centrations.104 The polymerization reaction of glu-
cose can also be catalyzed by another enzyme,
transglucosidase (EC 2.4.1.24), which is often
present in crude amyloglucosidase preparations.109
Amyloglucosidase acts synergistically with
a-amylase in raw starch hydrolysis.4849 The syn-
ergistic action is retarded not only by a decrease
in the molecular weight of the substrate but also
by an increase in the concentration of the sub-
strate.48
Several studies have been done on the immo-
bilization of amyloglucosidase on a variety of
supports by a variety of methods.29-47-144'145 As a
rule, immobilization brings about some changes
in enzyme properties such as kinetic, thermal sta-
bility, and optimum working conditions. More-
over, for immobilized enzymes, the restraint in
the diffusion of substrate molecules to its site of
reaction becomes an important factor. When
amyloglucosidase was immobilized on DEAE-
cellulose, there was an increase in K,,, and a de-
crease of Vmax for the hydrolysis of starch, which
could be partially explained by diffusion limita-
tions.144 On the other hand, thermostability of
amyloglucosidase increases with an increase in
the amount of enzyme adsorbed.145
 Amyloglucosidase immobilized on celite is
as efficient as the soluble form in catalyzing hy-
drolysis of maltose. However, it is less efficient
than the soluble enzyme in hydrolyzing 30% (w/w)
maltodextrin, giving a maximum dextrose equiva-
lent (DE) value of 96.0% instead of 98.2%.29
Such a low DE may result from the fact that the
accessibility of large oligosaccharide molecules
to the active site of immobilized enzyme is re-
tarded by the limited space inside the pore, and
the capability to form immobilized enzyme-sub-
strate complex is much lower than that of the
soluble enzyme when large oligosaccharide mol-
ecules are used as substrate.29-155
Interest in the cloning and expression of
amyloglucosidase has increased in recent years.
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Saha and Zeikus155 reported that amyloglucosidase
I from Aspergillus awamori was expressed in
Saccharomyces cerevisiae by means of the pro-
moter and termination region from the yeast eno-
lase gene efficiently secreting amyloglucosidase
to the medium.
The amyloglucosidase genes from S. diastat-
icus155 and Rhizopus sp.155 have been cloned and
their nucleotide sequences have been determined.
The use of the potent inducer cyclopentanone
resulted in maximum amyloglucosidase concen-
tration in a recombinant A. nidulans.103
The maltogenic exoamylase P-amylase (EC
3.2.1.2), of plant origin has been known for
many years. 30 Plant sources such as barley,
wheat, sweet potatoes, and soybeans have been
the most common sources of the enzyme.51-66152
However, its existence as an extracellular en-
zyme in Bacillus sp. and Pseudomonas sp. has
been established.66
P-Amylase acts in an exo-fashion, from the
nonreducing ends of the outer chains of starch or
related polysaccharides, and proceeds by gradual
removal of maltose units in the |3-anomeric form
(Figure 2). Amy lose is converted almost com-
pletely to maltose, whereas amylopectin and other
branched polymers are hydrolyzed to various
extents depending on the degree of branching;148
this hydrolysis results in about 50 to 60% conver-
sion to maltose. This is due to the inability of
P-amylase to hydrolyze a-1,6 linkages. The high-
est activity ocurrs at pH 5.0. The temperature of
optimum activity ranges between 55 and 60°C
(Table 1).
Saha and Zeikus156 have discovered a highly
thermoactive (75 to 85°C) Clostridium thermo-
sulfurogenes p-amylase. This enzyme makes it
possible to saccharify liquefied starch at higher
temperatures than have been used in the past.
Immobilization of P-amylase on alginate,
agarose, or ion-exchange carriers has been re-
ported.35 It was observed that the type of carrier
and the source of the P-amylase are important
factors for the required capacity of the continuous
process.
4. a-1,6 Enzymes
The presence of a-1,6 branches makes the
production of amylolytic derivatives of starch more
difficult than it would be in their absence. As
mentioned previously, amyloglucosidase can
cleave a-1,6 bonds, but rather slowly.51-108 On the
other hand, P- and oc-amylase have no activity on
these bonds.130 Hence, the use of enzymes able to
hydrolyze these links quickly is of increasing in-
terest to the modern starch industry.
Debranching enzymes, such as pullulanase
(EC 3.2.1.41) and isoamylase (EC 3.2.1.68), have
been known for more than a decade,129 but their
use has not gained widespread acceptance. In re-
cent years, the use of these enzymes has attracted
more attention as the efficiency of the hydrolysis
reaction could obviously be improved by incor-
porating a specific debranching enzyme in the
system.
Pullulanase was first discovered in 1961154
and attracted interest because of its specific
action on a-1,6 linkages in pullulan, a linear
D-glucose polymer consisting essentially of
maltotriosyl units joined by a-1,6 bonds (Fig-
ure 3),130 starch, amylopectin, and related oligosac-
charides.154 Pullulanase alone liberates products
of average DP, varying from 115 to 2300 after
hydrolysis of starch.169 This enzyme is generally
used in combination with amyloglucosidase, <x-
or p-amylase.51-129-130-154-169
Pullulanase is produced by mesophilic organ-
isms like Klebsiella aerogenes (Table I). 154 The
optimum temperature is around 60°C; this is very
important because in the glucose syrups industry,
saccharification is normally carried out at this
temperature. 51 The use of pullulanase with
amyloglucosidase simultaneously during saccha-
381

0-0-1u
L 0
L 0
L 0
0-0-0-1u
L 0
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FIGURE 3. Structure of pullulan. O, glucose unit; O-O,
oc-1,4 linkage; -> O, a-1,6 linkage. (Adapted from Ref-
erences 127, 128, and 151.)
rification reduces the amyloglucosidase require-
ment and the hydrolysis time.129-130 Nevertheless,
the determination of an inexpensive source of
an active thermostable pullulanase is under
study,78.90,122,154 because the enzymic conversion
of starch is preferentially carried out at high tem-
peratures. Under such conditions, contamination
is minimized, the reaction time is reduced, and
substrate solubility is enhanced.90 As a result, a
thermostable pullulanase from Thermus sp.
AMD33, which is capable of hydrolyzing both
a-1,6 and a-1,4 linkages, has been purified.122
Isoamylase, discovered in 1949,148 is elabo-
rated by a number of microorganisms like
K. aerogenes and Pseudomonas sp. (Table 1). This
enzyme hydrolyzes a-1,6 glycosidic linkages in
amylopectin and has a very low activity130 or no
activity66 toward pullulan.
When isoamylase is used before amylo-
glucosidase, the amylopectin fraction is depoly-
merized too rapidly and therefore is highly sus-
ceptible to retrogradation.130 In view of this, the
time of addition of isoamylase to the hydrolysis
system is very critical.
5. Isomerases
By far, the most important application of
immobilized enzymes in the food industry today
382
is the conversion of glucose to fructose by the
enzyme glucose isomerase (EC 5.3.1.5) (Fig-
ure 4).2654,ii5 The first of a series of commercial
glucose isomerases marketed was a soluble prod-
uct introduced in late 1973.26 It had a high pro-
duction cost and was used commercially for only
a short time. In 1974, an immobilized form of the
enzyme was introduced.4 Rapid development took
place and different immobilized isomerases have
been introduced since then.28-42'132-143-188
Cobalt is a well-known activator of glucose
isomerase enzymes; on the other hand, basic in-
vestigations have found that glucose isomerases
from some sources do not require cobalt ions.71-188
Magnesium is another activator of these enzymes
and there is a pH dependence on the required
level.188 Conversely, calcium and oxygen have an
inhibitory effect; the inhibitory effect of calcium
can be overcome by addition of magnesium,
whereas the oxygen effect is present at process
temperatures.6-188 Thus, the purity of the substrate
(glucose syrup) is important to enzyme stability.
6. Cyclodextrin Glycosyltransferases
Cyclodextrin glycosyltransferase (CGTase;
E.C. 2.4.1.19) is an enzyme capable of hydrolyz-
ing starch to a series of nonreducing cyclo-
maltooligosaccharides referred to as cyclodextrins,
which contain six, seven, or eight D-gluco-
pyranosyl residues.66-122 The CGTase enzymes
degrade starch by catalyzing cyclization and dis-
proportionation reactions (Figure 5).186
CGTase is produced mainly by Bacillus
strains. CGTase from B. macerans produces
oc-cyclodextrins as the major hydrolysis product
from starch,160 whereas thermostable CGTase from
B. stearothermophilus produces a- and p-cyclo-
dextrins50 and B. subtilis CGTase produces
y-cyclodextrins.160
IV. MALTODEXTRINS AND CORN
SYRUP SOLIDS
A. Introduction
The Food and Drug Administration (FDA)
defines maltodextrins (C6H10O5)n • H 2 O, as
nonsweet, nutritive saccharide polymers that con-
 CH OH
Glucose
i s oMe r a s e
0

HO 1 0H OH
OH

Glucose Fruofcose

FIGURE 4. Isomerization of glucose to fructose.

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ft 1 pha-cuc lodext.r-in

,0-Ov
O
\0

0-0-0-0-0-0-0 n
f

/
°
0 I t Beta-nyc 1 ortex +.r i n

0
0
Starch Molecule .O'°-O
\
X
— cycl o d e x t r i n

FIGURE 5. The proposed substrate-binding mechanism of cyclodextrin glyco-

syltransferases. -», cleavage sites; O, glucose unit; O-O, a-1,4 linkage; -> O, a-1,6 linkage.
(Adapted from Reference 157.)

sist of D-glucose units linked primarily by a-1,4 stance) can reduce the same amount of Cu2+ in
bonds having a DE <20.137 The term 20 DE Fehling solution to Cu+ as 20 parts of pure
signifies that 100 parts of the syrup (dry sub- D-glucose.23-36

383
 Maltodextrins are prepared as a white powder
STARCH
or concentrated solutions by partial hydrolysis of
SUSPENSION
starch with safe and suitable enzymes. Malto-
(30-4B V. « / u ; PH 6.5;
dextrins are generally recognized as safe (GRAS)
; alpha-attylase)
as a direct human food ingredient at levels consis-
tent with current good manufacturing practices. 63
On the other hand, corn syrup solids are defined \
by the FDA as dried glucose syrups in which the GELATINIZftTION
STEAM
reducing sugar content is 20 DE or higher. 137 ( 1 1 8 - 1 4 8 °C; 5 - 1 9 win)
Traditionally, maize starch has been used for
the manufacturing of these products. The work on
maize-based products has been continued in order
LIQUEFACTION
to extend the knowledge as to how a good starch
( A l p h a - a n y l a s e ; 98-95 °C;
product can be made from this important raw
90-120 Min)
material,11-21 but some attention has also gradu-
ally been given to the production of maltodextrins
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and other products from other types of starches, 107

namely, amaranth,14-15'61-67-69-70'139 cassava,11-44-56-61'96 EN2YME INftCTIUATION
potato, u ' 96 - 100 rice,1'22-65 sorghum, 1 and wheat.1-11 <pH 3 - 5 ; 5 nin b o i l e d )

B. Manufacturing Processes
FILTRATION AND
CARBON PURIFICATION
Maltodextrins and corn syrup solids are com-
monly prepared from regular and waxy corn starch8
by controlled enzymatic hydrolysis called lique-
faction (Figure 6).51-113-150-165 A starch slurry is
adjusted to the desired dry solid concentration (30
to 40% dry solid basis),7-165 pH adjusted at 6.5
with acid and then the enzyme (B. licheniformis
cc-amylase) and calcium ion are added. The starch
slurry is pumped through a jet-cooker where the
temperature is increased to 140°C and held for 5
to 10 min.148-165 Exiting the jet-cooker, the gelati- HALTODEXTRINS
nous starch is discharged into a liquefaction reac- OR
tor. At this point, the dispersed starch polymer COB! S¥KU? SOLIDS
material becomes more accessible to the en-
zymes.109 Then the gelatinized starch is vented to
the atmosphere and cooled to the liquefaction
temperature (90 to 95°C).148 A further addition of FIGURE 6. Liquefaction of starch to produce malto-
dextrins, and corn syrup solids. (Adapted from Refer-
bacterial oc-amylase takes place at this stage be- ences 51, 62, 104, and 107.)
cause the initial quantity of enzyme added has
been destroyed by high-temperture treatment.113
The liquefaction is allowed to continue until solids.51-69 Then, the pH value is decreased to 3.0
the required DE value is reached. Usually after to 5.0 and the slurry is boiled for about 5 min to
about 1 h, the DE is 10 to 12, and by 2 h it is 15 inactivate the enzyme.126 Color in the hydrolyzate
to 16.51113I5° By controlling the reaction time and/ is removed in granulated carbon columns. These
or by adjusting the enzyme dosage, it is possible columns are renewed by the removal of spent
to obtain a DE from 20 to 39 for corn syrup carbon and the addition of regenerated carbon on

384
 a regular basis.63-106 The carbon pulsed out of the
column is washed with water to remove the ad-
hering syrup, and the solution is returned to the
feed-line of the carbon column. The washed car-
bon is revivified by heating it in a gas-fired fur-
nace at approximately 920°C in an atmosphere
carefully controlled to maintain a low degree of
oxidation.109
The resulting syrup is then evaporated, spray
dried, and packaged.
Acid liquefaction requires the use of corro-
sion-resistant materials and gives rise to high-salt
ash contents.165 Originally, batch processing was
used in which a starch slurry was adjusted to a pH
of 1.8 to 2.0 with hydrochloric acid. The acidified
starch was heated at about 90°C for 30 min and
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then pressure cooked at 120 to 140°C to complete
starch breakdown. The DE of the liquefied prod-
uct was controlled within certain limits by adjust-
ment of the pressure cooking time.51 There is no
doubt that enzymatic liquefaction of starch is a
much more attractive process than acid liquefac-
tion regarding processing costs and quality of the
finished product.113
C. Functional Properties and
Applications
1. Maltodextrins
High-performance liquid chromatography
(HPLC) has been used to determine the carbohy-
drate profile of oligosaccharide mixtures.116-124-162
HPLC analysis can determine the DP, that is, the
number of glucose units joined in the molecule.112
Maltodextrins (DE < 20)107 as individual products
differ in their DP, but in general they present the
following typical carbohydrate profile: 0.3 to 1.6%
glucose (DP = 1), 0.9 to 5.8% maltose (DP = 2),
1.4 to 11.0% maltotriose (DP = 3), 1.4 to 6.1%
maltotetraose (DP = 4), and the rest of higher
saccharides (DP > 4) (75.5 to 96.0%).8-51-63 Some
of the most useful functional properties are de-
rived from this saccharide profile (Figure 7).
Practically all maltodextrins are supplied in
the dry form at a moisture level of about 4%.63 At
room temperature, a 40 to 70% solution generally
can be made with ease.107 Maltodextrins are rarely
sold in concentated solution form; there are fac-
tors affecting the storage stability of these prepa-
rations, that is, the stability of 50% (w/w) aque-
ous solution of low DE maltodextrins is frequently
poor, with precipitation occurring at 25°C.85
Some of the most useful functional properties
are low hygroscopicity, bland flavor, extremely
low sweetness, high viscosity, and the ability to
retard ice crystal growth in ice creams, ice milk,
and other frozen novelties.8'63'107 They contribute
chewiness, binding properties, and viscosity in
soft candy, and increase the shelf life and main-
tain moisture levels of hard candies.63 Malto-
dextrins also have application in pan coatings and
tableting,39 or can be used as partial replacements
for albumen in marshmallow formulation based
on albumen.110
Encapsulation methods involve the prepara-
tion of an emulsion or suspension of the flavor
and gelled or starch materials (i.e., maltodextrins)
followed by the rapid drying of suspended par-
ticles.32'120 Maltodextrins vary greatly in protect-
ing encapsulated flavors from oxidation. In phar-
maceuticals, maltodextrins are effective binders
for direct compression tableting and wet or dry
agglomeration processes.63
New products are emerging on the market
that can serve as fat and oil substitutes. Some of
these products are carbohydrates. Because reduc-
ing the risk of heart attack is possible by lowering
blood cholesterol levels,79 fat reduction has cap-
tured the interest of the public, and therefore the
attention of the food-processing industry. Several
companies are investing in the development of fat
replacements, and many have already responded
with new products.63-96-98
Some companies sell a number of malto-
dextrins.63'173 A low-DE maltodextrin containing
almost 98% penta- and higher saccharides is
claimed to provide a creamy, fat-like texture due
to a combination of good film-foaming character-
istics and low hygroscopicity while contributing
only 4 kcal/g; fats contribute 9 kcal/g.98 Another
company has launched a line of fat replacers called
N-Lite, designed to replace fats in a variety of
food applications.123 This company worked with
corn and other starches to develop what it calls a
"system approach" to fat replacement. This ap-
proach differentiates its products from those of
385
 Starch Haltodextrins Corn syrup
solids

Dextrose equivalent 5 18 15 18 25 36

Binding

Bodying agent

Browning reaction

Cohesiveness

Flavor enhancement
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Freezing point depression

Hygroscopicity

Prevention of coarse crystals

Solubility

Sweetness

Viscosity

FIGURE 7. Functional properties of maitodextrins and corn syrup solids.

(Adapted from References 62, 105, and 134.)

other firms, says the company, because it recog-
nizes the properties and roles of different fats in
foods and then tailors the starch to have the orga-
noleptic and functional properties needed for spe-
cific applications.
Other products contain proteins and surfac-
tants in addition to low-DE dextrins, which are
designed to replace up to 81% fat in cakes.96
Formulation of low-fat foods requires reformu-
lation of traditional products, sometimes with
different ingredients. When fat replacers are
used, the carbohydrate molecule is hydrated to
achieve fat-mimicking functionality. Addition-
ally, sensory attributes such as the degree of
cohesiveness, firmness, dryness, and juiciness
must also be considered.184 Making products
without fat is difficult and many may have se-
rious flavor defects. Some even suggest that
small amounts of fat, qualifying for low-fat
rather than fat-free standards, may creep back
into foods to compete with better tasting prod-
UCtS.79'96-98'173
2. Corn Syrup Solids
Corn syrup solids (DE > 20),107 like maito-
dextrins, as individual products differ in then-
degree of hydrolysis, but in general present the
following typical carbohydrate profile: 0.8 to
16.9% glucose (DP = 1), 5.5 to 26.9% maltose
(DP = 2), 9.1 to 16.4% maltotriose (DP = 3), 6.8

386
 to 10.0% maltotetraose (DP = 4), and the rest is
higher saccharides (DP > 4) (40.4 to 77.8%).51-63
Corn syrup solids are granular, crystalline,
or powdered forms of corn syrup.63 They pro-
vide a protective coating and sheen in panned
goods, and plasticity and viscosity in chewy con-
fections.39 They can be used in low-moisture
products, provide the ability to increase solids
without cooking, and make blending with dry
ingredients easier.8-107 Corn syrup solids are good
materials for flavor encapsulation and oxygen
uptake, that is, oxidation of orange oil decreases
as DE increases.149 Because there is a strong
dependence on oxidation on DE of the hydro-
lyzed starch,38'120-149 it has been observed that a
product with the highest DE (36.5) would be
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extremely stable and would have a shelf life of
years, without using an antioxidant.
V. OLIGOSACCHARIDES
Starch oligosaccharides are composed of
cc-D-glucopyranosyl units linked by a-1,4 and/or
a-1,6 bonds. The generic term "oligosaccharide"
is customarily used for saccharides having a DP
of 2 to 10. The large-scale production of malto-
oligosaccharides larger than maltotriose has not
been fully developed because these compounds
are rather difficult to manufacture. Oligosaccha-
rides with a DP > 3 have been considered only as
research chemicals and for medical use.133
The discovery of the microbial enzymes that
produce specific oligosaccharides, such as
maltotriose, maltotetraose, maltopentaose, and
maltohexaose, has made posible the manufacture
of syrups with a high content of malto-
oligosaccharide. These syrups are produced from
liquefied starch (DE 3) using the specific enzyme
in combination with debranching enzymes, such
as pullulanase or isoamylase.133 Such syrups are
new products having low sweetness. They pro-
vide resistance to retrogradation of starch gels
and prevent crystallization of sucrose and are now
being used as enhancers for various foods, pow-
dering materials, saccharides for dry milk, and
liquid diets for patients. High purity malto-
oligosaccharides are also used as substrates for
measuring oc-amylase levels in human serum and
urine in clinical tests.133
VI. SWEETENERS
A. Introduction
The development of corn sweeteners can be
traced back to the early nineteenth century, when
the German chemist Kirchhoff discovered that
starch yielded a sweet substance when heated
with acid.109-161 Nevertheless, the ancient Chi-
nese 1000 years before Christ made "Hung Fan,"
meaning "sweet from cereal."30 Kirchhoff s dis-
covery was not used industrially on a large
scale until 1850, and since then, the manufac-
ture of glucose and other syrups through acid
hydrolysis has expanded considerably.51 In
1815, de Saussure identified acid hydrolysis as
the underlying reaction of Kirchhoff s observa-
tion. He found that the end product of the hy-
drolytic reaction was dextrose.106
The discovery, isolation, and application of
various carbohydrase enzymes resulted in the
development of many new corn syrups.51-108 In
the early 1960s, the first commercially signifi-
cant quantities of crystalline dextrose were be-
ing marketed. It was produced from corn starch
by an all-enzyme process using bacterial a-
amylase for starch liquefaction and fungal
amyloglucosidase for saccharification of the
hydrolyzate to produce an approximately 95%
dextrose level.68-106
Hydrolysis with the P-amylase of barley malt
was a process established many years ago.51-127 In
1940, a new class of corn syrup sweeteners that
were clear, colorless, and exceptionally sweet
appeared on the market. These new syrups were
made with the bacterial enzyme P-amylase and
amyloglucosidase, which cause the production of
D-glucose and the concomitant formation of mal-
tose from oligosaccharides.109 The syrups imme-
diately enjoyed great demand because of their
enhanced sweetness.
The commercial development of glucose
isomerase, which converts glucose to its sweeter
isomer fructose,26 was a major milestone in the
corn sweetener industry. The first high-fruc-
tose corn syrup, commonly referred to as HFCS,
appeared in 1967 and contained 15% fruc-
tose.68'104 Further process improvements resulted
in HFCS products containing 42 and 55% fruc-
tose.24-68-106
387
 B. Dextrose Syrups tion is the hydrolysis of oligosaccharides or dex-
trins to low-molecular-weight sugars such as glu-
1. Manufacturing Processes cose, maltose, or a mixture of these and their by-
products.109
The manufacturing of dextrose feedstocks The starch slurry, pH 6.5, is gelatinized by
from starch is a multistage process involving ther- cooking at high temperature with cc-amylase. The
mostable cc-amylase for liquefaction and gelatinized starch is then liquefied by thermo-
amyloglucosidase for saccharification in succes- stable a-amylase. The product of this reaction is
sive enzymatic steps (Figure 8).137 Saccharifica- a soluble dextrin hydrolyzate with a DE of 5 to
15 5i,i29 After liquefaction, the pH of the slurry is
adjusted to 4.5 and the temperature lowered to
STARCH 60°C. Amyloglucosidase is now added and the
SUSPENSION
saccharification reaction is allowed to continue
(38-48 •/. u/v; pH £.5;
for 48 to 96 h in a stirred tank until the maximum
&**; alpha-anylase)
level of glucose is reached.106154'165

1 The aim of the saccharification process is to

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produce a syrup with the maximum amount of

STEAM
GELATIN1ZATI0N D-glucose. The reason for not obtaining higher
(118-148 °C; 5-lfl Hin) levels of glucose (>94%) is generally attributed to
reversion reactions catalyzed by amyloglucosi-
dase.148-154 With the advent of HPLC methods of
sugar analysis,116124162 it has been recognized that
LIQUEFACTION
BE < IS
an additional factor for not obtaining higher yields
of glucose is the formation of maltulose (4-OC-D-
( f l l p h a - a n s l a s e ; 90-95 °C;) glucopyranosyl-D-fructose).37-165 Once the maltu-
lose is formed, the final glucose yield is effec-
tively reduced because amyloglucosidase does not
SACCHARIFICATION
cleave the linkage between the glucose and fruc-
DE > 96
tose.37-148
(fluyloglucosidase; p u l l u l a n a s e j When the DE of the liquefied starch is high
pH 4 . 5 ; 55-69 °C; 48-96 h) (>20), more maltulose is produced than when a
low DE (7) is utilized.37 Maltulose formation is
eliminated when the saccharification is controlled
below pH 6.0.108165
FILTRATION AND
Following saccharification, the liquor is then
CARBON PURIFICATION
filtered to remove protein and fat and purified
with activated carbon to remove color and solubi-
lized protein. Carbon purification is normally fol-
ION-EXCHANGER lowed by an ion-exchange stage to remove
a s h 106,109,165 Then the glucose syrup is evaporated
i
and packaged.
The disadvantages of acid hydrolysis of glu-
EVAPORATION
cose are numerous. It requires the use of expen-
sive, acid-resistant equipment and corrosion prob-
lems are persistent. The nonspecificity of acid
hydrolysis results in the formation of consider-
GLUCOSE SYKUP able quantities of byproducts at the expense of
glucose yields, and the final product requires base
FIGURE 8. Production of glucose syrups. (Adapted
neutralization.142 Byproduct formation and base
from References 51, 104, 151, and 158.) neutralization result in a hydrolyzate high in ash

388
 and color content.137-165 Consequently, the extent
and expense of refining are increased.
If pullulanaseor isoamylase are used simulta-
neously with amyloglucosidase to saccharify a
partially hydrolyzed starch, a slight increase in
glucose yield can be obtained. Less saccharifica-
tion time and amyloglucosidase dosage are re-
quired; therefore, less isomaltose formation due
to reversion reactions takes place and the maxi-
mum glucose levels achieved are higher.129-130-154
The debranching enzyme takes care of the branch
points in the amylopectin so that the amylo-
glucosidase has to hydrolyze only the linear oli-
gosaccharides.13°-154
Replacing soluble amyloglucosidase with its
immobilized form allows for using the same en-
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zyme batch several times in order to shorten the
hydrolysis period and to apply a continuous pro-
cess. However, the use of immobilized amylo-
glucosidase is less efficient in the production of
glucose syrups than the soluble enzyme, giving a
maximum DE value of 96.0% instead of
98.2%.3-29-148 The reason for this lower maximum
DE is the increased formation of isomaltose and
the decreased hydrolysis of the high-molecular-
weight oligosaccharides. This phenomenon is
present due to the concentration gradients in the
enzyme layer of the particle caused by the diffu-
sion resistance.29'155 A successful application of
immobilized amyloglucosidase is in the produc-
tion of high conversion syrups, where the maxi-
mum DE required is only 60 to 70%.151
The continuous production of glucose syrups
in an ultrafiltration reactor has been developed
recently.86 The major components of the reactor
consist of a vessel, a hollow-fiber ultrafiltration
membrane module, a feed (liquefied starch) pump,
and a recycle pump. Concentration of both the
substrate and enzyme and reactor residence time
must be considered in order to maximize sub-
strate conversion and reactor capacity.87 Other
important considerations are the proper selection
and operation of a suitable membrane, the mo-
lecular weight cut-off of the membrane, and its
module design of configuration.89 Conversions of
DE 95 or greater were consistently obtained. The
formation of reversion products appeared to be
more dependent on the total quantity of enzyme
and substrate present in the ultrafiltration reactor.
For economical reasons, a commercial reactor
must be operated at the highest possible substrate
concentration, the lowest possible enzyme con-
centration, and the lowest possible residence
time.88
2. Functional Properties and
Applications
The carbohydrate composition of typical glu-
cose syrups are as follows: 94 to 98% glucose, 1
to 3% maltose, 0.3 to 0.5% maltotriose, and 1 to
2% higher saccharides.106-109 Glucose syrups are
used for nonyeast fermentation purposes (phar-
maceuticals, organic acids, or vitamins),51 for the
manufacture of crystalline D-glucose,109 or in the
production of fructose syrups by further continu-
ous processing.16-104-106
Glucose also provides a yeast-fermentable
source,96-137 and when heated in the presence of
amino acids, undergoes the Maillard-browning
process, thereby producing a brown color and
generating flavors.46 Perhaps one of the most stud-
ied chemical reactions in foods is the Maillard
reaction.58-107 The browning reaction has been stud-
ied from the dual standpoints of color and flavor
formation with almost equal attention to both,
indicating its importance in heat processing of
many kinds of foods.182-185
Glucose is used to produce polydextrose,
which is a nonsweet, randomly bonded glucan
containing small amounts of sorbitol and citric
acid.39-167 One of its most important properties is
the caloric value, which is only 1 cal/g, allowing
for the formulation of reduced-calorie products.8
When dextrose is treated with hydrogen un-
der pressure at elevated temperatures, it forms
sorbitol. This product has high resistance to color
development in storage or when heated. It also
offers good moisture retention. The dry form of
sorbitol is anhydrous and has a cooling effect
when eaten.8-110 Another use of this product is as
cryoprotectant for meats.111
C. Maltose Syrups
1. Introduction
There are three important types of maltose-
containing syrups: (1) high maltose syrups, (2)
389
 extremely high maltose syrups, and (3) high con-
version syrups.153 The general DE value of these
syrups and their saccharide composition is given
in Table 2. Starch hydrolyzates of 35 to 60%
maltose content on a dry substance basis, after
refining and concentration, are the so-called high
maltose syrups.126 Extremely high maltose syrups
have a 70 to 85% maltose content.125 High con-
version syrups are characterized by their high
glucose (35 to 43%) and maltose (30 to 47%)
contents; they have the highest DE (60 to 70).156
These syrups have a high fermentable sugar con-
tent (>85%).152-153
2. Manufacturing Process
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enzymes cannot act on a-1,6 linkages, the final
maltose content is limited to around 60%.148 As
the reaction is self-terminating, no specific en-
zyme inactivation step is required.109
In the case of extremely high maltose syrup
production, basically the same steps for the pro-
duction of high-maltose syrups are followed (Fig-
ure 9). However, one of the most important
diferences is that it is necessary to use a
debranching enzyme, such as pullulanase or
isoamylase, in addition to P-amylase or fungal oc-
amylase.125'126-129 Incorporating debranching en-
zymes into the saccharification system produces
higher maltose levels (Table 2).
The high-conversion syrups are produced by
saccharifying a liquefied starch by using both

p-amylase or fungal oc-amylase and amylo-

The production of various maltose syrups from
starch generally involves a two-step process. The
first step is the liquefaction of starch (Figure 9).
In this step, the starch usually is suspended in an
aqueous medium and gelatinized by heat and par-
tially hydrolyzed using a thermostable oc-amy-
lase.148 The second step in the preparation of
maltose syrups from starch substrates is sacchari-
fication. This is commercially done by a maltose-
producing enzyme such as microbial P-amylase
or fungal a-amylase.62-66-108'148
There are some variations in the production
of different maltose syrups (Figure 9). In order to
produce high maltose syrup, saccharification of
liquefied starch must be done by P-amylase or
fungal oc-amylase, for which the pH of the lique-
fied starch is adjusted to 5.0 to 5.5 and the tem-
perature is lowered to 50 to 55°C.125'148 The reac-
tion is continued until the desired level of maltose
is produced.126-152-153 Because these maltogenic
glucosidase.153 Once the saccharification is com-
plete, further glucose production is terminated by
inactivation of amyloglucosidase at 80 to 85°C.152
The maximum glucose concentration in these
syrups should not exceed 43% on a dry substance
basis.
The use of lower temperatures during saccha-
rification presents handling problems because of
high viscosities. C. thermosulfurogenes produces
an extracellular P-amylase that is stable and ac-
tive at 80 and 75°C.153'156 With this enzyme, it is
possible to produce maltose syrups by using higher
temperatures than those applied in the past, thus
avoiding low-temperature problems.
Purification and refining of maltose syrups
is accomplished by first filtering to remove in-
soluble matter such as fat and denatured pro-
tein. The syrup is then refined by means of
activated carbon and ion exchange, which re-
moves color, ash, and other minor impurities.

TABLE 2
The Composition of Maltose Syrups and Their Dextrose Equivalent (DE)

Saccharide High maltose Extremely high High conversion

(%, carbohydrate basis) syrup maltose syrup syrup

Glucose 0.5-3.0 1.0-3.0 35.0-43.0

Maltose 45.0-30.0 70.0-85.0 30.0-47.0
Maltotriose 6.0-25.0 8.0-21.0 8.0-15.0
Higher saccharides Balance Balance Balance
DE 35-50 45-60 60-70

Adapted from References 51, 149, and 150.

390
 STARCH
SUSPENSION
(38-40 X. w/v; pH £.5;
Ca++; alpha-aitylase)

J
GELATINIZATION
STEAM
(110-148 °C; 5-10 * i n )

LIQUEFACTION LIQUEFACTION
CE 5-18 1>E 38-48
(fllpha-amjlase; 98-95 °C; (flcid/alpha-aiwlase)
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90-128 Nin>

SACCHARIFICATIOH
SACCHARIFICATION SACCHARIFICATION BE 68-70
BE 38-35 BE 45-68 (Fungal alpba-aHylase or b e t a -
(Fungal alplia-awlase or beta- (Fungal alpha-anylase o r b e t a - aitylase; anuloglucosidase;
a n g l a s e ; ?H 5 . 8 - 5 . 5 ; 58-55 °C> anylase; pullulanase o r pH 6 . 8 ; 75 °C)
i s o a x y l a s e ; pH 6 . 0 ; 50-55 °C)

<
ENZYME INACTIUATION
FILTRATION FILTRATION (80-85 aC; 15 n i n )
AND AND
PURIFICATION PURIFICATION

FILTRATION
am
PURIFICATION

HIGH HAL70SE EXISEflELi1 KM

SYRUPS MALTOSE SYRUPS
HIGH CONVERSION
SYRUPS

FIGURE 9. Production of various maltose syrups by conventional processes. (Adapted from References 51,149,
and 150.)

The clarified solution may then be concentrated
by evaporation.
The development of immobilized enzyme
systems for maltose syrup production is advanta-
geous for several reasons, including enzyme re-
use and recovery, low cost and convenience of a
continuous reaction system, and higher reaction
rates due to high concentration of enzymes. Im-
mobilization of P-amylase on a variety of carriers
is documented.35 Although immobilized enzymes

391
 are not yet used in industry for starch hydroly-
sis,152 some attempts have been made to develop
a continuous maltose production process under
industrial-scale conditions.35 Making use of the
appropriate carrier and enzyme and applying the
correct immobilization procedure, a conjugate
with acceptable initial performance and stability
can be obtained.
Continuous production of high maltose syr-
ups in an ultrafiltration reactor has been devel-
oped by simultaneously using P-amylase and
isoamylase.77
3. Functional Properties and
Applications
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In general, various maltose syrups are used in
the brewing, baking, soft drink, canning, and con-
fectionery industries. These syrups can also be
used as moisture conditioners, stabilizers, carri-
ers, and bulking agents.153 They have a decreasing
tendency to crystallize and are relatively
nonhygroscopic. Hence, they are used in the manu-
facture of hard candies by the confectionery in-
dustry and in frozen desert formulations, where
they control ice crystal formation.51
In particular, the high maltose syrups are char-
acterized by their low viscosity of solution, low
hygroscopicity, good heat stability, and mild
sweetness.126 They may also be used as nutrients
for children,125 or to impart reduced browning
capacity.153
Extremely high maltose syrups are used to
produce pure crystalline maltose, which is of in-
creasing interest in the pharmaceutical industry,
where it may be used instead of D-glucose for
intravenous feeding of diabetics.126152 Crystalline
maltose would meet the requirements of those
applications due to its high purity.158
The catalytic hydrogenation of maltose gives
rise to maltitol, a polyhydric alcohol, which is a
low-calorie sweetener and is readily metabo-
lized.152 The isomerization of maltose produces
maltulose, which is on the same level as maltitol
in sweetness properties; it is 75% as sweet as
sucrose, resistant to crystallization, and useful as
a humectant. Chewy candies, fondant, and hard
candies are examples of its applications.39
The high conversion syrups have wide appli-
cations in many industries, for example, brewing,
392
baking, confectionery, canning, soft drinks, etc.156
Although these syrups have a high glucose con-
tent (35 to 42%), they are stable enough to crys-
tallization at temperatures as low as 4°C and at 80
to 83% dry substance.152
D. High-Fructose Corn Syrups
1. Introduction
Beet sugar usage grew along with that of
dextrose and corn syrup, but other sources of
nutritive sweeteners were still sought. Sucrose
was known to be a disaccharide consisting of
1 mol of D-glucose and 1 mol of D-fructose, linked
by an oxygen bridge at the respective aldose and
ketose functions. Fructose is also found in the free
state in certain fruits, vegetables, and honey.
As early as 1926, the Hon. Cyrenus Cole,
U.S. Representative in the House for the State of
Iowa, advocated growing the Jerusalem artichoke
(Helianthus tuberosus), a variety of sunflower, as
a crop because its tuber was rich in the polymer
inulin, which could be hydrolyzed to levulose.17
This term was used in preference to fructose to
designate the industrial product for the next 50
years. Levulose remained a target, and an elusive
one, until the commercialization of glucose
isomerase in the late 1960s and early 1970s.27
Then the name was changed and attention turned
to the economic potential of fructose.
The first shipment of HFCS was made in
1968, and in 1985 shipments exceeded 10 billion
lbs of dry solids.104 This shipment of HFCS was
the first generation of fructose syrups, which con-
tained 42% fructose and 50 to 55% dextrose.24-106
The syrup served as only a partial replacement for
sucrose. The use of HFCS in beverages did not
accelerate until the development of the second
generation in the mid-1970s. This product con-
tains 55% fructose and 40% dextrose.99-106 The
industry also produces a 90% fructose syrup.
The use of HFCS in the U.S. has increased
from 12.1 lb/person in 1978 to 42.0 lb/person in
1986.45 By 1990, the per capita intake of corn
sweeteners was 61.3 lb;53 a high percentage of
this was HFCS. This per capita increase has been
supported by one of its most desirable attributes:
sweetness. It has been reported that fructose has
a sweetness of up to 1.8 times that of sucrose.84-163
 2. Manufacturing Processes
Preparation of very high-quality dextrose feed-
stock for isomerization is first necessary because
of the very low color and ash specifications for
the finished HFCS. A high-purity feedstock is
also required for efficient utilization of the immo-
bilized isomerase enzyme column.
In the refining process, the high-dextrose
conversion liquor is filtered on rotary precoat
filters to remove coagulated protein and oil (Fig-
ure 10). This filtered liquor is then passed through
a series of check filters to remove traces of par-
ticulate matter.106 First, color is removed, and
then the carbon-treated liquor is filtered again and
passed through an ion-exchange system. It is then

HIGH DEXTROSE SYRUPS

O 9 S JO

FILTRATION
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PRIMARY CARBON TREATMENT;

ION EXCHANGE AND EVAPORATION

CHEMICAL TREATMENT

ISOMERIZATION
glucose isoiterase;
pH 8.5; 55-65 °C; 4h>

GLUCOSE
SECONDARY CARBON TREATMENT,
ION EXCHANGE AND EVAPORATION

CHR0MAT0GR81WC
42ZHFCS FRACTIONATION

I.
BLENDING S8ZHFCS

55ZKFCS

FIGURE 10. Flow chart for the manufacture of 42% high fructose
com syrup as well as 55 and 90% high fructose com syrup.
(Adapted from References 24, 102, and 140.)

393
 evaporated to a solid matter of 40 to 45%. Before
pH is adjusted, the refining glucose syrup is chemi-
cally treated by the addition of magnesium ions,42
which are activators of glucose isomerase and
also competitively inhibit the action of any re-
sidual calcium ions, which are potent inhibitors of
the enzyme.188
Byproduct formation at the isomerization
process is a function of temperature, pH, and
time. Under the conditions used, a residence
time of up to 4 h is considered safe, and the
small amount of color formed can be removed
by conventional carbon treatment after the
isomerization.24'106'132 Use of ion-exchange re-
fining in addition to carbon is necessary in order
to remove ash.
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The process used to produce the 55% HFCS
is also shown in Figure 10. This syrup is made
from the 42% syrup by a fractionation and blend-
ing process. It is pumped into the fractionation
unit, where calcium sites retain the fructose while
dextrose and the higher saccharides pass through.
The dextrose and higher saccharide stream is re-
turned to the 42% production channel. Water is
pumped through the fractionation unit to release
the fructose. The outlet stream generally is 80 to
90% fructose. It is then blended with the 42%
HFCS to make the 55% product.2451'104106
Besides sucrose and starch, other potential
commercial sources of fructose exist. One of these
is inulin. Inulin is a linear p-2,1-linked fructose
polymer terminated by a sucrose unit residue. It
occurs as an energy reserve in various plants,
particularly in those of the Compositae family;
good sources are chicory, Jerusalem artichoke,
and dahlia. The enzymatic hydrolysis of inulin,
using inulinase from yeast, has been reported as
an alternative way to produce fructose.187 There
have been other attempts to produce fructose from
other starch sources like cassava16-56 and broken
rice.31
As explained previously, fructose production
involves several separate enzymatic steps includ-
ing liquefaction, by cc-amylase, saccharification
by amyloglucosidase, and isomerization by glu-
cose isomerase; these steps require different reac-
tion conditions with their concomitant problems.
A single-step process for fructose production is
desirable. With this in mind, a single-step process
394
was developed to produce a 49:51 mixture of
glucose and fructose from liquefied starch.101
3. Functional Properties and
Applications
One of the most important characteristics
of HFCS is sweetness. In this respect, 42%
HFCS is about equal to sucrose (Table 3), 55%
HFCS is slightly higher, and 90% HFCS has
the greatest relative sweetness (180).58 Never-
theless, sweetness values vary with tempera-
ture, pH, and solids. In general, relative sweet-
ness of fructose is a function of concentration,
temperature, and pH, with the maximum sweet-
ness values being found in cooled, dilute solu-
tions at low pH.2484'104 Among other character-
istics of HFCS are the ability to produce food
systems with lower freezing points and higher
boiling points, thus effectively inhibiting crys-
tallization,8 and enhancing citrus flavor in fruit
products.46-75 There have been some reports
about the synergistic effects of HFCS on sweet-
ness. Increase in sweetness is perceived when
fructose is used with other nutritive and
nonnutritive sweeteners.24-75
The first-generation HFCS, containing 42%
fructose, 52% glucose, and 6% of higher sac-
charides (Table 3), found widespread use in
baking and other food products.96 However,
researchers have encountered problems that
limit the frequency and quantity of HFCS in
baked products.33 Excessive browning of the
TABLE 3
Typical Composition and Sweetness of High
Fructose Corn Syurps (HFCS)
42% 55% 90%
HFCS HFCS HFCS
Fructose (%) 42 55 90
Dextrose (%) 52 40 9
Higher saccharides (%) 6 5 1
Dry solid content (%) 75 77 80
Sweetness 8 100 100+ 160-180
a
Relative to sucrose = 100.
Adapted from References 24, 94, and 102.
 crust and crumb of cakes containing HFCS was
attributed to the Maillard-browning reactivity
of monosaccharides in the HFCS.114181 Johnson
et al.80 controlled the browning of cakes pre-
pared with up to 100% HFCS as a sucrose
replacement with the acidulant potassium acid
tartrate or cream of tartar. The total browning
and 5-hydroxymethylfurfural content of the
crust and crumb was altered as the amount of
HFCS replacement increased, indicating that
the acidulant reduces the browning reactivity
of monosaccharides by lowering the pH of the
batter prior to baking. The replacement of su-
crose by other sweeteners in cakes is not as
simple as it is in breads and requires special
reformulation of the system. The effect observed
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in cakes when sucrose has been replaced by
42% HFCS includes a decrease in baking time,
lower cake volume, change in taste and sweet-
ness, and gummy mouthfeel.96
The second generation HFCS, containing
55% fructose, 40% glucose, and 5% higher sac-
charides, has major use in beverages, especially
in soft drinks. Cola drinks account for approxi-
mately 63% of the soft drink volume. In 1980,
the 55% HFCS was first used in a major cola at
a 50% sweetener replacement level, and in 1984,
all cola manufacturers approved usage of the
syrup at a 100% replacement level. In the late
1970s and early 1980s, the noncola soft drink
producers switched to 100% replacement with
H F C S 68,104
The 55% HFCS was also used as replace-
ment for sucrose in sugar-snap cookies. It was
reported that 55% HFCS satisfactorily substi-
tuted for 10 or 30% of the sucrose in this type
of cookie.10 The cookies appear to dry out as
they cool because the water yielded by the su-
crose, as it crystallizes, is bound by the flour
components (starch and protein). HFCS acts as
a crystallization inhibitor and will keep sucrose
in solution, producing a cookie that retains its
soft texture.59-96-181
A very small amount of 90% HFCS is sold
commercially.104 It is available in liquid and
crystalline forms; the latter is a relatively new
product developed from advanced technology.75
Crystalline fructose, the P-pyranose form, has
the greatest relative sweetness (180), but this
decreases in aqueous solution to about 160.58
One of its applications is in food for diabetics39
because it is metabolized without insulin.
VII. CYCLODEXTRINS
A. Introduction
Cyclodextrins have been known for nearly
100 years. In 1891, Villiers discovered crystalline
dextrins, "cellulosines", in bacterial digests of
starch; as early as 1903, Schardinger demonstrated
the cyclic structure of these compounds.72-160 Be-
cause of Schardinger's studies, cyclodextrins were
initially referred to as Schardinger's dextrins; of
more significance was the fact that his work, al-
though crude, set the direction for future research,
pointing it toward a study of the structure of
cyclodextrins and their preparation. In 1938,
Freudenberg confirmed the ringed structure of
cyclodextrins and their ability to form inclusion
complexes.146
Three main types of cyclodextrins are known
(Figure 5): a-, P-, and y-cyclodextrins consisting
of six, seven, and eight glucose monomers, re-
spectively.72 These monomers are bound together
in a doughnut-shaped ring, giving the cyclodextrins
a molecular structure that is relatively rigid and
has a hollow cavity of a specific volume. These
cyclic molecules have neither a nonreducing nor
reducing end-group.
B. Manufacturing Processes
For commercial production of cyclodextrins,
two basic processes are used: the solvent and the
nonsolvent methods.72-176 In the solvent method, a
solvent directs the enzyme reaction to produce
predominantly one cyclodextrin. Using this
method, the reactor is filled with the starch sub-
strate, the enzyme, and the solvent. The substrate
is generally a partially hydrolyzed starch with a
DE <10.
At the end of the reaction period, the reactor
contains a mixture of an insoluble cyclodextrin
complexed with the solvent (i.e., oc-cyclodextrin
forms an insoluble complex with 1-decanol, and
395
 (5-cyclodextrin precipitates out with cyclo-
octane),128 starch hydrolysate produced by the
transglycosylation and hydrolysis reactions, and
the residual chemical. The cyclodextrin complex
is separated from the other components by cen-
trifugation or filtration. After removal of the
chemical from cyclodextrin, the soluble cyclo-
dextrin is refined like any other soluble starch-
derived product and crystallized. The cystals
are then recovered and dried. Because the sepa-
ration processes are highly efficient, the level of
residual chemical in the final product is <1 ppm.72
In the nonsolvent process, the reactor is
filled with a starch hydrolysate and the en-
zyme. As no organic chemicals are present to
prevent the growth of microorganisms, which
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could lead to contamination of the final product
with microbial metabolites, aseptic or sterile
conditions must be used. At the completion of
the reaction, the CDGTase is inactivated by
heat or acid. An oc-amylase unable to hydrolyze
the cyclodextrins is added to hydrolyze the
noncyclic dextrins.72
After refining, water is evaporated from the
reaction mixture to maximize the yield of the
(3-cyclodextrins, which is the least soluble and
generally the most readily crystallizable cyclo-
dextrin. If too much water is removed, more
than one cyclodextrin will be crystallized. The
crystals are collected by centrifugation or fil-
tration, washed with a small amount of water,
and dried.
C. Functional Properties and
Applications
The attraction of these cyclic, torus-shaped
maltooligosaccharides arises from their ability to
TABLE 4
Properties of Cyclodextrins
Molecular size
Type Glucose Inside Outside
units diameter diameter
a 6 5.7 13.7
P ' 1 7.8 15.3
Y 8 9.5 16.9
Adapted from Reference 143.
396
form inclusion complexes with a wide variety of
organic molecules of suitable size (guest mol-
ecules).
In aqueous solution, the slightly apolar
cyclodextrin cavity is occupied by water mol-
ecules. This polar-apolar interaction is energeti-
cally unfavorable and water can be readily dis-
placed by appropriate guest molecules that are
less polar, provided that their geometrical dimen-
sions match those of the cavity.146-174 This inclu-
sion complex formation is also known as "mo-
lecular encapsulation". The inclusion complexes
can be isolated as stable crystalline substances.
The most important parameter for complex
formation with hydrophobic substances is their
three-dimensional size (Table 4). Thus, for ex-
ample, oc-cyclodextrins typically form inclusion
complexes with aliphatic hydrocarbons, but, curi-
ously, also with gases like ethylene and carbon
dioxide. (3-Cyclodextrins form complexes with
molecules such as naphthols or steroids, whereas
y-cyclodextrins can accept bulky guests like vita-
min D2.160
Although cyclodextrins have been studied for
nearly a century, they were not utilized for food
applications until the last decade for several rea-
sons. First, cyclodextrins were expensive to pro-
duce and available in only limited amounts; sec-
ond, incomplete studies reported them to be toxic;
and third, there was not enough knowledge to
produce them on an industrial scale.146-160 How-
ever, because of continuing research, these barri-
ers have been gradually removed so that use is
now technologically possible in a variety of food
applications.
Perhaps the greatest potential for cyclodextrins
is in the area of flavors and spices. Flavor con-
stituents often have an unstable nature, which
(A)
Water solubility
Height (g/100 ml; 25°C)
7.0 14.50
7.0 1.85
7.0 23.20
 may restrict their application into formulated foods
and shorten the shelf life of the product.
Cyclodextrins, however, can stabilize volatile
materials.146
Cyclodextrins can also stabilize emulsions of
fats and oils, shielding them from oxidation and
thus preventing rancidity.175 They can also be
used to debitter citrus juices. This application is
extremely important because, previously, the cit-
rus industries have tried to mask the bitterness in
juice blends rather than remove it.94-164
With respect to bioconversions and fermenta-
tions, cyclodextrins apparently possess an advan-
tageous combination of properties. First, they
enhance solubilization of organic compounds.
Second, they reduce toxicity. Complexation of
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organic substrates and/or products may signifi-
cantly reduce their concentration.13 Third, they
are biocompatible. Cyclodextrins are known to
cause no damage either to free enzymes or to
microbes.13
Although the potential of cyclodextrins is
well documented, the market for these products
is still limited. Two important facts are respon-
sible for the present market situation: their high
cost and the limited availability of a- and y-
cyclodextrins.160 Cloning and overexpression of
CGTase genes provide a straightforward way to
satisfy the anticipated expansion of the
cyclodextrin market.160
A novel production of cyclodextrins in the
tubers of transgenic potato plants has been devel-
oped.131 This report declares that even though the
levels of cyclodextrins were low, this is the first
step in using genetic engineering for the "molecu-
lar farming" of novel carbohydrates.
VIII. CONCLUSIONS
Food processing through enzyme utilization
offers many advantages. Starch transformation is
an industry in which chemical processing has
yielded almost completely to enzyme processing.
Amylolytic enzymes have gained importance be-
cause of their ability to catalyze batch reactions
under moderate conditions, which are not reached
with chemical processes wherein nonspecific hy-
drolysis and formation of byproducts are present.
The technique of enzyme immobilization proved
to be a success, especially for fructose syrup pro-
duction. However, in order to improve the
economy of enzymatic catalysis in the production
of maltodextrins, glucose, or maltose, highly effi-
cient processes have been developed. Today,
specific amylases allow batch preparation of tai-
lor-made specific maltodextrin products, cyclo-
dextrins, or high glucose and maltose syrups.
Maltodextrins are important components of
foods, contributing a broad range of functionalities.
One of these is as a fat substitute. Financial ana-
lysts forecast that fat substitutes may match or
even surpass the success of sugar substitutes.57 In
1991, two thirds of the adult population, 124
million Americans, were using products with low
or reduced fat. These consumers included 72%
women and 61% men.121 There are now two dis-
tinct types of light food consumers: (1) dieters
and (2) calorie-, fat-, and health-conscious
nondieters. These consumers represent signifi-
cant markets.
As the demand for starch-based sweeteners
grows, high-maltose- and high-fructose-contain-
ing syrups will gain importance, principally as
industrial commodities for use as food ingredi-
ents. The production of maltose-containing syr-
ups has increased for the last several years in
Japan and the U.S. An increasing interest in mal-
tose products is also predicted in the European
markets.158 The market for high-fructose syrups
has grown dramatically in the United States espe-
cially for the soft drink industry.
The various applications for cyclodextrins
include pharmaceutical delivery systems, flavor
and odor enhancement, and the removal of un-
desirable compounds (such as caffeine) from
foods. In view of their high potential, as
cyclodextrins prices continue to drop, there will
undoubtedly be much research in this area. It is
necessary that the fine structure analysis of the
enzyme CGTase be completed. Then it may be
possible to engineer more specific a- or y-CGTases
or even CGTases that catalyze the synthesis of
cyclodextrins with more than eight glucose resi-
dues.
As the food industry continues to move to-
ward developing new amylolytic enzymes and
novel starch processes to provide products that
meet consumers' high expectations, sales will
continue to increase.
397
 ACKNOWLEDGMENT
This work was supported by Consejo Nacional
de Ciencia y Tecnologia (CONACYT).
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