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10 Chymosin in Cheese Making 245

trypsin and have an optimum pH activity between 7

and 8 (Green 1977, Kosikowski and Mistry 1997).

Microorganisms, including Bacillus subtilis, B.

cereus, B. polymyxa, Cryphonectria parasitica (for-

merly Endothia parasitica), and Mucor pusillus

Lindt (also known as Rhizomucor pusillus) and Rh.

miehei have been extracted for their protease

enzymes. The bacilli enzyme preparations were not

suited for cheese making because of excessive pro-

teolytic activity while the fungal-derived enzymes

gave good results, but not without off ﬂavors such as

bitter. Commercial enzyme preparations isolated

from Cr. parasitica led to good quality Emmental

cheese without bitter ﬂavor, but some cheeses, such

as Cheddar, that use lower cooking temperatures

reportedly showed bitterness. Enzyme preparations

of Rh. Miehei, at recommended levels, produced

Cheddar and other hard cheeses of satisfactory qual-

ity without bitter ﬂavor. Fungal enzyme preparations

are in commercial use, particularly in North Amer-

ica. In cottage cheese utilizing very small amounts

of coagulating enzymes, shattering of curd was min-

imum in starter-rennet set milk and maximum in

starter–microbial milk coagulating enzyme set milk

(Brown 1971).

Enzymes derived from Rh. miehei and Rh. pusillus

are not inactivated by pasteurization. Any residual

activity in whey led to hydrolysis of whey proteins

during storage of whey powder. This problem has

been overcome by treating these enzymes with per-

oxides to reduce heat stability. Commercial prepara-

tions of Rh. miehei and Rh. pusillus are inactivated

by pasteurization.The heat stability of Cr. parasitica–

derived enzyme is similar to that of chymosin.

These fungal enzymes are also milk-clotting as-

paratic enzymes, and all except Cr. parasitica clot

milk at the same peptide bond as chymosin. Cryphon-

ectria clots kappa-casein at the 104-105 bond.

CHYMOSIN ACTION ON MILK

Chymosin produces a smooth curd in milk, and it is

relatively insensitive to small shifts in pH that may

be found in milk due to natural variations, does not

cause bitterness over a wide range of addition, and is

not proteolytically active if the cheese supplements

other foods. Chymosin coagulates milk optimally at

pH 6.0–6.4 and at 20–30°C in a two-step reaction,

although the optimum pH of the enzyme is approx-

imately 4 (Kosikowski and Mistry 1997, Lucey

2003). Optimum temperature for coagulation is

approximately 40°C, but milk for cheese making is

coagulated with rennet at 31–32°C because at this

temperature the curd is rheologically most suitable

for cheese making. Above pH 7.0, activity is lost.

Thus, mastitic milk is only weakly coagulable, or

does not coagulate at all.

Chymosin is highly sensitive to shaking, heat,

light, alkali, dilutions, and chemicals. Stability is

highest when stored at 7°C and pH 5.4–6.0 under

dark conditions. Liquid rennet activity is destroyed

at 55°C, but rennet powders lose little or no activity

when exposed to 140°C. Standard single-strength

rennet activity deteriorates at about 1% monthly

when held cold in dark or plastic containers.

Single-strength rennet is usually added at 100–

200 mL per 1000 kg of milk. It serves to coagulate

milk and to hydrolyze casein during cheese ripening

for texture and ﬂavor development.

It should be noted that bovine chymosin has

greater speciﬁcity for cow’s milk than chymosin

derived from kid or lamb. Similarly, kid chymosin is

better suited for goat milk.

Milk contains fat, protein, sugar, salts, and many

minor components in true solution, suspension, or

emulsion. When milk is converted into cheese, some

of these components are selectively concentrated as

much as 10-fold, but some are lost to whey. It is the

fat, casein, and insoluble salts that are concentrated.

The other components are entrapped in the cheese

serum or whey, but only at about the levels at which

they existed in the milk. These soluble components

are retained, depending on the degree that the serum

or whey is retained in the cheese. For example, in a

fresh Cheddar cheese, the serum portion is lower in

volume than in milk. Thus, the soluble component

percentage of the cheese is smaller.

In washed curd cheeses such as Edam or Brick the

above relationship does not hold, and lactose, solu-

ble salts, and vitamins in the ﬁnal cheese are re-

duced considerably. Approximately 90% of living

bacteria in the cheese-milk, including starter bacte-

ria, are trapped in the cheese curd. Natural milk

enzymes and others are, in part, preferentially ab-

sorbed on fat and protein, and thus a higher concen-

tration remains with the cheese. In rennet coagula-

tion of milk and the subsequent removal of much of

the whey or serum, a selective separation of the milk

components occurs, and in the resulting concentrat-

ed curd mass, many biological agents become ac-

tive, marking the beginning of the ﬁnal product,

cheese.

10CH_Hui_277065 10/18/05 7:35 AM Page 245

246 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking

Milk for ripened cheese is coagulated at a pH

above the isoelectric point of casein by special pro-

teolytic enzymes, which are activated by small

amounts of lactic acid produced by added starter

bacteria. The curds are sweeter and more shrinkable

and pliable than those of fresh, unripened cheeses,

which are produced by isoelectric precipitation.

These enzymes are typiﬁed by chymosin that is found

in the fourth stomach, or abomasum, of a young calf.

The isoelectric condition of a protein is that at

which the net electric charge on a protein surface is

zero. In their natural state in milk, caseins are nega-

tively charged, and this helps maintain the protein in

suspension. Lactic acid neutralizes the charge on the

casein. The casein then precipitates as a curd at pH

4.6, the isoelectric point, as in cottage or cream

cheese and yogurt. For some types of cheeses this

type of curd is not desirable because it would be too

acid, and the texture would be too ﬁrm and grainy.

For ripened cheeses such as Cheddar and Swiss, a

sweeter curd is desired, so casein is precipitated as a

curd near neutrality, pH 6.2, by chymosin in 30–45

minutes at 30–32°C. This type of curd is signiﬁcant-

ly different than isoelectric curd and much more

suitable for ripened cheeses. Milk proteins other

than casein are not precipitated by chymosin. The

uniform gel formed is made up of modiﬁed casein

with fat entrapped within the gel.

MILK COAGULATION AND PROTEIN

HYDROLYSIS BY CHYMOSIN

The process of curd formation by chymosin is a

complex process and involves various interactions

of the enzyme with speciﬁc sites on casein, temper-

ature and acid conditions, and calcium. Milk is

coagulated by chymosin into a smooth gel capable

of extruding whey at a uniformly rapid rate. Other

common proteolytic enzymes such as pepsin, try-

psin, and papain coagulate milk too, but may cause

bitterness and loss of yield and are more sensitive to

pH and temperature changes.

An understanding of the structure of casein in

milk has contributed to the explanation of the mech-

anism of chymosin action in milk. Waugh and von

Hippel (1956) began to unravel the heterogeneous

nature of casein with their observations on kappa-

casein. Casein is comprised of 45–50% alpha-

s-casein, 25–35% beta-casein, 8–15% kappa-casein,

and 3–7% gamma-casein. Various subfractions of

alpha-casein have also been identiﬁed, as have gen-

etic variants. Each casein fraction differs in sensitiv-

ity to calcium, solubility, amino acid makeup and

electrophoretic mobility.

An understanding of the dispersion of casein in

milk has provoked much interest. Several models of

casein micelles have been proposed including the

coat-core, internal structure, and subunit models.

With the availability of newer analytical techniques

such as three-dimensional X-ray crystallography,

work on casein micelle modeling is continuing, and

additional understanding is being gained. The sub-

unit model (Rollema 1992) is widely accepted. It

suggests that the casein micelle is made up of small-

er submicelles that are attached by calcium phos-

phate bonds. Hence, the micelle consists of not only

pure casein (approximately 93%), but also minerals

(approximately 7%), primarily calcium and phos-

phate. Most of the kappa-casein is located on the

surface of the micelle. This casein has few phospho-

serine residues and hence is not affected by ionic

calcium. It is located on the surface of the micelle

and provides for stability of the micelle and protects

the calcium-sensitive and hydrophobic interior, in-

cluding alpha- and beta- caseins from ionic calcium.

These two caseins have phosphoserine residues and

high calcium-binding afﬁnity. The carbohydrate, N-

acetly neuramic acid, which is attached to kappa-

casein, makes this casein hydrophilic. Thus, the

casein micelle is suspended in milk as long as the

properties of kappa-casein remain unchanged. More

recently the dual-bonding model has been proposed

(Horne 1998).

Introducing chymosin to the cheese-milk, nor-

mally at about 32°C, destabilizes the casein micelle

in a two-step reaction, the ﬁrst of which is enzymat-

ic (or primary) and the second, nonenzymatic (or

secondary) (Kosikowski and Mistry 1997, Lucey

2003). These two steps are separate but cannot be

visually distinguished: only the appearance of a curd

signiﬁes the completion of both steps. The primary

phase was probably ﬁrst observed by Hammersten

in the late 1800s (Kosikowski and Mistry 1997) and

must occur before the secondary phase begins.

In the primary phase, chymosin cleaves the

phenylalanine-methionine bond (105-106) of kappa-

casein, thus eliminating its stabilizing action on

calcium-sensitive alpha-s- and beta-caseins. In the

secondary phase, the micelles without intact kappa-

casein aggregate in the presence of ionic calcium in

milk and form a gel (curd). This mechanism may be

summarized as follows (Fig. 10.1):

10CH_Hui_277065 10/18/05 7:35 AM Page 246

10 Chymosin in Cheese Making 247

The macropeptide, also known as caseinmacro-

peptide, contains approximately 30% amino sugar,

hence the name glycomacropeptide (Lucey 2003).

In the ultraﬁltration processes of cheese making, this

macropeptide is retained with whey proteins to in-

crease cheese yields signiﬁcantly. In the conventional

cheese-making process, the macropeptide is found

in whey.

The two-step coagulation does not fully explain

the presence of the smooth curd or coagulum. Beau,

many years ago, established that a strong milk gel

arose because the ﬁbrous ﬁlaments of paracasein

cross-linked to make a lattice. Bonding at critical

points between phosphorus, calcium, free amino

groups, and free carboxyl groups strengthened the

lattice, with its entrapped lactose and soluble salts.

A parallel was drawn between the gel formed when

only lactic acid was involved as in fresh, unripened

cheese and when considerable chymosin was in-

volved as in hard, ripened cheese. Both gels were

considered originally as starting with a ﬁbrous pro-

tein cross bonding, but the strictly lactic acid curd

was considered weaker, because not having been hy-

drolyzed by a proteolytic enzyme, it possessed less

bonding material. The essential conditions for a

smooth gel developing in either case were sufﬁcient

casein, a quiescent environment, moderate tempera-

ture, and sufﬁcient time for reaction.

FACTORS AFFECTING

CHYMOSIN ACTION IN MILK

Rennet coagulation in milk is inﬂuenced by many

factors that ultimately have an impact on cheese

characteristics (Kosikowski and Mistry 1997). The

cheese maker is usually able to properly control

these factors, some of which have a direct affect on

the primary phase and others on the secondary.

CHYMOSIN CONCENTRATION

Chymosin concentration affects gel ﬁrmness (Lom-

holt and Qvist 1999), and enzyme kinetics have

been used to study these effects. An increase in the

chymosin concentration (larger amounts of rennet

added to milk) reduces the total time required for

rennet clotting, measured by the appearance of the

ﬁrst curd particle. As a result, the secondary phase

of rennet action will also proceed much earlier, with

the net result of an increase in the rate of increase in

gel ﬁrmness. This property of chymosin is some-

times used to control the rennet coagulation in con-

centrated milks, which have a tendency to form

ﬁrmer curd because of the higher protein content.

Lowering the amount of rennet reduces the rate of

curd ﬁrming. It should also be noted that with an

alteration in the amount of rennet added to milk,

there also will be a change in the amount of residual

chymosin in the cheese.

TEMPERATURE

The optimal condition for curd formation in milk

with chymosin is 40–45°C, but this temperature is

not suitable for cheese making. Rennet coagulation

for cheese making generally occurs at 30—35°C for

proper ﬁrmness. At lower temperatures rennet clot-

ting rate is signiﬁcantly reduced, and at refrigeration

temperatures virtually no curd is obtained.

Chymosin action in milk is optimal at approximate-

ly pH 6. This is obtained with starter bacteria. If the

pH is lowered further rennet clotting occurs at a

faster rate, and curd ﬁrmness is reduced. Lowering

pH also changes the water-holding capacity, which

will have an impact on curd ﬁrmness.

Figure 10.1. Destabilization of the casein micelle by introduction of chymosin.

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248 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking

CALCIUM

Calcium has a signiﬁcant impact on rennet curd,

though it does not have a direct effect on the primary

phase of rennet action. Addition of ionic calcium, as

in the form of calcium chloride, for example, re-

duces the rate of rennet clot formation time and also

increases rennet curd ﬁrmness. Similarly if the calci-

um content of milk is lowered by approximately

30%, coagulation does not occur. Milks that have a

tendency to form weak curd may be fortiﬁed with

calcium chloride prior to the addition of rennet.

MILK PROCESSING

Heating milk to temperatures higher than approxi-

mately 70°C leads to delayed curd formation and

weak rennet curd (Vasbinder et al. 2003). In extreme

cases, there may be no curd at all. These effects are a

result of the formation of a complex involving disul-

phide linkages between kappa-casein and beta-

lactoglobulin under high heat treatment. Under these

conditions the 105-106 bond in kappa-casein is

inaccessible for chymosin action. This effect is gen-

erally not reversible. Under mild overheating condi-

tions, the addition of 0.02% calcium chloride may

help obtain ﬁrm rennet curd. Maubois et al. have

developed a process for reversing this effect by the

use of ultraﬁltration. Increasing the protein content

by ultraﬁltration before or after UHT treatment re-

stores curd-forming ability (Maubois et al. 1972).

According to Ferron-Baumy et al. (1991), such a

phenomenon results from lowering the zeta poten-

tial of casein micelles on ultraﬁltration.

Homogenization has a distinct impact on milk

rennet coagulation properties. Homogenized milk

produces softer curd, but when only the cream por-

tion of milk is homogenized, rennet curd becomes

ﬁrmer (Nair et al. 2000). This is possibly because of

the reduction of fat globule size due to homogeniza-

tion and coating of the fat globule surface with

casein. These particles then act as casein micelles.

Concentrating milk prior to cheese making is now

a common practice. Techniques include evapora-

tive concentration, ultraﬁltration, and microﬁltra-

tion. Each of these procedures increases the casein

concentration in milk; thus the rate of casein aggre-

gation during the secondary phase increases. Ren-

net coagulation usually occurs at a lower degree of

kappa-casein hydrolysis, and rennet curd is gener-

ally ﬁrmer. For example, in unconcentrated milk

approximately 90% kappa-casein must be hydro-

lyzed by chymosin before curd formation, but in

ultraﬁltered milk only 50% must be hydrolyzed.

(Dalgleish 1992). Under high concentration condi-

tions, rennet curd is extremely ﬁrm, and difﬁculties

are encountered in cutting curd using traditional

equipment. In cheeses manufactured from highly

concentrated milks, rennet should be properly mixed

in the milk mixture to prevent localization of rennet

action.

GENETIC VARIANTS

Protein polymorphism (genetic variants) of kappa-

casein has been demonstrated to have an effect on

rennet coagulation (Marzialli and Ng-Kwai-Hang

1986). Protein polymorphism refers to a small varia-

tion in the makeup of proteins due to minor differ-

ences in the amino acid sequence. Examples include

kappa-casein AA, AB, or BB, beta-lactoglobulin

AA, AB, and so on. Milk with kappa-casein BB

variants forms a ﬁrmer rennet curd because of in-

creased casein content associated with this variant of

kappa-casein. Some breeds of cows, Jerseys in par-

ticular, have larger proportions of this variant. The

BB variant of beta-lactoglobulin is also associated

with higher casein content in milk.

EFFECT OF CHYMOSIN ON

PROTEOLYSIS IN CHEESE

Once rennet curd has been formed and a fresh block

of cheese has been obtained, the ripening process

begins. During ripening, numerous biochemical re-

actions occur and lead to unique ﬂavor and texture

development. During this process residual chymosin

that is retained in the cheese makes important con-

tributions to the ripening process (Kosikowski and

Mistry 1997, Sousa et al. 2001). As the cheese takes

form, chymosin hydrolyzes the paracaseins into

peptides optimally at pH 5.6 and creates a peptide

pool for developing ﬂavor complexes (Kosikowski

and Mistry 1997).

The amount of rennet required to coagulate milk

within 30 minutes can be considerably below that

required to properly break down the paracaseins

during cheese ripening. Consequently, using too

little rennet in cheese making retards ripening, as

discerned by the appearance of the cheese and its

10CH_Hui_277065 10/18/05 7:35 AM Page 248

10 Chymosin in Cheese Making 249

end products. A lively, almost translucent looking

cheese, after ripening several months, indicates ade-

quate rennet; an opaque, dull looking cheese indi-

cates inadequate amounts. Apparently, conversion to

peptides and later hydrolysis to smaller molecules

by bacterial enzymes give the translucent quality.

Residual rennet in cheese hydrolyzes alpha-s-1-

casein to alpha-s-1-I-casein, which leads to a desir-

able soft texture in the aged cheese (Lawrence et al.

1987).

Electrophoretic techniques (Ledford et al. 1966)

have demonstrated that in most ripening Cheddar

cheese from milk coagulated by rennet, para-beta-

casein largely remains intact while para-alpha-

casein is highly degraded. According to Fox et al.

(1993), if there is too much moisture in cheese or

too little salt, the residual chymosin will produce

bitter peptides due to excessive proteolysis. In

cheeses with high levels of beta-lactoglobulin, as in

cheeses made by ultraﬁltration, proteolysis by resid-

ual chymosin is retarded because of partial inhibi-

tion of chymosin by the whey protein (Kosikowski

and Mistry 1997).

Edwards (1969) found that milk-coagulating en-

zymes from the mucors hydrolyze the paracaseins

somewhat similarly to rennet, while enzymes from

Cr. parasitica and the papaya plant completely

hydrolyze para-alpha- and para-beta-caseins during

ripening.

Proteolytic activity of rennet substitutes, especial-

ly microbial and fungal substitutes, is greater than

that of chymosin. Cheese yield losses, though small,

do occur with these rennets. Barbano and Ras-

mussen (1992) reported that fat and protein losses to

whey were higher with Rh. miehei and Rh. pusillus

compared with those for fermentation-derived chy-

mosin. As a result, the cheese yield efﬁciency was

higher with the latter than with the former two

microbial rennets.

EFFECT OF CHYMOSIN ON

CHEESE TEXTURE

The formation of a rennet curd is the beginning of

the formation of a cheese mass. Thus, a ripened

cheese assumes its initial biological identity in the

vat or press. This is where practically all of the criti-

cal components are assembled and the young cheese

becomes ready for further development.

Undisturbed fresh rennet curd is not cheese

because it still holds considerable amounts of water

and soluble constituents that must be removed

before any resemblance to a cheese occurs. This

block of curd is cut into thousands of small cubes.

Traditionally, this is done with wire knives (Fig.

10.2) but in large automated vats, built-in blades

rotate in one direction at a selected speed to system-

atically cut the curd. Then the whey, carrying with it

Figure 10.2. Cutting of rennet curd.

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250 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking

lactose, whey proteins, and soluble salts, streams

from the cut pores, accelerated by gentle agitation,

slowly increasing temperature, and a rising rate of

lactic acid production. These factors are manipu-

lated to the degree desired by the cheese maker.

Eventually the free whey is separated from curd.

Optimum acid development is essential for form-

ing rennet curd and creating the desired cheese mass.

This requires viable, active microbial starters to be

added to milk before the addition of rennet. These

bacteria should survive the cheese making process.

Beta-D-galactosidase (lactase) and phosphorylat-

ing enzymes of the starter bacteria hydrolyze lactose

and initiate the glucose-phosphate energy cycles for

the ultimate production of lactic acid through vari-

ous intermediary compounds. Lactose is hydrolyzed

to glucose and galactose. Simultaneously, the galac-

tose is converted in the milk to glucose, from which

point lactic acid is produced by a rather involved

glycolytic pathway. In most cheese milks, the con-

version of glucose to lactic acid is conducted homo-

fermentatively by lactic acid bacteria, and the con-

versions provide energy for the bacteria. For each

major cheese type, lactic acid must develop at the

correct time, usually not too rapidly, nor too slowly,

and in a speciﬁc concentration.

The cheese mass, which evolves as the whey

drains and the curds coalesce, contains the insoluble

salts, CaHPO

and MgHPO

, which serve as buffers

in the pressed cheese mass, and should the acid in-

crease too rapidly during cooking, they will dissolve

in the whey. In the drained curds or pressed cheese

mass under these circumstances, since no signiﬁcant

pool of insoluble divalent salt remains for conver-

sion into buffering salts, the pH will lower to 4.7—

4.8 from an optimum 5.2, leading to a sour acid–

ripened cheese. Thus, conservation of a reserve pool

of insoluble salts is necessary until the cheese mass

is pressed. Thereafter, the insoluble salts are changed

into captive soluble salts by the lactic acid and pro-

vide the necessary buffering power to maintain opti-

mum pH at a level that keeps the cheese sweet. Such

retention also is aided or controlled by the skills of

the cheese maker. For Cheddar, excess wetness of

the curd before pressing or too rapid an acid devel-

opment in the whey causes sour cheese. For a Swiss

or Emmental, the time period for acid development

differs; most of it takes place in the press, but the

principles remain the same (Kosikowski and Mistry

1997).

The development of proper acid in a curd mass

controls the microbial ﬂora. Sufﬁcient lactic acid

produced at optimum rates favors lactic acid bacte-

ria and discriminates against spoilage or food poi-

soning bacteria such as coliforms, clostridia, and

coagulase-positive staphylococci. But its presence

does more than that, for it transforms the chemistry

of the curd to provide the strong bonding that is nec-

essary for a smooth, integrated cheese mass.

As discussed earlier, chymosin action on milk

results in curd mass involving dicalcium paracasein.

Dicalcium paracasein is not readily soluble, stretch-

able, or possessive of a distinguished appearance.

However, if sufﬁcient lactic acid is generated, this

compound changes. The developing lactic acid solu-

bilizes considerable calcium, creating a new com-

pound, monocalcium paracasein. The change occurs

relatively quickly, but there is still a time require-

ment. For example, a Cheddar cheese curd mass,

salted prior to pressing, may show an 8:2 ratio of

dicalcium paracasein to monocalcium paracasein;

after 24 hours in the press, it is reversed, 2:8. Mono-

calcium paracasein has interesting properties. It is

soluble in warm 5% salt solution, it can be stretched

and pulled when warmed, and it has a live, glisten-

ing appearance. The buildup of monocalcium para-

casein makes a ripened cheese pliable and elastic.

Then, as more lactic acid continues to strip off calci-

um, some of the monocalcium paracasein changes to

free paracasein as follows:

Dicalcium paracasein  lactic acid → monocalcium

paracasein  calcium lactate

Monocalcium paracasein  lactic acid → free para-

casein  calcium lactate

Free paracasein is readily attacked by many

enzymes, contributing to a well-ripened cheese. The

curd mass becomes fully integrated upon the uni-

form addition of sodium chloride, the amount of

which varies widely for different cheese types. Salt

directly inﬂuences ﬂavor and arrests sharply the acid

production by lactic acid starter bacteria. Also, salt

helps remove excess water from the curd during

pressing and lessens the chances for a weak-bodied

cheese. Besides controlling the lactic acid fermenta-

tion, salt partially solubilizes monocalcium paraca-

sein. Thus, to a natural ripened cheese or to one that

is heat processed, salt helps give a smoothness and

plasticity of body that is not fully attainable in its

absence.

10CH_Hui_277065 10/18/05 7:35 AM Page 250

10 Chymosin in Cheese Making 251

Texture of cheese is affected signiﬁcantly during

ripening, especially during the ﬁrst 1–2 weeks ac-

cording to Lawrence et al. (1987). During this peri-

od, the alpha-s-1-casein is hydrolyzed to alpha-s-1-I

by residual chymosin, making the body softer and

smoother. Further proteolysis during ripening con-

tinues to inﬂuence texture.

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Starch Synthesis in the Potato Tuber

P. Geigenberger* and A. R. Fernie

253

Introduction

What is Starch?

Routes of Starch Synthesis and Degradation and Their

Regulation

Manipulation of Starch Yield

Manipulation of Starch Structure

Conclusions and Future Perspectives

References

INTRODUCTION

Starch is the most important carbohydrate used for

food and feed purposes and represents the major

resource of our diet. The total yield of starch in rice,

corn, wheat, and potato exceeds 10

tons per year

(Kossmann and Lloyd 2000, Slattery et al. 2000). In

addition to its use in a nonprocessed form, due to the

low cost incurred, extracted starch is processed in

many different ways. Processed starch is subse-

quently used in multiple forms, for example in high-

fructose syrup, as a food additive, or for various tech-

nical processes based on the fact that as a soluble

macromolecule it exhibits high viscosity and adhe-

sive properties (Table 11.1). The considerable im-

portance of starch has made increasing the content

and engineering the structural properties of plant

starches major goals of both classical breeding and

biotechnology over the last few decades (Smith et al.

1997, Sonnewald et al. 1997, Regierer et al. 2002).

Indeed, since the advent and widespread adoption of

transgenic approaches some 15 years ago gave rise to

the discipline of molecular plant physiology, much

information has been obtained concerning the po-

tential to manipulate plant metabolism. For this chap-

ter we intend to review genetic manipulation of

starch metabolism in potato (Solanum tuberosum).

Potato is one of the most important crops world-

wide, ranking fourth in annual production behind

the cereal species rice (Oryza sativa), wheat (Triti-

cum aestivum), and maize (Zea mais). Although in

Europe and North America the consumption of pota-

toes is mainly in the form of processed foodstuffs

such as fried potatoes and chips, in less developed

countries it represents an important staple food and

is grown by many subsistence farmers. The main

reasons for the increasing popularity of the potato in

developing countries are the high nutritional value

of the tubers combined with the simplicity of its

propagation by vegetative ampliﬁcation (Fernie and

Willmitzer 2001). Since all potato varieties are true

tetraploids and display a high degree of heterozy-

gosity, genetics have played only a minor role in

metabolic studies in this species. However, because

the potato is a member of the Solanaceae family, it

was amongst the ﬁrst crop plants to be accessible to

transgenic approaches. Furthermore, due to its rela-

tively largesize and metabolic homogeneity, the pota-

to tuber represents a convenient experimental sys-

tem for biochemical studies (Geigenberger 2003a).

In this chapter we will describe transgenic at-

tempts to modify starch content and structure in po-

tato tubers that have been carried out in the last two

decades. In addition to describing biotechnologic-

ally signiﬁcant results we will also detail fundamen-

tal research in this area that should enable future

*Corresponding author.

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Food Biochemistry and Food Processing

Edited by Y. H. Hui

254 Part II: Water, Enzymology, Biotechnology, and Protein Cross-linking

biotechnology strategies. However, we will begin by

brieﬂy describing starch, its structure, and its syn-

thesis.

WHAT IS STARCH?

For many years it has been recognized that the

majority of starches consist of two different macro-

molecules, amylose and amylopectin (Fig. 11.1),

which are both polymers of glucose and are organ-

ized into grains that range in size from 1 m to more

than 100 m. Amylose is classically regarded as an

essentially linear polymer wherein the glucose units

are linked through -1-4-glucosidic bonds. In con-

trast, although amylopectin contains -1-4-gluco-

sidic bonds, it also consists of a high proportion of

-1-6-glucosidic bonds. This feature of amylopectin

makes it a more branched, larger molecule than amy-

Table 11.1. Industrial Uses of Starch

Starch and Its Derivates Processing Industry Application

Amylose and amylopectin Food Thickener, texturants, extenders,

(polymeric starch) low calorie snacks

Paper Beater sizing, surface sizing, coating

Textile Wrap sizing, ﬁnishing, printing

Polymer Absorbents, adhesives,

biodegradable plastics

Products of starch hydrolysis, Food Sweeteners or stabilizing agents

such as glucose, maltose, Fermentation Feedstock to produce ethanol, liquors,

or dextrins spirits, beer, etc.

Pharmaceutical Feedstock to produce drugs and

medicine

Chemical Feedstock to produce organic solvents

or acids

Source: Adapted from Jansson et al. 1997, with modiﬁcation.

Figure 11.1. Starch structure: Amylose (A) is classically regarded as an essentially linear polymer wherein the glu-

cose units are linked through -1-4-glucosidic bonds. In contrast, although amylopectin contains -1-4-glucosidic

bonds, it also consists of a high proportion of -1-6-glucosidic bonds (B).

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