Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

Подождите немного. Документ загружается.

TRYPSIN INHIBITORS

G P Savage and S C Morrison, Lincoln University,

Canterbury, New Zealand

Introduction

0001 Trypsin inhibitors occur in a wide range of foods.

They have been studied extensively in plants

belonging to the Leguminoseae, Solanaceae, and Gra-

mineae families, as the majority of species in these

families are considered as important food sources; the

most important species are the grain legumes such as

chickpeas, mung beans, and soy beans. Grain legumes

are a major source of protein in the diets of many

people throughout the world and are thought to pro-

vide about 10% of the world’s dietary protein. They

are used extensively as a protein source for animal

feeds. The existence of a number of antinutritive

factors in legume plants, especially a group of prote-

ase inhibitors, has limited the use of legume seed

protein. Several different types of protease inhibitors

may be present in the same tissue.

Structure and Mechanism of Action

0002 The most common inhibitors in legumes act on serine

proteases, a group of proteolytic enzymes, including

trypsin and chymotrypsin. Serine protease inhibitors

are proteins that form very stable complexes with

these digestive enzymes, reducing their activity to

very low levels. Some nonprotein inhibitors have

also been identified.

Protease Inhibitors

0003 Protease inhibitors have been classified into families

based on the homologous sequences of amino acids at

the reactive inhibitory sites. The molecular structure

of the inhibitor affects both strength and specificity of

the inhibitor. The two main groups of protease inhibi-

tors found in legumes are the Kunitz soy bean trypsin

inhibitor and the Bowman–Birk soy bean protease

inhibitor.

0004 Protease inhibitors exist in several different iso-

forms, each with differing isoelectric points and

stability towards heat. In New Zealand peas, for

instance, between six and 10 isoinhibitors in each

cultivar have been observed, with isoelectric points

ranging from 4.6 to 7.6.

Kunitz Soy Bean Trypsin Inhibitor (KSTI)

0005The first protease inhibitor to be isolated and charac-

terized was KSTI. Molecular weights (mol wt)

ranging from 18 to 24 kDa and isoelectric points of

3.5–4.4 have been reported. This wide variation may

be attributed in part to isoforms of the inhibitor.

Taking into account all the variations, it appears that

the mol wt of most KSTIs is approximately 21 kDa.

KSTI consists of 181 amino acid residues linked by

two disulfide bonds; the reactive site is located at

residues 63 and 64, as shown in Figure 1.

0006A competitive inhibitor, KSTI binds to the reactive

site of trypsin, in a similar manner as it does to the

substrate protein, causing hydrolysis of the peptide

bonds between reactive site residues of the inhibitor

or substrate. Inhibitors differ from substrate proteins

in that the reactive site residues are held between

disulfide bonds. After hydrolysis, the modified inhibi-

tor is held together with the same conformation,

because of the disulfide bond. This forms a stable

enzyme–inhibitor complex.

Bowman–Birk Soy Bean Proteinase Inhibitor (BBI)

0007The second proteinase inhibitor to be isolated and

characterized, BBI, differs from KSTI in four ways.

It is a relatively small molecule of molecular weight

7–9 kDa, and is a ’double-headed’ inhibitor with in-

dependent binding sites for chymotrypsin and tryp-

sin. As it contains seven disulfide bonds, BBI is rich in

cysteine (20%). There have been conflicting reports

about the stability of this inhibitor. Bowman and Birk

commented that BBI displayed marked stability to-

wards heat, acid, alkali, and proteolytic enzymes such

as pepsin. It was initially thought that increased heat

stability was attributed to the stabilizing effect of the

disulfide bonds on the whole structure (Figure 2),

thus BBI would be more stable than KSTI. However,

some chymotrypsin inhibitors from potato and

barley, which display thermostability, contain no or

only one disulfide bridge. Other reports found BBI to

be more heat-labile than the Kunitz inhibitor. This

might be attributed to the many disulfide bonds,

which do not give BBI stability but rather make the

inhibitor susceptible to bind with other sulfhydryl

(-SH) groups other than those of the inhibitor. The

heat stability of BBI, however, has been shown to be

dependent on concentration.

0008Limited proteolysis results in separation of the

inhibitor into two active fragments, one with tryp-

sin inhibitor activity and the other with chymotrypsin

inhibitor activity. Further study of protease inhibitors

5878 TRYPSIN INHIBITORS

has revealed that the BBI is a prototype for a whole

family of homologous inhibitors found in grain

legumes and in some other plant families. The se-

quences of amino acids surrounding the two reactive

sites of the BBI are remarkably similar, not only to

each other but also to homologous active sites of

legume inhibitors isolated from other common grain

legumes. Comparison of the BBI with inhibitors

isolated from pea, groundnut, and chickpea show

that these are double-headed inhibitors, although

the mechanism by which they inhibit trypsin may

differ slightly owing to the spatial arrangement of

Asp

Glu

Asp Asp

Asp

Phe

Thr

Gln

Met

Arg

Glu

Ser

Cys

His

Cys

Lys

Arg

Cys

Lys

Thr

Gln

Pro

Asn

Ser

Ala

Tyr

Glu

Pro

Ser

Ala

Ser

Pro

Leu

Asn

Ser

Ile

Asp

Asn

Lys

Val

Cys

Glu

Phe

Cys

Gln

Ala

Pro

Tyr

Ser

Leu

IIe

fig0002 Figure 2 Structure of the Bowman–Birk inhibitor. From Odani S and Ikenaka T (1973) Studies on soybean trypsin inhibitors: VIII.

Disulfide bridges in soybean. Bowman–Birk protease inhibitors. Journal of Biochemistry 74: 697–715 with permission.

Asp

Phe

Val

Leu

Asp

Asn

Glu

Gly

Asn

Pro

Leu

Glu

Asn

Gly

Thr

Tyr

Tyr lle Leu Ser Asp Lle Thr Ala Phe Gly Gly lle Arg Ala Ala Pro Thr Gly Asn Glu

Arg

Cys

Pro

Leu

Thr

Val

Gln

Ser

Arg

Asn

Glu

Leu

Asp

Lys

GlylleGlyThrllelleSer

Pro

Ser

Tyr

Arg

lle

Arg

Phe

lle

Ala

Glu

LeuSerGluLysAspLeu

Lys

Gln

Phe

Gln

Val

LeuPro

Lys

Asn

Lys

Ser

Val

ValLeu

Arg

Thr

His

Gly

Asp

lle Ser lle Gly lle Asp

Gly

Cys

Lys

Asp

Glu

Ala

Gln

Pro

Cys

Phe

Val Leu Lys Tyr Asn Asn Phe

Glu

Asp

AspSerValArgGluLeuArgPheTrpGlyAspMetAlaAspLysAsnGlu

Gly

lle

Lys Val Ala Pro Gly Glu Pro Leu Asp Glu Val Val Ser Trp Glu Thr Pro lle

Gly

Val

Cys

Leu

Met

lle

Val

Ala

Phe

Ser

Asp

Phe

Lys

LeuSerLeuProHisGly

160

110

150

170

140

180

130

120

100

fig0001 Figure 1 Structure of the Kunitz soy bean trypsin inhibitor. From Koide T and Ikenaka T (1973) Studies on soybean trypsin inhibitors:

3. Amino acid sequence of the carboxyl-terminal region and the complete amino-acid sequence of soybean trypsin inhibitor (Kunitz).

European Journal of Biochemistry 32: 417–431, with permission.

TRYPSIN INHIBITORS 5879

the reactive sites. As many as five protease inhibitors

that have properties similar to the BBI may be present

in soy beans.

Determination of Activity

0009 The basic method of quantifying trypsin inhibitors

has been improved to increase its accuracy and sensi-

tivity and has been further simplified and miniatur-

ized. Many modifications to the basic method have

been made: the most common are the use of different

substrates, e.g., casein instead of the synthetic sub-

strate benzoyl-dl-arginine-p-nitroanilide, or the use

of other types of trypsin, e.g., porcine rather than

bovine. These modifications make it difficult to

compare the results published by different authors.

Assessing the variations in inhibitor activity between

legumes is made very difficult by the fact that trypsin

inhibitor activity (TIA) is not determined using a

standard procedure.

0010 The trypsin inhibitor affinity assay only determines

protein-type inhibitors forming complexes with

trypsin, but is highly sensitive at low concentrations.

0011 Immunological methods developed for the detec-

tion and characterization of specific types of protease

inhibitors involve the use of specific protease anti-

bodies. Recently, a specific immuno-(chymo)trypsin

inhibitor assay has been developed, which discrimin-

ates between the two main protease inhibitors in

soybeans, KSTI and BBI, unlike the Kakade enzymic

assay which does not.

0012 Several different units have been used when meas-

uring TIA, which also make comparisons between

data difficult. TIA is most commonly expressed as

trypsin inhibitor units (TIU) per milligram of sample

(TIU mg

1

DM) where one TIU is defined as a de-

crease of 0.01 absorbance units at 410 nm per 10 ml

assay solution under specified assay conditions. These

units make it possible to measure protease inhibitors

of any material without having to find a suitable

trypsin inhibitor standard.

Interfering Substances

0013 With samples which are low in TIA, the accuracy of

the values obtained can be distorted by nonspecific

interference from other proteins and compounds in

the reaction mixture. Interfering substances include

tannins in colored legume seed coats; free fatty acids

released in fermented products such as miso and

tempeh and indigestible polysaccharides can complex

with trypsin and contribute to the total TIA under

assay conditions.

0014 However, the presence of tannins and TIA in field

peas was found to be significantly negatively correl-

ated, indicating that tannins were unlikely to be

responsible for the additional inhibition of trypsin

in the Kakade assay. Maple and wrinkled peas have

high tannin levels but low TIA, irrespective of testa

or flower color. It would seem that those cultivars

exhibiting high tannin content have low levels of

TIA or vice versa, possibly to compensate for the

other compound, as both of these antinutritional

factors have been linked with disease resistance in

plants.

Distribution within Plants

0015Trypsin inhibitors are found most often in the seed

but their location is not necessarily restricted to this

part of the plant. In some legumes, such as the mung

bean and the field bean, high levels of TIA are found

in the leaves as well. They are present in many legumes

in varying amounts. They are also found in the leaves

and tubers of tuberous plants, such as potatoes

(Solanum tuberosum) and sweet potatoes (Ipomoea

batatas).

0016A higher level of TIA has been found in the outer

part of the cotyledon of soy beans, kidney beans, and

chickpeas, and in five different cultivars of peas four

times as much TIA was found in the cotyledon than in

the seed coat. Similar findings have been reported for

soy beans. In contrast, Vicia faba (faba bean) seeds

were found to have twice as much TIA in the hull than

the cotyledon.

Levels in Grain Legumes

0017Trypsin inhibitors are widely distributed across many

genera and species in the Leguminoseae family and

many other plant families; TIA has also been found in

a range of legumes, including red gram, kidney beans,

navy beans, black-eyed peas, peanuts, field beans,

French beans, and sweet peas, and in all varieties

tested of cowpeas, mung beans, lima beans, winged

beans, chickpeas, and rice beans. In addition, TIA

was found in lentils, but there was almost no TIA

in lupins.

0018Although trypsin inhibitors are found in most

legumes, the levels present tend to vary considerably.

Most legume species contain less than 50% of the

TIA of soy beans. Particularly low levels are present

in broad beans, peas, mung beans, lupins, and a few

varieties of kidney beans. Those species with at least

75% of the TIA in soy beans include cowpeas, pinto

beans, pigeon peas, kidney beans, moth beans, and

navy beans. Common legumes that contain levels

higher than those of soy bean include lima beans,

winged beans, and black beans.

5880 TRYPSIN INHIBITORS

0019 Differences in TIA between cultivars have been

observed, suggesting the possibility of breeding for

low TIA. For example, it was found that winter culti-

vars of peas were twice as active as summer cultivars

and that wrinkled cultivars were more active than

smooth cultivars. Considerable variability has also

been reported amongst different strains of cowpeas.

0020 Stage of maturity has also been shown to influence

TIA. As soy beans mature, the amount of TIA in-

creases, although the magnitude of increase differs

between varieties. The germination of a number of

grain legumes appears to increase their nutritive

value, although the effect on TIA seems to be quite

variable. There have been no reports of a significant

decrease in TIA on germination of lentils, chickpeas,

and navy beans but there was a decrease in TIA when

field peas were germinated. Other studies have found

that after germination of Vicia faba for 5 days TIA

decreased by 63%; TIA decreased by 50% in red

kidney beans after 10 days’ germination, and TIA in

three soy bean varieties decreased by an average of

13% over 3 days’ germination. The loss of TIA in

some cases may be accounted for by leaching during

soaking and washing procedures.

Effect of Processing

0021 Processing can generally be divided into methods that

are commonly used to prepare legumes for human

consumption, and those that are used in the produc-

tion of animal meals. The popularity of grain legumes

in human diets is no doubt attributable to the relative

ease with which TIA can be destroyed by many of

these methods. However, destruction of TIA inevit-

ably results in loss of some of the nutritive value of the

legume.

Boiling

0022Cooking presoaked (24-h) winged beans in boiling

water for 30 min was found to destroy most TIA. It

was also reported that mature soy beans required a

presoak of 24 h and cooking for at least 20 min to

eliminate TIA completely. Immature seeds do not

require presoaking to eliminate TIA. This, and the

fact that oven heating has very little effect on TIA,

suggests that the moisture content of the seed plays an

important role in the destruction of trypsin inhibitors.

Soaking chickpeas, broad beans, and mung beans

alone reduced TIA levels by 58–92%. Combined

soaking (18 h) and boiling (20 min) led to a 9.9–

56.7% reduction in peas, depending on cultivar.

0023It has been suggested that the extent to which TIA

is destroyed by heating is a function of the tempera-

ture, duration of heating, particle size, and moisture

content – variables that are closely controlled in the

industrial processing of soy bean meals in order to

obtain a product maximum nutritional value. The

nonprotein TIA content would also determine the

reduction in TIA achieved by the heating process.

Researchers have also found that the percentage of

TIA remaining in the cooked peas was found to

be negatively correlated to increased TIA levels in

the raw form, therefore it appears that the effect

of cooking is more effective in destroying TIA

when the original inhibitory activity (raw seed)

was higher than when it was low. A summary of the

effects of various heat treatments on TIA is given in

Table 1.

tbl0001 Table 1 Effect (percentages of original activity) of various types of heat treatment on the trypsin inhibitor activity (TIA) of legumes

Legume TIA Wet heat Autoclaved

extract:

103 kPa for

15 min

Dryheat

(TIunits

permg)

(%: soy

TIA ¼100%)

Heatingextract in

boiling-water bath

Roasting for 15 min at: Roasting for 2 min

at 200



30 min 60 min 75



C100



C125



Moth bean (Phaseolus aconitifolus) 1.44 27 57.8 11.6 2.4 80.8 42.5 Nil 7.6

Cow pea (Vigna catjung) 4.17 79 59.9 49.3 4.8 72.2 72.2 65.0 Nil

Cow pea (Vigna sinensis) 3.39 64 96.7 36.0 3.7 94.2 91.2 71.1 8.1

Red gram (Cajanus cajan) 3.19 60 71.5 64.5 12.3 80.5 54.5 17.9 11.7

French bean (Phaseolus vulgaris) 4.24 80 61.6 43.2 6.2 76.1 73.5 72.0 10.6

Pea (Pisum sativum) 1.33 25 20.8 12.5 Nil 42.1 27.6 4.3 Nil

Lentil (Lens culinaris) 1.31 25 8.5 Nil Nil 73.1 69.4 65.2 20.4

Green gram (Phaseolus aureus) 1.98 37 11.2 5.6 Nil 97.4 69.5 24.7 Nil

Black gram (Phaseolus mungo) 2.74 52 28.4 16.2 Nil 69.4 57.6 39.4 11.9

Chickpea (Cicer arietinum) 3.47 66 16.0 3.2 Nil 62.0 40.9 14.2 7.6

Soy bean (Glycine max) 5.30 100 61.9 22.1 58.9

TI, trypsin inhibitor.

From Rackis JJ, Wolf WJ and Baker EC (1986) Protease inhibitors in plant foods: content and inactivation. In: Friedman M (ed.) Advances in Experimental

Medicine and Biology: Nutritional and Toxicological Significance of Enzyme Inhibitors in Foods, pp. 299–337. New York: Plenum Press, with permission.

TRYPSIN INHIBITORS 5881

Other Cooking Methods

0024 Heating at temperatures below 75



C appears to have

no effect on winged beans. Increasing the temperature

to 100



C resulted in a 5% decrease in TIA of winged

beans. By increasing the temperature to 80



C, it was

found that TIA in white winged beans decreased by

25%, in 5 min and 45% after 30 min. Dry heating

(177



C for 20 min) peanuts and soy beans decreased

TIA by 7% and 20% respectively.

0025 Autoclaving has been shown to decrease TIA sig-

nificantly. In peanuts and soy beans autoclaved at

121



C for 20 min TIA decreased by 80% and 86%

respectively Autoclaving whole winged beans and

winged bean meal decreased TIA by 80%.

0026 Infrared treatment for 30 s on winged beans was

reported to have destroyed most TIA in the seeds.

0027 Microwave radiation of broad beans for 30 min

was found to destroy 90% of their TIA. It was

found that microwave treatment (time not known)

had no effect on TIA in winged beans. Microwave

treatment for 1.5–3.0 min of presoaked soy beans

destroyed 85–90% of the TIA.

Physiological Effects

0028 The majority of experiments which have considered

the physiological effects of grain legume TIA have

been carried out on animals and inferences made

about the effects on humans. The effects of TIA

found in soy beans have been predominantly studied

as the availability of pure forms of KSTI and BBI have

made the task of elucidating the inhibitor actions

much easier. The efficient digestion of proteins in

the digestive system requires the action of a number

of proteolytic enzymes, each hydrolyzing peptide

chains at specific points. These enzymes are produced

and stored in the pancreas in a precursor form known

as zymogens. The efficient digestion of proteins re-

quires simultaneous activation of all the zymogens.

Trypsin is thought to act as the common activator of

pancreatic zymogens.

Proteolytic Activity and Growth

0029 Because trypsin activates proteolytic enzymes, an

overall decrease in proteolytic activity would be

expected from the action of trypsin inhibitors.

In vitro studies have shown that KSTI, BBI, and

lima bean inhibitor completely inhibited trypsin and

chymotrypsin activity of human and rat pancreatic

juice. The TIA caused up to 50–60% reductions

in total proteolytic activity, and the residual activ-

ity observed was attributed to carboxypeptidase

activity.

0030Reduction in growth owing to loss of proteolytic

activity appears to vary between species fed raw soy

bean diets. Growth was reduced in the rat, mouse,

chick, and young guinea pig, whereas the adult guinea

pig, dog, pig, calf, monkey and, probably, the human

appeared to grow normally when fed these diets.

0031There are a number of factors that might contribute

to the differences in proteolytic activity and growth

between different species. One is that gastric juices

can inactivate protease inhibitors. In one study

human gastric juice almost eliminated the protease

inhibitor activity of KSTI when incubated for 24 h.

It was found that lima bean inhibitor activity was

only slightly affected by incubation in gastric juice,

suggesting that inhibitors homologous to lima bean

inhibitor are nutritionally more important than

inhibitors from the KSTI family.

0032Another reason for the varying activity of protease

inhibitors between species may be differences in the

specific activity of species-specific trypsin. In humans,

proteolytic enzymes hydrolyze casein at a lower rate

than in many animals, and the effect of inhibitors may

therefore be weaker in humans. Soy bean trypsin

inhibitor was shown to have a much greater effect

on trout trypsin than bovine trypsin, probably owing

to a higher specific activity of trout trypsin.

0033Human trypsin is known to exist in two forms. The

cationic form constitutes two-thirds of the total tryp-

sin secreted and is only very weakly inhibited, whereas

the anionic form is inhibited stoichiometrically. Infu-

sion of raw soy bean extract into the duodenum of rats

and humans caused the secretion of a modified inhibi-

tor-resistant trypsin, which was resistant to typical

serine protease inhibitors. The BBI was more potent

than KSTI in producing TIA-resistant enzymes.

Whether or not this was just the cationic form of

trypsin is unclear. The ability of the pancreas to

adapt to trypsin inhibitors may determine the effects

that they have on different species.

Toxicological Effects

0034The main toxicological effect observed from the in-

gestion of trypsin inhibitors is a marked hypertrophy

of the pancreas. Pancreatic hypertrophy is thought to

be caused by TIA stimulating excess enzyme secre-

tion, which draws essential amino acids away from

other body functions; in some cases this results in

death of the animal.

0035Negative feedback control of pancreatic secretion

is mediated by cholecystokinin (CCK). CCK is

released from the intestinal mucosa when levels of

trypsin or chymotrypsin are lowered, such as in

the formation of the trypsin–trypsin inhibitor com-

plex. The action of this complex is mediated by a

5882 TRYPSIN INHIBITORS

trypsin-sensitive CCK-releasing peptide, which inter-

acts with the luminal surface of the small intestine to

stimulate the release of CCK into the circulation.

CCK stimulates the secretion of the pancreozymins,

trypsinogen and chymotrypsinogen, the precursors to

trypsin and chymotrypsin respectively. Recently,

complexed as well as free inhibitors have been

found to stimulate the discharge of CCK in rats and

as a result have significantly increased the trypsino-

gen and chymotrypsinogen secretion rates. In normal

circumstances, when trypsin levels are adequate, tryp-

sin exerts a negative effect on this monitor peptide in

the gastrointestinal tract suppressing its pancreatic

secretion rate.

0036 The pancreas may therefore function abnormally

when these inhibitors are present as it produces more

enzymes to compensate for the loss of the enzyme via

the enzyme–inhibitor complex. Dietary amino acids

may be directed from other protein synthesis sites in

the body tissues for the synthesis of these additional

pancreatic enzymes. As pancreatic enzymes are rich in

the essential sulfur-containing amino acids, removal

of these amino acids from other sites of protein syn-

thesis involved in growth and maintenance is critical.

Continued ingestion of legumes would compound

these effects and, in small animals such as rats, have

been found to result in pancreatic hypertrophy,

growth depression, and failure to thrive.

0037 This negative-feedback control is known to occur

in the rat, pig, and calf, as well as in humans, suggest-

ing that other factors are also involved in pancreatic

hypertrophy.

See also: Plant Antinutritional Factors: Characteristics;

Fermented Foods: Fermentations of the Far East; Soy

(Soya) Sauce; Legumes: Legumes in the Diet; Dietary

Importance; Peanuts; Peas and Lentils; Protein:

Interactions and Reactions Involved in Food Processing;

Digestion and Absorption of Protein and Nitrogen

Balance; Heat Treatment for Food Proteins; Pulses; Soy

(Soya) Beans: Properties and Analysis; Dietary

Importance; Tannins and Polyphenols; Toxins in Food

– Naturally Occurring

Further Reading

Birk Y (1961) Purification and some properties of a highly

active inhibitor of trypsin and a-chymotrypsin from soy-

beans. Biochimica et Biophysica Acta 54: 378–381.

Bowman DE (1946) Fractions derived from soy beans and

navy beans which retard tryptic digestion of casein.

Proceedings of the Society of Experimental Biology

and Medicine 63: 547–550.

Brandon DL, Bates AH and Friedman M (1988) Enzyme-

linked immunoassay for soybean Kunitz trypsin

inhibitor using monoclonal antibodies. Journal of

Food Science 53: 102–106.

Gaborit T, Quillien L and Gue

guen J (1993) Determination

of trypsin inhibitor activity in seeds by a microtitre plate

method. In: van der Poel AFB, Huisman J and Saini HS

(eds) Recent Advances of Research in Antinutritional

Factors in Legume Seeds, pp. 37–40. Wageningen:

Wageningen Pers.

Kadam SS and Smithard RR (1987) Effects of heat treat-

ments on trypsin inhibitors and haemagglutinating-

activities in winged beans. Plant Foods for Human

Nutrition 37: 151–159.

Kakade ML, Rackis JJ, McGhee JE and Puski G (1974)

Determination of trypsin inhibitor activity of soy prod-

ucts: a collaborative analysis of an improved procedure.

Cereal Chemistry 51: 376–382.

Koide T and Ikenaka T (1973) Studies on soybean trypsin

inhibitors: 3. Amino acid sequence of the carboxyl-

terminal region and the complete amino-acid sequence

of soybean trypsin inhibitor (Kunitz). European Journal

of Biochemistry 32: 417–431.

Krogdahl A and Holm H (1981) Soybean proteinase inhibi-

tors and human proteolytic enzymes: selective inactiva-

tion of inhibitors by treatment with human gastric juice.

Journal of Nutrition 111: 2045–2051.

Liener IF (1980) Protease inhibitors. In: Liener IE (ed.)

Toxic Constituents of Plant Foodstuffs, pp. 7–57. New

York: Academic Press.

Noonan SC (1999) The nature and stability of trypsin

inhibitor isoforms in New Zealand grown pea (Pisum

sativum L.) cultivars. MSc thesis. New Zealand: Lincoln

University.

Odani S and Ikenaka T (1973) Studies on soybean trypsin

inhibitors: VIII. Disulfide bridges in soyabean. Bowman

Birk proteinase inhibitors. Journal of Biochemistry 74:

697–715.

Rackis JJ, Wolf WJ and Baker EC (1986) Protease inhibitors

in plant foods: content and inactivation. In: Friedman M

(ed.) Advances in Experimental Medicine and Biology:

Nutritional and Toxicological Significance of Enzyme

Inhibitors in Foods, pp. 299–337. New York: Plenum

Press.

Roozen JP and de Groot J (1988) Analysis of residual

inhibitor in feed flour. In: Huisman J, van der Poel TFB

and Liener IE (eds) Recent Advances of Research in

Antinutritional Factors in Legume Seeds, pp. 114–117.

Wageningen: Pudoc.

Savage GP (1988) The composition and nutritive value of

lentils (Lens culinaris). Nutrition Abstracts and Reviews

(Series A) 58: 319–343.

Valdebouze P, Bergeron E, Gaborit T and Delort-Laval J

(1980) Content and distribution of trypsin inhibitors in

some legume seeds. Canadian Journal of Plant Science

60: 695–701.

van Amerongen A, Ostafe V, Meijer MMT et al. (1998)

Specific-immuno-(chymo)trypsin-inhibitor assays for

determination of (residual) activity of Bowman–Birk or

Kunitz soybean trypsin inhibitors. In: Jansman AJM,

Hill GD, Huisman J and van der Poel AFB (eds) Recent

Advances of Research in Antinutritional Factors in

Legume Seeds and Rapeseed, pp. 33–37. Wageningen:

Wageningen Pers.

TRYPSIN INHIBITORS 5883

van Oort MG, Hamer RJ and Slager EA (1988) The trypsin

inhibitor assay: improvement of an existing method. In:

Huisman J, van der Poel TFB and Liener IE (eds) Recent

Advances of Research in Antinutritional Factors in

Legume Seeds, pp. 110–113. Wageningen: Pudoc.

Tuna See Fish: Introduction; Catching and Handling; Fish as Food; Demersal Species of Temperate Climates;

Pelagic Species of Temperate Climates; Tuna and Tuna-like Fish of Tropical Climates; Demersal Species of

Tropical Climates; Pelagic Species of Tropical Climates; Important Elasmobranch Species; Processing;

Miscellaneous Fish Products; Spoilage of Seafood; Dietary Importance of Fish and Shellfish; Fish Farming; Fish

Meal

Turkey See Poultry: Chicken; Ducks and Geese; Turkey

5884 TRYPSIN INHIBITORS

UHT See Heat Treatment: Ultra-high Temperature (UHT) Treatments; Chemical and Microbiological Changes;

Electrical Process Heating

Ultrafiltration See Membrane Techniques: Principles of Reverse Osmosis; Applications of Reverse

Osmosis; Principles of Ultrafiltration; Applications of Ultrafiltration

ULTRAVIOLET LIGHT

F Thompson, Halma PLC, London, UK

Introduction

0001 Since the turn of the century, when its existence was

proved experimentally, ultraviolet (UV) light has been

used to reduce microbiological contamination in

drinking water, process water, fluids, effluent, and

air, and on surfaces. The first municipal UV water

disinfection system was built in 1910 in Marseilles,

and since then, the technology has been refined and

developed, providing today’s reliable, computer-

controlled systems.

0002 This article reviews the technology of UV disinfec-

tion and outlines its industrial applications, especially

in food and drink manufacturing.

The Nature of UV Light

What is UV?

0003 The term ‘UV’ is applied to wavelengths of radiation

found in the electromagnetic spectrum between

visible light and X-rays (see Figure 1), from 100 to

400 nm. The region between 190 and 400 nm is

divided into three sections:

0004U1V-A, 320–400 nm. The longer wavelength limit

corresponds roughly to the start of visible light,

whereas the lower limit corresponds to the point

at which proteins and genetic material begin to

absorb significant amounts of UV energy.

0005UV-B, 290–320 nm. Ultraviolet light of these

wavelengths begins to damage biological mol-

ecules and is the region of the spectrum respon-

sible for sunburn and skin cancers.

0006UV-C, 200–290 nm. Far UV wavelengths are

highly destructive to biological molecules. In

nature, these wavelengths in sunlight are almost

totally absorbed by the ozone layer. UV-C wave-

lengths are mainly responsible for the germicidal

properties of UV light.

0007Commercial UV disinfection systems generate a

high proportion of UV-C, particularly at 254 nm,

which is very close to the most biologically damaging

wavelength of 260 nm (see Figure 2).

How UV Works

0008In sufficient doses, UV light of the wavelengths

mentioned is lethal to all microorganisms, including

bacteria, viruses, protozoa, molds, yeasts, fungal

spores, and algae.

0009The effectiveness of UV results from its ability

to cause molecular rearrangements in the genetic

material of microorganisms. This cripples the organ-

ism’s normal metabolism and prevents it from repro-

ducing. If a microorganism cannot reproduce, it is

considered dead.

0010The detailed mechanisms by which UV deactivates

microorganisms have only been fully understood re-

cently through advances in microbiological tech-

niques.

0011Deoxyribonucleic acid (DNA) is a helical macro-

molecule responsible for the storage, expression and

replication of a cell’s genetic information. (In some

cases, especially viruses, ribonucleic acid (RNA) is

the main carrier of genetic information, but as the

principle of UV damage is the same, we will limit the

description to DNA). Molecules of DNA are particu-

larly susceptible to damage by UV-C energy, and are

the primary site of absorption of UV photons.

0012Deoxyribonucleic acid is composed of four types of

deoxyribonucleotides, which differ only in their base

components. These are adenine, guanine, cytosine,

and thymine (which is replaced by uracil in RNA).

The main photochemical change to occur when DNA

is exposed to UV light is the dimerization of thymine

(and, less often, cytosine), where two of these mol-

ecules lie adjacent on a DNA strand. The effect of this

dimerization is to block the normal replication of

DNA, causing an irreversible breakdown in the cell’s

metabolism and replication.

Quantifying the Effectiveness of UV

0013The germicidal effectiveness of UV is expressed as the

number of organisms that survive after exposure.

Visible

Violet Red

Cosmic

rays

γ rays

X rays

Ultra-

violet

Infrared

Radio

waves

−7

m10

−3

Optical radiation

Wavelength, λ (m)

X rays

Vacuum

Short-

wave UV

(UV-C)

Middle-

wave UV

(UV-B)

Longwave

UV (UV-A)

Visible

light

100 200 300 400 800

λ (nm)

fig0001 Figure 1 Electromagnetic spectrum with expanded scale of UV radiation. Reproduced from Ultraviolet Light, Encyclopaedia of Food

Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press.

E. coli

killing

Nucleic acid

absorption

240 260 280 300

100

Relative units

Wavelength (nm)

fig0002 Figure 2 Similarity of the action spectrum for inactivation of

Escherichia coli cells, determined by FL Gates, to the absorption

spectrum of nucleic acids. Redrawn from Rupert CS (1960) In:

Burton M, Kirby-Smith JS and Magee JL (eds) Comparative

Effects of Radiation, pp. 49–61. New York: John Wiley with per-

mission.

5886 ULTRAVIOLET LIGHT

This depends on the intensity of UV (measured in

mW cm

2

), the number of microorganisms present

before disinfection, and the duration of exposure to

the UV source. These parameters are combined to

give the UV dose, the standard indicator of UV effect-

iveness. In its simplest terms, UV dose is a function of

intensity and time and can be illustrated as follows:

UV dose ¼ UV intensity  time

ðmJ cm

2

Þ¼ðmW cm

2

ÞðsÞ

1J¼ 1W s:

For water disinfection systems, the presence of mol-

ecules or suspended solids in the water, which may

absorb UV energy, must also be taken into account in

any calculations.

0014 The required UV dose differs for each type of

microorganism and is measured as a D

value,

i.e., the UV dose necessary for 1 log reduction in

concentration of any given microorganism. For

example, the D

value for Escherichia coli in water

is 5.4 mJ cm

2

. This means that a UV dose of

5.4 mJ cm

2

will reduce the population of E. coli to

10% of the original number, i.e., a 1 log reduction. A

dose of 10.8 mJ cm

2

(twice the D

) will give a 2 log

reduction, and so on (see Table 1).

Types of UV Systems

Lamp Type

0015 Ultraviolet energy for disinfection is produced using

mercury vapor lamps. These are similar in operation

and appearance to a fluorescent lamp. Ultraviolet

light is emitted as a current flows through vaporized

mercury. A protective quartz sleeve, which is trans-

parent to UV light, surrounds the lamp. Two types

of UV lamp are available – low-pressure lamps and

medium-pressure lamps.

0016 Low-pressure Lamps For low flow rates and surface

disinfection applications, low-pressure lamps are

most often used. These are highly efficient at

producing UV energy but are limited to a relatively

low UV output.

0017Medium-pressure Lamps Medium-pressure lamps

draw a much greater current than low-pressure

lamps, leading to a rapid rise in the mercury vapor

pressure. This gives a typical diffuse medium-pressure

UV spectrum (see Figure 3). Per unit area, the effect-

ive germicidal output of a medium-pressure lamp is

much greater (up to 30 times) than a low-pressure

lamp, allowing high flow rates to be treated using

fewer lamps. Medium-pressure lamps are used for

most industrial water and fluid disinfection systems

owing to their greater compactness and disinfection

capability. Unlike low-pressure lamps, which only

function efficiently within a limited temperature

range, medium-pressure systems are temperature-

independent, and therefore suitable for low-tempera-

ture and chilled-water applications.

System Design

0018The following parameters must be considered when

specifying a UV disinfection system:

0019The energy output of the UV source.

0020The flow rate of the fluid or air through the treat-

ment chamber, i.e., the time period of exposure to

UV.

0021The transmission value, i.e., the ability to transmit

UV light, of the fluid or air being treated.

0022The geometry of the irradiation chamber.

By optimizing these criteria, a UV system can be

tailored to treat virtually any water- or airflow, or

240 260 280

UV intensity

Wavelength (nm)

fig0003Figure 3 Energy distribution of medium- (h) and low-pressure

(&) UV lamps. Reproduced from Ultraviolet Light, Encyclopaedia

ofFood Science, FoodTechnology and Nutrition, Macrae R, Robinson

RK and Sadler MJ (eds), 1993, Academic Press.

tbl0001 Table 1 Dose–effect relationship for Escherichia coli (D

5.4 mJ cm

2

in water)

Dose

(mJ cm

2

)

Percentage of organisms

remaining

Log reduction

5.4 10 Log 1

10.8 1 Log 2

16.2 0.1 Log 3

21.6 0.01 Log 4

ULTRAVIOLET LIGHT 5887