Food Research International 43 (2010) 432–442

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Food Research International

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Review

Bioactive proteins and peptides in pulse crops: Pea, chickpea and lentil

F. Roy a, J.I. Boye b,*, B.K. Simpson a

a Food Science & Agricultural Chemistry Department, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9b Agriculture and Agri-Food Canada, 3600 Casavant Boul, West Saint-Hyacinthe, Quebec, Canada J2S 8E3

a r t i c l e i n f o a b s t r a c t

Keywords:Pulse cropLentilField peaChickpeaTrypsin inhibitorChymotrypsin inhibitorLectinAngiotensin I-converting enzyme

0963-9969/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodres.2009.09.002

* Corresponding author.E-mail address: boyej@agr.gc.ca (J.I. Boye).

Pulse crops are cool season, annually grown legume crops, which are harvested for their seeds. They areinvaluable agricultural commodities which are produced and imported by many regions of the world.Pulse seeds are a valuable source of dietary protein, carbohydrates, fiber and an important source ofessential vitamins and minerals. Their nutritional characteristics have been associated with a reductionin the incidence of various cancers, HDL cholesterol, type-2 diabetes and heart disease. Pulses also con-tain protein and non-protein antinutritional factors, which may cause deleterious effects on the hostwhen the seeds or processed seeds are consumed raw. Conversely, recent studies have demonstrated thatprotein antinutritional compounds such as lectins, protease inhibitors and the non-antinutritional com-ponent, angiotensin I-converting enzyme (ACE) inhibitor may have beneficial properties. Lectins havebeen associated with reducing certain forms of cancer, activating innate defense mechanisms and man-aging obesity. Protease inhibitors such as trypsin and chymotrypsin inhibitors have been demonstratedto reduce the incidence of certain cancers and demonstrate potent anti-inflammatory properties. Angio-tensin I-converting enzyme (ACE) inhibitor has been associated with a reduction in hypertension.

� 2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4322. Pea, chickpea and lentil: world production and consumption. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433

2.1. Dry pea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4332.2. Chickpea. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4332.3. Lentil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434

3. Protein content of pulses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4344. Antinutritional compounds of pulse crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434

4.1. Lectin characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4354.2. Deleterious effects of lectins on human health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4354.3. Nutraceutical potential of pulse lectins in human health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4364.4. Protease inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4364.5. Deleterious effects of protease inhibitors on human health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4374.6. Methods for reducing protease inhibitory compounds in pulse seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4384.7. Beneficial properties of denatured protease inhibitors on human health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438

5. Angiotensin I-converting enzyme (ACE) inhibitory peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4386. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440

ll rights reserved.

1. Introduction

Pulse crops belong to the family of cool season, annually grownleguminous crops (Maiti & Wesche-Ebeling, 2001). They arelegume crops that are harvested for their seed only and do notinclude legumes which are grown for oil, such as soybean (Nwok-

F. Roy et al. / Food Research International 43 (2010) 432–442 433

olo & Smartt, 1996). Consequently, pulse crops include dry pea,chickpea, lentil and lupin, along with various types of dry beansuch as kidney and lima bean. These crops are produced on manycontinents worldwide. North America, specifically Canada, andareas within Asia and the Middle East are responsible for themajority of pulse crop production and exportation. Importationof pulses occurs most frequently in populated countries such as In-dia and Egypt, where pulses are a staple of the diet.

Pulse crops are an excellent source of protein, carbohydrates,and fiber, and provide many essential vitamins and minerals. Theirhighly nutritional properties have been associated with many ben-eficial health-promoting properties, such as managing high choles-terol and type-2 diabetes and in the prevention of various forms ofcancer. However, pulse crops and other leguminous crops alsocontain many antinutritional proteins, such as lectins, proteaseinhibitors and the non-antinutritional compound, angiotensinI-converting enzyme (ACE) inhibitor. Various deleterious effectsmay occur following the ingestion of raw pulse seeds or flours,such as hemagglutination, bloating, vomiting and pancreaticenlargement, due to the activity of the antinutritional compoundsinside the host. Conversely, antinutritional compounds in pulsesmay have many beneficial properties in the treatment and/or pre-vention of disease when properly processed. This review will focuson the global production and exportation of dry pea, chickpea andlentil, and the deleterious and beneficial health effects of bioactiveproteins and peptides present in pulse crops.

2. Pea, chickpea and lentil: world production and consumption

2.1. Dry pea

Dry pea is among the world’s oldest crops, as records indicate itwas grown in the Middle East approximately 9000 years ago. Inaddition, it has been harvested in Europe for several thousandyears (Goodwin, 2003a,b), and has since been grown in 84 coun-tries including Australia, Canada, China and the United States(Smith & Jimmerson, 2005a,b; McKay, Schatz, & Enders, 2003).Dry pea is adapted to brown, dark brown and black soil zones insemi-arid and non-irrigated areas of the world, and has been pro-duced in the Canadian Prairies for over 100 years (Goodwin,2003a,b). The five main types of pea grown worldwide are Austrianwinter pea, green pea, maple pea, marrowfat pea and yellow pea.More than 60 varieties of pea have been developed for productionin Canada. Canada primarily produces green and yellow pea, withonly small quantities of maple, marrowfat and Austrian pea pro-duced (Pulse Canada, 2008a,b). In 2004, 12 million metric tonnesof dry pea were produced worldwide. Canada was the leadingcountry in pea production producing 28% of the total yield, fol-lowed by France and Russia at 14% and 10%, respectively (Smith& Jimmerson, 2005a,b). In Canada, Saskatchewan produces 68% ofCanada’s dry pea crop, while Alberta and Manitoba produceapproximately 22% and 10%, respectively (Goodwin, 2003a,b).

In addition to being the world’s largest producer of dry pea,Canada is also the leading exporter of dry pea. In 2007, Canada ex-ported over 2 million metric tonnes of dry pea, followed by theUnited States and France with 469 thousand and 350 thousandmetric tonnes, respectively. Conversely, in 2006, India was thelargest importer of dry pea as it imported over 1.1 million metrictonnes, followed by Spain and Belgium with 663 thousand and334 thousand metric tonnes imported, respectively. Presently,Canada and the United States are the 8th and 11th largest drypea importing countries, importing 77 thousand and 58 thousandmetric tonnes, respectively (Agriculture & Agri-Food Canada,2008).

Pea, similar to other pulse and grain commodities, is relativelyinexpensive and highly nutritious. It is high in fiber (soluble andinsoluble) and protein (especially rich in the essential amino acidstryptophan and lysine), low in sodium and fat, and is an excellentsource of complex carbohydrates, B vitamins, folate, and mineralssuch as calcium, iron, and potassium. In addition, a diet high indry pea has been demonstrated to be effective in lowering the inci-dence of colon cancer, type-2 diabetes, LDL-cholesterol and heartdisease (Agriculture & Agri-Food Canada, 2008). Due to the nutri-tional value described above, pea is considered to be an importantagricultural commodity. As such, it is commonly used in soups, orprocessed into pea flour, pea starch, or pea protein concentrates.The processed pea products can be used in baked goods, soupmixes, breakfast cereals, processed meats, health foods, pastasand purees. Commercially, dry pea is available canned or dried ineither whole or split pea varieties (Agriculture & Agri-Food Canada,2008; Slinkard, Bhatty, Drew, & Morrall, 1990). In addition, pea isone of the most commonly utilized pulse crops in animal feed forpoultry, sheep, cattle and swine feed rations, and is used as a feedadditive in the aquaculture industry (Schatz, 2002). Finally, dry peavarieties such as AC Trapper can be seeded as a green manure cropas an alternative to summer-fallow in organic farming practices(Lawley & Shirtliffe, 2004).

2.2. Chickpea

The chickpea is a member of the cool season Fabaceae(Leguminosae) family of legumes (Nwokolo & Smartt, 1996).Similar to dry pea, chickpea is one of the earliest cultivated vegeta-bles, as it is believed to have originated in the Middle East approx-imately 7450 years ago (Maiti & Wesche-Ebeling, 2001). Chickpeahas since been grown in temperate and semi-arid regions of theworld such as Asia, Europe, Australia and North America. In2004, 45 countries were actively producing chickpea, and togetherproduced a total of 8.6 million metric tonnes. India was the leadingproducer of chickpea accounting for approximately 66% of theworld’s production. Turkey was the second largest producer, pro-ducing approximately 7% of the world supply, followed by Pakistanand Iran at approximately 6% and 4%, respectively. In contrast, Can-ada and the United States contribute very little to the total quantityof chickpea produced worldwide, as they account for approxi-mately 1% and less than 1% of the world production, respectively(Smith & Jimmerson, 2005a,b). The majority of the Canadian pro-duction comes from the Prairie Provinces with Saskatchewan pro-ducing approximately 81% of Canada’s chickpeas, while Albertaproduces the rest.

Chickpea is the third most important pulse crop commodity inthe world based on total production (Yust et al., 2003). In 2003, In-dia was both the leading producer and importer of chickpea, withapproximately 259 thousand metric tonnes imported (30% of thetotal amount imported worldwide). Pakistan and Bangladesh werethe second and third largest importers of chickpea worldwide, withapproximately 123 thousand (14%) and 84 thousand metric tonn-nes (10%), respectively. Similar to chickpea production, the UnitedStates and Canada only marginally contributed to the total quan-tity of chickpea imported worldwide. In 2003–2004, the UnitedStates imported approximately 17 thousand metric tonnes (1%)(Smith & Jimmerson, 2005a,b), while Canada imported only 2 thou-sand metric tonnes (Agriculture & Agri-Food Canada, 2006a,b).

There are two main commercially available types of chickpeagrown worldwide: the desi and the kabuli chickpea. Desi chickpeaseed is small with a dark irregular-shaped seed coat and is grownon semi-arid land. Kabuli chickpea (Garbanzo beans) is larger thandesi chickpea, has a thin light-colored seed coat and is normallygrown in temperate regions of the world (Agriculture & Agri-FoodCanada, 2008). A variety of desi and kabuli chickpeas have been

434 F. Roy et al. / Food Research International 43 (2010) 432–442

developed and the characteristics of these cultivars may varydepending on the producing region. For example, some varietiessuch as CDC Frontier (Kabuli variety) have been developed for im-proved performance in the brown and dark soil zones of the Cana-dian Prairies. These varieties were specifically designed to expresscertain traits such as early maturation and the ability to resist par-ticular diseases, as this region in Canada has a short growing sea-son and a high incidence of the soil-borne disease AscochytaBlight (Barker, 2008). Regardless of the variety of chickpea seededin Canada, approximately 50% of Canada’s chickpea production isof the kabuli type, while the remaining 50% is of the desi type(Goodwin, 2003a,b).

There is a high demand for world production, exports and im-ports of chickpeas due to the crop’s nutritional value. Chickpea ishigh in protein, low in fat and sodium, cholesterol free and is anexcellent source of both soluble and insoluble fiber, complex car-bohydrates, vitamins, folate, and minerals, especially calcium,phosphorous, iron, and magnesium (Agriculture & Agri-Food Can-ada, 2006a,b; Nwokolo & Smartt, 1996). Chickpea is a good sourceof dietary protein due to its well-balanced amino acid compositionand protein bioavailability (Yust et al., 2003). It is relativelyinexpensive, and has been associated with the prevention of car-diovascular disease, managing type-2 diabetes and lowering LDL-cholesterol levels. Insoluble dietary fiber present in chickpea hasbeen associated with reducing the incidence of colon cancer,whereas soluble fiber has been demonstrated to have a beneficialeffect on weight loss and weight management (Agriculture &Agri-Food Canada, 2006a,b). The nutritional benefits of chickpeahave led to its use in various culinary applications such as hum-mus. In addition, chickpea is used in stews, soups and salads, andcan be processed into flour.

2.3. Lentil

Lentil (also known as red dahl, masur, massar, masuri tillseedand split pea) is grown annually on a variety of soil types rangingfrom sand to clay loan soils in semi-arid regions of the world. Len-til production originated in the Near East more than 8500 yearsago and has since spread to the Mediterranean, parts of Asia andwas subsequently introduced into North America by the early1900s (Oplinger, Hardman, Kaminski, Kelling, & Doll, 1990).Specifically, Canada began producing lentil in 1970 (Agriculture& Agri-Food Canada, 2006a,b). Lentil is now produced in over 48countries. In 2002, India, Turkey and Canada were the first,second, and third largest producers, of lentil, accounting forapproximately 33%, 16%, and 12%, respectively of the world’s pro-duction (Johnson & Jimmerson, 2003). Saskatchewan producesapproximately 95% of the Canadian lentil crop, with the remaining5% produced in Manitoba and Alberta (2Agriculture & Agri-FoodCanada, 2006a,b).

In 2001, Canada was the largest exporter of lentil with over 490thousand metric tonnes exported, which was approximately 70% ofthe total quantity of lentil produced in Canada that year. Egypt wasthe largest importer of lentil with over 113 thousand metric ton-nes. Turkey and Sri Lanka were the second and third largestimporters, with over 98 and 90 thousand metric tonnes imported,respectively in 2001 (Agriculture & Agri-Food Canada, 2006; John-son & Jimmerson, 2003).

Six types of lentil are commonly grown worldwide: Eston class,French green, Laird class, red, Richlea class and Spanish brown.These six types can be further subdivided into different varieties,which are adapted to various growing environments and condi-tions. Some varieties are better able to adapt to certain geograph-ical areas than others. For example, lentil varieties adapted to theWestern Canadian Prairies mature earlier due the short growingseason (Pulse Canada, 2008a,b). Canada produces mainly green

lentil (Laird varieties) accounting for approximately 70% of theworld’s green lentil production. However, it is estimated that 70%of the lentil produced worldwide is of the red lentil type, with25% being of the green type and 5% of the brown and other types(Agriculture & Agri-Food Canada, 2006a,b).

The nutritional value of lentil and its use in a variety of culinaryapplications make it an important commodity in terms of produc-tion, and trade. Lentil is high in protein especially rich in lysine andleucine, low in fat, and is an excellent source of dietary fiber andcomplex carbohydrates. Lentil also contains vitamins and mineralssuch as B vitamins, calcium, phosphorous and potassium, alongwith oleic, linoleic and palmitic acid (Adsule, 1996; Agriculture &Agri-Food Canada, 2006a,b). The nutritional characteristics of lentilhave been associated with cholesterol and lipid lowering effects inhumans, along with reducing the incidence of colon cancer andtype-2 diabetes (Agriculture & Agri-Food Canada, 2006a,b). Lentilis not commonly used in animal feed rations; however lentil strawis frequently used as animal feed (Saskatchewan Pulse Growers,2000). Similar to some dry pea varieties, lentil such as Indian Headblack lentil, is commonly grown as a green manure crop in organicfarming practices (Lawley & Shirtliffe, 2004).

3. Protein content of pulses

Pulse seeds accumulate protein throughout their development,hence mature pulses seeds are normally high in protein. Chickpea,lentil, and dry pea contain approximately 22%, 28.6%, and 23.3%protein, respectively on a dry weight basis (DW) (Pulse Canada,2004; Sotelo & Adsule, 1996). However, these percentages mayvary slightly depending on plant species, variety, maturity andgrowing conditions.

The majority of the protein found within pulse seeds is in theform of storage proteins, which are classified as albumins, globu-lins, and glutelins based on their solubility properties. Globulins,soluble in salt-water solutions, represent approximately 70% ofthe total protein found in pulses. One of two types of globulinusually predominates in pulses based on their sedimentation coef-ficient, vicilin or legumin which sediments at 7S or 11S, respec-tively. Albumins, soluble in water, account for 10–20% of thetotal protein in pulses. Finally, glutelins, soluble in dilute acidand base, account for 10–20% of the total protein found in the seedsof pulses (Duranti, 2006; Nwokolo & Smartt, 1996). Nutritionally,pulse storage proteins are relatively low in sulfur-containing ami-no acids, such as methionine, cysteine and tryptophan. However,the lysine content is relatively high compared to cereal crops. Forthis reason, combining pulse crops and grains such as rice, in thediet provides the essential amino acids required for proper humannutrition (Duranti, 2006).

There are many other types of protein found in legumes includ-ing various enzymes, protease inhibitors and lectins, which are col-lectively known as antinutritional compounds (ANCs). Themajority of these proteins are within the water-soluble albuminclass of legume proteins (Nwokolo & Smartt, 1996). In addition,they are distinct from other storage proteins with respect to func-tionality, as they have evolved within the seed as a protectivemechanism. The interest in the biological activity of pulse cropANCs and their potential use as a nutraceutical for promotinghealth and wellness and preventing or managing certain diseaseshas increased in recent years.

4. Antinutritional compounds of pulse crops

Antinutritional compounds (ANCs) are molecules that disruptthe digestion process when raw seed or flour is consumed bymonogastric species rendering the seed unpalatable (Domoney,

F. Roy et al. / Food Research International 43 (2010) 432–442 435

1999). On the other hand, ruminants are not affected by proteinANCs, as they have host-specific microorganisms capable of digest-ing feed rich in pulse seed or pulse flour. The accumulation of theseantinutritional compounds within the seeds of pulses is thought tohave evolved as a protective mechanism to help the plant completeits life cycle under adverse conditions, such as predation from par-asites, insects, fungi and herbivores, by causing sufficient intestinaltract discomfort within the host to deter predation (Duranti, 2006).

The antinutritional compounds found in pulse crops are classi-fied into two categories: protein ANCs and non-protein ANCs(Duranti & Gius, 1997), and range in effect from relatively inoffen-sive polyphenols to the relatively harmful protease inhibitors.Non-protein ANCs include alkaloids (Markievicz, Kolanowka, &Gulewics, 1988), phytic acid, phenolic compounds such as tannins(Davis, 1981) and saponins (Hudson & El-Difrawi, 1979). For anin-depth review of non-protein ANCs, the reader is referred tothe paper by Champ (2002) as a review of this type of ANC is be-yond the scope of this paper.

Protein ANCs commonly present in pulse crops include lectinsor agglutinins, trypsin inhibitors, chymotrypsin inhibitors, anti-fungal peptide (Ye & Ng, 2002) and ribosome-inactivating proteins(Wang & Ng, 2007). The protein ANCs from chickpea, dry pea andlentil may have mild to severe deleterious effects on human andanimal health. However, recent studies have identified that certainprotein ANCs may also have beneficial effects on human healthafter adequate processing procedures.

4.1. Lectin characteristics

Lectins (agglutinins) are ubiquitous carbohydrate-binding pro-teins which are found in a wide variety of important crop plants.Lectins are defined as ‘‘all plant protein possessing at least onenon-catalytic domain that binds reversibly to a specific mono- oroligosaccharide” (Ryan, 1990). Several hundred plant lectins havebeen identified, with most classified into four groups based ontheir molecular structure, carbohydrate binding characteristics,and biological activity (Peumans & Van Damme, 1996). The fourmain groups of lectins are: legume lectins, monocot mannose-binding lectins, chitin-binding lectins and the type-2 ribosome-inactivating proteins. Plant lectins are commonly found in foodconsumed without processing, such as fruit and vegetables(Nachbar & Oppenheim, 1980). Pea, chickpea, lentil, and otherpulse crops seeds, contain higher proportions of legume lectinsthan other lectin groups.

The biological role of legume lectins has yet to be elucidated;however it is thought that lectins act as a defense mechanism toprotect the plant from predation. These protective properties oflectins have resulted in several developments in plant biotechnol-ogy and genetic engineering in exploiting lectins in certain plantcultivars as bioactive insecticides (Sadeghi, Van Damme, Peumans,& Smagghe, 2006; Shukla, Arora, & Sharma, 2005). However, de-spite their beneficial properties as bioactive insecticides, whenpulses are consumed raw, they can resist gastrointestinal digestioncausing various adverse physiological effects in the host (Kelsallet al., 2002).

Table 1The agglutination activity of lectins from chickpea, lentil and field pea.

Common name Scientific name Part tested

Chickpea Cicer arietinum SeedLentil Lens culinaris SeedField pea Pisum sativum Seed

Adapted from Schuler, March 19, 2006 ABO blood type specific lectins in edible foods

4.2. Deleterious effects of lectins on human health

In humans and livestock, growth suppression, diarrhea, bloat-ing, vomiting and red blood cell agglutination, when sufficientquantities of raw seed or flour are consumed have been reported(Liener, Sharon, & Goldstein, 2002). While some lectins are heat-labile, others may be resistant to mild-heat treatments. Ingestionof active lectins can potentially cause red blood cell agglutination(Etzler, 1985), which may lead to complications such as hemolysisand in extreme cases, death (Liener et al., 2002). In addition to thetype of heat treatment (i.e., dry versus wet heating), the hemagglu-tinating activity (HA) of pulses depends on the temperature of theprocess and the crop variety. Bender and Reaidi (1982) have re-ported that the HA in pulse seeds may actually increase aftermild-heat treatment. In some instances, they found that heatingincreased the HA by sevenfold after 10 min at 80 �C. Hemaggluti-nins also show variable heat resistance depending on cultivar(Ayyagari, Narasinga, & Roy, 1989).

Lectins in chickpea, lentil and dry pea are hemagglutinins of allhuman blood types (A, B, AB, O), although they exhibit differentagglutination activity (Table 1). Specifically, the HA in lentil andfield pea is higher than in other pulse crops. Chickpea lectins haveno HA in red blood cells from humans (A, B, O types), rats, rabbitsor monkeys (Ayyagari et al., 1989). However, lectins from chickpeaagglutinated red blood cells from cows, although not at toxic levels(Contreras & Tagle, 1974). HA of lectins is affected by several fac-tors, such as the molecular properties of the lectin, cell surfaceproperties, metabolic state of the cells and the conditions of the as-say such as temperature and assay preparation (Lis & Sharon,1986). In addition, the cultivar, cultivation area, and method ofharvest may also affect the concentration of lectins within a partic-ular sample (Mekbungwan, 2007). These factors may contribute tothe discrepancy in the HA of chickpea compared to pea and lentil.To the authors’ knowledge, there have been no reports comparingthe concentration of lectins in pea, lentil and chickpea, or amongdifferent cultivars of these species. The concentration of lectins inraw lentil and pea samples was 0.1–1 (g/kg) and 0.2–2 (g/kg),respectively, according to Peumans and Van Damme (1996), how-ever chickpea was not evaluated in this study. Furthermore, theconcentration of lectins in any chickpea variety remains unclear.It is possible that a lower concentration of lectins in chickpea thaneither pea or lentil may be responsible for its low HA.

In addition to hemagglutination, mild food poisoning cases havecaused vomiting, bloating, and diarrhea in humans (Duranti & Gius,1997). Most cases are self-limiting, however some people may re-quire intravenous fluid replacement after consuming raw or inad-equately processed pulse seeds. In the livestock industry, feedingmonogastric animals raw pulse seeds reduces growth and perfor-mance as a result of lectin’s ability to bind to intestinal mucosalcells, which affects blood glucose response (Bardocz, Grant, &Pusztai, 1996). When ingested orally, lectins enhance the sheddingof brush border membranes and decrease villus length in the smallintestine, which in turn reduces the surface area and interfereswith normal gastric secretion for nutrient absorption (D’Mello,2002). Lectins also bind glycan receptors along the intestinal tract

Agglutination activity on blood type

A B AB O

+ + + ++++ +++ +++ ++++++ +++ +++ +++

. http://www.owenfoundation.com/Health_Science/Lectins_in_Foods.html.

436 F. Roy et al. / Food Research International 43 (2010) 432–442

causing discomfort and self-limiting ailments (Peumans & VanDamme, 1996).

Animals fed a soybean-rich diet can develop enlarged spleen,reduced serum insulin levels and have disruption of the normalprotein, fat and carbohydrate intermediary metabolism due tothe activity of lectin (Sharon & Lis, 2004). The lectin activity ofpea, chickpea and lentil has not been shown to produce the samepathological effects as raw soybean and kidney bean, althoughmild self-limiting ailments may arise in human populations andlivestock. However, there are conflicting reports on their toxicity.Peumans and Van Damme (1996) reported that lectins from lentiland field pea may be harmful to humans if consumed raw, whileothers have found that lectins from lentil, pea and chickpea arenon-toxic (Gonzalez, Mejia, & Prisecaru, 2005). A study on the lec-tin activity of several leguminous crops demonstrated that chick-pea and pigeon pea had lectin activity, although the activity wasreported to be below toxic levels (Singh, 1988). Although there isa risk of food poisoning from consuming raw pulse seeds, it shouldbe emphasized that under standard operating processing condi-tions the antinutritional effect of lectins is relatively minor.

4.3. Nutraceutical potential of pulse lectins in human health

Lectins present in pulses have long been considered antinutri-tional components, which must be denatured by thermal process-ing prior to human consumption. Over the past several years,scientific data has demonstrated that lectins may play a key rolein preventing certain cancers, and in the activation of certain in-nate defense mechanisms and can be utilized as a therapeuticagent for preventing or controlling obesity (Ewen, Bardocz, Pusztai,& Pryme, 2006; Hartmann & Meisel, 2007; Lima, Sampaio,Henriques, & Barja–Fidalgo, 1999; Pusztai & Bardocz 1996; Sameset al., 2001; Wang, Ng, Ooi, & Liu, 2000). The potential use of pulselectins as a nutraceutical for controlling obesity can be attributedto their ability to resist gastric digestion and by their ability tobe absorbed into the blood stream while remaining biologically ac-tive. These properties have also resulted in increased researchactivity in the use of pulse lectins as a cancer preventative.Although research remains in its infancy, several animal modelshave been described. Mice with non-Hodgkins lymphoma fed10 mg of mistletoe lectin per day were shown to have tumors with75% decrease in mitotic activity and 21% reduction in the nucleararea (Ewen et al., 2006). Lentil lectins have a strong effect onreducing the onset of human hepatoma (H3B) (Wang et al.,2000). Lentil agglutinins are also known to have considerable effecton human Merkel skin carcinomas (Sames et al., 2001). Pisumsativum agglutinin (PSA) has high affinity for tumor cells, whichmay in turn reduce the tumorigenicity of these cells (Bures, Moy-tycka, Bostik, Slavik, & Jirasek, 1986). Several factors can affectthe efficacy of lectins in reducing the onset of certain types oftumors.

The mode of action of lectins on tumor cells depends on thetype of tumor, the source of lectin and its biological activity. Theyare thought to bind to cell membranes or cell receptors causingcytotoxicity and apoptosis. Some theories suggest agglutinincauses reduction in cell division, increase in the number of macro-phages which increases the susceptibility of tumor cells to macro-phage attack, and improves the host immunocompetence (Barac,Stanojevic, & Pesic, 2005). Lectins may also penetrate the cell,resulting in cancer cell agglutination and many other antitumorproperties, such as the activation of certain protein kinases(Gonzalez et al., 2005). Most research on lectins/agglutinins as apotential adjunct to cancer treatment use lectin sources other thanpea, lentil and chickpea. Bean varieties, amaranth, wheat, potatoes,mistletoe, vetch, soybean and many animal lectins are commonlystudied. For further information regarding the use of lectins from

sources other than pulse crops, Gonzalez et al. (2005) provide a de-tailed review on the uses of important plant lectins as a potentialcancer treatment in their paper entitled, Lectins as bioactive plantproteins: a potential in cancer treatment. Most studies investigatinganticancer effects of lectins found in pulse crops have been per-formed with lectins from lentil and various pea varieties. In gen-eral, pea, lentil and chickpea are not well studied sources oflectins used as cancer therapeutic agents; however, these pulselectins may have great potential as a nutraceutical used to decreasethe risk of certain cancers.

Lectins from various sources have been identified as activeimmunomodulatory agents that can enhance the immune system,such as lymphocyte proliferation, natural killer cell activity, anti-body synthesis and cytokine regulation (Hartmann & Meisel,2007). Lectins from mistletoe induce cytokine activity by mononu-clear cells which can improve the effect of chemotherapeutic drugs(Ewen et al., 2006). The use of mistletoe lectin as an adjunct duringcancer treatment is currently commercially available in Europe.This has inspired research to discover lectins with immunomodu-latory properties from various other sources such as soybean, fungiand fruit. Soybean agglutinin lectins induce neutrophil andlymphocyte migration in vivo and activate mononuclear cells(Benjamin, Figueiredo, Henriques, & Barja-Fidalo, 1997). Recently,lectins from mushrooms (Dalloul, Lillehoj, Lee, Lee, & Chung,2005; Wang, Liu, Ng, Ooi, & Chang, 1996) and Jackfruit (ArtocarpusIntegrifolia) (Coltri, Casabona-Fortunato, Panuto-Castelo, 2004)have been demonstrated to have immunomodulatory properties.The current knowledge regarding pulse crops containing lectinswith immunomodulatory properties is based on pea. Pisum sativumagglutinin (PSA) studies (Lima et al., 1999) demonstrated that PSAinduces immunomodulatory effects, activating spleen lymphocytesin mice. More research is needed to characterize the effects of PSAin humans, as well as the use of lentil and chickpea agglutinins aspotential immunomodulatory compounds.

Obesity is often considered the root cause of other illness suchas heart disease, cancer and diabetes. Pulse seed lectins may alsobe efficacious in treating obesity. Pusztai and coworkers (Pusztaiet al., 1998) demonstrated that lectins from kidney bean could de-crease fat accumulation in rats, which was attributed to a decreasein insulin levels secondary to lectin activity. They also suggest thatit may be possible to use bean lectin as a dietary adjunct or pri-mary therapeutic agent in human trials to stimulate gastrointesti-nal function and reduce the incidence of obesity, if a safe andeffective dose range were to be established (Pusztai & Bardocz,1996). The main source of lectin used for treating/preventing obes-ity appears to be from bean. To the authors’ knowledge there areno reports suggesting that lectins isolated from chickpea, dry peaor lentil have a beneficial role in treating obesity. However, sincelectins isolated from closely related pulse crops such as bean havebeen demonstrated to treat and/or prevent obesity there may be apotential for the use of lectins isolated from other pulses as nutra-ceutical components.

4.4. Protease inhibitors

Similar to lectins, protease inhibitors are protein ANCs that havebeen isolated from a wide variety of fruit, vegetable and pulsecrops including pea, chickpea and lentil. In the 1970s and 1980s,considerable attention was given to protease inhibitors in pulse-based feed, as they interfered with digestion, growth and perfor-mance in domestic livestock (Champ, 2002). The level of proteaseinhibitors within pulses has since been considered an importantparameter in determining the quality of feed and food. Two well-characterized protease inhibitors found in pulse seeds are trypsinand chymotrypsin inhibitors, belonging to the Bowman-Birkinhibitor (BBI) family. Protease inhibitors from the BBI family con-

F. Roy et al. / Food Research International 43 (2010) 432–442 437

tain two active sites which inhibit the proteolytic enzymes trypsinand chymotrypsin. Due to the double inactivation of typsin andchymotrypsin, BBIs have been termed double-headed inhibitors(Domoney, Welham, & Sidebottom, 1993). When isolated from pi-geon pea, trypsin and chymotrypsin inhibitors had an averagemolecular mass of 10,500 and 15,000 Da, respectively (Godbole,Krishna, & Bhatia, 1994). However, the molecular mass may varybetween different species and environmental growing conditions.Ragg et al. (2006), Ferrasson, Quillien, and Gueguen (1997) andSmirnoff, Khalef, Birk, and Applebaum (1976), provide detailedinformation on the characteristics of trypsin and chymotrypsininhibitors from pea, chickpea and lentil for further review of thissubject.

All protease inhibitors present in pea, chickpea and lentil belongto the BBI family, whereas protease inhibitors from soybeans maybelong to the BBI or Kunitz family of protease inhibitors(Guillamon et al., 2008). The main difference between the BBIand the Kunitz inhibitors is the polypeptide make-up. The polypep-tide in Kunitz inhibitors consists of 181 residues with only twodisulfide bridges and one active site. The BBI polypeptide consistsof 70 residues and seven disulfide bridges with a double-headedinhibition mechanism (Laskowski & Kato, 1980).

The differences between the BBI and the Kunitz families of pro-tease inhibitors have a bearing on their respective inhibitory activ-ities. As protease inhibitors from pea, chickpea and lentil are fromthe BBI family, their inhibitory characteristics are relatively similar.However, differences in the concentration of inhibitor and theiractivity exist between pulse crop species (Champ, 2002) and vari-eties (Alonso, Orue, & Marzo, 1998). The concentration and inhib-itory activity of trypsin and chymotrypsin inhibitors in three peavarieties grown in Spain were found to vary (Table 2). Proteaseinhibitor concentrations also demonstrated varying degrees of sus-ceptibility to various processing treatments. This indicates that theprocessing technique and conditions used to denature proteaseantinutritional compounds may vary depending on the seed vari-ety being processed (Alonso et al., 1998). A similar study (Morri-son, Savage, Morton, & Russell, 2007) also demonstrated variabletrypsin inhibitory activity between cultivars of raw peas grownin New Zealand. This study also illustrated that the decrease inactivity varied between cultivars after thermal processing. Meanvalues of trypsin and chymotrypsin inhibitors in chickpea havealso been shown to vary. It was found that the concentration ofboth trypsin and chymotrypsin was higher in desi than in kabulichickpea (Singh & Jambunathan, 1981).

A review by Champ (2002) compared the trypsin (trypsin inhib-itory units; TIU/mg DM) and chymotrypsin (chymotrypsin inhibi-tory activity; CIA/g) inhibitory activity of various pulses. Theresults indicated that there was no TIA activity in chickpea orCIA activity in lentil or chickpea. However, other researchers foundthat the trypsin and chymotrypsin inhibitory activity in lentil seedmeal was 1.2 and 0.9 mg/g, respectively (Weder, Hegarty, Holzner,

Table 2Trypsin and chymotrypsin activity of three pea cultivars raw and after various processing

Treatment Trypsin inhibitor (IU mg� DM)

Renata Solara Ball

Raw seed 3.80 ± 0.24 2.80 ± 0.09 6.32Dehulling 4.02 ± 0.08 2.82 ± 0.06 6.47Soaking 3.74 ± 0.09 2.52 ± 0.06 5.56Germination (24 h) 3.57 ± 0.11 1.18 ± 0.02 4.64Germination (48 h) 3.32 ± 0.08 1.18 ± 0.02 2.81Germination (72 h) 2.76 ± 0.11 0.70 ± 0.03 1.55Extrusion 0.19 ± 0.02 0.16 ± 0.06 0.34

DM = dry matter.IU = international units.Adapted from: Alonso et al. (1998).

& Kern-Dirndorfer, 1983). Winter pulse crop varieties may alsohave more protease inhibitory activity than spring varieties(Champ, 2002). The results reported are variable when comparingspecies due to irregularities in units used and methods of analysis.A more comprehensive research is needed to analyze the activity ofprotease inhibition between species and varieties to further im-prove processing conditions and our understanding of the benefi-cial and detrimental effects of protease inhibitors in human andanimal nutrition. To date, the best characterized and studied BBIsare found in pea cultivars (Duranti & Gius, 1997), therefore moreresearch is needed on lentil and chickpea varieties.

4.5. Deleterious effects of protease inhibitors on human health

Although concentration and activity of protease inhibitors inpulse seeds are variable, detrimental effects of their inhibitoryproperties have been demonstrated (Champ, 2002; Duranti,2006; Messina, 1999). Similar to lectins, protease inhibitors inter-fere with digestion when pulse seeds or pulse flour are consumedraw. Protease inhibitors are resistant to pepsin and the acidic pH ofthe human digestive tract and, therefore, interfere with digestionby inhibiting trypsin and chymotrypsin through irreversible bind-ing to the enzymes. The mechanism by which BBI and Kunitzinhibitor act has been described by Guillamon et al. (2008) as ‘‘sup-pressing the negative feedback regulation of pancreatic secretionsthrough the release of the hormone cholecystokinin from the intes-tinal mucosa”. The negative feedback regulation may stimulatepancreatic hypertrophy and decrease growth and performance ofthe host due to reduced ability of the digestive enzymes to prop-erly hydrolyze dietary protein, thereby decreasing the amino acidabsorption and de novo proteins synthesis. The reduction ingrowth and performance can also be attributed to the loss of thesulfur-rich components of trypsin and chymotrypsin due to theirinhibition by protease inhibitors. In addition to pancreatic enlarge-ment secondary to the presence of protease inhibitors in raw pulseseeds, chemically induced pancreatic tumors have also been re-ported in some animal species from soybean trypsin inhibitors(Messina, 1999). Unlike lectins, gastrointestinal ailments such asdiarrhea, bloating and vomiting associated with protease inhibitorshave not been reported, and the only harmful effects on humansoccurred when seeds were consumed raw or processed inade-quately. The deleterious effects of protease inhibitors on humansare usually performed in in vitro trials (Duranti, 2006), howeverthe potential benefits of denatured trypsin and chymotrypsininhibitors are being studied in in vivo models (Kennedy, 1993). Inaddition to affecting the host, protease inhibitors can negatively af-fect the properties of foodstuffs where protease enzymes are com-monly used. Gel-formation, water-holding capacity, foaming andthe whipping ability of a product are all negatively affected by pro-tease inhibitors from pulse seeds (Garcia-Cerreno, 1996).

treatments.

Chymotrypsin inhibitor (IU mg� DM)

et Renata Solara Ballet

± 0.23 2.88 ± 0.05 2.73 ± 0.10 4.85 ± 0.09± 0.08 2.92 ± 0.03 2.76 ± 0.05 4.91 ± 0.12± 0.19 2.49 ± 0.06 2.25 ± 0.07 4.20 ± 0.08± 0.14 2.38 ± 0.07 2.20 ± 0.08 4.09 ± 0.05± 0.06 2.01 ± 0.06 2.12 ± 0.02 3.27 ± 0.19± 0.09 1.80 ± 0.05 1.87 ± 0.05 2.28 ± 0.08± 0.02 1.05 ± 0.02 0.96 ± 0.04 1.68 ± 0.07

438 F. Roy et al. / Food Research International 43 (2010) 432–442

4.6. Methods for reducing protease inhibitory compounds in pulseseeds

As previously mentioned, denaturation of antinutritional prote-ase inhibitory compounds is dependant on processing conditions,pulse species and variety. Germination, extrusion cooking, dehul-ling and hydrothermal processing are common commercial pro-cesses used to inactivate protease inhibitors (Table 2). Studies onchicks fed processed soybean demonstrated that extrusion cookingof soybean meal at temperatures between 104 �C and 120 �C for30–60 s resulted in an increase in growth rates in chicks while pan-creas weights remained low. The increased growth rate and the ab-sence of pancreatic hypertrophy indicated that a specific time andtemperature range was sufficient to denature the protease inhibi-tor in that particular soybean variety. Combining soaking with heattreatment has also been successful in decreasing the concentrationof protease inhibitors. After soaking 17 different pea varieties for18 h and then boiling them for 20 min, trypsin inhibitor contentwas reduced in all of the varieties by 42–92% (TIU/mg DM), withan average reduction of 78% (TIU/mg DM) (Morrison et al., 2007).Other studies demonstrate variability in protease inhibitor activityafter utilizing different time and temperature combinations,depending on the species, cultivar and treatment method (Alonsoet al., 1998; El-Adawy, Rahma, El-Bedawy, & Sobihah, 1999;Marquez & Alonso, 1999).

Agricultural practices may also have an effect on the proteaseinhibitor content of pulse seeds. Direct versus two-phase harvest-ing of different variety of pea, chickling vetch, lentil and soybeanshowed that two-phase harvesting reduced the trypsin inhibitorycontent in pea by up to 44% and up to 25% in chickling vetch.The reduction in trypsin inhibitor concentration in lentil andsoybean was too small to be considered statistically relevant(Pisulewska & Pisulewski, 2000). The reduction in protease inhibi-tor concentrations in two-phase harvesting may be an indicationthat the seed accumulates protease inhibitory compounds as itreaches maturity. In a two-phase harvesting system, the plant iscut and then left in the field to dry before the plant seed has anopportunity to accumulate the inhibitory compounds.

Plant breeding programs have attempted to decrease proteaseinhibitor levels in pulses through genetic manipulation. Francehas established a protease inhibitor threshold and does not permitthe registration of pea varieties with levels of trypsin inhibitorstwo units higher than the two official control cultivars (Morrisonet al., 2007). Although this policy decreases the antinutritional con-tent in the seeds, the crops may not perform as well, resulting indecreased yields, since protease inhibitors also protect the plantfrom predation. Similarly, protease inhibitors also protect the seedpost-harvest by protecting against fungi and other microorganismsduring storage (Garcia-Cerreno, 1996). Therefore, decreasing theprotease inhibitor content of pulse crops through plant breedingprograms may actually be more detrimental than growing plantswith a higher protease inhibitory content and subsequently inacti-vating the inhibitors post-farm gate through established process-ing techniques.

4.7. Beneficial properties of denatured protease inhibitors on humanhealth

Despite the negative effects of protease inhibitors, denaturedprotease inhibitors have several beneficial properties on humanhealth. In 1992 BBI proteases from legume crops achieved investi-gational new drug status by the FDA due to their health-promotingbenefits in a denatured form (Kennedy, 1995). Since then numer-ous reports have been published on the potential health-promotingbenefits of protease inhibitors, such as their potential use as ananti-inflammatory agent. Most research on the health benefits of

protease inhibitors has been performed with soybean. A soybeanextract containing soybean BBI that suppressed carcinogenesis inseveral animal models was developed with chymotrypsin proteaseinhibitor as the active component responsible for the anticarcino-genic behaviour (Kennedy, 1993). Soybean BBI suppressed colon,esophageal, liver, lung and oral carcinogenesis, and it has also beenproven efficient in suppressing radiation and chemically inducedcarcinogenesis in in vitro transformation assays (Moy & Bilings,1994). Studies in mice have confirmed that BBI activity was widelydistributed in various tissues (blood, kidney, liver and lungs) inmice after ingestion. Human and animal fibroblast and epithelialcells have also been shown to internalize protease inhibitors(Moy & Bilings, 1994). These findings indicate that BBIs are ableto resist digestion and be transported to various tissues in the host.

Anti-inflammatory properties of protease inhibitors in pulseshave also been demonstrated. Mice fed BBI concentrate demon-strated decreased inflammation of the colon when ulcerative coli-tis was induced in comparison to mice on a standard diet (Ware,Wan, Newberne, & Kennedy, 1999). The beneficial effects of BBIor other protease inhibitors as an anti-inflammatory or therapeuticcancer agent depend on the pulse species and seed variety used.Protease inhibitors are suggested as potential drugs for treatingvarious diseases such as human immunodeficiency virus (HIV),hypertension and neurodegenerative disease, along with variousinfectious diseases, however these are often treated with syntheticpeptidomimetic inhibitors rather than with natural protease inhib-itors from pulses. To the best of our knowledge there have been noreports confirming the successful use of protease inhibitors frompulse crops in the treatment of the above diseases. Currently, pub-lished information regarding the use of protease inhibitors frompulse seeds as therapeutic agents is limited to the treatment of cer-tain cancers or as an anti-inflammatory agent. Further research isneeded to determine if protease inhibitors derived from pulsecrops can also be used in the treatment of various other diseases.

Once the protease inhibitor reaches the target tissue throughthe blood stream, the precise mechanism of cancer and inflamma-tory suppression is not well understood. Moy and Bilings (1994)suggest that the protease inhibitors ‘‘suppress malignant transfor-mation by inhibiting cellular enzymes involved in the inductionand/or expression of the transformed phenotype”. Other research-ers have hypothesized that ‘‘protease inhibitors suppress carcino-genesis by altering the levels of certain types of proteolyticactivities, hydrolyzing activities and the expression of certain typesof oncogenes that play an important role in carcinogenesis”(Kennedy, 1993). Therefore, further research is needed to charac-terize the precise mechanism of cancer and inflammationsuppression.

5. Angiotensin I-converting enzyme (ACE) inhibitory peptides

Angiotensin I-converting enzyme (ACE) is widely distributed inmammalian tissues, predominantly as membrane bound ectoen-zymes in vascular and endothelial cells, along with several othercell types such as absorptive epithelial, neuroepithelial and malegerminal cells. ACE is a zinc metallopeptidase, which is activatedby chloride and has broad in vitro substrate specificity (Li, Le, Shi,& Shrestha, 2004). ACE causes high blood pressure by convertingthe biologically inactive angiotensin I to the potent vasoconstrictorangiotensin II, and also inactivates the vasodilator bradykinin(Hong et al., 2008) (Fig. 1). Drugs containing ACE inhibitory pep-tides are commonly used for treating hypertension and regulatingblood pressure, however they are costly and have been associatedwith numerous side effects (Erdmann, Cheung, & Schroder, 2008).

Since the discovery of ACE inhibitory peptides from snake ve-nom in 1971, there has been a renewed interest in isolating ACE

Angiotensinogen

Angiotensin I

Angiotensin II

ACE INHIBITORS

Vasoconstriction

ACE activity

Inactivated peptide

Bradykinin

Fig. 1. Simplified schematic demonstrating the mechanism of action of ACE.

F. Roy et al. / Food Research International 43 (2010) 432–442 439

inhibitors from natural sources. ACE inhibitory peptides have beenisolated from various food sources, including dairy products, caseinand whey protein, soybean, eggs, sake, porcine muscle, wheat, peaand chickpea (Erdmann et al., 2008; Meisel, Walsh, Murray, &FitzGerals., 2005, chap. 13; Pedroche et al., 2002; Vermeirssenet al., 2005). ACE inhibitory peptides have proven to be effectivein the prevention and treatment of hypertension, heart failure,myocardial infarctions and diabetic nephropathy in both humanand animal models (Meisel et al., 2005). The efficacy of ACE inhib-itory peptides is dose dependent and, therefore, dependent on theirpotency, which is normally measured as an IC50 value (concentra-tion of inhibitory peptide needed for 50% inhibition of ACE activ-ity). For example, inhibitory peptides isolated from rice, mungbean, and pea have IC50 of 18,200, 26.5 and, 0.15–0.23 lM, respec-tively (Hong et al., 2008). However, the inhibitory activity of thesepeptides in vitro may not have the same potency in vivo. When in-gested orally, some ACE inhibitory peptides within the food matrixmay be latent and must be fully or partially digested by gastroin-testinal enzymes such as trypsin, chymotrypsin and pepsin. Thedigestion of ACE inhibitors by these enzymes may result in eitherinactivation or activation of the inhibitory peptide, or the peptidemay remain in the latent form. Therefore, the potency measuredin vitro may be vastly different from the potency in vivo. Li et al.(2004) discovered that inhibitory peptides may not always haveantihypertensive activities. Peptides with a high inhibitory activitydid not always translate to a decrease in blood pressure (Li et al.,2004). In addition, the amino acid sequence and the length of theinhibitory peptide chain affect the efficacy of the peptide’s antihy-pertensive properties. Inhibitory peptides with short peptidechains, usually 2–5 amino acids, with C-terminal proline orhydroxyproline residues have stronger inhibitory effects, as theyhave been shown to bind the ACE more strongly. Proline, lysineand arginine are the preferred C-terminal substrate for ACE, con-tributing to a more potent ACE inhibition (Erdmann et al., 2008).In addition, short peptides are more readily absorbed by the car-diovascular system than free amino acids. Larger peptides (10–51amino acids) are readily absorbed, however their ACE inhibitor po-tency is greatly reduced (Erdmann et al., 2008).

Many food peptides and proteins derived by enzymatic hydro-lysis with antihypertensive properties in vitro and in vivo havebeen discovered. Many in vivo studies have been performed in hu-mans and spontaneous hypertensive rats (SHR) by intravenous andoral administration of the bioactive peptides and/or hydrolysate.Most studies have focused on the use of peptides derived fromdairy products, fish, sesame seed, eggs and mung bean, amongmany others (Hong et al., 2008). For example, a group of hyperten-sive humans consuming 95 ml of Calpis sour milk daily showed

significant reduction in blood pressure compared to placebo (Hata,Yamamoto, Ohni, Nakajima, & Takano, 1996). Fujita and Yoshikawa(1999) isolated two different peptides from thermolysin-digest of‘‘Katsuo-bushi”, a traditional Japanese dish created from dried bo-nito. The LKPNM (100 mg/kg) and LKP (30 mg/kg) peptides de-creased blood pressure in SHR by 30 and 50 mm Hg, respectively,after intravenous administration. Minimum effective doses ofLKPNM and LKP were 8 and 2.25 mg/kg. Purified peptides from ses-ame peptide powder have been shown to substantially decreasesystolic blood pressure in SHR after a single orally administereddose of either 1 or 10 mg/kg (Nakano et al., 2006).

Several studies continue to demonstrate the antihypertensivecharacteristics of various peptides isolated from a wide variety offoodstuffs and food ingredients (Erdmann et al., 2008; Honget al., 2008; Li et al., 2004; Meisel et al., 2005). However, to datea comparison of the ACE inhibitory properties of peptides derivedfrom pea, chickpea and lentil to that of dairy proteins has not beenadequately investigated, and further research is warranted. Inves-tigation of pulses as a potential source of ACE inhibitory peptideshas primarily focused on soybean (Kuba, Tana, Tawata, & Yasuda,2005; Mallikarjun, Gowda, Appu, & Prakash, 2006; Wu & Ding,2002) and mung bean, to a lesser extent (Li, Shi, Liu, & Le, 2006).However, recent research has focused on pea and chickpea (Aluko,2008; Humiski & Aluko, 2007; Pedroche et al., 2002; Vermeirssenet al., 2005). The treatment of legumin from chickpea with alcalseproduced a hydrolysate with ACE inhibitory (IC50) activity of0.18 mg/ml. Fractionation of the hydrolysate by reverse phasechromatography also yielded six inhibitory peptides ranging ininhibitory activity (IC50) from 0.011 to 0.021 mg/ml (Yust et al.,2003). A study by Vermeirssen et al. (2005) evaluated the antihy-pertensive effect of pea and whey digests and investigated the suc-cess of ACE inhibitory compounds in reaching the systemiccirculatory system in vitro. After intravenous administration of50 mg protein kg -1 body weight in SHR, ACE inhibitory compoundsfrom pea showed a transient but strong antihypertensive effect of44.4 mm Hg, while whey digest had no effect at an equivalent dose.Peptides derived from whey were more susceptible to digestion bygastric enzymes than pea-derived ACE inhibitory peptides. In addi-tion, pea ACE inhibitory peptides were converted to other activepeptides upon gastrointestinal digestion with similar ACE inhibi-tory activity rather than being denatured (Vermeirssen et al.,2005). Research on the isolation and characterization of ACE inhib-itory compounds from lentil varieties is limited. Work is currentlyongoing in the author’s laboratory to identify ACE inhibitory pep-tides in lentil. Presence of ACE inhibitor peptides in chickpea andpea cultivars has provided a solid foundation for the potentialuse of pulse seeds or pulse seed extracts in treating and preventinghypertension. However, more research is needed regarding the iso-lation and characterization of ACE inhibitory compounds derivedfrom pulse crops using both in vitro and in vivo models.

In addition to possessing bioactive properties that reducehypertension, the onset of heart failure, the risk of myocardialinfarctions and diabetic nephropathy, ACE inhibitors have beendemonstrated to have antioxidative properties. Limited studieshave demonstrated that antihypertensive drugs containing ACEinhibitors such as enalaprilat, lisinopril and captopril have antiox-idative properties. These three drugs showed oxygen free radicalscavenging properties by the chemiluminescence assay of oxida-tion of hypoxanthine by xanthine oxidase in the presence of lumi-nal (Mira, Silva, Queiroz, & Manso, 1993). The structure of thesedrugs (Fig. 2) illustrates that the compounds have functionalgroups that react with oxygen, resulting in the scavenging of reac-tive hydroxyl ions through a decarboxylation reaction of the car-boxyl group. Furthermore, enalaprilat and lisinopril havearomatic rings which may contribute to the compound’s antioxida-tive actions, whereas some of the oxygen scavenging properties

Fig. 2. Chemical structure of three common antihypertensive drugs. Source: http://www.drugbank.ca (Wishart et al., 2008).

440 F. Roy et al. / Food Research International 43 (2010) 432–442

from captopril may result from the oxidation of its sulfur group(Mira et al., 1993). In a related study of six antihypertension drugs(captopril, enalapril, fosinopril, perindopril, quinapril and ramip-ril), only captopril had significant antioxidative properties whenassayed by the ferric reducing antioxidant power (FRAP) assay(Benzie & Tomlinson, 1998).

The comparison of antioxidative properties found in ACE inhib-itor drugs to those found in raw pulse seeds or pulse flour may bedifficult, as ACE inhibitor potencies of pharmaceutical compoundsare generally expressed in IC50 values rather than concentration.Therefore, it is difficult to definitively determine if pharmaceuticalACE inhibitors are more potent antioxidants than those derivedfrom pulse seeds based on current information. However, it canbe hypothesized that ACE inhibitor containing drugs have a higherconcentration of ACE inhibitors compared to chickpea, pea andlentil, as pharmaceutical compounds are sold in capsules in a con-centrated form. Furthermore, the antioxidative properties of phar-maceuticals may have a higher potency and may be metabolizedmore readily, as pharmaceutical compounds are typically encapsu-lated to protect the active ingredient from adverse conditions suchas microbial activity, protease enzymes and low stomach pH. Inaddition, the antioxidative properties of the ACE inhibitor peptidesderived from pulses may be lost due to modifications to the pep-tide prior to being absorbed by the host.

There are other components of pulse seeds, such as genistein,daidzein and other phytoestrogens, which also have been demon-strated to have strong antioxidative properties. Therefore, some ofthe antioxidative effects in vivo may be predominantly the result ofother phenolic components and not ACE inhibitors (Mitchell,2001). Although a small number of studies have identified certainACE inhibitor containing drugs as having antioxidative properties,further research is needed to characterize ACE inhibitors isolatedfrom pulse seeds. Moreover, most of the antioxidative studies de-scribed above have been performed in vitro. Further researchshould be undertaken to examine antioxidative potencies in vivo,

with and without encapsulation. In conclusion, additional researchis needed to further investigate the antioxidative properties ofpulse seeds and their potential role in human health and disease.

6. Conclusion

The abundance and availability of pulse crops worldwide, cou-pled with their high nutritional value, have resulted in pulse cropsbeing one of the most widely produced and consumed agriculturalcommodities. Pulse crops have long been known for their nutri-tional and health-promoting properties, such as being an excellentsource of protein, fiber, carbohydrates, and for their role indecreasing the risk of certain cancers, managing obesity, loweringcholesterol and type-2 diabetes. Recently, the bioactive propertiesof proteins and peptides derived from pulse seeds have gained in-creased recognition in the areas of food science and nutrition fortheir potential benefits in treating and/or reducing the onset of dis-ease. Lectins and protease inhibitors which were traditionally con-sidered as protein antinutritional compounds have shownpotential in the treatment and/or prevention of various cancers,obesity and hypertension which has necessitated a reconsiderationof the use of the term ‘‘antinutritional”. Additionally, the ACEinhibitory properties of pulse peptides could make them primarytherapeutic agents or adjuncts to treatment for certain cardiovas-cular diseases. Pulse seeds may, therefore, be potentially excellentsources of beneficial bioactive proteins and peptides, and tech-niques for the efficient extraction and fractionation of these pro-teins and peptides are needed. Further research is also needed toimprove our understanding of the mechanisms involved in theabsorption into the blood stream, target sites and activity in vari-ous tissues of biologically active compounds derived from dry peas,chickpeas and lentils. Another area requiring research is compara-tive studies of the bioactivities of various pulse cultivars of thesame species and between different pulse crops, in order to deter-mine the cultivar of a specific pulse that has the greatest concen-tration and activity of a desired biologically active compound.

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