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This article is downloaded from

http://researchoutput.csu.edu.au It is the paper published as:

Author: S. Agboola, O. Mofolasayo, B. Watts and R. Aluko

Title: Functional properties of yellow field pea (Pisum sativum L) seed flours and the in vitro bioactive properties of their polyphenols

Journal: Food Research International ISSN: 0963-9969

Year: 2010

Volume: 43

Issue: 2

Pages: 582-588

Abstract: Functional and bioactive properties of yellow field pea (Pisum sativum L) seed flour, protein isolate (PPI), two high fibre products (Centara III, Centara IV), and one high fibre-starch ingredient (Uptake 80), were determined. The whole seed flour had superior water and oil absorption capacities but the high fibre flours had significantly higher (p<0.05) swelling ability. Centara IV and Uptake 80 had the highest gel clarity while Centara IV gel was the most resistant to freeze-thaw treatment. Polyphenolic constituents were extracted singly or sequentially with aqueous methanol and acetone; the whole pea seed flour and the pea protein isolate had significantly more polyphenolic constituents than the fibre products, which also resulted in higher in vitro antioxidant activities (trolox equivalent antioxidant capacity and diphenyl-picryl hydrazyl free radical scavenging ability). Results of renin- and ACE-inhibitory activities were mixed and did not correspond to the overall polyphenolic content and antioxidant test results, probably indicating the importance of components specific to individual extracts.

Author Address: sagboola@csu.edu.au

URL: http://dx.doi.org/10.1016/j.foodres.2009.07.013

http://researchoutput.csu.edu.au/R/-?func=dbin-jump-full&object_id=11948&local_base=GEN01-CSU01

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CRO Number: 11948

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Some functional properties of yellow field pea (Pisum sativum L.) seed flours and the in

vitro bioactive properties of their polyphenols

Samson O. Agboolaa,*, Olawunmi A. Mofolasayob, Beverley M. Wattsb, and Rotimi E.

Alukob,c

aSchool of Agricultural and Wine Sciences, Charles Sturt University, Private Bag 588,

Wagga Wagga 2678, Australia

bDepartment of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba,

Canada R3T 2N2

cThe Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba,

Winnipeg, Manitoba, Canada R3T 6C5

Running title: Functional and bioactive properties of pea flours

*Corresponding author: Tel.: + 61-2-6933-4041; Fax: +61-2-6933-2866; E-mail address:

sagboola@csu.edu.au (S.O. Agboola)

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Abstract

Functional and bioactive properties of yellow field pea (Pisum sativum L) seed flour, protein

isolate (PPI), two high fibre products (Centara III, Centara IV), and one high fibre-starch

ingredient (Uptake 80), were determined. The whole seed flour had superior water and oil

absorption capacities but the high fibre flours had significantly higher (p<0.05) swelling

ability. Centara IV and Uptake 80 had the highest gel clarity while Centara IV gel was the

most resistant to freeze-thaw treatment. Polyphenolic constituents were extracted singly or

sequentially with aqueous methanol and acetone; the whole pea seed flour and the pea protein

isolate had significantly more polyphenolic constituents than the fibre products, which also

resulted in higher in vitro antioxidant activities (trolox equivalent antioxidant capacity and

diphenyl-picryl hydrazyl free radical scavenging ability). Results of renin- and ACE-

inhibitory activities were mixed and did not correspond to the overall polyphenolic content

and antioxidant test results, probably indicating the importance of components specific to

individual extracts.

Keywords: phenolics; flavonoids; functional properties; antioxidant activity; pea seed; fibre

products

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1. Introduction

Yellow field peas (Pisum sativum L.) are a major pulse crop in western Canada, and

Canada is a world leader in field pea export (Wang, Daun, & Malcolmson, 2003). Yellow

field peas are rich in protein, starch, fibre, vitamins and minerals, and thus serve as a good

source of commonly used food ingredients such as protein isolates and starches, as well of

fibre ingredients which are of increasing importance in the design of health and diet foods.

Fibre is now well-established as a desirable component of health foods due to its beneficial

effect on intestinal motility. The seed coats of leguminous plants have also been shown to

contain a significant amount of polyphenols (Barampama & Simard, 1993; Elias, Fernandez,

& Bressami, 1979), and there is increasing evidence for the therapeutic effects of plant

polyphenols present in human diets (Beninger & Hosfield, 2003; Salah, Miller, Paganga,

Tijburg, Bolwell, & Rice-Evans, 1995). Ingredients obtained from the pea hulls are therefore

a source of both dietary fibre and polyphenols, enhancing the health promoting potential of

these flours.

The therapeutic effects of the polyphenols have been attributed to their antioxidant

properties. Reactive oxygen species (ROS) generated in vivo in humans has been implicated

in the pathogenesis of a variety of disorders including cancers, artherosclerosis, hypertension

and aging (Halliwell & Gutteridge, 1999). Antioxidants have also been shown to protect the

human body from the ROS damage, reducing their activity by scavenging the free radicals

generated, complexing pro-oxidant metals and quenching singlet oxygen (Tachakittirungrod,

Okonogi, & Chowwanapoonpohn, 2007). Plant constituents that can contribute to antioxidant

activity have thus received considerable attention, and many food plants have been analysed

for total phenols, total flavonoids and free radical scavenging ability (Antolovic, Prenzler,

Pastilides, McDonald, & Robards, 2002).

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Human studies using polyphenol-rich foods such as cocoa, tea and grapes have

indicated that high flavonoid levels can be associated with reduction in blood pressure (Actis-

Goretta, Ottaviani, & Fraga, 2006; Diebolt, Bucher, & Andriantsitohaina, 2001; Duffy et al.,

2001; Taubert, Berkels, Roesen, & Klaus, 2003). Studies on the health benefits of natural

products such as peas, should investigate polyphenolic constituents both for general

antioxidant capacity as well as for inhibition capacity targeted at promoters of specific

disorders, such as angiotensin converting enzyme (ACE) and renin. ACE is critical for the

operation of renin-angiotensin system (RAS) and the administration of ACE inhibitors has

been positively linked to the regulation of blood pressure in humans (Brunner, Gavras, &

Wacber, 1979; Corvol, Williams, & Soubrier, 1995). Additionally, renin-inhibitory agents

have been shown to produce a highly selective inhibition of RAS, significantly enhancing the

side-effect profile of other therapeutic agents (Stanton, 2003).

Nutritional guidelines emphasizing the importance of fibre for intestinal health, and

the potential benefits of polyphenols, have increased the demand for high fibre and

antioxidant–rich food products by consumers. To take advantage of this demand, food

manufacturers must be able to incorporate the pea protein, fibre and starch-fibre ingredients

into acceptable food products. Knowledge of the functional properties of these flours is

critical for their successful application. Initial work on the functional properties of pea seed

protein isolate has demonstrated functional properties that are equivalent, or superior, to those

of soybean protein isolate (Sosulski & McCurdy, 1987; Sumner, Nielsen, & Youngs, 1981).

However, to date there is very little information on the functional properties of the other types

of pea ingredients. Therefore, some of the functional properties of commercial pea seed

flours were determined in the research reported here.

In addition, since some functional properties have been shown to be extraction solvent

dependent, the effects of solvents on the profiles and in vitro bioactive properties of

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polyphenols obtained from yellow pea seed flour products were determined. A number of

different extraction fluids, including water, organic solvents (usually methanol and acetone)

and their aqueous mixtures have been employed for polyphenol extraction from plant sources

(Akowuah, Ismail, Norhayati, & Sadikun, 2005; Chavan, Shahidi & Naczk, 2001; Sripad,

Prakash, & Narasinga Rao, 1982; Troszyska, Estrella, Lopez-Amores, Hernandez, 2002;

Yilmaz & Toledo, 2006). Comparison of the amounts of extracted polyphenols and

evaluation of the antioxidant and antihypertensive activities of these products, should aid in

the subsequent development and formulation of functional foods containing active

polyphenolic constituents.

2. Materials and Methods

2.1. Materials

Industrially produced samples of pea seed flour, protein isolate (PPI or PropulseTM),

two very high fibre ingredients (Centara IIITM, Centara IVTM) and one starch-fibre flour

(Uptake 80TM), were supplied by Nutri-Pea Ltd (Portage la Prairie, Manitoba, Canada).

Purified rabbit lung ACE and hippuryl-histidyl-leucine (HHL) were purchased from Sigma

Chemicals (St. Louis, MO). Renin (human recombinant), purity ≥ 99% by SDS-PAGE, was

purchased from Cayman Chemical (Ann Arbor, MI, USA). Fluorescent renin peptide

substrate 1, Arg-Glu(EDANS)-lle-His-Pro-Phe-His-Leu-Val-lle-His-Thr-Lys(DABCYL)-

Arg, was purchased from Molecular Probes (Carlsbad, CA, USA), while EDANS, 5-[(2-

aminoethyl)amino]-naphthalene-1-sulfonic acid; DABCYL, 4-(4-

dimethylaminophenylazo)benzoic acid. HPLC-grade solvents and all analytical-grade

reagents and chemicals were purchased from Fisher Scientific (Oakville, ON, Canada).

2.2. Determination of functional properties

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Functional properties were determined according to the following previously

published methods: water holding and fat absorption capacities (Tang, Ten, Wang, & Yang,

2006); soluble solids and swelling power (Torruco-Uco & Betancur-Ancona, 2007); least

gelation concentration (Severin & Xia, 2006); freeze-thaw stability (Eliasson & Kim, 1992);

starch gel clarity (Torruco-Uco & Betancur-Ancona, 2007) and resistant starch (Goni,

Garcia-Diz, Manas, & Saura-Calioxto, 1996).

2.3. Extraction of polyphenols from pea seed products

Flour samples were extracted in three ways, first using 80% methanol only, secondly

sequentially using 80% methanol followed by 50% methanol, and thirdly using, in sequence,

80% methanol, 50% methanol and 70% acetone. For each extraction, dried sample was added

to the extraction solvent at a 1:10 ratio, and after stirring for one hour the mixture was

centrifuged at 10,000g for 20 minutes, the supernatant decanted and then concentrated on a

rotary evaporator, under vacuum at less than 30°C. The concentrate was subsequently freeze-

dried and weighed for total dry matter yield. For the sequential extractions, supernatants from

each step were pooled, similarly concentrated, freeze-dried and weighed.

2.4. Analysis of total phenolics, total flavonoids and antioxidant activities

The total phenolic (Folin-Ciocalteu procedure), flavonoid and antioxidant capacity

assays {trolox equivalent antioxidant capacity (TEAC) and diphenyl-picryl hydrazyl (DPPH.)

free radical scavenging ability} were determined as outlined by Michalska et al. (2007). The

content of total phenolics in each extract was expressed as mmol of gallic acid (GA) per 100

gram of dry matter in the sample, while the flavonoid content was expressed as mmol of (±)-

catechin equivalent per 100 gram of dry matter in the sample. The total antioxidant capacity

was based on the ability of extracts to inhibit ABTS and DPPH free radicals. The results were

compared to extinction of 6-hydroxy-2,5,7,8-tetra-methylchromane-2-carboxylic acid

8

(Trolox) reacting with both types of radicals and expressed in mmol trolox per 100 gram of

dry matter in the extracted sample.

2.5. Analysis of antihypertensive activities

ACE-inhibitory activity of the extracts was determined using the method of Cushman

& Cheung (1971) with some modifications. Briefly, a 50 µL aliquot of sample (10 mg/mL)

was dissolved in 100 mM borate buffer containing 0.3 M NaCl (pH 8.3) and mixed with 50

µL ACE solution (50 mU/mL). The mixture was then pre-incubated at 37°C in an Eppendorf

thermomixer for 10 min, after which 150 µL of 4.15 mM hippuryl-histidyl-leucine substrate

was added. The enzyme-substrate mixture was incubated for 30 min at 37°C, and then 500

µL of 1 M HCl was added to stop the reaction. The hippuric acid produced was extracted by

adding 1.5 mL ethyl acetate and vortexing for 1 min. After separation by standing for 5 min,

800 µL of the ethyl acetate layer was transferred into a 2 mL Eppendorf tube and dried in a

hot water bath with the aid of nitrogen. After drying, 1 mL of distilled water was added and

the absorbance determined on an Ultrospec 4000 UV-visible spectrophotometer (Pharmacia

Biotech, Montreal, PQ) at 228 nm. A blank sample containing 50 µL of 1 M HCl in place of

sample was also prepared and used in the calculation of the % inhibition as follows:

% ACE inhibition = (Blank Abs – Sample Abs) x 100/Blank Abs

Renin-inhibition was determined by following the fluorescence method of Wang, et

al. (1993) as modified by Yuan et al. (2006). The peptide substrate and fresh renin were

diluted to 95 µM or to 0.5 mg/mL respectively, using 50 mM Tris-HCl buffer (pH 8.0)

containing 100 mM NaCl. The control (blank) reaction mixture contained 20 µL of substrate,

and 160 µL buffer and was pre-incubated for 10 min at 37°C to reach equilibrium before 10

µL renin was added to initiate the reaction. The inhibition reaction mixture contained the

same reagents, but the buffer was reduced to 150 µL and 10 µL of 1 mg/mL extract was

9

added. Time dependent increase in fluorescence intensity was monitored for at least 10 min

on a Jasco FP-6300 spectrofluorimeter (Japan Spectroscopic Company, Tokyo) equipped

with a thermostated microcuvette (100 µL) cell holder which was maintained at 37°C.

Maximum fluorescence intensity at excitation and emission wavelengths of 340 nm and 490

nm respectively was recorded for all samples and compared to the blank sample for the

calculation of % renin inhibition.

2.6. High Performance Liquid Chromatography (HPLC) profiling

Freeze dried extracts were dissolved in water-acetic acid (98:2) to obtain 3 mg/mL

solution. Each solution was then analysed on a reverse-phase C18 column (5 µm particle size;

250 x 10 mm) using a Waters Delta 600 preparatory-scale HPLC system equipped with a

2996 photodiode array detector (Waters Corp., Milford, MA, USA). The mobile phase

consisted of solvent A, methanol-acetonitrile (50:50), and solvent B, water-acetic acid (98:2).

An aliquot (1 mL) of extract was injected and eluted with a gradient of 95%-52% solvent A

in 65 min at a flow-rate of 2.5 mL/min; UV spectra were recorded at 280 nm.

2.7. Statistical analysis

All experiments were performed in triplicate and mean results (with standard deviation)

are reported. Unless otherwise stated, analysis of variance (ANOVA) was used to compare

the means and Fisher’s least significant difference (LSD) test was used to separate the means

that were significantly different (P ≤ 0.05), using SPSSTM Statistical software version 1.1

(SPSS Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Functional properties

Comparison of the functional properties of pea flours (Table 1) showed that water holding

capacity (WHC) and fat holding capacity (FHC) values for the whole flour were significantly

10

higher (p<0.05) than values obtained for the other flour products. The PPI (Propulse) had the

least values for WHC and FHC, which may be due to the lower levels of starch and fibre

when compared to the fibre products or whole flour. The results are different from a previous

work (Sosulski & McCurdy, 1987) that showed higher WHC and FHC values for pea protein

isolate when compared to the values of for pea seed flour. However, the flour used in that

study was produced from dehulled pea seeds, and in this study the flour was milled from

whole seeds including the hulls. Since hulls are largely fibre that adsorbs water and oil,

higher values of WHC and FHC are to be expected.

The PPI contained 30% soluble solids, a significantly higher (p<0.05) amount than those

from the other flours. This could be attributed partly to the sugar content, but also to the

soluble proteins in the PPI. Although the whole flour had similar sugar content, and was 25%

protein, these components were apparently complexed sufficiently to resist extraction under

these conditions.

Uptake 80 had significantly highest (p<0.05) swelling power of the flours as determined

at 90oC, which could have resulted from having higher levels of protein and starch than the

fibre products (Centara III & IV), and higher levels of fibre than whole seed flour. The PPI

had the second highest swelling power, although levels of fibre and starch were much less

than for the Uptake 80. Starch is known to exhibit high swelling power in water (Torruco-

Uco & Betancur-Ancona, 2007) and when combined with the swelling abilities of proteins

and fibre could have contributed to the superior swelling ability of Uptake 80. The fact that

PPI had higher swelling ability than whole seed flour and fibre products suggests proteins

also contribute to swelling of seed flours especially when the product is heated. When gels

were formed at room temperature, Centara IV and PPI produced a paste rather than a

cohesive gel, an indication that the intensity of inter-molecular interactions was not strong

enough to overcome repulsive forces. This could have been due to the relatively high levels

11

of sugars in the PPI but other factors must have influenced the lack of gelation of the Centara

IV. Though the whole flour also had high levels of sugars it was still able to form a gel at

room temperature, probably as a result of the high level of starch. Uptake 80 had the least

gelation concentration (LGC) suggesting that the presence of moderate amounts of starch and

fibre could provide molecular interactions that favour gel formation at room temperature. In

contrast, Centara IV had a desirably and significantly low (p<0.05) value of LGC at 4oC

(compared to lack of gelation at room temperature), which indicates that intermolecular

interactions were substantially enhanced at cold temperatures when compared to room

temperature. The increase in gelling ability upon cooling probably results from a decrease in

mobility of the molecules with decreasing temperature, which enhanced bond formation

within and between the biopolymer molecules (Renkema & van Vliet, 2002). The favourable

effect of cold temperature is evident in the decreased values of LGC for whole flour and

Centara III in addition to the ability of PPI to form a gel at 4oC (compared to lack of gelation

at room temperature).

Gel clarity was indirectly proportional to LGC probably because high levels of flour

produce dense gels that limit transmission of light. The gel clarity values for the pea fibre

products are similar to those that have been reported for pure starches (Torruco-Uco &

Betancur-Ancona, 2007). Since gel clarity is very important to give shine and opacity to

foods, the pea fibre products may be useful in the manufacture of fruit pie fillings and candies

(Torruco-Uco & Betancur-Ancona, 2007).

The gel produced by Centara IV was the most stable to one cycle of freeze-thaw

treatment, while Uptake 80 gel was the least stable. The presence of starch (specifically

amylose molecules) enhances retrogradation and reduces stability to freeze-thaw treatment.

Centara IV has no starch content, which may be responsible for the ability of the gel to resist

freeze-thaw treatment better than the other starch-containing flours. However, it is possible

12

that fibre also contributes to freeze-thaw stability since Centara IV had the highest level of

fibre. Overall these pea flours do not appear to be suitable for the manufacture of frozen

products, due to their high levels of water expulsion during the freeze-thaw treatment.

The resistant starch level in whole flour was higher than that in Uptake 80, which

suggests a loss in resistant starch molecules during processing of the seed flour. A previous

report (Goni et al., 1996) has shown that cooked pea flour had resistant starch content of

~11%, which is half the level found in the raw pea flour used in this work but similar to the

amount present in Uptake 80.

3.2. Extraction of polyphenols

Table 2 shows the yield of the five samples based on extraction with 80% methanol only

and sequentially with the other aqueous solvents. In general, repeated extractions yielded

more dry matter. Methanol alone extracted over 50% of the whole pea seed solids, and almost

14% of the PPI solids. Sequential extractions with the extra steps of 50% methanol (Met/Met)

and 70% acetone (Met/Met/Ace) produced average increases of about 6% for the whole pea

seed flour and increases of 41% and 13% respectively for the PPI. Fan, Sosulski, & Hamon

(1976) reported that repeated extractions were necessary to fully extract the wide range of

polyphenols in sunflower seed. Furthermore, Chavan et al. (2001) and Troszynska et al.

(2002) reported maximization of extracted polyphenols from beach pea and yellow pea seed

coat, respectively, when acetone was used compared to methanol. However, the fibre

products had very low extractable materials even with the sequential extractions, showing a

maximum of less than 10% yield (97 mg/g sample) for the Met/Met/Ace extract from Uptake

80. This could be related to the very low solubility of fibre materials in aqueous polar

solvents that were predominantly employed in this study. Thus, it is highly likely that the

extracts obtained from pea seed flours and PPI contained more non-phenolic compounds than

those obtained from the fibre samples. Sripad et al. (1982) reported the concomitant

13

extraction of proteins with the polyphenols from sunflower seed using various aqueous

solvents including methanol and acetone.

3.3. Total phenolics and in vitro bioactive properties

Table 3 shows the results of total phenolics assay, while Table 4 shows antioxidant and

antihypertensive properties of Met/Met sequential extracts obtained from all samples. Our

studies showed that there was no significant difference in either the HPLC profile (see section

3.4. below) or activity of the extracts obtained using only 80% ethanol or those obtained from

sequential extractions. This result suggests that while more materials were extracted with

repeated extractions, the nature of the extract did not change significantly, even when using

different solvents with varying polarity. Conversely, Akowuah et al. (2005) reported

increased antioxidant activity (DPPH free radical scavenging) in aqueous acetone extracts of

Orthosiphon stamineus leaf compared to aqueous methanolic extracts. Siah, Agboola, Wood,

& Blanchard (2008) also reported higher phenolic content and increased antioxidant activity

(TEAC and DPPH) in faba bean samples (seed coat and cotyledon) extracted with 70%

acetone compared to those extracted with 80% methanol. The difference may be related to

the fact that extractions in those studies were carried out individually with different solvents

whereas subsequent extractions were employed in this study based on the initial aqueous

methanolic residue.

The fibre samples had the lowest total phenolics, total flavonoids and antioxidant

properties compared to the PPI and whole pea seed flour extracts. Significantly, however,

there does not seem to be any relationship between the phenolic and flavonoid contents,

whereas, the total phenolics seemed to correspond in general to the antioxidant activities.

This is in agreement with results obtained by Anton, Ross, Beta, Fulcher, & Arntfield (2008)

who reported positive correlation between total phenolic content and DPPH antioxidant

activity in navy and pinto beans. Comparatively, the antihypertensive activities of ACE and

14

renin inhibition do not correlate with each other as significant renin inhibition was recorded

for all extract samples while Centara III and PPI polyphenolics did not show any ACE-

inhibitory property. Our results are similar to a previous report that showed in vitro ACE-

inhibition by peptides does not always correspond to renin inhibition as different mechanisms

control the action of the two enzymes (Yuan, Wu, & Aluko, 2007).

These results also suggest specificity in the activities of the extracts rather than generic

properties of either phenolics or flavonoids. Furthermore, the significant differences between

the extracts obtained from the fibre products may be related to the specificity of their

manufacturing process whereby different traditional products, e.g., proteins and starches,

were targeted. Although Uptake 80 appears to be the best among the products overall in terms

of polyphenol content, antioxidant and antihypertensive activities, the values were much

lower than those obtained for whole pea seed extracts. Hung & Morita (2008) showed that the

outer coats of buckwheat grains contained more polyphenols and therefore had higher

antioxidant properties compared to the inner part of the grain. DueOas, Hernandez, & Estrella

(2006) studied in vitro antioxidant capacities in legumes and made a similar conclusion.

Therefore, it would be useful to assess the seed coat components of yellow field peas that

have not been subjected to pre-processing as in these fibre products in order to make stronger

conclusions about their nature with respect to polyphenolic content and health functionality.

3.4. HPLC profiles

Figure 1 shows the reverse phase HPLC profiles of 80% methanolic extracts of pea seed,

protein isolate and a typical fibre product sample (Centara III). The other two fibre samples

(Centara IV and Uptake 80) showed profiles that are very similar to that of Centara III,

though they lack the peak that was obtained at about 25 min elution. All extracts show major

peaks at the beginning of the elution, suggesting that they were mostly hydrophilic species

consistent with the polar solvents employed for their extraction. The PPI also showed the

15

least variety in the range of peaks throughout the elution, albeit having larger peaks between

5 and 12 minutes. This is most likely as a result of the protein materials being almost

exclusively obtained from the cotyledons of the seed, which has been confirmed to have

much lower levels of polyphenols compared to the seed coat of leguminous seeds (Elias et

al., 1979; Barampama & Simaud, 1993). Furthermore, protein isolates are typically processed

in such a way as to reduce the level of non-protein phenolic bodies (contaminants). The pea

seed flour extract on the other hand clearly had much more polyphenolic moieties across the

whole elution spectrum with a few significant peaks in the 15-25 minute range. This may

indicate the full range of materials present in both seed coat and cotyledon. Siah et al. (2008)

reported a similar range of peaks in the HPLC profile of faba bean, with the majority being in

the seed coat fraction.

4. Conclusions

The functional properties of whole pea flour, high fibre, fibre-starch and high protein

ingredients, derived from yellow field peas, indicate that these products could contribute

desirable functional characteristics to a wide range of food products. High levels of water and

oil absorption, good gelation capabilities, and gel clarity should make these ingredients

useful in a variety of applications. The concentrated pea products appeared to have relatively

low amounts of polyphenols compared to the whole pea flour. Future research on the

polyphenols of field peas should focus on analysis of the hulls and of the cotyledon fractions

separately. It can be inferred from the preceding results that not all polyphenolic species

obtained from the solvent extracts were active, and only some moieties were responsible for

antioxidant and antihypertensive activities. Therefore, future research should also focus on

the purification and identification of active compounds using post-HPLC activity testing.

16

Acknowledgements

Funding for this work was provided by operating and equipment grants to REA from

the Advanced Foods and Materials Network of Centres of Excellence (AFMnet), the Natural

Sciences and Engineering Research Council of Canada (NSERC) and Manitoba Association

of Agricultural Societies under the Agri-Food Research & Development Initiative (ARDI)

program. We thank our industrial partner, Nutri-Pea Ltd (Portage la Prairie, Manitoba) for

supply of the yellow pea seed flours.

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21

Table 1. Selected functional properties of commercial yellow pea seed flours1 1

Property Whole flour Centara III Centara IV Uptake 80 Propulse 2

Water holding capacity (g/g flour) 4.04 + 0.23a 2.79 + 0.05b 2.72 + 0.01b 2.40 + 0.01c 1.70 + 0.04d 3

Fat holding capacity (g/g flour) 2.49 + 0.22a 1.59 + 0.10b 1.63 + 0.14b 1.57 + 0.12b 0.95 + 0.05c 4

Swelling power at 90oC (g water/g flour) 6.41 + 0.32c 6.21 + 0.38c 6.26 + 0.31c 9.22 + 0.04a 8.25 + 0.19b 5

Soluble solids at 90oC (%) 1.33 + 0.58b 2.33 + 0.58b 2.50 + 0.71b 1.33 + 0.58b 30.00 + 4.58a 6

Least gelation concentration (%) 7

20oC 14.00 + 0.00a 14.00 + 0.00a no gel 10.00 + 0.00b no gel 8

4oC 12.00 + 0.00b 12.00 + 0.00b 8.00 + 0.00d 10.00 + 0.00c 16.00 + 0.00a 9

Gel clarity (% Transmission) 10.73 + 0.31c 36.77+ 2.75b 56.80 + 0.50a 53.93 + 2.97a 0.00 + 0.00d 10

Freeze-thaw stability of gels 11

(% water released) 27.82 + 0.33c 29.66 + 0.02b 18.93 + 0.04e 30.36 + 0.03a 24.95 + 0.03d 12

Resistant starch (%) 22.03 + 0.64a - - 15.28 + 0.23b - 13

14

1 Major composition (dry weight basis): 15

Whole pea seed flour: 25.0% protein, 50.0% starch, 8.0% fibre, 12.5% sugars, 2.0% lipids, 2.5% ash 16

Centara III: 6.0% protein, 90.0% fibre, 0.5% lipids, 0.5% sugars, 1.0% starch, 2.0% ash 17

22

Centara IV: 3.0% protein, 93.0% fibre, 0.5% lipids, 1.5% sugars, 0.0% starch, 2.0% ash 18

Uptake 80: 10.0% protein, 35.0% fibre, 0.5% lipids, 50.0% starch, 2.5% sugars, 2.0% ash 19

Propulse (pea protein isolate): 82.0% protein, 3.0% lipids, 0.0% fibre, 0.7% starch, 11.3% sugars, 3.0% ash 20

a-e Within each row, data with the same superscript are not significantly different from each other (P ≥ 0.05). 21

22

23

24

25

26

27

28

23

29

Table 2. Yielda of extracts (mg/g sample dry weight) obtained from field pea seed and its 30

products using 80% methanol only (Met), sequentially with 80% methanol and 50% 31

methanol (Met/Met), and 80% methanol, 50 % methanol and 70% acetone (Met/Met/Ace) 32

Product Met Met/Met Met/Met/Ace

Whole pea seedb 516.00 ± 12.04 546.20 ± 13.37 581.23 ± 14.48

Pea protein isolateb 138.10 ± 4.37 194.35 ± 6.48 219.44 ± 9.44

Centara IIIb 31.66 ± 1.67 49.96 ± 2.11 67.23 ± 4.37

Centara IVb,c 26.13 ± 0.97 34.72 ± 1.44 86.54 ± 5.34

Uptake 80b,c 23.30 ± 0.88 33.27 ± 2.27 97.00 ± 8.37

33

a Data are means of at least three replicates ± standard deviation. 34

b Within the row, each yield value for each extraction procedure is significantly different 35

(P ≤ 0.05) from the other two. 36

c Data for Centara IV and Uptake 80 within each column are not significantly different 37

from each other (P ≥ 0.05). 38

39

40

41

24

42

43

Table 3. Content of total phenolic compounds and total flavonoids in pea seed and its 44

productsa 45

Product Total phenolic compounds

(mmol gallic acid/100g sample

dry weight)

Total flavonoids

(mmol ±-catechin/100g

sample dry weight)

Whole pea seed 5.67 ± 0.47a 1.35 ± 0.30a

Pea protein isolate 2.84 ± 0.23b 0.44 ± 0.02b

Centara III 0.56 ± 0.08c 0.81 ± 0.10c

Centara IV 0.47 ± 0.05c 0.16 ± 0.01d

Uptake 80 2.36 ± 0.33b 0.24 ± 0.01e

46

a Data are means of triplicates ± standard deviation. Within each column, means with the 47

same superscript are not significantly different (P ≥ 0.05). 48

49

25

Table 4. In vitro antioxidant and antihypertensive activities of extracts obtained from pea 50

seed and its productsa 51

Product Antioxidant activities

(mmol Trolox/100g sample dry weight)

Antihypertensive activities

TEAC DPPH % ACE

inhibition

% renin

inhibition

Whole pea seed 15.62 ± 1.04a 10.89 ± 0.81a 65.28 ± 2.57a 48.69 ± 2.53a

Pea protein isolate

4.13 ± 0.39b 1.89 ± 0.23b None detected 67.58 ± 3.03b

Centara III 1.56 ± 0.21c 0.63 ± 0.09c None detected 26.61 ± 1.40c

Centara IVb 0.54 ± 0.06d 0.24 ± 0.03d 27.78 ± 1.37b 67.25 ± 2.86b

Uptake 80b 3.54 ± 0.30e 2.79 ± 0.27e 26.13 ± 1.23b 24.31 ± 1.41c

52

a Data are means of triplicates ± standard deviation. Within each column, means with the 53

same superscript are not significantly different (P ≥ 0.05). 54

55

26

Figure Legend 56

Figure 1. Reverse phase HPLC profiles of 80% methanolic extracts of pea seed flour (A) and 57

its products including pea protein isolate (B) and a typical fibre sample- Centara III (C). The 58

vertical axis in each figure is designated in Absorbance Units (AU), measured at 280 nm. 59

60

61

27

62

63

64

65

C

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

B

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

A

AU

0.00

0.02

0.04

0.06

0.08

0.10

Minutes 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00