The effects of various processing conditions on a protein isolate from Lupinus angustifolius

Food Chemistry 120 (2010) 496–504

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

The effects of various processing conditions on a protein isolate from Lupinus angustifolius Elena Sirtori a,*, Donatella Resta b, Francesca Brambilla a, Christian Zacherl c, Anna Arnoldi a a

Laboratorio di Chimica degli Alimenti e Spettrometria di Massa, Dipartimento di Endocrinologia, Fisiopatologia e Biologia Applicata, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy b HPF-Nutraceutics, Via Balzaretti 9, 20133 Milan, Italy c Fraunhofer Institut für Verfahrenstechnik und Verpackung, Giggenhauser Straße 35, 85354 Freising, Germany

a r t i c l e

i n f o

Article history: Received 13 May 2009 Received in revised form 25 August 2009 Accepted 14 October 2009

Keywords: 2D-electrophoresis De novo sequencing Differential scanning calorimetry Shotgun proteomic Thermal treatment

a b s t r a c t Lupin protein is a promising ingredient in functional foods because of its purported hypocholesterolaemic and hypotensive activities. In this study a lupin protein isolate from Lupinus angustifolius was thermally and mechanically treated and the effects on its protein profile were determined. As a preliminary step, the main protein components of L. angustifolius were identified, using the canonical proteomic approach, including 2D-separation and mass spectrometry and, whenever necessary, also ‘‘de novo peptide sequencing”. Most of the main spots were assigned to the major lupin storage proteins: a-conglutin, b-conglutin, c-conglutin, and d-conglutin. The protein degradation induced by the different treatments was studied via differential scanning calorimetry (DSC), 2D-electrophoresis, and mass spectrometry, in order to get the fingerprint of the intact peptides after processing. The results indicate that, even after harsh industrial processing, a-, b- and d-conglutin are still able to release stable peptides, although they are completely or partially degraded, as shown by the 2D protein profiles and the DSC graphs. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction During the last decade functional foods have gained attention from the food industry because of their possible role in the maintenance of health and wellbeing. Plant proteins are valuable ingredients in the preparation of functional foods for the prevention of cardiovascular disease (FDA, 1999; Sirtori, Eberini, & Arnoldi, 2007). Although soybean is still the major plant protein used in these foods, other grain legumes are currently in rapid development as protein sources, in particular pea and sweet lupin (Dijkstra, Linnemann, & van Boekel, 2003). Sweet lupin has some characteristics that may be appreciated by both consumers and the food industry: it has a low content of anti-nutritional factors (Muzquiz et al., 1998; Resta, Boschin, D’Agostina, & Arnoldi, 2008), is phytoestrogen-free (Sirtori et al., 2004), and has some positive technological properties. In addition, several studies have shown that this legume is characterised by hypocholesterolaemic (Bettzieche et al., 2008; Sirtori et al., 2004; Spielmann et al., 2007), anti-atherogenic (Marchesi et al., 2008), hypotensive (Lee et al., 2009; Pilvi et al., 2006), and hypoglycaemic activities (Hall, Thomas, & Johnson, 2005; Lee et al., 2006).

* Corresponding author. Tel.: +39 02 503 18210; fax: +39 02 503 18204. E-mail address: [email protected] (E. Sirtori). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.10.043

Lupin is a common name that represents four domestic species, Lupinus albus (white lupin), Lupinus angustifolius (narrow-leaf lupin), Lupinus luteus (yellow lupin), and Lupinus mutabilis (pearl lupin). White lupin is currently the most important in human nutrition, but the use of narrow-leaf lupin is increasing. The recent use of this seed limits the available knowledge about its technological and functional properties. Since data regarding the consequences of industrial processing on the quality of narrow-leaf lupin protein are still not available, the present investigation was designed to evaluate the effects of mild and harsh thermal and mechanical treatments on a lupin protein isolate. In fact, industrial processing is known to cause protein denaturation and a certain degree of insolubilisation (Porres, Aranda, López-Jurado, & Urbano, 2005; Zheng, Fasina, Sosulski, & Tyler, 1998). Denaturation may cause either deleterious (Carbonaro, Grant, & Cappelloni, 2005; Porres, Aranda, LópezJurado, & Urbano, 2006) or beneficial (Hancock, Peo, Lewis, & Crenshaw, 1990) effects on the nutritive value and nutraceutical properties of proteins. In this study, differential scanning calorimetry and proteomic techniques, such as 2D-electrophoresis and mass spectrometry, were used to investigate the effects of different processing conditions on the protein profile and to assess the availability of resistant peptides after treatments. A preliminary phase of the investigation was dedicated to the identification of the main spots

E. Sirtori et al. / Food Chemistry 120 (2010) 496–504

in the 2D-map of L. angustifolius seed protein, since the literature was incomplete. 2. Materials and methods 2.1. Laboratory procedure for the preparation of total protein extract (TPE) from lupin seeds Lupin seeds (L. angustifolius cv Boregine) were de-hulled and the dry cotyledons were milled (60-mesh sieve); the resulting flour was defatted with n-hexane (20 ml/g of flour) for 24 h with agitation, decanted and air-dried. The globulin fraction was extracted by stirring with 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate hydrate (CHAPS), and 65 mM 1,4-dithiothreitol (DTT), for 2 h at room temperature (RT). The slurry was centrifuged at 6000g and 4 °C for 30 min and the proteins extracted in the supernatant were immediately analysed or stored at 80 °C until use. 2.2. 2D-electrophoresis Protein sample (20 ll; about 7 lg/ll) was diluted in 130 ll of IEF solubilisation buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% ampholyte pH 3–10 and pH 4–6); the proteins were reduced with 65 mM DTT and alkylated with 200 mM iodoacetamide (IAM), both steps for 1 h at (RT) in the dark. Isoelectric focusing was performed on 7 cm, pH 3–10 non-linear IPG strips (Bio-Rad Laboratories Inc., Hercules, CA). The strips were rehydrated overnight with the above solution and focused for 20,000 V/h, with a maximum of 2500 V using the Protean IEF Cell (Bio-Rad). Prior to the second dimension, strips were incubated in equilibration buffer (6 M urea, 30% glycerol, 1% SDS, 0.1 mM EDTA, 50 mM Tris–HCl pH 8.8) for 25 min at RT. The separation was performed on 13% SDS– PAGE gels, using the Mini Protean 3 Dodeca Cell (Bio-Rad). The gels were stained with Bio-Safe Coomassie (Bio-Rad), that detects proteins without the use of methanol or acetic acid. This staining produces a white background on the gel, with greater band/spot intensity, in comparison with Coomassie Blue G-250 staining, allowing a good qualitative/quantitative analysis by the software. Gels were scanned with a VersaDoc 3000 (Bio-Rad), and the PDQuest software (Bio-Rad) was used to compare the different 2D-maps. All samples were run in duplicate. 2.3. Tryptic in-gel digestion for the identification of the spots from 2D gels The spots were excised from the 2D gel of L. angustifolius total protein extract, washed with water at 56 °C for 10 min and dehydrated with 100 ll of CH3CN at 56 °C for 20 min; the procedure was repeated three times, in order to shrink the gel pieces. The CH3CN was removed and the spots were dried in air for 10 min. The spots were reduced with 60 ll of 10 mM DTT/0.1 M NH4HCO3 (freshly prepared), for 30 min at 56 °C, and subsequently alkylated with 60 ll of 55 mM IAM/0.1 M NH4HCO3, for 20 min at RT, in the dark. The spots were washed three times with 200 ll of 0.1 M NH4HCO3 and dehydrated with 300 ll of CH3CN. The gel pieces were rehydrated in 20 ll of trypsin solution (12.5 ng/ll sequencing-grade trypsin, 50 mM NH4HCO3/5 mM CaCl2) at 4 °C for 50 min and then incubated at 37 °C overnight. In order to improve the peptides’ excision, the gel pieces were sonicated for 10 min and centrifuged briefly. The supernatants were cleaned with a Microspin Column (Harvard Apparatus Ltd., Edenbridge, UK) and the peptides were further fragmented and analysed using a HPLCChip-Ion Trap or a HPLC-Chip-qTOF (Agilent Technologies, Palo Alto, CA).

497

2.4. Preparation of the lupin protein isolate (LPI) in a pilot plant Lupin seeds from L. angustifolius (cv Boregine) were supplied by Saatzucht Steinach GmbH (Steinach, Germany), de-hulled with an underflow peeler (Streckel & Schrader KG, Hamburg, Germany) and sieved in an air-lift system (Alpine Hosakawa AG & Co. OHG, Augsburg, Germany). The kernels were flaked in a flaking mill (Streckel & Schrader KG). The resulting yellow flakes were defatted with nhexane and flash-dried in a solvent de-oiling system (E&E Verfahrenstechnik GmbH, Warendorf, Germany). The flakes were dissolved in water in the ratio 1:10 (w/v) and stirred at 30 °C for 1 h. The solution was adjusted with 3 N NaOH to pH 8.0. Afterwards, insoluble substances were removed by decanter centrifugation at 30 °C (Westfalia decanter centrifuge). The supernatant was adjusted to pH 4.6 with 3 N HCl, stirred for 2 h at 10 °C and constant pH, then centrifuged at 10 °C. The precipitate was washed twice in water at a ratio of 1:8 (w/v), and then dissolved again in water at a ratio of 1:8 (w/v) at 30 °C, adjusted to pH 7.0 and spray-dried (D’Agostina et al., 2006; Holley et al., 2000). 2.5. Processing of the lupin protein isolate 2.5.1. Thermal treatment Lupin protein isolate (LPI) was mixed with water in a ratio of 1:2 (protein:water). After 5 min soaking, small amounts of the slurry were placed into aluminium boxes and heated in an oven at 100, 150, and 200 °C, respectively. Samples were taken after 5, 15 and 30 min; each sample was cooled to RT and lyophilised. 2.5.2. Mechanical process in an Ultraturrax apparatus In order to simulate mechanical stress, 200 ml of a 10% LPI solution (in water) were mixed in an Ultraturrax (Janke & Kunkel IKA Labortechnik, Staufen, Germany) for 1 min at 8000, 13,500 and 24,000 rpm, respectively. After treatment, the samples were lyophilised. 2.5.3. High-pressure homogeniser The simulation of mechanical stress caused by extruders or homogenisers was performed using a high-pressure homogeniser (APV-2000, APV, Unna, Germany): solutions of LPI in water (10%) were homogenised at 100, 300 and 1000 bar, respectively. After treatment, the samples were lyophilised. 2.6. Differential scanning calorimetry (DSC) analysis DSC was carried out according to Sousa, Mitchell, Ledward, and Hill (1995). The pre-treated, lyophilised protein isolates were dissolved in distilled water at a concentration of 20% (w/v) by stirring for 10 min at RT. Small amounts of the protein solutions were weighed in Tzero Hermetic pans (TA Instruments – Waters LLC, Waltham, MA) and the pans were sealed hermetically. Thermograms were obtained by heating from 313 K (40 °C) to 393 K (120 °C) at a heating rate of 2 K/min, using a DSCQ 2000 system (TA Instruments – Waters LLC). All samples were immediately re-scanned after cooling to 40 °C, to investigate reversibility. Besides the onset point temperatures of the protein denaturation peak of the samples, the software calculated the enthalpy, DH (in J/g), from the peak areas. As the reference an empty, sealed pan was used. 2.7. 2D-electrophoresis of the processed LPIs The treated and untreated LPIs were extracted with the same procedure applied for the protein extraction from the seeds and submitted to 2D-electrophoresis as indicated in Section 2.2.

498

E. Sirtori et al. / Food Chemistry 120 (2010) 496–504

2.8. Tryptic digestion of untreated and treated LPIs for peptide fingerprint The protein samples were reduced with 1 M DTT (in the ratio 50 mol of DTT to 1 mol of Cys), and alkylated with 1 M IAM (in the ratio 200 mol of IAM to 1 mol of Cys); each step was performed for 1 h at RT. The proteins were then digested with sequencinggrade trypsin (0.5 lg/ll) (Promega Corporation, Madison, WI) in the ratio of 1:50 (w/w) enzyme/protein for 16 h at 37 °C. The peptide mixtures obtained were analysed using the HPLC-Chip-Ion Trap (Agilent Technologies).

2.9.4. De novo sequencing of the unmatched peptide The qTOF MS/MS spectra corresponding to the spots from 1 to 18 were subjected to a manual ‘‘de novo sequencing” in order to increase the legumin coverage. The mass tolerance of the precursor ions was 20 ppm; the mass tolerance of the product ions was 50 ppm. The ‘‘de novo sequenced” peptides were aligned against the NCBInr protein database in order to evaluate their homologies with respect to the amino acid sequence of other legumins (BLASTp Program).

3. Results and discussion 2.9. HPLC-Chip-Ion Trap and HPLC-Chip-qTOF analyses 3.1. Complete 2D-map of L. angustifolius seed protein All experiments were performed on an Agilent 1200 LC connected to an Agilent Ion Trap (SL series) mass spectrometer or to an Agilent G6520A quadrupole time-of-flight (qTOF) mass spectrometer (Agilent Technologies). The ionisation system was the HPLC-Chip Cube working in nanoflow electrospray positive ion mode. Solvents and sample delivery to the chip, high-pressure switching of flows, automated chip loading and positioning in the MS source were all accomplished by this interface. The chromatographic separation of the tryptic digest was performed on a biocompatible LC Chip containing a 40-nl enrichment column (Zorbax 300SB; C18, 5 mm), a 0.075  43 mm analytical column (Zorbax 300SB; C18, 5 mm), capillary tubing connections, and a nano-electrospray capillary. The chromatographic separations were performed with a linear gradient, using (A) 95% water, 5% CH3CN, 0.1% formic acid and (B) 95% CH3CN, 5% water, 0.1% formic acid as mobile phases; solvent B was increased from 3% to 50% for 50 min, then increased from 50% to 80% in 10 min and returned to 3% after 10 min. The enrichment of the sample prior to starting the gradient was performed at 4 ll/min in 100% A using the loading pump. 2.9.1. Ion trap conditions Capillary voltage 1850 V, endplate offset of 500 V, drying gas flow 3 l/min, drying gas temperature 300 °C, scan range m/z 300– 2200, target mass m/z 700, average of 2 spectra, ICC target 30.000, maximum accumulation time 150 ms. The MS/MS analyses were performed in Auto MS(n) mode. 2.9.2. Quadrupole time-of-flight (qTOF) conditions Drying gas flow 3 l/min, drying gas temperature 300 °C, fragmentor 175 V, skimmer 65 V, collision energy: slope 3.7 V, offset 2.5 V, MS scan range and rate m/z 300–2000 at 3 Hz, MS/MS scan range and rate m/z 50–3000 at 3 Hz, auto MS/MS: 8 precursors, active exclusion on with 1 repeat and release after 0.17 min. Preferred charge state: 2, 3.

Fig. 1 shows the 2D-map of the reduced total protein extract of L. angustifolius seed. IPG were run in the presence of 7 M urea, hence non-covalently bound oligomers were dissociated in all cases. The total spots detected by PDQuest Software were 202. In most cases rows rather than single spots were observed; in particular, series of components with similar molecular weights (MWs) but varying pI values. These spots are different isoforms of the same protein with different degrees of post-translational phosphorylation and glycosylation. Often these spot rows are further arranged into clusters of rows of closely spaced MW values. In fact, all the lupin globulins derived from a unique common ancestor polypeptide, that undergoes proteolytic cleavage, giving a complex mixture of polypeptides, which aggregate to form globulins. In addition to this, the immature proteins are subjected to many post-translational modifications, as already mentioned. Owing to these phenomena, globulins are an extremely heterogeneous class of proteins. All sectors of the map (from acidic to basic pI and from high to low MWs) contain prominent spots. We used both HPLC-Chip-Ion Trap and HPLC-Chip-qTOF for protein identification. The HPLC-Chip-Ion Trap permitted the identification of the L. angustifolius proteins whose partial or complete sequences are deposited in the NCBI database. The 57 spots picked for MS/MS analysis are numbered in Fig. 1 and their identities are listed in Table 1.

250 150 100 75 -

48 49 47 50 51

1

50 -

2

3

53 54

4

37 -

5

52

55 56

57 39 40 41

6

42 43

7 34 35 36

25 -

15 14 13 12

20 26

27

28 29

20

21

17 32 33

18

31 8

22 23 24 25

15 -

45 46

38

37 16

2.9.3. Database searches Protein database searches were performed with Spectrum Mill MS Proteomic Workbench (Rev A.03.03). All searches used the NCBInr protein database biweekly updated with trypsin specificity, 1 missed cleavage. The ‘‘forward” database search of the ion trap data was performed using a mass tolerance of ±2.5 Da for precursor ions and a tolerance of ±0.7 Da for fragment ions, while the qTOF search used 20 ppm precursor and 40 ppm product ion tolerance. A reverse database search (Brambilla, Resta, Isak, Zanotti, & Arnoldi, 2009) was also performed. In order to validate the peptide and protein matches, the Spectrum Mill default value for the peptide scores, forward–reversed scores, rank 1–rank 2 scores, percentage scored peak intensity (SPI%) and protein scores were considered (Brambilla et al., 2009).

44

30

9

10

11

19

10 3

pI

10

Fig. 1. 2D-map of the reduced total protein extract of L. angustifolius cv Boregine. The proteins were run in the first dimension on 7 cm, pH 3–10 non-linear (NL) IPG strips (Bio-Rad); the second dimension was performed on 13% denaturing SDS– PAGE gel. Standard marker (Precision Plus Protein; Bio-Rad) is indicated in kDa on the left. Solid line rectangles (—) indicate the acidic and basic subunits of aconglutin. Dash-dotted rectangles (---) indicate b-conglutin. The dotted oval (- -) highlights the heavy chain of d-conglutin. The solid line ovals enclose the large and small subunits of c-conglutin. The peptide sequences and the protein identification of each numbered spot are listed in Table 1.

499

E. Sirtori et al. / Food Chemistry 120 (2010) 496–504

Table 1 Identification of lupin proteins excised from 2D gels. The peptide sequence matches were determined using Spectrum Mill software. The NCBInr of the protein sequences are listed (b-conglutin or vicilin-like protein and a-conglutin or legumin-like protein are different names to indicate the same proteins). Several organisms have a positive match with the same peptide sequence (Lupinus albus, Pisum sativum, Vicia faba and Vicia sativa). The proteins that did not show any match with the NCBI database were identified via HPLCChip-qTOF (de novo sequencing). No. spot

Species

Protein identification

NCBInr accession

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18

Lupinus angustifolius Lupinus angustifolius Lupinus albus Lupinus albus Lupinus albus Pisum sativum Vicia sativa Vicia faba Vicia faba Lupinus angustifolius Lupinus angustifolius LUPAN (L. angustifolius) Lupinus angustifolius Lupinus angustifolius Lupinus angustifolius Lupinus albus Lupinus albus Lupinus angustifolius

a-Conglutin

2313076 De novo sequenced peptide 85361412 62816184 62816188 126161 483449 81988 259475 662366 223005 116181 149208403 149208401 169950562 46451223 89994190 23194375

19, 20, 21 22, 23, 24, 25 From 26 to 57 (except for 37 and 38)

37, 38

Most of the spots were positively identified by their correlation to b-, d- and c-conglutin amino acid sequences: 30 spots corresponded to fragments of b-conglutin, three belonged to c-conglutin (2 to the large subunit, same MW – 47 kDa – but different pI, and 1 to the small subunit – 17 kDa), and four spots were identified as d-conglutin. Furthermore, two spots in the vicilin area were identified as putative triacylglycerol factor protein, a protein responsible for the accumulation of triacylglycerols (Francki, Whitaker, Smith, & Atkins, 2002). One of the main storage proteins in all legume seeds is a-conglutin (legumin-like protein); this is a hexameric protein consisting of six disulphide-linked subunits synthesised as a precursor, comprising a N-terminal acidic alpha-chain (with greater MW) and a Cterminal basic beta chain (with lower MW) (Shutov, Kakhovskaya, Braun, Bäumlein, & Müntz, 1995). Unfortunately, the L. angustifolius amino acid sequence of this protein is still lacking in the databases, only a very short fragment of 131 amino acids being available (conglutin alpha, NCBInr 2313076). It was observed, nevertheless, that some spots belonging to the gel regions characteristic for this protein class gave positive matches with the L. albus legumin-like proteins (NCBInr 85361412, 62816184, 62816188), Pisum sativum legumin A2 precursor (NCBInr 126161), Vicia sativa legumin A (NCBInr 483449), Vicia faba legumin B (NCBInr 81988) and legumin propolypeptide beta chain (NCBInr 259475). Therefore, in order to improve the legumin coverage, the spots numbered from 1 to 18 were submitted to HPLC-Chip-qTOF analysis, which permitted the ‘‘de novo sequencing” of the good-quality MS/MS spectra that did not match against any legumin protein sequence in the database. ‘‘De novo peptide sequencing” is peptide sequencing performed without prior knowledge of the amino acid sequence. When specific species are not in the public databases, or when the reported sequences are uncompleted, the ‘‘de novo sequencing” approach is the only way to get valuable peptide sequence information. Goodquality spectra are required for a reliable ‘‘de novo sequencing”; the MS/MS spectra acquired with the qTOF mass spectrometer are suitable to obtain a reliable ‘‘de novo sequencing”, because of the high mass resolution and wide MS and MS/MS scan range of the system (Frank, Savitski, Nielsen, Zubarev, & Pevzner, 2007). A charged peptide may be fragmented into two pieces in three different ways, which may produce a pair of a- and x-ions, a pair of b- and y-ions, or a pair of c- and z-ions. Double charged peptides provide better fragmentation spectra for interpretation. The most

Legumin Legumin-like protein Legumin-like protein Legumin-like protein Legumin A2 precursor Legumin A Legumin B Legumin propolypeptide beta chain c-Conglutin c-Conglutin smaller subunit d-Conglutin-2 large chain b-Conglutin b-Conglutin b-Conglutin b-Conglutin precursor Vicilin-like protein Putative TAG factor protein

common peptide fragments observed in low energy collisions are a-, b-, and y-ions. The b-ions appear to extend from the amino terminus (N-terminus) and y-ions appear to extend from the carboxyl terminus (C-terminus). The most abundant ions in double charged peptides are y-ions, which often form the complete series in an MS/ MS spectrum. The next are a- and b-ions, of which many are not observed. The c-, x-, and z-ions occur much less frequently (Frank et al., 2007). In addition, these ions can often form new ions due to loss of water or ammonia. In the low mass region of the MS/MS spectrum, the amino acid immonium ions may be present. From the differences of the m/z ratios of consecutive ions belonging to the same series, it is possible to obtain the amino acid sequence of the peptide. The following peptides were ‘‘de novo sequenced”: NGI/LEETI/ LCTAI/LI/LR, ADI/LYNPTAGR, TNDQATTSPI/LK, VQVVNSQGNSVF NDDI/LR, GI/LPAEVI/LANAFR, I/LSI/LNQVSEI/LK, and VEEGI/LGVI/ LSPK. As usual, since leucine (L) and isoleucine (I) have the same residue mass (113.08407 Da), a correct attribution along the peptide sequence is very difficult. The mass tolerance between measured and expected precursor ion was 20 ppm; the mass tolerance between measured and expected product ions in the MS/MS peptide spectrum was 50 ppm. These peptides were selected on the basis of the high homology of their amino acid sequences with those of the legumins of other grain legumes. The ‘‘de novo sequencing” of peptide ADI/LYNPTAGR is shown as an example in Fig. 2. 3.2. Effect of thermal processing on the protein isolate With the aim of mimicking industrial food processing, the LPI was treated thermally and the effects on the protein quality were determined using different experimental approaches. As a preliminary step, DSC experiments were used to determine the feature of native/degraded proteins in the treated samples, in comparison with untreated LPI. The thermally-induced transitions of the proteins are plotted in the DSC thermograms shown in Fig. 3, and the corresponding denaturation temperatures (Td) are listed in Table 2. The two peaks in the thermogram of the untreated sample indicate the presence of two major protein classes: the vicilins (bconglutin) and the legumins (a-conglutin). Fig. 3 shows that both peaks were measurable at low temperatures and short heating

500

E. Sirtori et al. / Food Chemistry 120 (2010) 496–504

x10 4 Product Ion (8.283 min) (539.2708[z=2]) 86.0970 I/L

1.4

R

A

G

T

N

P

Y

l/L

D

1.3 1.2

AD

Y

I/L

N

1.1 1

501.2786 y5

0.9 0.8 0.7 615.3224 y6

0.6 0.5 136.0767 Y 187.0711 b2

0.4

778.3839 y7 300.1568 b3

0.3 0.2 70.0650 P

0.1

175.1202 232.1419 y1 y2 159.0774 a2

303.1789 y3

404.2222 463.2186 y4 b4

891.4728 y8

1006.4430 y9

577.2625 b5

0 0

50

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050

Countsvs. Mass-to-Charge(m/z) Fig. 2. The MS/MS sequencing of ADI/LYNPTAGR (m/z 539.2708, error ppm 3.8): in the low mass region the amino acid immonium ions of P, I/L and Y are identified; the complete y-ion series (y1–y9) and a partial b-ion series (b1–b5) are reported.

a

Heat Flow (W/g)

100°C 30min

100°C 15min

100°C 5min

untreated 60

80

Exo Up

100

Temperature (°C)

120 Universal V4.3A TA Instruments

b

Heat Flow (W/g)

200°C 30min

200°C 15min

200°C 5min

untreated

40 Exo Up

60

80

Temperature (°C)

100

120

Universal V4.3A TA Instruments

Fig. 3. Exemplary DSC thermograms of the thermally treated and untreated LPI (30% solution), at a scanning rate of 2 °C/min at pH 7.0: (a) mild treatment at 100 °C for 5, 15, and 30 min; (b) harsh treatment at 200 °C for 5, 15, and 30 min.

times (5 min, 100 °C); whereas only the legumin peak could be detected at medium heating conditions (30 min at 100 °C and 5 min at 200 °C). Harsher thermal conditions (15–30 min at 200 °C) caused a decrease in native protein, since no peaks could be observed. These endothermic transitions were caused by the denaturation of vicilin at lower temperatures (71.49 °C) and legumin at higher temperatures (87.00 °C). According to the denaturation behaviour of other legume proteins, such as pea protein or soy protein, the vicilin fraction showed lower heat stability than the legumin fraction, which denatured at higher temperatures. The transition of any protein from native to denatured conformation depends on its 3D structure and is accompanied by the cleavage of inter- and intra-molecular bonds. The vicilin tertiary structure is mainly bound by weak non-covalent bonds as hydrogen bonds, hydrophobic interactions and electrostatic forces. These forces are more heat labile than the covalent bonds, i.e., the disulphide bridges, which form the tertiary protein structure of the legumin, and show higher stability at low and medium thermal treatments, because they require more energy to break. Furthermore, the LPI slurries heated for a second time did not show any peaks, meaning that the observed transitions for the vicilin denaturation were irreversible. This is not a surprise, because at high protein concentrations (20–30%) denaturation becomes an irreversible process, since extensive intermolecular interactions are favoured with aggregation of the unfolded protein (Biliaderis, 1983). In this case, the protein aggregation is considered as an exothermic process and it is the product of a positive (denaturation) and a negative (aggregation) contributor. Analysis of the DSC thermogram also enabled the determination of the denaturation enthalpy (DH) of the protein samples (Table 2). The DH value, calculated from the area under the transition peak, is correlated with the content of secondary structure of a protein (Koshiyama, Hamano, & Fukushima, 1981). It is a net value from a combination of endothermic reactions, such as the disruption of hydrogen bonds, and exothermic processes, including protein aggregation and the break-up of hydrophobic interactions (Arnt-

501

E. Sirtori et al. / Food Chemistry 120 (2010) 496–504 Table 2 Denaturation enthalpy (DH) and onset point of protein denaturation (Td) of treated LPI. Td1 and DH1 refer to vicilin peak; Td2 and DH2 to legumin peak. Untreated sample was used as reference. Onset point protein denaturation peak (°C) Td1 Untreated 71.49 Thermal treatments 100 °C, 5 min 72.72 100 °C, 15 min No peak 100 °C, 30 min No peak 150 °C, 5 min No peak 150 °C, 15 min No peak 150 °C, 30 min No peak 200 °C, 5 min No peak 200 °C, 15 min No peak 200 °C, 30 min No peak Ultraturrax 8000 rpm 13,500 rpm 24,000 rpm

72.04 73.41 72.77

High-pressure homogeniser 100 bar 71.79 300 bar 71.88 1000 bar 78.59

24000rpm

Heat Flow (W/g)

Sample

Denaturation enthalpy (J/g)

Td2

D H1

D H2

90.92

0.6987

0.8465

91.08 91.89 91.94 89.36 89.51 91.90 87.00 91.48 no peak

0.7218 No peak No peak No peak No peak No peak No peak No peak No peak

0.6909 0.3250 0.2269 0.6573 0.2037 0.1832 0.7386 0.0638 no peak

91.03 91.44 91.24

0.9327 0.8667 0.7573

0.9183 0.7633 0.7207

88.20 90.21 88.27

0.9617 0.8849 0.7895

0.8166 0.9481 0.9190

field & Murray, 1981). The DSC results showed a decreasing denaturation enthalpy of the treated LPIs with increased intensity of the thermal treatment. In particular after a 5 min treatment, the DH value was very high in comparison with longer treatments (15

13500rpm

8000rpm

untreated 60

80

Exo Up

100

Temperature (°C)

120 Universal V4.3A TA Instruments

Fig. 5. Exemplary DSC graphs of the mechanically treated samples (10% solution), at a scanning rate of 2 °C/min at pH 7.0, with the untreated LPI used as reference. The samples were treated at 8000, 13,500, and 24,000 rpm with Ultraturrax. Denaturation enthalpy and onset point data of protein denaturation are shown in Table 2.

and 30 min), which were more similar. The DH decreasing trend is in agreement with the lower content of native protein observed. The extent of protein degradation induced by processing was also determined by 2D gels (Fig. 4). Attention was focused on the relative stability of the major storage proteins. Long times and high temperatures denatured the majority of the proteins. Nevertheless, both the basic and acidic subunits of the legumin (11S protein) and d-conglutin (2S globulin) were still clearly visible on the 2D gels even after a severe treatment (200 °C), although their intensities

Untreated

kDa 75 50 37 25 20 15 kDa 75 50 37

100°C, 5 min

150°C, 5 min

200°C, 5 min

100°C, 15 min

150°C, 15 min

200°C, 15 min

100°C, 30 min

150°C, 30 min

200°C, 30 min

25 20 15 kDa 75 50 37 25 20 15 kDa 75 50 37 25 20 15 3

pI

10

3

pI

10

3

pI

10

Fig. 4. 2D electrophoretic analysis of thermally treated LPI at different times of exposure. Isoelectric focusing was performed on 7 cm, pH 3–10 non-linear IPG strips (Bio-Rad), and the second dimension was done on 13% SDS–PAGE gel. Standard markers are indicated (in kDa) on the left.

502 Table 3 Peptide sequences obtained after tryptic digestion of the samples treated at 100 and 200 °C, for 5 and 30 min. Untreated LPI (UN) was used as reference. The protein identification with the corresponding NCBInr, the MH+ value, the pI and the position of the peptides are reported. Peptide

Protein

pI

MH+

Start aa position

UN

100 °C 5 min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52

(R)IVEFQSNPNTLILPK (K)HSDADYILVVLNGR (R)ATITIVNPDKR (R)LPAGTTSYILNPDDNQNLR (K)DQQSYFSGFSK (R)NTLEATFNTR (R)LLGFGINANENQR (R)NFLAGSEDNVISQLDR (K)ELTFPGSAQDVER (K)NQQQSYFANAQPQQKQQR (R)IIEFQSKPNTLILPK (R)ATITIVNPDKR (R)QVYNLEQGDALR (R)LPAGTTSYILNPDDNQNLR (K)LYDFYPSTTK (K)DQQSYFSGFSK (R)NTLEATFNTR (K)HAQSSSGEGKPSESGPFNLR (R)SNKPIYSNK (R)TNRLENLQNYR (R)IIEFQSKPNTLILPK (R)ATITIVNPDKR (R)QVYNLEQGDALR (R)LPAGTTSYILNPDDNQNLR (K)LYDFYPSTTK (K)DQQSYFSGFSK (R)NTLEATFNTR (K)HAQSSSGEGKPSESGPFNLR (R)SNKPIYSNK (R)LLGFGINANENQR (R)NFLAGGSEDNVIK (K)ELTFPGSIEDVER (K)NQQQSYFANAQPQQQQQR (R)ALQQIYESQSEQCEGR (R)QQEQQLEGELEKLPR (R)QQEQQLEGELEK (R)ICGFGPLR (R)QLQQVNLR (R)HNIGQSTSPDAYNPQAGR (K)TLTSLDFPILR (R)NTLEATFNTR (R)LLGFGINADENQR (R)NFLAGSKDNVIR (R)NTLEATFNTR (R)NFLAGSEDNVIR (R)SSQESEELDQCCEQLNELNSQR (R)RPFYTNAPQEIYIQQGR ADI/LYNPTAGR VQVVNSQGNSVFNDDI/LR GI/LPAEVI/LANAFR I/LSI/LNQVSEI/LK VEEGI/LGVI/LSPK

Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208401] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 149208403] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin beta [L. angustifolius, 169950562] Conglutin delta-2 large chain [LUPAN, 116181] Conglutin delta-2 large chain [LUPAN, 116181] Conglutin delta-2 large chain [LUPAN, 116181] Conglutin delta-2 large chain [LUPAN, 116181] Delta small chain [L. angustifolius, 116182] Conglutin alpha [L. angustifolius, 2313076] Conglutin alpha [L. angustifolius, 2313076] Beta conglutin precursor [L. albus, 46451223] Beta conglutin precursor [L. albus, 46451223] Beta conglutin precursor [L. albus, 46451223] Vicilin-like protein [L. albus, 89994190] Vicilin-like protein [L. albus, 89994190] Conglutin delta [L. albus, 80221495] Legumin-like protein [L. albus, 85361412] Legumin [L. angustifolius, de novo peptide] Legumin [L. angustifolius, de novo peptide] Legumin [L. angustifolius, de novo peptide] Legumin [L. angustifolius, de novo peptide] Legumin [L. angustifolius, de novo peptide]

6.00 5.21 8.79 4.21 5.83 6.00 6.00 4.03 4.14 9.99 8.59 8.79 4.37 4.21 5.83 5.83 6.00 6.76 9.70 8.41 8.59 8.79 4.37 4.21 5.83 5.83 6.00 6.76 9.70 6.00 4.37 4.00 8.75 4.25 4.49 4.09 8.25 9.75 6.74 5.50 6.00 4.37 8.75 6.00 4.37 3.83 8.59 5.88 4.21 6.00 6.00 4.53

1712.958 1571.818 1227.706 2102.51 1293.575 1166.580 1445.750 1777.872 1448.702 2192.059 1741.026 1227.706 1405.707 2102.51 1234.599 1293.575 1166.580 2071.979 1050.558 1420.729 1741.026 1227.706 1405.707 2102.51 1234.599 1293.575 1166.580 2071.979 1050.558 1445.750 1306.664 1491.733 2192.023 1925.866 1824.945 1458.707 919.482 998.574 1912.890 1275.731 1166.580 1446.734 1333.722 1166.580 1334.670 2683.121 2081.056 1077.532 1890.930 1257.695 1130.642 1127.631

139 154 167 191 233 259 436 449 468 484 225 254 265 277 309 319 330 387 407 214 225 254 265 277 309 319 330 387 407 526 539 558 574 33 49 49 64 11 67 87 259 451 464 259 464 75 86 – – – –

                                            

 

     

100 °C 30 min

       

      

    

 

  



    

 

200 °C 5 min

200 °C 30 min







        

       

      

      

     

    



 

 







  



E. Sirtori et al. / Food Chemistry 120 (2010) 496–504

No.

E. Sirtori et al. / Food Chemistry 120 (2010) 496–504

were reduced after 15 and 30 min heating. On the contrary, the vicilins (7S protein) appeared to be more heat labile, since after only a short heat treatment (15 min at 100 °C), the corresponding spots were faint and no longer visible at successive times and temperatures. These results were in agreement with those of the DSC analysis. 3.3. Effect of mechanical stress on LPI The mechanical stress was applied using an Ultraturrax (8000, 13,500, and 24,000 rpm) and a homogeniser (100, 300 and 1000 bar), in order to simulate different mixing and extrusion devices. The DSC results (Fig. 5) did not show any relevant difference in the curves of the analysed samples. In all samples treated in the Ultraturrax the two peaks for the vicilins and legumins could be observed, and there was no significant reduction of the denaturation peak area or of the onset temperature of the protein denaturation (Table 2). Thus, this mechanical treatment did not affect the native structures of the protein fractions. On the contrary, in the samples treated with the homogeniser at moderate pressure, a slight increase of the DH2 value could be observed. This may be related to an increased protein solubility, which led to higher endothermic transitions in the DSC. These results are in agreement with the protein profiles shown by the gels: neither the Ultraturrax nor the homogeniser produced any obvious differences in the protein profile vs. untreated sample and the spot intensities remained constant (data not shown). High-pressure treatments induce protein modifications, due to disruption of hydrophobic and electrostatic interactions. The exact modifications depend on the pressure applied, duration of treatment and temperature of pressurisation. According to literature data (Chapleau & de Lamballerie-Anton, 2003), lupin proteins are sensitive to pressure ranging from 200 to 600 MPa: the legumins tend to form pressure-induced aggregates and the vicilins are denatured. The samples analysed in this work, instead, were treated less severely (max 1000 bar = 100 MPa). Another investigation on lupin proteins (Masson, 1992) has shown that pressures higher than 200 MPa modify the electrostatic and hydrophobic interactions, which lead to structure modification as well as protein aggregation induced by tertiary and secondary structure changes. Since high-pressure processing cleaves noncovalent interactions within proteins, new protein complexes may form. Application of high pressure on globular proteins, such as lupin proteins, has also been shown to involve a decrease of volume linked with the compression of the internal cavities (Masson, 1992). 3.4. Peptide fingerprint of the treated samples In order to identify the peptides/proteins resistant and still potentially releasable by digestion after processing, the untreated and treated samples were digested with trypsin and analysed via mass spectrometry. The peptide sequences identified in the treated LPI are listed in Table 3 (only some exemplary thermal treatments are reported). The majority of the peptides belonging to d-conglutin and vicilin proteins (b-conglutin) appeared to be resistant to 100 °C treatments, both at short and long time exposures together with eight peptides of the legumin family (a-conglutin and the ‘‘de novo sequenced” peptides). After harsh thermal treatments 10 peptides (nos. 6, 17, 27, 31, 32, 34, 41, 44, 46 and 47) remained intact, 4 out of which were resistant even after 30 min at 200 °C. Peptide 46 was not detected in the untreated sample, possibly because the trypsin cleavage site of this peptide is hidden by protein folding and it becomes accessible to the enzyme only after heat induced protein denaturation.

503

These data indicate that, even after drastic industrial processing conditions, the vicilin proteins are still able to release intact peptides after incubation with proteases, although their 2D protein profiles and DSC behaviour are completely modified. Eventually, it may be interesting to note that most of the resistant peptides showed a high homology sequence with some allergenic peptides reported in literature. Several peptides from 48, 43 and 41 kDa SDS–PAGE bands were identified in literature (Lewis, Grimshaw, Warner, & Hourihane, 2005) as subunits of Ara h 1 and Arah 3/Arah 4, the major peanut allergens. When these peptides were aligned with the lupin peptides, high sequence identities were found. In particular, the peptides 5, 6, 16, 17, 26, 27, 41, 44 and 47 are homologous to DQSSYLQGFSR (allergen Ara h 1, NCBInr 1168391, 70% identity), NTLEAAFNAEFNEIR (allergen Ara h 1, NCBInr 1168391, 87% identity), and RPFYSNAPQEIFIQQGR (allergen Arah3/Arah4, NCBInr 21314465, 88% identity). In general, the resistance of the proteins to digestion and processing is thought to be an important characteristic of food allergens (Moreno, 2007).

4. Conclusions Industrial protein processing covers a wide range of applications, all characterised by the need to ensure the best protein viability. It is thus necessary to optimise the technological conditions, in order to achieve the desired sensory characteristics and safety and, in the meantime, induce the smallest denaturation or insolubilisation, phenomena that could have deleterious effects on the nutritive value and nutraceutical properties of proteins. This is particularly true in case of functional foods, whose activity depends on the actual presence of specific proteins/peptides in the final products. Only a few papers have dealt with this issue. A study (AlvarezAlvarez et al., 2005), in which whole lupin seeds were boiled, microwave heated and autoclaved, has shown that the processing procedures produce minimum changes on the SDS–PAGE protein profile; only autoclaving at 138 °C produced an important effect on the integrity and structure of lupin proteins. Our investigation, by combining the proteomic techniques and DSC analysis, allowed us to highlight the modification of the protein profile of a lupin protein isolate submitted to different treatments selected to mimic industrial processing conditions. We have demonstrated that selected treatments (mild temperatures and/or short times) modify the protein profile only marginally. These conditions should be applied in food production. Another very important result of our investigation, especially considering the potential applications to functional foods, was the demonstration that even in the most drastic conditions some relevant peptides are still released intact after hydrolysis with trypsin. In particular, several peptides belonging to b- and d-conglutin sequences are resistant to processing and are potentially still releasable by digestion. b-Conglutin especially is a good candidate for being the bioactive lupin component, since this protein has high identity (from 52% to 57%) and similarity (from 73% to 79%) with the a0 subunit of b-conglycinin (NCBInr 9967361), the soybean vicilin which, in agreement with available literature (Duranti et al., 2004), seems to be one of the main hypocholesterolaemic components of this seed. A recent research has identified some soybean peptides able to stimulate LDL-receptor transcription: the most active derive from the a or a0 -chain of b-conglycinin (Cho, Juillerat, & Lee, 2008); these peptides are highly homologous to some stable lupin peptides. Other resistant peptides are derived from lupin d-conglutin, a 2S sulphur-rich globulin. Interestingly, the heavy chain of this protein shows high sequence homology with lunasin (NCBInr

504

E. Sirtori et al. / Food Chemistry 120 (2010) 496–504

31693820), a cancer-preventive peptide recently isolated from the 2S protein fraction of soybean. Lunasin is a heat-stable peptide, which retains its bioactivity even after boiling (de Lumen, 2005). The fact that several lupin intact peptides deriving from these proteins may still be easily released by digestion even after processing is certainly a relevant issue for the quality of the functional foods based on lupin proteins. Acknowledgements We are grateful to Dr. P. Traldi (CNR, Istituto di Scienze e Tecnologie Molecolari, Padova, Italy) for the HPLC-Chip-qTOF analysis and to Dr. M. Zanotti (Agilent Technologies, Cernusco SN, Italy) for his precious help in the ‘‘de novo sequencing” analysis. This work was supported by Fondazione Cariplo, Project ‘‘Novel Methodologies for the quality control of the production of lupin and lupin based food products” (2005-1803), by the European Commission, Project ‘‘Bioprofibre” (COOP-CT-2006-032075) and by MIUR ‘‘Metodologie innovative per la quantificazione dei principali allergeni in alimenti complessi” (PRIN 2006-075417). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2009.10.043. References Alvarez-Alvarez, J., Guillamón, E., Crespo, J., Cuadrado, C., Burbano, C., Rodríguez, J., et al. (2005). Effects of extrusion, boiling, autoclaving, and microwave heating on lupine allergenicity. Journal of Agricultural and Food Chemistry, 53(4), 1294–1298. Arntfield, S. D., & Murray, E. D. (1981). The influence of processing parameters of food protein functionality I. Differential scanning calorimetry as an indicator of protein denaturation. Canadian Institute of Food Science and Technology Journal, 14. Bettzieche, A., Brandsch, C., Weiße, K., Hirche, F., Eder, K., & Stangl, G. I. (2008). Lupin protein influences the expression of hepatic genes involved in fatty acid synthesis and triacylglycerol hydrolysis of adult rats. The British Journal of Nutrition, 99(5), 952–962. Biliaderis, C. G. (1983). Differential scanning calorimetry in food research. A review. Food Chemistry, 10, 239–265. Brambilla, F., Resta, D., Isak, I., Zanotti, M., & Arnoldi, A. (2009). A label-free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC-Chip coupled with ion trap mass spectrometry. Proteomics, 9(2), 272–286. Carbonaro, M., Grant, G., & Cappelloni, M. (2005). Heat-induced denaturation impairs digestibility of legume (Phaseolus vulgaris L. and Vicia faba L.) 7S and 11S globulins in the small intestine of rat. Journal of the Science of Food and Agriculture, 85, 65–72. Chapleau, N., & de Lamballerie-Anton, M. (2003). Improvement of emulsifying properties of lupin proteins by high pressure induced aggregation. Food Hydrocolloids, 17, 273–280. Cho, S., Juillerat, M., & Lee, C. (2008). Identification of LDL-receptor transcription stimulating peptides from soybean hydrolysate in human hepatocytes. Journal of Agricultural and Food Chemistry, 56(12), 4372–4376. D’Agostina, A., Antonioni, C., Resta, D., Arnoldi, A., Bez, J., Knauf, U., et al. (2006). Optimization of a pilot-scale process for producing lupin protein isolates with valuable technological properties and minimum thermal damage. Journal of Agricultural and Food Chemistry, 54(1), 92–98. de Lumen, B. (2005). Lunasin: A cancer-preventive soy peptide. Nutrition Reviews, 63(1), 16–21. Dijkstra, D., Linnemann, A., & van Boekel, T. (2003). Towards sustainable production of protein-rich foods: Appraisal of eight crops for Western Europe. PART II: Analysis of the technological aspects of the production chain. Critical Reviews in Food Science and Nutrition, 43(5), 481–506. Duranti, M., Lovati, M. R., Dani, V., Barbiroli, A., Scarafoni, A., Castiglioni, S., et al. (2004). The alpha’ subunit from soybean 7S globulin lowers plasma lipids and upregulates liver beta-VLDL receptors in rats fed a hypercholesterolemic diet. Journal of Nutrition, 134(6), 1334–1339.

FDA (1999). Food labeling health claims: Soybean protein and coronary heart disease. Final rule. Federal Register, 64, 57699–57733. Francki, M., Whitaker, P., Smith, P., & Atkins, C. (2002). Differential expression of a novel gene during seed triacylglycerol accumulation in lupin species (Lupinus angustifolius L. and L. mutabilis L.). Functional Integrative Genomics, 2(6), 292–300. Frank, A., Savitski, M., Nielsen, M., Zubarev, R., & Pevzner, P. (2007). De novo peptide sequencing and identification with precision mass spectrometry. Journal of Proteome Research, 6(1), 114–123. Hall, R. S., Thomas, S. J., & Johnson, S. K. (2005). Australian sweet lupin flour addition reduces the glycaemic index of a white bread breakfast without affecting palatability in healthy human volunteers. Asia Pacific Journal of Clinical Nutrition, 14(1), 91–97. Hancock, J., Peo, E. J., Lewis, A., & Crenshaw, J. (1990). Effects of ethanol extraction and duration of heat treatment of soybean flakes on the utilization of soybean protein by growing rats and pigs. Journal of Animal Science, 68(10), 3233–3243. Holley, W., Müller, K., Kamal, H., Wäsche, A., Borcherding, A., & Luck, T. (2000). Method for treating and processing lupin seeds containing alkaloid, oil and protein. Patent WO 0054608. Koshiyama, I., Hamano, M., & Fukushima, D. (1981). A heat denaturation study of the 11S globulin in soybean seeds. Food Chemistry, 6, 309–322. Lee, Y., Mori, T., Puddey, I., Sipsas, S., Ackland, T., Beilin, L., et al. (2009). Effects of lupin kernel flour-enriched bread on blood pressure: A controlled intervention study. The American Journal of Clinical Nutrition, 89(3), 766–772. Lee, Y. P., Mori, T. A., Sipsas, S., Barden, A., Puddey, I. B., Burke, V., et al. (2006). Lupin-enriched bread increases satiety and reduces energy intake acutely. The American Journal of Clinical Nutrition, 84(5), 975–980. Lewis, S., Grimshaw, K., Warner, J., & Hourihane, J. (2005). The promiscuity of immunoglobulin E binding to peanut allergens, as determined by Western blotting, correlates with the severity of clinical symptoms. Clinical and Experimental Allergy, 35(6), 767–773. Marchesi, M., Parolini, C., Diani, E., Rigamonti, E., Cornelli, L., Arnoldi, A., et al. (2008). Hypolipidaemic and anti-atherosclerotic effects of lupin proteins in a rabbit model. The British Journal of Nutrition, 4, 1–4. Masson, P. (1992). Pressure denaturation of proteins. In C. Balny, R. Hayashi, K. Heremans, & P. Masson (Eds.), High pressure and biotechnology (Vol. 224). Colloque INSERM/John Libbey Eurotext Ltd. Moreno, F. (2007). Gastrointestinal digestion of food allergens: Effect on their allergenicity. Biomedicine and Pharmacotherapy, 61(1), 50–60. Muzquiz, M., Pedrosa, M. M., Cuadrado, C., Ayet, G., Burbano, C., & Brenes, A. (1998). Variation of alkaloids, alkaloids esters, phytic acid and phytase activity in germinated seed of Lupinus albus and L. luteus. Wageningen (Netherlands): Wageningen Pers. Pilvi, T. K., Jauhiainen, T., Cheng, Z. J., Mervaala, E. M., Vapaatalo, H., & Korpela, R. (2006). Lupin protein attenuates the development of hypertension and normalises the vascular function of NaCl-loaded Goto-Kakizaki rats. Journal of Physiology and Pharmacology, 57(2), 167–176. Porres, J., Aranda, P., López-Jurado, M., & Urbano, G. (2005). Nutritional potential of raw and free alpha-galactosides lupin (Lupinus albus Var. multolupa) seed flours. Effect of phytase treatment on nitrogen and mineral dialyzability. Journal of Agricultural and Food Chemistry, 53(8), 3088–3094. Porres, J. M., Aranda, P., López-Jurado, M., & Urbano, G. (2006). Nutritional evaluation of protein, phosphorus, calcium and magnesium bioavailability from lupin (Lupinus albus var. multolupa)-based diets in growing rats: Effect of alpha-galactoside oligosaccharide extraction and phytase supplementation. The British Journal of Nutrition, 95(6), 1102–1111. Resta, D., Boschin, G., D’Agostina, A., & Arnoldi, A. (2008). Evaluation of total quinolizidine alkaloids content in lupin flours, lupin-based ingredients, and foods. Molecular Nutrition and Food Research, 52(4), 490–495. Shutov, A., Kakhovskaya, I., Braun, H., Bäumlein, H., & Müntz, K. (1995). Leguminlike and vicilin-like seed storage proteins: Evidence for a common singledomain ancestral gene. Journal of Molecular Evolution, 41(6), 1057–1069. Sirtori, C. R., Eberini, I., & Arnoldi, A. (2007). Hypocholesterolaemic effects of soya proteins: Results of recent studies are predictable from the Anderson metaanalysis data. British Journal of Nutrition, 97(5), 816–822. Sirtori, C. R., Lovati, M. R., Manzoni, C., Castiglioni, S., Duranti, M., Magni, C., et al. (2004). Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells. Journal of Nutrition, 134(1), 18–23. Sousa, I. M. N., Mitchell, J. R., Ledward, D. A., & Hill, S. (1995). Differential scanning calorimetry of lupin and soy proteins. Lebensmittel Untersuchung und Forschung, 201, 566. Spielmann, J., Shukla, A., Brandsch, C., Hirche, F., Stangl, G. I., & Eder, K. (2007). Dietary lupin protein lowers triglyceride concentrations in liver and plasma in rats by reducing hepatic gene expression of sterol regulatory element-binding protein-1c. Annals of Nutrition and Metabolism, 51(4), 387–392. Zheng, G. H., Fasina, O., Sosulski, F. W., & Tyler, R. T. (1998). Nitrogen solubility of cereals and legumes subjected to micronization. Journal of Agricultural and Food Chemistry, 46, 4150–4157.