Preparation, composition and functional properties of pennycress (Thlaspi arvense L.) seed protein isolates

Industrial Crops and Products 55 (2014) 173–179

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Preparation, composition and functional properties of pennycress (Thlaspi arvense L.) seed protein isolates夽 Mila P. Hojilla-Evangelista a,∗ , Gordon W. Selling a , Mark A. Berhow b , Roque L. Evangelista c a Plant Polymer Research Unit, National Center for Agricultural Utilization Research (NCAUR), USDA Agricultural Research Service (ARS), 1815 N. University St., Peoria, IL 61604, United States b Functional Foods Research Unit, NCAUR, USDA ARS, 1815 N. University St., Peoria, IL 61604, United States c Bio-Oils Research Unit, NCAUR, USDA ARS, 1815 N. University St., Peoria, IL 61604, United States

a r t i c l e

i n f o

Article history: Received 30 September 2013 Received in revised form 12 February 2014 Accepted 15 February 2014 Available online 12 March 2014 Keywords: Pennycress Pennycress protein isolates Protein extraction Protein functionality

a b s t r a c t This study evaluated two methods, saline extraction (SE) and conventional acid precipitation (AP), to recover proteins from pennycress (Thlaspi arvense L.) seed meal. SE was done using 0.1 M NaCl at 50 ◦ C while AP involved alkaline extraction (pH 10) first followed by protein precipitation at pH 4. Composition, amino acid profiles, and functional properties (solubility, foaming, emulsification, water-holding capacity, heat coagulability) of the resultant protein extracts were compared. SE and AP produced pennycress protein extracts that were sinigrin-free and containing at least 90% (db) crude protein, which classifies the extracts as protein isolates (PI). Extraction method had major influence on the amino acid profiles and functional properties of the protein isolates. Pennycress SEPI was markedly more soluble (68–91% solubility at pH 2 and ≥7) and had excellent emulsifying properties that were clearly superior to those of APPI. On the other hand, APPI had better foaming properties and was more stable to heating than SEPI. These results strongly demonstrate that high-purity pennycress seed protein isolates can be produced by either saline extraction or acid precipitation and have functional properties that are desirable for non-food uses. Published by Elsevier B.V.

1. Introduction Pennycress (Thlaspi arvense L.; Brassicaceae) is a common agricultural weed, classified as an annual or winter annual crop that grows extensively in temperate North America. Oil in pennycress seed (36%; Isbell, 2009) is considered a highly promising alternative feedstock for biofuel production, hence, the current interest in commercial development of the crop. Additionally, pennycress has several agronomic traits that are advantageous to a feedstock, such as high seed yield, high oil content in seeds, and early harvest date that allows for two-crop rotation with soybeans grown in upper midwestern region of the United States (Isbell, 2009; Evangelista et al., 2012).

夽 Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable. USDA is an equal opportunity provider and employer. ∗ Corresponding author. Tel.: +1 309 681 6350; fax: +1 309 681 6691. E-mail address: [email protected] (M.P. Hojilla-Evangelista). http://dx.doi.org/10.1016/j.indcrop.2014.02.016 0926-6690/Published by Elsevier B.V.

Evangelista et al. (2012) established the processing parameters for oil pressing of pennycress seed, while Moser et al. (2009) evaluated and confirmed the suitability of pennycress seed oil for biodiesel production. As with corn and soybean processing, the profitability of pennycress based-biodiesel industry would be enhanced by utilizing its co-products. The seed meal has already shown potential as a biofumigant for horticultural crops (Vaughn et al., 2005) and pyrolysis-induced liquid jet fuel intermediates (Boateng et al., 2010). Protein in pennycress seed meal is substantial (20–27%; Selling et al., 2013; Hojilla-Evangelista et al., 2013) and could be developed as a major co-product of oil processing. Recently, our research group was first to report that pennycress seed proteins were comprised primarily of albumins and globulins (42% of total protein) and had eight major polypeptides within M.W. 5–41 kDa. Amino acid contents were not negatively affected by oil processing conditions and, based on amino acid scoring patterns, the nutritional quality of pennycress seed and press cake proteins was superior to those of meals from soybean and canola. Pennycress crude protein extract had poor solubility (35–45% soluble protein at pH greater than 4), but showed excellent foaming capacity (120 mL foam), foam stability (93–97% remaining

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foam), and emulsification ability (EAI > 150 m2 /g protein) (HojillaEvangelista et al., 2013; Selling et al., 2013). The identity and proportion of soluble protein classes, along with protein solubility profile, are essential to defining the conditions for optimal extraction and recovery of proteins. For example, several studies on canola and rapeseed (which also belong to the Brassicaceae family like pennycress) isolated the protein by using the typical technique of alkali solubilization followed by acid precipitation at isoelectric pH (Xu and Diosady, 1994; Klockeman et al., 1997; Aluko and McIntosh, 2001). The recovered protein product contained 76–80% crude protein (Aluko and McIntosh, 2001), which increased greatly (88–100% crude protein) when ultrafiltration was included in the procedure (Rubin et al., 1990; Xu and Diosady, 1994). Ultrafiltration was also found to nearly eliminate glucosinolates and phytates in canola and rapeseed protein isolates (Rubin et al., 1990). The steps and conditions evaluated by these extraction studies would likely be applicable to pennycress protein. Continuing the pennycress protein research, the present work evaluated a saline-based extraction (SE) method to recover higherpurity protein product from pennycress seed meal and compared the yield and product quality with that obtained by conventional acid precipitation (AP). This report also presents the composition, amino acid profiles, SDS-PAGE data, and functional properties (solubility, foaming, emulsification, water-holding capacity, heat coagulability) of the protein extracts obtained by the two methods. 2. Materials and methods 2.1. Seed preparation before protein extraction Field pennycress (T. arvense L.) seeds used in this research were harvested in 2010 from a single location near Peoria, Illinois. After cleaning by screening and aspiration, seeds were dried to ca. 10% moisture content by using a fan-equipped box dryer. Pennycress seed meal was prepared and defatted by following exactly the procedure described by Hojilla-Evangelista et al. (2013). Seeds were cracked (Allis Chalmers roller mill, Milwaukee, WI), ground into 0.35 mm particle size, and then hexane-defatted at 25 ◦ C (HojillaEvangelista and Evangelista, 2006) until oil content was less than 10%. The meal was air-dried in a fume hood and ground again to a final particle size of 0.25 mm (60-mesh) by using a pin mill (Alpine Model 160z, Augsburg, Germany). The 0.25 mm pennycress meal sample was then defatted a second time with hexane at ambient conditions to attain residual oil content of <0.5% (db), a level that we had established previously as having no significant influence on results of functionality tests (Hojilla-Evangelista and Evangelista, 2006). Pennycress meal samples were stored in screwcapped polyethylene containers at room temperature (21–24 ◦ C) until use.

second cycle of mixing with pre-heated 0.1 M NaCl (10 mL:1 g starting sample) and stirring following conditions described previously. The spent solids were collected in a tared glass dish, frozen overnight, freeze-dried in a tray drier and then stored for further analyses. Freeze-drying prevented mold growth that would have occurred during air-drying in a fume hood. Protein extraction efficiencies were calculated based on the protein contents of the starting meal and spent solids. The combined extracts were centrifuged at 25 ◦ C and 10,000 × g for 20 min. Solids were discarded, while the supernatant then underwent two cycles of vacuumfiltration, standing overnight in the refrigerator, and decanting. The filtered extract was placed into SpectraPor molecular porous membrane tubings (MWCO 3500 Da) and dialyzed against deionized water at 4 ◦ C for 3–4 days with twice-a-day water changes. The dialyzed extracts were freeze-dried and then stored for compositional and protein functionality testing. Extractions were done in duplicate. 2.2.2. Acid precipitation The conventional procedure for extracting proteins was based on the commercial method of producing soybean protein isolates (Wolf, 1983; Lusas and Rhee, 1997). One-hundred grams of defatted pennycress seed meal was mixed with 1.0 L water pre-heated to 50 ◦ C and then its pH was adjusted to a final value of 10.0 by addition of NaOH (1.00–1.25 g). The container with sample mixture was placed in a water-bath maintained at 50 ◦ C. The overhead stirrer (Caframo Model BDC 1850, Caframo Ltd., Wiarton, Ontario, Canada) mixed the hydrated sample at 250 rpm for 90 min. The mixture was then centrifuged at 10,000 × g and 25 ◦ C for 20 min. Solids were collected in a tared glass dish, frozen overnight, freezedried, and stored for further analyses as described in the preceding section. The supernatant was adjusted to pH 4.5 by addition of 1 M HCl, after which it was centrifuged at 10,000 × g and 25 ◦ C for 20 min. Solubles were discarded. The precipitated protein was redissolved by addition of 500–600 mL water and neutralization with 2 M NaOH. The mixture was then centrifuged at 25 ◦ C and 10,000 × g for 20 min. Any solids were discarded, while the supernatant was dialyzed in the same manner as with the saline-extracted protein. The retentate was then freeze-dried to recover the protein product. Extractions were performed in duplicate. 2.3. Composition and amino acids Moisture, crude oil, and crude protein (Dumas %N × 6.25) contents and amino acid profiles of the seed meal and recovered protein extracts were determined by following exactly the standard methods of AOCS (2009) and AOAC (2003) that were described in our earlier work on pennycress seed and press cake crude proteins (Hojilla-Evangelista et al., 2013). 2.4. Determination of sinigrin content

2.2. Production of protein isolates 2.2.1. Saline extraction Based on our previous finding that the dominant soluble protein classes in pennycress seed were albumins and globulins (HojillaEvangelista et al., 2013), we adapted the saline extraction method that we used for corn germ protein (Hojilla-Evangelista, 2012), with minor modifications. One-hundred grams of ground defatted pennycress seed meal were mixed with 1.0 L of pre-heated (50 ◦ C) 0.1 M NaCl in a stainless steel beaker. The mixture was placed in a water-bath maintained at 50 ◦ C and stirred for 2 h at 250 rpm using an overhead motorized stirrer (Caframo Real Torque Digital Stirrer, Model BDC 1850, Caframo Ltd., Wiarton, Ontario, Canada). The mixture was filtered through layers of cheese cloth and the extract was set aside. The solids were subjected to a

Pennycress seed meal and freeze-dried protein extracts were weighed, placed in filter paper packets and defatted overnight in a Soxhlet extractor with hexane. For HPLC analysis, typically about 0.25 g of defatted meal was placed in a capped vial with 2 mL of methanol. The vials were sonicated for 15 min in a sonicating water bath then allowed to stand overnight. After a brief sonication, a portion of this extract was filtered through a 0.45 ␮m filter into an auto sampler vial. For glucosinolate quantitation, a modification of HPLC method developed by Betz and Fox (1994) was used. The extract was run on a Shimadzu (Columbia, MD) HPLC System (two LC 20AD pumps, SIL 20A autoinjector, DGU 20As degasser, SPD-20A UVVIS detector, and a CBM-20A communication BUS module) running under the Shimadzu LC Solutions software. The column was a C18

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Inertsil reverse phase column (250 mm × 4.6 mm; RP C-18, ODS-3, 5 ␮m; Varian, Torrance, CA). The glucosinolates were detected by monitoring at 237 nm. The initial mobile phase conditions were 12% methanol/88% aqueous 0.005 M tetrabutylammonium bisulfate (TBS) at a flow rate of 1 mL/min. After injection of 15 ␮L of sample, the binary gradient was held at the initial conditions for 2 min, developed to 34% methanol/66% aqueous 0.005 M THS over and additional 20 min, developed to 50% methanol/50% aqueous 0.005 M THS over another 18 min, then finally developed to 100% methanol over another 10 min. Concentrations of glucosinolates in the samples were calculated from a standard curve of freshly prepared pure sinigrin (Sigma) on a nmoles injected basis. The relative concentrations were calculated from the sinigrin standard curve on a nmolar basis and converted to mg/g dry weight. The concentrations of the glucosinolates were then back calculated to determine the concentration in the whole fat seeds. 2.5. Infrared analysis of protein FTIR spectra were obtained by using a Thermo Nicolet Avatar 370 FTIR spectrometer (West Palm Beach, FL). The infrared (IR) spectra were analyzed with the Omnic software package provided by Thermo. Two milligrams of sample and 250 mg KBr (Thermo Spectratech IR grade, Waltham, MA) were mixed in the wig-L-Bug for 30 s. The mixture was pressed into a pellet using a Carver Model 4350.L Press (Carver Inc., Wabash, IN). The KBr pellet was removed from the mold and placed in the FTIR spectrometer set in transition mode to obtain the IR spectra of the protein samples. 2.6. SDS-PAGE Electrophoresis of reduced proteins from defatted pennycress seed meal, freeze-dried protein extracts, and spent solids after protein extraction was done according to the procedure of Wu and Hojilla-Evangelista (2005), which we described in detail previously (Hojilla-Evangelista et al., 2013).

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at 500 nm were used to calculate emulsification activity index (EAI, in m2 /g), a measure of the area of oil–water interface stabilized by a unit weight of protein, and emulsion stability index (ESI, in min). 2.7.4. Water-holding capacity Water-holding capacity (WHC) of the samples was determined by following the method of Balmaceda et al. (1984) for partly soluble materials, with modifications. One-gram protein sample was measured into a pre-weighed, dry 50-mL polycarbonate centrifuge tube (Wi ). Distilled water (30 mL) was added, the tube was capped, and then placed on a platform shaker for 15 min to disperse the sample. After shaking, the sample pH was adjusted to 2.0, 7.0 or 10.0 by adding 1.0 M HCl or 1.0 M NaOH. The centrifuge tube containing the sample was heated for 30 min in a water-bath maintained at 60 ◦ C, then was cooled in tap water for 30 min. The sample was centrifuged at 18,000 × g and 25 ◦ C for 10 min. The supernatant was decanted carefully and the weight of the centrifuge tube + sample was determined (Wf ). The amount of water held per gram protein sample was calculated as: WHC = [Wf − Wi – [(100 − A)/100]]/1 g sample, where A = % solubility × % protein in dry sample. 2.7.5. Heat coagulability Heat coagulability of the protein sample was determined based on the method described by Myers et al. (1994). Sample solutions were prepared to contain 5% protein (dry basis) and their pH values adjusted to 2.0, 7.0 or 10.0 by adding 1 M HCl or 1 M NaOH. Solutions were centrifuged at 10,000 × g and 25 ◦ C for 30 min and protein content of the supernatant was then determined by the Biuret method. A 20-mL aliquot of the supernatant was pipetted into a centrifuge tube, which was then placed in a 90–100 ◦ C water-bath for 30 min, cooled to room temperature, and then centrifuged at 10,000 × g and 25 ◦ C for 15 min. Supernatant was poured through a Whatman No. 2 filter paper and the amount of protein in the filtrate was determined by the Biuret method. Heat coagulability was the percentage loss in protein solubility after heating. 2.8. Statistical analyses

2.7. Functionality tests 2.7.1. Protein solubility Protein solubility profiles were determined according to the method of Balmaceda et al. (1984). Starting samples contained 10 mg protein/mL. The sample mixtures had their pH adjusted to 2.0, 4.0, 5.5, 7.0, 8.5, or 10.0 by adding 0.1 M or 1 M HCl or NaOH. The mixture was then centrifuged at 10,000 × g and 25 ◦ C for 20 min. The supernatant was collected and the amount of soluble protein was determined by the Biuret method. The standard curve was generated by using bovine serum albumin. 2.7.2. Foaming properties Samples containing 10 mg protein/mL were prepared for the determination of foam capacity and stability according to the method described by Myers et al. (1994) Tests were done at pH 7 (0.01 M phosphate buffer) and the pH where protein solubility was greatest. Twenty milliliters of protein solution was carefully pipetted into a graduated column fitted with a coarse fritted disk at the bottom. Air was introduced into the column through the stem at a flow rate of 100 mL/min at 20 psi. Timing began at the first appearance of bubbles. The volume (mL) of foam generated in 1 min was foam capacity, while the % foam remaining after standing in the foaming column for 15 min indicated foam stability. 2.7.3. Emulsification properties Emulsifying properties were determined according to the method of Wu et al. (1998), which we described in detail previously (Hojilla-Evangelista et al., 2013). Absorbance readings taken

Statistical analyses were performed by using the SAS® Systems for Windows software (SAS Institute Inc., Cary, NC). Significant differences among the treatments (p < 0.05) were detected by using analysis of variance (ANOVA) and Duncan’s Multiple Range tests on duplicate or triplicate replications of data. 3. Results and discussion 3.1. Moisture, oil, protein and sinigrin contents Pennycress seeds have been reported to contain 28–33% (db) oil, and 27% (db, fat-free basis) crude protein (Evangelista et al., 2012; Selling et al., 2013). The crude protein content of the pennycress seed meal that we used as starting material (Table 1) was higher than the literature values, which may be related to the different harvest batch and more immediate completion of the cleaning and drying steps that reduced the risk of seed deterioration during storage prior to oil pressing. The freeze-dried extract from saline extraction had higher protein purity than the extract recovered by acid precipitation (Table 1), but both would be classified as protein isolates because their protein contents are at least 90% (db) (Lusas and Rhee, 1997). In addition, the saline extraction method recovered more protein from pennycress seed meal than did the acid precipitation method, as shown by its higher protein extraction efficiency (Table 1). Other studies that produced protein isolates from related Brassicaceae seed meals (canola and rapeseed) by using methods similar to our procedures reported protein contents ranging from 76 to 99% (Rubin et al., 1990; Xu and Diosady, 1994;

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Table 1 Moisture, oil, and protein contents of defatted pennycress seed meal, recovered protein extracts, and spent solids, and extraction efficiencies of protein recovery methods. Pennycress sample

Moisture content %

Crude fat % (db)

Crude protein % (db)

Extraction efficiencya (%)

Seed meal, defatted Saline extraction (SE) SE Protein extract SE Spent solids Acid precipitation (AP) AP Protein extract AP Spent solids

9.63 ± 0.03 a

0.34 ± 0.08 a

33.44 ± 1.52 c

None

1.87 ± 0.08 c 2.13 ± 0.37 c

0.03 ± 0.02 c 0.37 ± 0.07 a

97.06 ± 3.42 a 20.05 ± 0.16 d

40.0 ± 0.5 a

3.66 ± 1.29 c 6.30 ± 2.91 b

0.27 ± 0.01 b 0.09 ± 0.01 c

90.40 ± 1.23 b 21.80 ± 0.07 d

34.8 ± 0.2 b

Means ± standard deviations of duplicate extraction trials and triplicate analyses for composition data per trial. Means in the same column followed by different letters are significantly different (p < 0.05). a Calculated based on the protein contents of the defatted starting material and corresponding spent solids.

Aluko and McIntosh, 2001). Protein recoveries were generally in the low-to-moderate range (33–65%; Gillberg and Tornell, 1976; Diosady et al., 1984; Xu and Diosady, 1994), although Klockeman et al. (1997) reported 87.5% protein recovery in their process for canola meal protein isolate. A major concern for plants belonging to the Cruciferae family is the presence of glucosinolates. These compounds and their degradation products are considered antinutrients because they impart undesirable properties, such as poor digestibility, poor physicochemical properties, off-color, and bad taste (Wu and Muir, 2008), as well as toxic effects, thus relegating the seed meals (canola, rapeseed, or mustard) to feed or fertilizer use (Shahidi and Naczk, 1990; Xu and Diosady, 1994). Pennycress seed and press cake, however, contain only sinigrin (allyl-glucosinolate), which is also present in other edible plants such as horseradish and brown mustard (Vaughn et al., 2006). The sinigrin content of our defatted pennycress seed meal was 36.71 ± 0.41 mg/g sample, while in acidprecipitated protein isolate (APPI), sinigrin content was found to be one-hundred times less than that in the seed meal (0.37 ± 0.03 mg/g sample). We did not detect sinigrin in the saline-extracted protein isolate (SEPI). The absence or trace amounts of sinigrin in the pennycress protein isolates was not surprising, as it has been reported previously that membrane filtration techniques were particularly effective in reducing glucosinolate contents owing to their lower molecular weights compared to proteins (Diosady et al., 1984; Rubin et al., 1990; Tan et al., 2011). The lack of sinigrin in pennycress SEPI and its minimal amount in APPI could boost their potential for food and feed applications.

3.3. Infrared analysis of protein The FTIR spectra of SEPI and APPI (Fig. 1), which have been adjusted to have the same absorbance at 1570 cm−1 , indicate that there are some differences between the isolates in the 1200–400 cm−1 region, where the peaks for SEPI were apparently higher than those of APPI. The increased absorbance at 1100 cm−1 is suggestive of more C-O bonds, which may be associated with carbohydrates. In contrast to our previous extractions of pennycress protein (Selling et al., 2013), SEPI and APPI, in general, have less carbohydrate impurities as indicated by the greatly reduced C O absorbance (Silverstein et al., 1981) between 930 and 1250 cm−1 . Both pennycress protein isolates had very little oil present based on the lack of a peak at 1740 cm−1 , which is the location for the C O absorbance of an ester (Silverstein et al., 1981). These results validate the data in Table 1 that show high protein purity and minimal residual oil in pennycress SEPI and APPI. 3.4. SDS-PAGE results The band pattern of protein in defatted pennycress seed meal (Fig. 2) replicated our previous result, which detected eleven polypeptide bands that resolved between <6.5 to ca. 100 kDa Table 2 Amino acid compositions (g/100 g protein) of defatted pennycress seed meal, salineextracted (SE) or acid precipitated (AP) protein isolates (PI) from pennycress, and soybean and canola protein isolates.a Amino acid

3.2. Amino acid contents Protein in defatted pennycress seed meal, SEPI, and APPI had similar contents for most of the amino acids (Table 2). Glutamic acid + glutamine, aspartic acid + asparagine, arginine, and leucine were present in the greatest amounts. This result supports our previous findings for ground pennycress seed and press cakes (Hojilla-Evangelista et al., 2013; Selling et al., 2013). SEPI had the most amounts of tryptophan, glutamic acid + glutamine, and proline among the pennycress protein samples, but its lysine, alanine and glycine contents were less than those found in the seed meal. On the other hand, APPI contained the greatest amount of serine but the least amounts of lysine, cysteine, and glutamic acid + glutamine among all samples. Tan et al. (2011) also observed high similarities in amino acid contents between canola protein isolate and its meal. They postulated that the sharp decrease in cysteine content of canola protein isolate may have been caused by degradation due to high pH and long processing time in the NaOH-extraction method. The pennycress SEPI and APPI amino acids were also very comparable with those reported for soybean protein isolate (Wolf, 1983) and generally superior to those in canola protein isolate (Rubin et al., 1990).

Pennycress

Seed meal Histidinec 2.79 a 4.43 ab Isoleucine 7.77 ab Leucine 5.96 a Lysine 1.53 a Methionine 4.98 a Phenylalanine 4.83 ab Threonine 1.29 b Tryptophan 3.33 b Tyrosine Valinec 6.28 a 5.14 a Alanine 7.85 b Arginine Aspartic + asparagine 8.79 a 2.04 a Cysteine 16.32 b Glutamic acid + glutamine 6.83 a Glycine 6.04 b Proline 3.81 b Serine

Soybean PIb

Canola PIb

SEPI

APPI

2.80 a 4.09 b 7.18 b 5.21 b 1.41 a 4.86 a 4.35 b 1.68 a 2.95 b 5.67 b 4.67 b 8.17 ab 8.85 a 2.20 a 19.14 a

2.80 a 4.73 a 8.84 a 3.63 c 1.58 a 5.23 a 5.20 a 1.15 b 4.15 a 6.40 a 5.55 a 8.32 a 9.04 a 1.62 b 15.12 c

2.70 4.90 7.70 6.40 1.10 5.40 3.70 1.40 3.70 4.80 3.90 7.80 11.90 1.30 20.50

3.35 3.46 7.17 7.33 2.36 4.00 4.16 1.30 3.10 4.67 4.15 4.70 6.82 4.23 22.38

6.15 b 6.17 b 4.32 a

4.10 5.30 5.50

4.70 7.86 4.16

6.15 b 7.22 a 3.42 b

a Values for pennycress seed meal and protein isolates are means of duplicate determinations. Means across columns followed by different letters are significantly different (p < 0.05). b Data for soybean protein isolate from Wolf (1983); for canola protein isolate from Rubin et al. (1990). c Histidine through valine are essential amino acids.

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Pennycress APPI

1.8

Pennycress SEPI

1.6

Absorbance

1.4 1.2 1.0

100 Protein Solubility, %

2.0

177

Pennycress Seed Meal SE Protein Isolate

80

AP Protein Isolate

60 40 20

0.8

0

0.6

2.0

0.2 0.0

4.0

6.0

8.0

10.0

pH

0.4

Fig. 3. Solubility profiles of protein in pennycress seed meal and saline (NaCl)extracted (SE) or acid precipitated (AP) isolates. 3900

3400

2900

2400

1900

1400

900

400

cm-1 Fig. 1. FT-IR spectra of pennycress saline-extracted protein isolate (SEPI) and acidprecipitated protein isolate (APPI).

(Hojilla-Evangelista et al., 2013). Both seed protein isolates (SEPI and APPI) showed band patterns that were nearly identical to that of the seed meal, but the seven dominant polypeptides that resolved between 6.5 and 41 kDa were displayed as darker and wider bands (implying greater quantities) in the protein isolates (Fig. 2). The low M.W. range of the pennycress protein isolates is nearly similar to that of canola protein isolate, 14–59 kDa, within which was detected as eight major bands (Wu and Muir, 2008). SEPI appears to have more bands between 50 and 100 kDa compared with APPI. We also noted the presence of these bands in the water- and NaClsoluble protein fractions in pennycress seed protein in our previous work (Hojilla-Evangelista et al., 2013). Faint bands in the spent solids indicated that the major polypeptides were present in substantially less quantities. 3.5. Protein solubility We reported previously that pennycress seed protein generally had poor solubility based on 13–22% soluble protein at pH 2–7 and 43% soluble protein at pH 10, with the least amount of

soluble protein (ca. 10%) at pH 4 (Hojilla-Evangelista et al., 2013). The isoelectric precipitation pH is similar to those of several Brassica seed protein (Wanasundara et al., 2012) but pennycress protein solubility was lower than that of protein in mustard seed meal (Wanasundara et al., 2012). Pennycress SEPI was highly soluble at pH 2 (68%) and at pH ≥ 7 (74–91%) (Fig. 3). As with the defatted pennycress seed meal, the least amount of soluble protein was detected at pH 4; however, at pH below or above 4, SEPI was substantially more soluble than the meal. Canola protein isolate produced by a method also involving dilute NaCl and ultrafiltration was reported to have a similarly high solubility (52–97%) at pH 3–9, with >90% solubility observed at pH 5–9 (Yoshie-Stark et al., 2008). The solubility behavior of pennycress SEPI in acidic, pH-neutral, and alkaline media would open up several potential applications simply based on these three distinctly different environments. Pennycress APPI was most soluble (50%) at pH 2 and pH 10 (Fig. 3). This behavior appears to follow the solubility profile obtained by Klockeman et al. (1997) for acid-precipitated canola protein isolate, which showed less than 60% solubility at pH 2–10. In contrast to SEPI and the seed meal, APPI had a higher isoelectric precipitation pH at 5.5. In their process to produce Chinese rapeseed protein isolates, Xu and Diosady (1994) observed a broad range of pH (5.5–8) for precipitation, which they attributed to the presence of proteins having varying isoelectric points, given the complicated protein composition of rapeseed. APPI was far less soluble than SEPI at nearly all pH points tested, with the greatest differences in solubility occurring at pH ≥ 7 (Fig. 3). SEPI would be comprised mainly of albumins and globulins, which typically show high solubilities in aqueous media. The solubility data for APPI (Fig. 3) imply that potential uses may be in highly acidic and highly alkaline applications. Solubility properties are useful for designing methods for extracting proteins and fractionating their sub-units, as well as for identifying the potential applications of the protein. Insolubility is often associated with denaturation, which impairs other functional properties (Cheftel et al., 1985). 3.6. Foaming properties

Fig. 2. SDS-PAGE band patterns of protein in defatted pennycress seed meal, saline-extracted protein isolate (SEPI), acid-precipitated protein isolate (APPI), and corresponding spent solids (SS). Concentration = 4 mg protein/mL; 15 ␮L sample load volume.

Soluble proteins in pennycress seed meal and APPI produced notable foam volumes and highly stable foams that were apparently not affected by pH of analysis (Table 3). SEPI, on the other hand, produced less foam than the meal and APPI, with its highest and comparable foam stability recorded at pH 2 only. The lower foam stabilities of SEPI at pH 7 and 10 indicated that protein–protein interactions at the air–water interface were weak and unable to form interfacial membranes (Tan et al., 2011). Foaming capacity and foam stability of pennycress APPI were nearly identical to values obtained for AP soybean protein (131 mL and

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Table 3 Foaming properties, emulsion activity and stability indices (EAI, ESI), water-holding capacities (WHC), and heat coagulability at pH 2, pH 7 and pH 10 of soluble proteins in pennycress seed meal, saline-extracted (SE) protein isolate and acid precipitated (AP) protein isolate.a Pennycress sample and pH of analysis

Seed meal, pH 2 Seed meal, pH 7 Seed meal, pH 10 – SEPI, pH 2 SEPI, pH 7 SEPI, pH 10 – APPI, pH 2 APPI, pH 7 APPI, pH 10 a b

Functional properties

Foaming capacity (mL)

Foam stability (% foam left)

EAI (m2 /g)

ESI (min)

WHC (g water/g protein)

Heat coagulability % loss of solubility

ndb 113 ± 5 b 122 ± 1 a

nd 94 ± 3 a 96 ± 2 a

nd 147 ± 1 d 197 ± 3 b

nd 17.0 ± 0.7 bc 16.8 ± 0.7 bc

nd 3.51 ± 0.10 d 2.84 ± 0.02 e

0.0 ± 0.0 d 80.2 ± 0.7 a 77.6 ± 1.5 a

107 ± 4 c 102 ± 1 d 101 ± 2 d

95 ± 1 a 39 ± 6 c 69 ± 2 b

141 ± 5 d 192 ± 6 b 208 ± 8 a

13.9 ± 0.4 cd 21.2 ± 0.7 b 34.2 ± 1.2 a

6.63 ± 1.39 b 2.48 ± 0.10 e 6.22 ± 0.56 b

11.7 ± 0.1 c 60.7 ± 0.3 b 63.7 ± 0.1 b

127 ± 6 a 127 ± 5 a 128 ± 1 a

92 ± 3 a 98 ± 1 a 95 ± 1 a

88 ± 2 f 105 ± 2 e 178 ± 5 c

12.4 ± 0.1 d 14.2 ± 0.5 cd 15.8 ± 0.5 cd

7.52 ± 0.19 a 4.30 ± 0.01 c 4.53 ± 0.19 c

0.0 ± 0.0 d 3.7 ± 0.4 d 0.0 ± 0.0 d

Values are means ± standard deviations of duplicate determinations. Means within a column followed by different letters are significantly different (p < 0.05). nd means not determined.

95% remaining foam, respectively; Hojilla-Evangelista et al., 2004). In previous research on canola protein isolate, Pedroche et al. (2004) and Aluko et al. (2005) reported that the alkali extraction-acid precipitation method of producing their isolate reduced the foaming capacity and stability of the isolates compared with the meal. This behavior was displayed by pennycress SEPI but not by APPI.

assumed a gel-like appearance at the end of the analyses done at pH 2 and 10 and the gelation may have affected the reliability of the WHC results for APPI. Heat-induced gelation has been observed and studied in rapeseed and canola protein isolates; gels formed under both acidic and alkaline conditions, but gel strength was found to be strongest at pH > 9 (Léger and Arntfield, 1993; Schwenke et al., 1998).

3.7. Emulsifying properties 3.9. Heat coagulability EAI values for all pennycress protein samples increased with pH (Table 3), indicating that emulsifying capacity was enhanced as pH moved toward the alkaline range. This result was also noted for soluble proteins in pennycress seed meal and press cakes (HojillaEvangelista et al., 2013), which could be attributed to greater participation of proteins in oil–water interfacial reactions brought on by alkali-induced formation of more soluble proteins through unfolding of polypeptide chains. Emulsifying capacity of SEPI was clearly superior to those of the defatted meal and APPI at all pH values tested (Table 3), as well as, to that of soybean protein (56–99 m2 /g protein; Hojilla-Evangelista et al., 2004) extracted by methods similar to what we used in the current work. Emulsions formed by pennycress SEPI were the most stable, especially at pH 7 and 10, but also the most affected by changes in pH (Table 3). ESI values for SEPI at pH 7 were notably greater than the 15 min reported for soybean protein at the same pH (Hojilla-Evangelista et al., 2004). The lower emulsifying capacity and stability of APPI are similar to that of canola protein isolate prepared by alkali extraction and acid precipitation, which had poorer emulsifying properties compared to its meal (Pedroche et al., 2004). Tan et al. (2011) suggested that ultrafiltered canola protein isolate had considerably better emulsifying properties than acid-precipitated canola protein isolate, which they associated with greater overall solubility of the ultrafiltered protein sample. 3.8. Water-holding capacity (WHC) WHC is related to properties such as texture, body, viscosity, and adhesion (Cheftel et al., 1985). Pennycress seed meal and protein isolates showed significantly different WHC values at the pH conditions tested (Table 3). SEPI and APPI had highest WHC at pH 2, while at pH 10, both had WHC greater than that of the seed meal. At pH 7, APPI WHC was greatest among the pennycress protein samples. The unusually high WHC values (>6.0 g water/g protein) shown by SEPI and APPI are very similar to those we reported for lesquerella meal and press cake (6–8 g water/g protein; Hojilla-Evangelista and Evangelista, 2009). However, we observed that the APPI solids

Heat coagulability measures protein susceptibility to heating, which is typically demonstrated by substantial and irreversible reduction in solubility caused by the aggregation of unfolded protein molecules (Kinsella, 1976; Cheftel et al., 1985). These structural changes consequently impart significant influences on protein functionality (Cheftel et al., 1985). The extent of a protein’s susceptibility to heat treatment is influenced by the nature of the protein, protein concentration, pH, ionic strength, and water activity (Cheftel et al., 1985). APPI showed minimal (<4%) loss of solubility at all pH levels tested (Table 3), indicating high stability when subjected to heating. In contrast, heating at ca. 100 ◦ C was highly detrimental to pennycress seed meal protein and SEPI at pH 7 and 10 (60–80% loss of soluble protein). Both seed meal protein and SEPI were most stable to heating when tested at pH 2. The severe loss of solubility may be related to substantial presence of the NaCl- and water-soluble proteins. In previous work on pennycress seed and press cakes, the amounts of saline-soluble proteins were reduced drastically when extraction was done at 77 ◦ C (Selling et al., 2013) or when seeds were subjected to cooking at 82 ◦ C before oil pressing (Hojilla-Evangelista et al., 2013). 4. Conclusions Saline extraction and acid precipitation produced pennycress protein extracts that were sinigrin-free and with very high protein content (at least 90% db), which classifies the extracts as protein isolates. The extraction method had significant impact on the amino acid profiles and functional properties of the resultant protein isolates. Pennycress SEPI and APPI amino acid profiles were comparable with those reported for soybean protein isolate and superior to those in canola protein isolate. SEPI showed greater solubility and had excellent emulsifying properties that were clearly superior to those of APPI. Despite poorer solubility, APPI had better foaming properties and markedly greater stability to heating than did SEPI. These results strongly demonstrated that pennycress seed protein isolates produced by either saline extraction or acid

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precipitation have desirable functional properties that could be exploited for non-food uses, such as pressurized foams or whipped products and emulsions. Acknowledgments We are grateful to Debra Flickinger, Mardell Schaer, Kelly Utt, Ray Holloway, and Jeff Forrester of NCAUR for their assistance in the preparation and analyses of the samples. References Aluko, R.E., McIntosh, T., 2001. Polypeptide profile and functional properties of defatted meals and protein isolates of canola seeds. J. Sci. Food Agric. 81, 391–396. Aluko, R.E., McIntosh, T., Katepa-Mupondwa, F., 2005. Comparative study of the polypeptide profiles and functional properties of Sinapis alba and Brassica juncea seed meals and protein concentrates. J. Sci. Food Agric. 85, 1931–1937. AOAC, 2003. Official Methods of Analysis of AOAC International, seventeenth ed., revision 2. AOAC International, Gaithersburg, MD. AOCS, 2009. Official Methods and Recommended Practices of the American Oil Chemists’ Society, sixth ed. AOCS Press, Urbana, IL. Balmaceda, E.A., Kim, M.K., Franzen, R., Mardones, B., Lugay, J.C., 1984. Protein functionality methodology – standard tests. In: Regenstein, J.M., Regenstein, C.E. (Eds.), Food Protein Chemistry. Academic Press, NY, pp. 278–291. Betz, J.M., Fox, W.D.,1994. High-performance liquid chromatographic determination of glucosinolates in Brassica vegetables. In: Food Phytochemicals I: Fruits and Vegetables. American Chemical Society, Washington, DC, pp. 181–196. Boateng, A.A., Mullen, C.A., Goldberg, N.M., 2010. Producing stable pyrolysis liquids from oilseed presscakes of mustard family plants: pennycress (Thlaspi arvense L.) and camelina (Camelina sativa). Energy fuels 24, 6624–6632. Cheftel, J.C., Cuq, J.L., Lorient, D., 1985. Amino acids, peptides, and proteins. In: Fennema, O.R. (Ed.), Food Chemistry. , second ed. Marcel Dekker Inc., NY, pp. 275, 286, 298–308, 351–355. Diosady, L.L., Tzeng, Y.-M., Rubin, L.J., 1984. Preparation of rapeseed protein concentrates and isolates using ultrafiltration. J. Food Sci. 49, 768–770, 776. Evangelista, R.L., Isbell, T.A., Cermak, S.C., 2012. Extraction of pennycress (Thlaspi arvense L.) seed oil by full pressing. Ind. Crops Prod. 37, 76–81. Gillberg, L., Tornell, B., 1976. Preparation of rapeseed protein isolates: Dissolution and precipitation behavior of rapeseed proteins. J. Food Sci. 41, 1063–1069. Hojilla-Evangelista, M.P., 2012. Extraction and functional properties of non-zein proteins in corn germ from wet-milling. J. Am. Oil Chem. Soc. 89, 167–174. Hojilla-Evangelista, M.P., Evangelista, R.L., 2006. Effects of cooking and screwpressing on functional properties of Cuphea PSR23 seed proteins. J. Am. Oil Chem. Soc. 83, 713–718. Hojilla-Evangelista, M.P., Evangelista, R.L., 2009. Functional properties of protein from Lesquerella fendleri seed and press cake from oil processing. Ind. Crops Prod. 29, 466–472. Hojilla-Evangelista, M.P., Sessa, D.J., Mohamed, A., 2004. Functional properties of soybean and lupin protein concentrates produced by ultrafiltration-diafiltration. J. Am. Oil Chem. Soc. 81, 1153–1157. Hojilla-Evangelista, M.P., Evangelista, R.L., Isbell, T.A., Selling, G.W., 2013. Effects of cold-pressing and seed cooking on functional properties of protein in pennycress (Thlaspi arvense L.) seed and press cakes. Ind. Crops Prod. 45, 223–229. Isbell, T.A., 2009. US effort in the development of new crops (lesquerella, pennycress, coriander and cuphea). Oleagineux Corps Gras Lipides 16, 205–210. Kinsella, J.E., 1976. Functional properties of proteins in foods: a survey. Crit. Rev. Food Sci. Nutr. 7, 219–280.

179

Léger, L.W., Arntfield, S.D., 1993. Thermal gelation of the 12S canola globulin. J. Am. Oil Chem. Soc. 70, 853–861. Klockeman, D.M., Toledo, R., Sims, K.A., 1997. Isolation and characterization of defatted canola meal protein. J. Agric. Food Chem. 45, 3867–3870. Lusas, E.W., Rhee, K.C., 1997. Soybean processing and utilization. In: Erickson, D.R. (Ed.), Practical Handbook of Soybean Processing and Utilization. AOCS Press, Champaign, IL, pp. 117–160. Moser, B.R., Knothe, G., Vaughn, S.F., Isbell, T.A., 2009. Production and evaluation of biodiesel from field pennycress (Thlaspi arvense L.) oil. Energy fuels 23, 4149–4155. Myers, D.J., Hojilla-Evangelista, M.P., Johnson, L.A., 1994. Functional properties of protein extracted from flaked, defatted, whole corn by ethanol/alkali during sequential extraction processing. J. Am. Oil Chem. Soc. 71, 1201–1204. Pedroche, J., Yust, M.M., Lqari, H., Giron-Calle, J., Alaiz, M., Vioque, J., Millan, F., 2004. Brassica carinata protein isolates: chemical composition, protein characterization and improvement of functional properties by protein hydrolysis. Food Chem. 88, 337–346. Rubin, L.J., Diosady, L.L., Tzeng, Y.-M., 1990. Ultrafiltration in rapeseed processing. In: Shahidi, F. (Ed.), Canola and Rapeseed: Production, Chemistry, Nutrition, and Processing Technology. AVI-Van Nostrand Reinhold, NY, pp. 307–330. Schwenke, K.D., Dahme, A., Wolter, T., 1998. Heat-induced gelation of rapeseed proteins: effect of protein interaction and acetylation. J. Am. Oil Chem. Soc. 75, 83–87. Selling, G.W., Hojilla-Evangelista, M.P., Evangelista, R.L., Isbell, T.A., Price, N., Doll, K.M., 2013. Extraction of proteins from pennycress seeds and press cake. Ind. Crops Prod. 41, 113–119. Shahidi, F., Naczk, M., 1990. Removal of glucosinolates and other antinutirents from canola and rapeseed by methanol/ammonia processing. In: Shahidi, F. (Ed.), Canola and Rapeseed: Production, Chemistry, Nutrition, and Processing Technology. AVI-Van Nostrand Reinhold, NY, pp. 291–306. Silverstein, R.M., Bassler, G.C., Morrill, T.C.,1981. Infrared spectroscopy. In: Spectrometric Identification of Organic Compounds. John Wiley and Sons, NY, pp. 95–135. Tan, S.H., Mailer, R.J., Blanchard, C.L., Agboola, S.O., 2011. Canola proteins for human consumption: extraction profile, and functional properties. J. Food Sci. 76, R16–R28. Vaughn, S.F., Isbell, T.A., Weisleder, D., Berhow, M.A., 2005. Biofumigant compounds released by field pennycress (Thlaspi arvense L.) seedmeal. J. Chem. Ecol. 31, 167–177. Vaughn, S.F., Palmquist, D.E., Duval, S.M., Berhow, M.A., 2006. Herbicidal activity of glucosinolate-containing seedmeals. Weed Sci. 54, 743–748. Wanasundara, J.P.D., Abeysekara, S.J., McIntosh, T.C., Falk, K.C., 2012. Solubility differences of major storage proteins in Brassicaceae oilseeds. J. Am. Oil Chem. Soc. 89, 869–881. Wolf, W.J., 1983. Edible soybean protein products. In: Wolff, I.A. (Ed.), CRC Handbook of Processing and Utilization in Agriculture. Vol. 2, Part 2: Plant Products. CRC Press, NY, pp. 23–55. Wu, J., Muir, A.D., 2008. Comparative structural, emulsifying, and biological properties of two major canola proteins, cruciferin and napin. J. Food Sci. 73, C210–C216. Wu, W.U., Hettiarachchy, N.S., Qi, M., 1998. Hydrophobicity, solubility, and emulsifying properties of soy protein peptides prepared by papain modification and ultrafiltration. J. Am. Oil Chem. Soc. 75, 845–850. Wu, Y.V., Hojilla-Evangelista, M.P., 2005. Lesquerella fendleri protein fractionation and characterization. J. Am. Oil Chem. Soc. 82, 53–56. Yoshie-Stark, Y., Wada, Y., Wasche, A., 2008. Chemical composition, functional properties, and bioactivities of rapeseed protein isolates. Food Chem. 107, 32–39. Xu, L., Diosady, L.L., 1994. Functional properties of Chinese rapeseed protein isolates. J. Food Sci. 59, 1127–1130.