Article pubs.acs.org/JAFC
Characterization of Heat-Set Gels from RuBisCO in Comparison to Those from Other Proteins Anneke H. Martin,* Maaike Nieuwland, and Govardus A. H. de Jong Functional Ingredients, TNO, P.O. Box 360, 3700 AJ Zeist, The Netherlands ABSTRACT: To anticipate a future shortage in functional proteins, it is important to study the functionality of new alternative protein sources. Native RuBisCO was extracted from spinach, and its gelation behavior was compared to other native proteins from animal and plant origins. Protein gels were analyzed for their mechanical gel properties during small and large deformation and for their microstructure. Heat-induced aggregation and network formation of RuBisCO resulted in gels with unique characteristics compared to, for example, whey protein and egg white protein. Having a very low critical gelling concentration and low denaturation temperature, RuBisCO readily forms a network with a very high gel strength (G′, fracture stress), but upon deformation it has a brittle character (low critical strain, low fracture strain). This breakdown behavior can be explained by the dominant role of hydrophobic and hydrogen bonds between RuBisCO molecules during network formation and by the coarse microstructure. RuBisCO was shown to exhibit high potential as a functional ingredient giving opportunities for the design of new textures at low protein concentration. KEYWORDS: RuBisCO, extraction, gelation, fracture properties, protein interactions, plant proteins
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INTRODUCTION To counter future shortages in functional proteins, there is a need for efficient, sustainable, and cost-effective use of protein with regard to functionality. Biorefinery and extraction of functional proteins is required to broaden the applicability of alternative protein sources. To date, most attention has been given to proteins from seeds or plants, for example, soy, pea, and, to a lesser extent, lupine, sunflower, and rapeseed.1,2 Novel sources that are not commercially utilized yet are leaf proteins, for example, from alfalfa (Lucerne) or grass.3,4 In addition, waste side streams such as beet leaf or sources such as algae are possibly important new sources of protein and could offer potential economic benefit when the protein fraction is valorized. The similarity between all of these leaf sources is that they all contain RuBisCO and similar types of membrane proteins. Moreover, the amino acid sequence of the large subunits of RuBisCO from spinach are identical to those from beet leaf as derived through queries to the Uniprot database, making spinach a good model system for research purposes. RuBisCO (1,5-biphosphate carboxylase/oxygenase) is an enzyme essential in the first step of photosynthesis and present in leaves of numerous plants, making it the most abundant protein in the world.5 It has a molecular weight of ∼550 kDa and is composed of eight large (L) (55 kDa) and eight small (S) (15 kDa) polypeptide chains (form I RuBisCO). The core of the protein structure is built of four dimers (L2) and is stabilized by eight salt links per dimer−dimer interface.6−8 Under optimal conditions up to 50% of the protein in the leaf is RuBisCO,9,10 although this percentage varies with species and growing conditions. In some algae only 16% of the total protein is RuBisCO.9 The optimal purification process of RuBisCO varies with the plant source and was most extensively investigated for tobacco,11−15 spinach,15−17 and alfalfa,18−22 but also sources such as beet leaves,23,24 algae,9,25,26 and soybean leaves27 were studied among others. Challenges during © 2014 American Chemical Society
purification are the removal of unwanted components in combination with mild treatment to preserve functionality of the isolated protein. Unwanted components include thylakoid fragments, which are bound to membrane proteins containing chlorophyll, which gives the undesired green color, and components such as phenols and off-flavors. Various procedures have been utilized to extract protein from leaves, and the functionality of these fractions was found to be determined mostly by the ratio between RuBisCO and other soluble proteins and by the presence of polyphenols and charged carbohydrates.22,26 Polyphenols are generally difficult to remove as they tend to bind to the proteins during extraction in a reversible or irreversible manner through the action of polyphenol oxidase.28 To overcome this, we have developed an improved isolation procedure using anion exchange and size exclusion chromatography delivering colorless and highly soluble, native RuBisCO. Although these chromatography steps are frequently used for the isolation of active RuBisCO in proteomic research,29,30 it is uncommon for research on protein functionality as chromatography is relatively costly on a larger scale. The advantage, however, of this method is that it delivers an almost 100% pure RuBisCO fraction without other soluble proteins and polyphenols. The functional properties of RuBisCO are highly dependent on the extraction process as this determines the composition, solubility, and denaturation state of the protein fraction.31,32 Generally, however, protein isolates (containing RuBisCO) from different species (tobacco, alfalfa, soy, and sugar beet leaves) have been shown to possess functional properties similar to commercially applied protein isolates. Tobacco leaf Received: Revised: Accepted: Published: 10783
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The protein content of this source is 22% of proteins. The dried peas were ground to get a flour. Then, the flour was sieved, and only the fraction with size smaller than 180 μm was used for extraction. Lupine was extracted from a lupine flour (Lupinus angustifolius, unknown cultivar) product called FRALU CON, from L. I. Frank (Twello, The Netherlands). Soy was extracted from defatted soybean flour (Cargill, Amsterdam, The Netherlands) having a protein content of ∼50 wt %. For all plant proteins the following steps were performed. Flour was dissolved in Milli-Q water, ratio 1:10. The pH was adjusted to 8, and the mixture was incubated for 1 h at room temperature while stirring. Centrifugation of the suspension was done at 6000g for 30 min at 7 °C, and the supernatant was taken for isoelectric precipitation at pH 4.5 (lupine and soy) or pH 4.8 (pea). Incubation was performed for at least 2 h (overnight in the case of soy) at 4 °C. After centrifugation (6000g for 30 min at 7 °C), the pellet was washed with cold Milli-Q water and dispersed using an Ultra Rurrax at low speed. For soy, three wash steps (1:3 volume ratio with Milli-Q water) were carried out, whereas for pea and lupine only one wash step (1:10 volume ratio) was done. Centrifugation and collection of the pellet were repeated followed by dispersion of the pellet in 3 volumes of Milli-Q water and adjustment of the pH to 7. Protein solutions were stirred overnight for complete dissolution. A final centrifugation step (10000g, 30 min at 10 °C) was performed to get rid of the insoluble material. Sodium azide (0.02%) was added as preservative, and the samples were kept at 4 °C until further use. All protein solutions were set at pH 7. Differential Scanning Calorimetry (DSC). Thermal analysis was performed with a TA Instruments type Q200 modulated differential scanning calorimeter. About 30 mg samples (containing 10% protein) were weighed in MD cups, which were hermetically sealed. The temperature scan in linear mode was performed from 20 to 140 °C with increasing temperature with a rate of 2 °C/min. During this scan the heat flow was measured. Measurements were performed in duplicate, and results are presented as mean values. Gelation. Gelation kinetics were studied as a function of time using a stress-controlled AR2000 rheometer (TA Instruments, Etten-Leur, The Netherlands). A concentric cylinder system with a bob diameter of 14 mm was used. A temperature range was applied in which the temperature increased from 25 to 95 °C with a heating rate of 5 °C min−1. It was kept at this temperature for 1 h (strain 10−3, 6.27 rad s−1, frequency 1 Hz) before cooling back to 25 °C. The applied strain was within linear region. G′, G″, and tan δ were measured during both steps as a function of time. At the end of the measurement a strain sweep was performed from 0.001 to 1 (25 °C). Samples were covered with a thin layer of paraffin oil to prevent evaporation. Measurements were performed in duplicate, and results are presented as mean values ± standard deviation (SD). Texture Analysis. Samples for large deformation were prepared by heating 10−15% protein solutions (in a skin similar to that of a sausage) in a water bath (95 °C) for 45 min. Heated protein gels were prepared 1 day prior to texture analysis and stored overnight in the refrigerator. Gels were cut into cylindrical pieces (2 cm × 2 cm) using a steel wire. Samples were compressed in a single compression test to 90% of their initial height at a compression speed of 1 mm/s using a Texture Analyzer (TA Instruments) with a flat plate probe (diameter = 75 mm). The compressive stress and strain were determined and recalculated to true Hencky’s stress and strain. Young’s modulus was calculated from the linear part of the stress over strain curve. Each measurement was performed in triplicate, and results are presented as mean values ± standard deviation (SD). Microscopy. Microstructure was visualized using confocal laser scanning microscopy (CLSM). Protein gels were cut using a wet knife, and protein was colored with a Rhodamine B solution (0.2%). Measurements were performed on a TCS SP2 AOBS Leica Confocal (Leica-microsystems, Mannheim, Germany) mounted on a DM IRE2. Two objectives were used, a 20× objective (HC PL apo CS 20×/0.70 D) and a 63× objective (HCX Pl apo 63×/1.40). An excitation wavelength of 561 nm and an emission wavelength of 571−660 nm were employed. Four pictures were averaged, and a scanning speed of 400 Hz was used.
protein isolate was compared for its water-binding, emulsification, and gelation properties with egg white and soy protein isolates11 and was found to have similar or better properties compared to egg white protein,33 for example, in higher foam stability.11 With regard to gelation, RuBisCO from alfalfa, tobacco, and spinach were reported to yield strong gels.6 Libouga et al.34 determined the critical gelation concentration of RuBisCO (from alfalfa) by heating sealed tubes. Unfortunately, this method yields no information on gelation kinetics. Lamsal et al.31 performed stress relaxation tests on heat-set soluble leaf protein gels (alfalfa) that were found softer and faster to relax compared to whey protein. Up to now, the gelation properties of RuBisCO were studied only for limited conditions (concentrations, pH) and experimental setup. No information is available on large deformation, texture properties, or microstructure of heatinduced gels of RuBisCO. For application purposes in food products, information on these properties and comparison with existing (e.g., whey protein, egg white protein) and other plant proteins (e.g., soy, pea, lupine) is essential. Hence, the objective of this study is to obtain insight into the gelation behavior of RuBisCO and the subsequent gel properties with regard to fracture behavior and microstructure. In addition, the heatinduced gelation of RuBisCO is compared to whey protein and egg white protein, which have been studied extensively for their gelation behavior, and to plant proteins such as soy, pea, and lupine. This study aims to show the potential of RuBisCO as a functional ingredient, giving opportunities for the design of new textures at low protein concentration.
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MATERIAL AND METHODS
Materials. WPI (BiPRO) was obtained from Davisco (Eden Prairie, MN, USA). Egg white protein was kindly supplied by Bouwhuis Enthoven (Raalte, The Netherlands). Water was purified using a Milli-Q system (Millipore Systems, Molsheim, France). Polyvinylpolypyrrolidone (PVPP), sodium metabisulfite, and Tris-base were obtained from Sigma-Aldrich (St. Louis, MO, USA). QSepharose, Sephadex G75, and Superdex 200 were obtained from GE Healthcare (The Netherlands). Extraction RuBisCO. Fresh spinach was pressed using an Angel juicer (Slowjuice, Naarden, The Netherlands); the juice was collected into a container and mixed with wet PVPP and 0.1% sodium metabisulfite. During collection the juice was stirred and cooled in ice water and stored at 4 °C until further use. To remove the chlorophyll, the collected juice was heated at 50 °C for 30 min while stirring and flushing the headspace with nitrogen. After heating, the juice was cooled to 4−6 °C while stirring in a cold room and centrifuged (15000g, 35 min, 6−8 °C). The clear juice was set to pH 8 with solid Tris-base (4 g/L) and 4 M NaOH. The juice was filtered over a 0.45 μm filter before being loaded on a Q-Sepharose fast flow (FF). The clear juice containing the RuBisCO was diluted with Milli-Q water and eluens until conductivity was below 6.5 mS/cm. The resulting solution was loaded on a Q-sepharose FF column (1.4 L, diameter = 10 cm, height = 23 cm) previously equilibrated with 20 mM Tris/HCl, pH 8.0. Immediately after the Q-Sepharose, soluble fractions containing RuBisCO were desalted into 20 mM Tris/HCl, pH 8, using a Sephadex G75 column. The desalted fractions were colorless, treated with a 0.22 μm sterile filter, and stored as a protein concentrate (12.4 mg/mL, 20 mM Tris/HCl, pH 8) at 4 °C until further use. For gelation experiments, this solution was diafiltrated against demineralized water and concentrated to the desired concentration using an Amicon 8200 stirred cell (Merck Millipore, Darmstadt Germany) with a 3K membrane filter. pH was set at pH 7. Yield of RuBisCO from spinach was 10 g/10 kg of spinach. Extraction of Plant Proteins. Pea was extracted from a commercial brand of dry peas (HAK, Almkerk, The Netherlands). 10784
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Figure 1. Characterization of RuBisCO using (A) size exclusion chromatography and (B) SDS-PAGE, with the left lane being molecular weight markers and the right lane being RuBisCO. Protein Interactions. Solutions of 10% RuBisCO, 10% WPI with 0.2 M NaCl, and 10% egg white protein were heated for 30 min 90 °C in a microwell plate. After heating, the gels were removed from the microwell plate and rolled (roller mixer) in a 15 mL greiner tuber for 24 h with the following solutions: (1) Milli-Q water, (2) Milli-Q + 2% β-mercaptoethanol, (3) 2 M NaCl, (4) 2 M NaCl + 2% βmercaptoethanol, (5) 8 M urea, (6) 8 M urea +2% β-mercaptoethanol, (7) 2% SDS, or (8) 2% SDS + 2% β-mercaptoethanol, as previously described by Liu et al.35 Gels were visually inspected whether they were solubilized or remained intact.
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RESULTS AND DISCUSSION
Characterization RuBisCO Isolate. Following the isolation procedure, the purity of the obtained protein isolate was measured with size exclusion chromatography on a Superdex 200 column and gel electrophoresis (see Figure 1). Size exclusion results showed one peak with a narrow distribution indicating a 95% purity of RuBisCO. In addition, the electrophoresis shows two bands at 12.5 and 55 kDa indicating the presence of the small and large subunits of RuBisCO, respectively. Both analyses show that a pure RuBisCO isolate was obtained using the current extraction procedure. UV−vis analysis of the final RuBisCO solution showed no clear sign of any polyphenol absorbance at 340 nm (data not shown). In fact, the ratio between the absorbance at 280 nm and that at 340 nm was >40, indicative of the almost complete absence of polyphenols. To verify the nativity of the isolated RuBisCO as well as the other plant proteins that were isolated, DSC was performed. Figure 2 shows the thermograms for RuBisCO, isolated plant proteins from soy (SPI), pea (PPI), and lupine (LPI), and, in addition, egg white protein (EWP) and whey protein isolate (WPI). For all proteins clear enthalpic peaks were found indicating the presence of native protein. For RuBisCO the denaturation temperature (Td) was found to be 64.9 °C, which is slightly lower than values found by Libouga et al.34 and
Figure 2. DSC thermograms for isolated proteins from (top to bottom) EWP, LPI, WPI, SPI, PPI, and RuBisCO (measured at 10% protein concentration).
Beghin et al.36 Libouga et al.34 and Beghin et al.35 report Td values of 66.5 and 67.5 °C, respectively, for RuBisCO isolated from alfalfa. The difference is caused by the known higher thermostability of RuBisCO from alfalfa. Compared to WPI, EWP, and especially plant proteins such as SPI, RuBisCO has a relatively low Td. For WPI and EWP, Td was found to be 72 and 78 °C, similar to the literature.37,38 For SPI two major peaks were found at ∼73 and ∼90 °C assigned to the main fractions β-conglycinin and glycinin, respectively, similar to values found by Renkema et al.39 For pea protein the individual proteins of legumins and vicilins overlap and one endothermic peak was observed with a Td of 82 °C, which is slightly lower compared to values reported by Shand et al.40 but similar to those reported by Munialo et al.41 and Mession et al.42 For lupine protein also different protein fractions are expected, that is, vicilin (β-conglutin) and legumin (α-conglutin), but the 10785
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Figure 3. Storage modulus G′ (Pa) as a function of time during a heating−cooling profile (··· temperature) for (A) ()10% RuBisCO, (− −) 5% RuBisCO, and (−·−) 2.5% RuBisCO, and (B) (−·−) 2.5% RuBisCO, () 10% EWP, and (- - -) 12.5% WPI.
Table 1. Onset Gelation Time tg, Estimated Infinite G∞ ′ at 95 °C, and Critical Strain γc for Different Concentrations of RuBisCO
thermogram could not distinguish between these fractions with separate peaks and shows one broad endothermic peak between 66 and 85 °C. Sirtori et al.,43 however, found two peaks, at 71.5 and 91 °C for the vicilin and legumin, respectively. Gelation Properties. The gelation of RuBisCO was followed by measuring the storage modulus (G′) as a function of time during a given heating profile. For 2.5, 5, and 10% protein concentrations, the increase in G′ with time is given in Figure 3A. Having a low denaturation temperature, RuBisCO readily gels as shown by the steep increase in G′ at short time scale, after which a plateau value is quickly reached. When cooled to room temperature, a further increase in G′ is observed, which generally can be appointed to hydrophobic and electrostatic interactions and hydrogen bonds. Already at very low protein concentration (2.5%) RuBisCO was found to form gels with a G′ of 5 kPa. With increasing protein concentration G′ increases to 104 kPa for 10% RuBisCO, which is extremely high compared to whey protein and egg white protein. Moreover, Figure 3B shows that G′ values were obtained in the same order of magnitude for 2.5% RuBisCO, 10% EWP, and 12.5% WPI. The onset in gelation is directly related to the denaturation temperature of the protein as the point at which G′ starts to increase (onset gelation time) from RuBisCO to WPI to EWP agrees with Td of 65, 72, and 78 °C, respectively. The increase in G′ during heating, however, is much larger for WPI and EWP than for RuBisCO, whereas during cooling the opposite occurred, in the end leading to similar values for G′. Typically, during heating covalent bonds are formed, strengthening the network, whereas during cooling hydrogen bonds and hydrophobic interactions play a role. It can be postulated that for RuBisCO the latter type of interactions are more dominant in determining the final gel strength. The gelation behavior of RuBisCO was fitted using firstorder kinetics ′ (1 − e−k(t − tg)) G′(t ) = G∞
RBC concn (%)
tg (s)
G′∞ (kPa)
γc (-)
2.5 5 10
630 594 546
0.5 ± 0.1 2.4 ± 0.3 10.4 ± 0.2
0.0436 ± 0.004 0.0397 ± 0.003 0.0374 ± 0.002
given in Table 2. At a protein concentration of 10%, RuBisCO has the lowest tg and the highest G′∞ followed by EWP and SPI. Table 2. Effect of 0.2 M NaCl on Onset Gelation Time tg and Estimated G∞ ′ at 95 °C of RuBisCO in Comparison to Other Proteins 0 M NaCl
+0.2 M NaCl
protein concn (%)
tg (s)
G′∞ (kPa)
tg (s)
10% RBC 10% WPI 10% EWP 10% SPI 15% PPI 21.5% LPI
546 no gel 740 790 no gel 1038
10.4 ± 0.2
590 726 720 798 438 768
4.1 ± 0.2 0.40 ± 0.01 0.34 ± 0.03
G′∞ (kPa) 6.6 7.7 4.4 0.81 2.8 1.8
± ± ± ± ± ±
0.4 0.1 0.2 0.04 0.1 0.1
Note that WPI does not form a gel at 10% protein concentration, and neither does PPI give a significant value for G∞ ′ at 95 °C at a concentration of 15%. For the latter, gel formation did occur during cooling. Upon the addition of 0.2 M NaCl, tg decreased and G′∞ increased for all proteins except for RuBisCO, and now WPI and PPI form a gel as well. The addition of small amounts of salt to globular proteins promotes aggregation due to reduced steric repulsion. Hence, network formation is enhanced and stronger networks are formed. Apparently, the effect of salt on the gelation behavior of RuBisCO is opposite, as tg increased and G′ at infinite time decreased upon addition of 0.2 M NaCl. During cooling from 95 to 25 °C, G′ further increased for all proteins. However, differences in the increase in G′ during cooling were observed, and values for G′ at 95 and 25 °C are given in Table 3. Two important features can be observed here, that is, a much higher increase in G′ during cooling for RuBisCO compared to the other proteins and the higher tan δ of the same gel when cooled. As postulated before, RuBisCO
(1)
in which G′∞ is the estimated G′ value at infinite time after the heating cycle (at 95 °C), k is a constant, and tg is the onset gelation time. Table 1 summarizes the findings for G∞ ′ and tg for the three different protein concentrations. With increasing protein concentration, G∞ ′ and tg increase and decrease, respectively. Similarly, the gelation behaviors of WPI, EWP, SPI, PPI, and LPI were fitted, and the results for tg and G∞ ′ are 10786
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a gel at pH 7 starts from ∼5% (SPI) and the order in which it increases follows SPI < EWP < WPI < PPI < LPI. For SPI the highest concentration that could be measured was 10% as above that concentration the (native) protein solution became too viscous to handle. Critical gelation concentrations for SPI, EWP, and WPI agree with literature values.44,45 After a heating and cooling profile had been applied, a strain sweep was performed to study the linear region and breakdown behavior of RuBisCO gels. Figure 5A shows the decrease in G′ when the strain increased from 0.001 to 1 for three concentrations of RuBisCO (2.5, 5, and 10%). For all concentrations, it appeared that the linear region for RuBisCO is quite small and, as a result, the critical strain (γc) is low as well. Values for γc are given in Table 1 and show that with increasing RuBisCO concentration, γc decreases. In comparison to gels made from WPI and EWP, RuBisCO exhibits a brittle character prone to early breakdown. Figure 5B clearly shows the early breakdown of RuBisCO compared to protein gels made from WPI and EWP and indicates once more that the type of interactions in a RuBisCO gel are of a different, weaker kind. In fact, covalent bonds between protein molecules and aggregates (as commonly found in WPI and EWP gels) tend to be stronger than hydrophobic interactions and hydrogen bonds. In summary, RuBisCO typically has a very low critical gelling concentration and a low tg, resulting in gels that have a very high G′ but also a high tan δ. In addition, early breakdown of RuBisCO gels during the strain sweep was observed. It is postulated that the type of interactions that are formed during aggregation and network formation play a role in RuBisCO’s specific behavior. To further investigate this, fracture properties and microstructure were determined. As WPI, EWP, and SPI form self-supporting gels at much lower concentrations than PPI and LPI, the latter two are not further taken into account for comparison with RuBisCO. Fracture Properties. Fracture properties were determined to establish information on (true) fracture stress and (true) fracture strain during compression, as is relevant for textural and sensory properties. Fracture properties of RuBisCO were compared with those of WPI, EWP, and SPI, and true fracture stress (σf) was plotted against true fracture strain (εf) for protein gels without and with 0.2 M NaCl (see Figure 6). RuBisCO (10%) has a higher σf (125 kPa) compared to 10% WPI (no gel), 10% EWP (90 kPa), and 10% SPI (31 kPa) but a similar εf to SPI. The relatively low εf indicates that RuBisCO forms a brittle gel as is commonly observed for SPI.44 As for 10% WPI no self-supporting gel is formed, a 15% concentration was used for comparison. The higher σf of 15% WPI compared to that of 10% RuBisCO gels is most probably caused by the higher protein concentration. In addition, WPI (and also EWP) forms more elastic gels. For EWP, WPI, and SPI, the addition of 0.2 M NaCl led to similar or slightly increased values for σf and lower εf. For RuBisCO, however, both σf and εf decreased drastically when salt was added, similar to the reduction in gel strength observed with small deformation measurements (G′ values). In addition, the brittleness of RuBisCO in large deformation is in line with the early breakdown observed for RuBisCO with small deformation experiments. Microstructure. To understand the differences in small and large deformation properties between RuBisCO and other proteins, the microstructure of RuBisCO is probed with CLSM as a function of NaCl concentration for 5 and 10% RuBisCO (see Figure 7) and compared to 10% WPI. Whereas WPI and also EWP and SPI form transparent and hence fine-stranded
Table 3. Estimated G∞ ′ at 95 °C (End of Heating) and 25 °C (End of Cooling) and tan δ for Proteins Gels Made of RuBisCO (RBC), Whey Protein Isolate (WPI), Egg White Protein (EWP), Soy Protein Isolate (SPI), Pea Protein Isolate (PPI), and Lupine Protein Isolate (LPI) protein concn (%) 2.5% RBC 5% RBC 10% RBC 12.5% WPI 15% WPI 10% EWP 12.5% EWP 10% SPI 17.5% PPI 21.5% LPI
G∞ ′ (kPa) at 95 °C 0.5 2.4 10.4 1.9 5.0 4.1 10.2 0.40 1.0 0.34
± ± ± ± ± ± ± ± ± ±
0.1 0.3 0.2 0.2 0.1 0.2 0.4 0.01 0.2 0.03
G∞ ′ (kPa) at 25 °C
increase in G∞ ′ at 25/95 °C
tan δ at 25 °C
± ± ± ± ± ± ± ± ± ±
∼10 ∼10 ∼10 ∼3 ∼2 ∼2 ∼2 ∼4 ∼3 ∼30
0.21 0.22 0.20 0.09 0.07 0.09 0.10 0.11 0.08 0.15
5.0 26.8 104.2 6.3 11.5 8.2 20.4 1.5 3.1 9.7
0.9 7.9 10.8 0.2 0.4 0.9 0.3 0.1 0.2 0.2
forms a network in which different types of interactions play a role, giving it its specific behavior. After cooling to 25 °C, G′ reached a steady state value for all proteins. Plotting this G′ value as a function of protein concentration for RuBisCO in comparison to other proteins, it is obvious that the gelation potential of RuBisCO exceeds that of WPI and EWP and by far that of plant proteins such as SPI, LPI, and PPI (see Figure 4). For each protein an increase in G′
Figure 4. Storage modulus G′ as a function of protein concentration for (◆) RuBisCO, (■) WPI, (△) EWP, (×) SPI, (●) LPI, and (○) PPI. Dotted lines are to guide the eye.
was found with increasing protein concentration, and the critical gelation concentration for each protein could be estimated from the graph. For RuBisCO the critical gelation concentration (cgel) is 10 times higher than that for WPI (with β-lactoglobulin of 18 kDa in monomeric form as most prevalent protein) and EWP (containing predominantly ovalbumin of 44 kDa) and that reported values for hydrodynamic radius, Rg, for RuBisCO range from 5 to 7 nm,7 where ovalbumin and β-lactoglobulin, the major proteins of EWP and WPI, have values of ∼3 and ∼2 nm, respectively.45,46 With regard to the microstructure as observed with CLSM, the density of the RuBisCO network increased with increasing 10788
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Figure 7. Microstructure of RuBisCO gels (A, 5% protein concentration; B, 10% protein concentration) and WPI (C, 10%) as a function of NaCl concentration (1, no salt; 2, 0.2 M; 3, 0.5 M) as measured by CLSM. The scale bar presented in image B1 applies to all images.
phobic interactions and hydrogen bonds come into play. In small deformation rheology a distinction can be made between the increase in G′ during the heating phase and the second increase in G′ during the cooling phase. From Table 3 it is clear that for RuBisCO the increase in G′ during cooling is much higher than for the other proteins, suggesting that noncovalent interactions play a much larger role in strengthening the protein network. To further investigate this, RuBisCO gels were subjected to a series of solvents that each break specific interactions in proteins gels (see Table 4). β-Mercaptoethanol
(ME), NaCl, urea, SDS, and combinations of these solvents were used to distinguish between disulfide bridges, hydrogen bonds, hydrophobic, and electrostatic interactions. Both WPI and EWP gels stayed intact when dissolved in water, ME, 2 M NaCl, or 2 M NaCl+ME. RuBisCO gels, however, changed in size. Upon addition of NaCl, both in the absence and in the presence of ME, the gels shrank, indicating contraction of the protein network due to enhanced hydrophobic interactions. Addition of ME alone resulted in swelling of the RuBisCO gel. As disulfide bridges are present within the large subunits, ME may disrupt these when accessible, leading to a looser and open structure. Urea and SDS are known to disrupt hydrogen bonds and/or hydrophobic interactions. When added to RuBisCO, these gels are partially dissolved, suggesting that hydrogen bonds and hydrophobic interactions play a role in holding the protein network together. The falling apart by urea or SDS is confirmed by Douillard et al.,7 who reported on dissociation of small subunits from the large ones for spinach RuBisCO already at 3 M urea. As only the small subunits are dissociated, this may explain the partial dissolvement of the gel as opposed to complete dissolvement. Addition of urea or SDS to WPI or EWP led to swelling of the gel. Here, internal hydrogen bonds and hydrophobic interactions are broken, but the presence of disulfide bridges holds the protein network together, acting as a frame with more open pores. For both WPI and EWP the
Table 4. Solvents to Break up Protein−Protein Interactions type of interaction solvent (1) (2) (3) (4) (5) (6) (7) (8) a
water MEa 2 M NaCl 2 M NaCl + ME 8 M urea 8 M urea + ME 2% SDSb 2% SDS + ME
S−S
H-bonds
hydrophobic
electrostatic
× ×
× ×
× × × ×
× × × ×
× ×
ME, β-mercaptoethanol. bSDS, sodium dodecyl sulfate. 10789
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importance of disulfide bridges in aggregation was frequently studied,47,48 and hence this confirms the results obtained here. A combination of urea and ME or SDS and ME, targeting all interactions, dissolved all three protein gels. Hence, no differences were observed between RuBisCO, EWP, and WPI. Using solvents to study the type of interactions resulted in a validation of the hypothesis that hydrophobic interactions and H-bonds play a larger role in RuBisCO gels than in WPI or EWP gels. This explains the higher increase in G′ upon cooling (Table 3) and the higher brittleness as these bonds are weaker than the covalent bonds that occur with WPI and EWP. In conclusion, heat-induced aggregation and network formation of RuBisCO resulted in gels with unique characteristics compared to well-known proteins such as WPI and EWP and plant proteins such as SPI, PPI, and LPI. Having a very low critical gelling concentration and low denaturation temperature, RuBisCO could be applied in a wide range of products that need mild processing conditions. Product categories of interest include meat replacers, either as a bulk ingredient or as a binder, but it could also be applied as a meat improvement agent. The gelation functionality could also be applied in potato starch based products, surimi, or fish cakes. In these applications RuBisCO would be a more sustainable alternative to soy or egg white protein. In general, RuBisCO would be very suitable to partially replace proteins with poorer functional properties to enhance, for example, the gelation behavior. Moreover, the unique set of properties of RuBisCO allows for expansion of the application area, as it forms a strong network at low concentrations (high G′, high fracture stress) but upon deformation (oral processing) it has a brittle character (low critical strain, low fracture strain). In relation to fracture properties of food gels made from different types of hydrocolloids or mixtures thereof, the fracture strain of RuBisCO is in the same region as different tofu model systems44 and of mixtures of whey protein and κ-carrageenan, where κ-carrageenan forms the continuous phase.49 Selectively mixing RuBisCO with other types of biopolymers would give the opportunity to deliver new textures at low concentrations, similarly to mixtures of soy protein and gelatin.50 The typical breakdown behavior of RuBisCO can be explained by the dominant role of hydrophobic and electrostatic interactions and H-bonds and by the coarse microstructure, as opposed to disulfide bridges and (more) fine-stranded networks for WPI and EWP. These findings enhance the knowledge currently available on RuBisCO functionality, especially in the area of understanding the mechanical and microstructural properties of RuBisCO gels.
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REFERENCES
(1) Wanadundra, J. P. Proteins of Brassicaceae oilseeds and their potential as a plant protein source. Crit. Rev. Food Sci. Nutr. 2011, 51, 635−677. (2) Day, L. Proteins from land plants − potential resources for human nutrition and food security. Trends Food Sci. Technol. 2013, 32, 25−42. (3) Dijkstra, D. S.; Linneman, A. R.; van Boekel, T. A. J. S. 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. Crit. Rev. Food Sci. Nutr. 2003, 43, 481−506. (4) Boland, M. J.; Rae, A. N.; Vereijken, J. M.; Meuwissen, M. P. M.; Fischer, A. R. H.; van Boekel, M. A. J. S.; Rutherfurd, S. M.; Gruppen, H.; Moughan, P. J.; Hendriks, W. H. The future supply of animalderived protein for human consumption. Trends Food Sci. Technol. 2013, 29, 62−73. (5) Ellis, R. J. The most abundant protein in the world. Trends Biochem. Sci. 1979, 4, 241−244. (6) Barbeau, W. E.; Kinsella, J. E. Ribulose biphosphate carboxylase/ oxygenase (rubisco) from green leaves − potential as food protein. Food Rev. Int. 1988, 4, 93−127. (7) Douillard, R.; de Mathan, O. Leaf protein for food use: potential of Rubisco. In New and Developing Sources of Proteins; Hudson, B. J. F., Ed.; Springer: New York, 1994; pp 307−342. (8) Hartman, F. C.; Harpel, M. R. Structure, function, regulation, and assembly of D-ribulose-1,5-bisphosphatecarboxylase oxygenase. Annu. Rev. Biochem. 1994, 63, 197−234. (9) Hilditch, C. M.; Jones, P. B.; Balding, P.; Smith, A. J.; Rogers, L. J. Ribulose biphosphate carboxylases from macroalgae − proteolysis during extraction and properties of the enzyme from Porphyra umbilicalis. Phytochemicals 1991, 30, 745−750. (10) Patel, M.; Berry, J. O. Rubisco gene expression in C4 plants. J. Exp. Bot. 2008, 59, 1625−1634. (11) Sheen, S. J.; Sheen, V. L. Functional properties of fraction-1 protein from tobacco leaf. J. Agric. Food Chem. 1985, 33, 79−83. (12) Hassiotou, E.; Tsaftaris, A. Comparison of three procedures for rubisco purification from green tobacco leaves. Chim. Chronica 1995, 24, 147−158. (13) Fantozzi, P.; Sensidoni, A. Protein extraction from tobaccoleaves − technological, nutritional and agronomical aspects. Qual. Plant. − Plant Foods Hum. Nutr. 1983, 32, 351−368. (14) Fu, H.; Machado, P. A.; Hahm, T. S.; Kratochvil, R. J.; Wei, C. I.; Lo, Y. M. Recovery of nicotine-free proteins from tobacco leaves using phosphate buffer system under controlled conditions. Bioresour. Technol. 2010, 101, 2034−2042. (15) Carmo-Silva, A. E.; Barta, C.; Salvucci, M. E. Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase from leaves. Methods Mol. Biol. 2011, 684, 339−347. (16) Salvucci, M. E.; Portis, A. R.; Ogren, W. L. Purification of ribulose-1,5-bisphosphate carboxylase oxygenase with high specific activity by fast protein liquid-chromatography. Anal. Biochem. 1986, 153, 97−101. (17) Paulsen, J. M.; Lane, M. D. Spinach ribulose diphosphate carboxylase. I. Purification and properties of enzyme. Biochemistry 1966, 5, 2350−2357. (18) Betschar, A.; Kinsella, J. E. Extractability and solubility of leaf protein. J. Agric. Food Chem. 1973, 21, 60−65. (19) Knuckles, B. E.; Kohler, G. O. Functional properties of edible protein concentrates from alfalfa. J. Agric. Food Chem. 1982, 30, 748− 752. (20) Lu, P. S.; Kinsella, J. E. Extractability and properties of protein from alfalfa leaf meal. J. Food Sci. 1972, 37, 94−99. (21) Edwards, R. H.; Miller, R. E.; Fremery, D. D.; Knuckles, B. E.; Bickoff, E. M.; Kohler, G. O. Pilot-plant production of an edible white fraction leaf protein concentrate from alfalfa. J. Agric. Food Chem. 1975, 23, 620−626. (22) D’Alvise, N.; Lesueur-Lambert, C.; Fertin, B.; Dhulster, P.; Guillochon, D. Removal of polyphenols and recovery of proteins from
AUTHOR INFORMATION
Corresponding Author
*(A.H.M.) E-mail: [email protected]. Phone: +31 (0)88 866 1815. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Helene Mocking and Ron van den Dool are acknowledged for their help in extraction of RuBisCO, soy protein, pea protein, and lupine protein. 10790
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alfalfa white protein concentrate by ultrafiltration and adsorbent resin separations. Sep. Sci. Technol. 2000, 35, 2453−2472. (23) Jwanny, E. W.; Montanari, L.; Fantozzi, P. Protein-production for human use from sugar-beet byproducts. Bioresour. Technol. 1993, 43, 67−70. (24) Merodio, C.; Sabater, B. Preparation of a white protein fraction in high yield from sugar beet (Beta vulgaris L) leaves. J. Sci. Food Agric. 1988, 44, 237−243. (25) Schwenzfeier, A.; Wierenga, P. A.; Gruppen, H. Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp. Bioresour. Technol. 2011, 102, 9121−9127. (26) Satoh, H.; Okada, M.; Nakayama, K.; Miyaji, K. Purification and further characterization of pyrenoid proteins and ribulose-1,5bisphosphate carboxylase-oxygenase from the green-alga Bryopsis maxima. Plant Cell Physiol. 1984, 25, 1205−1214. (27) Sheen, S. J. Whole-plant utilization − the potential of soybean. Biomass 1986, 10, 195−206. (28) Barbeau, W. E.; Kinsella, J. E. Factors affecting the binding of chlorogenic acid to fraction 1 leaf protein. J. Agric. Food Chem. 1983, 31, 993−998. (29) Wang, D.; Naidu, S. L.; Portis, A. R., Jr.; Moose, S. P.; Long, S. P. Can the cold tolerance of C4 photosynthesis in Miscanthus giganteus relative to Zea mays be explained by differences in activities and thermal properties of Rubisco? J. Exp. Bot. 2008, 59, 1779−1787. (30) Whitney, S. M.; Kane, H. J.; Houtz, R. L.; Sharwood, R. E. Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2. Plant Physiol. 2009, 149, 1887−1895. (31) Lamsal, B. P.; Koegel, R. G.; Gunasekaran, S. Some physicochemical and functional properties of alfalfa soluble leaf proteins. LWT−Food Sci. Technol. 2007, 40, 1520−1526. (32) Zhong, C.; Wang, R.; Zhou, Z.; Jia, S.-R.; Tan, Z.-L.; Han, P.-P. Functional properties of protein isolates from Caragana korshinskii Kom. extracted by three different methods. J. Agric. Food Chem. 2012, 60, 10337−10342. (33) Barbeau, W. E. Functional properties of leaf proteins: criteria required in food applications. Ital. J. Food Sci. 1990, 4, 213−225. (34) Libouga, D. G.; AguieBeghin, V.; Douillard, R. Thermal denaturation and gelation of Rubisco: effects of pH and ions. Int. J. Biol. Macromol. 1996, 19, 271−277. (35) Liu, K.; Hsieh, F.-H. Protein−protein interactions during highmoisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems. J. Agric. Food Chem. 2008, 56, 2681−2687. (36) Beghin, V.; Bizot, H.; Audebrand, M.; Lefebvre, J.; Libouga, D. G.; Douillard, R. Differential scanning calorimetric studies of the effects of ions and pH on ribulose 1,5-bisphosphate carboxylase/ oxygenase. Int. J. Biol. Macromol. 1993, 15, 195−200. (37) Verheul, M.; Roefs, S. P. F. M.; de Kruif, C. G. Kinetics of heatinduced aggregation of β-lactoglobulin. J. Agric. Food Chem. 1998, 46, 896−903. (38) de Groot, J.; de Jongh, H. H. J. The presence of heat-stable conformers of ovalbumin affects properties of thermally formed aggregates. Protein Eng. 2003, 16, 1035−1040. (39) Renkema, J. M. S.; Lakemond, C.; de Jongh, H. H. J.; Gruppen, H.; van Vliet, T. The effect of pH on heat denaturation and gel forming properties of soy proteins. J. Biotechnol. 2000, 79, 223−230. (40) Shand, P. J.; Ya, H.; Pietrasik, Z.; Wanasundara, P. K. J. P. D. Physicochemical and textural properties of heat-induced pea protein isolate gels. Food Chem. 2007, 102, 1119−1130. (41) Munialo, C. D.; van der Linden, E.; de Jongh, H. H. J. The ability to store energy in pea protein gels is set by network dimensions smaller than fifty nanometers. Food Res. Int. 2014, 64, 482−491. (42) Mession, J.-L.; Sok, N.; Assifaoui, A.; Saurel, R. Thermal denaturation of pea globulins (Pisum sativum L.) − molecular interactions leading to heat-induced protein aggregation. J. Agric. Food Chem. 2013, 61, 1196−1204.
(43) Sirtori, E.; Resta, D.; Brambilla, F.; Zacherl, C.; Arnoldi, A. The effects of various processing conditions on a protein isolate from Lupinus angustifolius. Food Chem. 2010, 120, 496−504. (44) Urbonaite, V.; de Jongh, H. H. J.; van der Linden, E.; Pouvreau, L. Origin of water loss from soy protein gels. J. Agric. Food Chem. 2014, 62, 7550−7558. (45) Weijers, M.; Nicolai, T.; Visschers, R. W. Influence of the ionic strength on the structure of heat-set globular protein gels at pH 7. Ovalbumin. Macromolecules 2004, 37, 8709−8714. (46) Verheul, M.; Roefs, S. P. F. M. Structure of particulate whey protein gels: effect of NaCl concentration, pH, heating temperature and protein composition. J. Agric. Food Chem. 1998, 46, 4909−4916. (47) Hoffmann, M. A. M.; van Mil, P. J. J. M. Heat-induced aggregation of β-lactoglobulin: role of the free thiol group and disulphide bonds. J. Agric. Food Chem. 1997, 45, 2942−2948. (48) Arntfield, S. D.; Murray, E. D.; Ismond, M. A. H. Role of disulfide bonds in determining the rheological and microstructural properties of heat-induced protein networks from ovalbumin and vicilin. J. Agric. Food Chem. 1991, 39, 1378−1385. (49) Ç akir, E.; Foegeding, E. A. Combining protein micro-phase separation and protein-polysaccharide segregative phase separation to produce gel structures. Food Hydrocolloids 2011, 25, 1538−1546. (50) Ersch, E.; ter Laak, I.; van der Linden, E.; Venema, P.; Martin, A. H. Modulating fracture properties of mixed protein systems. Food Hydrocolloids 2015, 44, 59−65.
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Characterization of Heat-Set Gels from RuBisCO in Comparison to Those from Other Proteins Anneke H. Martin,* Maaike Nieuwland, and Govardus A. H. de Jong Functional Ingredients, TNO, P.O. Box 360, 3700 AJ Zeist, The Netherlands ABSTRACT: To anticipate a future shortage in functional proteins, it is important to study the functionality of new alternative protein sources. Native RuBisCO was extracted from spinach, and its gelation behavior was compared to other native proteins from animal and plant origins. Protein gels were analyzed for their mechanical gel properties during small and large deformation and for their microstructure. Heat-induced aggregation and network formation of RuBisCO resulted in gels with unique characteristics compared to, for example, whey protein and egg white protein. Having a very low critical gelling concentration and low denaturation temperature, RuBisCO readily forms a network with a very high gel strength (G′, fracture stress), but upon deformation it has a brittle character (low critical strain, low fracture strain). This breakdown behavior can be explained by the dominant role of hydrophobic and hydrogen bonds between RuBisCO molecules during network formation and by the coarse microstructure. RuBisCO was shown to exhibit high potential as a functional ingredient giving opportunities for the design of new textures at low protein concentration. KEYWORDS: RuBisCO, extraction, gelation, fracture properties, protein interactions, plant proteins
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INTRODUCTION To counter future shortages in functional proteins, there is a need for efficient, sustainable, and cost-effective use of protein with regard to functionality. Biorefinery and extraction of functional proteins is required to broaden the applicability of alternative protein sources. To date, most attention has been given to proteins from seeds or plants, for example, soy, pea, and, to a lesser extent, lupine, sunflower, and rapeseed.1,2 Novel sources that are not commercially utilized yet are leaf proteins, for example, from alfalfa (Lucerne) or grass.3,4 In addition, waste side streams such as beet leaf or sources such as algae are possibly important new sources of protein and could offer potential economic benefit when the protein fraction is valorized. The similarity between all of these leaf sources is that they all contain RuBisCO and similar types of membrane proteins. Moreover, the amino acid sequence of the large subunits of RuBisCO from spinach are identical to those from beet leaf as derived through queries to the Uniprot database, making spinach a good model system for research purposes. RuBisCO (1,5-biphosphate carboxylase/oxygenase) is an enzyme essential in the first step of photosynthesis and present in leaves of numerous plants, making it the most abundant protein in the world.5 It has a molecular weight of ∼550 kDa and is composed of eight large (L) (55 kDa) and eight small (S) (15 kDa) polypeptide chains (form I RuBisCO). The core of the protein structure is built of four dimers (L2) and is stabilized by eight salt links per dimer−dimer interface.6−8 Under optimal conditions up to 50% of the protein in the leaf is RuBisCO,9,10 although this percentage varies with species and growing conditions. In some algae only 16% of the total protein is RuBisCO.9 The optimal purification process of RuBisCO varies with the plant source and was most extensively investigated for tobacco,11−15 spinach,15−17 and alfalfa,18−22 but also sources such as beet leaves,23,24 algae,9,25,26 and soybean leaves27 were studied among others. Challenges during © 2014 American Chemical Society
purification are the removal of unwanted components in combination with mild treatment to preserve functionality of the isolated protein. Unwanted components include thylakoid fragments, which are bound to membrane proteins containing chlorophyll, which gives the undesired green color, and components such as phenols and off-flavors. Various procedures have been utilized to extract protein from leaves, and the functionality of these fractions was found to be determined mostly by the ratio between RuBisCO and other soluble proteins and by the presence of polyphenols and charged carbohydrates.22,26 Polyphenols are generally difficult to remove as they tend to bind to the proteins during extraction in a reversible or irreversible manner through the action of polyphenol oxidase.28 To overcome this, we have developed an improved isolation procedure using anion exchange and size exclusion chromatography delivering colorless and highly soluble, native RuBisCO. Although these chromatography steps are frequently used for the isolation of active RuBisCO in proteomic research,29,30 it is uncommon for research on protein functionality as chromatography is relatively costly on a larger scale. The advantage, however, of this method is that it delivers an almost 100% pure RuBisCO fraction without other soluble proteins and polyphenols. The functional properties of RuBisCO are highly dependent on the extraction process as this determines the composition, solubility, and denaturation state of the protein fraction.31,32 Generally, however, protein isolates (containing RuBisCO) from different species (tobacco, alfalfa, soy, and sugar beet leaves) have been shown to possess functional properties similar to commercially applied protein isolates. Tobacco leaf Received: Revised: Accepted: Published: 10783
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The protein content of this source is 22% of proteins. The dried peas were ground to get a flour. Then, the flour was sieved, and only the fraction with size smaller than 180 μm was used for extraction. Lupine was extracted from a lupine flour (Lupinus angustifolius, unknown cultivar) product called FRALU CON, from L. I. Frank (Twello, The Netherlands). Soy was extracted from defatted soybean flour (Cargill, Amsterdam, The Netherlands) having a protein content of ∼50 wt %. For all plant proteins the following steps were performed. Flour was dissolved in Milli-Q water, ratio 1:10. The pH was adjusted to 8, and the mixture was incubated for 1 h at room temperature while stirring. Centrifugation of the suspension was done at 6000g for 30 min at 7 °C, and the supernatant was taken for isoelectric precipitation at pH 4.5 (lupine and soy) or pH 4.8 (pea). Incubation was performed for at least 2 h (overnight in the case of soy) at 4 °C. After centrifugation (6000g for 30 min at 7 °C), the pellet was washed with cold Milli-Q water and dispersed using an Ultra Rurrax at low speed. For soy, three wash steps (1:3 volume ratio with Milli-Q water) were carried out, whereas for pea and lupine only one wash step (1:10 volume ratio) was done. Centrifugation and collection of the pellet were repeated followed by dispersion of the pellet in 3 volumes of Milli-Q water and adjustment of the pH to 7. Protein solutions were stirred overnight for complete dissolution. A final centrifugation step (10000g, 30 min at 10 °C) was performed to get rid of the insoluble material. Sodium azide (0.02%) was added as preservative, and the samples were kept at 4 °C until further use. All protein solutions were set at pH 7. Differential Scanning Calorimetry (DSC). Thermal analysis was performed with a TA Instruments type Q200 modulated differential scanning calorimeter. About 30 mg samples (containing 10% protein) were weighed in MD cups, which were hermetically sealed. The temperature scan in linear mode was performed from 20 to 140 °C with increasing temperature with a rate of 2 °C/min. During this scan the heat flow was measured. Measurements were performed in duplicate, and results are presented as mean values. Gelation. Gelation kinetics were studied as a function of time using a stress-controlled AR2000 rheometer (TA Instruments, Etten-Leur, The Netherlands). A concentric cylinder system with a bob diameter of 14 mm was used. A temperature range was applied in which the temperature increased from 25 to 95 °C with a heating rate of 5 °C min−1. It was kept at this temperature for 1 h (strain 10−3, 6.27 rad s−1, frequency 1 Hz) before cooling back to 25 °C. The applied strain was within linear region. G′, G″, and tan δ were measured during both steps as a function of time. At the end of the measurement a strain sweep was performed from 0.001 to 1 (25 °C). Samples were covered with a thin layer of paraffin oil to prevent evaporation. Measurements were performed in duplicate, and results are presented as mean values ± standard deviation (SD). Texture Analysis. Samples for large deformation were prepared by heating 10−15% protein solutions (in a skin similar to that of a sausage) in a water bath (95 °C) for 45 min. Heated protein gels were prepared 1 day prior to texture analysis and stored overnight in the refrigerator. Gels were cut into cylindrical pieces (2 cm × 2 cm) using a steel wire. Samples were compressed in a single compression test to 90% of their initial height at a compression speed of 1 mm/s using a Texture Analyzer (TA Instruments) with a flat plate probe (diameter = 75 mm). The compressive stress and strain were determined and recalculated to true Hencky’s stress and strain. Young’s modulus was calculated from the linear part of the stress over strain curve. Each measurement was performed in triplicate, and results are presented as mean values ± standard deviation (SD). Microscopy. Microstructure was visualized using confocal laser scanning microscopy (CLSM). Protein gels were cut using a wet knife, and protein was colored with a Rhodamine B solution (0.2%). Measurements were performed on a TCS SP2 AOBS Leica Confocal (Leica-microsystems, Mannheim, Germany) mounted on a DM IRE2. Two objectives were used, a 20× objective (HC PL apo CS 20×/0.70 D) and a 63× objective (HCX Pl apo 63×/1.40). An excitation wavelength of 561 nm and an emission wavelength of 571−660 nm were employed. Four pictures were averaged, and a scanning speed of 400 Hz was used.
protein isolate was compared for its water-binding, emulsification, and gelation properties with egg white and soy protein isolates11 and was found to have similar or better properties compared to egg white protein,33 for example, in higher foam stability.11 With regard to gelation, RuBisCO from alfalfa, tobacco, and spinach were reported to yield strong gels.6 Libouga et al.34 determined the critical gelation concentration of RuBisCO (from alfalfa) by heating sealed tubes. Unfortunately, this method yields no information on gelation kinetics. Lamsal et al.31 performed stress relaxation tests on heat-set soluble leaf protein gels (alfalfa) that were found softer and faster to relax compared to whey protein. Up to now, the gelation properties of RuBisCO were studied only for limited conditions (concentrations, pH) and experimental setup. No information is available on large deformation, texture properties, or microstructure of heatinduced gels of RuBisCO. For application purposes in food products, information on these properties and comparison with existing (e.g., whey protein, egg white protein) and other plant proteins (e.g., soy, pea, lupine) is essential. Hence, the objective of this study is to obtain insight into the gelation behavior of RuBisCO and the subsequent gel properties with regard to fracture behavior and microstructure. In addition, the heatinduced gelation of RuBisCO is compared to whey protein and egg white protein, which have been studied extensively for their gelation behavior, and to plant proteins such as soy, pea, and lupine. This study aims to show the potential of RuBisCO as a functional ingredient, giving opportunities for the design of new textures at low protein concentration.
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MATERIAL AND METHODS
Materials. WPI (BiPRO) was obtained from Davisco (Eden Prairie, MN, USA). Egg white protein was kindly supplied by Bouwhuis Enthoven (Raalte, The Netherlands). Water was purified using a Milli-Q system (Millipore Systems, Molsheim, France). Polyvinylpolypyrrolidone (PVPP), sodium metabisulfite, and Tris-base were obtained from Sigma-Aldrich (St. Louis, MO, USA). QSepharose, Sephadex G75, and Superdex 200 were obtained from GE Healthcare (The Netherlands). Extraction RuBisCO. Fresh spinach was pressed using an Angel juicer (Slowjuice, Naarden, The Netherlands); the juice was collected into a container and mixed with wet PVPP and 0.1% sodium metabisulfite. During collection the juice was stirred and cooled in ice water and stored at 4 °C until further use. To remove the chlorophyll, the collected juice was heated at 50 °C for 30 min while stirring and flushing the headspace with nitrogen. After heating, the juice was cooled to 4−6 °C while stirring in a cold room and centrifuged (15000g, 35 min, 6−8 °C). The clear juice was set to pH 8 with solid Tris-base (4 g/L) and 4 M NaOH. The juice was filtered over a 0.45 μm filter before being loaded on a Q-Sepharose fast flow (FF). The clear juice containing the RuBisCO was diluted with Milli-Q water and eluens until conductivity was below 6.5 mS/cm. The resulting solution was loaded on a Q-sepharose FF column (1.4 L, diameter = 10 cm, height = 23 cm) previously equilibrated with 20 mM Tris/HCl, pH 8.0. Immediately after the Q-Sepharose, soluble fractions containing RuBisCO were desalted into 20 mM Tris/HCl, pH 8, using a Sephadex G75 column. The desalted fractions were colorless, treated with a 0.22 μm sterile filter, and stored as a protein concentrate (12.4 mg/mL, 20 mM Tris/HCl, pH 8) at 4 °C until further use. For gelation experiments, this solution was diafiltrated against demineralized water and concentrated to the desired concentration using an Amicon 8200 stirred cell (Merck Millipore, Darmstadt Germany) with a 3K membrane filter. pH was set at pH 7. Yield of RuBisCO from spinach was 10 g/10 kg of spinach. Extraction of Plant Proteins. Pea was extracted from a commercial brand of dry peas (HAK, Almkerk, The Netherlands). 10784
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Figure 1. Characterization of RuBisCO using (A) size exclusion chromatography and (B) SDS-PAGE, with the left lane being molecular weight markers and the right lane being RuBisCO. Protein Interactions. Solutions of 10% RuBisCO, 10% WPI with 0.2 M NaCl, and 10% egg white protein were heated for 30 min 90 °C in a microwell plate. After heating, the gels were removed from the microwell plate and rolled (roller mixer) in a 15 mL greiner tuber for 24 h with the following solutions: (1) Milli-Q water, (2) Milli-Q + 2% β-mercaptoethanol, (3) 2 M NaCl, (4) 2 M NaCl + 2% βmercaptoethanol, (5) 8 M urea, (6) 8 M urea +2% β-mercaptoethanol, (7) 2% SDS, or (8) 2% SDS + 2% β-mercaptoethanol, as previously described by Liu et al.35 Gels were visually inspected whether they were solubilized or remained intact.
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RESULTS AND DISCUSSION
Characterization RuBisCO Isolate. Following the isolation procedure, the purity of the obtained protein isolate was measured with size exclusion chromatography on a Superdex 200 column and gel electrophoresis (see Figure 1). Size exclusion results showed one peak with a narrow distribution indicating a 95% purity of RuBisCO. In addition, the electrophoresis shows two bands at 12.5 and 55 kDa indicating the presence of the small and large subunits of RuBisCO, respectively. Both analyses show that a pure RuBisCO isolate was obtained using the current extraction procedure. UV−vis analysis of the final RuBisCO solution showed no clear sign of any polyphenol absorbance at 340 nm (data not shown). In fact, the ratio between the absorbance at 280 nm and that at 340 nm was >40, indicative of the almost complete absence of polyphenols. To verify the nativity of the isolated RuBisCO as well as the other plant proteins that were isolated, DSC was performed. Figure 2 shows the thermograms for RuBisCO, isolated plant proteins from soy (SPI), pea (PPI), and lupine (LPI), and, in addition, egg white protein (EWP) and whey protein isolate (WPI). For all proteins clear enthalpic peaks were found indicating the presence of native protein. For RuBisCO the denaturation temperature (Td) was found to be 64.9 °C, which is slightly lower than values found by Libouga et al.34 and
Figure 2. DSC thermograms for isolated proteins from (top to bottom) EWP, LPI, WPI, SPI, PPI, and RuBisCO (measured at 10% protein concentration).
Beghin et al.36 Libouga et al.34 and Beghin et al.35 report Td values of 66.5 and 67.5 °C, respectively, for RuBisCO isolated from alfalfa. The difference is caused by the known higher thermostability of RuBisCO from alfalfa. Compared to WPI, EWP, and especially plant proteins such as SPI, RuBisCO has a relatively low Td. For WPI and EWP, Td was found to be 72 and 78 °C, similar to the literature.37,38 For SPI two major peaks were found at ∼73 and ∼90 °C assigned to the main fractions β-conglycinin and glycinin, respectively, similar to values found by Renkema et al.39 For pea protein the individual proteins of legumins and vicilins overlap and one endothermic peak was observed with a Td of 82 °C, which is slightly lower compared to values reported by Shand et al.40 but similar to those reported by Munialo et al.41 and Mession et al.42 For lupine protein also different protein fractions are expected, that is, vicilin (β-conglutin) and legumin (α-conglutin), but the 10785
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Figure 3. Storage modulus G′ (Pa) as a function of time during a heating−cooling profile (··· temperature) for (A) ()10% RuBisCO, (− −) 5% RuBisCO, and (−·−) 2.5% RuBisCO, and (B) (−·−) 2.5% RuBisCO, () 10% EWP, and (- - -) 12.5% WPI.
Table 1. Onset Gelation Time tg, Estimated Infinite G∞ ′ at 95 °C, and Critical Strain γc for Different Concentrations of RuBisCO
thermogram could not distinguish between these fractions with separate peaks and shows one broad endothermic peak between 66 and 85 °C. Sirtori et al.,43 however, found two peaks, at 71.5 and 91 °C for the vicilin and legumin, respectively. Gelation Properties. The gelation of RuBisCO was followed by measuring the storage modulus (G′) as a function of time during a given heating profile. For 2.5, 5, and 10% protein concentrations, the increase in G′ with time is given in Figure 3A. Having a low denaturation temperature, RuBisCO readily gels as shown by the steep increase in G′ at short time scale, after which a plateau value is quickly reached. When cooled to room temperature, a further increase in G′ is observed, which generally can be appointed to hydrophobic and electrostatic interactions and hydrogen bonds. Already at very low protein concentration (2.5%) RuBisCO was found to form gels with a G′ of 5 kPa. With increasing protein concentration G′ increases to 104 kPa for 10% RuBisCO, which is extremely high compared to whey protein and egg white protein. Moreover, Figure 3B shows that G′ values were obtained in the same order of magnitude for 2.5% RuBisCO, 10% EWP, and 12.5% WPI. The onset in gelation is directly related to the denaturation temperature of the protein as the point at which G′ starts to increase (onset gelation time) from RuBisCO to WPI to EWP agrees with Td of 65, 72, and 78 °C, respectively. The increase in G′ during heating, however, is much larger for WPI and EWP than for RuBisCO, whereas during cooling the opposite occurred, in the end leading to similar values for G′. Typically, during heating covalent bonds are formed, strengthening the network, whereas during cooling hydrogen bonds and hydrophobic interactions play a role. It can be postulated that for RuBisCO the latter type of interactions are more dominant in determining the final gel strength. The gelation behavior of RuBisCO was fitted using firstorder kinetics ′ (1 − e−k(t − tg)) G′(t ) = G∞
RBC concn (%)
tg (s)
G′∞ (kPa)
γc (-)
2.5 5 10
630 594 546
0.5 ± 0.1 2.4 ± 0.3 10.4 ± 0.2
0.0436 ± 0.004 0.0397 ± 0.003 0.0374 ± 0.002
given in Table 2. At a protein concentration of 10%, RuBisCO has the lowest tg and the highest G′∞ followed by EWP and SPI. Table 2. Effect of 0.2 M NaCl on Onset Gelation Time tg and Estimated G∞ ′ at 95 °C of RuBisCO in Comparison to Other Proteins 0 M NaCl
+0.2 M NaCl
protein concn (%)
tg (s)
G′∞ (kPa)
tg (s)
10% RBC 10% WPI 10% EWP 10% SPI 15% PPI 21.5% LPI
546 no gel 740 790 no gel 1038
10.4 ± 0.2
590 726 720 798 438 768
4.1 ± 0.2 0.40 ± 0.01 0.34 ± 0.03
G′∞ (kPa) 6.6 7.7 4.4 0.81 2.8 1.8
± ± ± ± ± ±
0.4 0.1 0.2 0.04 0.1 0.1
Note that WPI does not form a gel at 10% protein concentration, and neither does PPI give a significant value for G∞ ′ at 95 °C at a concentration of 15%. For the latter, gel formation did occur during cooling. Upon the addition of 0.2 M NaCl, tg decreased and G′∞ increased for all proteins except for RuBisCO, and now WPI and PPI form a gel as well. The addition of small amounts of salt to globular proteins promotes aggregation due to reduced steric repulsion. Hence, network formation is enhanced and stronger networks are formed. Apparently, the effect of salt on the gelation behavior of RuBisCO is opposite, as tg increased and G′ at infinite time decreased upon addition of 0.2 M NaCl. During cooling from 95 to 25 °C, G′ further increased for all proteins. However, differences in the increase in G′ during cooling were observed, and values for G′ at 95 and 25 °C are given in Table 3. Two important features can be observed here, that is, a much higher increase in G′ during cooling for RuBisCO compared to the other proteins and the higher tan δ of the same gel when cooled. As postulated before, RuBisCO
(1)
in which G′∞ is the estimated G′ value at infinite time after the heating cycle (at 95 °C), k is a constant, and tg is the onset gelation time. Table 1 summarizes the findings for G∞ ′ and tg for the three different protein concentrations. With increasing protein concentration, G∞ ′ and tg increase and decrease, respectively. Similarly, the gelation behaviors of WPI, EWP, SPI, PPI, and LPI were fitted, and the results for tg and G∞ ′ are 10786
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a gel at pH 7 starts from ∼5% (SPI) and the order in which it increases follows SPI < EWP < WPI < PPI < LPI. For SPI the highest concentration that could be measured was 10% as above that concentration the (native) protein solution became too viscous to handle. Critical gelation concentrations for SPI, EWP, and WPI agree with literature values.44,45 After a heating and cooling profile had been applied, a strain sweep was performed to study the linear region and breakdown behavior of RuBisCO gels. Figure 5A shows the decrease in G′ when the strain increased from 0.001 to 1 for three concentrations of RuBisCO (2.5, 5, and 10%). For all concentrations, it appeared that the linear region for RuBisCO is quite small and, as a result, the critical strain (γc) is low as well. Values for γc are given in Table 1 and show that with increasing RuBisCO concentration, γc decreases. In comparison to gels made from WPI and EWP, RuBisCO exhibits a brittle character prone to early breakdown. Figure 5B clearly shows the early breakdown of RuBisCO compared to protein gels made from WPI and EWP and indicates once more that the type of interactions in a RuBisCO gel are of a different, weaker kind. In fact, covalent bonds between protein molecules and aggregates (as commonly found in WPI and EWP gels) tend to be stronger than hydrophobic interactions and hydrogen bonds. In summary, RuBisCO typically has a very low critical gelling concentration and a low tg, resulting in gels that have a very high G′ but also a high tan δ. In addition, early breakdown of RuBisCO gels during the strain sweep was observed. It is postulated that the type of interactions that are formed during aggregation and network formation play a role in RuBisCO’s specific behavior. To further investigate this, fracture properties and microstructure were determined. As WPI, EWP, and SPI form self-supporting gels at much lower concentrations than PPI and LPI, the latter two are not further taken into account for comparison with RuBisCO. Fracture Properties. Fracture properties were determined to establish information on (true) fracture stress and (true) fracture strain during compression, as is relevant for textural and sensory properties. Fracture properties of RuBisCO were compared with those of WPI, EWP, and SPI, and true fracture stress (σf) was plotted against true fracture strain (εf) for protein gels without and with 0.2 M NaCl (see Figure 6). RuBisCO (10%) has a higher σf (125 kPa) compared to 10% WPI (no gel), 10% EWP (90 kPa), and 10% SPI (31 kPa) but a similar εf to SPI. The relatively low εf indicates that RuBisCO forms a brittle gel as is commonly observed for SPI.44 As for 10% WPI no self-supporting gel is formed, a 15% concentration was used for comparison. The higher σf of 15% WPI compared to that of 10% RuBisCO gels is most probably caused by the higher protein concentration. In addition, WPI (and also EWP) forms more elastic gels. For EWP, WPI, and SPI, the addition of 0.2 M NaCl led to similar or slightly increased values for σf and lower εf. For RuBisCO, however, both σf and εf decreased drastically when salt was added, similar to the reduction in gel strength observed with small deformation measurements (G′ values). In addition, the brittleness of RuBisCO in large deformation is in line with the early breakdown observed for RuBisCO with small deformation experiments. Microstructure. To understand the differences in small and large deformation properties between RuBisCO and other proteins, the microstructure of RuBisCO is probed with CLSM as a function of NaCl concentration for 5 and 10% RuBisCO (see Figure 7) and compared to 10% WPI. Whereas WPI and also EWP and SPI form transparent and hence fine-stranded
Table 3. Estimated G∞ ′ at 95 °C (End of Heating) and 25 °C (End of Cooling) and tan δ for Proteins Gels Made of RuBisCO (RBC), Whey Protein Isolate (WPI), Egg White Protein (EWP), Soy Protein Isolate (SPI), Pea Protein Isolate (PPI), and Lupine Protein Isolate (LPI) protein concn (%) 2.5% RBC 5% RBC 10% RBC 12.5% WPI 15% WPI 10% EWP 12.5% EWP 10% SPI 17.5% PPI 21.5% LPI
G∞ ′ (kPa) at 95 °C 0.5 2.4 10.4 1.9 5.0 4.1 10.2 0.40 1.0 0.34
± ± ± ± ± ± ± ± ± ±
0.1 0.3 0.2 0.2 0.1 0.2 0.4 0.01 0.2 0.03
G∞ ′ (kPa) at 25 °C
increase in G∞ ′ at 25/95 °C
tan δ at 25 °C
± ± ± ± ± ± ± ± ± ±
∼10 ∼10 ∼10 ∼3 ∼2 ∼2 ∼2 ∼4 ∼3 ∼30
0.21 0.22 0.20 0.09 0.07 0.09 0.10 0.11 0.08 0.15
5.0 26.8 104.2 6.3 11.5 8.2 20.4 1.5 3.1 9.7
0.9 7.9 10.8 0.2 0.4 0.9 0.3 0.1 0.2 0.2
forms a network in which different types of interactions play a role, giving it its specific behavior. After cooling to 25 °C, G′ reached a steady state value for all proteins. Plotting this G′ value as a function of protein concentration for RuBisCO in comparison to other proteins, it is obvious that the gelation potential of RuBisCO exceeds that of WPI and EWP and by far that of plant proteins such as SPI, LPI, and PPI (see Figure 4). For each protein an increase in G′
Figure 4. Storage modulus G′ as a function of protein concentration for (◆) RuBisCO, (■) WPI, (△) EWP, (×) SPI, (●) LPI, and (○) PPI. Dotted lines are to guide the eye.
was found with increasing protein concentration, and the critical gelation concentration for each protein could be estimated from the graph. For RuBisCO the critical gelation concentration (cgel) is 10 times higher than that for WPI (with β-lactoglobulin of 18 kDa in monomeric form as most prevalent protein) and EWP (containing predominantly ovalbumin of 44 kDa) and that reported values for hydrodynamic radius, Rg, for RuBisCO range from 5 to 7 nm,7 where ovalbumin and β-lactoglobulin, the major proteins of EWP and WPI, have values of ∼3 and ∼2 nm, respectively.45,46 With regard to the microstructure as observed with CLSM, the density of the RuBisCO network increased with increasing 10788
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Figure 7. Microstructure of RuBisCO gels (A, 5% protein concentration; B, 10% protein concentration) and WPI (C, 10%) as a function of NaCl concentration (1, no salt; 2, 0.2 M; 3, 0.5 M) as measured by CLSM. The scale bar presented in image B1 applies to all images.
phobic interactions and hydrogen bonds come into play. In small deformation rheology a distinction can be made between the increase in G′ during the heating phase and the second increase in G′ during the cooling phase. From Table 3 it is clear that for RuBisCO the increase in G′ during cooling is much higher than for the other proteins, suggesting that noncovalent interactions play a much larger role in strengthening the protein network. To further investigate this, RuBisCO gels were subjected to a series of solvents that each break specific interactions in proteins gels (see Table 4). β-Mercaptoethanol
(ME), NaCl, urea, SDS, and combinations of these solvents were used to distinguish between disulfide bridges, hydrogen bonds, hydrophobic, and electrostatic interactions. Both WPI and EWP gels stayed intact when dissolved in water, ME, 2 M NaCl, or 2 M NaCl+ME. RuBisCO gels, however, changed in size. Upon addition of NaCl, both in the absence and in the presence of ME, the gels shrank, indicating contraction of the protein network due to enhanced hydrophobic interactions. Addition of ME alone resulted in swelling of the RuBisCO gel. As disulfide bridges are present within the large subunits, ME may disrupt these when accessible, leading to a looser and open structure. Urea and SDS are known to disrupt hydrogen bonds and/or hydrophobic interactions. When added to RuBisCO, these gels are partially dissolved, suggesting that hydrogen bonds and hydrophobic interactions play a role in holding the protein network together. The falling apart by urea or SDS is confirmed by Douillard et al.,7 who reported on dissociation of small subunits from the large ones for spinach RuBisCO already at 3 M urea. As only the small subunits are dissociated, this may explain the partial dissolvement of the gel as opposed to complete dissolvement. Addition of urea or SDS to WPI or EWP led to swelling of the gel. Here, internal hydrogen bonds and hydrophobic interactions are broken, but the presence of disulfide bridges holds the protein network together, acting as a frame with more open pores. For both WPI and EWP the
Table 4. Solvents to Break up Protein−Protein Interactions type of interaction solvent (1) (2) (3) (4) (5) (6) (7) (8) a
water MEa 2 M NaCl 2 M NaCl + ME 8 M urea 8 M urea + ME 2% SDSb 2% SDS + ME
S−S
H-bonds
hydrophobic
electrostatic
× ×
× ×
× × × ×
× × × ×
× ×
ME, β-mercaptoethanol. bSDS, sodium dodecyl sulfate. 10789
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importance of disulfide bridges in aggregation was frequently studied,47,48 and hence this confirms the results obtained here. A combination of urea and ME or SDS and ME, targeting all interactions, dissolved all three protein gels. Hence, no differences were observed between RuBisCO, EWP, and WPI. Using solvents to study the type of interactions resulted in a validation of the hypothesis that hydrophobic interactions and H-bonds play a larger role in RuBisCO gels than in WPI or EWP gels. This explains the higher increase in G′ upon cooling (Table 3) and the higher brittleness as these bonds are weaker than the covalent bonds that occur with WPI and EWP. In conclusion, heat-induced aggregation and network formation of RuBisCO resulted in gels with unique characteristics compared to well-known proteins such as WPI and EWP and plant proteins such as SPI, PPI, and LPI. Having a very low critical gelling concentration and low denaturation temperature, RuBisCO could be applied in a wide range of products that need mild processing conditions. Product categories of interest include meat replacers, either as a bulk ingredient or as a binder, but it could also be applied as a meat improvement agent. The gelation functionality could also be applied in potato starch based products, surimi, or fish cakes. In these applications RuBisCO would be a more sustainable alternative to soy or egg white protein. In general, RuBisCO would be very suitable to partially replace proteins with poorer functional properties to enhance, for example, the gelation behavior. Moreover, the unique set of properties of RuBisCO allows for expansion of the application area, as it forms a strong network at low concentrations (high G′, high fracture stress) but upon deformation (oral processing) it has a brittle character (low critical strain, low fracture strain). In relation to fracture properties of food gels made from different types of hydrocolloids or mixtures thereof, the fracture strain of RuBisCO is in the same region as different tofu model systems44 and of mixtures of whey protein and κ-carrageenan, where κ-carrageenan forms the continuous phase.49 Selectively mixing RuBisCO with other types of biopolymers would give the opportunity to deliver new textures at low concentrations, similarly to mixtures of soy protein and gelatin.50 The typical breakdown behavior of RuBisCO can be explained by the dominant role of hydrophobic and electrostatic interactions and H-bonds and by the coarse microstructure, as opposed to disulfide bridges and (more) fine-stranded networks for WPI and EWP. These findings enhance the knowledge currently available on RuBisCO functionality, especially in the area of understanding the mechanical and microstructural properties of RuBisCO gels.
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REFERENCES
(1) Wanadundra, J. P. Proteins of Brassicaceae oilseeds and their potential as a plant protein source. Crit. Rev. Food Sci. Nutr. 2011, 51, 635−677. (2) Day, L. Proteins from land plants − potential resources for human nutrition and food security. Trends Food Sci. Technol. 2013, 32, 25−42. (3) Dijkstra, D. S.; Linneman, A. R.; van Boekel, T. A. J. S. 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. Crit. Rev. Food Sci. Nutr. 2003, 43, 481−506. (4) Boland, M. J.; Rae, A. N.; Vereijken, J. M.; Meuwissen, M. P. M.; Fischer, A. R. H.; van Boekel, M. A. J. S.; Rutherfurd, S. M.; Gruppen, H.; Moughan, P. J.; Hendriks, W. H. The future supply of animalderived protein for human consumption. Trends Food Sci. Technol. 2013, 29, 62−73. (5) Ellis, R. J. The most abundant protein in the world. Trends Biochem. Sci. 1979, 4, 241−244. (6) Barbeau, W. E.; Kinsella, J. E. Ribulose biphosphate carboxylase/ oxygenase (rubisco) from green leaves − potential as food protein. Food Rev. Int. 1988, 4, 93−127. (7) Douillard, R.; de Mathan, O. Leaf protein for food use: potential of Rubisco. In New and Developing Sources of Proteins; Hudson, B. J. F., Ed.; Springer: New York, 1994; pp 307−342. (8) Hartman, F. C.; Harpel, M. R. Structure, function, regulation, and assembly of D-ribulose-1,5-bisphosphatecarboxylase oxygenase. Annu. Rev. Biochem. 1994, 63, 197−234. (9) Hilditch, C. M.; Jones, P. B.; Balding, P.; Smith, A. J.; Rogers, L. J. Ribulose biphosphate carboxylases from macroalgae − proteolysis during extraction and properties of the enzyme from Porphyra umbilicalis. Phytochemicals 1991, 30, 745−750. (10) Patel, M.; Berry, J. O. Rubisco gene expression in C4 plants. J. Exp. Bot. 2008, 59, 1625−1634. (11) Sheen, S. J.; Sheen, V. L. Functional properties of fraction-1 protein from tobacco leaf. J. Agric. Food Chem. 1985, 33, 79−83. (12) Hassiotou, E.; Tsaftaris, A. Comparison of three procedures for rubisco purification from green tobacco leaves. Chim. Chronica 1995, 24, 147−158. (13) Fantozzi, P.; Sensidoni, A. Protein extraction from tobaccoleaves − technological, nutritional and agronomical aspects. Qual. Plant. − Plant Foods Hum. Nutr. 1983, 32, 351−368. (14) Fu, H.; Machado, P. A.; Hahm, T. S.; Kratochvil, R. J.; Wei, C. I.; Lo, Y. M. Recovery of nicotine-free proteins from tobacco leaves using phosphate buffer system under controlled conditions. Bioresour. Technol. 2010, 101, 2034−2042. (15) Carmo-Silva, A. E.; Barta, C.; Salvucci, M. E. Isolation of ribulose-1,5-bisphosphate carboxylase/oxygenase from leaves. Methods Mol. Biol. 2011, 684, 339−347. (16) Salvucci, M. E.; Portis, A. R.; Ogren, W. L. Purification of ribulose-1,5-bisphosphate carboxylase oxygenase with high specific activity by fast protein liquid-chromatography. Anal. Biochem. 1986, 153, 97−101. (17) Paulsen, J. M.; Lane, M. D. Spinach ribulose diphosphate carboxylase. I. Purification and properties of enzyme. Biochemistry 1966, 5, 2350−2357. (18) Betschar, A.; Kinsella, J. E. Extractability and solubility of leaf protein. J. Agric. Food Chem. 1973, 21, 60−65. (19) Knuckles, B. E.; Kohler, G. O. Functional properties of edible protein concentrates from alfalfa. J. Agric. Food Chem. 1982, 30, 748− 752. (20) Lu, P. S.; Kinsella, J. E. Extractability and properties of protein from alfalfa leaf meal. J. Food Sci. 1972, 37, 94−99. (21) Edwards, R. H.; Miller, R. E.; Fremery, D. D.; Knuckles, B. E.; Bickoff, E. M.; Kohler, G. O. Pilot-plant production of an edible white fraction leaf protein concentrate from alfalfa. J. Agric. Food Chem. 1975, 23, 620−626. (22) D’Alvise, N.; Lesueur-Lambert, C.; Fertin, B.; Dhulster, P.; Guillochon, D. Removal of polyphenols and recovery of proteins from
AUTHOR INFORMATION
Corresponding Author
*(A.H.M.) E-mail: [email protected]. Phone: +31 (0)88 866 1815. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Helene Mocking and Ron van den Dool are acknowledged for their help in extraction of RuBisCO, soy protein, pea protein, and lupine protein. 10790
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alfalfa white protein concentrate by ultrafiltration and adsorbent resin separations. Sep. Sci. Technol. 2000, 35, 2453−2472. (23) Jwanny, E. W.; Montanari, L.; Fantozzi, P. Protein-production for human use from sugar-beet byproducts. Bioresour. Technol. 1993, 43, 67−70. (24) Merodio, C.; Sabater, B. Preparation of a white protein fraction in high yield from sugar beet (Beta vulgaris L) leaves. J. Sci. Food Agric. 1988, 44, 237−243. (25) Schwenzfeier, A.; Wierenga, P. A.; Gruppen, H. Isolation and characterization of soluble protein from the green microalgae Tetraselmis sp. Bioresour. Technol. 2011, 102, 9121−9127. (26) Satoh, H.; Okada, M.; Nakayama, K.; Miyaji, K. Purification and further characterization of pyrenoid proteins and ribulose-1,5bisphosphate carboxylase-oxygenase from the green-alga Bryopsis maxima. Plant Cell Physiol. 1984, 25, 1205−1214. (27) Sheen, S. J. Whole-plant utilization − the potential of soybean. Biomass 1986, 10, 195−206. (28) Barbeau, W. E.; Kinsella, J. E. Factors affecting the binding of chlorogenic acid to fraction 1 leaf protein. J. Agric. Food Chem. 1983, 31, 993−998. (29) Wang, D.; Naidu, S. L.; Portis, A. R., Jr.; Moose, S. P.; Long, S. P. Can the cold tolerance of C4 photosynthesis in Miscanthus giganteus relative to Zea mays be explained by differences in activities and thermal properties of Rubisco? J. Exp. Bot. 2008, 59, 1779−1787. (30) Whitney, S. M.; Kane, H. J.; Houtz, R. L.; Sharwood, R. E. Rubisco oligomers composed of linked small and large subunits assemble in tobacco plastids and have higher affinities for CO2 and O2. Plant Physiol. 2009, 149, 1887−1895. (31) Lamsal, B. P.; Koegel, R. G.; Gunasekaran, S. Some physicochemical and functional properties of alfalfa soluble leaf proteins. LWT−Food Sci. Technol. 2007, 40, 1520−1526. (32) Zhong, C.; Wang, R.; Zhou, Z.; Jia, S.-R.; Tan, Z.-L.; Han, P.-P. Functional properties of protein isolates from Caragana korshinskii Kom. extracted by three different methods. J. Agric. Food Chem. 2012, 60, 10337−10342. (33) Barbeau, W. E. Functional properties of leaf proteins: criteria required in food applications. Ital. J. Food Sci. 1990, 4, 213−225. (34) Libouga, D. G.; AguieBeghin, V.; Douillard, R. Thermal denaturation and gelation of Rubisco: effects of pH and ions. Int. J. Biol. Macromol. 1996, 19, 271−277. (35) Liu, K.; Hsieh, F.-H. Protein−protein interactions during highmoisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems. J. Agric. Food Chem. 2008, 56, 2681−2687. (36) Beghin, V.; Bizot, H.; Audebrand, M.; Lefebvre, J.; Libouga, D. G.; Douillard, R. Differential scanning calorimetric studies of the effects of ions and pH on ribulose 1,5-bisphosphate carboxylase/ oxygenase. Int. J. Biol. Macromol. 1993, 15, 195−200. (37) Verheul, M.; Roefs, S. P. F. M.; de Kruif, C. G. Kinetics of heatinduced aggregation of β-lactoglobulin. J. Agric. Food Chem. 1998, 46, 896−903. (38) de Groot, J.; de Jongh, H. H. J. The presence of heat-stable conformers of ovalbumin affects properties of thermally formed aggregates. Protein Eng. 2003, 16, 1035−1040. (39) Renkema, J. M. S.; Lakemond, C.; de Jongh, H. H. J.; Gruppen, H.; van Vliet, T. The effect of pH on heat denaturation and gel forming properties of soy proteins. J. Biotechnol. 2000, 79, 223−230. (40) Shand, P. J.; Ya, H.; Pietrasik, Z.; Wanasundara, P. K. J. P. D. Physicochemical and textural properties of heat-induced pea protein isolate gels. Food Chem. 2007, 102, 1119−1130. (41) Munialo, C. D.; van der Linden, E.; de Jongh, H. H. J. The ability to store energy in pea protein gels is set by network dimensions smaller than fifty nanometers. Food Res. Int. 2014, 64, 482−491. (42) Mession, J.-L.; Sok, N.; Assifaoui, A.; Saurel, R. Thermal denaturation of pea globulins (Pisum sativum L.) − molecular interactions leading to heat-induced protein aggregation. J. Agric. Food Chem. 2013, 61, 1196−1204.
(43) Sirtori, E.; Resta, D.; Brambilla, F.; Zacherl, C.; Arnoldi, A. The effects of various processing conditions on a protein isolate from Lupinus angustifolius. Food Chem. 2010, 120, 496−504. (44) Urbonaite, V.; de Jongh, H. H. J.; van der Linden, E.; Pouvreau, L. Origin of water loss from soy protein gels. J. Agric. Food Chem. 2014, 62, 7550−7558. (45) Weijers, M.; Nicolai, T.; Visschers, R. W. Influence of the ionic strength on the structure of heat-set globular protein gels at pH 7. Ovalbumin. Macromolecules 2004, 37, 8709−8714. (46) Verheul, M.; Roefs, S. P. F. M. Structure of particulate whey protein gels: effect of NaCl concentration, pH, heating temperature and protein composition. J. Agric. Food Chem. 1998, 46, 4909−4916. (47) Hoffmann, M. A. M.; van Mil, P. J. J. M. Heat-induced aggregation of β-lactoglobulin: role of the free thiol group and disulphide bonds. J. Agric. Food Chem. 1997, 45, 2942−2948. (48) Arntfield, S. D.; Murray, E. D.; Ismond, M. A. H. Role of disulfide bonds in determining the rheological and microstructural properties of heat-induced protein networks from ovalbumin and vicilin. J. Agric. Food Chem. 1991, 39, 1378−1385. (49) Ç akir, E.; Foegeding, E. A. Combining protein micro-phase separation and protein-polysaccharide segregative phase separation to produce gel structures. Food Hydrocolloids 2011, 25, 1538−1546. (50) Ersch, E.; ter Laak, I.; van der Linden, E.; Venema, P.; Martin, A. H. Modulating fracture properties of mixed protein systems. Food Hydrocolloids 2015, 44, 59−65.
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