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Modulation of the rheological properties and microstructure of collagen by addition of co-gelling proteins
Food Hydrocolloids 49 (2015) 118e126
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Modulation of the rheological properties and microstructure of collagen by addition of co-gelling proteins €upler a, b, Monika Gibis a, Reinhard Kohlus c, Anja Maria Oechsle a, Michaela Ha Jochen Weiss a, * a
Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany €t Berlin, Koenigin-Luise-Strasse 22, 14195 Department of Food Technology and Food Chemistry, Chair of Food Process Engineering, Technische Universita Berlin, Germany c Department of Food Process Engineering and Food Powders, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 December 2014 Received in revised form 9 February 2015 Accepted 17 March 2015 Available online 24 March 2015
Collagen gels were modified by addition of co-gelling proteins to obtain gels with new functionalities. Microstructure and rheology of these mixed telopeptide-poor collagen gels were assessed. It was assumed that proteins with a low molecular weight strengthen the collagen structure by embedding themselves in the matrix, while high molecular weight proteins weaken the structure by interfering with the assembly of the network. Different combinations of collagen (2.8e5.19% (w/w)) and co-gelling protein concentrations (0.36 e2.14% (w/w)) were applied. Blood plasma protein, soy protein isolate, whey protein isolate, and gluten were used as co-gelling proteins. The storage modulus was measured as an indicator of the gel strength. Frequency sweeps (0.1e10 Hz) at 1% strain were conducted in the linear viscoelastic region. The position of the co-gelling proteins in the collagen matrix was examined via both confocal laser scanning and scanning electron microscopy. The results showed weakening effects for whey protein isolate. The addition of blood plasma protein did not affect the rheology, but the microstructure was influenced, featuring more fibrillar structures in the pores compared to the reference gels. Gluten seemed to lead to phase separation, forming a separate layer without interacting with the collagen matrix. The greatest impact was found for soy protein isolate, with a strengthening effect indicated by increased storage moduli and well distributed and embedded soy protein isolate in the collagen network. In conclusion, co-gelling proteins display a suitable approach to modify collagen strength in order to create matrices with new functionalities, such as co-extruded sausage casings with modified knack or snap. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Filled collagen type I gel Soy protein isolate Whey protein isolate Gluten Blood plasma protein
1. Introduction Filled gels, defined as macromolecular gels with dispersed particles, are widely used in the food industry (Banerjee & rez-Mateos, 2000; Bhattacharya, 2012; Montero, Hurtado, & Pe van Vliet, 1988). The particles influence the properties of the gel,
* Corresponding author. Tel.: þ49 711 459 24415; fax: þ49 711 459 24446. E-mail address: [email protected] (J. Weiss). http://dx.doi.org/10.1016/j.foodhyd.2015.03.013 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
depending on their interactions with the matrix, their size and lam, Venema, de Vries, van den Berg, & van der concentration (Sag Linden, 2014). Two effects have been reviewed for small deformations of filled gels: Either there is no interaction of particles and gel, leading to a decrease in the modulus (inactive filler), or an interaction exists between filler and matrix (active filler), which leads to an increase of the modulus (van Vliet, 1988). Proteins and polysaccharides can act as such fillers. The addition of polysaccharides has been studied extensively (Foegeding & Davis, 2011; Nicoleti & Telis, 2009). Furthermore, the influence of proteins on
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myofibrillar proteins was reviewed (Sun & Holley, 2011). The present study covers the impact of high and low molecular weight cogelling proteins on collagen gels. Collagen is the main protein of connective tissue in mammals, has a fibrillar structure and its gels have been studied extensively for application in the food and pharma industry (Barbut, 2010; €schl, & Aigner, 2003; Harper, Barbut, Lim, Friess, 1998; Gelse, Po & Marcone, 2012; Stenzel, Miyata, & Rubin, 1974; Wallace & Rosenblatt, 2003). Collagen type I is rod-shaped with a length and width of about 300 nm and 1.5 nm, respectively (Dekker, 1997; Traub & Piez, 1971). It is composed of a triple helix with three a-chains, two a1 and one a2, and has an approximate molecular weight of 300 kDa (Friess, 1998; Stenzel et al., 1974). Each a-chain forms a left-handed helix, which is integrated into the right-handed triple helix (Freudenberg et al., 2007). The appearance of various amino acids (glycine, proline, 4-hydroxyproline) enables the helical alignment and highly ordered structure by electrostatic and hydrophobic interactions, as well as hydrogen bonds and hydration forces (Bella, Brodsky, & Berman, 1995; Gelse et al., 2003; Komsa-Penkova, Koynova, Kostov, & Tenchov, 1996; Lee, Singla, & Lee, 2001; Stenzel et al., 1974; Wallace & Rosenblatt, 2003; Zanaboni, Rossi, Onana, & Tenni, 2000). After the alignment into fibrils and, subsequently, into fibers, lysine and hydroxylysine residues form different crosslinks of varying stability between helical regions and non-helical amino and carboxyl terminals, the mez-Guille n, so-called telopeptides (Gelse et al., 2003; Go nez, Lo pez-Caballero, & Montero, 2011; Reiser, McCormick, Gime & Rucker, 1992). Collagen gels show viscous behavior and a shear-thinning effect under stress, which makes the gels suitable for the extrusion process (Wallace & Rosenblatt, 2003). Bovine collagen is applied in the food industry within the co-extrusion process displaying a decent alternative, compared to the relatively high cost of using natural casings and the growing demand for sausage guts (Barbut, 2010). Furthermore, sausages can be produced continuously. Although the patent was established 30 years ago and collagen casings are used extensively in the meat industry today, there is very little published research focusing on the physicochemical properties of collagen formulations and its impact on the mechanical properties of the co-extruded casings (Harper et al., 2012). In addition to the impact of various acids and pH values, as studied by Oechsle et al. (Oechsle, Wittmann, Gibis, Kohlus, & Weiss, 2014), co-gelling proteins affect collagen gel properties that might improve the film quality of the co-extruded collagen casings. In this study, food grade proteins, namely whey protein isolate, blood plasma protein, soy protein isolate, and gluten, were incorporated into the collagen gel matrix to modify microstructure and rheology. The proteins were selected according to their molecular size (small or large), shape (globular or extended), and surface properties (hydrophilic or hydrophobic). We hypothesized that proteins with a small molecular weight (whey and blood plasma protein) strengthen the network structure, while proteins with a high molecular weight (soy protein isolate and gluten) disturb the gel formation. These presumptions were made due to different surface characters and molecular sizes of the proteins. Small globular proteins were supposed to fit in between the meshes of the telopeptide-poor collagen matrix due to their small molecular weight. By contrast, a lack of network-forming ability was hypothesized for gluten, because it is known for its bonding via disulfide bridges, but collagen does not provide cysteine residues (Shewry & Tatham, 1997). Soy protein isolate was expected to disturb the network formation due to its size and hydrophobic surface, which predicts that soy protein isolate would behave like an inactive filler (Chen & Dickinson, 1999). The study enables targeted modification of collagen rheology and microstructure to
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form co-extruded sausage casings according to the consumer's demand. 2. Materials and methods 2.1. Materials Telopeptide-poor collagen was kindly provided by Kalle (Wiesbaden, Germany) and Protein Consulting (Singhofen, Germany) by splitting off telopeptides and intermolecular crosslinks from native collagen to obtain single collagen triple helices (Bueker, Bueker, & Grolig, 2009). Hereafter, telopetide-poor collagen is referred to as collagen. Whey protein isolate was received from Davisco (BiPRO®, Davisco Food Internationals Inc., Eden Prairie, Minnesota, USA). Blood plasma protein was provided by TasteMakers (Tastemakers GmbH, Stuttgart, Germany). Soy protein isolate was used from ADM (PRO-FAM 974 Isolated Soy Protein, ADM Speciality Products€ner Oilseeds, Chicago, Illinois, USA). Gluten was received from Kro €rke (Weizengluten vital HP, Hermann Kro € ner GmbH, IbbenbüSta ren, Germany). 2.2. Experimental plan The filled gels were prepared according to a statistical design of experiments using the SAS/QC® software (SAS 9.3 for Windows, SAS Institute Inc., Cary, North Carolina, USA). The small composite design (Hartley Method) was used as a response surface in order to generate a predictive model according to Eq. (1) with nine runs, including three center points. Five concentrations of collagen (2.8%, 3%, 4%, 5%, and 5.19%, all (w/w)) and co-gelling protein (0.36%, 0.5%, 1.25%, 2%, and 2.14%, all (w/w)) led to nine samples for each cogelling protein. According to the usage in industry, 4% (w/w) collagen containing 1.25% co-gelling protein was used as a center point. 0
G ¼ A þ B$cC þ C$cCP þ D$c2C þ E$cC $cCP þ F$c2CP ;
(1)
with the storage modulus (G0 ), the collagen concentration (cC), the co-gelling protein concentration (cCP), and the terms A-F affecting either collagen, the co-gelling protein, or the combination of both. 2.3. Sample preparation The protein concentrations were calculated based on the protein content of the samples. For telopeptide-poor collagen the protein concentration of the gel was determined in a former study (Oechsle et al., 2014). For the other proteins this information was deduced from the technical data sheet. The specimens were diluted according to the experimental plan with phosphoric acid (ChemSolute, Renningen, Germany), mixed with an Artisan 5KSM 150 and a flex edge beater 5KFE5T (KitchenAid, St. Joseph, Minnesota, USA) for 5 min at the highest setting, and adjusted to pH 3. 2.4. z-potential The z-potential was measured between pH 3 and 7 to determine the isoelectric point with a particle electrophoresis instrument (Nano, ZS, Malvern Instruments, Malvern, UK). The titration was conducted automatically with a multipurpose titrator (MPT-2, Malvern Instruments, UK). Different concentrations were applied for each protein to get the best dilution factor for each sample: 2% (w/v) whey protein isolate solution, 0.05% (w/v) soy protein isolate solution, 0.025% (w/v) gluten solution, 0.2% (w/v) blood plasma protein solution, and 0.025% (w/v) collagen suspension. The samples were diluted with phosphoric acid, adjusted to pH 3, and
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homogenized using a Stomacher Circulator 400 from Seward (West Sussex, UK) for 5 min (300 rpm). 2.5. SDS-PAGE A Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad Laboratories, Hercules, California, USA) was used for the electrophoresis. The SDS-PAGE was conducted to determine the different molecular weights of the protein subunits and to verify the collagen type. The method developed by Laemmli was applied in this study (Laemmli, 1970). All protein samples were diluted to a concentration of 1% (w/w) with double-distilled water. Sample buffer (950 mL) and b-mercaptoethanol (50 mL) were mixed. Subsequently, each protein was diluted to an appropriate concentration with sample buffer: 0.1% (w/w) for whey protein isolate, blood plasma protein, soy protein isolate, and collagen. A concentration of 0.25% (w/w) was used for gluten. The samples were incubated at 95 C for 4 min. Finally, 10 mL of each protein solution was transferred into the wells of the gel, and the electrophoresis was ran for 40 min (200 V). Afterwards, the gel was stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad Laboratories, Hercules, California, USA) for 40 min and destained overnight in 15% methanol (Carl Roth, Karlsruhe, Germany) and 10% sodium acetate (Carl Roth, Karlsruhe, Germany) in double-distilled water until the bands were clearly visible. Roti-Mark prestained T852 Marker (Carl Roth, Karlsruhe, Germany) was utilized to determine the molecular weight of the proteins. 2.6. Rheology A rheometer (Physica MCR 502, Anton Paar, Graz, Austria) equipped with a plateeplate geometry with a diameter of 25 mm was chosen for the rheological measurements of the protein gels prepared. The samples were characterized via amplitude sweeps (1 Hz, 0.1e100% strain) and frequency sweeps in the linear viscoelastic region (1% strain, 0.1e100 Hz) and the storage modulus G’ was measured as indicator for the gel strength, while the loss modulus indicates the viscous behavior of the viscoelastic gel. An amount of 20 g of sample was prepared for each of the nine combinations defined by the experimental plan of collagen and cogelling protein.
2.8. Statistical analysis All measurements were repeated at least three times using duplicate samples. Means and standard deviations were calculated from these measurements using Excel (Microsoft, Redmond, VA, USA).
3. Results and discussion 3.1. z-potential Collagen displayed an isoelectric point at pH 4.0e4.9 (Fig. 1). The expected isoelectric point of collagen was 4.7, which matched the data collected (Highberger, 1939). The isoelectric point for blood plasma protein was found to be at pH 5.4e5.6. The literature states an isoelectric point at pH 4.8 for albumin from blood plasma and at pH 5.0e7.0 for globulins (Highberger, 1939). This could explain the high isoelectric point. An isoelectric point at pH 5.0e5.2 was found for whey protein isolate. According to the literature, the isoelectric point of b-lactoglobulin is at pH 5.2 and of a-lactalbumin at pH 4.1 (Klein, Aserin, Ishai, & Garti, 2010; Phan-Xuan et al., 2013). The whey protein isolate applied in this study contained 75% of blactoglobulin. This could be the reason why the isoelectric point was located in the region of pure b-lactoglobulin and above the one of a-lactalbumin. The isoelectric points of soy protein isolate and gluten were located at pH 4.9e5.1 and pH 6.7e6.9, respectively. The isoelectric point of soy protein isolate was expected at around pH 4.6 (Jaramillo, Roberts, & Coupland, 2011). For gluten, an isoelectric point of pH 7.3 was postulated by the literature (Nordqvist, € m, 2010). The divergent isoelectric points in Khabbaz, & Malmstro comparison to the data in references could appear due to a varying amino acid composition of the proteins, depending on origin and extraction conditions. This might have led to divergent isoelectric points. Nevertheless, it can be concluded that all proteins feature a positive charge at pH 3. Thus, the attractive collageneco-gelling protein interactions in the gel are based on water-mediated hydrogen-bonds rather than the repulsive electrostatic interactions (Leikin, Rau, & Parsegian, 1995).
2.7. Microscopy A Nikon Eclipse C1 (Nikon GmbH Mikroskope, Düsseldorf, Germany) combined with an inverted microscope (Nikon Eclipse Ti, Düsseldorf, Germany) was used for the confocal laser scanning microscopy (CLSM). Lasers with wavelengths of 488 nm and 543 nm from Melles Griot (Carlsbad, California, USA) were used. The samples prepared according to the experimental plan were dyed with 50 mL of 0.1% (v/v) Calcofluor (SigmaeAldrich, Buchs, Switzerland) and 50 mL of 0.1% (w/v) Eosin B (Sigma Aldrich, Buchs, Switzerland). Two emission band-pass filters were used and a magnification of 60x was applied. The samples were prepared according to the experimental plan and dipped into liquid nitrogen and freeze-dried (Lyovac GT 2, Finn Aqua Santasalo-Sohlberg, Hürth, Germany and Lyovac GT 4, LH €ln, Germany) for approximately 48 h for the scanning Leybold, Ko electron microscopy (SEM). The samples were sputtered with gold and palladium (20:80) for about 8 min with a sputtering system (SCD 040, Balzers, Bingen, Germany). The microscopy images were taken with a scanning electron microscope from Zeiss (DSM 940, Zeiss, Hamburg, Germany) under vacuum at 5000 kV. Magnifications of 500x, 1000x and 5000x were applied.
Fig. 1. z-potential of collagen, blood plasma protein, whey protein isolate, soy protein isolate, and gluten as a function of the pH.
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3.2. SDS-PAGE SDS-PAGE was performed in order to identify the molecular weight distribution of the proteins. The gel is shown in Fig. 2. The fractions of gluten can be slightly seen in lane 2. The largest fraction is below 17 kDa and led to the conclusion that the protein was hydrolysed during the treatment. Slight bands are also visible at around 50 kDa, which may belong to the u-gliadin fraction (50e65 kDa). A small fraction at ~33 kDa could corresponds to the a-, b- and g-gliadin fractions (33e40 kDa). Furthermore, this fraction could represent the low molecular weight glutenins (30e40 kDa). The bands at 95 kDa and above may refer to aggremez-Guille n et al., 2011). gates formed by the protein fractions (Go Soy protein isolate showed four bands: two at ~95 kDa, one at ~33 kDa, and one at ~17 kDa. The first two could indicate a0 , a, b, and g subunits of b-conglycinin and their aggregates. The fractions at around 33 kDa and 17 kDa may belong to subunits of the 11 S globulin (Petruccelli & Anon, 1995). Blood plasma protein showed three obvious bands between 50 and 95 kDa, which are probably due to albumin. The two bands between 17 and 23 kDa may occur due to the protein breakdown and hydrolysis during the treatment with sodium dodecyl sulfate and could represent parts of albumin s, Cuvelier, & Relkin, 2007). The band between 123 and (D avila, Pare 245 kDa might correspond to globulins with a molecular weight of about 150 kDa (Haurowitz, 2014). Whey protein isolate featured two bands around the marker band of 17 kDa. These bands probably refer to b-lactoglobulin (18.3 kDa) and a-lactalbumin (14.2 kDa) (Dekker, 1997). For collagen, both a1 and a2 bands were
Fig. 2. SDS polyacrylamide gel electrophoresis pattern of the molecular weight markers (Roti-Mark prestained T852 Marker, Carl Roth, Karlsruhe, Germany), gluten, soy protein isolate, blood plasma protein, whey protein isolate, and collagen.
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visible (~100 kDa), as well as the b-band (~200 kDa) and the g-band (~300 kDa). Hereby, the a-bands represent monomers, the b-bands stand for dimers, and the g-bands correspond to trimers of collagen molecules. This leads to the conclusion that the sample consists mostly of collagen type I (Friess, 1998). Overall, collagen displayed the largest molecular weight, followed by blood plasma protein, gluten, and soy protein isolate. Whey protein isolate, as expected, featured the smallest molecular weight. However, blood plasma protein featured large structures which were not expected. This might be due to high temperatures during the drying step where large and fibillar structures can be formed (Wang, Xu, Huang, Huang, & Zhou, 2014). Moreover, the proteins provided a range of fractions with different molecular weights. This can be partly ascribed to degradation and polymerization occurring during the SDS-PAGE procedure. 3.3. Rheology The frequency sweeps of the different gels were performed in the linear viscoelastic region and 2.8% collagen containing 1.25% cogelling protein and 4.05% collagen as reference are shown exemplarily in Fig. 3. All samples displayed a higher storage modulus (G0 ) than loss modulus (G00 ), which means that all samples featured a gel-like behavior (Almdal, Dyre, Hvidt, & Kramer, 1993). It displays the influence of 1.25% (w/w) co-gelling protein on 2.8% (w/w) collagen, and 4.05% (w/w) collagen was used as a reference with an equal total protein content. In the range of small deformation (0.1e1 Hz), whey protein isolate and soy protein isolate showed higher storage moduli than the reference. Moreover, the addition of blood plasma protein displayed nearly no effect, whereas gluten weakened the gel, resulting in lower storage and loss moduli compared to the reference. According to the hypothesis, this was expected for the small whey protein isolate and the large gluten molecules. However, the addition of the extended soy protein isolate increased the gel strength, while the small-sized blood plasma protein weakened the structure slightly. This indicates that not only the size influences the interactions between the co-gelling proteins and collagen, but also the amino acid residues that are crucial for the surface properties and collageneco-gelling protein interactions. However, the effects discussed are only true for 2.8% (w/w) collagen concentrations. Therefore, the impact of 1.25% (w/ w) co-gelling protein on 4% (w/w) collagen was investigated and demonstrated that all co-gelling proteins had a weakening effect on the collagen structure (data not shown). Thus, soy and whey protein isolate which initially increased the gel strength of 2.8% (w/w) collagen, decreased the storage modulus of 4% (w/w) collagen. This demonstrates that the collageneco-gelling protein interactions are also concentration dependent. Therefore, Fig. 4 depicts the storage modulus of 4% (w/w) collagen containing various concentrations of co-gelling proteins at 1 Hz in order to demonstrate the impact of the co-gelling protein concentration. The dashed line represents the reference sample of 4% (w/w) collagen. Soy protein isolate showed a higher storage modulus if 2.14% (w/w) or even 4% (w/w) was added. This might be ascribed to the ability of soy protein isolate to bind to the collagen structure. The storage modulus of the gel with blood plasma protein also increased, but had a large standard deviation, indicating an inhomogeneous matrix. The addition of gluten and whey protein isolate had hardly any influence on the gel strength over the whole concentration range. This indicates a weakening effect for high whey protein isolate concentrations. To the best of our knowledge, no studies have been conducted on the influence of co-gelling proteins on collagen gels. Therefore, literature concerning the interactions of gelatin with other proteins was considered. However, care has to be taken with the interpretations, because gelatin features a globular structure, as
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Fig. 3. Storage (G0 ) and loss modulus (G00 ) of 4.05% (w/w) collagen and 2.8% (w/w) collagen containing 1.25% (w/w) co-gelling subject to the frequency f.
Fig. 4. Storage modulus G0 at 1 Hz of 4% collagen (dashed line) containing co-gelling proteins at different concentrations (cco-gelling protein).
well as different intra- and intermolecular interactions, thus, exhibiting other properties than collagen itself. Nevertheless, Pang et al. showed a weakening effect of whey protein isolate on gelatin depending on the gelatin concentration (Pang, Deeth, Sopade, Sharma, & Bansal, 2014). This was in agreement with the data of this study for 4% collagen containing 1.25% whey protein isolate. Soy protein isolate was found to exhibit proteineprotein interactions with gelatin (Denavi et al., 2009). It was also found to participate in the network formation of myofibrillar proteins (Sun & Holley, 2011). This could be an explanation for the higher storage modulus for 2.8% collagen containing 1.25% soy protein isolate. Gluten hydrolysates were reported to disturb the gelation of myofibrillar proteins (Sun & Holley, 2011). The experiments conducted also suggest weakening interactions between gluten and collagen. Furthermore, almost no effects were observed for blood plasma protein. Neither the storage nor loss modulus of collagen containing blood plasma protein was affected. This was also shown
by Hurtado et al. for the impact of blood plasma protein on frank, Pare s, & Carretero, furter style sausages (Hurtado, Saguer, Toldra 2012). Fig. 5 depicts the response surface calculated by SAS with the corresponding equation and the data points represent the storage moduli measured at 1 Hz. Only significant terms (p < 0.05) were used for the predictive model. No significant factors could be determined for blood plasma protein. Hence, the master model solely consists of terms which are not significant (p > 0.05). Therefore, the results should be considered with caution. The wide variations of the triple point might be attributed to a lack of homogenization for this mixture and the formation of an inhomogeneous structure triggered by the addition of blood plasma protein. For soy protein isolate, the storage modulus increased with increasing soy protein isolate concentration, particularly at low collagen concentrations. This is in accordance with the conclusion drawn earlier regarding soy protein isolate interacting with the collagen network and, thus, supporting the gel strength. By contrast, whey protein isolate increased the storage modulus at low collagen concentrations, whereas at higher collagen concentrations, weakening effects could be observed. These opposite effects might be attributed to whey protein isolate interfering with the collagenecollagen interactions. At lower collagen concentrations, the strengthening effect can be ascribed to the increase of the whey protein isolate content itself. Gluten did not affect the collagen structure, signified by the predictive model that only depends on the collagen concentration. The exclusion of the non-significant terms in the predictive model resulted in a lower coefficient of determination (R2; ¼ 83.06%) than to the master model that contained all terms and featured a R2; of 97.86%. Nevertheless, gluten exhibited a relatively high coefficient of determination when taking into consideration that merely one term was applied for the predictive model. The lack of influence of gluten can be accredited to the poor gelling properties at the prevalent conditions. Gluten gels are formed via a heating step and the formation of disulfide bridges (Chen & Dickinson, 1999). Collagen does not exhibit cysteine residues, nor was a heating step carried out. Thus, repulsive electrostatic forces might prevail over the attractive interactions. 3.4. Microscopy 3.4.1. Confocal laser scanning microscopy Fig. 6 depicts the confocal laser scanning microscopy images of 2.8% (w/w) collagen containing 1.25% (w/w) co-gelling protein.
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Fig. 5. The predictive model and response surface according to Eq. (1) of the storage modulus (G0 ) at 1 Hz subject to the collagen concentration (cC) and blood plasma protein (cBBP), soy protein isolate (cSPI), whey protein isolate (cWPI), and gluten (cGLU) concentration deduced from the statistical analysis.*The master model containing no significant terms was applied.
Eosin B was used to stain connective tissue and appeared red (in web version). Calcofluor was applied and generally dyed proteins that emitted in the blue (in web version) and red spectrum. Thus, selective dying of either collagen or co-gelling protein was not feasible. Nevertheless, the combination of both dying agents improved the resolution of the gel matrices compared to a single dying agent. 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein implies fibers covered by globular and extended structures. Collagen containing soy protein isolate only featured a violet network, suggesting that either only collagen is displayed or soy protein isolate is embedded in the collagen matrix and a mixed network is formed. The strengthening effects proved by the rheological measurements indicate the latter. Whey protein isolate represented an interrupted globular structure of collagen containing. This is in accordance with the conclusions drawn before with whey protein isolate disturbing the collagen network formation. For collagen containing gluten a planar extended structure was visible. Whether it corresponds to collagen or to gluten needs further investigation. Nevertheless, fibers or an interwoven network could not be observed indicating that phase separation occurred and no collagen interconnections were formed. 3.4.2. Scanning electron microscopy Scanning electron microscopy images in Fig. 7 indicated a wellordered structure for 2.8% (w/w) collagen, while the 4% (w/w) collagen structure was arranged in a crater-shaped form. The 5.19%
(w/w) collagen displayed a more chaotic structure, with the fibers forming interconnections within the pores, which also led to a decrease of the pore size. It seemed that the collagen did not have enough space to assemble in an ordered structure. Furthermore, the additional collagenecollagen interactions might explain the higher storage modulus with increasing collagen concentration. For 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein strands were visible within the pores and the matrix resembled the 5.19% (w/w) collagen sample. However, blood plasma protein affected the gel strength sparsely according to the rheological measurements. Thus, not only the presence of the interconnections within the pores might be responsible for the strengthening effects, but also the co-gelling protein network strength itself. Thus, blood plasma protein networks might be weaker than collagen matrices based on the globular and smaller structure of the protein molecules. Consequently, the addition of blood plasma protein has almost no effect on the collagen strength. The addition of soy protein isolate increased the size of the collagen pores and additional networks were visible. Moreover, the overall network structure seems to be less homogeneous than collagen containing blood plasma protein. Considering the strengthening effects of soy protein isolate determined via the rheological measurements and the predictive model, a mixed interwoven network might have been formed that results in an increase of the gel strength. The network structure was also visible in the confocal laser scanning microscopy images. Furthermore, the participation of soy protein
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Fig. 6. Confocal laser scanning microscopy images of 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein, soy protein isolate, whey protein isolate, and gluten (scale bar ¼ 50 mm).
isolate in network formation had already been found to occur during the gelation of myofibrillar proteins (Sun & Holley, 2011). For collagen containing whey protein isolate no interconnections or additional networks were visible. Moreover, a smooth and homogeneous structure with smaller pores than collagen itself could be observed. With the rheological analysis and confocal laser scanning images in mind, whey protein isolate seems to interact with collagen through hydrophobic interactions. Hence, a uniform cogelled network was formed. Therefore, whey protein isolate might have covered the collagen molecules, occupied the binding sites of collagenecollagen interactions, and thus, decreased the storage modulus. Sarbon et al. also reported that whey protein interacts with chicken skin gelatin featuring synergistic effects at low whey protein concentrations (Sarbon, Badii, & Howell, 2015). However, at higher whey protein concentrations the storage
modulus decreased. This is in accordance with the observations made in this study since the addition of whey protein upon 1.25% (w/w) weakened the collagen structure, whereas below strengthening effects could be observed. Nevertheless, gelatin features a different structure and gelling mechanism and should be therefore considered with caution. On the contrary, collagen containing gluten exhibited a separate layer on top of the collagen matrix. This conforms to the conclusion drawn earlier from the predictive model with gluten having no influence on the storage modulus. Moreover, confocal laser scanning images revealed a planar structure that could correspond to phase-separated gluten layers. Furthermore, the collagen matrix below the layer displays the same pore size as 2.8% (w/w) collagen. This also supports the hypothesis, that collagen is not affected by the addition of gluten, and neither weakening nor strengthening effects occurred.
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Fig. 7. Scanning electron microscopy images of 2.8% (w/w), 4% (w/w), and 5.19% (w/w) collagen, and 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein, soy protein isolate, whey protein isolate, and gluten (scale bar ¼ 100 mm, 10 mm).
Fig. 8. Schematic mechanism illustrating the influence of the co-gelling proteins on the storage modulus (G0 ) of collagen matrices: The addition of whey protein isolate led to the formation of a co-gelled network with whey protein isolate interrupting the collagen interconnections and weakening the structure. Blood plasma protein featured additional strands within the collagen pores, but strengthening effects could not be observed based on the weak structure of blood plasma proteins. The incorporation of gluten resulted in phase separation and the collagen strength and structure were affected. The addition of soy protein isolate led to strengthening effects based on the formation of mixed interwoven networks.
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4. Conclusion The addition of co-gelling proteins can modify collagen strength and microstructure. The gel can be weakened or strengthened if the collageneco-gelling interactions are guaranteed and phase separation does not occur (Fig. 8). Thus, weakening effects might enable the flowability of the collagen matrix. On the contrary, increased elasticity involving strengthening effects enables shrinkage and expansion without irreversible deformation occurring in the gel matrix. Hence, the addition of co-gelling proteins displays a convenient tool to modify the collagen structure, rheology, and functionality for various applications. Possible synergistic effects caused by heat treatment of the co-gelling proteins to unfold the hydrophobic domains and to form disulfide bridges need further investigation. Acknowledgements We greatfully acknowledge Davisco Food Internationals Inc., € ner Tastemakers GmbH, ADM Foods & Wellness, and Hermann Kro GmbH for providing the protein samples. Furthermore, we would like to thank Robert Wilfer and Katja Mader (Kalle GmbH) and Gert and Marion Büker (Protein Consulting) for providing the collagen samples, Barbara Maier for her assistance with the SEM imaging, and Stefan B. Irmscher for his support in all fields. This research project was supported by the German Ministry of Economics and Energy (via AiF) and the FEI (Forschungskreis der Ern€ ahrungsindustrie e.V., Bonn): Project AiF 17478 N. References Almdal, K., Dyre, J., Hvidt, S., & Kramer, O. (1993). Towards a phenomenological definition of the term ‘gel’. Polymer Gels and Networks, 1(1), 5e17. Banerjee, S., & Bhattacharya, S. (2012). Food gels: gelling process and new applications. Critical Reviews in Food Science and Nutrition, 52(4), 334e346. Barbut, S. (2010). Microstructure of natural, extruded and co-extruded collagen casings before and after heating. Italian Journal of Food Science, 22(2), 126e133. Bella, J., Brodsky, B., & Berman, H. M. (1995). Hydration structure of a collagen peptide. Structure, 3(9), 893e906. Bueker, M., Bueker, G., & Grolig, G. (2009). In USPTO (Ed.), Collagen concentrate, use thereof and also process for production thereof. Chen, J., & Dickinson, E. (1999). Effect of surface character of filler particles on rheology of heat-set whey protein emulsion gels. Colloids and Surfaces B: Biointerfaces, 12(3), 373e381. s, D., Cuvelier, G., & Relkin, P. (2007). Heat-induced gelation of D avila, E., Pare porcine blood plasma proteins as affected by pH. Meat Science, 76(2), 216e225. Dekker, M. (1997). Structure function relationship of muscle proteins. In S. Damodaran, & A. Paraf (Eds.), Food proteins and their application (pp. 341e392). New York: CRC Press. rez-Mateos, M., An ~o n, M. C., Montero, P., Mauri, A. N., & Go mezDenavi, G. A., Pe n, M. C. (2009). Structural and functional properties of soy protein isolate Guille and cod gelatin blend films. Food Hydrocolloids, 23(8), 2094e2101. Foegeding, E. A., & Davis, J. P. (2011). Food protein functionality: a comprehensive approach. Food Hydrocolloids, 25(8), 1853e1864. Freudenberg, U., Behrens, S. H., Welzel, P. B., Muller, M., Grimmer, M., Salchert, K., et al. (2007). Electrostatic interactions modulate the conformation of collagen I. Biophysical Journal, 92(6), 2108e2119. Friess, W. (1998). Collagenebiomaterial for drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 45(2), 113e136. €schl, E., & Aigner, T. (2003). Collagensdstructure, function, and Gelse, K., Po biosynthesis. Advanced Drug Delivery Reviews, 55(12), 1531e1546. mez-Guille n, M. C., Gime nez, B., Lo pez-Caballero, M. E., & Montero, M. P. (2011). Go Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocolloids, 25(8), 1813e1827.
Harper, B. A., Barbut, S., Lim, L. T., & Marcone, M. F. (2012). Microstructural and textural investigation of various manufactured collagen sausage casings. Food Research International, 49(1), 494e500. Haurowitz, F. (2014). Proteins of the blood serum. Retrieved April 12, 2014, from http://www.britannica.com/EBchecked/topic/479680/protein/72559/Proteinsof-the-blood-serum. Highberger, J. H. (1939). The isoelectric Point of collagen. Journal of the American Chemical Society, 61(9), 2302e2303. s, D., & Carretero, C. (2012). Porcine plasma as Hurtado, S., Saguer, E., Toldr a, M., Pare polyphosphate and caseinate replacer in frankfurters. Meat Science, 90(3), 624e628. Jaramillo, D. P., Roberts, R. F., & Coupland, J. N. (2011). Effect of pH on the properties of soy proteinepectin complexes. Food Research International, 44(4), 911e916. Klein, M., Aserin, A., Ishai, P. B., & Garti, N. (2010). Interactions between whey protein isolate and gum Arabic. Colloids and Surfaces B: Biointerfaces, 79(2), 377e383. Komsa-Penkova, R., Koynova, R., Kostov, G., & Tenchov, B. G. (1996). Thermal stability of calf skin collagen type I in salt solutions. Biochimica et Biophysica Acta, 1297(2), 171e181. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227(5259), 680e685. Lee, C. H., Singla, A., & Lee, Y. (2001). Biomedical applications of collagen. International Journal of Pharmaceutics, 221(1e2), 1e22. Leikin, S., Rau, D. C., & Parsegian, V. A. (1995). Temperature-favoured assembly of collagen is driven by hydrophilic not hydrophobic interactions. Nature Structural Biology, 2(3), 205e210. rez-Mateos, M. (2000). Microstructural behaviour Montero, P., Hurtado, J. L., & Pe and gelling characteristics of myosystem protein gels interacting with hydrocolloids. Food Hydrocolloids, 14(5), 455e461. Nicoleti, J. F., & Telis, V. R. N. (2009). Viscoelastic and thermal properties of collagenexanthan gum and collagenemaltodextrin suspensions during heating and cooling. Food Biophysics, 4(3), 135e146. €m, E. (2010). Comparing bond strength and Nordqvist, P., Khabbaz, F., & Malmstro water resistance of alkali-modified soy protein isolate and wheat gluten adhesives. International Journal of Adhesion and Adhesives, 30(2), 72e79. Oechsle, A. M., Wittmann, X., Gibis, M., Kohlus, R., & Weiss, J. (2014). Collagen entanglement influenced by the addition of acids. European Polymer Journal, 58(0), 144e156. Pang, Z., Deeth, H., Sopade, P., Sharma, R., & Bansal, N. (2014). Rheology, texture and microstructure of gelatin gels with and without milk proteins. Food Hydrocolloids, 35(0), 484e493. Petruccelli, S., & Anon, M. C. (1995). Soy protein isolate components and their interactions. Journal of Agricultural and Food Chemistry, 43(7), 1762e1767. Phan-Xuan, T., Durand, D., Nicolai, T., Donato, L., Schmitt, C., & Bovetto, L. (2013). Tuning the structure of protein particles and gels with calcium or sodium ions. Biomacromolecules, 14(6), 1980e1989. Reiser, K., McCormick, R. J., & Rucker, R. B. (1992). Enzymatic and nonenzymatic cross-linking of collagen and elastin. Federation of American Societis for Experimental Biology, 6(7), 2439e2449. Sarbon, N. M., Badii, F., & Howell, N. K. (2015). The effect of chicken skin gelatin and whey protein interactions on rheological and thermal properties. Food Hydrocolloids, 45(0), 83e92. lam, D., Venema, P., de Vries, R., van den Berg, M., & van der Linden, E. (2014). Sag Whey protein particles modulate mechanical properties of gels at high protein concentrations. Food Hydrocolloids, 38(0), 163e171. Shewry, P. R., & Tatham, A. S. (1997). Disulphide bonds in wheat gluten proteins. Journal of Cereal Science, 25(3), 207e227. Stenzel, K. H., Miyata, T., & Rubin, A. L. (1974). Collagen as a biomaterial. Annual Review of Biophysics and Bioengineering, 3(1), 231e253. Sun, X. D., & Holley, R. A. (2011). Factors influencing gel formation by myofibrillar proteins in muscle foods. Comprehensive Reviews in Food Science and Food Safety, 10(1), 33e51. Traub, W., & Piez, K. A. (1971). The chemistry and structure of collagen. Advances in Protein Chemistry, 25, 243e352. van Vliet, T. (1988). Rheological properties of filled gels. Influence of filler matrix interaction. Colloid and Polymer Science, 266(6), 518e524. Wallace, D. G., & Rosenblatt, J. (2003). Collagen gel systems for sustained delivery and tissue engineering. Advanced Drug Delivery Reviews, 55(12), 1631e1649. Wang, P., Xu, X., Huang, M., Huang, M., & Zhou, G. (2014). Effect of pH on heatinduced gelation of duck blood plasma protein. Food Hydrocolloids, 35(0), 324e331. Zanaboni, G., Rossi, A., Onana, A. M. T., & Tenni, R. (2000). Stability and networks of hydrogen bonds of the collagen triple helical structure: influence of pH and chaotropic nature of three anions. Matrix Biology, 19(6), 511e520.
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Modulation of the rheological properties and microstructure of collagen by addition of co-gelling proteins €upler a, b, Monika Gibis a, Reinhard Kohlus c, Anja Maria Oechsle a, Michaela Ha Jochen Weiss a, * a
Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany €t Berlin, Koenigin-Luise-Strasse 22, 14195 Department of Food Technology and Food Chemistry, Chair of Food Process Engineering, Technische Universita Berlin, Germany c Department of Food Process Engineering and Food Powders, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 December 2014 Received in revised form 9 February 2015 Accepted 17 March 2015 Available online 24 March 2015
Collagen gels were modified by addition of co-gelling proteins to obtain gels with new functionalities. Microstructure and rheology of these mixed telopeptide-poor collagen gels were assessed. It was assumed that proteins with a low molecular weight strengthen the collagen structure by embedding themselves in the matrix, while high molecular weight proteins weaken the structure by interfering with the assembly of the network. Different combinations of collagen (2.8e5.19% (w/w)) and co-gelling protein concentrations (0.36 e2.14% (w/w)) were applied. Blood plasma protein, soy protein isolate, whey protein isolate, and gluten were used as co-gelling proteins. The storage modulus was measured as an indicator of the gel strength. Frequency sweeps (0.1e10 Hz) at 1% strain were conducted in the linear viscoelastic region. The position of the co-gelling proteins in the collagen matrix was examined via both confocal laser scanning and scanning electron microscopy. The results showed weakening effects for whey protein isolate. The addition of blood plasma protein did not affect the rheology, but the microstructure was influenced, featuring more fibrillar structures in the pores compared to the reference gels. Gluten seemed to lead to phase separation, forming a separate layer without interacting with the collagen matrix. The greatest impact was found for soy protein isolate, with a strengthening effect indicated by increased storage moduli and well distributed and embedded soy protein isolate in the collagen network. In conclusion, co-gelling proteins display a suitable approach to modify collagen strength in order to create matrices with new functionalities, such as co-extruded sausage casings with modified knack or snap. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Filled collagen type I gel Soy protein isolate Whey protein isolate Gluten Blood plasma protein
1. Introduction Filled gels, defined as macromolecular gels with dispersed particles, are widely used in the food industry (Banerjee & rez-Mateos, 2000; Bhattacharya, 2012; Montero, Hurtado, & Pe van Vliet, 1988). The particles influence the properties of the gel,
* Corresponding author. Tel.: þ49 711 459 24415; fax: þ49 711 459 24446. E-mail address: [email protected] (J. Weiss). http://dx.doi.org/10.1016/j.foodhyd.2015.03.013 0268-005X/© 2015 Elsevier Ltd. All rights reserved.
depending on their interactions with the matrix, their size and lam, Venema, de Vries, van den Berg, & van der concentration (Sag Linden, 2014). Two effects have been reviewed for small deformations of filled gels: Either there is no interaction of particles and gel, leading to a decrease in the modulus (inactive filler), or an interaction exists between filler and matrix (active filler), which leads to an increase of the modulus (van Vliet, 1988). Proteins and polysaccharides can act as such fillers. The addition of polysaccharides has been studied extensively (Foegeding & Davis, 2011; Nicoleti & Telis, 2009). Furthermore, the influence of proteins on
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myofibrillar proteins was reviewed (Sun & Holley, 2011). The present study covers the impact of high and low molecular weight cogelling proteins on collagen gels. Collagen is the main protein of connective tissue in mammals, has a fibrillar structure and its gels have been studied extensively for application in the food and pharma industry (Barbut, 2010; €schl, & Aigner, 2003; Harper, Barbut, Lim, Friess, 1998; Gelse, Po & Marcone, 2012; Stenzel, Miyata, & Rubin, 1974; Wallace & Rosenblatt, 2003). Collagen type I is rod-shaped with a length and width of about 300 nm and 1.5 nm, respectively (Dekker, 1997; Traub & Piez, 1971). It is composed of a triple helix with three a-chains, two a1 and one a2, and has an approximate molecular weight of 300 kDa (Friess, 1998; Stenzel et al., 1974). Each a-chain forms a left-handed helix, which is integrated into the right-handed triple helix (Freudenberg et al., 2007). The appearance of various amino acids (glycine, proline, 4-hydroxyproline) enables the helical alignment and highly ordered structure by electrostatic and hydrophobic interactions, as well as hydrogen bonds and hydration forces (Bella, Brodsky, & Berman, 1995; Gelse et al., 2003; Komsa-Penkova, Koynova, Kostov, & Tenchov, 1996; Lee, Singla, & Lee, 2001; Stenzel et al., 1974; Wallace & Rosenblatt, 2003; Zanaboni, Rossi, Onana, & Tenni, 2000). After the alignment into fibrils and, subsequently, into fibers, lysine and hydroxylysine residues form different crosslinks of varying stability between helical regions and non-helical amino and carboxyl terminals, the mez-Guille n, so-called telopeptides (Gelse et al., 2003; Go nez, Lo pez-Caballero, & Montero, 2011; Reiser, McCormick, Gime & Rucker, 1992). Collagen gels show viscous behavior and a shear-thinning effect under stress, which makes the gels suitable for the extrusion process (Wallace & Rosenblatt, 2003). Bovine collagen is applied in the food industry within the co-extrusion process displaying a decent alternative, compared to the relatively high cost of using natural casings and the growing demand for sausage guts (Barbut, 2010). Furthermore, sausages can be produced continuously. Although the patent was established 30 years ago and collagen casings are used extensively in the meat industry today, there is very little published research focusing on the physicochemical properties of collagen formulations and its impact on the mechanical properties of the co-extruded casings (Harper et al., 2012). In addition to the impact of various acids and pH values, as studied by Oechsle et al. (Oechsle, Wittmann, Gibis, Kohlus, & Weiss, 2014), co-gelling proteins affect collagen gel properties that might improve the film quality of the co-extruded collagen casings. In this study, food grade proteins, namely whey protein isolate, blood plasma protein, soy protein isolate, and gluten, were incorporated into the collagen gel matrix to modify microstructure and rheology. The proteins were selected according to their molecular size (small or large), shape (globular or extended), and surface properties (hydrophilic or hydrophobic). We hypothesized that proteins with a small molecular weight (whey and blood plasma protein) strengthen the network structure, while proteins with a high molecular weight (soy protein isolate and gluten) disturb the gel formation. These presumptions were made due to different surface characters and molecular sizes of the proteins. Small globular proteins were supposed to fit in between the meshes of the telopeptide-poor collagen matrix due to their small molecular weight. By contrast, a lack of network-forming ability was hypothesized for gluten, because it is known for its bonding via disulfide bridges, but collagen does not provide cysteine residues (Shewry & Tatham, 1997). Soy protein isolate was expected to disturb the network formation due to its size and hydrophobic surface, which predicts that soy protein isolate would behave like an inactive filler (Chen & Dickinson, 1999). The study enables targeted modification of collagen rheology and microstructure to
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form co-extruded sausage casings according to the consumer's demand. 2. Materials and methods 2.1. Materials Telopeptide-poor collagen was kindly provided by Kalle (Wiesbaden, Germany) and Protein Consulting (Singhofen, Germany) by splitting off telopeptides and intermolecular crosslinks from native collagen to obtain single collagen triple helices (Bueker, Bueker, & Grolig, 2009). Hereafter, telopetide-poor collagen is referred to as collagen. Whey protein isolate was received from Davisco (BiPRO®, Davisco Food Internationals Inc., Eden Prairie, Minnesota, USA). Blood plasma protein was provided by TasteMakers (Tastemakers GmbH, Stuttgart, Germany). Soy protein isolate was used from ADM (PRO-FAM 974 Isolated Soy Protein, ADM Speciality Products€ner Oilseeds, Chicago, Illinois, USA). Gluten was received from Kro €rke (Weizengluten vital HP, Hermann Kro € ner GmbH, IbbenbüSta ren, Germany). 2.2. Experimental plan The filled gels were prepared according to a statistical design of experiments using the SAS/QC® software (SAS 9.3 for Windows, SAS Institute Inc., Cary, North Carolina, USA). The small composite design (Hartley Method) was used as a response surface in order to generate a predictive model according to Eq. (1) with nine runs, including three center points. Five concentrations of collagen (2.8%, 3%, 4%, 5%, and 5.19%, all (w/w)) and co-gelling protein (0.36%, 0.5%, 1.25%, 2%, and 2.14%, all (w/w)) led to nine samples for each cogelling protein. According to the usage in industry, 4% (w/w) collagen containing 1.25% co-gelling protein was used as a center point. 0
G ¼ A þ B$cC þ C$cCP þ D$c2C þ E$cC $cCP þ F$c2CP ;
(1)
with the storage modulus (G0 ), the collagen concentration (cC), the co-gelling protein concentration (cCP), and the terms A-F affecting either collagen, the co-gelling protein, or the combination of both. 2.3. Sample preparation The protein concentrations were calculated based on the protein content of the samples. For telopeptide-poor collagen the protein concentration of the gel was determined in a former study (Oechsle et al., 2014). For the other proteins this information was deduced from the technical data sheet. The specimens were diluted according to the experimental plan with phosphoric acid (ChemSolute, Renningen, Germany), mixed with an Artisan 5KSM 150 and a flex edge beater 5KFE5T (KitchenAid, St. Joseph, Minnesota, USA) for 5 min at the highest setting, and adjusted to pH 3. 2.4. z-potential The z-potential was measured between pH 3 and 7 to determine the isoelectric point with a particle electrophoresis instrument (Nano, ZS, Malvern Instruments, Malvern, UK). The titration was conducted automatically with a multipurpose titrator (MPT-2, Malvern Instruments, UK). Different concentrations were applied for each protein to get the best dilution factor for each sample: 2% (w/v) whey protein isolate solution, 0.05% (w/v) soy protein isolate solution, 0.025% (w/v) gluten solution, 0.2% (w/v) blood plasma protein solution, and 0.025% (w/v) collagen suspension. The samples were diluted with phosphoric acid, adjusted to pH 3, and
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homogenized using a Stomacher Circulator 400 from Seward (West Sussex, UK) for 5 min (300 rpm). 2.5. SDS-PAGE A Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad Laboratories, Hercules, California, USA) was used for the electrophoresis. The SDS-PAGE was conducted to determine the different molecular weights of the protein subunits and to verify the collagen type. The method developed by Laemmli was applied in this study (Laemmli, 1970). All protein samples were diluted to a concentration of 1% (w/w) with double-distilled water. Sample buffer (950 mL) and b-mercaptoethanol (50 mL) were mixed. Subsequently, each protein was diluted to an appropriate concentration with sample buffer: 0.1% (w/w) for whey protein isolate, blood plasma protein, soy protein isolate, and collagen. A concentration of 0.25% (w/w) was used for gluten. The samples were incubated at 95 C for 4 min. Finally, 10 mL of each protein solution was transferred into the wells of the gel, and the electrophoresis was ran for 40 min (200 V). Afterwards, the gel was stained with Coomassie Brilliant Blue R-250 Staining Solution (Bio-Rad Laboratories, Hercules, California, USA) for 40 min and destained overnight in 15% methanol (Carl Roth, Karlsruhe, Germany) and 10% sodium acetate (Carl Roth, Karlsruhe, Germany) in double-distilled water until the bands were clearly visible. Roti-Mark prestained T852 Marker (Carl Roth, Karlsruhe, Germany) was utilized to determine the molecular weight of the proteins. 2.6. Rheology A rheometer (Physica MCR 502, Anton Paar, Graz, Austria) equipped with a plateeplate geometry with a diameter of 25 mm was chosen for the rheological measurements of the protein gels prepared. The samples were characterized via amplitude sweeps (1 Hz, 0.1e100% strain) and frequency sweeps in the linear viscoelastic region (1% strain, 0.1e100 Hz) and the storage modulus G’ was measured as indicator for the gel strength, while the loss modulus indicates the viscous behavior of the viscoelastic gel. An amount of 20 g of sample was prepared for each of the nine combinations defined by the experimental plan of collagen and cogelling protein.
2.8. Statistical analysis All measurements were repeated at least three times using duplicate samples. Means and standard deviations were calculated from these measurements using Excel (Microsoft, Redmond, VA, USA).
3. Results and discussion 3.1. z-potential Collagen displayed an isoelectric point at pH 4.0e4.9 (Fig. 1). The expected isoelectric point of collagen was 4.7, which matched the data collected (Highberger, 1939). The isoelectric point for blood plasma protein was found to be at pH 5.4e5.6. The literature states an isoelectric point at pH 4.8 for albumin from blood plasma and at pH 5.0e7.0 for globulins (Highberger, 1939). This could explain the high isoelectric point. An isoelectric point at pH 5.0e5.2 was found for whey protein isolate. According to the literature, the isoelectric point of b-lactoglobulin is at pH 5.2 and of a-lactalbumin at pH 4.1 (Klein, Aserin, Ishai, & Garti, 2010; Phan-Xuan et al., 2013). The whey protein isolate applied in this study contained 75% of blactoglobulin. This could be the reason why the isoelectric point was located in the region of pure b-lactoglobulin and above the one of a-lactalbumin. The isoelectric points of soy protein isolate and gluten were located at pH 4.9e5.1 and pH 6.7e6.9, respectively. The isoelectric point of soy protein isolate was expected at around pH 4.6 (Jaramillo, Roberts, & Coupland, 2011). For gluten, an isoelectric point of pH 7.3 was postulated by the literature (Nordqvist, € m, 2010). The divergent isoelectric points in Khabbaz, & Malmstro comparison to the data in references could appear due to a varying amino acid composition of the proteins, depending on origin and extraction conditions. This might have led to divergent isoelectric points. Nevertheless, it can be concluded that all proteins feature a positive charge at pH 3. Thus, the attractive collageneco-gelling protein interactions in the gel are based on water-mediated hydrogen-bonds rather than the repulsive electrostatic interactions (Leikin, Rau, & Parsegian, 1995).
2.7. Microscopy A Nikon Eclipse C1 (Nikon GmbH Mikroskope, Düsseldorf, Germany) combined with an inverted microscope (Nikon Eclipse Ti, Düsseldorf, Germany) was used for the confocal laser scanning microscopy (CLSM). Lasers with wavelengths of 488 nm and 543 nm from Melles Griot (Carlsbad, California, USA) were used. The samples prepared according to the experimental plan were dyed with 50 mL of 0.1% (v/v) Calcofluor (SigmaeAldrich, Buchs, Switzerland) and 50 mL of 0.1% (w/v) Eosin B (Sigma Aldrich, Buchs, Switzerland). Two emission band-pass filters were used and a magnification of 60x was applied. The samples were prepared according to the experimental plan and dipped into liquid nitrogen and freeze-dried (Lyovac GT 2, Finn Aqua Santasalo-Sohlberg, Hürth, Germany and Lyovac GT 4, LH €ln, Germany) for approximately 48 h for the scanning Leybold, Ko electron microscopy (SEM). The samples were sputtered with gold and palladium (20:80) for about 8 min with a sputtering system (SCD 040, Balzers, Bingen, Germany). The microscopy images were taken with a scanning electron microscope from Zeiss (DSM 940, Zeiss, Hamburg, Germany) under vacuum at 5000 kV. Magnifications of 500x, 1000x and 5000x were applied.
Fig. 1. z-potential of collagen, blood plasma protein, whey protein isolate, soy protein isolate, and gluten as a function of the pH.
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3.2. SDS-PAGE SDS-PAGE was performed in order to identify the molecular weight distribution of the proteins. The gel is shown in Fig. 2. The fractions of gluten can be slightly seen in lane 2. The largest fraction is below 17 kDa and led to the conclusion that the protein was hydrolysed during the treatment. Slight bands are also visible at around 50 kDa, which may belong to the u-gliadin fraction (50e65 kDa). A small fraction at ~33 kDa could corresponds to the a-, b- and g-gliadin fractions (33e40 kDa). Furthermore, this fraction could represent the low molecular weight glutenins (30e40 kDa). The bands at 95 kDa and above may refer to aggremez-Guille n et al., 2011). gates formed by the protein fractions (Go Soy protein isolate showed four bands: two at ~95 kDa, one at ~33 kDa, and one at ~17 kDa. The first two could indicate a0 , a, b, and g subunits of b-conglycinin and their aggregates. The fractions at around 33 kDa and 17 kDa may belong to subunits of the 11 S globulin (Petruccelli & Anon, 1995). Blood plasma protein showed three obvious bands between 50 and 95 kDa, which are probably due to albumin. The two bands between 17 and 23 kDa may occur due to the protein breakdown and hydrolysis during the treatment with sodium dodecyl sulfate and could represent parts of albumin s, Cuvelier, & Relkin, 2007). The band between 123 and (D avila, Pare 245 kDa might correspond to globulins with a molecular weight of about 150 kDa (Haurowitz, 2014). Whey protein isolate featured two bands around the marker band of 17 kDa. These bands probably refer to b-lactoglobulin (18.3 kDa) and a-lactalbumin (14.2 kDa) (Dekker, 1997). For collagen, both a1 and a2 bands were
Fig. 2. SDS polyacrylamide gel electrophoresis pattern of the molecular weight markers (Roti-Mark prestained T852 Marker, Carl Roth, Karlsruhe, Germany), gluten, soy protein isolate, blood plasma protein, whey protein isolate, and collagen.
121
visible (~100 kDa), as well as the b-band (~200 kDa) and the g-band (~300 kDa). Hereby, the a-bands represent monomers, the b-bands stand for dimers, and the g-bands correspond to trimers of collagen molecules. This leads to the conclusion that the sample consists mostly of collagen type I (Friess, 1998). Overall, collagen displayed the largest molecular weight, followed by blood plasma protein, gluten, and soy protein isolate. Whey protein isolate, as expected, featured the smallest molecular weight. However, blood plasma protein featured large structures which were not expected. This might be due to high temperatures during the drying step where large and fibillar structures can be formed (Wang, Xu, Huang, Huang, & Zhou, 2014). Moreover, the proteins provided a range of fractions with different molecular weights. This can be partly ascribed to degradation and polymerization occurring during the SDS-PAGE procedure. 3.3. Rheology The frequency sweeps of the different gels were performed in the linear viscoelastic region and 2.8% collagen containing 1.25% cogelling protein and 4.05% collagen as reference are shown exemplarily in Fig. 3. All samples displayed a higher storage modulus (G0 ) than loss modulus (G00 ), which means that all samples featured a gel-like behavior (Almdal, Dyre, Hvidt, & Kramer, 1993). It displays the influence of 1.25% (w/w) co-gelling protein on 2.8% (w/w) collagen, and 4.05% (w/w) collagen was used as a reference with an equal total protein content. In the range of small deformation (0.1e1 Hz), whey protein isolate and soy protein isolate showed higher storage moduli than the reference. Moreover, the addition of blood plasma protein displayed nearly no effect, whereas gluten weakened the gel, resulting in lower storage and loss moduli compared to the reference. According to the hypothesis, this was expected for the small whey protein isolate and the large gluten molecules. However, the addition of the extended soy protein isolate increased the gel strength, while the small-sized blood plasma protein weakened the structure slightly. This indicates that not only the size influences the interactions between the co-gelling proteins and collagen, but also the amino acid residues that are crucial for the surface properties and collageneco-gelling protein interactions. However, the effects discussed are only true for 2.8% (w/w) collagen concentrations. Therefore, the impact of 1.25% (w/ w) co-gelling protein on 4% (w/w) collagen was investigated and demonstrated that all co-gelling proteins had a weakening effect on the collagen structure (data not shown). Thus, soy and whey protein isolate which initially increased the gel strength of 2.8% (w/w) collagen, decreased the storage modulus of 4% (w/w) collagen. This demonstrates that the collageneco-gelling protein interactions are also concentration dependent. Therefore, Fig. 4 depicts the storage modulus of 4% (w/w) collagen containing various concentrations of co-gelling proteins at 1 Hz in order to demonstrate the impact of the co-gelling protein concentration. The dashed line represents the reference sample of 4% (w/w) collagen. Soy protein isolate showed a higher storage modulus if 2.14% (w/w) or even 4% (w/w) was added. This might be ascribed to the ability of soy protein isolate to bind to the collagen structure. The storage modulus of the gel with blood plasma protein also increased, but had a large standard deviation, indicating an inhomogeneous matrix. The addition of gluten and whey protein isolate had hardly any influence on the gel strength over the whole concentration range. This indicates a weakening effect for high whey protein isolate concentrations. To the best of our knowledge, no studies have been conducted on the influence of co-gelling proteins on collagen gels. Therefore, literature concerning the interactions of gelatin with other proteins was considered. However, care has to be taken with the interpretations, because gelatin features a globular structure, as
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Fig. 3. Storage (G0 ) and loss modulus (G00 ) of 4.05% (w/w) collagen and 2.8% (w/w) collagen containing 1.25% (w/w) co-gelling subject to the frequency f.
Fig. 4. Storage modulus G0 at 1 Hz of 4% collagen (dashed line) containing co-gelling proteins at different concentrations (cco-gelling protein).
well as different intra- and intermolecular interactions, thus, exhibiting other properties than collagen itself. Nevertheless, Pang et al. showed a weakening effect of whey protein isolate on gelatin depending on the gelatin concentration (Pang, Deeth, Sopade, Sharma, & Bansal, 2014). This was in agreement with the data of this study for 4% collagen containing 1.25% whey protein isolate. Soy protein isolate was found to exhibit proteineprotein interactions with gelatin (Denavi et al., 2009). It was also found to participate in the network formation of myofibrillar proteins (Sun & Holley, 2011). This could be an explanation for the higher storage modulus for 2.8% collagen containing 1.25% soy protein isolate. Gluten hydrolysates were reported to disturb the gelation of myofibrillar proteins (Sun & Holley, 2011). The experiments conducted also suggest weakening interactions between gluten and collagen. Furthermore, almost no effects were observed for blood plasma protein. Neither the storage nor loss modulus of collagen containing blood plasma protein was affected. This was also shown
by Hurtado et al. for the impact of blood plasma protein on frank, Pare s, & Carretero, furter style sausages (Hurtado, Saguer, Toldra 2012). Fig. 5 depicts the response surface calculated by SAS with the corresponding equation and the data points represent the storage moduli measured at 1 Hz. Only significant terms (p < 0.05) were used for the predictive model. No significant factors could be determined for blood plasma protein. Hence, the master model solely consists of terms which are not significant (p > 0.05). Therefore, the results should be considered with caution. The wide variations of the triple point might be attributed to a lack of homogenization for this mixture and the formation of an inhomogeneous structure triggered by the addition of blood plasma protein. For soy protein isolate, the storage modulus increased with increasing soy protein isolate concentration, particularly at low collagen concentrations. This is in accordance with the conclusion drawn earlier regarding soy protein isolate interacting with the collagen network and, thus, supporting the gel strength. By contrast, whey protein isolate increased the storage modulus at low collagen concentrations, whereas at higher collagen concentrations, weakening effects could be observed. These opposite effects might be attributed to whey protein isolate interfering with the collagenecollagen interactions. At lower collagen concentrations, the strengthening effect can be ascribed to the increase of the whey protein isolate content itself. Gluten did not affect the collagen structure, signified by the predictive model that only depends on the collagen concentration. The exclusion of the non-significant terms in the predictive model resulted in a lower coefficient of determination (R2; ¼ 83.06%) than to the master model that contained all terms and featured a R2; of 97.86%. Nevertheless, gluten exhibited a relatively high coefficient of determination when taking into consideration that merely one term was applied for the predictive model. The lack of influence of gluten can be accredited to the poor gelling properties at the prevalent conditions. Gluten gels are formed via a heating step and the formation of disulfide bridges (Chen & Dickinson, 1999). Collagen does not exhibit cysteine residues, nor was a heating step carried out. Thus, repulsive electrostatic forces might prevail over the attractive interactions. 3.4. Microscopy 3.4.1. Confocal laser scanning microscopy Fig. 6 depicts the confocal laser scanning microscopy images of 2.8% (w/w) collagen containing 1.25% (w/w) co-gelling protein.
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Fig. 5. The predictive model and response surface according to Eq. (1) of the storage modulus (G0 ) at 1 Hz subject to the collagen concentration (cC) and blood plasma protein (cBBP), soy protein isolate (cSPI), whey protein isolate (cWPI), and gluten (cGLU) concentration deduced from the statistical analysis.*The master model containing no significant terms was applied.
Eosin B was used to stain connective tissue and appeared red (in web version). Calcofluor was applied and generally dyed proteins that emitted in the blue (in web version) and red spectrum. Thus, selective dying of either collagen or co-gelling protein was not feasible. Nevertheless, the combination of both dying agents improved the resolution of the gel matrices compared to a single dying agent. 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein implies fibers covered by globular and extended structures. Collagen containing soy protein isolate only featured a violet network, suggesting that either only collagen is displayed or soy protein isolate is embedded in the collagen matrix and a mixed network is formed. The strengthening effects proved by the rheological measurements indicate the latter. Whey protein isolate represented an interrupted globular structure of collagen containing. This is in accordance with the conclusions drawn before with whey protein isolate disturbing the collagen network formation. For collagen containing gluten a planar extended structure was visible. Whether it corresponds to collagen or to gluten needs further investigation. Nevertheless, fibers or an interwoven network could not be observed indicating that phase separation occurred and no collagen interconnections were formed. 3.4.2. Scanning electron microscopy Scanning electron microscopy images in Fig. 7 indicated a wellordered structure for 2.8% (w/w) collagen, while the 4% (w/w) collagen structure was arranged in a crater-shaped form. The 5.19%
(w/w) collagen displayed a more chaotic structure, with the fibers forming interconnections within the pores, which also led to a decrease of the pore size. It seemed that the collagen did not have enough space to assemble in an ordered structure. Furthermore, the additional collagenecollagen interactions might explain the higher storage modulus with increasing collagen concentration. For 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein strands were visible within the pores and the matrix resembled the 5.19% (w/w) collagen sample. However, blood plasma protein affected the gel strength sparsely according to the rheological measurements. Thus, not only the presence of the interconnections within the pores might be responsible for the strengthening effects, but also the co-gelling protein network strength itself. Thus, blood plasma protein networks might be weaker than collagen matrices based on the globular and smaller structure of the protein molecules. Consequently, the addition of blood plasma protein has almost no effect on the collagen strength. The addition of soy protein isolate increased the size of the collagen pores and additional networks were visible. Moreover, the overall network structure seems to be less homogeneous than collagen containing blood plasma protein. Considering the strengthening effects of soy protein isolate determined via the rheological measurements and the predictive model, a mixed interwoven network might have been formed that results in an increase of the gel strength. The network structure was also visible in the confocal laser scanning microscopy images. Furthermore, the participation of soy protein
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Fig. 6. Confocal laser scanning microscopy images of 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein, soy protein isolate, whey protein isolate, and gluten (scale bar ¼ 50 mm).
isolate in network formation had already been found to occur during the gelation of myofibrillar proteins (Sun & Holley, 2011). For collagen containing whey protein isolate no interconnections or additional networks were visible. Moreover, a smooth and homogeneous structure with smaller pores than collagen itself could be observed. With the rheological analysis and confocal laser scanning images in mind, whey protein isolate seems to interact with collagen through hydrophobic interactions. Hence, a uniform cogelled network was formed. Therefore, whey protein isolate might have covered the collagen molecules, occupied the binding sites of collagenecollagen interactions, and thus, decreased the storage modulus. Sarbon et al. also reported that whey protein interacts with chicken skin gelatin featuring synergistic effects at low whey protein concentrations (Sarbon, Badii, & Howell, 2015). However, at higher whey protein concentrations the storage
modulus decreased. This is in accordance with the observations made in this study since the addition of whey protein upon 1.25% (w/w) weakened the collagen structure, whereas below strengthening effects could be observed. Nevertheless, gelatin features a different structure and gelling mechanism and should be therefore considered with caution. On the contrary, collagen containing gluten exhibited a separate layer on top of the collagen matrix. This conforms to the conclusion drawn earlier from the predictive model with gluten having no influence on the storage modulus. Moreover, confocal laser scanning images revealed a planar structure that could correspond to phase-separated gluten layers. Furthermore, the collagen matrix below the layer displays the same pore size as 2.8% (w/w) collagen. This also supports the hypothesis, that collagen is not affected by the addition of gluten, and neither weakening nor strengthening effects occurred.
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Fig. 7. Scanning electron microscopy images of 2.8% (w/w), 4% (w/w), and 5.19% (w/w) collagen, and 2.8% (w/w) collagen containing 1.25% (w/w) blood plasma protein, soy protein isolate, whey protein isolate, and gluten (scale bar ¼ 100 mm, 10 mm).
Fig. 8. Schematic mechanism illustrating the influence of the co-gelling proteins on the storage modulus (G0 ) of collagen matrices: The addition of whey protein isolate led to the formation of a co-gelled network with whey protein isolate interrupting the collagen interconnections and weakening the structure. Blood plasma protein featured additional strands within the collagen pores, but strengthening effects could not be observed based on the weak structure of blood plasma proteins. The incorporation of gluten resulted in phase separation and the collagen strength and structure were affected. The addition of soy protein isolate led to strengthening effects based on the formation of mixed interwoven networks.
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4. Conclusion The addition of co-gelling proteins can modify collagen strength and microstructure. The gel can be weakened or strengthened if the collageneco-gelling interactions are guaranteed and phase separation does not occur (Fig. 8). Thus, weakening effects might enable the flowability of the collagen matrix. On the contrary, increased elasticity involving strengthening effects enables shrinkage and expansion without irreversible deformation occurring in the gel matrix. Hence, the addition of co-gelling proteins displays a convenient tool to modify the collagen structure, rheology, and functionality for various applications. Possible synergistic effects caused by heat treatment of the co-gelling proteins to unfold the hydrophobic domains and to form disulfide bridges need further investigation. Acknowledgements We greatfully acknowledge Davisco Food Internationals Inc., € ner Tastemakers GmbH, ADM Foods & Wellness, and Hermann Kro GmbH for providing the protein samples. Furthermore, we would like to thank Robert Wilfer and Katja Mader (Kalle GmbH) and Gert and Marion Büker (Protein Consulting) for providing the collagen samples, Barbara Maier for her assistance with the SEM imaging, and Stefan B. Irmscher for his support in all fields. This research project was supported by the German Ministry of Economics and Energy (via AiF) and the FEI (Forschungskreis der Ern€ ahrungsindustrie e.V., Bonn): Project AiF 17478 N. References Almdal, K., Dyre, J., Hvidt, S., & Kramer, O. (1993). Towards a phenomenological definition of the term ‘gel’. Polymer Gels and Networks, 1(1), 5e17. Banerjee, S., & Bhattacharya, S. (2012). Food gels: gelling process and new applications. Critical Reviews in Food Science and Nutrition, 52(4), 334e346. Barbut, S. (2010). Microstructure of natural, extruded and co-extruded collagen casings before and after heating. Italian Journal of Food Science, 22(2), 126e133. Bella, J., Brodsky, B., & Berman, H. M. (1995). Hydration structure of a collagen peptide. Structure, 3(9), 893e906. Bueker, M., Bueker, G., & Grolig, G. (2009). In USPTO (Ed.), Collagen concentrate, use thereof and also process for production thereof. Chen, J., & Dickinson, E. (1999). Effect of surface character of filler particles on rheology of heat-set whey protein emulsion gels. Colloids and Surfaces B: Biointerfaces, 12(3), 373e381. s, D., Cuvelier, G., & Relkin, P. (2007). Heat-induced gelation of D avila, E., Pare porcine blood plasma proteins as affected by pH. Meat Science, 76(2), 216e225. Dekker, M. (1997). Structure function relationship of muscle proteins. In S. Damodaran, & A. Paraf (Eds.), Food proteins and their application (pp. 341e392). New York: CRC Press. rez-Mateos, M., An ~o n, M. C., Montero, P., Mauri, A. N., & Go mezDenavi, G. A., Pe n, M. C. (2009). Structural and functional properties of soy protein isolate Guille and cod gelatin blend films. Food Hydrocolloids, 23(8), 2094e2101. Foegeding, E. A., & Davis, J. P. (2011). Food protein functionality: a comprehensive approach. Food Hydrocolloids, 25(8), 1853e1864. Freudenberg, U., Behrens, S. H., Welzel, P. B., Muller, M., Grimmer, M., Salchert, K., et al. (2007). Electrostatic interactions modulate the conformation of collagen I. Biophysical Journal, 92(6), 2108e2119. Friess, W. (1998). Collagenebiomaterial for drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 45(2), 113e136. €schl, E., & Aigner, T. (2003). Collagensdstructure, function, and Gelse, K., Po biosynthesis. Advanced Drug Delivery Reviews, 55(12), 1531e1546. mez-Guille n, M. C., Gime nez, B., Lo pez-Caballero, M. E., & Montero, M. P. (2011). Go Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocolloids, 25(8), 1813e1827.
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