Impact of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) on functional characteristics of emulsified sausages

Meat Science 93 (2013) 240–247

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Impact of carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC) on functional characteristics of emulsified sausages Valerie Schuh a, Karin Allard a, Kurt Herrmann a, Monika Gibis a, Reinhard Kohlus b, Jochen Weiss a,⁎ a b

Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany Dept. of Food Process Engineering, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstr. 25, 70599 Stuttgart, Germany

a r t i c l e

i n f o

Article history: Received 12 April 2012 Received in revised form 29 August 2012 Accepted 30 August 2012 Keywords: Carboxymethyl cellulose Microcrystalline cellulose Lyoner-style sausage Fibers Meat Fat replacer

a b s t r a c t Inclusion of fibers, such as carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC), at the expense of fat or protein in meat batters could be used to produce healthier sausages while lowering production costs. To study the impact of CMC/MCC on structural/functional characteristics of emulsified sausages, standard-fat Lyoner-style sausages were formulated with CMC/MCC at concentrations of 0.3–2.0%. Methods of analysis included rheology, water binding capacity (WBC), texture measurements, and Confocal Laser Scanning Microscopy (CLSM). WBC, texture measurements, and rheology all indicated that addition of CMC (>0.7%) led to destabilization of the batter, which upon heating could no longer be converted into a coherent protein network, a fact that was also revealed in CLSM images. In contrast, MCC was highly compatible with the matrix and improved firmness (1405–1651 N/100 g) with increasing concentration compared to control (1381 N/100 g) while keeping WBC (4.6–5.9%) with b 2% MCC at the level of the control (4.8%). Results were discussed in terms of molecular interactions of meat proteins with celluloses. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Emulsified meat batters contain meat, fat, salt, ice, and spices. Recently, the meat industry has been searching for additives that render meat products nutritionally more beneficial (e.g. by reducing their caloric content, or by adding fibers, minerals or bioactive compounds). This reduction in meat content comes also in the context of demands to reduce the CO2-footprint of end products. Components that are being used to replace meat include non-meat proteins such as for example milk, soy, wheat, and lupine proteins (Alamanou, Bloukas, Paneras, & Doxastakis, 1996; Homco-Ryan et al., 2004). However, a substantial problem of replacing meat with non-meat proteins is that many of these other proteins have a substantial food allergen potential and thus require declaration. Alternative components that can be used as functional ingredients to replace meat are hydrocolloids such as for example carrageen (Candogan & Kolsarici, 2003; Hsu & Chung, 2000), xanthan (Ramírez, Barrera, Morales, & Vázquez, 2002), and guar gum (Ulu, 2006). In contrast to the above-listed non-meat proteins, they do not have an allergen potential and thus do not need to be declared as such. Moreover, they may afford products with better water binding capacities than non-meat proteins in particular in low-fat products, where water content per overall mass of product increases. An improved texture of low-fat products was achieved with using carrageenan (Ayadi, Kechaou, Makni, & Attia, 2009). Authors found an increase in water binding

⁎ Corresponding author. Tel.: +49 711 459 24415; fax: +49 711 459 24446. E-mail address: [email protected] (J. Weiss). 0309-1740/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2012.08.025

capacity, firmness and cohesiveness of turkey meat sausages when low concentrations were added. At levels of 0.2–0.5 wt.% carrageenan an increase in gel elasticity of sausages was observed. Microcrystalline cellulose (MCC) and carboxymethyl cellulose (CMC) are both cellulose derivatives that have been approved for use in foods as fiber additives (Holtzapple, 2003). MCC is mostly produced from wood pulp although cotton and liner can also be used by treatment with mineral acid to obtain alpha-cellulose. The alpha cellulose is then partially depolymerized and purified to yield MCC. The properties of MCC depend on the depolymerization process which often involves application of mechanical forces to generate cellulose crystals of specific sizes. CMC is obtained from alkali-cellulose after reaction with monochloroacetic acid. Its properties depend on the degree of chain polymerization and the degree of substitution of the basic glucose blocks in cellulose with glycopyranose. MCC has been successfully used in fruit juices as thickeners and in low-fat products to impart a creamy mouth feel. CMC has been used in various dairy products (Bayarri, González-Tomás, & Costell, 2009), specifically in ice cream (Regand & Goff, 2002) and in bakery products (Xue & Ngadi, 2009). The functionality of hydrocolloids in meat batter depends on the possible interaction scenarios between proteins and hydrocolloids as reported in numerous studies (Doublier, Garnier, Renard, & Sanchez, 2000; Grinberg & Tolstoguzov, 1997). Those studies however have typically been carried out in solutions that contained only low concentrations of each of the polymers. In contrast, many meat products such as for example emulsified sausages contain large concentrations of proteins that may exceed 15% proteins (Sampaio, Castellucci, Pinto

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e Silva, & Torres, 2004). Thus only few studies on the interaction of hydrocolloids with proteins have been conducted in highly concentrated systems and even less studies have carried out in-depth physiochemical investigations with CMC or MCC in meat products (Barbut & Mittal, 1996; Mittal & Barbut, 1993; Ordóñez, Rovira, & Jaime, 2001). The objective of this study was therefore to assess the impact of addition of MCC and CMC to meat products. Since 70% of all consumed meats in Germany are meat products and amongst those roughly 30% are emulsified sausages, we chose to use Lyoner-style sausages as a model meat system. CMC and MCC at various concentrations were added to a standard emulsified sausage formulation and the structural and functional properties of the generated batters and sausages (after heating of the batter) assessed using Confocal Laser Scanning Microscopy, dynamic rheology, texture analysis, color, and water binding capacity measurements. Since CMC is a charged hydrocolloid while MCC is an uncharged one, we hypothesized that the two hydrocolloids may show profound differences in their interaction behavior with an emulsified meat batter. 2. Materials and methods 2.1. Materials Medium molecular weight carboxymethyl cellulose (CMC) from wood pulp with a degree of substitution (°DS) of 0.55–0.75 and microcrystalline cellulose (MCC) from wood pulp obtained by spray drying with a low content of 8.4–13.7 wt.% of CMC was provided by Danisco (Aarhus, Denmark). Both materials were used without further purification. Distilled water was used in the preparation of CMC and MCC dispersions for addition to sausage formulations. Fluorescent stains Calcofluor White and Nile Red and acetone as solvent for the stains were purchased from Fluka Chemie AG (Buchs, Switzerland). For the standard-fat Lyoner sausages, meat and back fat were obtained from a local retailer (Mega, Stuttgart, Germany). The meat contained after standardization by a master butcher approximately 19 wt.% protein, 73 wt.% water, and 8 wt.% fat. The back fat contained around 2 wt.% protein, 8 wt.% water, and 90 wt.% of fat. 2.2. Preparation of hydrocolloid dispersion Concentrated CMC and MCC dispersions containing 10 wt.% CMC or MCC in 90 wt.% water were prepared by adding water to a 13-liter bowl chopper equipped with 6 rotating knives (Mado Garant, Maschinenfabrik Dornhan GmbH, Dornhan, Germany). CMC or MCC was added during the first minute of the chopping process at low rotational knife speeds (knife speed: 1400 rpm, bowl speed: 12 rpm). The bowl chopper was then set to the highest speed (knife speed: 2800 rpm, bowl speed: 24 rpm) to disperse the hydrocolloids for 4 min. 2.3. Sausage preparation Sausages were prepared following the German “Magerbrät” process for emulsified sausages. Different concentrations of CMC and MCC (0, 0.3, 0.5, 0.7, 1.0, 1.5 and 2 wt.%) were added to the basic recipe that consisted of 50 wt.% meat, 28 wt.% fat and 22 wt.% water. In batches containing cellulose derivatives the amount of ice added during the meat chopping process (see below) was adjusted. This was done because the cellulose derivatives were added to the batters in a pre-hydrated gel form that already contained water. The amount of ice was accordingly varied such that all batches had similar final water contents. Minced meat (3 mm) was added to the bowl chopper (see above). During the first minute of chopping, curing salt (1.8%), diphosphate (0.2%), and half of the ice were added. After 120 s, the hydrated CMC and MCC dispersions, fat, and spices were added to the batter. The bowl chopper was set to operate at high speed (knife

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speed: 2800 rpm, bowl speed: 24 rpm) for 60 s and the remaining ice was added to the batter. The emulsified batter was generated by applying another 150 s of high speed chopping (knife speed: 2800 rpm, bowl speed: 24 rpm). Temperatures of the meat batters after chopping ranged from 11.2 to 14.8 °C. The pH of the batters ranged from 5.7 to 5.8 and was not affected by hydrocolloid addition. The batter was then stuffed with a piston-driven filler (Mado Patron, Maschinenfabrik Dornhan GmbH, Dornhan, Germany) into impermeable fibrous casings (70 mm diameter, Nalo Top, Kalle GmbH, Wiesbaden, Germany). The stuffed sausages were then heated in a universal smoking and heating chamber (Unigar 1800 BE Compact with software Ness Digitronic 4, Ness & Co. GmbH, Remshalden, Germany) to a core temperature of 72 °C following industrial standard procedures in Germany. The heated sausages were then cooled down and stored at 4 °C until use in subsequent analysis. 2.4. Dynamic rheology Fresh meat batters were subjected to dynamic rheological measurements using a rotational rheometer (MCR 300, Anton Paar, Stuttgart, Germany) operated with a Rheoplus/32 V3.31 software immediately after preparation of the fresh batter. Measurements were carried out with a plate–plate geometry measurement system (diameter: 25 mm, gap width: 1.5 mm). The temperature was maintained at 25 °C during an initial frequency sweep measurement of storage (G′) and loss (G″) moduli at frequencies from 0.1 to 100 Hz at a strain of 0.01 (1%). After the initial sweep, samples were heated from 25 to 80 °C on the rheometer using a Peltier-type heating system to induce gelation and then cooled back to 25 °C. The sample was then again subjected to a second frequency sweep using the same conditions as in the first sweep. All measurements were repeated three times using duplicate samples. 2.5. Water binding capacity Fresh meat batters (22–24 g) were filled in eight containers (diameter: 3.5 cm, height: 5 cm). Containers were sealed and their weight recorded. Containers were then heated in boiling water for 45 min to a uniform temperature (~ 98 °C) to induce gel formation in the batter following the procedure suggested by Honikel (1982). Samples were finally cooled and removed from the containers to again record their weight. The water loss percentage WTLoss, an expression of the water holding capacity of the heated batter, was then determined as

WT Loss ½%  ¼

Minitial −Mend  100 Minitial

ð2:1Þ

with Minitial being the weight of the sample prior to heating and Mend after heating. 2.6. Firmness The firmness of the heated meat batter was determined using a Kramer shear cell (Instron Model 1011, Instron Corporation Ltd., Canton, Massachusetts, USA) which imitates biting into a sausage slice. For each sample, 10 slices of 4 mm thickness were prepared and cut into disks with 4.5 mm diameter. The weight of each slice (~5.9–6.1 g) was recorded. To prevent drying, slices were wrapped into a plastic foil and stored at 7 °C for 1 h to ensure that all samples had the same temperature. Samples were then subjected to a shear test in the Kramer shear cell and the maximum force needed to penetrate the sample was recorded. A normalized force (N/100 g) was calculated from the maximum force using the recorded weight of the samples.

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2.7. Colorimetery Color of all samples was measured using a chromatometer (Typ CR 200, Konica Minolta Sensing Europe B.V., Nieuwegein, Netherlands). Results were recorded as L*, a*, and b* color values. For each sample, eight slices of 3 mm thickness were cut and analyzed. The colorimeter was calibrated against a standard white plate prior to each measurement. Illumination was carried out with a D65 light source. 2.8. Confocal Laser Scanning Microscopy (CLSM) Structural alterations in the sausage matrix were assessed by Confocal Laser Scanning Microscopy. Sausages were cut into disks of 2 cm diameter and 7.2 mm slice thickness to fit into custom made object slides. Samples for CLSM analysis were taken from different sausages of the same batch randomly at different points throughout the sausage. 10 pictures per slide were recorded. Half the samples were stained with 50 μl Calcofluor White (emission maximum at 433 nm), while the other half was stained with 50 μl Nile Red. Samples were kept in the dark under an aluminum foil to prevent photo-bleaching and imaged within 20 min after the application of the fluorescent stains using a confocal laser scanning microscope (Nikon D-Eclipse C1, Nikon GmbH Mikroskope, Düsseldorf, Germany), attached to a Nikon Eclipse Ti inverted microscope (pinhole of 30 μm). Samples were excited with two laser beams, a red diode laser (638 nm, 25 mW) and a blue argon-ion laser (single line laser 488 nm, 10 mW). Images were recorded at a magnification of 20× (Planapochromat 20×/0.75/1.0 mm), and analyzed with the EZ-C1 (Gold version 3.70) program of Nikon. 2.9. Statistical analysis Experiments were repeated once. Sample measurements were performed in multiples (3× rheology, 8× WBC, 10× firmness and color). Means and standard deviations were calculated from these measurements using Excel (Microsoft, Redmond, VA, USA). Statistical analysis was carried-out on firmness measurements, water losses and color values using the Statistical Analysis System (SAS 9.2, SAS Institute Inc., North Carolina, USA). In a first step, data was tested for normal distribution. In a second step, a variance analysis using a Tukey-Test with the “Generalized Linear Model” (GLM) procedure was conducted to determine significant differences (α= 0.05) between sample and control batches. 3. Results and discussion

Fig. 1. Dynamic oscillatory rheology (G′ and G″) of unheated and heated meat batters (25 °C, 1% strain); number of samples tested: n= 3. (A) Unheated batter containing 0 wt.% cellulose (control), 2 wt.% CMC or MCC; (B) Heated batter containing 0 wt.% cellulose (control), 2 wt.% CMC or MCC.

3.1. Rheological properties of unheated and heated meat batters Fig. 1 shows exemplary results of a frequency scan of heated and unheated meat batters containing either no hydrocolloid (control), 2 wt.% CMC or 2 wt.% MCC. Even prior to heating, all samples displayed a strong gel-like character (Fig. 1A). All samples had elastic moduli that were larger than their viscous moduli. Upon heating, the rheological properties of all samples changed significantly (Fig. 1B). Both G′ and G″ increased by more than one magnitude when compared at similar oscillation frequencies with heated samples. The change in the frequency dependent slope of G′ and G″ suggests that gels became substantially more elastic, which may be attributed to crosslinking of solubilized proteins in the meat batter. Samples that contained MCC had similar or higher elastic and loss moduli when compared to the control while samples containing 2 wt.% CMC were noticeably weaker than controls both in their heated and unheated states. Fig. 2 shows G′ and G″ at an oscillation frequency of 1 Hz and 10 Hz for all samples containing between 0 and 2 wt.% CMC and MCC prior to and after heating. Two very different effects are visible from the figure, namely, that upon addition of increasing concentrations of MCC in the heated and the unheated batter, both storage and loss moduli increased

with increasing concentration (Fig. 2A), while in the case of CMC, the opposite effect, that is a decreasing storage and loss moduli with increasing hydrocolloid concentration can be observed (Fig. 2B). A decrease in the gel strength of a meat product by addition of CMC has recently been reported by Chattong and co‐authors (Chattong, Apichartsrangkoon, & Bell, 2007), albeit the authors did not suggest what may have caused this decrease. Addition of low quantities of hydrocolloid (0–1 wt.%) led to significant losses in both storage and loss modulus though no changes in loss tangent were observed. 3.2. Firmness Fig. 3 shows the results of measurements of the firmness of heated samples containing 0–2 wt.% CMC or MCC. Firmness initially increased by 306 N/100 g (235–254) upon addition of 0.3 wt.% CMC before slightly decreasing. At 0.5 and 0.7 wt.% CMC, respectively, firmness was 254 and 236 N/100 g higher than the control batch (1250 N/100 g) before decreasing sharply to less than 1/3 of the peak firmness when 2 wt.% CMC had been added. This is in contrast with findings of Lin, Keeton,

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Fig. 3. Texture analysis (firmness) of heated meat batters containing CMC and MCC at various hydrocolloid concentrations (0–2 wt.%) (number of samples tested: n = 10).

Addition of 1 wt.% CMC caused a sudden loss of firmness of sausages. Sausages that contained 2 wt.% CMC had a firmness of 486 N/100 g while the control batch had a firmness of 1250 N/100 g. Change of firmness in all samples upon addition of CMC was statistically significantly different from the control. In contrast, firmness increased when MCC was present in the heated meat batter. Samples containing 2 wt.% MCC had a firmness that was approximately 20% higher than that of the control. All samples with the exception of 0.3 wt.% MCC were statistically significantly different from the control (Table 1). In the study of Barbut and Mittal (1996), MCC had also been used to formulate low fat frankfurters at a concentration of 0.5 wt.%. The authors similarly found that MCC reinforced the firmness of the product compared to the control batch. 3.3. Water loss of heated batters

Fig. 2. G′ and G″ (storage and loss modulus) of unheated and heated meat batters at 1 Hz and 10 Hz (25 °C; 1% strain); number of samples tested: n = 3. (A) CMC, (B) MCC; bh = before heating; ah = after heating (number of samples tested: n= 3).

Gilchrist, and Cross (1988) who assessed the impact of addition of different CMCs with different degrees of substitution. At a concentration of 0.25 wt.% CMC added to low-fat frankfurter formulations Lin found an immediate decrease in firmness when compared to control batches. Moreover, no significant difference in firmness was found when CMCs with different degrees of substitution (low: 0.65–0.9 °DS, high: 0.85– 0.95 °DS) and molecular weights (low: 250 kDa, high: 700 kDa) were compared with each other. A possible explanation could be that the concentration used in the study of Lin et al. (1988) was possibly too low to observe the effects reported in this study. An earlier study (Barbut & Mittal, 1996) found no changes in the texture with the addition of 0.35 wt.% CMC in a low fat-frankfurter. It is not quite clear where the reported differences in results stem from, but production processes, base formulations and hydrocolloids differed, which may contribute to the disagreements. However, authors had evacuated all meat batters after chopping prior to filling in casings, a process which has been known to cause a hardening of sausages that may have overridden the effect of CMC onto the batters' structures and qualities.

Table 1 shows the water loss of CMC and MCC containing batters after heating as a function of hydrocolloid concentration. All batches except the sample that contained 0.7 wt.% CMC were significantly different from the control. Addition of CMC of 0.3 wt.% and 0.5 wt.% increased the water loss by 60% and 90%, respectively, in comparison with the control. A decrease of water loss and thus an increase of water binding was found when more than 0.7 wt.% CMC were added. Sausages that contained 1–2 wt.% had half the water losses and thus double the water holding capacity than controls. The reason for the increased water loss at 0.3– 0.5 wt.% CMC has yet to be found. Potentially, the salt concentration in the meat batter was high enough to have influenced the molecular conformation of CMC, which may have affected its ability to bind water. Rosilio reported that addition of salt inhibits the water absorption of CMC (Rosilio, Albrecht, Baszkin, & Merle, 2000). Sodium ions may cause an aggregation of alkyl chains which may decrease the effective volume of the hydrocolloid, which reduces the total volume of water that the hydrocolloid can sweep out. At higher CMC concentrations and increasing CMC to salt weight ratios, the effect of salt may diminish and CMC molecules may retain their original conformation allowing them to bind higher amounts of water. Here, the degree of substitution may play a role as the interaction of CMC with water depends on the number of functionalized side groups on the cellulose backbone. Clearly more in-depth molecular studies may be required to elucidate the observed results. The effect of addition of MCC on water loss was much less pronounced than when CMC was added. Only the samples containing 1 wt.% and

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Table 1 Effect of the addition of CMC and MCC on water binding capacity, firmness and L*, a*, b* color values of Lyoner (WT = water; n = number of samples tested; results displayed as means ± standard deviation; α = 0.05). Sample

WTloss (%), n = 8

Firmness (N/100 g), n = 10

Color values, n= 10 L*

Control 1 0.3 wt.% 0.5 wt.% 0.7 wt.% 1.0 wt.% 1.5 wt.% 2.0 wt.% Control 2 0.3 wt.% 0.5 wt.% 0.7 wt.% 1.0 wt.% 1.5 wt.% 2.0 wt.%

c

CMC CMC CMC CMC CMC CMC MCC MCC MCC MCC MCC MCC

5.5 ± 1.02 9.2 ± 0.7b 11.0 ± 0.8a 6.3 ± 0.6c 3.7 ± 0.5d 3.5 ± 1.1ed 2.2 ± 1e 4.8 ± 0.8cd 5.3 ± 0.6cbd 4.6 ± 0.5d 5.8 ± 0.5cb 5.9 ± 1.2b 5.5 ± 0.6cbd 8.3 ± 0.5a

c

1250 ± 41 1557 ± 60a 1510 ± 46ab 1485 ± 62b 1111 ± 45d 730 ± 31e 487 ± 20f 1381 ± 34c 1405 ± 69c 1524 ± 34b 1509 ± 65b 1514 ± 60b 1631 ± 45a 1651 ± 59a

a* b

71.05 ± 0.43 73.68 ± 0.48a 73.68 ± 0.8a 74.47 ± 0.61a 74.00 ± 0.58a 73.45 ± 0.77a 74.09 ± 0.75a 69.71 ± 0.61e 70.67 ± 1.02d,e 71.98 ± 0.91d,c 72.23 ± 1.19c 72.64 ± 0.81b,c 73.59 ± 0.61ab 74.39 ± 0.74a

b* a

9.94 ± 0.18 10.32 ± 0.34a 9.91 ± 0.43a 10.76 ± 0.28a 9.91 ± 0.45a 10.40 ± 0.35a 10.29 ± 0.40a 10.93 ± 0.33a,b 10.67 ± 0.46a,b 11.26 ± 0.36a 11.01 ± 0.46a,b 10.81 ± 0.45a,b 10.66 ± 0.27a,b 10.49 ± 0.42b

8.32 ± 0.08b 8.37 ± 0.06a 8.23 ± 0.07a 8.49 ± 0.19a 8.52 ± 0.15a 8.51 ± 0.06a 8.53 ± 0.13a 8.28 ± 0.14b,c 9.48 ± 0.10a,b,c 9.39 ± 0.18c 9.53 ± 0.13a,b 9.44 ± 0.20a 9.34 ± 0.19a 9.32 ± 0.09a

a–f

Means with the same letter within the same column of CMC/MCC are not significantly different (α b 0.05).

2 wt.% MCC were significantly firmer than the control. Upon addition of 2 wt.% MCC the water loss increased from 4.8% (control) to 8.3%. The high water loss at the largest concentration of MCC could be due to the alterations in the protein gelation process. Studies have shown that crystalline cellulose may provide a heat stabilizing effect that in the case of sausages may have prevented a thorough protein gel network to be formed (Akimoto, 2008; Nishimura, Morita, Funemi, & Yoshihira, 2009; Thomas, 1986). 3.4. Colorimetry Table 1 shows results of the color measurement of samples containing CMC or MCC after having been heated. The a*-value is a particularly suitable indicator of changes to the red coloring of meat products. However, no significant differences of CMC and MCC containing samples and the control were found. Both CMC and MCC did not show a major impact on the red color of the sausages. The lightness values (L*) of the sausage did not change significantly upon addition of CMC, L*-values ranged within 73.45–74.47 which is a change of about one unit. Only L*-value changes of above one unit are detectable by the human eye (Poynton, 1996). Since no extensive water losses in hydrocolloid-containing sausages were observed, this is not truly surprisingly. If L*-values increase in products with the same base formulation, it is often a sign for a pronounced water loss. This is for example the case for PSE meat (pale, soft, and exudative) were the L*-value may be 10 units higher than in normal pork meat (pH 5.6–6) (Barbut, 1993; Chmiel, Słowiński, & Dasiewicz, 2011). Within MCC containing batches rising L*-values can be observed with rising MCC concentration. Changes should be too small to influence consumer choice though. Changes in the color of heated meat batters were only found for b*-values, where MCC influenced the yellow color of sausages. Typically yellowness in sausages corresponds to concentrations of fat present in the product. Sausages with a higher amount of fat have commonly higher b*-values. This increase in yellowness was also observed upon the addition of MCC. The b*-value slightly increased by roughly 1, which is however close to the limit that can be detected by the human eye (Mendoza, Dejmek, & Aguilera, 2006). 3.5. Structure of heated meat batters CLSM images were recorded to assess whether changes to the structure of the heated meat products occurred when cellulose derivatives were added. Goal of the visualization was to determine changes in the structure of the protein matrix. CLSM is an effective method to visualize compartmentalization in multicomponent system. For example, CLSM

images of gelatin–maltodextrin mixed system have previously shown phase separation of protein–polysaccharide matrix when salt was added or pH and temperature altered (Lorén, Langton, & Hermansson, 1999). Figs. 4 and 5 show CLSM images of the control (Figs. 4A–B and 5A–B), as well as samples containing CMC and MCC at concentrations of 0.5 wt.% and 2.0 wt.%, stained with two different fluorescence colors, Calcofluor White and Nile Red. Fig. 5A shows the control sample which was stained with Calcofluor White, indicative of the protein matrix. The black voids in the image are mostly fat or in some rare cases air. The second stain Nile Red (Control: Fig. 5B) was used to visualize the location of the dispersed fat particles. This stain also slightly stained the protein matrix which allowed the position of fat to be distinguished from potential air or water voids. Moreover, this stain allowed an assessment of the degree of emulsification of fat in the protein matrix. Addition of CMC (Fig. 4C–F) caused increasing discontinuities in the protein matrix to appear. CLSM images of samples containing 0.5 wt.% CMC (Fig. 4C–D) show that the protein-matrix is gradually being disrupted. The protein matrix is no longer one interconnected network but begins to be an assembly of smaller protein aggregates that however occasionally still connect. In other words the network attains an increasingly fractal dimension. Addition of 1 wt.% CMC caused a nearly complete breakdown of the previously continuous network (data not shown). Upon addition of 2 wt.% CMC (Fig. 4E–F) only individual small protein aggregates that are no longer interconnected are visible. Fundamentally, the heated batter has transitioned from a network to a dispersion of solid protein particles dispersed in water and mixed with fat particles. The network-disrupting effect of CMC has been reported by Chattong et al. (2007) as well. The authors speculated that CMC may have acted as a “detergent” in the matrix and interfered with protein crosslinking. The interaction between CMC and meat proteins has also been investigated by Morin, Temelli, and McMullen (2004). They analyzed an uncooked and cooked meat matrix by Scanning Electron Microscopy. There, CMC was located in the water pockets that were present in the protein network with accumulations at the protein–water interface. They concluded that the negatively charged carboxyl-groups of CMC were binding to the positively charged amino-groups of proteins. However, after heating, a well ordered protein matrix was visible and the CMC was no longer unevenly distributed. Likely, this lack of a visible structure change was due to the low concentration of 0.3 wt.% CMC used in their study. While addition of less than 2 wt.% MCC (Fig. 5C–D) did not noticeably impact the structure of the heated meat batter, addition of 2 wt.% MCC led to alterations in the meat batter organization (Fig. 5E–F). Little void spaces are becoming apparent that could be associated with a

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Fig. 4. CLSM images of Lyoner containing different concentrations of CMC (0–2 wt.%), stained with Calcofluor White (Emax = 600 nm) and Nile Red (Emax = 433 nm). Objective 20×/ 0.75/1.0; (A) control, Calcofluor White; (B) control, Nile Red; (Ca and Db) 0.5 wt.% CMC; (Ea and Fb) 2 wt.% CMC; aCalcofluor White, bNile Red.

Fig. 5. CLSM images of Lyoner containing different concentrations of MCC (0–2 wt.%), stained with Calcofluor White (Emax = 600 nm) and Nile Red (Emax = 433 nm). Objective 20 ×/0.75/1.0 mm; (A) control, Calcofluor White; (B) control, Nile Red; (Ca and Db) 0.5 wt.% MCC; (Ea and Fb) 2 wt.% MCC; aCalcofluor White, bNile Red.

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swelling of the MCC in the batter and a subsequent shrinkage during heating associated with a liberation of water. 3.6. Mechanistic insights and potential explanation of results Proteins and hydrocolloids are known to interact in a variety of ways. Firstly, hydrocolloids in general may carry positive or negative charges or be uncharged. It should be pointed out though that few hydrocolloids carry positive charges, and most hydrocolloids are either negatively charged or uncharged. A noticeable exception is chitosan, a polysaccharide that can be obtained by alkaline deacetylation from chitin. Proteins on the other hand are positively charged below their isoelectric point, are neutral at the isoelectric point and negatively charged above it. Upon mixing of solutions of hydrocolloids and proteins, a number of different phenomena may therefore be observed depending on the chemical nature (molecular structure, weight, and charge) and concentration of proteins and hydrocolloids. (1) Proteins and hydrocolloids may be co-soluble. In this case, a single phase system that contains both polymers simultaneously is formed. (2) Proteins and hydrocolloids may be thermodynamically incompatible. In this case, the two polymer phase separate into two phases, one that is rich in hydrocolloids and one that is rich in just proteins. (3) Proteins and hydrocolloids may aggregate and form insoluble complexes. In most cases, the aggregated complexes will over time precipitate and form a flocculated polymer complex layer at the bottom of the test container. Environmental conditions such as pH, temperature, and ionic strength also influence the interaction (V.B.Tolstoguzov, 1991). The specific influence of these three parameters on the magnitude and nature of the interactions depends on the molecular characteristics of the polymers. Cellulose for example is a nonionic polymer. Since it does not carry a charge, the polymer is not affected by changes in pH unless the pH is low enough to induce hydrolysis. Carboxymethyl cellulose contains a percentage of anionically charged monomers with an approximate pKa value of 4.0. Thus, above a pH of 4.0, CMC is consistently negatively charged. In contrast, the pH of meat proteins depends strongly upon the pH, i.e. they carry an overall positive charge below their isoelectric point (approx. 5.0– 5.1) and a negative charge above their isoelectric point. Thus, one can expect that there is no overall electrostatic interaction between cellulose and meat proteins regardless of pH, but that there will be an attractive electrostatic interaction between CMC and meat proteins below a pH of 5.0 and a repulsive interaction above a pH of 5.0. Electrostatic interactions are also typically modulated by the presence of ions in the system. Characteristically, at higher salt concentrations, the magnitude of the electrostatic interactions (e.g. between CMC and meat proteins) may decrease due to the added ions shielding the surface charges of the respective polymers. As a consequence, both polymers experience lower attractive or repulsive forces. Since temperature may impact both electrostatic and Van der Waals interactions, it may affect interactions in combinations of both uncharged and charged polymers (e.g. cellulose and meat proteins, or CMC and meat proteins). Clearly because of the complexity of the nature of the interactions, additional experiments would be required to quantify and qualify the specific influence of temperature, ionic strength and pH on the observed phenomena (Turgeon, Beaulieu, Schmitt, & Sanchez, 2003). Moreover, other additives such as e.g. sugars may also impact the interactions. For example sucrose has shown to improve the solubility of proteins and as such also the co-solubility of protein–polysaccharide solutions (Antipova & Semenova, 1995). Finally, combinations of changes of the different parameters max lead to an unexpected behavior e.g. the addition of salt to an acid gelatin/iota-carrageenan system has shown to improve compatibility of the two polymers at pH 6.5 (Michon, Cuvelier, Launay, Parker, & Takerkart, 1995).

To date the microstructure changes in the meat batters and possible hydrocolloid–meat protein interactions still have not been studied closely. For this reason and to fully understand our results further more detailed investigations are underway at the moment. 4. Conclusion CLSM analysis proved to be a useful method for the determination of changes to the structure of the meat batter matrix. It was feasible to visualize both the structure of the dispersed fat particles as well as the nature of the protein network. The CLSM images showed that profound alterations to the structure of the heated meat batter occurred when CMC was added while only little change occurred when MCC was added. This correlated with the subsequent food quality attributes of the batter and the generated sausage. Particularly firmness and viscoelasticity measurements showed that the mechanical strength of the meat batter prior to and after heating was greatly reduced when CMC was added. In contrast, firmness increases could be observed when MCC was added. Results have important implications for meat scientists and the meat industry. They demonstrate that molecular interactions are of key importance when using novel ingredients such as charged and uncharged fibers. Depending on the molecular nature and the concentration of the added ingredient, structure of the product may change in unexpected ways, which affects a whole host of food quality attributes. Nevertheless, results with CMC and MCC are very promising, as CMC may decrease the texture firmness of high-protein low-fat sausage formulations and MCC may improve the integrity of the protein gel network and thus offers a mean to lower protein concentration while representing compounds with known physiologically beneficial functionalities. Acknowledgments This research has been supported by DANISCO A/S, Copenhagen, Denmark. The authors also wish to specially thank Jesper Kampp, Hermann Sloot, Christian Vogel, and Karen Marie Sondergaard for their help. References Akimoto, M. (2008). Composition composed of highly dispersible cellulose complex and polysaccharide. Alamanou, S., Bloukas, J. G., Paneras, E. D., & Doxastakis, G. (1996). Influence of protein isolate from lupin seeds (Lupinus albus ssp. Graecus) on processing and quality characteristics of frankfurters. Meat Science, 42, 79–93. Antipova, A. S., & Semenova, M. G. (1995). Effect of sucrose on the thermodynamic incompatibility of different biopolymers. Carbohydrate Polymers, 28, 359–365. Ayadi, M. A., Kechaou, A., Makni, I., & Attia, H. (2009). Influence of carrageenan addition on turkey meat sausages properties. Journal of Food Engineering, 93, 278–283. Barbut, S. (1993). Colour measurements for evaluating the pale soft exudative (PSE) occurrence in turkey meat. Food Research International, 26, 39–43. Barbut, S., & Mittal, G. S. (1996). Effects of three cellulose gums on the texture profile and sensory properties of low fat frankfurters. International Journal of Food Science and Technology, 31, 241–247. Bayarri, S., González-Tomás, L., & Costell, E. (2009). Viscoelastic properties of aqueous and milk systems with carboxymethyl cellulose. Food Hydrocolloids, 23, 441–450. Candogan, K., & Kolsarici, N. (2003). Storage stability of low-fat beef frankfurters formulated with carrageenan or carrageenan with pectin. Meat Science, 64, 207–214. Chattong, U., Apichartsrangkoon, A., & Bell, A. E. (2007). Effects of hydrocolloid addition and high pressure processing on the rheological properties and microstructure of a commercial ostrich meat product “Yor” (Thai sausage). Meat Science, 76, 548–554. Chmiel, M., Słowiński, M., & Dasiewicz, K. (2011). Lightness of the color measured by computer image analysis as a factor for assessing the quality of pork meat. Meat Science, 88, 566–570. Doublier, J. L., Garnier, C., Renard, D., & Sanchez, C. (2000). Protein–polysaccharide interactions. Current Opinion in Colloid & Interface Science, 5, 202–214. Grinberg, V. Y., & Tolstoguzov, V. B. (1997). Thermodynamic incompatibility of proteins and polysaccharides in solutions. Food Hydrocolloids, 11, 145–158. Holtzapple, M. T. (2003). Cellulose. In C. Benjamin (Ed.), Encyclopedia of food sciences and nutrition (pp. 998–1007). Oxford: Academic Press. Homco-Ryan, C. L., Ryan, K. J., Wicklund, S. E., Nicolalde, C. L., Lin, S., McKeith, F. K., et al. (2004). Effects of modified corn gluten meal on quality characteristics of a model emulsified meat product. Meat Science, 67, 335–341. Honikel, K. O. (1982). Wasserbindung und “Fettemulgierung”bei der Verarbeitung zu Brühwurstbräten. Fleischwirtschaft, 62, 16–22.

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