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Influence of calcium on white efflorescence formation on dry fermented sausages with co-extruded alginate casings
Food Research International 131 (2020) 109012
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Influence of calcium on white efflorescence formation on dry fermented sausages with co-extruded alginate casings Jonas Hilbig, Katrin Hartlieb, Kurt Herrmann, Jochen Weiss, Monika Gibis
T
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Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: White efflorescence Calcium concentration Alginate casing Dry fermented sausage
The effect of the concentration of calcium in the crosslinking solution during co-extrusion of dry fermented sausages with calcium alginate casing on the white efflorescence was investigated. With the co-extrusion technology, a continuous sausage production is possible and, furthermore, snack products with very small calibers can be produced. Therefore, crosslinking solutions with different concentrations of CaCl2 (15, 20, 25, and 30%) were used during the production. High concentrations of calcium led to a very rapid and intensive white efflorescence formation; the efflorescences covered ~90% of the surface of the samples produced with 20–30% of CaCl2 after 8 weeks of storage. However, the batch produced with the lowest amount of CaCl2 showed a slow efflorescence formation and a significantly decreased area covered by it (~70% after 8 weeks). The differences in the formation were attributed to the excess of calcium on the surface of the sausages (saturated calcium alginate film), therefore leading to rapid complexation of lactate to mostly calcium lactate. Whereas with 15% of CaCl2 in the solution, only small amounts of calcium are not bound by the alginate film, and the formation of white efflorescence is due to the time-delayed formation with magnesium, which diffuses from the core to the surface.
1. Introduction Co-extrusion of calcium alginate casings on food, especially on dry fermented sausages, is an emerging technology, which is due to the fact that producers can move from batch processing to continuous processing with increased product volume (Harper, Barbut, Smith, & Marcone, 2015). Moreover, the production of meat snack products with very small calibers is possible, which would not be possible with natural casings (Gennadios, Hanna, & Kurth, 1997). For example, in meat products they are used on dry fermented sausages with small calibers (Hilbig, Murugesan, Gibis, Herrmann, & Weiss, 2019; Walz, Gibis, Lein, et al., 2018) or on breakfast pork sausages (Liu, Kerry, & Kerry, 2007). During the co-extrusion process, a thin film of alginate is applied to the surface of the product, and subsequently crosslinked in a calcium chloride bath (Harper et al., 2015). The divalent calcium cations applied now bind to the guluronate blocks of the alginate, thereby forming junctions between different alginate chains, which results in a gel structure, the so-called egg-box model (Grant, Morris, Rees, Smith, & Thom, 1973). The disadvantage of the application of calcium alginate casings on meat products is the formation of white crystals on the surface, the socalled white efflorescence. The formation is a physical mass transport phenomenon of dissociated acids and minerals from the core of the ⁎
sausages to the surface. It is a big issue for the meat processing industry, because the affected products are rejected by the consumer because of misjudging it as microbial spoilage (Walz, Gibis, Herrmann, Hinrichs, & Weiss, 2017). Dry fermented sausages are mainly affected by the formation of the crystals (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Fritz, et al., 2018; Walz, Gibis, Herrmann, et al., 2017; Walz, Gibis, Lein, et al., 2018), and so are dry cured hams (Arnau, Gou, & Guerrero, 1994; Arnau, Guerrero, Hortós, and García-Regueiro, 1996; Arnau, Maneja, Guerrero, & Monfort, 1993). Efflorescence normally occurs on meat products which are stored at low temperatures and are packed under modified atmosphere (Arnau, Guerrero, & Gou, 1998). There are three types of efflorescences: efflorescences consisting of disodium phosphate heptahydrate (Arnau et al., 1998), efflorescences consisting of creatine monohydrate (Kröckel, Jira, Kühne, & Müller, 2003), and efflorescences consisting of magnesium or calcium lactate (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017). Walz, Gibis, Herrmann, et al. (2017) showed that magnesium was identified as one of the main efflorescence-causing substances, resulting in the irreversible efflorescence type II. In the study of Hilbig, Murugesan, et al. (2019), it was shown that the application of calcium alginate casings cause a fast formation of white efflorescence on the surface of dry fermented sausages. This effect was attributed to the excess of calcium on the surface because of the crosslinking of the alginate gel
Corresponding author. E-mail address: [email protected] (M. Gibis).
https://doi.org/10.1016/j.foodres.2020.109012 Received 7 October 2019; Received in revised form 10 January 2020; Accepted 15 January 2020 Available online 21 January 2020 0963-9969/ © 2020 Elsevier Ltd. All rights reserved.
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308, mixture of Lactobacillus sakei, Staphylococcus carnosus, Staphylococcus xylosus, DuPont Nutrition & Health, Brabrand, Denmark) were added during processing. The sausage batter was produced in a bowl chopper (K64 DC, Seydelmann GmbH, Aalen, Germany). The alginate gel was produced with a 10% alginate powder (Protanal® ME 5147, DuPont Nutrition & Health, Brabrand, Denmark) and 90% water solution in a bowl chopper, and the crosslinking solutions were produced with 15, 20, 25, or 30% of CaCl2 (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and water. Both were prepared one day prior to the sausage production (storage at 15 °C). The sausages were produced with the ConPro co-extrusion system from Albert Handtmann Holding GmbH & Co. KG (Biberach, Germany). The meat batter was extruded through the co-extrusion head and the alginate gel is applied through an annular gap around the pipe for the meat batter. The sausages had a caliber of 19, a length of 270 mm and a casing thickness of 0.2 mm. Directly after the co-extrusion, the sausages were crosslinked in a CaCl2 bath (residence time of 9 s) with the different CaCl2 solutions and a total of four batches were produced. Subsequently, the sausages were fermented (24 h at 24 °C), lightly smoked to prevent spoiling, and dried to a weight loss of 44% in a climatic chamber (Ness Wärmetechnik GmbH, Remshalden, Germany). The dried sausages were packaged (vacuum bags, SL 135 × 180 PA/PE 90, MEGA eG, Stuttgart, Germany) under modified atmosphere (80% N2, 20% CO2, Protadur® C20, Westfalen AG, Münster, Germany) and stored at 4 °C up to 8 weeks. 28 randomly selected sausages were chosen every second week (0, 2, 4, 6, and 8 weeks) for chemical and optical analyses, and the surface analyzed. For this purpose, a layer of 0.6 mm thickness was cut off the sausages with a food slicer (VS8A, Bizerba, Balingen, Germany) and the slices were homogenized using a kitchen blender (Moulinette Moulinex D56, Braun, Frankfurt, Germany), vacuum packed, and stored at −20 °C prior to the chemical analyses. The sausages for all the batches for one experiment were produced from the same raw materials and the experiments were done in duplicate.
with calcium chloride (Rhim, 2004). To inhibit the formation of white efflorescences on dry fermented sausages with collagen casings different methods were investigated, such as the application of phosphates (Walz, Gibis, Fritz, et al., 2018; Walz, Gibis, Schrey, et al., 2017), the smoke intensity (Walz, Gibis, Herrmann, & Weiss, 2019), and the variation of the drying conditions (Walz, Gibis, Koummarasy, et al., 2018). The application of phosphates and strong smoking inhibited successfully the white efflorescence formation; however, the strong smoking of the product influenced the sensory negatively. Compared to that, on dry fermented sausages with calcium alginate casings an excess of calcium is present on the surface due to the calcium chloride crosslinking solution forming a stable polymer film. The white efforescences on the surface of these sausages consisted mainly of calcium and no magnesium ions. This suggests that a different mechanism of formation is at play, and it puts at question whether the previously developed solutions are applicable to these products. Moreover, the stability of the casing needs to be considered. For example the application of a phosphate surface treatment successfully inhibited the crystal formation however the calcium alginate casing was destabilized due to the removement of calcium (Hilbig, Murugesan, et al., 2019). Furthermore, in the study of Hilbig, Hartlieb, Herrmann, Weiss, and Gibis (2019) different chelators were introduced into the calcium alginate casing and only the application of polyphosphates and citric acid could reduce the crystal formation but did not completely inhibit it. Since the influence of magnesium on white efflorescence formation on dry fermented sausages with collagen casings is well understood (Walz, Gibis, Herrmann, et al., 2017) the effect of calcium ions on the crystal formation is largely unknown. However, previous studies indicated that calcium could influence the white efflorescence formation (Hilbig, Hartlieb, et al., 2019; Hilbig, Murugesan, et al., 2019). Therefore, information is needed to what extent calcium is influencing the crystallization and if magnesium or complexes of magnesium lactate are needed to induce the crystallization (for example as crystallization nucleus). For the mechanistic context, it is important to consider the individual cations separately and independently of each other in a model system. Therefore, the purpose of the current study was to investigate the influence of different calcium chloride concentrations in the crosslinking solution during dry fermented sausage production on the white efflorescence formation. To do so, four different concentrations of calcium chloride solution (15, 20, 25, and 30%) were applied. These concentrations were selected because 25% and 30% are common concentrations used in the industrial settings of crosslinking baths, and due to limits in casing stability; i.e. a stable coextrusion and crosslinking process for casings can no longer be ensured below concentrations of 15%. The hypothesis was that with increasing concentrations of calcium in the crosslinking solution the white efflorescence formation would be more intensive and faster compared to lower concentrations. In addition, there would be an optimal concentration with reduced formation of efflorescence as well, as without loss of the stability of the casings.
2.2. Production of meat substitute sausage
2. Materials and methods
To investigate if white efflorescences are formed without the divalent cation magnesium in the system, meat substitute sausages were produced either with magnesium or only with calcium. The meat substitute was composed of 77.3% water, 20.6% cellulose fibers (LC200, JRS GmbH & Co. KG, Rosenberg, Germany), and 2.1% guar gum powder (Ruitenberg, Twello, Netherlands). Additionally, 10 g/kg lactate (added as calcium or magnesium lactate), 5 g/kg creatine, 28 g/kg nitrite curing salt, 3 g/kg coloring agent (for better visibility of the crystals, Carmirose, Frutarom Savory Solutions GmbH, KorntalMünchingen, Germany), and 5 g/kg magnesium (as magnesium lactate) were added to the batches with magnesium. Furthermore, the pH value was adjusted to 5 with lactic acid. The mass was produced in a bowl chopper (3000 rpm for 120 s). The sausages were vacuum packaged and stored for 8 weeks. The sausages were optically analyzed every second week and additionally after 8 weeks; the cations on the surface were determined by removing the casings from the sausages.
2.1. Production of sausage and sample preparation
2.3. Tensile test
The meat for the experiments was standardized according to the meat classification system to SII (pork shoulder without fat tissue where the sinews have been largely removed with a maximal visual fat content of 5%) and SVIII (back fat without rind) (Hack, Gerhardt, & Staffe, 1976). The meat was purchased from a local wholesaler (MEAG eG, Stuttgart, Germany). The meat batter was composed of 35% minced lean pork shoulder SII (3 mm whole plate, WD 114, Seydelmann GmbH, Aalen, Germany; 2 °C), 45% frozen lean pork shoulder SII (−10 °C), and 20% frozen pork back fat SVIII (−18 °C). Furthermore, 28 g/kg nitrite curing salt (0.5% sodium nitrite), 5 g/kg dextrose, 3 g/kg white pepper, 0.5 g/kg ascorbic acid, 0.1 g/kg starter culture (TEXEL® SA-
For the tensile test, sausages were produced with the substitute mass (Section 2.2) and, after crosslinking, the casing was gently removed, and pieces of 45 × 100 mm were cut out with the help of a template. The tensile tests were performed with the Instron Model 3365 Tensile Tester (Instron GmbH, Darmstadt, Germany). For the measurements, the casings were fixed in the device and elongated until the film ruptured. The test was repeated at least 15 times. Moreover, the tensile strength (TS) of the films was calculated according to Formula (1):
TS = 2
Fmax A cross
(1)
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(Section 2.5.1) were cut in half lengthwise (n = 16 halves) and subsequently scanned and digitalized (Perfection V 100 Photo, Epson, Suwa, Nagano, Japan). The pictures were analyzed with ImageJ (NIH, Bethesda, MD, USA). First, the white background of the pictures was removed. Subsequently, the total surface area of the sausages was determined by setting the saturation to 0–255, the brightness to 38–255, and the hue to 0–255. To determine the white efflorescence area, the saturation was set to 0–93, the brightness to 38–255, and the hue to 0–255. The relative area of the white efflorescences were calculated by dividing the white efflorescence area by the total area of the sausages. The determination was done in groups of 4, obtaining a fourfold determination.
Fmax: maximum force (N) before rupture Across: cross-sectional area of the films (m2; 0.2 mm thickness and 45 mm length) 2.4. Chemical analyses 2.4.1. Dry matter and moisture content The moisture content and dry matter were determined according to the standard method (AOAC, 1990). 5 g of the homogenized sample was mixed with 30–35 g sea sand and dried at 103 °C until a constant weight was reached. The determination was done in duplicate and other analyses were related to it. 2.4.2. Lactate and creatine content The determination of lactate and creatine was done according to the method described in Walz, Gibis, Herrmann, et al. (2017). Briefly, fat and protein of the samples were precipitated with Carrez I and II solutions. Subsequently, the samples were filtered with folded filter and then through membrane filters into HPLC vials and analyzed with an isocratic HPLC (Agilent 1100 Series, Agilent Technologies Inc., Santa Clara, CA, USA). A silica column with a pore size of 5 µm heated to 35 °C was used for the separation of the compounds in the samples (Reprosil-Pur 120 C18 AQ, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany). The flow rate was set to 0.7 ml/min and the compounds were detected with an UV detector set to 230 nm. The concentrations were determined by comparison with calibration curves (lactate 0.05–5 g/L; creatine 0.01–1 g/L). The limit of quantification (LOQ) was 5 mg/kg sausages and the limit of detection (LOD) was 2 mg/kg for the organic acids. The samples were prepared in duplicate and injected two times with a volume of 20 µL (n = 4).
2.6. Statistical analysis The correlation between the sensory and image analysis was calculated by determining the Pearson product-moment correlation coefficient. In addition, a one-way analysis of variance (ANOVA) was performed with a post-hoc Tukey test to determine significant differences (p < 0.05) using SPSS (IBM SPSS Statistics 24, IBM, Germany). The assumptions of normality or homoscedasticity were tested. Means and standard deviations were calculated for each measurement using Excel (Microsoft, Redmond, WA, USA). 3. Results and discussion 3.1. Appearance, sensory and image analysis of the dry-fermented sausages The calcium alginate casings produced were all stable independent of the calcium chloride concentration used for the crosslinking. Tensile tests of the different casings showed only minor differences between the concentrations (Table 1). In Fig. 1, the appearance of the different batches during the storage of 8 weeks is shown. The batches produced with 15 and 20% of CaCl2 solutions showed no efflorescence formation immediately after the production (0 weeks), whereas in the batches produced with the higher amounts of 25 and 30% after the production efflorescence are already visible. High amounts of efflorescence could be seen after 2 weeks of storage for the sausages manufactured with 20, 25, and 30% of CaCl2 solution, whereas the batch produced with 15% CaCl2 solution only showed high amounts of efflorescence after 8 weeks of storage. However, the visual analysis of the batch produced with 15% CaCl2 solution led to very high standard deviations of the scores after 2, 4, 6 and 8 weeks. One reason for this was the uneven distribution of the efflorescences on the surface of the sausages, which led to some subjectivity in the assessment of the degree of efflorescence formation by panelists. For a better comparison of the samples, sensory evaluation and image analysis were conducted (Fig. 2). Moreover, the correlation coefficient was calculated, to compare the results of the image analysis and sensory evaluation. The samples produced with 15, 20, and 25% CaCl2 solutions showed high correlations (15% r = 0.92; 20% r = 0.89; 25% r = 0.82), whereas the correlation was lower, at r = 0.61, for the batch produced with 30% CaCl2 solution, which can be explained by the high grades in the sensory evaluation after 0 weeks. However, a high correlation of the sensory and image analysis has been found in raw fermented sausage (Hilbig, Murugesan, et al., 2019), low-
2.4.3. Total mineral content A standard method was used to determine the total mineral content (AOAC, 1990). 1 ml of magnesium acetate solution was added to 5 g of the homogenized samples and well mixed. Subsequently, the samples were incinerated for 3 days at 600 °C in a muffle furnace (M 110, Heraeus, Fellbach, Germany). Furthermore, 1 ml of the magnesium acetate solution used was incinerated for the calculation of the total mineral content of the sample. The determination was done in duplicate. 2.4.4. Cation content The samples were incinerated as described in Section 2.4.3; however, no magnesium acetate solution was added to the homogenized samples. Afterwards, the ash was microwave digested and the elements were detected by inductively coupled plasma optical emission spectrometry (ICP-OES) (VDLUFA, 2011). The elements calcium, magnesium, potassium, and sodium were quantified in duplicate. 2.5. Optical analyses 2.5.1. Sensory analysis Two pairs (n = 4 sausages) of the dry fermented sausages were hung inside one glass carafe (#902.797.19, IKEA Deutschland GmbH & Co. KG, Munich, Germany) for the sensory evaluation. The humidity was set to 68% by 150 ml of a 28.52% calcium chloride solution, and the sausages were stored for 24 h at 14 °C to induce the white efflorescence formation (Walz, Gibis, Herrmann, et al., 2017). At least 20 test persons had to optically evaluate the white efflorescence amount on the surface of the sausages. The sausages had to be graded from 1 (no white efflorescence visible) to 10 (high amount of white efflorescence visible). Due to a double determination, a total of 8 sausages were graded.
Table 1 Tensile strengths of the co-extruded films with different concentrations of CaCl2 in the crosslinking solution (15, 20, 25, and 30%).
2.5.2. Image analysis For the image analysis, the sausages from the sensory evaluation
Calcium chloride concentration
Tensile strength (MPa)
15% 20% 25% 30%
1.00 1.02 1.12 1.07
a–b
3
± ± ± ±
0.10b 0.07b 0.12a 0.08ab
Significant differences (p < 0.05) between the different concentrations.
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Fig. 1. Visual appearance of the sausages during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2.
alginate casing. However, the drier surface of the sausages could lead to an accelerated formation of the crystals on the surface.
fat yoghurt and cream cheese (Johansen, Laugesen, Janhøj, Ipsen, & Frøst, 2008), and beef meat (Wu & Sun, 2013). In all samples, a significant increase of the amount of white efflorescence are seen from 0 to 2 weeks of storage. After approximately 6 weeks of storage, the amount is maximal; however, the sample produced with 15% of CaCl2 solution showed a steady increase during the 8 weeks of storage. Compared to the other samples, the amount of white efflorescence was always significantly lower. In the image analysis, a maximal amount of 72.08% was found after 8 weeks, which was almost 20% lower than that of the other samples (20% CaCl2 90.21%; 25% CaCl2 88.14%; 30% CaCl2 91.27%). Compared to sausages with natural or collagen casings, the white efflorescence formation occurs very rapidly on sausages with calcium alginate casings (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017; Walz, Gibis, Lein, et al., 2018). Due to the very high concentrations of divalent calcium ions on the surface of the sausage which could not be bound in the egg-box model of the alginate gel, the formation of complexes with lactate is accelerated (Fang et al., 2007). Another factor could be that the pore diameter in alginate gels is, compared to natural (4.90 nm) or collagen casings (5.10 nm), much higher, at 234.50 µm (Ledesma, Rendueles, & Díaz, 2015). Due to that, the mass transport of efflorescence-causing substances (magnesium, lactate, and creatine) towards the surface could be much faster (Walz, Gibis, Lein, et al., 2018). Moreover, compared to the study of Hilbig, Murugesan, et al. (2019) the white efflorescence formation in the present study is more intensive at 25% of CaCl2. This could be due to different weight losses during the drying. In the present study, the sausages were dried to a weight loss of 44% compared to 42.5% in Hilbig, Murugesan, et al. (2019) to increase the stability of the calcium
3.2. Creatine and lactate content The creatine and lactate content determined in the surface layer during the storage of 8 weeks is shown in Fig. 3. These two compounds are known for their involvement in the white efflorescence formation (Kröckel, 2004; Kröckel et al., 2003). The concentration of creatine in the samples, independent of the CaCl2 solution used, was more or less constant (between 10 and 15 mg/g DM) during the storage of 8 weeks. This shows that the creatine content did not influence the white efflorescence formation during the storage of the sausages, because the amount of white efflorescence increased, whereas the creatine content remained constant. Similar results were seen in the study of Hilbig, Murugesan, et al. (2019). In contrast, the concentration of lactate increased significantly in the surface layer during the storage for the batches produced with 20, 25, and 30% of CaCl2 in the crosslinking solution. The values increased the most in the sample produced with 30% CaCl2, from 14.81 mg/g DM to 33.14 mg/g DM. However, the content in the surface layer of the batch produced with the 15% CaCl2 solution showed no significant increase or decrease of lactate (between 22.36 and 30.69 mg/g DM) during the storage, and furthermore, the concentration was, in some cases, significantly lower compared to the samples produced with higher amounts of CaCl2 solutions. Lactate as the counterpart of magnesium and calcium is an important factor for the white efflorescence formation in raw fermented sausages. It is formed during the fermentation of fermentable sugars (in this case Fig. 2. Image analysis (A) and optical sensory (B) of the sausage during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2. Capital letters indicate significant differences (p < 0.05) between the batches, and lowercase letters indicate significant differences (p < 0.05) during storage of the single batch.
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Fig. 3. Creatine and lactate contents (mean ± standard deviation mg/g DM) of the sausages during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2. Capital letters indicate significant differences (p < 0.05) between the different layers of one batch, and lowercase letters indicate significant differences (p < 0.05) between the storage times of one layer during the storage of 8 weeks.
significantly with increasing concentration of CaCl2 solution during the crosslinking of the batches, because more calcium could deposit on the surface of the sausages. The concentration of calcium is very high and so the alginate gel is saturated and unbound calcium ions are accumulated on the surface, which now can form crystalline complexes with lactate, thereby forming the efflorescence. The fastest formation was seen with 30% of CaCl2 in the crosslinking solution, which also led to the highest concentrations on the surface, which also increased significantly during the storage (3.76 up to 6.12 mg/g DM). The lowest concentration and increase were seen in the batch produced with 15% CaCl2. In this batch, the amount was 2.10 mg/g DM and increased only 0.88 mg/g DM (2.98 mg/g DM) during the storage, whereas, for 20% CaCl2, the amount increased by 2.00 mg/g DM, for 25% 2.45 mg/g DM, and for 30% 2.36 mg/g DM. These increases could explain the faster and more pronounced white efflorescence formation on the sausages with higher CaCl2 concentrations. There, the calcium formed complexes with lactate and, because of that calcium was removed from the equilibrium, which led to increased diffusion to the surface (Hilbig, Murugesan, et al., 2019). The magnesium contents showed reversed results, because the contents were the highest in the samples produced with 15% CaCl2, and the lowest in the sample produced with 30% CaCl2 (after 8 weeks 1.58 mg/g DM for 15% and 1.40 mg/g DM for 30%). This shows that the efflorescence formation at lower calcium concentrations in the crosslinking solution is a combined effect of calcium and magnesium complexation with lactate. This mechanism is described in detail below.
dextrose) by lactic acid bacteria which are part of the starter culture used (Leroy & De Vuyst, 2004). The increase of lactate in the surface layer could be due to an increased mass transport because, in the formation of complexes with calcium and magnesium on the surface, lactate is removed from the diffusion equilibrium, thus leading to an increased diffusion towards the surface of the sausages. The same result was shown in dry fermented sausages produced with collagen casings (Walz, Gibis, Herrmann, et al., 2017). With the lower concentration of CaCl2 in the crosslinking solution (15%), this effect is not so pronounced, because less lactate is removed from the equilibrium and, hence, less calcium is available on the surface of the sausage. The formation of the efflorescences in this case could resemble the slower formation with divalent magnesium ions in sausages produced with collagen casings because magnesium first has to diffuse to the surface to form the crystalline complexes (Walz, Gibis, Herrmann, et al., 2017). 3.3. Cation content The contents of the monovalent cations potassium and sodium showed an increase from 0 to 2 weeks of storage; however, the contents in all the batches were the same, so the influence on the white efflorescence formation could be ignored (Appendix: Fig. A1). However, former studies showed that the concentrations of magnesium and calcium are crucial for the formation of white efflorescences (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017), which is why the concentrations in the surface layer during the storage of 8 weeks are shown in Fig. 4. The concentration of calcium increased
Fig. 4. Calcium and magnesium contents (mean ± standard deviation mg/g DM) of the sausages during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2. Capital letters indicate significant differences (p < 0.05) between the different layers of one batch, and lowercase letters indicate significant differences (p < 0.05) between the storage times of one layer during the storage of 8 weeks.
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are formed: i. High concentration of calcium in the crosslinking solution At high concentrations of calcium in the crosslinking solution, the gel is saturated with calcium and high amounts of unbound calcium accumulates on the surface of the sausage. During the storage, lactate is complexed by the calcium ions, thereby forming the white crystals. This leads to the removal of calcium and lactate from the diffusion equilibrium and, due to that, more of these substances are transported to the surface, leading to a rapid and high formation of white efflorescences. This also happens with magnesium to some extent, but it does not influence the amount of efflorescences because of the excess of calcium ions. ii. Low concentration of calcium in the crosslinking solution At low concentrations of calcium in the crosslinking solution, almost all calcium ions applied to the surface of the product are bound by the alginate gel, and only a very small amount of the calcium ions is unbound and can form only low amounts of white efflorescence. Because of that, the main formation of the crystals is due to the slow diffusion of magnesium from the core to the surface, which results in a slower formation process of efflorescences. It was also shown that the extent of white efflorescence formed by magnesium is lower compared to the rapid formation with calcium (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017).
Fig. 5. Visual appearance of the alginate casings of the meat substitute sausages produced either with magnesium (indicated as Mg2+) or calcium lactate (indicated as Ca2+) after 8 weeks of storage.
3.4. Effect of magnesium and calcium on white efflorescence formation in a model system A model system is necessary to investigate if calcium alone is able to form white efflorescence with lactate. For this issue, meat substitute sausages were produced. This was done due to the fact that meat itself contains magnesium with concentrations up to 0.2 g/kg (Toldrá, Hui, Astiasaran, Sebranek, & Talon, 2007), and thus a system was needed without magnesium to study the effect of calcium alone on the white efflorescence formation: One batch with, and one without, the addition of magnesium. The sausages produced showed white efflorescence formation during the storage of the samples independent of magnesium. Efflorescences are already visible after 2 weeks of storage; the amount did not differ between the sausages with or without the addition of magnesium (Fig. 5). To confirm that no magnesium was present in the complexes without the addition of magnesium, the casing was removed and the cation content was determined. The results of the determination are shown in Table 2. The determination showed contents of 5.80 mg/g DM for the sausages with magnesium and, for the sausages without magnesium, ignorable contents of 0.09 mg/g DM, which could be due to the use of tap water to produce the alginate gel. Furthermore, the concentration of calcium in the crosslinking seems crucial for the process of white efflorescence formation. Therefore, it could be concluded that magnesium or complexes of magnesium lactate, for example as crystallization nuclei, are not necessary for the white efflorescence formation on dry fermented sausages with calcium alginate casing.
5. Conclusion The present study showed that the amount of calcium in the crosslinking solution has an impact on the amount, speed of formation, and type of white efflorescence, which is formed during the storage. High concentrations promote the formation of complexes of calcium and lactate and, because of the excess of calcium, the formation happens quite fast. However, at low concentrations of calcium, the formation is slower due to the complexation of magnesium with lactate instead of calcium. The diffusion of magnesium to the surface takes time and the concentration is lower compared to the calcium amount present on the surface at higher concentrations in the crosslinking solutions. Therefore, a good way to reduce or inhibit the white efflorescences on the surface of dry fermented sausages with calcium alginate casing is to reduce the concentration of calcium in solution to the minimum, which is required for the crosslinking of the casing. Furthermore, combined with the addition of polyphosphates, the formation could be completely inhibited. Future research may focus on an investigation of optimal calcium concentrations needed to ensure crosslinking of alginate gels while minimizing efflorescence formation. This also offers options for a combinatorial approach analogous to the hurdle methodology, e.g. a minimization of calcium chloride contents in crosslinking baths combined with the use of suitable chelators.
4. Proposed mechanism
CRediT authorship contribution statement
Fig. 6 illustrates the proposed mechanism of the formation. Furthermore, the investigation showed two ways the crystals on the surface
Jonas Hilbig: Conceptualization, project administration, data curation, writing - original draft. Katrin Hartlieb: Investigation (master thesis). Kurt Herrmann: Supervision - pilot plant. Jochen Weiss: Conceptualization, supervision, writing - review & editing. Monika Gibis: Conceptualization, project administration, supervision, writing - review & editing.
Table 2 Magnesium and calcium contents (mg/g DM) in the alginate casings of the meat substitute sausages produced either with magnesium (indicated as Mg2+) or with calcium lactate (indicated as Ca2+) after 8 weeks of storage. Sample 2+
+ CaCl2 Mg Ca2+ + CaCl2 a–b
Calcium content (mg/g DM) b
11.56 ± 0.07 34.13 ± 0.48a
Declaration of Competing Interest
Magnesium content (mg/g DM) a
5.80 ± 0.09 0.09 ± 0.01b
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Significant differences (p < 0.05) between the different concentrations. 6
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Fig. 6. Schematic illustration of assumed diffusion of the efflorescence-causing components (magnesium, calcium, and lactate) to the surface of the sausages during the storage time.
Acknowledgement
within the program for promoting the Industrial Collective Research (IGF) of the German Ministry of Economic Affairs and Energy (BMWi), based on a resolution of the German Parliament.
This IGF Project (AiF 19689N) of the FEI is/was supported via AiF Appendix A See Fig. A1.
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Fig. A1. Potassium and sodium contents (mean ± standard deviation mg/g DM) of the sausages crosslinked with either 15, 20, 25, or 30% of CaCl2 during 8 weeks of storage.
Kulmbach, 43(166), 355–362. Kröckel, L., Jira, W., Kühne, D., & Müller, W.-D. (2003). Creatine blooms on the surface of prepacked fermented sausages. European Food Research and Technology, 217(1), 1–3. Ledesma, E., Rendueles, M., & Díaz, M. (2015). Characterization of natural and synthetic casings and mechanism of BaP penetration in smoked meat products. Food Control, 51, 195–205. Leroy, F., & De Vuyst, L. (2004). Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends in Food Science & Technology, 15(2), 67–78. Liu, L., Kerry, J. F., & Kerry, J. P. (2007). Application and assessment of extruded edible casings manufactured from pectin and gelatin/sodium alginate blends for use with breakfast pork sausage. Meat Science, 75(2), 196–202. Rhim, J.-W. (2004). Physical and mechanical properties of water resistant sodium alginate films. LWT - Food Science and Technology, 37(3), 323–330. Toldrá, F., Hui, Y. H., Astiasaran, I., Sebranek, J., & Talon, R. (2007). Handbook of fermented meat and poultry. Wiley Online Library. VDLUFA (2011). Methodenbuch Band VII Umweltanalytik (4th ed., Vol. VII). Bonn: VDLUFA-Verlag. Walz, F. H., Gibis, M., Fritz, M., Herrmann, K., Hinrichs, J., & Weiss, J. (2018). Optimum hexametaphosphate concentration to inhibit efflorescence formation in dry fermented sausages. Meat Science, 139, 35–43. Walz, F. H., Gibis, M., Herrmann, K., Hinrichs, J., & Weiss, J. (2017). Chemical and optical characterization of white efflorescences on dry fermented sausages under modified atmosphere packaging. Journal of the Science of Food and Agriculture, 97(14), 4872–4879. Walz, F. H., Gibis, M., Herrmann, K., & Weiss, J. (2019). Impact of smoking on efflorescence formation on dry-fermented sausages. Food Structure, 20, 100111. Walz, F. H., Gibis, M., Koummarasy, S., Reichert, C. L., Herrmann, K., Hinrichs, J., & Weiss, J. (2018). Influence of different drying rates on mass transport of efflorescence-causing substances in thin caliber salamis during refrigerated storage in N 2/CO 2 MAP. European Food Research and Technology, 244(4), 663–674. Walz, F. H., Gibis, M., Lein, M., Herrmann, K., Hinrichs, J., & Weiss, J. (2018). Influence of casing material on the formation of efflorescences on dry fermented sausages. LWT - Food Science and Technology, 89, 434–440. Walz, F. H., Gibis, M., Schrey, P., Herrmann, K., Reichert, C. L., Hinrichs, J., & Weiss, J. (2017). Inhibitory effect of phosphates on magnesium lactate efflorescence formation in dry-fermented sausages. Food Research International, 100, 352–360. Wu, D., & Sun, D.-W. (2013). Advanced applications of hyperspectral imaging technology for food quality and safety analysis and assessment: A review — Part II: Applications. Innovative Food Science & Emerging Technologies, 19, 15–28.
References AOAC (1990). Official methods of analysis (15th ed.). Arlington, VA: Association of Official Analytical Chemists. Arnau, J., Gou, P., & Guerrero, L. (1994). The effects of freezing, meat pH and storage temperature on the formation of white film and tyrosine crystals in dry-cured hams. Journal of the Science of Food and Agriculture, 66(3), 279–282. Arnau, J., Guerrero, L., & Gou, P. (1998). The precipitation of phosphates in meat products. Fleischwirtschaft International, 3, 46–47. Arnau, J., Guerrero, L., Hortós, M., & García-Regueiro, J. A. (1996). The composition of white film and white crystals found in dry-cured hams. Journal of the Science of Food and Agriculture, 70(4), 449–452. Arnau, J., Maneja, E., Guerrero, L., & Monfort, J. (1993). Phosphate crystals in raw cured ham. Fleischwirtschaft, 73(8), 875–876. Fang, Y., Al-Assaf, S., Phillips, G. O., Nishinari, K., Funami, T., Williams, P. A., & Li, L. (2007). Multiple steps and critical behaviors of the binding of calcium to alginate. The Journal of Physical Chemistry B, 111(10), 2456–2462. Gennadios, A., Hanna, M. A., & Kurth, L. B. (1997). Application of edible coatings on meats, poultry and seafoods: A review. LWT - Food Science and Technology, 30(4), 337–350. Grant, G. T., Morris, E. R., Rees, D. A., Smith, P. J., & Thom, D. (1973). Biological interactions between polysaccharides and divalent cations: The egg-box model. FEBS letters, 32(1), 195–198. Hack, K.-H., Gerhardt, U., & Staffe, E. (1976). Verarbeitungsmaterial-Atlas für die Fleischund Wurstwarenproduktion. Stuttgart: Gewürzmüller. Harper, B. A., Barbut, S., Smith, A., & Marcone, M. F. (2015). Mechanical and microstructural properties of “wet” alginate and composite films containing various carbohydrates. Journal of Food Science, 80(1), E84–E92. Hilbig, J., Hartlieb, K., Herrmann, K., Weiss, J., & Gibis, M. (2019). Influence of phosphates and pH value on white efflorescence formation on dry fermented sausages. European Food Research and Technology. Hilbig, J., Murugesan, V., Gibis, M., Herrmann, K., & Weiss, J. (2019). Surface treatment with condensed phosphates reduced efflorescence formation on dry fermented sausages with alginate casings. Journal of Food Engineering, 262, 189–199. Johansen, S. M. B., Laugesen, J. L., Janhøj, T., Ipsen, R. H., & Frøst, M. B. (2008). Prediction of sensory properties of low-fat yoghurt and cream cheese from surface images. Food Quality and Preference, 19(2), 232–246. Kröckel, L. (2004). Influence of fermentation on creatine and lactate concentrations in white efflorescences on dry fermented sausages. Mitteilungsblatt der Fleischforschung
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Influence of calcium on white efflorescence formation on dry fermented sausages with co-extruded alginate casings Jonas Hilbig, Katrin Hartlieb, Kurt Herrmann, Jochen Weiss, Monika Gibis
T
⁎
Department of Food Physics and Meat Science, Institute of Food Science and Biotechnology, University of Hohenheim, Garbenstrasse 21/25, 70599 Stuttgart, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: White efflorescence Calcium concentration Alginate casing Dry fermented sausage
The effect of the concentration of calcium in the crosslinking solution during co-extrusion of dry fermented sausages with calcium alginate casing on the white efflorescence was investigated. With the co-extrusion technology, a continuous sausage production is possible and, furthermore, snack products with very small calibers can be produced. Therefore, crosslinking solutions with different concentrations of CaCl2 (15, 20, 25, and 30%) were used during the production. High concentrations of calcium led to a very rapid and intensive white efflorescence formation; the efflorescences covered ~90% of the surface of the samples produced with 20–30% of CaCl2 after 8 weeks of storage. However, the batch produced with the lowest amount of CaCl2 showed a slow efflorescence formation and a significantly decreased area covered by it (~70% after 8 weeks). The differences in the formation were attributed to the excess of calcium on the surface of the sausages (saturated calcium alginate film), therefore leading to rapid complexation of lactate to mostly calcium lactate. Whereas with 15% of CaCl2 in the solution, only small amounts of calcium are not bound by the alginate film, and the formation of white efflorescence is due to the time-delayed formation with magnesium, which diffuses from the core to the surface.
1. Introduction Co-extrusion of calcium alginate casings on food, especially on dry fermented sausages, is an emerging technology, which is due to the fact that producers can move from batch processing to continuous processing with increased product volume (Harper, Barbut, Smith, & Marcone, 2015). Moreover, the production of meat snack products with very small calibers is possible, which would not be possible with natural casings (Gennadios, Hanna, & Kurth, 1997). For example, in meat products they are used on dry fermented sausages with small calibers (Hilbig, Murugesan, Gibis, Herrmann, & Weiss, 2019; Walz, Gibis, Lein, et al., 2018) or on breakfast pork sausages (Liu, Kerry, & Kerry, 2007). During the co-extrusion process, a thin film of alginate is applied to the surface of the product, and subsequently crosslinked in a calcium chloride bath (Harper et al., 2015). The divalent calcium cations applied now bind to the guluronate blocks of the alginate, thereby forming junctions between different alginate chains, which results in a gel structure, the so-called egg-box model (Grant, Morris, Rees, Smith, & Thom, 1973). The disadvantage of the application of calcium alginate casings on meat products is the formation of white crystals on the surface, the socalled white efflorescence. The formation is a physical mass transport phenomenon of dissociated acids and minerals from the core of the ⁎
sausages to the surface. It is a big issue for the meat processing industry, because the affected products are rejected by the consumer because of misjudging it as microbial spoilage (Walz, Gibis, Herrmann, Hinrichs, & Weiss, 2017). Dry fermented sausages are mainly affected by the formation of the crystals (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Fritz, et al., 2018; Walz, Gibis, Herrmann, et al., 2017; Walz, Gibis, Lein, et al., 2018), and so are dry cured hams (Arnau, Gou, & Guerrero, 1994; Arnau, Guerrero, Hortós, and García-Regueiro, 1996; Arnau, Maneja, Guerrero, & Monfort, 1993). Efflorescence normally occurs on meat products which are stored at low temperatures and are packed under modified atmosphere (Arnau, Guerrero, & Gou, 1998). There are three types of efflorescences: efflorescences consisting of disodium phosphate heptahydrate (Arnau et al., 1998), efflorescences consisting of creatine monohydrate (Kröckel, Jira, Kühne, & Müller, 2003), and efflorescences consisting of magnesium or calcium lactate (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017). Walz, Gibis, Herrmann, et al. (2017) showed that magnesium was identified as one of the main efflorescence-causing substances, resulting in the irreversible efflorescence type II. In the study of Hilbig, Murugesan, et al. (2019), it was shown that the application of calcium alginate casings cause a fast formation of white efflorescence on the surface of dry fermented sausages. This effect was attributed to the excess of calcium on the surface because of the crosslinking of the alginate gel
Corresponding author. E-mail address: [email protected] (M. Gibis).
https://doi.org/10.1016/j.foodres.2020.109012 Received 7 October 2019; Received in revised form 10 January 2020; Accepted 15 January 2020 Available online 21 January 2020 0963-9969/ © 2020 Elsevier Ltd. All rights reserved.
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308, mixture of Lactobacillus sakei, Staphylococcus carnosus, Staphylococcus xylosus, DuPont Nutrition & Health, Brabrand, Denmark) were added during processing. The sausage batter was produced in a bowl chopper (K64 DC, Seydelmann GmbH, Aalen, Germany). The alginate gel was produced with a 10% alginate powder (Protanal® ME 5147, DuPont Nutrition & Health, Brabrand, Denmark) and 90% water solution in a bowl chopper, and the crosslinking solutions were produced with 15, 20, 25, or 30% of CaCl2 (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and water. Both were prepared one day prior to the sausage production (storage at 15 °C). The sausages were produced with the ConPro co-extrusion system from Albert Handtmann Holding GmbH & Co. KG (Biberach, Germany). The meat batter was extruded through the co-extrusion head and the alginate gel is applied through an annular gap around the pipe for the meat batter. The sausages had a caliber of 19, a length of 270 mm and a casing thickness of 0.2 mm. Directly after the co-extrusion, the sausages were crosslinked in a CaCl2 bath (residence time of 9 s) with the different CaCl2 solutions and a total of four batches were produced. Subsequently, the sausages were fermented (24 h at 24 °C), lightly smoked to prevent spoiling, and dried to a weight loss of 44% in a climatic chamber (Ness Wärmetechnik GmbH, Remshalden, Germany). The dried sausages were packaged (vacuum bags, SL 135 × 180 PA/PE 90, MEGA eG, Stuttgart, Germany) under modified atmosphere (80% N2, 20% CO2, Protadur® C20, Westfalen AG, Münster, Germany) and stored at 4 °C up to 8 weeks. 28 randomly selected sausages were chosen every second week (0, 2, 4, 6, and 8 weeks) for chemical and optical analyses, and the surface analyzed. For this purpose, a layer of 0.6 mm thickness was cut off the sausages with a food slicer (VS8A, Bizerba, Balingen, Germany) and the slices were homogenized using a kitchen blender (Moulinette Moulinex D56, Braun, Frankfurt, Germany), vacuum packed, and stored at −20 °C prior to the chemical analyses. The sausages for all the batches for one experiment were produced from the same raw materials and the experiments were done in duplicate.
with calcium chloride (Rhim, 2004). To inhibit the formation of white efflorescences on dry fermented sausages with collagen casings different methods were investigated, such as the application of phosphates (Walz, Gibis, Fritz, et al., 2018; Walz, Gibis, Schrey, et al., 2017), the smoke intensity (Walz, Gibis, Herrmann, & Weiss, 2019), and the variation of the drying conditions (Walz, Gibis, Koummarasy, et al., 2018). The application of phosphates and strong smoking inhibited successfully the white efflorescence formation; however, the strong smoking of the product influenced the sensory negatively. Compared to that, on dry fermented sausages with calcium alginate casings an excess of calcium is present on the surface due to the calcium chloride crosslinking solution forming a stable polymer film. The white efforescences on the surface of these sausages consisted mainly of calcium and no magnesium ions. This suggests that a different mechanism of formation is at play, and it puts at question whether the previously developed solutions are applicable to these products. Moreover, the stability of the casing needs to be considered. For example the application of a phosphate surface treatment successfully inhibited the crystal formation however the calcium alginate casing was destabilized due to the removement of calcium (Hilbig, Murugesan, et al., 2019). Furthermore, in the study of Hilbig, Hartlieb, Herrmann, Weiss, and Gibis (2019) different chelators were introduced into the calcium alginate casing and only the application of polyphosphates and citric acid could reduce the crystal formation but did not completely inhibit it. Since the influence of magnesium on white efflorescence formation on dry fermented sausages with collagen casings is well understood (Walz, Gibis, Herrmann, et al., 2017) the effect of calcium ions on the crystal formation is largely unknown. However, previous studies indicated that calcium could influence the white efflorescence formation (Hilbig, Hartlieb, et al., 2019; Hilbig, Murugesan, et al., 2019). Therefore, information is needed to what extent calcium is influencing the crystallization and if magnesium or complexes of magnesium lactate are needed to induce the crystallization (for example as crystallization nucleus). For the mechanistic context, it is important to consider the individual cations separately and independently of each other in a model system. Therefore, the purpose of the current study was to investigate the influence of different calcium chloride concentrations in the crosslinking solution during dry fermented sausage production on the white efflorescence formation. To do so, four different concentrations of calcium chloride solution (15, 20, 25, and 30%) were applied. These concentrations were selected because 25% and 30% are common concentrations used in the industrial settings of crosslinking baths, and due to limits in casing stability; i.e. a stable coextrusion and crosslinking process for casings can no longer be ensured below concentrations of 15%. The hypothesis was that with increasing concentrations of calcium in the crosslinking solution the white efflorescence formation would be more intensive and faster compared to lower concentrations. In addition, there would be an optimal concentration with reduced formation of efflorescence as well, as without loss of the stability of the casings.
2.2. Production of meat substitute sausage
2. Materials and methods
To investigate if white efflorescences are formed without the divalent cation magnesium in the system, meat substitute sausages were produced either with magnesium or only with calcium. The meat substitute was composed of 77.3% water, 20.6% cellulose fibers (LC200, JRS GmbH & Co. KG, Rosenberg, Germany), and 2.1% guar gum powder (Ruitenberg, Twello, Netherlands). Additionally, 10 g/kg lactate (added as calcium or magnesium lactate), 5 g/kg creatine, 28 g/kg nitrite curing salt, 3 g/kg coloring agent (for better visibility of the crystals, Carmirose, Frutarom Savory Solutions GmbH, KorntalMünchingen, Germany), and 5 g/kg magnesium (as magnesium lactate) were added to the batches with magnesium. Furthermore, the pH value was adjusted to 5 with lactic acid. The mass was produced in a bowl chopper (3000 rpm for 120 s). The sausages were vacuum packaged and stored for 8 weeks. The sausages were optically analyzed every second week and additionally after 8 weeks; the cations on the surface were determined by removing the casings from the sausages.
2.1. Production of sausage and sample preparation
2.3. Tensile test
The meat for the experiments was standardized according to the meat classification system to SII (pork shoulder without fat tissue where the sinews have been largely removed with a maximal visual fat content of 5%) and SVIII (back fat without rind) (Hack, Gerhardt, & Staffe, 1976). The meat was purchased from a local wholesaler (MEAG eG, Stuttgart, Germany). The meat batter was composed of 35% minced lean pork shoulder SII (3 mm whole plate, WD 114, Seydelmann GmbH, Aalen, Germany; 2 °C), 45% frozen lean pork shoulder SII (−10 °C), and 20% frozen pork back fat SVIII (−18 °C). Furthermore, 28 g/kg nitrite curing salt (0.5% sodium nitrite), 5 g/kg dextrose, 3 g/kg white pepper, 0.5 g/kg ascorbic acid, 0.1 g/kg starter culture (TEXEL® SA-
For the tensile test, sausages were produced with the substitute mass (Section 2.2) and, after crosslinking, the casing was gently removed, and pieces of 45 × 100 mm were cut out with the help of a template. The tensile tests were performed with the Instron Model 3365 Tensile Tester (Instron GmbH, Darmstadt, Germany). For the measurements, the casings were fixed in the device and elongated until the film ruptured. The test was repeated at least 15 times. Moreover, the tensile strength (TS) of the films was calculated according to Formula (1):
TS = 2
Fmax A cross
(1)
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(Section 2.5.1) were cut in half lengthwise (n = 16 halves) and subsequently scanned and digitalized (Perfection V 100 Photo, Epson, Suwa, Nagano, Japan). The pictures were analyzed with ImageJ (NIH, Bethesda, MD, USA). First, the white background of the pictures was removed. Subsequently, the total surface area of the sausages was determined by setting the saturation to 0–255, the brightness to 38–255, and the hue to 0–255. To determine the white efflorescence area, the saturation was set to 0–93, the brightness to 38–255, and the hue to 0–255. The relative area of the white efflorescences were calculated by dividing the white efflorescence area by the total area of the sausages. The determination was done in groups of 4, obtaining a fourfold determination.
Fmax: maximum force (N) before rupture Across: cross-sectional area of the films (m2; 0.2 mm thickness and 45 mm length) 2.4. Chemical analyses 2.4.1. Dry matter and moisture content The moisture content and dry matter were determined according to the standard method (AOAC, 1990). 5 g of the homogenized sample was mixed with 30–35 g sea sand and dried at 103 °C until a constant weight was reached. The determination was done in duplicate and other analyses were related to it. 2.4.2. Lactate and creatine content The determination of lactate and creatine was done according to the method described in Walz, Gibis, Herrmann, et al. (2017). Briefly, fat and protein of the samples were precipitated with Carrez I and II solutions. Subsequently, the samples were filtered with folded filter and then through membrane filters into HPLC vials and analyzed with an isocratic HPLC (Agilent 1100 Series, Agilent Technologies Inc., Santa Clara, CA, USA). A silica column with a pore size of 5 µm heated to 35 °C was used for the separation of the compounds in the samples (Reprosil-Pur 120 C18 AQ, Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany). The flow rate was set to 0.7 ml/min and the compounds were detected with an UV detector set to 230 nm. The concentrations were determined by comparison with calibration curves (lactate 0.05–5 g/L; creatine 0.01–1 g/L). The limit of quantification (LOQ) was 5 mg/kg sausages and the limit of detection (LOD) was 2 mg/kg for the organic acids. The samples were prepared in duplicate and injected two times with a volume of 20 µL (n = 4).
2.6. Statistical analysis The correlation between the sensory and image analysis was calculated by determining the Pearson product-moment correlation coefficient. In addition, a one-way analysis of variance (ANOVA) was performed with a post-hoc Tukey test to determine significant differences (p < 0.05) using SPSS (IBM SPSS Statistics 24, IBM, Germany). The assumptions of normality or homoscedasticity were tested. Means and standard deviations were calculated for each measurement using Excel (Microsoft, Redmond, WA, USA). 3. Results and discussion 3.1. Appearance, sensory and image analysis of the dry-fermented sausages The calcium alginate casings produced were all stable independent of the calcium chloride concentration used for the crosslinking. Tensile tests of the different casings showed only minor differences between the concentrations (Table 1). In Fig. 1, the appearance of the different batches during the storage of 8 weeks is shown. The batches produced with 15 and 20% of CaCl2 solutions showed no efflorescence formation immediately after the production (0 weeks), whereas in the batches produced with the higher amounts of 25 and 30% after the production efflorescence are already visible. High amounts of efflorescence could be seen after 2 weeks of storage for the sausages manufactured with 20, 25, and 30% of CaCl2 solution, whereas the batch produced with 15% CaCl2 solution only showed high amounts of efflorescence after 8 weeks of storage. However, the visual analysis of the batch produced with 15% CaCl2 solution led to very high standard deviations of the scores after 2, 4, 6 and 8 weeks. One reason for this was the uneven distribution of the efflorescences on the surface of the sausages, which led to some subjectivity in the assessment of the degree of efflorescence formation by panelists. For a better comparison of the samples, sensory evaluation and image analysis were conducted (Fig. 2). Moreover, the correlation coefficient was calculated, to compare the results of the image analysis and sensory evaluation. The samples produced with 15, 20, and 25% CaCl2 solutions showed high correlations (15% r = 0.92; 20% r = 0.89; 25% r = 0.82), whereas the correlation was lower, at r = 0.61, for the batch produced with 30% CaCl2 solution, which can be explained by the high grades in the sensory evaluation after 0 weeks. However, a high correlation of the sensory and image analysis has been found in raw fermented sausage (Hilbig, Murugesan, et al., 2019), low-
2.4.3. Total mineral content A standard method was used to determine the total mineral content (AOAC, 1990). 1 ml of magnesium acetate solution was added to 5 g of the homogenized samples and well mixed. Subsequently, the samples were incinerated for 3 days at 600 °C in a muffle furnace (M 110, Heraeus, Fellbach, Germany). Furthermore, 1 ml of the magnesium acetate solution used was incinerated for the calculation of the total mineral content of the sample. The determination was done in duplicate. 2.4.4. Cation content The samples were incinerated as described in Section 2.4.3; however, no magnesium acetate solution was added to the homogenized samples. Afterwards, the ash was microwave digested and the elements were detected by inductively coupled plasma optical emission spectrometry (ICP-OES) (VDLUFA, 2011). The elements calcium, magnesium, potassium, and sodium were quantified in duplicate. 2.5. Optical analyses 2.5.1. Sensory analysis Two pairs (n = 4 sausages) of the dry fermented sausages were hung inside one glass carafe (#902.797.19, IKEA Deutschland GmbH & Co. KG, Munich, Germany) for the sensory evaluation. The humidity was set to 68% by 150 ml of a 28.52% calcium chloride solution, and the sausages were stored for 24 h at 14 °C to induce the white efflorescence formation (Walz, Gibis, Herrmann, et al., 2017). At least 20 test persons had to optically evaluate the white efflorescence amount on the surface of the sausages. The sausages had to be graded from 1 (no white efflorescence visible) to 10 (high amount of white efflorescence visible). Due to a double determination, a total of 8 sausages were graded.
Table 1 Tensile strengths of the co-extruded films with different concentrations of CaCl2 in the crosslinking solution (15, 20, 25, and 30%).
2.5.2. Image analysis For the image analysis, the sausages from the sensory evaluation
Calcium chloride concentration
Tensile strength (MPa)
15% 20% 25% 30%
1.00 1.02 1.12 1.07
a–b
3
± ± ± ±
0.10b 0.07b 0.12a 0.08ab
Significant differences (p < 0.05) between the different concentrations.
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Fig. 1. Visual appearance of the sausages during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2.
alginate casing. However, the drier surface of the sausages could lead to an accelerated formation of the crystals on the surface.
fat yoghurt and cream cheese (Johansen, Laugesen, Janhøj, Ipsen, & Frøst, 2008), and beef meat (Wu & Sun, 2013). In all samples, a significant increase of the amount of white efflorescence are seen from 0 to 2 weeks of storage. After approximately 6 weeks of storage, the amount is maximal; however, the sample produced with 15% of CaCl2 solution showed a steady increase during the 8 weeks of storage. Compared to the other samples, the amount of white efflorescence was always significantly lower. In the image analysis, a maximal amount of 72.08% was found after 8 weeks, which was almost 20% lower than that of the other samples (20% CaCl2 90.21%; 25% CaCl2 88.14%; 30% CaCl2 91.27%). Compared to sausages with natural or collagen casings, the white efflorescence formation occurs very rapidly on sausages with calcium alginate casings (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017; Walz, Gibis, Lein, et al., 2018). Due to the very high concentrations of divalent calcium ions on the surface of the sausage which could not be bound in the egg-box model of the alginate gel, the formation of complexes with lactate is accelerated (Fang et al., 2007). Another factor could be that the pore diameter in alginate gels is, compared to natural (4.90 nm) or collagen casings (5.10 nm), much higher, at 234.50 µm (Ledesma, Rendueles, & Díaz, 2015). Due to that, the mass transport of efflorescence-causing substances (magnesium, lactate, and creatine) towards the surface could be much faster (Walz, Gibis, Lein, et al., 2018). Moreover, compared to the study of Hilbig, Murugesan, et al. (2019) the white efflorescence formation in the present study is more intensive at 25% of CaCl2. This could be due to different weight losses during the drying. In the present study, the sausages were dried to a weight loss of 44% compared to 42.5% in Hilbig, Murugesan, et al. (2019) to increase the stability of the calcium
3.2. Creatine and lactate content The creatine and lactate content determined in the surface layer during the storage of 8 weeks is shown in Fig. 3. These two compounds are known for their involvement in the white efflorescence formation (Kröckel, 2004; Kröckel et al., 2003). The concentration of creatine in the samples, independent of the CaCl2 solution used, was more or less constant (between 10 and 15 mg/g DM) during the storage of 8 weeks. This shows that the creatine content did not influence the white efflorescence formation during the storage of the sausages, because the amount of white efflorescence increased, whereas the creatine content remained constant. Similar results were seen in the study of Hilbig, Murugesan, et al. (2019). In contrast, the concentration of lactate increased significantly in the surface layer during the storage for the batches produced with 20, 25, and 30% of CaCl2 in the crosslinking solution. The values increased the most in the sample produced with 30% CaCl2, from 14.81 mg/g DM to 33.14 mg/g DM. However, the content in the surface layer of the batch produced with the 15% CaCl2 solution showed no significant increase or decrease of lactate (between 22.36 and 30.69 mg/g DM) during the storage, and furthermore, the concentration was, in some cases, significantly lower compared to the samples produced with higher amounts of CaCl2 solutions. Lactate as the counterpart of magnesium and calcium is an important factor for the white efflorescence formation in raw fermented sausages. It is formed during the fermentation of fermentable sugars (in this case Fig. 2. Image analysis (A) and optical sensory (B) of the sausage during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2. Capital letters indicate significant differences (p < 0.05) between the batches, and lowercase letters indicate significant differences (p < 0.05) during storage of the single batch.
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Fig. 3. Creatine and lactate contents (mean ± standard deviation mg/g DM) of the sausages during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2. Capital letters indicate significant differences (p < 0.05) between the different layers of one batch, and lowercase letters indicate significant differences (p < 0.05) between the storage times of one layer during the storage of 8 weeks.
significantly with increasing concentration of CaCl2 solution during the crosslinking of the batches, because more calcium could deposit on the surface of the sausages. The concentration of calcium is very high and so the alginate gel is saturated and unbound calcium ions are accumulated on the surface, which now can form crystalline complexes with lactate, thereby forming the efflorescence. The fastest formation was seen with 30% of CaCl2 in the crosslinking solution, which also led to the highest concentrations on the surface, which also increased significantly during the storage (3.76 up to 6.12 mg/g DM). The lowest concentration and increase were seen in the batch produced with 15% CaCl2. In this batch, the amount was 2.10 mg/g DM and increased only 0.88 mg/g DM (2.98 mg/g DM) during the storage, whereas, for 20% CaCl2, the amount increased by 2.00 mg/g DM, for 25% 2.45 mg/g DM, and for 30% 2.36 mg/g DM. These increases could explain the faster and more pronounced white efflorescence formation on the sausages with higher CaCl2 concentrations. There, the calcium formed complexes with lactate and, because of that calcium was removed from the equilibrium, which led to increased diffusion to the surface (Hilbig, Murugesan, et al., 2019). The magnesium contents showed reversed results, because the contents were the highest in the samples produced with 15% CaCl2, and the lowest in the sample produced with 30% CaCl2 (after 8 weeks 1.58 mg/g DM for 15% and 1.40 mg/g DM for 30%). This shows that the efflorescence formation at lower calcium concentrations in the crosslinking solution is a combined effect of calcium and magnesium complexation with lactate. This mechanism is described in detail below.
dextrose) by lactic acid bacteria which are part of the starter culture used (Leroy & De Vuyst, 2004). The increase of lactate in the surface layer could be due to an increased mass transport because, in the formation of complexes with calcium and magnesium on the surface, lactate is removed from the diffusion equilibrium, thus leading to an increased diffusion towards the surface of the sausages. The same result was shown in dry fermented sausages produced with collagen casings (Walz, Gibis, Herrmann, et al., 2017). With the lower concentration of CaCl2 in the crosslinking solution (15%), this effect is not so pronounced, because less lactate is removed from the equilibrium and, hence, less calcium is available on the surface of the sausage. The formation of the efflorescences in this case could resemble the slower formation with divalent magnesium ions in sausages produced with collagen casings because magnesium first has to diffuse to the surface to form the crystalline complexes (Walz, Gibis, Herrmann, et al., 2017). 3.3. Cation content The contents of the monovalent cations potassium and sodium showed an increase from 0 to 2 weeks of storage; however, the contents in all the batches were the same, so the influence on the white efflorescence formation could be ignored (Appendix: Fig. A1). However, former studies showed that the concentrations of magnesium and calcium are crucial for the formation of white efflorescences (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017), which is why the concentrations in the surface layer during the storage of 8 weeks are shown in Fig. 4. The concentration of calcium increased
Fig. 4. Calcium and magnesium contents (mean ± standard deviation mg/g DM) of the sausages during 8 weeks of storage. The alginate gel was crosslinked with either 15, 20, 25, or 30% of CaCl2. Capital letters indicate significant differences (p < 0.05) between the different layers of one batch, and lowercase letters indicate significant differences (p < 0.05) between the storage times of one layer during the storage of 8 weeks.
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are formed: i. High concentration of calcium in the crosslinking solution At high concentrations of calcium in the crosslinking solution, the gel is saturated with calcium and high amounts of unbound calcium accumulates on the surface of the sausage. During the storage, lactate is complexed by the calcium ions, thereby forming the white crystals. This leads to the removal of calcium and lactate from the diffusion equilibrium and, due to that, more of these substances are transported to the surface, leading to a rapid and high formation of white efflorescences. This also happens with magnesium to some extent, but it does not influence the amount of efflorescences because of the excess of calcium ions. ii. Low concentration of calcium in the crosslinking solution At low concentrations of calcium in the crosslinking solution, almost all calcium ions applied to the surface of the product are bound by the alginate gel, and only a very small amount of the calcium ions is unbound and can form only low amounts of white efflorescence. Because of that, the main formation of the crystals is due to the slow diffusion of magnesium from the core to the surface, which results in a slower formation process of efflorescences. It was also shown that the extent of white efflorescence formed by magnesium is lower compared to the rapid formation with calcium (Hilbig, Murugesan, et al., 2019; Walz, Gibis, Herrmann, et al., 2017).
Fig. 5. Visual appearance of the alginate casings of the meat substitute sausages produced either with magnesium (indicated as Mg2+) or calcium lactate (indicated as Ca2+) after 8 weeks of storage.
3.4. Effect of magnesium and calcium on white efflorescence formation in a model system A model system is necessary to investigate if calcium alone is able to form white efflorescence with lactate. For this issue, meat substitute sausages were produced. This was done due to the fact that meat itself contains magnesium with concentrations up to 0.2 g/kg (Toldrá, Hui, Astiasaran, Sebranek, & Talon, 2007), and thus a system was needed without magnesium to study the effect of calcium alone on the white efflorescence formation: One batch with, and one without, the addition of magnesium. The sausages produced showed white efflorescence formation during the storage of the samples independent of magnesium. Efflorescences are already visible after 2 weeks of storage; the amount did not differ between the sausages with or without the addition of magnesium (Fig. 5). To confirm that no magnesium was present in the complexes without the addition of magnesium, the casing was removed and the cation content was determined. The results of the determination are shown in Table 2. The determination showed contents of 5.80 mg/g DM for the sausages with magnesium and, for the sausages without magnesium, ignorable contents of 0.09 mg/g DM, which could be due to the use of tap water to produce the alginate gel. Furthermore, the concentration of calcium in the crosslinking seems crucial for the process of white efflorescence formation. Therefore, it could be concluded that magnesium or complexes of magnesium lactate, for example as crystallization nuclei, are not necessary for the white efflorescence formation on dry fermented sausages with calcium alginate casing.
5. Conclusion The present study showed that the amount of calcium in the crosslinking solution has an impact on the amount, speed of formation, and type of white efflorescence, which is formed during the storage. High concentrations promote the formation of complexes of calcium and lactate and, because of the excess of calcium, the formation happens quite fast. However, at low concentrations of calcium, the formation is slower due to the complexation of magnesium with lactate instead of calcium. The diffusion of magnesium to the surface takes time and the concentration is lower compared to the calcium amount present on the surface at higher concentrations in the crosslinking solutions. Therefore, a good way to reduce or inhibit the white efflorescences on the surface of dry fermented sausages with calcium alginate casing is to reduce the concentration of calcium in solution to the minimum, which is required for the crosslinking of the casing. Furthermore, combined with the addition of polyphosphates, the formation could be completely inhibited. Future research may focus on an investigation of optimal calcium concentrations needed to ensure crosslinking of alginate gels while minimizing efflorescence formation. This also offers options for a combinatorial approach analogous to the hurdle methodology, e.g. a minimization of calcium chloride contents in crosslinking baths combined with the use of suitable chelators.
4. Proposed mechanism
CRediT authorship contribution statement
Fig. 6 illustrates the proposed mechanism of the formation. Furthermore, the investigation showed two ways the crystals on the surface
Jonas Hilbig: Conceptualization, project administration, data curation, writing - original draft. Katrin Hartlieb: Investigation (master thesis). Kurt Herrmann: Supervision - pilot plant. Jochen Weiss: Conceptualization, supervision, writing - review & editing. Monika Gibis: Conceptualization, project administration, supervision, writing - review & editing.
Table 2 Magnesium and calcium contents (mg/g DM) in the alginate casings of the meat substitute sausages produced either with magnesium (indicated as Mg2+) or with calcium lactate (indicated as Ca2+) after 8 weeks of storage. Sample 2+
+ CaCl2 Mg Ca2+ + CaCl2 a–b
Calcium content (mg/g DM) b
11.56 ± 0.07 34.13 ± 0.48a
Declaration of Competing Interest
Magnesium content (mg/g DM) a
5.80 ± 0.09 0.09 ± 0.01b
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Significant differences (p < 0.05) between the different concentrations. 6
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Fig. 6. Schematic illustration of assumed diffusion of the efflorescence-causing components (magnesium, calcium, and lactate) to the surface of the sausages during the storage time.
Acknowledgement
within the program for promoting the Industrial Collective Research (IGF) of the German Ministry of Economic Affairs and Energy (BMWi), based on a resolution of the German Parliament.
This IGF Project (AiF 19689N) of the FEI is/was supported via AiF Appendix A See Fig. A1.
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Fig. A1. Potassium and sodium contents (mean ± standard deviation mg/g DM) of the sausages crosslinked with either 15, 20, 25, or 30% of CaCl2 during 8 weeks of storage.
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