Food Chemistry 145 (2014) 840–844
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Proteolytic characterisation in grass carp sausage inoculated with Lactobacillus plantarum and Pediococcus pentosaceus Xiaohua Nie ⇑, Shengli Lin, Qilin Zhang College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
a r t i c l e
i n f o
Article history: Received 7 May 2013 Received in revised form 13 August 2013 Accepted 23 August 2013 Available online 5 September 2013 Keywords: Grass carp sausage Lactobacillus plantarum Pediococcus pentosaceus Proteolysis
a b s t r a c t The proteolysis in grass carp sausages inoculated with Lactobacillus plantarum ZY40 and Pediococcus pentosaceus GY23 was investigated. As fermentation progressed, sarcoplasmic and myofibrillar proteins in both sausages were obviously degraded, and the proteolytic process was more intense in sausages inoculated with P. pentosaceus GY23. The increases in a-amino nitrogen, trichloroacetic acid (TCA)-soluble peptides and free amino acids were also detected in both sausages. The differences in a-amino nitrogen content and free amino acids concentration were due to the activity of inoculated lactic acid bacteria, while endogenous enzymes contributed to the release of TCA-soluble peptides. Our findings indicate that lactic acid bacteria influence proteolytic characterisation in fermented fish sausage, with strain-dependent activity. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In China, the gross yield of the low-valued freshwater fish such as grass carp, silver carp and bighead carp, is over 60% of the total freshwater fishery yield. However, the potential of these fish species has not yet been fully explored, due to the limited storage period and strong earthy/musty taste and odour. Fermented fish sausages are popular in oriental countries and also in parts of the Western countries. The common ingredients in these products include minced fish, salt, sugar and spices (Aryanta, Fleet, & Buckle, 1991). Lactic acid bacteria are the most important microorganisms as starter cultures for sausage fermentation. In recent years there has been an increasing interest in the development of fermented fish sausage with lactic acid bacteria. Although microbial and physical properties of lactic acid bacteria fermented fish sausages have been reported (Glatman, Drabkin, & Gelman, 2000; Riebroy, Benjakul, & Visessanguan, 2008; Xu, Xia, Yang, & Nie, 2010), fundamental aspects including proteolysis are less studied. Proteolysis or protein degradation is one of the most important biochemical changes during sausage fermentation. It can influence final texture and flavour by forming low-molecular-weight compounds such as peptides, amino acids and their derivatives (Benito, Rodríguez, Córdoba, Andrade, & Córdoba, 2005; Casaburi et al., 2008; Hughes et al., 2002). Proteolysis is attributed to the action of endogenous and microbial enzymes, although the main role of microorganisms seems to be in participating in the secondary hydrolysis of small proteins and ⇑ Corresponding author. Tel./fax: +86 571 88320345. E-mail address: [email protected] (X. Nie). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.096
peptides (Candogan, Wardlaw, & Acton, 2009; Fadda, Oliver, & Vignolo, 2002; Flores & Toldrá, 2011). Lactic acid bacteria usually do not possess strong proteolytic properties. However, some Lactobacillus sake, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus curvatus and Lactobacillus pentsus strains have been reported to intensify the hydrolysis of muscle proteins (Castellano, Aristoy, Sentandreu, Vignolo, & Toldrá, 2012; Fadda et al., 2002; Sanz et al., 1999; Sriphochanart & Skolpap, 2010). Several peptidases have been described in vitro for L. sake, L. curvatus and L. plantarum (Ammor & Mayo, 2007; Christensen, Dudley, Pederson, & Steele, 1999; Sanz & Toldrá, 2002). In a previous study, Lactobacillus plantarum ZY40 and Pediococcus pentosaceus GY23 were isolated from traditional fish products and selected as possible starter cultures (Zhang, Lin, & Nie, 2013). The objective of this study was to prepare grass carp (Ctenopharyngodon idellus) sausage with the two lactic acid bacteria and evaluate the proteolytic changes during the fermentation.
2. Materials and methods 2.1. Starter culture L. plantarum ZY40 and P. pentosaceus GY23 were selected from traditional fish products as possible starter cultures. Each strain were cultured twice in MRS broth at 30 °C for 24 h. The cells were collected by centrifugation (5000g, 5 min), washed twice in saline solution (0.85% NaCl) and resuspended in the same solution at a level of 1010 cfu/ml. These suspensions were used as starter cultures.
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2.3. Determination of pH and titratable acid Each ten-gram samples was homogenised in 100 ml of distiled water for 1 min, followed by centrifugation at 10,000g for 15 min. Using the supernatant, the pH was measured with a digital pH metre (Mettler Toledo, Schwerzenbach, Switzerland); titratable acid was determined by the AOAC (2002) method and expressed as a percentage of lactic acid. All determinations were performed in triplicate. 2.4. Protein composition analysis The protein components in sausage were fractionated according to the procedure of Visessanguan et al. (2006) with some modification. Sausage sample (5 g) was homogenised with 50 ml of 0.025 M potassium phosphate buffer (pH 7.2) for 1 min, followed by centrifugation at 10,000g for 15 min. The extraction was repeated twice and the supernatants were combined as sarcoplasmic protein fraction. The pellet was then extracted with 50 ml of 0.025 M potassium phosphate buffer (pH 7.2) containing 0.6 M NaCl by the same process. The supernatants were combined as myofibrillar protein fraction. The final residue was the salt-insoluble protein fraction. The nitrogen content of all protein fractions and total protein was determined by the Kjeldahl method (AOAC, 2002). Each protein fraction was expressed as a percentage of total protein. All determinations were performed in triplicate. 2.5. SDS–PAGE electrophoresis The protein concentrations of sarcoplasmic and myofibrillar fractions were adjusted to 6 mg/ml in a solution 0.025 M potassium phosphate buffer (pH 7.2) containing 2% sodium dodecyl sulphate (SDS). Each protein fraction was mixed with an equal volume of SDS–PAGE sample buffer (0.125 M Tris–HCl, 4% SDS, 20% glycerol, 10% b-mercaptoethanol, 0.005% bromophenol blue, pH 6.8), then heated at 100 °C for 5 min. SDS–PAGE was performed on 4% stacking gel and 10% separating gel, and electrophoresis was done at a constant voltage of 120 V. After electrophoresis, the gels were stained with Coomasie Brilliant Blue R-250 (0.1%) in 25% methanol and 10% acetic acid, and subsequently destained with 40% methanol and 10% acetic acid. Standard proteins from BioRad (Hercules, CA, USA) were run simultaneously for molecular mass identification.
2.7. Free amino acid analysis Sample for free amino acid analysis were prepared according to the method described by Aro Aro et al. (2010). Sample (10 g) was homogenised with 100 ml distiled water for 1 min, followed by centrifugation at 10,000g for 15 min. The supernatant was diluted 1:1 with 10% trichloroacetic acid, then kept for 30 min. After filtration, free amino acid in the filtrate was analysed using a fully automated amino acid analyser HITACHI l-8800A (Hitachi Ltd., Tokyo, Japan). The concentration of free amino acids in sausage was calculated by calibrating with standard amino acids and expressed as mg/100 g sample. 2.8. Statistical analysis The data were analysed statistically by one-way ANOVA using SPSS 17.0. S and means were compared by Duncan’s multiple range tests. 3. Result and discussion 3.1. pH and titratable acid The changes of pH value and titratable acid content in grass carp sausages are displayed in Fig. 1. There was a rapid decrease in pH in both sausages, reaching the values of 4.6–4.7 after 24 h and subsequently trending toward constancy until the end of fermentation. The pH decrease is generally associated with the organic acids produced by lactic acid bacteria. Low pH is an important factor in the control of pathogenic and spoilage bacteria (Glatman et al., 2000; Riebroy et al., 2008). Consistent with the pH change, titratable acid contents sharply increased and then remained constant afterward. An immediate and rapid production of titratable acid at initial stage of fermentation was found in the sausage inoculated with P. pentosaceus GY23. It seemed that P. pentosaceus GY23 had a better acidification capacity than L. plantarum ZY40. These tritatable acid contents were lower than those reported by Xu et al. (2010), probably due to the addition of sugars in fermentation (González-Fernández, Santos, Rovira, & Jaime, 2006). 3.2. Protein composition The evolutions of protein composition in grass carp sausages are reported in Fig. 2. Myofibrillar protein was the main protein 1.0
7.5 L. plantarum ZY-40 P. pentosaceus GY-23
7.0
0.8
6.5 6.0
0.6
5.5
0.4
5.0 0.2
4.5
2.6. Assay of a-amino nitrogen and trichloroacetic acid (TCA)-soluble peptides
a-Amino nitrogen was analysed by titration with formaldehyde (AOAC, 2002) and expressed as mg/100 g sample. The extraction of TCA-soluble peptides was performed as described by Riebroy et al.
titratable acid (%)
Fresh grass carp (C. idellus) were purchased from a local market (Hangzhou, Zhejiang, China), and immediately deheaded, gutted, scaled and filleted. The fillets were minced using a meat bone separator (drum with 5 mm diameter perforations). The mince was mixed with 2% NaCl, 0.5% glucose and 0.5% sucrose, then inoculated with L. plantarum ZY40 or P. pentosaceus GY23 at a final level of 5 107 cfu/g. The resulting mixtures were stuffed into collagen casings (25 mm diameter), at approximately 100 g each. The two batches of 15 sausages were then stored at 30 °C and 90% relative humidity. Three sausages from each batch were taken every 12 h for chemical analyses. After removing the casing, sausages were thoroughly cut up and ground in a meat grinder until the homogenous sample was obtained.
(2008). Sausage sample (5 g) was homogenised with 50 ml of 5% trichloroacetic acid and then kept at 4 °C for 1 h. After filtration, the soluble peptides in the filtrate was measured by Lowry method (Lowry, Rosebrough, Farr, & Randle, 1951) and expressed as lmol tyrosine/g sample. All determinations were performed in triplicate.
pH
2.2. Manufacture of grass carp sausage
0.0
4.0 0
12
24
36
48
Fermentaion time (h) Fig. 1. Changes of pH value and titratable acid content in grass carp sausage during the fermentation. (—): pH value; (- - -): titratable acid.
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component in initial sausages, followed by sarcoplasmic protein and salt-insoluble protein. As fermentation proceeded, a progressive decrease in sarcoplasmic and mypfibrillar proteins was accompanied by an strong increase in salt-insoluble protein. Similar changes were observed in Nham (Visessanguan et al., 2006) and silver carp sausages (Xu et al., 2010). It is possible that both sarcoplasmic and myofibrillar proteins had been degraded or became insoluble due to acid denaturation. At the end of fermentation, myofibrillar protein in sausages inoculated with L. plantarum ZY40 and P. pentosaceus GY23 were reduced by 76.23 and 67%, respectively.
3.3. SDS–PAGE profiles SDS–PAGE profiles of sarcoplasmic and myofibrillar proteins in grass carp sausages are shown in Fig. 3. The band intensity of these proteins decreased markedly or even disappeared throughout the fermentation, due to proteolysis and acid induced denaturation. Significant degradation of sarcoplasmic proteins was observed in both sausages. Proteolysis was much faster in the sausage inoculated with P. pentosaceus GY23, a marked difference notable after 24 h fermentation. Similar results were documented by Aro Aro et al. (2010) and Fadda et al. (2002). The intensity of the bands between 39 and 105 kDa markedly decreased, followed by the appearance of the bands at 37 kDa and 70 kDa. The protein fraction at 36 kDa remained stable during fermentation. Myofibrillar profiles of both sausages were identical, but the proteolysis was more intense in sausages inoculated with P. pentosaceus GY23. There was a significant reduction in the 200 kDa (myosin) and 45 kDa (actin) bands with the simultaneous appearance of several bands in the range of 70–150 kDa. Similar changes of myosin and actin were reported by Casaburi et al. (2007), Visessanguan et al. (2006) and Riebroy et al. (2008). The degradation
40
3.4. a-Amino nitrogen and TCA-soluble peptides
20
0 sarcoplasmic protein myofibrillar protein salt-insoluble protein (a) sausage inoculated with L. plantarumZY40
Relative protein content (%)
80
60
0h 12 h 24 h 36 h 48 h
40
20
0 sarcoplasmic protein myofibrillar protein salt-insoluble protein (b) sausage inoculated with pentosaceus GY23 Fig. 2. Change of protein composition in grass carp sausage during the fermentation.
The changes of proteolytic parameters such as a-amino nitrogen and TCA-soluble peptides are presented in Fig. 4. A gradual increase in the content of a-amino nitrogen and TCA-soluble peptides was observable in both sausages throughout fermentation. These changes coincided with the continuous degradation of sarcoplasmic and myofibrillar proteins (Fig. 3). These results 5
250 L. plantarum ZY-40 P. pentosaceus GY-23
4
200
3
150
2
100
1
50
0
0
12 24 36 Fermentaion time (h)
48
0
TCA-soluble peptide (μ mol tyrosine/g)
60
0h 12 h 24 h 36 h 48 h
products between 70 and 150 kDa may be generated from myosin degradation, as found by other authors during processing of salami (Mauriello, Casaburi, & Villani, 2002) and fermented sausages (Roseiro et al., 2008). The SDS–PAGE profiles suggested that lactic acid bacteria affected the breakdown of myofibrillar and sarcoplasmic proteins, with a strain-dependant activity. This is in agreement with the available literature by other authors, who have reported that some L. casei, L. curvatus, L. plantarum and P. pentosaceus strains intensify the proteolytic effects of muscle proteins degradation caused by muscle proteinases (Fadda et al., 2002; Sanz et al., 1999; Sriphochanart & Skolpap, 2010).
α-amino nitrogen (mg/100g)
Relative protein content (%)
80
Fig. 3. SDS–PAGE profiles of sarcoplasmic and myofibrillar proteins in grass carp sausage. Lane std.: molecular weight standards; Line 1, initial sausage; line 2, 4, 6, 8, sausage inoculated with L. plantarum ZY40 after 12, 24, 36, 48 h fermentation; line 3, 5, 7, 9, sausage inoculated with P. pentosaceus GY23 after 12, 24, 36, 48 h fermentation.
Fig. 4. Changes of a-amino nitrogen and TCA-soluble peptide in grass carp sausage. (—): a-amino nitrogen; (- - -): TCA-soluble peptide.
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X. Nie et al. / Food Chemistry 145 (2014) 840–844 Table 1 Free amino acid concentration in fermented grass carp sausage. Free amino acid (mg/100 g)
Initial sausage
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total concentration
0.70a 7.75a 5.66b 2.97a 115.24a 24.76a 1.73a 2.85a 2.05a 2.18a 3.93a 1.06a 1.58a 117.15a 8.20a 0.00a 297.81a
Fermented sausage L. plantarum ZY40
P. pentosaceus GY23
2.77b 6.77a 2.60a 9.00b 122.31a 26.93a 4.70b 61.32c 7.42b 3.75a 14.62b 1.85a 6.47b 125.93a 8.89a 11.23b 416.56b
18.35c 15.37b 10.95c 21.98c 123.85a 40.98b 15.11c 17.93b 23.69c 17.15b 51.07c 13.94b 27.41c 133.14b 16.44b 20.28c 567.67c
a, b, c
Values with unlike superscript letters in the same line are significantly different (P < 0.05).
agree with several studies demonstrating that proteolysis during sausage fermentation is reflected by an increase in a-amino nitrogen and TCA-soluble peptides content (Riebroy et al., 2008; Xu et al., 2010). Higher increase in a-amino nitrogen content was observed in sausages inoculated with P. pentosaceus GY23 throughout fermentation. Increases in TCA-soluble peptides were very similar for both sausages with no significant differences in their contents. Similar patterns were reported for som-fug inoculated with Pediococcus acidilactici (Riebroy et al., 2008) and Nham inoculated with Lactobacillus curvatus (Visessanguan et al., 2006). Numerous studies have concluded that initial breakdown of muscle protein is mainly attributed to endogenous proteinases, followed by the action of microbial peptidases which further degrade the protein fragments to small peptides and free amino acids (Candogan et al., 2009; Fadda et al., 2002; Molly et al., 1997). It might be suggested that P. pentosaceus GY23 may have more exopeptidases, flavouring the generation of free amino acids from the N-amino terminal of muscle proteins and peptides as compared to L. plantarum ZY40. 3.5. Free amino acids The concentrations of free amino acids in grass carp sausages are shown in Table 1. Histidine and glycine were the predominant free amino acids in initial sausages, representing 39.34 and 38.70% of total free amino acids concentration. An intense increase in the concentration of most free amino acids was observed in the sausages with L. plantarum ZY40 and P. pentosaceus GY23 with a final concentration of 416.56 and 567.67/100 g, respectively. Similar increases were reported by Candogan et al. (2009) and Aro Aro et al. (2010). The release of free amino acids is attributable to the proteolytic action of endogenous and microbial enzymes (Candogan et al., 2009; Fadda et al., 2002). Some strains of lactic acid bacteria are endowed with proteolytic activity, mainly intracellular amino, di- and tripeptidases (Fadda, López, & Vignolo, 2010; Papamanoli, Tzanetakis, Litopoulou-Tzanetaki, & Kotzekidou, 2003; Sanz & Toldrá, 2002). In general, these endo- and exo-peptidases contribute to increase the concentration of free amino acids that affect flavour development. Sausages inoculated with P. pentosaceus GY23 exhibited a significant increase in all the free amino acids, whereas sausages inoculated with L. plantarum ZY40 showed the increase in valine, arginine, phenylalanine, leucine, methionine and glutamic acid. A higher concentration of most free amino acids was observed in
sausages inoculated with P. pentosaceus GY23. These findings are in accordance with the change of a-amino nitrogen content (Fig. 4), and further confirm that P. pentosaceus GY23 had a more active proteolytic capacity to generate free amino acids during sausage fermentation. High concentrations of valine and leucine were found in sausages inoculated with L. plantarum ZY40 and P. pentosaceus GY23, respectively. These amino acids are well known to be the precursors of flavour compounds in fermented foods (Ammor et al., 2005; Montel, Masson, & Talon, 1998). 4. Conclusion In the present study, two batch sausages were prepared with L. plantarum ZY40 and P. pentosaceus GY23. During the fermentation, sarcoplasmic and myofibrillar proteins in both sausages were obviously degraded with a concomitant increase in a-amino nitrogen, TCA-soluble peptides and free amino acids. Moreover, there were apparent differences in proteolytic characterisations between the two sausages. The sausage inoculated with P. pentosaceus GY23 had a more intense proteolysis, as indicated by the greater rate of protein degradation and higher accumulation of a-amino nitrogen and free amino acids. TCA-soluble peptides were similar in both sausages. Therefore, endogenous enzymes were responsible for the initial degradation of muscle proteins, while lactic acid bacteria mainly participated in the generation of free amino acids. Acknowledgements This study was financially supported by the National 863 Project of China (Serial Number 2007AA09Z442). We are grateful to J.M. Xu technical help with free amino acid analysis. References Ammor, S., Dufour, E., Zagorec, M., Chaillou, S., Chaillou, S., & Chevallier, I. (2005). Characterisation and selection of Lactobacillus sake strains isolated from traditional dry sausage for their potential use as starter cultures. Food Microbiology, 22, 529–538. Ammor, M. S., & Mayo, B. (2007). Selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: An update. Meat Science, 76, 138–146. AOAC (2002). Official methods of analysis (17th ed.). Arlington, VA, USA: Association of Official Analytical Chemists, International, Inc.. Aro Aro, J. M., Nyam-Osor, P., Tsuji, K., Shimada, K., Fukushima, M., & Sekikawa, M. (2010). The effect of starter cultures on proteolytic changes and amino acid content in fermented sausages. Food Chemistry, 119, 279–285.
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Aryanta, R. W., Fleet, G. H., & Buckle, K. A. (1991). The occurrence and growth of microorganisms during the fermentation of fish sausage. International Journal of Food Microbiology, 13, 143–155. Benito, M. J., Rodríguez, M., Córdoba, M. G., Andrade, M. J., & Córdoba, J. J. (2005). Effect of the fungal protease EPg222 on proteolysis and texture in the dry fermented sausage ‘salchichón’. Journal of the Science of Food and Agriculture, 85, 273–280. Candogan, K., Wardlaw, F. B., & Acton, J. C. (2009). Effect of starter culture on proteolytic changes during processing of fermented beef sausage. Food Chemistry, 116, 731–737. Casaburi, A., Aristoy, M. C., Cavella, S., Monaco, R. D., Ercolini, D., Toldra, F., et al. (2007). Biochemical and sensory characteristics of traditional fermented sausages of Vallo di Diano (Southern Italy) as affected by the use of starter cultures. Meat Science, 76, 295–307. Casaburi, A., Monaco, R. D., Cavella, S., Toldrá, F., Ercolini, D., & Villani, F. (2008). Proteolytic and lipolytic starter cultures and their effect on traditional fermented sausages ripening and sensory traits. Food Microbiology, 25, 334–335. Castellano, P., Aristoy, M. C., Sentandreu, M. A., Vignolo, G., & Toldrá, F. (2012). Lactobacillus sakei CRL1862 improves safety and protein hydrolysis in meat systems. Journal of Applied Microbiology, 113, 1407–1416. Christensen, J. E., Dudley, E. G., Pederson, J. A., & Steele, J. L. (1999). Peptidases and amino acid catabolism in lactic acid bacteria. Antonie van Leeuwenhoek, 76, 217–246. Fadda, S., López, C., & Vignolo, G. (2010). Role of lactic acid bacteria during meat conditioning and fermentation: Peptides generated as sensorial and hygienic biomarkers. Meat Science, 86, 66–79. Fadda, S., Oliver, G., & Vignolo, G. (2002). Protein degradation by Lactobacillus plantarum and Lactobacillus casei in a sausage model system. Journal of Food Science, 67, 1179–1183. Flores, M., & Toldrá, F. (2011). Microbial enzymatic activities for improved fermented meats. Trends in Food Science & Technology, 22, 81–90. Glatman, L., Drabkin, L. V., & Gelman, A. (2000). Using lactic acid bacteria for developing novel fish food products. Journal of the Science of Food and Agriculture, 80, 375–380. González-Fernández, C., Santos, E. M., Rovira, J., & Jaime, I. (2006). The effect of sugar concentration and starter culture on instrumental and sensory textural properties of chorizo-Spanish dry-cured sausage. Meat Science, 74, 467–475. Hughes, M. C., Kerry, J. P., Arendt, E. K., Kenneally, P. M., McSweeney, P. L. H., et al. (2002). Characterisation of proteolysis during the ripening of semi-dry fermented sausages. Meat Science, 62, 205–216.
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Proteolytic characterisation in grass carp sausage inoculated with Lactobacillus plantarum and Pediococcus pentosaceus Xiaohua Nie ⇑, Shengli Lin, Qilin Zhang College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
a r t i c l e
i n f o
Article history: Received 7 May 2013 Received in revised form 13 August 2013 Accepted 23 August 2013 Available online 5 September 2013 Keywords: Grass carp sausage Lactobacillus plantarum Pediococcus pentosaceus Proteolysis
a b s t r a c t The proteolysis in grass carp sausages inoculated with Lactobacillus plantarum ZY40 and Pediococcus pentosaceus GY23 was investigated. As fermentation progressed, sarcoplasmic and myofibrillar proteins in both sausages were obviously degraded, and the proteolytic process was more intense in sausages inoculated with P. pentosaceus GY23. The increases in a-amino nitrogen, trichloroacetic acid (TCA)-soluble peptides and free amino acids were also detected in both sausages. The differences in a-amino nitrogen content and free amino acids concentration were due to the activity of inoculated lactic acid bacteria, while endogenous enzymes contributed to the release of TCA-soluble peptides. Our findings indicate that lactic acid bacteria influence proteolytic characterisation in fermented fish sausage, with strain-dependent activity. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction In China, the gross yield of the low-valued freshwater fish such as grass carp, silver carp and bighead carp, is over 60% of the total freshwater fishery yield. However, the potential of these fish species has not yet been fully explored, due to the limited storage period and strong earthy/musty taste and odour. Fermented fish sausages are popular in oriental countries and also in parts of the Western countries. The common ingredients in these products include minced fish, salt, sugar and spices (Aryanta, Fleet, & Buckle, 1991). Lactic acid bacteria are the most important microorganisms as starter cultures for sausage fermentation. In recent years there has been an increasing interest in the development of fermented fish sausage with lactic acid bacteria. Although microbial and physical properties of lactic acid bacteria fermented fish sausages have been reported (Glatman, Drabkin, & Gelman, 2000; Riebroy, Benjakul, & Visessanguan, 2008; Xu, Xia, Yang, & Nie, 2010), fundamental aspects including proteolysis are less studied. Proteolysis or protein degradation is one of the most important biochemical changes during sausage fermentation. It can influence final texture and flavour by forming low-molecular-weight compounds such as peptides, amino acids and their derivatives (Benito, Rodríguez, Córdoba, Andrade, & Córdoba, 2005; Casaburi et al., 2008; Hughes et al., 2002). Proteolysis is attributed to the action of endogenous and microbial enzymes, although the main role of microorganisms seems to be in participating in the secondary hydrolysis of small proteins and ⇑ Corresponding author. Tel./fax: +86 571 88320345. E-mail address: [email protected] (X. Nie). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.08.096
peptides (Candogan, Wardlaw, & Acton, 2009; Fadda, Oliver, & Vignolo, 2002; Flores & Toldrá, 2011). Lactic acid bacteria usually do not possess strong proteolytic properties. However, some Lactobacillus sake, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus curvatus and Lactobacillus pentsus strains have been reported to intensify the hydrolysis of muscle proteins (Castellano, Aristoy, Sentandreu, Vignolo, & Toldrá, 2012; Fadda et al., 2002; Sanz et al., 1999; Sriphochanart & Skolpap, 2010). Several peptidases have been described in vitro for L. sake, L. curvatus and L. plantarum (Ammor & Mayo, 2007; Christensen, Dudley, Pederson, & Steele, 1999; Sanz & Toldrá, 2002). In a previous study, Lactobacillus plantarum ZY40 and Pediococcus pentosaceus GY23 were isolated from traditional fish products and selected as possible starter cultures (Zhang, Lin, & Nie, 2013). The objective of this study was to prepare grass carp (Ctenopharyngodon idellus) sausage with the two lactic acid bacteria and evaluate the proteolytic changes during the fermentation.
2. Materials and methods 2.1. Starter culture L. plantarum ZY40 and P. pentosaceus GY23 were selected from traditional fish products as possible starter cultures. Each strain were cultured twice in MRS broth at 30 °C for 24 h. The cells were collected by centrifugation (5000g, 5 min), washed twice in saline solution (0.85% NaCl) and resuspended in the same solution at a level of 1010 cfu/ml. These suspensions were used as starter cultures.
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2.3. Determination of pH and titratable acid Each ten-gram samples was homogenised in 100 ml of distiled water for 1 min, followed by centrifugation at 10,000g for 15 min. Using the supernatant, the pH was measured with a digital pH metre (Mettler Toledo, Schwerzenbach, Switzerland); titratable acid was determined by the AOAC (2002) method and expressed as a percentage of lactic acid. All determinations were performed in triplicate. 2.4. Protein composition analysis The protein components in sausage were fractionated according to the procedure of Visessanguan et al. (2006) with some modification. Sausage sample (5 g) was homogenised with 50 ml of 0.025 M potassium phosphate buffer (pH 7.2) for 1 min, followed by centrifugation at 10,000g for 15 min. The extraction was repeated twice and the supernatants were combined as sarcoplasmic protein fraction. The pellet was then extracted with 50 ml of 0.025 M potassium phosphate buffer (pH 7.2) containing 0.6 M NaCl by the same process. The supernatants were combined as myofibrillar protein fraction. The final residue was the salt-insoluble protein fraction. The nitrogen content of all protein fractions and total protein was determined by the Kjeldahl method (AOAC, 2002). Each protein fraction was expressed as a percentage of total protein. All determinations were performed in triplicate. 2.5. SDS–PAGE electrophoresis The protein concentrations of sarcoplasmic and myofibrillar fractions were adjusted to 6 mg/ml in a solution 0.025 M potassium phosphate buffer (pH 7.2) containing 2% sodium dodecyl sulphate (SDS). Each protein fraction was mixed with an equal volume of SDS–PAGE sample buffer (0.125 M Tris–HCl, 4% SDS, 20% glycerol, 10% b-mercaptoethanol, 0.005% bromophenol blue, pH 6.8), then heated at 100 °C for 5 min. SDS–PAGE was performed on 4% stacking gel and 10% separating gel, and electrophoresis was done at a constant voltage of 120 V. After electrophoresis, the gels were stained with Coomasie Brilliant Blue R-250 (0.1%) in 25% methanol and 10% acetic acid, and subsequently destained with 40% methanol and 10% acetic acid. Standard proteins from BioRad (Hercules, CA, USA) were run simultaneously for molecular mass identification.
2.7. Free amino acid analysis Sample for free amino acid analysis were prepared according to the method described by Aro Aro et al. (2010). Sample (10 g) was homogenised with 100 ml distiled water for 1 min, followed by centrifugation at 10,000g for 15 min. The supernatant was diluted 1:1 with 10% trichloroacetic acid, then kept for 30 min. After filtration, free amino acid in the filtrate was analysed using a fully automated amino acid analyser HITACHI l-8800A (Hitachi Ltd., Tokyo, Japan). The concentration of free amino acids in sausage was calculated by calibrating with standard amino acids and expressed as mg/100 g sample. 2.8. Statistical analysis The data were analysed statistically by one-way ANOVA using SPSS 17.0. S and means were compared by Duncan’s multiple range tests. 3. Result and discussion 3.1. pH and titratable acid The changes of pH value and titratable acid content in grass carp sausages are displayed in Fig. 1. There was a rapid decrease in pH in both sausages, reaching the values of 4.6–4.7 after 24 h and subsequently trending toward constancy until the end of fermentation. The pH decrease is generally associated with the organic acids produced by lactic acid bacteria. Low pH is an important factor in the control of pathogenic and spoilage bacteria (Glatman et al., 2000; Riebroy et al., 2008). Consistent with the pH change, titratable acid contents sharply increased and then remained constant afterward. An immediate and rapid production of titratable acid at initial stage of fermentation was found in the sausage inoculated with P. pentosaceus GY23. It seemed that P. pentosaceus GY23 had a better acidification capacity than L. plantarum ZY40. These tritatable acid contents were lower than those reported by Xu et al. (2010), probably due to the addition of sugars in fermentation (González-Fernández, Santos, Rovira, & Jaime, 2006). 3.2. Protein composition The evolutions of protein composition in grass carp sausages are reported in Fig. 2. Myofibrillar protein was the main protein 1.0
7.5 L. plantarum ZY-40 P. pentosaceus GY-23
7.0
0.8
6.5 6.0
0.6
5.5
0.4
5.0 0.2
4.5
2.6. Assay of a-amino nitrogen and trichloroacetic acid (TCA)-soluble peptides
a-Amino nitrogen was analysed by titration with formaldehyde (AOAC, 2002) and expressed as mg/100 g sample. The extraction of TCA-soluble peptides was performed as described by Riebroy et al.
titratable acid (%)
Fresh grass carp (C. idellus) were purchased from a local market (Hangzhou, Zhejiang, China), and immediately deheaded, gutted, scaled and filleted. The fillets were minced using a meat bone separator (drum with 5 mm diameter perforations). The mince was mixed with 2% NaCl, 0.5% glucose and 0.5% sucrose, then inoculated with L. plantarum ZY40 or P. pentosaceus GY23 at a final level of 5 107 cfu/g. The resulting mixtures were stuffed into collagen casings (25 mm diameter), at approximately 100 g each. The two batches of 15 sausages were then stored at 30 °C and 90% relative humidity. Three sausages from each batch were taken every 12 h for chemical analyses. After removing the casing, sausages were thoroughly cut up and ground in a meat grinder until the homogenous sample was obtained.
(2008). Sausage sample (5 g) was homogenised with 50 ml of 5% trichloroacetic acid and then kept at 4 °C for 1 h. After filtration, the soluble peptides in the filtrate was measured by Lowry method (Lowry, Rosebrough, Farr, & Randle, 1951) and expressed as lmol tyrosine/g sample. All determinations were performed in triplicate.
pH
2.2. Manufacture of grass carp sausage
0.0
4.0 0
12
24
36
48
Fermentaion time (h) Fig. 1. Changes of pH value and titratable acid content in grass carp sausage during the fermentation. (—): pH value; (- - -): titratable acid.
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component in initial sausages, followed by sarcoplasmic protein and salt-insoluble protein. As fermentation proceeded, a progressive decrease in sarcoplasmic and mypfibrillar proteins was accompanied by an strong increase in salt-insoluble protein. Similar changes were observed in Nham (Visessanguan et al., 2006) and silver carp sausages (Xu et al., 2010). It is possible that both sarcoplasmic and myofibrillar proteins had been degraded or became insoluble due to acid denaturation. At the end of fermentation, myofibrillar protein in sausages inoculated with L. plantarum ZY40 and P. pentosaceus GY23 were reduced by 76.23 and 67%, respectively.
3.3. SDS–PAGE profiles SDS–PAGE profiles of sarcoplasmic and myofibrillar proteins in grass carp sausages are shown in Fig. 3. The band intensity of these proteins decreased markedly or even disappeared throughout the fermentation, due to proteolysis and acid induced denaturation. Significant degradation of sarcoplasmic proteins was observed in both sausages. Proteolysis was much faster in the sausage inoculated with P. pentosaceus GY23, a marked difference notable after 24 h fermentation. Similar results were documented by Aro Aro et al. (2010) and Fadda et al. (2002). The intensity of the bands between 39 and 105 kDa markedly decreased, followed by the appearance of the bands at 37 kDa and 70 kDa. The protein fraction at 36 kDa remained stable during fermentation. Myofibrillar profiles of both sausages were identical, but the proteolysis was more intense in sausages inoculated with P. pentosaceus GY23. There was a significant reduction in the 200 kDa (myosin) and 45 kDa (actin) bands with the simultaneous appearance of several bands in the range of 70–150 kDa. Similar changes of myosin and actin were reported by Casaburi et al. (2007), Visessanguan et al. (2006) and Riebroy et al. (2008). The degradation
40
3.4. a-Amino nitrogen and TCA-soluble peptides
20
0 sarcoplasmic protein myofibrillar protein salt-insoluble protein (a) sausage inoculated with L. plantarumZY40
Relative protein content (%)
80
60
0h 12 h 24 h 36 h 48 h
40
20
0 sarcoplasmic protein myofibrillar protein salt-insoluble protein (b) sausage inoculated with pentosaceus GY23 Fig. 2. Change of protein composition in grass carp sausage during the fermentation.
The changes of proteolytic parameters such as a-amino nitrogen and TCA-soluble peptides are presented in Fig. 4. A gradual increase in the content of a-amino nitrogen and TCA-soluble peptides was observable in both sausages throughout fermentation. These changes coincided with the continuous degradation of sarcoplasmic and myofibrillar proteins (Fig. 3). These results 5
250 L. plantarum ZY-40 P. pentosaceus GY-23
4
200
3
150
2
100
1
50
0
0
12 24 36 Fermentaion time (h)
48
0
TCA-soluble peptide (μ mol tyrosine/g)
60
0h 12 h 24 h 36 h 48 h
products between 70 and 150 kDa may be generated from myosin degradation, as found by other authors during processing of salami (Mauriello, Casaburi, & Villani, 2002) and fermented sausages (Roseiro et al., 2008). The SDS–PAGE profiles suggested that lactic acid bacteria affected the breakdown of myofibrillar and sarcoplasmic proteins, with a strain-dependant activity. This is in agreement with the available literature by other authors, who have reported that some L. casei, L. curvatus, L. plantarum and P. pentosaceus strains intensify the proteolytic effects of muscle proteins degradation caused by muscle proteinases (Fadda et al., 2002; Sanz et al., 1999; Sriphochanart & Skolpap, 2010).
α-amino nitrogen (mg/100g)
Relative protein content (%)
80
Fig. 3. SDS–PAGE profiles of sarcoplasmic and myofibrillar proteins in grass carp sausage. Lane std.: molecular weight standards; Line 1, initial sausage; line 2, 4, 6, 8, sausage inoculated with L. plantarum ZY40 after 12, 24, 36, 48 h fermentation; line 3, 5, 7, 9, sausage inoculated with P. pentosaceus GY23 after 12, 24, 36, 48 h fermentation.
Fig. 4. Changes of a-amino nitrogen and TCA-soluble peptide in grass carp sausage. (—): a-amino nitrogen; (- - -): TCA-soluble peptide.
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Initial sausage
Aspartic acid Threonine Serine Glutamic acid Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Total concentration
0.70a 7.75a 5.66b 2.97a 115.24a 24.76a 1.73a 2.85a 2.05a 2.18a 3.93a 1.06a 1.58a 117.15a 8.20a 0.00a 297.81a
Fermented sausage L. plantarum ZY40
P. pentosaceus GY23
2.77b 6.77a 2.60a 9.00b 122.31a 26.93a 4.70b 61.32c 7.42b 3.75a 14.62b 1.85a 6.47b 125.93a 8.89a 11.23b 416.56b
18.35c 15.37b 10.95c 21.98c 123.85a 40.98b 15.11c 17.93b 23.69c 17.15b 51.07c 13.94b 27.41c 133.14b 16.44b 20.28c 567.67c
a, b, c
Values with unlike superscript letters in the same line are significantly different (P < 0.05).
agree with several studies demonstrating that proteolysis during sausage fermentation is reflected by an increase in a-amino nitrogen and TCA-soluble peptides content (Riebroy et al., 2008; Xu et al., 2010). Higher increase in a-amino nitrogen content was observed in sausages inoculated with P. pentosaceus GY23 throughout fermentation. Increases in TCA-soluble peptides were very similar for both sausages with no significant differences in their contents. Similar patterns were reported for som-fug inoculated with Pediococcus acidilactici (Riebroy et al., 2008) and Nham inoculated with Lactobacillus curvatus (Visessanguan et al., 2006). Numerous studies have concluded that initial breakdown of muscle protein is mainly attributed to endogenous proteinases, followed by the action of microbial peptidases which further degrade the protein fragments to small peptides and free amino acids (Candogan et al., 2009; Fadda et al., 2002; Molly et al., 1997). It might be suggested that P. pentosaceus GY23 may have more exopeptidases, flavouring the generation of free amino acids from the N-amino terminal of muscle proteins and peptides as compared to L. plantarum ZY40. 3.5. Free amino acids The concentrations of free amino acids in grass carp sausages are shown in Table 1. Histidine and glycine were the predominant free amino acids in initial sausages, representing 39.34 and 38.70% of total free amino acids concentration. An intense increase in the concentration of most free amino acids was observed in the sausages with L. plantarum ZY40 and P. pentosaceus GY23 with a final concentration of 416.56 and 567.67/100 g, respectively. Similar increases were reported by Candogan et al. (2009) and Aro Aro et al. (2010). The release of free amino acids is attributable to the proteolytic action of endogenous and microbial enzymes (Candogan et al., 2009; Fadda et al., 2002). Some strains of lactic acid bacteria are endowed with proteolytic activity, mainly intracellular amino, di- and tripeptidases (Fadda, López, & Vignolo, 2010; Papamanoli, Tzanetakis, Litopoulou-Tzanetaki, & Kotzekidou, 2003; Sanz & Toldrá, 2002). In general, these endo- and exo-peptidases contribute to increase the concentration of free amino acids that affect flavour development. Sausages inoculated with P. pentosaceus GY23 exhibited a significant increase in all the free amino acids, whereas sausages inoculated with L. plantarum ZY40 showed the increase in valine, arginine, phenylalanine, leucine, methionine and glutamic acid. A higher concentration of most free amino acids was observed in
sausages inoculated with P. pentosaceus GY23. These findings are in accordance with the change of a-amino nitrogen content (Fig. 4), and further confirm that P. pentosaceus GY23 had a more active proteolytic capacity to generate free amino acids during sausage fermentation. High concentrations of valine and leucine were found in sausages inoculated with L. plantarum ZY40 and P. pentosaceus GY23, respectively. These amino acids are well known to be the precursors of flavour compounds in fermented foods (Ammor et al., 2005; Montel, Masson, & Talon, 1998). 4. Conclusion In the present study, two batch sausages were prepared with L. plantarum ZY40 and P. pentosaceus GY23. During the fermentation, sarcoplasmic and myofibrillar proteins in both sausages were obviously degraded with a concomitant increase in a-amino nitrogen, TCA-soluble peptides and free amino acids. Moreover, there were apparent differences in proteolytic characterisations between the two sausages. The sausage inoculated with P. pentosaceus GY23 had a more intense proteolysis, as indicated by the greater rate of protein degradation and higher accumulation of a-amino nitrogen and free amino acids. TCA-soluble peptides were similar in both sausages. Therefore, endogenous enzymes were responsible for the initial degradation of muscle proteins, while lactic acid bacteria mainly participated in the generation of free amino acids. Acknowledgements This study was financially supported by the National 863 Project of China (Serial Number 2007AA09Z442). We are grateful to J.M. Xu technical help with free amino acid analysis. References Ammor, S., Dufour, E., Zagorec, M., Chaillou, S., Chaillou, S., & Chevallier, I. (2005). Characterisation and selection of Lactobacillus sake strains isolated from traditional dry sausage for their potential use as starter cultures. Food Microbiology, 22, 529–538. Ammor, M. S., & Mayo, B. (2007). Selection criteria for lactic acid bacteria to be used as functional starter cultures in dry sausage production: An update. Meat Science, 76, 138–146. AOAC (2002). Official methods of analysis (17th ed.). Arlington, VA, USA: Association of Official Analytical Chemists, International, Inc.. Aro Aro, J. M., Nyam-Osor, P., Tsuji, K., Shimada, K., Fukushima, M., & Sekikawa, M. (2010). The effect of starter cultures on proteolytic changes and amino acid content in fermented sausages. Food Chemistry, 119, 279–285.
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