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Flavour profiles of dry sausages fermented by selected novel meat starter cultures
Meat Science 58 (2001) 111±116
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Flavour pro®les of dry sausages fermented by selected novel meat starter cultures S. ErkkilaÈ a,*, E. PetaÈjaÈ a, S. Eerola b, L. Lilleberg b, T. Mattila-Sandholm c, M-L. Suihko c a
Department of Food Technology, University of Helsinki, PO Box 27, 00014 Helsinki, Finland Department of Chemistry, National Veterinary and Food Research Institute, Helsinki, Finland c VTT Biotechnology, Espoo, Finland
b
Received 3 July 2000; received in revised form 13 September 2000; accepted 4 October 2000
Abstract Probiotic or bioprotective Lactobacillus rhamnosus strains GG, LC-705 and E-97800 as well as Pediococcus pentosaceus E-90390 and Lactobacillus plantarum E-98098 were studied for their ability to act as main fermenting organisms in the manufacturing process of dry sausages. In the preliminary tests, their abilities to produce lactic acid and biogenic amines, histamine or tyramine, were studied in MRS broth and analysed by high-performance liquid chromatography. The strains produced higher or equal amounts of lactic acid compared to control and were amine negative. During the actual fermentation process of dry sausages the numbers of inoculated bacteria increased from the level 6.5±7.0 log cfu/g to 8.0±9.0 log cfu/g. The most fast growing strains were P. pentosaceus E-90390 and the control while the growth of L. plantarum E-98098 and L. rhamnosus LC-705 were the slowest. The pH value of the sausages decreased from 5.6 to 4.9±5.0. The presence of these experimental strains as major organisms in the sausages after fermentation and ripening was con®rmed on the bases of their genetic ®ngerprints. The ¯avour pro®les of the experimental sausages produced by these probiotic or protective strains were similar with that produced by the commercial meat starter culture and commercial North European dry sausage recipe. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Probiotic; Dry sausage; Flavour pro®le; Ribotyping
1. Introduction Functional foods are designed to maintain or even promote human health. Their positive eects are due to either additions of active ingredients or removal or replacement of undesirable substances. It has been predicted that functional foods will account for 5% of total food consumption in Europe (Hilliam, 1998). Probiotic cultures are designated to balance the indigenous micro¯ora of the host and may have several health promoting eects (Salminen, Deighton, Benno, & Gorbach, 1998). In addition to the dairy industry, researchers in the meat industry have also recently begun to ®nd novel starter cultures with additional value. Since the 1950s and the times of Professor F.P. Niinivaara, meat starter cultures have ensured the quality and safety of * Corresponding author. Tel.: +358-2-62037334; fax: +358-262037399. E-mail address: [email protected].® (S. ErkkilaÈ).
the fermented products. Starter strains used in dry sausage manufacture process in the north of Europe produce sucient amounts of lactic acid in order to decrease the pH of meat to values 4.8±5.0. The low pH value is the basis for the safety as well as texture and colour of dry sausages (KroÈckel, 1995). Recently dry sausages fermented by probiotic cultures have been studied by Andersen (1998), Arihara et al. (1998), Sameshima, Magome, Takeshita, Arihara, Itoh, and Kondo (1998) and ErkkilaÈ, Suihko, Eerola, PetaÈjaÈ, and MattilaSandholm (2000), ErkkilaÈ, VenaÈlaÈinen, Hielm, PetaÈjaÈ, Puolanne, and Mattila-Sandholm (2000). The typical ¯avour of a dry sausage is a result of the bacterial and also muscle enzyme metabolism of carbohydrates, proteins and lipids. In combination with spices, all metabolism products result in the sensory pro®le of a dry sausage. For North European sausages lactic acid is the main ¯avour component, but acetic acid is also present and actually needed at low concentrations for full dry sausage ¯avour. The amount of other acids
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is typically extremely low. The amount of free amino acids and fatty acids increases during the ripening process, which may slightly aect the ¯avour of dry sausage (Dainty & Blom, 1995; LuÈcke, 1986). It is generally believed that dierent starter cultures result in the dierent sensory characteristics of dry sausages. This means that when novel strains are selected, the ability of the strains to contribute to the high quality sensory properties of the end product should be studied (Holzapfel, Haberer, Snel, Schillinger, & Huis in't Veld., 1998). The objective of the present work was to study probiotic or bioprotective Lactobacillus rhamnosus GG, E97800 and LC-705 as well as Pediococcus pentosaceus E-90390 and Lactobacillus plantarum E-98098 as dry sausage starter cultures. Preliminary studies were focused on their ability to form sucient amounts of lactic acid and not to form biogenic amines. The eventual decarboxylation process of amino acids by lactic acid bacteria may result high amounts of toxic biogenic amines (Shalaby, 1996). The monitoring of dry sausage fermentation and ripening was focused on the growth of selected strains and on tracing them from the sausages, as well as on the development of pH value in dry sausages. The principal aim was to characterise any eventual dierences in ¯avour pro®les between dry sausages fermented by dierent strains. 2. Materials and methods 2.1. Strains The strains used in this study were kindly provided by Valio Ltd., VTT Biotechnology and GewuÈrzmuÈller GmbH (Table 1). The bene®cial eects of probiotic L. rhamnosus GG are well documented (Saxelin, 1997) while L. rhamnosus E-97800 is a novel potential probiotic strain (Kontula et al., 2000; Kontula, Suihko, von Wright, & Mattila-Sandholm, 1999). L. rhamnosus LC705 is a bioprotective lactic acid bacterium for fermented dairy products (MaÈyraÈ-MaÈkinen & Suomalainen,
Table 1 Bacterial strain used for fermentation Species
Code
Supplier
Lactobacillus rhamnosus L. rhamnosus L. rhamnosus Lactobacillus plantarum Pediococcus pentosaceus P. pentosaceusa Lactobacillus sakeib
GG LC-705 E-97800 E-98098 E-90390 Condi Rasant LS-25
Valio Ltd. Valio Ltd. VTT Biotechnology VTT Biotechnology VTT Biotechnology GewuÈrzmuÈller GmbH GewuÈrzmuÈller GmbH
a b
Used as a control in preliminary tests. Used as a control in dry sausage fermentation trials.
1995) while P. pentosaceus E-90390 and L. plantarum E98098 are protective cultures for malting procedure (Haikara & Mattila-Sandholm, 1993). Recently L. rhamnosus LC-705 (Tuomola & Salminen, 1998) and L. plantarum E-98098 (Alander, De Smet, Nollet, Verstraete, von Wright, & Matilla-Sandholm, 1999; Miettinen et al., 1998) have been studied also for their probiotic properties. The commercial starter strains P. pentosaceus (isolated from a commercial mixed culture of P. pentosaceus and Staphylococcus carnosus named Condi Rasant, GewuÈrzmuÈller, Germany) and Lactobacillus sakei (isolated from a commercial mixed culture of L. sakei and S. carnosus named LS-25, GewuÈrzmuÈller, Germany) was used as the controls in preliminary tests and in the actual dry sausage manufacturing, respectively. 2.2. Production of lactic acid in MRS broth Strains were cultivated on MRS agar (pH 5.6, Difco) for 3 days at 30 C. Three colonies of each strain were picked and inoculated in 10 ml MRS broths (Difco). The purity of broth cultures were checked by Gram staining and microscoping. After 48 h incubation at 22 C, the broths were puri®ed with strong anionexchange solid phase extraction cartridges. Lactic acid was separated with a reversed-phase column (Spherisorb S5ODS2 254.6 mm, 5 mm, Phase separations Ltd, UK) in a high-performance liquid chromatography (HPLC) system. Lactic acid concentrations of the samples were determined at a wavelength of 214 nm by using the external standard method with 0.5 mM, 1.0 mM, 5.0 mM and 10.0 mM standards. The mobile phase 50 mM potassium phosphate buer (pH 2.4) was pumped at 1 ml/min and the column temperature was 30 C. The relative retention value for lactic acid was 1.3. Standards were run eight times and each sample twice. 2.3. Formation of biogenic amines in MRS broth Strains were cultivated on MRS agar (pH 5.6, Difco) for 3 days at 30 C. Three colonies of each strain were picked from pure culture MRS agar plates and inoculated into 10 ml MRS broths (Difco) containing 0.5% histidine and 0.5% tyrosine. Since the maximum histamine and tyramine production in MRS broth takes place after 24 h of incubation (Chandler, Batish, Babu, & Singh, 1989) the inoculated broths were incubated for 48 h at 22 C. Histamine and tyramine were extracted from 10 ml of MRS-broth and detected as their dansyl derivatives at 254 nm by a HPLC-system (HP 1090 Series M LC, diode-array detector including gradient module, binary DR5 pumps, autoinjector system with 25 ml loop) by the method of Eerola, Hinkkanen, Lindfors, and Hirvi (1993). The limit of determination for histamine was 1 mg/ml and for tyramine 2 mg/ml.
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2.4. Preparation of sausages Raw sausage material, which was 18% of protein and 13% of fat (pork, beef and pork fat) as well as salt (2.7%, analysed by sodium-ions) and spices, e.g. white pepper and cardamom (Finnish commercial formulation), was divided into six separate batches and inoculated by selected ®ve experimental probiotic or bioprotective strains. L. sakei (isolated from LS-25, GewuÈrzmuÈller, Germany) was used as the control. LS-25 and Condi Rasant used in preliminary tests are both meat starter cultures for fast acidifyed dry sausages with similar acidifying capacities (GewuÈrzmuÈller, Germany). Lactic acid bacteria were inoculated as pure MRS broth cultures grown at 30 C for 48 h aiming at 7 log cfu/g raw material (100 ml/10 kg). Normally, staphylococci are used in combination with lactic acid bacteria. In this study, a commercial staphylococci starter culture, PoÈkelferment 77 (S. carnosus, Chr. Hansen, Denmark), was used in each batch aiming at 6 log cfu/g raw material (1 g/10 kg). Three dierent experimental series with each strain were prepared by using 65 mm diameter casings. The series were fermented and ripened with the same program: for two days at 23±22 C and rH 95% followed by a gradual reduction of temperature (from 21 to 19 C) and relative humidity (from rH 94% to rH 90%) during the next 4 days. Sausages were also smoked (2 h) twice a day between the second and ®fth day of fermentation. After 7 days of fermentation, sausages were ripened for 28 days at 17 C (rH 75%). 2.5. Microbiological determinations, pH and weight losses of dry sausages Each batch was examined microbiologically after manufacture (0 day) and after 7, 14, 28 and 35 days of fermentation and ripening. The numbers of staphylococci were determined on Baird Parker agar (2 days at 37 C) and the numbers of lactic acid bacteria on MRS agar (pH 5.6; 3 days at 30 C). Characterisation of the inoculated experimental pure strains (0 day) and of the sausage isolates after 35 days were carried out using the automated RiboPrinter1 Microbial Characterisation System (QualiconTM, USA) according to the instructions of the manufacturer (Bruce, 1996). Cells from the MRS agar plates were suspended in buer. DNA was released by two lysing agents and cut with EcoR1 restriction enzyme. Resulted fragments were electrophoretically separated according to their size, transferred to a nylon membrane, and hybridized with a chemically labelled rRNA operon from Escherichia coli. The luminescent fragments of membranes were photographed with a CCD camera and converted to digital information. Analysis software automatically processed the digitalized image producing a RiboPrint1 pattern (genetic ®ngerprints) which was used to characterise the
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samples. The RiboPrint1 patterns of the isolates were compared to patterns from the inoculated strains. pH (Ingold S 2518 electrode) was measured directly from the sausage as a mean value of three measurements and the weight losses (%) were calculated as a mean value of two selected sausages of the raw sausage (0 day) and sausages after 7, 14, 28 and 35 days of fermentation and ripening. 2.6. Flavour pro®le The ¯avour pro®le of dry sausages were evaluated by following the principles presented in ISO 11035:1994. The panellists (n=17) were provided with a wide range of commercial Finnish dry sausages and they generated descriptive words independently. The words were collected by the panel leader and the ®ve most descriptive words (Table 2) were selected by consensus. For selected descriptive words panel leader selected reference standards (Table 2). Since the actual chemical substance for the reference standard is not important as long as the panellists are comfortable with them (Stampanoni, 1994) the standards as well as their concentrations of chemical dilutions were selected only for their ¯avour impression. The applicable concentrations for each dilution were selected by consensus in order to expresses the degree of characteristics analysed. It was pointed out to the panellists that the dilutions did not describe the actual concentration of chemical substance in the dry sausages. The meaning and quantitative scale of descriptive words for each panel member was discussed during the training sessions as advised by Civille and Lawless (1986). Training was followed by evaluation of commercial dry sausages with a score sheet, which included, in addition to descriptive words, after-taste and persistence as well as the overall impression (Table 2). After-taste was determined after 1 min from ejection and persistence was described as the persistence of after-taste. Overall impression was understood as quality of dry sausage and the success of fermentation process. It was evaluated at the end of evaluation session. Each of the descriptive words, after-taste and persistence were evaluated from 1 to 7 (0=not detected) while overall impression was evaluated from ``excellent quality'' (3) via ``good quality'' (2) to ``satisfactory quality`` (1). Table 2 The score sheet for ¯avour evaluation Flavour
Chemical
Concentration (v/v)
Lactic acid Acetic acid Bitter Sweet Salty After-taste Persistence Over-all-impression
Lactic acid Acetic acid Arginine Alanine NaCl
0.10 0.05 0.30 0.60 0.70
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The panellists independently performed the evaluation of the experimental sausages. The reference standards for each descriptive word were present during the evaluation to help in identifying and scaling the sensory attribute found in the samples as advised by Rainey (1986). The samples (2-mm slices) were coded with three-digit random numbers, serving sequences were randomised and the panel members used individual booths and score sheets. 2.7. Statistical analyses The results were analysed by one way analysis of variance (Statgraphics, Win 32). 3. Results and discussion 3.1. Production of lactic acid The numbers of lactic acid bacteria increased in the broth cultures to 9.5±9.9 log cfu/ml. The lowest number was in E-90390 broth culture and the highest number in the control broth culture. The pH values in the broths were 4.1±4.3. The amount of lactic acid in MRS broths varied between the strains (Fig. 1) but all experimental strains produced sucient enough lactic acid in order to act as main fermenting organisms in the manufacturing of dry sausages.
fermented by E-98098 and LC-705 the number of lactic acid bacteria increased at a slower rate compared to sausages fermented by other strains. The number of staphylococci remained at the inoculated number (6 log cfu/g) to the end of the ripening period. Surprisingly, the dierences in numbers of lactic acid bacteria did not eect the pH of the dry sausages, which decreased from 5.6 to the low values 4.9±5.0 in all sausages in 7 days of fermentation. The pH remained at that level to the end of the ripening period in all sausages. The dierences in ability to produce lactic acid were tested in preliminary tests (Fig. 1) which might have been the reason for the low pH even in the sausages fermented by E-98098 and LC-705. The weight losses of the sausages were 45±48% at the end of the ripening period. These high weight losses could be expected since these low fat dry sausages were ripened for a relatively long time period (35 days) to simulate industrial scale. 3.4. Ribotyping The presence of experimental bacterial strains in the sausages from the pure cultures (0 day) until the end of ripening (35 days) was traced by ribotyping (Fig. 3). A total of 18 colonies of six bacterial pure cultures and 125 colonies isolated from 36 sausages were ribotyped by
3.2. Formation of biogenic amines Biogenic amines are generally present in dry sausages (Eerola, Roig Sagues, & Hirvi, 1998; Maijala, 1994) because the conditions in dry sausage manufacturing favour decarboxylase reactions caused by amine positive microorganisms Ð the temperature is between +20 C and +37 C, the pH value is between 5 and 7, and decarboxylating bacteria may be present at high levels (Beutling, 1996). The highest concentrations of biogenic amines found in dry sausages are high enough to result in food toxi®cation (Shalaby, 1996). Therefore, the main fermentative organisms should be decarboxylase negative (BuckenhuÈskes, 1993). In the study of Maijala (1993) of the most commonly used lactic acid bacteria in meat starter cultures, all strains were amine negative and regarded as safe. In the present study the analysis of MRS broths clearly showed that the strains form neither histamine nor tyramine. The level of histamine was <10 mg/ml and the level of tyramine <15 mg/ml.
Fig. 1. The amount of lactic acid (mmol/l) in MRS broths after incubating for 48 h at 22 C.
3.3. Microbiological analysis, pH and weight losses of dry sausages The numbers of lactic acid bacteria in dry sausages increased in sausages (Fig. 2) during the fermentation and ripening as expected. However, in the sausages
Fig. 2. The numbers of lactic acid bacteria (log cfu/g) during fermentation (0±7 days) and ripening (7±35 days).
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Consequently, the ¯avour pro®les are not markedly dierent. In the present study Ð similar to normal procedure in industrial scale Ð spices were added in to all sausages. It could be supposed that the commercial spice mixture might have overruled the slight dierences in ¯avour pro®le caused by dierent organisms. As studied by numerous researchers (Dainty & Blom, 1995; Hammes & Hertel, 1998) the biological activity varies between strains resulting in dierent ¯avour pro®les in dry sausage. But it is also possible that the strains studied in the present study do not result dierent ¯avour pro®le by their nature. The genetic ®ngerprints of the three L. rhamnosus strains are very similar (ErkkilaÈ, Suihko, Eerola, PetaÈjaÈ, & Mattila-Sandholm, 2001) which gave reason to expect similar ¯avour pro®les for the dry sausages fermented by these strains.
Fig. 3. The Riboprint patterns of the strains.
4. Conclusions The present study indicates that dry sausages can be manufactured by probiotic or bioprotective L. rhamnosus GG, LC-705 and E-97800 as well as P. pentosaceus E-90390 and L. plantarum E-98098. The number of lactic acid bacteria, whether it is 8 log cfu/g or 9 log cfu/g in the ®nal product, does not eect the technological or sensory properties of the commercial dry sausage. There are no marked dierences in pH, in the weight losses or even in the ¯avour pro®les of the ®nal North European commercial dry sausage product fermented by these strains. Fig. 4. Flavour pro®les of dry sausages fermented by dierent strains.
References 1
1
the automated RiboPrinter System. The RiboPrint patterns (genetic ®ngerprints) generated are illustrated in Fig. 3. It was possible to trace the strains using this technique since three or more colonies of each sample were picked from the MRS agar plate (highest dilutions) and ribotyped. The same RiboPrint1 patterns resulted after ripening (35 days) as from the pure cultures of the experimental strains. This indicates that these organisms were responsible for the fermentation of these trial sausages. They were also most probably responsible for the formation of lactic acid and other ¯avour compounds. 3.5. Flavour pro®le of the sausages The ¯avour pro®les of dry sausages fermented by ®ve experimental strains and one commercial strain were almost identical (Fig. 4). The after-taste was weaker (P<0.05) in sausages fermented by GG and the persistence weaker (P<0.05) in sausages fermented by LC705 compared to control sausages. The overall impressions were evaluated as being between ``good quality'' and ``excellent quality'' in all sausages.
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Eerola, S., Hinkkanen, R., Lindfors, E., & Hirvi, T. (1993). Liquid chromatographic determination of biogenic amines in dry sausage. Journal of AOAC International, 76, 575±577. Eerola, S., Roig Sagues, A.-X., & Hirvi, T. (1998). Biogenic amines in Finnish dry sausages. Journal in Food Safety, 18, 127±138. ErkkilaÈ, S., Suihko, M-L., Eerola, S., PetaÈjaÈ, E., & Mattila-Sandholm, T. (2001). Dry sausages fermented by Lactobacillus rhamnosus strains. International Journal of Food Microbiology (in press). ErkkilaÈ, S., VenaÈlaÈinen, M., Hielm, S., PetaÈjaÈ, E., Puolanne, E., & Mattila-Sandholm, T. (2000). Survival of Escherichia coli O157:H7 in dry sausage fermented by probiotic lactic acid bacteria. Journal of the Science of Food and Agriculture, 80, 2101±2104. Haikara, A., & Mattila-Sandholm, T. (1993). Procedure for treatment of seed material to be germinated. PLT/FI93/00388, Publication No. WO 94/16053. Hammes, W., & Hertel, C. (1998). New developments in meat starter cultures. Meat Science, 49(supplement), 125±138. Hilliam, M. (1998). The market of functional foods. International Dairy Journal, 8, 349±353. Holzapfel, W., Haberer, P., Snel, J., Schillinger, U., & Huis in't Veld, J. (1998). Overview of gut ¯ora and probiotics. International Journal of Food Microbiology, 41, 85±101. Kontula, P., Nollet, L., Saarela, M., Vilpponen-Salmela, T., Verstraete, W., von Wright, A., & Mattila-Sandholm, T. (2001). The eect of lactulose on the survival of Lactobacillus rhamnosus in the Simulator of Human Intestinal Microbiota Ecosystem (SHIME) and in vivo. Microbial Ecology in Health and Disease (in press). Kontula, P., Suihko, M.-L., von Wright, A., & Mattila-Sandholm, T. (1999). The eect of lactose derivatives on lactic acid bacteria. Journal of Dairy Science, 82, 249±256. KroÈckel, L. (1995). Bacterial fermentation of meats. In G. CampbellPlatt, & P. E. Cook, Fermented meats (pp. 69±109). UK: Blackie Academic & Professional. LuÈcke, F-K. (1986). Microbiological processes in the manufacture of dry sausage and raw ham. Fleischwirtschaft, 66, 1505±1509.
Maijala, R. (1993). Formation of histamine and tyramine by some lactic acid bacteria in MRS-broth and modi®ed decarboxylation agar. Letters in Applied Microbiology, 17, 40±43. Maijala, R. (1994). Formation of biogenic amines in dry sausages with special reference to raw materials, lactic acid bacteria, pH decrease, temperature and time. Academic dissertation, Helsinki, Finland. MaÈyraÈ-MaÈkinen, A., & Suomalainen, T. (1995). Lactobacillus casei ssp. rhamnosus, bacterial preparations comprising said strain and preparations for the controlling of yeast and moulds. United States Patent US 5 378 458. Miettinen, M., Alander, M., von Wright, A., Vuopio-Varkila, J., Marteau, P., Huis in't Veld, J., & Mattila-Sandholm, T. (1998). The survival of and cytokine induction by lactic acid bacteria after passage through a gastrointestinal model. Microbial Ecology in Health and Disease, 10, 141±147. Rainey, B. (1986). Importance of reference standards in training panellists. Journal of Sensory Studies, 1, 149±154. Salminen, S., Deighton, M., Benno, Y., & Gorbach, S. (1998). Lactic acid bacteria in health and disease. In S. Salminen, & A. von Wright, Lactic acid bacteria. (2nd. ed) (pp. 211±254). New York: Marcel Dekker. Saxelin, M. (1997). Lactobacillus GG Ð a human probiotic strain with thorough clinical documentation. Food Reviews International, 13, 293±313. Sameshima, T., Magome, C., Takeshita, K., Arihara, K., Itoh, M., & Kondo, Y. (1998). Eect of intestinal Lactobacillus starter cultures on the behaviour of Staphylococcus aureus in fermented sausage. International Journal of Food Microbiology, 41, 1±7. Shalaby, A. (1996). Signi®cance of biogenic amines to food safety and human health. Food Research International, 29, 675±690. Stampanoni, C. (1994). Quantitative ¯avor pro®ling technique for ¯avored dairy products. Journal of Sensory Studies, 9, 383±400. Tuomola, E., & Salminen, S. (1998). Adhesion of some probiotic and dairy Lactobacillus stains to Caco-2 cell cultures. International Journal of Food Microbiology, 41, 45±51.
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Flavour pro®les of dry sausages fermented by selected novel meat starter cultures S. ErkkilaÈ a,*, E. PetaÈjaÈ a, S. Eerola b, L. Lilleberg b, T. Mattila-Sandholm c, M-L. Suihko c a
Department of Food Technology, University of Helsinki, PO Box 27, 00014 Helsinki, Finland Department of Chemistry, National Veterinary and Food Research Institute, Helsinki, Finland c VTT Biotechnology, Espoo, Finland
b
Received 3 July 2000; received in revised form 13 September 2000; accepted 4 October 2000
Abstract Probiotic or bioprotective Lactobacillus rhamnosus strains GG, LC-705 and E-97800 as well as Pediococcus pentosaceus E-90390 and Lactobacillus plantarum E-98098 were studied for their ability to act as main fermenting organisms in the manufacturing process of dry sausages. In the preliminary tests, their abilities to produce lactic acid and biogenic amines, histamine or tyramine, were studied in MRS broth and analysed by high-performance liquid chromatography. The strains produced higher or equal amounts of lactic acid compared to control and were amine negative. During the actual fermentation process of dry sausages the numbers of inoculated bacteria increased from the level 6.5±7.0 log cfu/g to 8.0±9.0 log cfu/g. The most fast growing strains were P. pentosaceus E-90390 and the control while the growth of L. plantarum E-98098 and L. rhamnosus LC-705 were the slowest. The pH value of the sausages decreased from 5.6 to 4.9±5.0. The presence of these experimental strains as major organisms in the sausages after fermentation and ripening was con®rmed on the bases of their genetic ®ngerprints. The ¯avour pro®les of the experimental sausages produced by these probiotic or protective strains were similar with that produced by the commercial meat starter culture and commercial North European dry sausage recipe. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Probiotic; Dry sausage; Flavour pro®le; Ribotyping
1. Introduction Functional foods are designed to maintain or even promote human health. Their positive eects are due to either additions of active ingredients or removal or replacement of undesirable substances. It has been predicted that functional foods will account for 5% of total food consumption in Europe (Hilliam, 1998). Probiotic cultures are designated to balance the indigenous micro¯ora of the host and may have several health promoting eects (Salminen, Deighton, Benno, & Gorbach, 1998). In addition to the dairy industry, researchers in the meat industry have also recently begun to ®nd novel starter cultures with additional value. Since the 1950s and the times of Professor F.P. Niinivaara, meat starter cultures have ensured the quality and safety of * Corresponding author. Tel.: +358-2-62037334; fax: +358-262037399. E-mail address: [email protected].® (S. ErkkilaÈ).
the fermented products. Starter strains used in dry sausage manufacture process in the north of Europe produce sucient amounts of lactic acid in order to decrease the pH of meat to values 4.8±5.0. The low pH value is the basis for the safety as well as texture and colour of dry sausages (KroÈckel, 1995). Recently dry sausages fermented by probiotic cultures have been studied by Andersen (1998), Arihara et al. (1998), Sameshima, Magome, Takeshita, Arihara, Itoh, and Kondo (1998) and ErkkilaÈ, Suihko, Eerola, PetaÈjaÈ, and MattilaSandholm (2000), ErkkilaÈ, VenaÈlaÈinen, Hielm, PetaÈjaÈ, Puolanne, and Mattila-Sandholm (2000). The typical ¯avour of a dry sausage is a result of the bacterial and also muscle enzyme metabolism of carbohydrates, proteins and lipids. In combination with spices, all metabolism products result in the sensory pro®le of a dry sausage. For North European sausages lactic acid is the main ¯avour component, but acetic acid is also present and actually needed at low concentrations for full dry sausage ¯avour. The amount of other acids
0309-1740/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(00)00135-2
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is typically extremely low. The amount of free amino acids and fatty acids increases during the ripening process, which may slightly aect the ¯avour of dry sausage (Dainty & Blom, 1995; LuÈcke, 1986). It is generally believed that dierent starter cultures result in the dierent sensory characteristics of dry sausages. This means that when novel strains are selected, the ability of the strains to contribute to the high quality sensory properties of the end product should be studied (Holzapfel, Haberer, Snel, Schillinger, & Huis in't Veld., 1998). The objective of the present work was to study probiotic or bioprotective Lactobacillus rhamnosus GG, E97800 and LC-705 as well as Pediococcus pentosaceus E-90390 and Lactobacillus plantarum E-98098 as dry sausage starter cultures. Preliminary studies were focused on their ability to form sucient amounts of lactic acid and not to form biogenic amines. The eventual decarboxylation process of amino acids by lactic acid bacteria may result high amounts of toxic biogenic amines (Shalaby, 1996). The monitoring of dry sausage fermentation and ripening was focused on the growth of selected strains and on tracing them from the sausages, as well as on the development of pH value in dry sausages. The principal aim was to characterise any eventual dierences in ¯avour pro®les between dry sausages fermented by dierent strains. 2. Materials and methods 2.1. Strains The strains used in this study were kindly provided by Valio Ltd., VTT Biotechnology and GewuÈrzmuÈller GmbH (Table 1). The bene®cial eects of probiotic L. rhamnosus GG are well documented (Saxelin, 1997) while L. rhamnosus E-97800 is a novel potential probiotic strain (Kontula et al., 2000; Kontula, Suihko, von Wright, & Mattila-Sandholm, 1999). L. rhamnosus LC705 is a bioprotective lactic acid bacterium for fermented dairy products (MaÈyraÈ-MaÈkinen & Suomalainen,
Table 1 Bacterial strain used for fermentation Species
Code
Supplier
Lactobacillus rhamnosus L. rhamnosus L. rhamnosus Lactobacillus plantarum Pediococcus pentosaceus P. pentosaceusa Lactobacillus sakeib
GG LC-705 E-97800 E-98098 E-90390 Condi Rasant LS-25
Valio Ltd. Valio Ltd. VTT Biotechnology VTT Biotechnology VTT Biotechnology GewuÈrzmuÈller GmbH GewuÈrzmuÈller GmbH
a b
Used as a control in preliminary tests. Used as a control in dry sausage fermentation trials.
1995) while P. pentosaceus E-90390 and L. plantarum E98098 are protective cultures for malting procedure (Haikara & Mattila-Sandholm, 1993). Recently L. rhamnosus LC-705 (Tuomola & Salminen, 1998) and L. plantarum E-98098 (Alander, De Smet, Nollet, Verstraete, von Wright, & Matilla-Sandholm, 1999; Miettinen et al., 1998) have been studied also for their probiotic properties. The commercial starter strains P. pentosaceus (isolated from a commercial mixed culture of P. pentosaceus and Staphylococcus carnosus named Condi Rasant, GewuÈrzmuÈller, Germany) and Lactobacillus sakei (isolated from a commercial mixed culture of L. sakei and S. carnosus named LS-25, GewuÈrzmuÈller, Germany) was used as the controls in preliminary tests and in the actual dry sausage manufacturing, respectively. 2.2. Production of lactic acid in MRS broth Strains were cultivated on MRS agar (pH 5.6, Difco) for 3 days at 30 C. Three colonies of each strain were picked and inoculated in 10 ml MRS broths (Difco). The purity of broth cultures were checked by Gram staining and microscoping. After 48 h incubation at 22 C, the broths were puri®ed with strong anionexchange solid phase extraction cartridges. Lactic acid was separated with a reversed-phase column (Spherisorb S5ODS2 254.6 mm, 5 mm, Phase separations Ltd, UK) in a high-performance liquid chromatography (HPLC) system. Lactic acid concentrations of the samples were determined at a wavelength of 214 nm by using the external standard method with 0.5 mM, 1.0 mM, 5.0 mM and 10.0 mM standards. The mobile phase 50 mM potassium phosphate buer (pH 2.4) was pumped at 1 ml/min and the column temperature was 30 C. The relative retention value for lactic acid was 1.3. Standards were run eight times and each sample twice. 2.3. Formation of biogenic amines in MRS broth Strains were cultivated on MRS agar (pH 5.6, Difco) for 3 days at 30 C. Three colonies of each strain were picked from pure culture MRS agar plates and inoculated into 10 ml MRS broths (Difco) containing 0.5% histidine and 0.5% tyrosine. Since the maximum histamine and tyramine production in MRS broth takes place after 24 h of incubation (Chandler, Batish, Babu, & Singh, 1989) the inoculated broths were incubated for 48 h at 22 C. Histamine and tyramine were extracted from 10 ml of MRS-broth and detected as their dansyl derivatives at 254 nm by a HPLC-system (HP 1090 Series M LC, diode-array detector including gradient module, binary DR5 pumps, autoinjector system with 25 ml loop) by the method of Eerola, Hinkkanen, Lindfors, and Hirvi (1993). The limit of determination for histamine was 1 mg/ml and for tyramine 2 mg/ml.
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2.4. Preparation of sausages Raw sausage material, which was 18% of protein and 13% of fat (pork, beef and pork fat) as well as salt (2.7%, analysed by sodium-ions) and spices, e.g. white pepper and cardamom (Finnish commercial formulation), was divided into six separate batches and inoculated by selected ®ve experimental probiotic or bioprotective strains. L. sakei (isolated from LS-25, GewuÈrzmuÈller, Germany) was used as the control. LS-25 and Condi Rasant used in preliminary tests are both meat starter cultures for fast acidifyed dry sausages with similar acidifying capacities (GewuÈrzmuÈller, Germany). Lactic acid bacteria were inoculated as pure MRS broth cultures grown at 30 C for 48 h aiming at 7 log cfu/g raw material (100 ml/10 kg). Normally, staphylococci are used in combination with lactic acid bacteria. In this study, a commercial staphylococci starter culture, PoÈkelferment 77 (S. carnosus, Chr. Hansen, Denmark), was used in each batch aiming at 6 log cfu/g raw material (1 g/10 kg). Three dierent experimental series with each strain were prepared by using 65 mm diameter casings. The series were fermented and ripened with the same program: for two days at 23±22 C and rH 95% followed by a gradual reduction of temperature (from 21 to 19 C) and relative humidity (from rH 94% to rH 90%) during the next 4 days. Sausages were also smoked (2 h) twice a day between the second and ®fth day of fermentation. After 7 days of fermentation, sausages were ripened for 28 days at 17 C (rH 75%). 2.5. Microbiological determinations, pH and weight losses of dry sausages Each batch was examined microbiologically after manufacture (0 day) and after 7, 14, 28 and 35 days of fermentation and ripening. The numbers of staphylococci were determined on Baird Parker agar (2 days at 37 C) and the numbers of lactic acid bacteria on MRS agar (pH 5.6; 3 days at 30 C). Characterisation of the inoculated experimental pure strains (0 day) and of the sausage isolates after 35 days were carried out using the automated RiboPrinter1 Microbial Characterisation System (QualiconTM, USA) according to the instructions of the manufacturer (Bruce, 1996). Cells from the MRS agar plates were suspended in buer. DNA was released by two lysing agents and cut with EcoR1 restriction enzyme. Resulted fragments were electrophoretically separated according to their size, transferred to a nylon membrane, and hybridized with a chemically labelled rRNA operon from Escherichia coli. The luminescent fragments of membranes were photographed with a CCD camera and converted to digital information. Analysis software automatically processed the digitalized image producing a RiboPrint1 pattern (genetic ®ngerprints) which was used to characterise the
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samples. The RiboPrint1 patterns of the isolates were compared to patterns from the inoculated strains. pH (Ingold S 2518 electrode) was measured directly from the sausage as a mean value of three measurements and the weight losses (%) were calculated as a mean value of two selected sausages of the raw sausage (0 day) and sausages after 7, 14, 28 and 35 days of fermentation and ripening. 2.6. Flavour pro®le The ¯avour pro®le of dry sausages were evaluated by following the principles presented in ISO 11035:1994. The panellists (n=17) were provided with a wide range of commercial Finnish dry sausages and they generated descriptive words independently. The words were collected by the panel leader and the ®ve most descriptive words (Table 2) were selected by consensus. For selected descriptive words panel leader selected reference standards (Table 2). Since the actual chemical substance for the reference standard is not important as long as the panellists are comfortable with them (Stampanoni, 1994) the standards as well as their concentrations of chemical dilutions were selected only for their ¯avour impression. The applicable concentrations for each dilution were selected by consensus in order to expresses the degree of characteristics analysed. It was pointed out to the panellists that the dilutions did not describe the actual concentration of chemical substance in the dry sausages. The meaning and quantitative scale of descriptive words for each panel member was discussed during the training sessions as advised by Civille and Lawless (1986). Training was followed by evaluation of commercial dry sausages with a score sheet, which included, in addition to descriptive words, after-taste and persistence as well as the overall impression (Table 2). After-taste was determined after 1 min from ejection and persistence was described as the persistence of after-taste. Overall impression was understood as quality of dry sausage and the success of fermentation process. It was evaluated at the end of evaluation session. Each of the descriptive words, after-taste and persistence were evaluated from 1 to 7 (0=not detected) while overall impression was evaluated from ``excellent quality'' (3) via ``good quality'' (2) to ``satisfactory quality`` (1). Table 2 The score sheet for ¯avour evaluation Flavour
Chemical
Concentration (v/v)
Lactic acid Acetic acid Bitter Sweet Salty After-taste Persistence Over-all-impression
Lactic acid Acetic acid Arginine Alanine NaCl
0.10 0.05 0.30 0.60 0.70
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The panellists independently performed the evaluation of the experimental sausages. The reference standards for each descriptive word were present during the evaluation to help in identifying and scaling the sensory attribute found in the samples as advised by Rainey (1986). The samples (2-mm slices) were coded with three-digit random numbers, serving sequences were randomised and the panel members used individual booths and score sheets. 2.7. Statistical analyses The results were analysed by one way analysis of variance (Statgraphics, Win 32). 3. Results and discussion 3.1. Production of lactic acid The numbers of lactic acid bacteria increased in the broth cultures to 9.5±9.9 log cfu/ml. The lowest number was in E-90390 broth culture and the highest number in the control broth culture. The pH values in the broths were 4.1±4.3. The amount of lactic acid in MRS broths varied between the strains (Fig. 1) but all experimental strains produced sucient enough lactic acid in order to act as main fermenting organisms in the manufacturing of dry sausages.
fermented by E-98098 and LC-705 the number of lactic acid bacteria increased at a slower rate compared to sausages fermented by other strains. The number of staphylococci remained at the inoculated number (6 log cfu/g) to the end of the ripening period. Surprisingly, the dierences in numbers of lactic acid bacteria did not eect the pH of the dry sausages, which decreased from 5.6 to the low values 4.9±5.0 in all sausages in 7 days of fermentation. The pH remained at that level to the end of the ripening period in all sausages. The dierences in ability to produce lactic acid were tested in preliminary tests (Fig. 1) which might have been the reason for the low pH even in the sausages fermented by E-98098 and LC-705. The weight losses of the sausages were 45±48% at the end of the ripening period. These high weight losses could be expected since these low fat dry sausages were ripened for a relatively long time period (35 days) to simulate industrial scale. 3.4. Ribotyping The presence of experimental bacterial strains in the sausages from the pure cultures (0 day) until the end of ripening (35 days) was traced by ribotyping (Fig. 3). A total of 18 colonies of six bacterial pure cultures and 125 colonies isolated from 36 sausages were ribotyped by
3.2. Formation of biogenic amines Biogenic amines are generally present in dry sausages (Eerola, Roig Sagues, & Hirvi, 1998; Maijala, 1994) because the conditions in dry sausage manufacturing favour decarboxylase reactions caused by amine positive microorganisms Ð the temperature is between +20 C and +37 C, the pH value is between 5 and 7, and decarboxylating bacteria may be present at high levels (Beutling, 1996). The highest concentrations of biogenic amines found in dry sausages are high enough to result in food toxi®cation (Shalaby, 1996). Therefore, the main fermentative organisms should be decarboxylase negative (BuckenhuÈskes, 1993). In the study of Maijala (1993) of the most commonly used lactic acid bacteria in meat starter cultures, all strains were amine negative and regarded as safe. In the present study the analysis of MRS broths clearly showed that the strains form neither histamine nor tyramine. The level of histamine was <10 mg/ml and the level of tyramine <15 mg/ml.
Fig. 1. The amount of lactic acid (mmol/l) in MRS broths after incubating for 48 h at 22 C.
3.3. Microbiological analysis, pH and weight losses of dry sausages The numbers of lactic acid bacteria in dry sausages increased in sausages (Fig. 2) during the fermentation and ripening as expected. However, in the sausages
Fig. 2. The numbers of lactic acid bacteria (log cfu/g) during fermentation (0±7 days) and ripening (7±35 days).
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Consequently, the ¯avour pro®les are not markedly dierent. In the present study Ð similar to normal procedure in industrial scale Ð spices were added in to all sausages. It could be supposed that the commercial spice mixture might have overruled the slight dierences in ¯avour pro®le caused by dierent organisms. As studied by numerous researchers (Dainty & Blom, 1995; Hammes & Hertel, 1998) the biological activity varies between strains resulting in dierent ¯avour pro®les in dry sausage. But it is also possible that the strains studied in the present study do not result dierent ¯avour pro®le by their nature. The genetic ®ngerprints of the three L. rhamnosus strains are very similar (ErkkilaÈ, Suihko, Eerola, PetaÈjaÈ, & Mattila-Sandholm, 2001) which gave reason to expect similar ¯avour pro®les for the dry sausages fermented by these strains.
Fig. 3. The Riboprint patterns of the strains.
4. Conclusions The present study indicates that dry sausages can be manufactured by probiotic or bioprotective L. rhamnosus GG, LC-705 and E-97800 as well as P. pentosaceus E-90390 and L. plantarum E-98098. The number of lactic acid bacteria, whether it is 8 log cfu/g or 9 log cfu/g in the ®nal product, does not eect the technological or sensory properties of the commercial dry sausage. There are no marked dierences in pH, in the weight losses or even in the ¯avour pro®les of the ®nal North European commercial dry sausage product fermented by these strains. Fig. 4. Flavour pro®les of dry sausages fermented by dierent strains.
References 1
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the automated RiboPrinter System. The RiboPrint patterns (genetic ®ngerprints) generated are illustrated in Fig. 3. It was possible to trace the strains using this technique since three or more colonies of each sample were picked from the MRS agar plate (highest dilutions) and ribotyped. The same RiboPrint1 patterns resulted after ripening (35 days) as from the pure cultures of the experimental strains. This indicates that these organisms were responsible for the fermentation of these trial sausages. They were also most probably responsible for the formation of lactic acid and other ¯avour compounds. 3.5. Flavour pro®le of the sausages The ¯avour pro®les of dry sausages fermented by ®ve experimental strains and one commercial strain were almost identical (Fig. 4). The after-taste was weaker (P<0.05) in sausages fermented by GG and the persistence weaker (P<0.05) in sausages fermented by LC705 compared to control sausages. The overall impressions were evaluated as being between ``good quality'' and ``excellent quality'' in all sausages.
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