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Meat starters have individual requirements for Mn2+
Meat Science 55 (2000) 161±168
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Meat starters have individual requirements for Mn2+ Beate F. Hagen, Helga Nñs, Askild L. Holck* MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 AÊs, Norway Received 26 August 1999; received in revised form 11 October 1999; accepted 11 October 1999
Abstract The eect of dierent Mn2+ concentrations on sausage fermentation was evaluated. A screening experiment was carried out with six lactobacilli starters in a sausage model. To further investigate the eects found, two selected lactobacilli strains were tested in pilot-scale sausage production. For all starters an increased fermentation rate was observed after Mn2+ addition. Dierences in the development of microbial, textural and sensory parameters were observed in the sausages. For one of the cultures these dierences levelled out during sausage production yielding identical end products with and without Mn2+, for the other strain the dierences due to Mn2+ addition in the sausages remained throughout the production process yielding sausages with dierent properties. Knowing a starter culture's requirements for Mn2+ will allow optimisation of dry fermented sausage production in order to increase reliability and reproducibility of production decrease fermentation time and ensure microbial safety of the ®nal product. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Dry sausage; Starter culture; Accelerated ripening
1. Introduction A challenge for the fermented meat industry is to produce more economically a dry fermented sausage of stable, high quality, but with the ¯avour, appearance and texture of traditional sausages. Many strategies for accelerating the fermentation and/or ripening processes by manipulating sausage formulation have been studied and suggested. Several groups have reported on the addition of enzymes, both proteolytic and lipolytic to improve speed. Zapelena, Zalacain, Ansorena and co-workers could not ®nd any accelerating eect by the addition of lipases, but suggest that careful selection of the correct protease could accelerate maturation without texture deterioration (Ansorena, Zapalena, AstiasaraÂn & Bello, 1998a,b; Zalacain, Zapelena, AstiasaraÂn & Bello, 1995, 1996; Zalacain, Zapelena, de Pena, AstiasaraÂn & Bello, 1997; Zapelena, Ansorena, Zalacain, AstiasaraÂn & Bello, 1998; Zapelena, Zalacain, Paz de Dena, AstiasaraÂn & Bello, 1997a,b). Likewise, the group of DõÂaz have found possible means to accelerate proteolysis and lipolysis,
* Corresponding author. Tel.: +47-6497-0100; fax: +47-6497-0333. E-mail address: [email protected] (A.L. Holck).
thus providing substrates for rapid tranformation into aromatic compounds, however they have not as yet found satisfactory, practical protocols (DõÂaz, Fernandez, Garcia de Fernando, de la Hoz & Ordonez, 1993, 1996, 1997; FernaÂndez, de la Hoz, DõÂaz, Cambero & OrdoÂnez, 1995a,b). Previous work in our group have shown that addition of a bacterial protease extract in pilot scale salami production shortens the ripening time by 50% (Hagen, BerdagueÂ, Holck, Nñs & Blom, 1996; Nñs, Holck, Axelsson, Andersen & Blom, 1995). Addition of a soy protein isolate has also been shown to stimulate growth of the starter culture and thereby accelerate fermentation. A more convenient and less expensive approach to the fermentation time would be to optimise the level of ingredients already present in the sausage formulation. Although the purpose of adding spices to sausage formulations is to achieve the desired ¯avour, they also have other functions; serving as a source of fermentable and reducing sugars, nitrates and nitrites, colourings, metallic ions etc. Spices are known to have an in¯uence on the development of texture, ¯avour and colour during the ripening of sausages (Aguirrezabal, Mateo, Dominguez & Zumalacarregui, 1998). It has long been known that components in spices are stimulatory to acid production of starter cultures (Nes
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(99)00138-2
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& SkjelkvaÊle, 1982) and Zaika and Kissinger (1984) showed that one factor in spices that has this property is Mn2+. It is established that the metabolism of many lactobacilli is dependent on high levels of Mn2+ (Archibald, 1986), and that the requirement for Mn2+ diers among dierent lactobacilli. Meat has low levels of Mn2+, and addition of Mn2+ may thus stimulate lactobacilli during meat fermentation. Raccach and Marshall state that lactic acid fermentation by Lactobacillus plantarum in beaker sausage was stimulated by Mn2+ and Coventry and Hickey (1993) have previously shown that adding Mn2+ to a salami model stimulates the fermentation rate of a Pediococcus starter culture. The objective of this work was to evaluate the eect of dierent concentrations of Mn2+ on sausage fermentation. A screening experiment was carried out with six lactobacilli starters in a sausage model. To further investigate the eect found, two selected lactobacilli were tested in pilot-scale sausage production. 2. Materials and methods 2.1. Mn2+ determination A commercial salami spice mixture was purchased from a local supplier. The concentration of Mn2+ in the spices was determined by ¯ame atomic absorption spectrometry in a laboratory performing accredited chemical analyses (Sero AS, 1361 Billingstad, Norway). 2.2. Starter cultures The starter cultures used in these experiments are listed in Table 1.The non-commercial strain Lactobacillus sp. (M1) was isolated from dry fermented sausages, where this culture was the predominating species F1 was a starter culture, closely related to Lactobacillus farciminis, used by a local factory. The four commercial starters were all supplied as mixed cultures from the producers, with at least one Staphylococcus strain in addition to the Lactobacillus strains. All mixtures were seeded onto MRS (De Man, Rogosa, Sharpe) (Oxoid) agar plates and the Lactobacillus strains were isolated before use. Cultures were stored at ÿ80 C in MRS
medium supplemented with glycerol (18% v/v). For all experiments, the starter cultures were precultivated in MRS for 16 h at 30 C, transferred once in MRS under the same conditions, washed with peptone water [0.1% (w/v) Bacto Peptone (Oxoid) and 0.85% (w/v) NaCl (p.a.) in distilled water] and resuspended in peptone water diluted to desired OD600. Optical density (OD600) was determined at 600 nm with Ultrospec 3000 (Pharmacia Biotech). 2.3. Sausage model fermentations A 30-kg sausage mixture which contained (% w/w): beef (51.8), pork (18.8), lard (25.6), sodium chloride (3.3), nitrite (0.02), glucose (0.4) and ascorbic acid (0.04) was chopped and blended in a cutter. 150 g (1 g) aliquots of this batter were put in plastic boxes (dia. 68 mm) and stored at ÿ20 C. To the thawed samples was added 0.5 ml distilled water containing various concentrations of Mn2+ sulphate (MnSO4H2O p.a. Merck) and 0.25 ml of starter culture (OD600 4.00.2). The additions were mixed into the sausage batter with a blender (Braun Multimix Duo M810) and the boxes were centrifuged (1000 rpm, 2 min, High Speed Sentrifuge, Sorvall RC 5C, rotor SH 3000, Sorvall Inc.) to mimic the eect of vacuum stung. pH electrodes (Xerolyt, Mettler-Toledo, Process, Analytical Inc.) were inserted into the middle of each batter through a tight hole in the lid and the samples were incubated at 22 C for 120 h. pH was recorded every hour by an automatic logging system (Intab AAC-2, Interface-Teknik AB, Sweden) controlled by a software package (Easy View 2.12, Interface-Teknik AB, Sweden). The model experiments were performed in a two-step procedure: in step 1, spices (0.5% w/w) were added to the sausage batter (with exception of the control batter) together with manganese solutions to achieve the following ®nal concentrations of Mn2+, including the Mn2+ present in the spices: 0, 0.7, 1, 1.2, 1.7, 2.7, 5.7 and 10.7 ppm. Step 1 was a screening step with only one experiment per starter in order to decide the appropriate concentrations of Mn2+ for further studies. In step 2, the Mn2+ levels 0, 0.25, 0.5, 1.0 and 2.5 ppm were selected and all starters were investigated for pH-development in two independent experiments in batters without spices added.
Table 1 Starter cultures for screening of Mn2+ requirements
2.4. Sausage production
Strain
Name
Supplier
Lactobacillus sp. Lactobacillus sp. Lactobacillus plantarum Lactobacillus sp. Lactobacillus sakei Lactobacillus curvatus
M1 F1 Duploferment Biostart 741 LS-25 LS-3
MATFORSK Local factory Chr. Hansen Raps GewuÈrzmuÈller GmbH GewuÈrzmuÈller GmbH
Salami sausages (without spices and not subjected to smoking) were made according to the above recipe in a designed experiment with two factors; starter culture (two types) and Mn2+ addition (four levels). Two batters of 50 kgs were made. To batter 1, Lactobacillus M1 was added as starter culture and supplied at 6106 cells/ g sausage mixture. To batter 2, Lactobacillus F1 was
B.F. Hagen et al. / Meat Science 55 (2000) 161±168
added as starter culture and supplied at 4106 cells/g. For each batter the ingredients were mixed and divided into ®ve batches (10.0 kg each). To batch 1 was added 7.7 ml distilled water. To batch 2 was added 7.7 ml of a solution of MnSO4 in distilled water that gave a ®nal concentration in the sausage mixture of 0.25 ppm Mn2+. To batches 3 and 4 were added MnSO4 to give a ®nal concentration of 1.0 ppm Mn2+, and likewise, batch 5 had a ®nal concentration of 2.5 ppm Mn2+. From each batch, 25 sausages of 400 g were prepared. The sausages were stued in arti®cial casings (50 mm diam.) and placed in a ripening chamber for 3 days at 24 C, 96% relative humidity (RH), 2 days at 16 C, 92% RH, 2 days at 16 C, 88% RH and ®nally the sausages were allowed to ripen at 16 C, 85% RH until mature. Sausage samples were collected for analyses after 0, 2, 3, 6, 9, 14, and 23 days. All analyses were performed on two individual sausages. The weight of each sausage was determined before and after ripening and the weight loss calculated. 2.5. pH, water activity and colony counts pH was assayed on 10 g sausage homogenized in 90 ml peptone water. Water activity was measured by an electronic hygrometer (Novasina aw-center, sensor: enRSK-4/CT-4, Novasina AG, PhaÈcon, Switzerland) on 10 g sausages cut into 5 mm cubes. Growth of the added starter cultures was assayed by plating appropriate dilutions of sausage homogenised in peptone water on MRS agar (Oxoid). The same dilutions were also plated on blood agar (Difco) to check for possible contamination. The plates were incubated at 30 C for 2 days. 2.6. Texture analysis Texture of the sausages was evaluated by Texture Pro®le Analysis (TPA) using a Texture Analyser TAXT2 (Stable Micro Systems). A bite size sample was compressed to 50% of its original height, twice, in a reciprocating motion that imitated the action of the jaw. Analyses of force±time curves led to the identi®cation of four textural parameters (hardness, springiness, cohesiveness and resilience) and two calculated parameters (gumminess and chewiness) (Bourne, 1978). 2.7. Sensory analysis Sensory descriptive pro®ling was carried out by 10 trained assessors at 14 and 23 days after production. The ¯avour pro®le of the sausages was determined using an unstructured line with end points (1±9) where 1 denoted low intensity and 9 high intensity for each of the following 18 characteristics: Odour intensity, acidic odour, colour tone of fat, whiteness, overall colour tone,
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colour intensity, ¯avour intensity, maturity ¯avour, fresh ¯avour, acidic taste, sour taste, salty taste, bitter taste, rancid ¯avour, hardness, fattiness, juiciness and stickiness. Dierences in sensory score were evaluated using Tukey's test. 2.8. Data analysis Statistical signi®cances of observed dierences among means of experimental results were evaluated by analysis of variance (ANOVA) with Tukey's test for pairwise comparison of means using Minitab for Windows release 12.1 (Minitab Inc., PA, USA) to detect the eect of the two treatments (starter culture, Mn2+ addition). Results from the two duplicate samples were averaged. 3. Results The concentration of Mn2+ in the commercial spice mixture was 131 mg Mn2+/kg. As the recommended addition of the spices to salami sausages is 0.5±1.0%, the corresponding addition of Mn2+ would be 0.66± 1.31 ppm. In the ®rst screening experiment, we studied the eects of Mn2+ levels equal to and higher than commonly found in sausages, so levels ranging from 0.66 to 10 ppm Mn2+ were evaluated. In this preliminary step of the pH-logging experiment, we found that for each starter, pH development did not change when the Mn2+ concentration exceeded 2.7 ppm (results not shown). Hence, in step two, we studied in more detail the eect of adding from 0 to 2.5 ppm Mn2+. For the four commercial starters and M1, there was a pronounced dierence in pH-drop between the samples without added Mn2+ and the samples with as little as 0.25 ppm Mn2+. Fig. 1 shows the pH development with varying Mn2+ concentration for M1 that was typical for these ®ve starters. Adding Mn2+ to cultures of F1 did not signi®cantly accelerate the initial pH decline (results not shown). However, when Mn2+ was added at 1 ppm and above the ®nal pH value was reached in a much shorter time (40 h). On the other hand, when Mn2+ was not added, or present at a low level (0.25 ppm), the fermentation rate gradually slowed down and the pH dropped slowly towards its ®nal value (60 h). From the sausage model fermentations, the two noncommercial meat starter cultures (M1 and F1) were selected for further study in the sausage production experiment; as these two were the two starters that showed dierent responses to Mn2+ addition. The sausages produced had weight loss and water activities at levels normal for Norwegian sausages; all sausages had a water activity lower than 0.90 at the end of ripening, and a weight loss of about 35%. For these parameters, there were no signi®cant dierences
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between the dierent levels of Mn2+ added or the different cultures used. The pH development (Fig. 2) and lactobacilli counts (Fig. 3) in sausages were in¯uenced by the concentration of Mn2+. For M1 sausages, the pH was signi®cantly lower (P<0.05) and the lactobacilli counts
signi®cantly higher throughout sampling in sausages with added Mn2+. In F1 sausages, the pH was signi®cantly lower from day 3 to day 9 in the sausages with added Mn2+, but in ®nished sausages the pH was not dierent. Moreover, the cell counts in F1 fermented sausages did not increase on addition of Mn2+ and, on
Fig. 1. pH decrease for M1 in salami model system with increased addition of Mn2+ (&) 0 ppm; (~) 0.25 ppm, (&) 0.5 ppm, (*) 1.0 ppm and (^) 2.5 ppm. Each point is the mean of two separate experiments.
Fig. 2. pH development in sausages fermented with M1 (full line) and F1 (dashed line) with increasing addition of Mn2+ (&) 0 ppm; (~) 0.25 ppm, (*) 1.0 ppm and (^) 2.5 ppm. Each point is the mean of (2 productions2 sausages)=4 measurements (apart from 1.0 ppm where each point is the mean of (2 productions2 replicates2 sausages)=8 measurements).
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Hardness and chewiness were more pronounced in the M1 sausages (as compared to the F1) at the highest Mn2+ addition, and cohesiveness and resilience were more pronounced in the M1 sausages without Mn2+ addition. Sensory analyses showed that the sausages were not mature after 14 days, as all sensory attributes (except odour intensity) developed further until 23 days (P<0.05) (Table 3). However, after 14 days M1 fermented sausages with Mn2+ added were evaluated as more mature than sausages without Mn2+ whereas the F1 fermented sausages showed no dierences on Mn2+ addition. These dierences levelled out so that after 23 days there were no signi®cant dierences (P<0.05) between sausages with dierent additions of Mn2+ apart from the colour of the fat that was more pronounced in M1 sausages with Mn2+ added. Sensory dierences between sausages fermented with the two dierent starters were generally small. Rancid ¯avour
the contrary, the cells died o at the end of ripening (days 14 and 23) in the sausages where Mn2+ was added (P<0.05). Instrumental measurements of texture showed that sausages fermented with F1 gave more cohesiveness, springiness and resilience than M1 sausages after 3 days, these dierences lasted until 9 days of ripening but after 14 days of ripening there were no dierences (results not shown). After 3 days of ripening, F1 fermented sausages did not show any dierences from Mn2+ addition, whereas for all textural parameters, M1 fermented sausages without added Mn2+ had lower values than the M1 sausages with Mn2+ (results not shown). For the ®nished sausages (after 23 days) there were no dierences in textural properties with varying Mn2+ concentration for the F1 fermented sausages, whereas the M1 sausages showed increasing hardness and gumminess and decreasing cohesiveness, springiness and resilience with increasing Mn2+ addition (Table 2).
Fig. 3. Lactobacilli colony forming units (cfu) in sausages fermented with M1 (full line) and F1 (dashed line) with increasing addition of Mn2+ (&) 0 ppm; (~) 0.25 ppm, (*) 1.0 ppm and (^) 2.5 ppm. Each point is the mean of (2 productions2 replicates)=4 measurements (except for 1.0 ppm where each point is the mean of (2 productions2 replicates2 sausages)=8 measurements).
Table 2 Textural properties of sausages fermented with two dierent starters and containing dierent levels of Mn2+a,b
2+
Hardness
Cohesiveness
Springiness
Resilience
Gumminess
Chewiness
Culture
Culture
Culture
Culture
Culture
Culture
Mn
M1
F1
M1
F1
M1
F1
M1
F1
M1
F1
M1
F1
0 0.25 1 2.5
9777a 11948ab 13742b 13946by
10539a 11634a 12482a 111503az
0.467ay 0.443ab 0.433b 0.434b
0.449az 0.441a 0.437a 0.447a
0.600a 0.532b 0.538b 0.547b
0.568ay 0.540a 0.535a 0.545a
0.198ay 0.161b 0.161b 0.163b
0.161az 0.156a 0.155a 0.159a
4561a 5282ab 5909b 6049b
4722a 5094a 5441a 5136a
2741a 2812a 3173a 3303ay
2681a 2746a 2909a 2792az
a b
All values for texture responses are the average of 2 productions3 measurements =6 evaluations after 23 days of ripening. Dierent letters (a,b) in the same column and dierent letters (y,z) in the same row indicate signi®cant dierences (P<0.05).
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Table 3 Sensory analyses after 14 and 23 days of ripening of sausages fermented with two dierent starters and containing dierent levels of Mn2+a,b Days of ripening
14
23
Mn2+ (ppm)
Culture
0
0.25
1
2.5
0
0.25
1
2.5
Odour intensity
M F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1
5.99 5.95 4.79 4.18 5.45 5.47 4.93 5.07 4.59 4.52 4.79 4.74 5.79 5.74 3.15a 4.00 6.01y 4.93z 4.45 4.35 2.60 2.65 5.31 5.30 3.81 4.01 1.63 2.18 3.85 3.88 5.64 5.45 5.44 5.50 4.20 3.90
5.71 5.85 4.69 4.35 5.54 5.46 5.02 5.02 4.87 4.87 4.96 5.02 5.76 5.69 4.04a 4.00 5.25 5.24 4.82 4.79 2.80 2.62 5.43 5.62 3.92 4.00 1.35 1.68 4.30 4.21 5.26 5.15 5.23 5.47 3.56 3.57
5.64 5.80 4.38 4.25 5.30 5.26 5.07 5.09 4.63 4.55 4.73 4.80 5.73 5.80 4.04b 4.11 5.06 5.06 4.59 4.46 2.69 2.52 5.62 5.64 3.98 3.97 1.53 1.77 4.35 4.19 5.07 5.26 5.10 5.38 3.48 3.72
5.66 5.91 4.09 4.54 5.22 5.40 5.01 5.20 4.55 4.59 4.78 4.92 5.84 5.86 3.87ab 3.84 5.40 5.47 4.62 4.70 2.40 2.40 5.52 5.48 3.90 3.79 1.18y 2.15z 4.06 3.92 5.09 5.35 5.30 5.57 3.54 3.85
5.99 5.71 4.23 3.82 5.46ay 5.88z 4.73y 4.21z 5.26y 5.78z 5.39 5.95 6.09 6.10 5.21 5.69 3.43 3.19 3.98 4.25 2.98 2.89 5.66 5.67 4.18 4.34 2.02 2.29 4.88 4.89 4.95 4.81 4.90 4.72 3.24y 2.70z
5.85 5.78 4.30 3.70 6.02b 5.60 4.52 4.27 5.74 5.40 5.79 5.69 6.09 6.05 5.57 5.77 3.65 3.19 4.45 4.06 3.00 2.69 5.83 5.69 4.36 4.37 1.91y 2.53z 5.26 5.09 4.77 4.80 4.93 4.69 2.93 2.67
5.85 5.85 4.06 3.78 5.88ab 5.97 4.461y 4.112z 5.68 5.78 5.75 5.84 6.13 6.12 5.78 6.03 3.48 3.05 4.11 4.11 2.92 2.89 5.85 5.90 4.33 4.26 1.90 2.30 5.26 5.40 4.56 4.87 4.70 4.68 2.80 2.61
5.84 5.81 3.84 4.01 5.79ab 5.65 4.35 4.30 5.54 5.21 5.74 5.49 5.88 5.90 5.76 5.40 3.39 3.79 4.27 4.25 2.73 2.67 5.80 5.66 4.21 4.19 1.70 1.96 5.12 4.75 4.85 4.88 4.65 4.89 2.66 2.84
Acidic odour Fat colour Whiteness Colour tone Colour intensity Flavour intensity Maturity ¯avour Fresh ¯avour Acidic taste Sour taste Salty taste Bitter taste Rancid ¯avour Hardness Fattiness Juiceness Stickiness a b
All values for sensory responses are the average of 2 measurements11 assessors =22 evaluations. Dierent letters (a,b) in the same row and dierent letters (y,z) in the same column indicate signi®cant dierences (P<0.05).
was more pronounced in F1 sausages for some of the concentrations of Mn2+ and after 23 days of ripening some colour parameters of the F1 sausages were dierent from those of the M1 sausages. 4. Discussion The two dierent starter cultures chosen for sausage production (Lactobacillus sp. M1 and F1) showed a more pronounced pH-drop with increasing Mn2+ addition in the fermentation period (0±3 days). However, for F1, the dierences levelled out after 9 days, whereas for M1 the mature products (after 23 days) with and without Mn2+ were dierent, giving a sausage with lower pH and more starter bacteria when Mn2+ was added. The accelerated fermentation in the M1 sausages with added Mn2+ was
also evident from the dierences found in textural and sensory parameters. This suggests that the requirements for Mn2+ for these two starters are dierent. According to Archibald (1986), many lactobacilli require and contain high levels of divalent Mn2+. The fact that the ®nal product of M1 fermented sausages is dierent if Mn2+ is added, shows that the concentration of Mn2+ is of great importance for successful sausage fermentation. A rapid, reliable and reproducible fermentation process will ensure that each production spends less time in the fermentation chamber which will result in increased production capacity and better production planning. In addition rapid fermentation might ensure the microbial safety of the ®nal products since it has been shown that Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes may grow during the early stages of the fermentation process if the pH is too high
B.F. Hagen et al. / Meat Science 55 (2000) 161±168
(Arinder & Borch, 1999; Nissen & Holck, 1998). Moreover, for summer sausage, fermentation to pH 4.6 before cooking gives a 7 log reduction of Escherichia coli O157:H7 whereas fermentation to pH 5.0 before heating only results in a 3.2 log reduction (Calicioglu, Faith, Buege & Luchansky, 1997). To the extent that texture is a parameter used for judging whether a sausage is ready for the market, Mn2+ addition may also contribute to an overall shorter production time, as was evident from the increased hardness of the sausages made using Mn2+ and Lactobacillus M1. Mn2+ is a ubiquitous substance, present in most foods of vegetable and animal origin (Pennington, Schoen, Salmon, Young, Johnson & Marts, 1995), however the amount in vegetables is about 10 times that in meat. The Mn2+ content in animals may vary from 0.06 to 5.0 ppm, depending on species, muscle or organ and feeding of the animal (Mikulik, Vavrova, Zima & Sucmanova, 1977). In plant material, Mn2+ levels are high due to a high concentration in the chloroplasts of the plants (Archibald, 1986). Spices, one of the ingredients in the dry sausage recipe, act as a source of Mn2+ and the study reported by Zaika and Kissinger (1984) shows that the Mn2+ concentration of various dry spices used for salami production ranged from 10 to 660 ppm. This means that many commercial spice mixtures will not contain sucient Mn2+ for optimal growth of all starter cultures in sausages. The results from this work show that the level of manganese present in the raw materials for dry sausage production (meat, spices) is not always sucient to achieve optimal fermentation and in extreme cases, the fermentation might not proceed at all, if no extra Mn2+ is provided. The fact that lactobacilli may accumulate high intracellular levels of Mn2+, might in part explain the accelerating eect on sausage fermentation found on adding a cell-free extract from a protease producing Lactobacillus paracasei to a sausage batter (Hagen et al., 1996). Later investigations have shown that the accelerating eect could not solely be due to the enzyme added, but also in part to the presence of Mn2+ in the extract. In this case the Mn2+ would be co-extracted with the enzyme from the cells. 5. Conclusions As is evident from the present work, dierent starter cultures have dierent requirements for Mn2+. In all cases increased fermentation activity was observed after Mn2+ addition, with concomitant dierences in the development of microbiological, textural and sensory parameters being observed in the sausages. Sometimes, as for Lactobacillus F1, these dierences will level out
167
during production resulting in identical products at the end of production regardless of whether Mn2+ is added. For other strains such as Lactobacillus M1 dierences due to Mn2+ addition will remain throughout the production process giving sausages with changed properties. For a producer, knowing his starter cultures' requirements for Mn2+ will allow for optimisation of dry fermented sausage production, both in terms of producing sausages with desired sensory and textural properties and in terms of increasing reliability, reproducibility and safety of the process. Acknowledgements The authors wish to thank Birgitta Baardsen and Brit OppegaÊrd Pedersen for skillful technical assistance, and Hans Blom for stimulating discussions and Lars Axelsson for critical reading of the manuscript. The European Commission is acknowledged for ®nancial support through EU-FAIR project no. CT97-3227.
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FernaÂndez, M., de la Hoz, L., DõÂaz, O., Cambero, M. I., & OrdoÂnez, J. A. (1995a). Eect of the addition of pancreatic lipase on the ripening of dry±fermented sausages Ð 1. Microbial, physico-chemical and lipolytic changes. Meat Science, 40, 159±170. FernaÂndez, M., de la Hoz, L., DõÂaz, O., Cambero, M. I., & OrdoÂnez, J. A. (1995b). Eect of the addition of pancreatic lipase on the ripening of dry-fermented sausages Ð Part 2. Free fatty acids, short-chain fatty acid, carbonyls and sensory quality. Meat Science, 40, 351±362. Hagen, B. F., BerdagueÂ, J.-L., Holck, A. L., Nñs, H., & Blom, H. (1996). Bacterial proteinase reduces maturation time of dry fermented sausages. Journal of Food Science, 61, 1024±1029. Mikulik, A., Vavrova, M., Zima, S., & Sucmanova, M. (1977). Contents of copper, manganese, zinc and iron in raw, cooked and cured pork. Proceedings of the European Meeting of Meat Research Workers, 23, E5:1. Nñs, H., Holck, A. L., Axelsson, L., Andersen, H. J., & Blom, H. (1995). Accelerated ripening of dry fermented sausage by addition of a Lactobacillus proteinase. International Journal of Food Science and Technology, 29, 651±659. Nes, I. F., & SkjelkvaÊle, R. (1982). Eect of natural spices and oleoresins on Lactobacillus plantarum in the fermentation of dry sausage. Journal of Food Science, 47, 1618±1621,1625. Nissen, H., & Holck, A. (1998). Survival of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella kentucky in Norwegian fermented, dry sausage. Food Microbiology, 15, 273±279. Pennington, J. A. T., Schoen, S. A., Salmon, G. D., Young, B., Johnson, R. D., & Marts, R. W. (1995). Composition of core foods of the U.S. food supply, 1982±1991. III. Copper, manganese, selenium, and iodine. Journal of Food Composition and Analysis, 8, 171±217. Raccach, M., & Marshall, P. S. (1985). Eect of manganese ions on
the fermentative activity of frozen-thawed lactobacilli. Journal of Food Science, 50, 665±668. Zaika, L. L., & Kissinger, J. C. (1984). Fermentation enhancement by spices: Identi®cation of the active component. Journal of Food Science, 49, 5±9. Zalacain, I., Zapelena, J., AstiasaraÂn, I., & Bello, J. (1995). Dry fermented sausages elaborated with lipase from Candida cylindracea. Comparison with traditional formulations. Meat Science, 40, 55±61. Zalacain, I., Zapelena, M. J., Astiasaran, I., & Bello, J. (1996). Addition of lipase from Candida cylindracea to a traditional formulation of a dry fermented sausage. Meat Science, 42, 155±163. Zalacain, I., Zapelena, M. J., de Pena, M. P., AstiasaraÂn, I., & Bello, J. (1997). Use of lipase from Rhizomucor miehei in dry fermented sausages elaboration. Microbial, chemical and sensory analysis. Meat Science, 45, 99±105. Zapelena, M. J., Ansorena, D., Zalacain, I., AstiasaraÂn, I., & Bello, J. (1998). Dry fermented sausages manufactured with dierent amounts of commercial proteinases: evolution of total free a-NH2± N groups and sensory evaluation of the texture. Meat Science, 49, 213±221. Zapelena, M. J., Zalacain, I., Paz de Dena, M., Astiasaran, I., & Bello, J. (1997a). Eect of the addition of a neutral proteinase from Bacillus subtilis (Neutrase) on nitrogen fractions and texture of Spanish fermented sausage. Journal of Agricultural and Food Chemistry, 45, 2798±2801. Zapelena, M. J., Zalacain, I., Paz De Pena, M., Astiasaran, I., & Bello, J. (1997b). Addition of a neutral proteinase from Bacillus subtilis (Neutrase) together with a starter to a dry fermented sausage elaboration and its eect on the amino acid pro®le and the ¯avor development. Journal of Agricultural and Food Chemistry, 45, 472± 475.
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Meat starters have individual requirements for Mn2+ Beate F. Hagen, Helga Nñs, Askild L. Holck* MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 AÊs, Norway Received 26 August 1999; received in revised form 11 October 1999; accepted 11 October 1999
Abstract The eect of dierent Mn2+ concentrations on sausage fermentation was evaluated. A screening experiment was carried out with six lactobacilli starters in a sausage model. To further investigate the eects found, two selected lactobacilli strains were tested in pilot-scale sausage production. For all starters an increased fermentation rate was observed after Mn2+ addition. Dierences in the development of microbial, textural and sensory parameters were observed in the sausages. For one of the cultures these dierences levelled out during sausage production yielding identical end products with and without Mn2+, for the other strain the dierences due to Mn2+ addition in the sausages remained throughout the production process yielding sausages with dierent properties. Knowing a starter culture's requirements for Mn2+ will allow optimisation of dry fermented sausage production in order to increase reliability and reproducibility of production decrease fermentation time and ensure microbial safety of the ®nal product. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Dry sausage; Starter culture; Accelerated ripening
1. Introduction A challenge for the fermented meat industry is to produce more economically a dry fermented sausage of stable, high quality, but with the ¯avour, appearance and texture of traditional sausages. Many strategies for accelerating the fermentation and/or ripening processes by manipulating sausage formulation have been studied and suggested. Several groups have reported on the addition of enzymes, both proteolytic and lipolytic to improve speed. Zapelena, Zalacain, Ansorena and co-workers could not ®nd any accelerating eect by the addition of lipases, but suggest that careful selection of the correct protease could accelerate maturation without texture deterioration (Ansorena, Zapalena, AstiasaraÂn & Bello, 1998a,b; Zalacain, Zapelena, AstiasaraÂn & Bello, 1995, 1996; Zalacain, Zapelena, de Pena, AstiasaraÂn & Bello, 1997; Zapelena, Ansorena, Zalacain, AstiasaraÂn & Bello, 1998; Zapelena, Zalacain, Paz de Dena, AstiasaraÂn & Bello, 1997a,b). Likewise, the group of DõÂaz have found possible means to accelerate proteolysis and lipolysis,
* Corresponding author. Tel.: +47-6497-0100; fax: +47-6497-0333. E-mail address: [email protected] (A.L. Holck).
thus providing substrates for rapid tranformation into aromatic compounds, however they have not as yet found satisfactory, practical protocols (DõÂaz, Fernandez, Garcia de Fernando, de la Hoz & Ordonez, 1993, 1996, 1997; FernaÂndez, de la Hoz, DõÂaz, Cambero & OrdoÂnez, 1995a,b). Previous work in our group have shown that addition of a bacterial protease extract in pilot scale salami production shortens the ripening time by 50% (Hagen, BerdagueÂ, Holck, Nñs & Blom, 1996; Nñs, Holck, Axelsson, Andersen & Blom, 1995). Addition of a soy protein isolate has also been shown to stimulate growth of the starter culture and thereby accelerate fermentation. A more convenient and less expensive approach to the fermentation time would be to optimise the level of ingredients already present in the sausage formulation. Although the purpose of adding spices to sausage formulations is to achieve the desired ¯avour, they also have other functions; serving as a source of fermentable and reducing sugars, nitrates and nitrites, colourings, metallic ions etc. Spices are known to have an in¯uence on the development of texture, ¯avour and colour during the ripening of sausages (Aguirrezabal, Mateo, Dominguez & Zumalacarregui, 1998). It has long been known that components in spices are stimulatory to acid production of starter cultures (Nes
0309-1740/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0309-1740(99)00138-2
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& SkjelkvaÊle, 1982) and Zaika and Kissinger (1984) showed that one factor in spices that has this property is Mn2+. It is established that the metabolism of many lactobacilli is dependent on high levels of Mn2+ (Archibald, 1986), and that the requirement for Mn2+ diers among dierent lactobacilli. Meat has low levels of Mn2+, and addition of Mn2+ may thus stimulate lactobacilli during meat fermentation. Raccach and Marshall state that lactic acid fermentation by Lactobacillus plantarum in beaker sausage was stimulated by Mn2+ and Coventry and Hickey (1993) have previously shown that adding Mn2+ to a salami model stimulates the fermentation rate of a Pediococcus starter culture. The objective of this work was to evaluate the eect of dierent concentrations of Mn2+ on sausage fermentation. A screening experiment was carried out with six lactobacilli starters in a sausage model. To further investigate the eect found, two selected lactobacilli were tested in pilot-scale sausage production. 2. Materials and methods 2.1. Mn2+ determination A commercial salami spice mixture was purchased from a local supplier. The concentration of Mn2+ in the spices was determined by ¯ame atomic absorption spectrometry in a laboratory performing accredited chemical analyses (Sero AS, 1361 Billingstad, Norway). 2.2. Starter cultures The starter cultures used in these experiments are listed in Table 1.The non-commercial strain Lactobacillus sp. (M1) was isolated from dry fermented sausages, where this culture was the predominating species F1 was a starter culture, closely related to Lactobacillus farciminis, used by a local factory. The four commercial starters were all supplied as mixed cultures from the producers, with at least one Staphylococcus strain in addition to the Lactobacillus strains. All mixtures were seeded onto MRS (De Man, Rogosa, Sharpe) (Oxoid) agar plates and the Lactobacillus strains were isolated before use. Cultures were stored at ÿ80 C in MRS
medium supplemented with glycerol (18% v/v). For all experiments, the starter cultures were precultivated in MRS for 16 h at 30 C, transferred once in MRS under the same conditions, washed with peptone water [0.1% (w/v) Bacto Peptone (Oxoid) and 0.85% (w/v) NaCl (p.a.) in distilled water] and resuspended in peptone water diluted to desired OD600. Optical density (OD600) was determined at 600 nm with Ultrospec 3000 (Pharmacia Biotech). 2.3. Sausage model fermentations A 30-kg sausage mixture which contained (% w/w): beef (51.8), pork (18.8), lard (25.6), sodium chloride (3.3), nitrite (0.02), glucose (0.4) and ascorbic acid (0.04) was chopped and blended in a cutter. 150 g (1 g) aliquots of this batter were put in plastic boxes (dia. 68 mm) and stored at ÿ20 C. To the thawed samples was added 0.5 ml distilled water containing various concentrations of Mn2+ sulphate (MnSO4H2O p.a. Merck) and 0.25 ml of starter culture (OD600 4.00.2). The additions were mixed into the sausage batter with a blender (Braun Multimix Duo M810) and the boxes were centrifuged (1000 rpm, 2 min, High Speed Sentrifuge, Sorvall RC 5C, rotor SH 3000, Sorvall Inc.) to mimic the eect of vacuum stung. pH electrodes (Xerolyt, Mettler-Toledo, Process, Analytical Inc.) were inserted into the middle of each batter through a tight hole in the lid and the samples were incubated at 22 C for 120 h. pH was recorded every hour by an automatic logging system (Intab AAC-2, Interface-Teknik AB, Sweden) controlled by a software package (Easy View 2.12, Interface-Teknik AB, Sweden). The model experiments were performed in a two-step procedure: in step 1, spices (0.5% w/w) were added to the sausage batter (with exception of the control batter) together with manganese solutions to achieve the following ®nal concentrations of Mn2+, including the Mn2+ present in the spices: 0, 0.7, 1, 1.2, 1.7, 2.7, 5.7 and 10.7 ppm. Step 1 was a screening step with only one experiment per starter in order to decide the appropriate concentrations of Mn2+ for further studies. In step 2, the Mn2+ levels 0, 0.25, 0.5, 1.0 and 2.5 ppm were selected and all starters were investigated for pH-development in two independent experiments in batters without spices added.
Table 1 Starter cultures for screening of Mn2+ requirements
2.4. Sausage production
Strain
Name
Supplier
Lactobacillus sp. Lactobacillus sp. Lactobacillus plantarum Lactobacillus sp. Lactobacillus sakei Lactobacillus curvatus
M1 F1 Duploferment Biostart 741 LS-25 LS-3
MATFORSK Local factory Chr. Hansen Raps GewuÈrzmuÈller GmbH GewuÈrzmuÈller GmbH
Salami sausages (without spices and not subjected to smoking) were made according to the above recipe in a designed experiment with two factors; starter culture (two types) and Mn2+ addition (four levels). Two batters of 50 kgs were made. To batter 1, Lactobacillus M1 was added as starter culture and supplied at 6106 cells/ g sausage mixture. To batter 2, Lactobacillus F1 was
B.F. Hagen et al. / Meat Science 55 (2000) 161±168
added as starter culture and supplied at 4106 cells/g. For each batter the ingredients were mixed and divided into ®ve batches (10.0 kg each). To batch 1 was added 7.7 ml distilled water. To batch 2 was added 7.7 ml of a solution of MnSO4 in distilled water that gave a ®nal concentration in the sausage mixture of 0.25 ppm Mn2+. To batches 3 and 4 were added MnSO4 to give a ®nal concentration of 1.0 ppm Mn2+, and likewise, batch 5 had a ®nal concentration of 2.5 ppm Mn2+. From each batch, 25 sausages of 400 g were prepared. The sausages were stued in arti®cial casings (50 mm diam.) and placed in a ripening chamber for 3 days at 24 C, 96% relative humidity (RH), 2 days at 16 C, 92% RH, 2 days at 16 C, 88% RH and ®nally the sausages were allowed to ripen at 16 C, 85% RH until mature. Sausage samples were collected for analyses after 0, 2, 3, 6, 9, 14, and 23 days. All analyses were performed on two individual sausages. The weight of each sausage was determined before and after ripening and the weight loss calculated. 2.5. pH, water activity and colony counts pH was assayed on 10 g sausage homogenized in 90 ml peptone water. Water activity was measured by an electronic hygrometer (Novasina aw-center, sensor: enRSK-4/CT-4, Novasina AG, PhaÈcon, Switzerland) on 10 g sausages cut into 5 mm cubes. Growth of the added starter cultures was assayed by plating appropriate dilutions of sausage homogenised in peptone water on MRS agar (Oxoid). The same dilutions were also plated on blood agar (Difco) to check for possible contamination. The plates were incubated at 30 C for 2 days. 2.6. Texture analysis Texture of the sausages was evaluated by Texture Pro®le Analysis (TPA) using a Texture Analyser TAXT2 (Stable Micro Systems). A bite size sample was compressed to 50% of its original height, twice, in a reciprocating motion that imitated the action of the jaw. Analyses of force±time curves led to the identi®cation of four textural parameters (hardness, springiness, cohesiveness and resilience) and two calculated parameters (gumminess and chewiness) (Bourne, 1978). 2.7. Sensory analysis Sensory descriptive pro®ling was carried out by 10 trained assessors at 14 and 23 days after production. The ¯avour pro®le of the sausages was determined using an unstructured line with end points (1±9) where 1 denoted low intensity and 9 high intensity for each of the following 18 characteristics: Odour intensity, acidic odour, colour tone of fat, whiteness, overall colour tone,
163
colour intensity, ¯avour intensity, maturity ¯avour, fresh ¯avour, acidic taste, sour taste, salty taste, bitter taste, rancid ¯avour, hardness, fattiness, juiciness and stickiness. Dierences in sensory score were evaluated using Tukey's test. 2.8. Data analysis Statistical signi®cances of observed dierences among means of experimental results were evaluated by analysis of variance (ANOVA) with Tukey's test for pairwise comparison of means using Minitab for Windows release 12.1 (Minitab Inc., PA, USA) to detect the eect of the two treatments (starter culture, Mn2+ addition). Results from the two duplicate samples were averaged. 3. Results The concentration of Mn2+ in the commercial spice mixture was 131 mg Mn2+/kg. As the recommended addition of the spices to salami sausages is 0.5±1.0%, the corresponding addition of Mn2+ would be 0.66± 1.31 ppm. In the ®rst screening experiment, we studied the eects of Mn2+ levels equal to and higher than commonly found in sausages, so levels ranging from 0.66 to 10 ppm Mn2+ were evaluated. In this preliminary step of the pH-logging experiment, we found that for each starter, pH development did not change when the Mn2+ concentration exceeded 2.7 ppm (results not shown). Hence, in step two, we studied in more detail the eect of adding from 0 to 2.5 ppm Mn2+. For the four commercial starters and M1, there was a pronounced dierence in pH-drop between the samples without added Mn2+ and the samples with as little as 0.25 ppm Mn2+. Fig. 1 shows the pH development with varying Mn2+ concentration for M1 that was typical for these ®ve starters. Adding Mn2+ to cultures of F1 did not signi®cantly accelerate the initial pH decline (results not shown). However, when Mn2+ was added at 1 ppm and above the ®nal pH value was reached in a much shorter time (40 h). On the other hand, when Mn2+ was not added, or present at a low level (0.25 ppm), the fermentation rate gradually slowed down and the pH dropped slowly towards its ®nal value (60 h). From the sausage model fermentations, the two noncommercial meat starter cultures (M1 and F1) were selected for further study in the sausage production experiment; as these two were the two starters that showed dierent responses to Mn2+ addition. The sausages produced had weight loss and water activities at levels normal for Norwegian sausages; all sausages had a water activity lower than 0.90 at the end of ripening, and a weight loss of about 35%. For these parameters, there were no signi®cant dierences
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between the dierent levels of Mn2+ added or the different cultures used. The pH development (Fig. 2) and lactobacilli counts (Fig. 3) in sausages were in¯uenced by the concentration of Mn2+. For M1 sausages, the pH was signi®cantly lower (P<0.05) and the lactobacilli counts
signi®cantly higher throughout sampling in sausages with added Mn2+. In F1 sausages, the pH was signi®cantly lower from day 3 to day 9 in the sausages with added Mn2+, but in ®nished sausages the pH was not dierent. Moreover, the cell counts in F1 fermented sausages did not increase on addition of Mn2+ and, on
Fig. 1. pH decrease for M1 in salami model system with increased addition of Mn2+ (&) 0 ppm; (~) 0.25 ppm, (&) 0.5 ppm, (*) 1.0 ppm and (^) 2.5 ppm. Each point is the mean of two separate experiments.
Fig. 2. pH development in sausages fermented with M1 (full line) and F1 (dashed line) with increasing addition of Mn2+ (&) 0 ppm; (~) 0.25 ppm, (*) 1.0 ppm and (^) 2.5 ppm. Each point is the mean of (2 productions2 sausages)=4 measurements (apart from 1.0 ppm where each point is the mean of (2 productions2 replicates2 sausages)=8 measurements).
B.F. Hagen et al. / Meat Science 55 (2000) 161±168
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Hardness and chewiness were more pronounced in the M1 sausages (as compared to the F1) at the highest Mn2+ addition, and cohesiveness and resilience were more pronounced in the M1 sausages without Mn2+ addition. Sensory analyses showed that the sausages were not mature after 14 days, as all sensory attributes (except odour intensity) developed further until 23 days (P<0.05) (Table 3). However, after 14 days M1 fermented sausages with Mn2+ added were evaluated as more mature than sausages without Mn2+ whereas the F1 fermented sausages showed no dierences on Mn2+ addition. These dierences levelled out so that after 23 days there were no signi®cant dierences (P<0.05) between sausages with dierent additions of Mn2+ apart from the colour of the fat that was more pronounced in M1 sausages with Mn2+ added. Sensory dierences between sausages fermented with the two dierent starters were generally small. Rancid ¯avour
the contrary, the cells died o at the end of ripening (days 14 and 23) in the sausages where Mn2+ was added (P<0.05). Instrumental measurements of texture showed that sausages fermented with F1 gave more cohesiveness, springiness and resilience than M1 sausages after 3 days, these dierences lasted until 9 days of ripening but after 14 days of ripening there were no dierences (results not shown). After 3 days of ripening, F1 fermented sausages did not show any dierences from Mn2+ addition, whereas for all textural parameters, M1 fermented sausages without added Mn2+ had lower values than the M1 sausages with Mn2+ (results not shown). For the ®nished sausages (after 23 days) there were no dierences in textural properties with varying Mn2+ concentration for the F1 fermented sausages, whereas the M1 sausages showed increasing hardness and gumminess and decreasing cohesiveness, springiness and resilience with increasing Mn2+ addition (Table 2).
Fig. 3. Lactobacilli colony forming units (cfu) in sausages fermented with M1 (full line) and F1 (dashed line) with increasing addition of Mn2+ (&) 0 ppm; (~) 0.25 ppm, (*) 1.0 ppm and (^) 2.5 ppm. Each point is the mean of (2 productions2 replicates)=4 measurements (except for 1.0 ppm where each point is the mean of (2 productions2 replicates2 sausages)=8 measurements).
Table 2 Textural properties of sausages fermented with two dierent starters and containing dierent levels of Mn2+a,b
2+
Hardness
Cohesiveness
Springiness
Resilience
Gumminess
Chewiness
Culture
Culture
Culture
Culture
Culture
Culture
Mn
M1
F1
M1
F1
M1
F1
M1
F1
M1
F1
M1
F1
0 0.25 1 2.5
9777a 11948ab 13742b 13946by
10539a 11634a 12482a 111503az
0.467ay 0.443ab 0.433b 0.434b
0.449az 0.441a 0.437a 0.447a
0.600a 0.532b 0.538b 0.547b
0.568ay 0.540a 0.535a 0.545a
0.198ay 0.161b 0.161b 0.163b
0.161az 0.156a 0.155a 0.159a
4561a 5282ab 5909b 6049b
4722a 5094a 5441a 5136a
2741a 2812a 3173a 3303ay
2681a 2746a 2909a 2792az
a b
All values for texture responses are the average of 2 productions3 measurements =6 evaluations after 23 days of ripening. Dierent letters (a,b) in the same column and dierent letters (y,z) in the same row indicate signi®cant dierences (P<0.05).
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Table 3 Sensory analyses after 14 and 23 days of ripening of sausages fermented with two dierent starters and containing dierent levels of Mn2+a,b Days of ripening
14
23
Mn2+ (ppm)
Culture
0
0.25
1
2.5
0
0.25
1
2.5
Odour intensity
M F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1 M1 F1
5.99 5.95 4.79 4.18 5.45 5.47 4.93 5.07 4.59 4.52 4.79 4.74 5.79 5.74 3.15a 4.00 6.01y 4.93z 4.45 4.35 2.60 2.65 5.31 5.30 3.81 4.01 1.63 2.18 3.85 3.88 5.64 5.45 5.44 5.50 4.20 3.90
5.71 5.85 4.69 4.35 5.54 5.46 5.02 5.02 4.87 4.87 4.96 5.02 5.76 5.69 4.04a 4.00 5.25 5.24 4.82 4.79 2.80 2.62 5.43 5.62 3.92 4.00 1.35 1.68 4.30 4.21 5.26 5.15 5.23 5.47 3.56 3.57
5.64 5.80 4.38 4.25 5.30 5.26 5.07 5.09 4.63 4.55 4.73 4.80 5.73 5.80 4.04b 4.11 5.06 5.06 4.59 4.46 2.69 2.52 5.62 5.64 3.98 3.97 1.53 1.77 4.35 4.19 5.07 5.26 5.10 5.38 3.48 3.72
5.66 5.91 4.09 4.54 5.22 5.40 5.01 5.20 4.55 4.59 4.78 4.92 5.84 5.86 3.87ab 3.84 5.40 5.47 4.62 4.70 2.40 2.40 5.52 5.48 3.90 3.79 1.18y 2.15z 4.06 3.92 5.09 5.35 5.30 5.57 3.54 3.85
5.99 5.71 4.23 3.82 5.46ay 5.88z 4.73y 4.21z 5.26y 5.78z 5.39 5.95 6.09 6.10 5.21 5.69 3.43 3.19 3.98 4.25 2.98 2.89 5.66 5.67 4.18 4.34 2.02 2.29 4.88 4.89 4.95 4.81 4.90 4.72 3.24y 2.70z
5.85 5.78 4.30 3.70 6.02b 5.60 4.52 4.27 5.74 5.40 5.79 5.69 6.09 6.05 5.57 5.77 3.65 3.19 4.45 4.06 3.00 2.69 5.83 5.69 4.36 4.37 1.91y 2.53z 5.26 5.09 4.77 4.80 4.93 4.69 2.93 2.67
5.85 5.85 4.06 3.78 5.88ab 5.97 4.461y 4.112z 5.68 5.78 5.75 5.84 6.13 6.12 5.78 6.03 3.48 3.05 4.11 4.11 2.92 2.89 5.85 5.90 4.33 4.26 1.90 2.30 5.26 5.40 4.56 4.87 4.70 4.68 2.80 2.61
5.84 5.81 3.84 4.01 5.79ab 5.65 4.35 4.30 5.54 5.21 5.74 5.49 5.88 5.90 5.76 5.40 3.39 3.79 4.27 4.25 2.73 2.67 5.80 5.66 4.21 4.19 1.70 1.96 5.12 4.75 4.85 4.88 4.65 4.89 2.66 2.84
Acidic odour Fat colour Whiteness Colour tone Colour intensity Flavour intensity Maturity ¯avour Fresh ¯avour Acidic taste Sour taste Salty taste Bitter taste Rancid ¯avour Hardness Fattiness Juiceness Stickiness a b
All values for sensory responses are the average of 2 measurements11 assessors =22 evaluations. Dierent letters (a,b) in the same row and dierent letters (y,z) in the same column indicate signi®cant dierences (P<0.05).
was more pronounced in F1 sausages for some of the concentrations of Mn2+ and after 23 days of ripening some colour parameters of the F1 sausages were dierent from those of the M1 sausages. 4. Discussion The two dierent starter cultures chosen for sausage production (Lactobacillus sp. M1 and F1) showed a more pronounced pH-drop with increasing Mn2+ addition in the fermentation period (0±3 days). However, for F1, the dierences levelled out after 9 days, whereas for M1 the mature products (after 23 days) with and without Mn2+ were dierent, giving a sausage with lower pH and more starter bacteria when Mn2+ was added. The accelerated fermentation in the M1 sausages with added Mn2+ was
also evident from the dierences found in textural and sensory parameters. This suggests that the requirements for Mn2+ for these two starters are dierent. According to Archibald (1986), many lactobacilli require and contain high levels of divalent Mn2+. The fact that the ®nal product of M1 fermented sausages is dierent if Mn2+ is added, shows that the concentration of Mn2+ is of great importance for successful sausage fermentation. A rapid, reliable and reproducible fermentation process will ensure that each production spends less time in the fermentation chamber which will result in increased production capacity and better production planning. In addition rapid fermentation might ensure the microbial safety of the ®nal products since it has been shown that Escherichia coli O157:H7, Staphylococcus aureus and Listeria monocytogenes may grow during the early stages of the fermentation process if the pH is too high
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(Arinder & Borch, 1999; Nissen & Holck, 1998). Moreover, for summer sausage, fermentation to pH 4.6 before cooking gives a 7 log reduction of Escherichia coli O157:H7 whereas fermentation to pH 5.0 before heating only results in a 3.2 log reduction (Calicioglu, Faith, Buege & Luchansky, 1997). To the extent that texture is a parameter used for judging whether a sausage is ready for the market, Mn2+ addition may also contribute to an overall shorter production time, as was evident from the increased hardness of the sausages made using Mn2+ and Lactobacillus M1. Mn2+ is a ubiquitous substance, present in most foods of vegetable and animal origin (Pennington, Schoen, Salmon, Young, Johnson & Marts, 1995), however the amount in vegetables is about 10 times that in meat. The Mn2+ content in animals may vary from 0.06 to 5.0 ppm, depending on species, muscle or organ and feeding of the animal (Mikulik, Vavrova, Zima & Sucmanova, 1977). In plant material, Mn2+ levels are high due to a high concentration in the chloroplasts of the plants (Archibald, 1986). Spices, one of the ingredients in the dry sausage recipe, act as a source of Mn2+ and the study reported by Zaika and Kissinger (1984) shows that the Mn2+ concentration of various dry spices used for salami production ranged from 10 to 660 ppm. This means that many commercial spice mixtures will not contain sucient Mn2+ for optimal growth of all starter cultures in sausages. The results from this work show that the level of manganese present in the raw materials for dry sausage production (meat, spices) is not always sucient to achieve optimal fermentation and in extreme cases, the fermentation might not proceed at all, if no extra Mn2+ is provided. The fact that lactobacilli may accumulate high intracellular levels of Mn2+, might in part explain the accelerating eect on sausage fermentation found on adding a cell-free extract from a protease producing Lactobacillus paracasei to a sausage batter (Hagen et al., 1996). Later investigations have shown that the accelerating eect could not solely be due to the enzyme added, but also in part to the presence of Mn2+ in the extract. In this case the Mn2+ would be co-extracted with the enzyme from the cells. 5. Conclusions As is evident from the present work, dierent starter cultures have dierent requirements for Mn2+. In all cases increased fermentation activity was observed after Mn2+ addition, with concomitant dierences in the development of microbiological, textural and sensory parameters being observed in the sausages. Sometimes, as for Lactobacillus F1, these dierences will level out
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during production resulting in identical products at the end of production regardless of whether Mn2+ is added. For other strains such as Lactobacillus M1 dierences due to Mn2+ addition will remain throughout the production process giving sausages with changed properties. For a producer, knowing his starter cultures' requirements for Mn2+ will allow for optimisation of dry fermented sausage production, both in terms of producing sausages with desired sensory and textural properties and in terms of increasing reliability, reproducibility and safety of the process. Acknowledgements The authors wish to thank Birgitta Baardsen and Brit OppegaÊrd Pedersen for skillful technical assistance, and Hans Blom for stimulating discussions and Lars Axelsson for critical reading of the manuscript. The European Commission is acknowledged for ®nancial support through EU-FAIR project no. CT97-3227.
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