Scientific Publications - Work Done by Microbiology Reader Bioscreen C

Journal of Applied Microbiology, 2000, Mar, Vol. 88, No. 3, pp. 536-545

Production of sakacin P  by Lactobacillus sakei  in a completely defined medium

Moretro T, Aasen IM, Storro I, Axelsson L.
 

ABSTRACT

In order to investigate factors influencing the production of the bacteriocin, sakacin P, Lactobacillus sakei CCUG 42687 was grown in a completely defined medium (DML-B) with 33 components. Although the maximum sakacin P concentration obtained was higher on a complex medium due to higher cell mass, the production per cell mass was higher in DML-B. Sakacin P was produced at 4-30 degrees C, with the highest specific production at low temperatures. More sakacin P was produced at uncontrolled pH compared with cultivation at pH 6.3. Tween-80 had a positive effect on sakacin P production, while addition of sodium chloride and trace metals had negative effects. The decrease in sakacin P concentration during the late growth and stationary phases was shown to be cell-independent and promoted at high temperature and pH. Some differences in production levels of sakacin P were found among six strains of Lactobacillus sakei tested.

INTRODUCTION

Many lactic acid bacteria produce ribosomally-synthesized antimicrobial peptides called bacteriocins, which can be divided into classes based on structure, size and mode of action ( Nes et al. 1996). Class II bacteriocins comprises small, heat-stable cationic peptides which contain no modified amino acids. One subgroup, class IIa, contains bacteriocins with certain sequence motifs in their N-terminal halves and are active against the food pathogen, Listeria moncytogenes ( Nes et al. 1996). For commercial use of bacteriocins in food systems, optimization of their production is necessary. Most bacteriocins are produced as primary metabolites during growth ( De Vuyst et al. 1996; Nilsen et al. 1998). The lactic acid bacteria are fastidious organisms and good growth usually requires complex media. The effects of growth conditions on production have been studied for many bacteriocins in such media, i.e. amylovorin L471 ( De Vuyst et al. 1996; Lejeune et al. 1998), pediocin PA-1 ( Biswas et al. 1991), lactocin S ( Mørtvedt-Abildgaard et al. 1995), nisin ( De Vuyst & Vandamme 1993), sakacin K ( Leroy & De Vuyst 1999), enterocin 1146 ( Parente & Hill 1992) and enterocin A and B ( Nilsen et al. 1998). Production of some bacteriocins increases under unfavourable conditions, such as at low growth rates in continuous culture and the presence of ethanol ( De Vuyst et al. 1996), while production of other bacteriocins decreases under such conditions ( Nilsen et al. 1998). For many bacteriocins, the concentration in the culture supernatant fluid decreases in the late stages of a batch culture. This effect has been suggested to be due to proteolytic degradation, aggregation, or adsorption to cells ( De Vuyst et al. 1996).

Sakacin P is a 43 amino acid class IIa bacteriocin produced by several strains of Lactobacillus sakei ( Tichaczek et al. 1992; Larsen et al. 1993; Holck et al. 1994; Trüper & De Clari 1997). The bacteriocin production is regulated by a three-component system consisting of an induction factor, a histidine kinase and a response regulator ( Eijsink et al. 1996; Hühne et al. 1996; Brurberg et al. 1997). The induction factor is a bacteriocin-like peptide secreted by the producing organism itself and serves as the signal for transcriptional activation of the genes required for bacteriocin production. Compared with other studied bacteriocins, sakacin P has higher activity against the food pathogen, L. monocytogenes, and could thus be promising for use in the food industry ( Eijsink et al. 1998). Complex media do not allow examination of all individual medium factors affecting bacteriocin production. However, this is feasible using a completely defined medium, and a minimal medium for bacteriocin production can thus be established. Only a few bacteriocins from lactic acid bacteria have been studied with regard to production in a defined medium, e.g. nisin ( De Vuyst 1995). This work describes the first study of bacteriocin production in a defined medium by a Lactobacillus species. Lactobacillus sakei CCUG 42687, which produces sakacin P, was studied in DML, a defined medium developed for meat lactobacilli ( Møretrøet al. 1998). Various medium components and growth conditions were tested for their effect on cell growth and sakacin P production. Bacteriocin production in a defined and a complex medium was compared. Growth and production of sakacin P by five other Lact. sakei strains were also compared with Lact. sakei CCUG 42687.

MATERIALS AND METHODS

Organisms and media

The strains used in this work are listed in Table 1. All Lact. sakei strains were confirmed to produce sakacin P. Lactobacillus sakei CCUG 42687 was the principal strain studied. The organisms were maintained as stock cultures in MRS (Oxoid) with 18% glycerol at 80 °C. Growth and sakacin P production were tested on a completely defined medium (DML, composed of 18 amino acids, three nucleotide bases, three mineral salts, seven vitamins, glucose, Tween-80 and succinate as a buffer), originally developed for six strains of meat lactobacilli including Lact. sakei CCUG 42687 ( Møretrøet al. 1998). For cultivation with and without pH control, 20 and 100 mmol l ¹ succinate were used, respectively. Growth and production of sakacin P in a defined medium in a fermentor was compared with that in a complex medium (Reference medium) used for optimization of growth and sakacin P production of Lact. sakei CCUG 42687, with the following composition (l ¹): 10 g yeast extract (Oxoid), 10 g tryptone (Oxoid), 30 g glucose, 0·05 g MnSO₄.H₂O, 0·2 g MgSO₄.7H₂O, 1 ml Tween-80 (Sigma) and 2·7 g KH₂PO₄ ( Aasen et al. 2000).The effect of addition of a trace mineral solution was tested. When used, 1 ml l ¹ was added to the defined medium of a solution that contained (mg l ¹): FeSO₄.7H₂O 50, CuSO₄.5H₂O 3·9, ZnSO₄.7H₂O 4·4, MnSO₄.H₂O 1·5, CoCl₂.6H₂O 0·2 and Na₂MoO₄.2H₂O 0·1. Synthetic inducing peptide (IP-673, 2 mg ml ¹ dissolved in 0·1% trifluoracetic acid) was a kind gift from Vincent G.H. Eijsink ( Eijsink et al. 1996). Purified sakacin P (282 mg l ¹) was obtained as described previously ( Holck et al. 1994).

Fermentation, microtitre plate cultures and growth measurements

To study growth and production of sakacin P in a fermentor, a CF 3000 D fermentor (CHEMAP AG, Volketswil, Switzerland) with 2 l culture volume was used. The fermentor was inoculated (1% v/v) with an overnight culture grown on DML or MRS for cultivation on defined or complex media, respectively. When the cells were grown with controlled pH, the pH was monitored and maintained by automatic addition of 2 mol l ¹ KOH for DML and 2 mol l ¹ NaOH for complex media. The fermentor was operated without aeration and with slow agitation (100 rev min ¹) to keep the fermentation broth homogenous. The optical densities (O.D.), at 600 nm, of samples from the fermentor were measured with an Ultraspec 3000 spectrophotometer (Pharmacia Biotech Ltd, Cambrigde, UK). Cell dry mass was determined at the end of growth by washing cells once with distilled water and drying at 105 °C for 6 h.

To study the effects of the individual medium components on growth and sakacin P production, the organisms were cultured in microtitre plates with a 400 l culture volume in each well in Bioscreen C (Labsystems, Helsinki, Finland), a microtitre plate reader measuring growth as O.D. For each condition to be tested, inoculated culture was divided and transferred to 10 parallel wells, and samples of 400 l were removed from the plates during growth for bacteriocin determination. Calibration curves were made to correlate Bioscreen O.D. values, O.D. values from the Ultraspec 3000 spectrophotometer and cell dry mass (CDM). These were used to convert all the O.D. values to CDM values. The following terms are used in this work to describe the production of sakacin P: volumetric production sakacin P production per volume culture (mg l ¹); and specific production production per cell mass (mg g ¹).

Bacteriocin measurement

Cell supernatant fluids were collected by centrifugation (5 min, 6000 g, 20 °C) and the supernatant fluids were frozen at 20 °C until bacteriocin measurement. Sakacin P was measured using a microtitre plate method described previously with L. ivanovii Li4(pVS2) (erythromycin- and chloramphenicol-resistant) as indicator ( Axelsson et al. 1998). Erythromycin and chloramphenicol were added to the assay medium, both to a final concentration of 10 g ml ^1. Thawed supernatant fluid was diluted in assay medium and assayed without filtration as the producing organism would not grow in this medium. The use of a standard of purified sakacin P of known concentration on every plate resulted in quantitative measurement (mg l ¹) of sakacin P with a coefficient of variation less than 15%. The sakacin P producers most likely produce at least one additional bacteriocin ( Eijsink et al. 1998). It is therefore important that an indicator strain is specifically sensitive to sakacin P for correct quantification. Comparisons between the activity of crude (supernatant fluid) and pure sakacin P for several strains, according to Eijsink (Eijsink, personal communication; Eijsink et al. 1998), showed that L. ivanovii Li4 was insensitive to other putative bacteriocins produced under the conditions of the assay and thus, specific for sakacin P (Axelsson and Møretrø, unpublished data).

RESULTS

Effects of temperature and pH

Initially, NaOH was used to adjust the pH of the defined medium. However, the growth rate () and the maximum optical density at 600 nm were increased by about 10%, and the volumetric production of sakacin P was increased by 40-50%, by using KOH instead. Based on this observation, KOH was used for all pH adjustments in DML. Biotin is present in DML because of its stimulatory effect on Lact. plantarum and Lact. pentosus ( Møretrøet al. 1998), but it could be omitted from DML without affecting growth or sakacin P production of Lact. sakei CCUG 42687, resulting in a minimal medium for growth and sakacin P production with 33 components (DML-B).

When Lact. sakei CCUG 42687 was cultured in DML-B (containing 0·3% Tween-80) in the fermentor with controlled pH 6·3, the increase in sakacin P activity paralleled the increase in cell mass, indicating primary metabolite kinetics ( Fig. 1a). Growth ceased when about half the glucose had been consumed, as can be seen from the KOH consumption, which corresponded to the glucose consumption. The production of sakacin P stopped at the same time. Glucose consumption continued further until depletion, but at a lower rate. After sakacin P concentration had reached its peak, a gradual decrease in concentration was evident. This decrease continued further until the fermentation was terminated, and it is hereafter referred to as the inactivation phase (see below). In general, both volumetric and specific sakacin P production was increased by decreasing temperatures of growth ( Table 2). Repeated fermentations of Lact. sakei CCUG 42687 in DML-B with pH 6·3 at 15 °C resulted in poor growth compared with all other conditions tested.

When Lact. sakei CCUG 42687 was grown in the fermentor, without pH control, in DML-B (0·3% Tween-80), growth ceased at pH 5·1-5·2. The pH continued to drop further but at a lower rate until 4·8-4·9, and glucose was not completely consumed. Sakacin P production followed the same kinetics without pH control as with controlled pH, except that there was a broader peak in total activity without pH control ( Fig. 1a,b). Both volumetric and specific production of sakacin P was higher without pH control, compared with pH 6·3, at the corresponding temperatures ( Table 2). The specific production at pH 6·3 was higher in DML-B than in the complex Reference medium. However, the volumetric production was higher in the complex medium due to a higher cell mass ( Table 2). For culture without pH control, both volumetric and specific production were higher in DML-B compared with the complex MRS medium. Growth of Lact. sakei CCUG 42687 and production of sakacin P in DML-B were also tested in still cultures in tubes at 4 and 37 °C. At 4 °C, a sakacin P concentration of 2·5 mg l ¹ was obtained with a cell dry mass of 0·25 g l ^1. No growth was obtained in DML-B at 37 °C. Growth was obtained in MRS at 37 °C but sakacin P production was negligible (less than 1 g l ¹).

Effect of pH and heat on the inactivation phase

Samples were collected, at a time near to maximum activity, from the Lact. sakei CCUG 42687 culture at 20 °C in the fermentor, with controlled pH 6·3 and without pH control (pH 5·1 at sampling). The samples were subjected to various treatments as shown in Table 3. As noted above, the decrease in activity in the fermentor was fastest at pH 6·3 ( Table 3). When a sample from a culture grown at controlled pH 6·3 was incubated further without pH control, the pH had decreased to 4·9 at end of the incubation and the decrease in sakacin P concentration was less than in the fermentor. Incubation of supernatant fluid from cultures grown at pH 6·3 led to a decrease in activity similar to that in the fermentor. Heat treatment of the supernatant fluid from a culture grown at controlled pH 6·3 resulted in a 50% loss of activity after 20 min. However, further incubation of heat-treated supernatant fluid did not lead to further loss in activity. The activity was fairly stable in all samples from a culture grown without pH control. After the inactivation phase, no sakacin P potentially adsorbed to cells could be released from cells by treatment with 100 mmol l ¹ NaCl at pH 2, according to Yang et al. (1992). The supernatant fluid from a culture grown at 20 °C with controlled pH 6·3 was tested for non-specific proteolytic activity using casein as a substrate, as described previously ( Nrs & Nissen-Meyer 1992). No proteolytic activity was detected.

Effects of medium components

Tween-80 stimulates production of several bacteriocins ( Parente & Hill 1992; Huot et al. 1996). To examine whether it has similar effects on sakacin P production, Lact. sakei CCUG 42687 was grown at 20 °C, in a Bioscreen C in DML-B, with Tween-80 concentrations of 0, 0·025, 0·055, 0·075, 0·11, 0·2, 0·3 and 0·5% (v/v). No growth was obtained without Tween-80 and 0·11% Tween-80 was required for optimal growth ( Fig. 2). The highest sakacin P activity was obtained with 0·3% Tween-80, and this concentration was chosen for the fermentor experiments and for further optimization in the Bioscreen C.

The effects of the concentrations of other compounds present in DML-B were tested by growing Lact. sakei CCUG 42687, at 20 and 30 °C, in eight different DML-B based defined media composed after a fractional factorial 2⁷⁴ experimental design with the seven variables amino acids, vitamins, bases, salts, Tween-80, glucose and succinate. This experimental design requires the variables to be present in two concentration levels, a 'low' and a 'high' level. The low level equals the concentrations in DML-B except 0·2% for Tween-80. The high level equals the DML-B concentration times three, except for double concentration of glucose and succinate and 0·3% for Tween-80. At 20 °C, no effects on sakacin P production were observed for any of the variables. At 30 °C, high levels of amino acids had a positive effect on both volumetric and specific sakacin P production (data not shown). The volumetric production of sakacin P at 30 °C was 40% higher in the rich medium with concentrations of all compounds at the high level, compared with DML-B (0·3% Tween-80).

The addition of sodium chloride and ethanol had negative effects on production of enterocin A and B, but for these bacteriocins, the production can be partly restored by the addition of the inducing peptide ( Nilsen et al. 1998). To investigate whether similar effects could be obtained for sakacin P, Lact. sakei CCUG 42687 was grown at 20 and 30 °C in a Bioscreen C in DML-B (0·3% Tween-80) and in DML-B with added sodium chloride, ethanol and a trace mineral solution (see MATERIALS and METHODS for composition and amount used). At 20 °C, addition of sodium chloride and ethanol reduced both growth and specific bacteriocin production, with an increased effect at higher concentrations ( Table 4). Addition of trace metals had a positive effect on growth, although sakacin P production was reduced by approximately 50%. At 30 °C, the effects of sodium chloride and trace metals were essentially the same. However, ethanol had a slightly positive effect on sakacin P production and a less negative effect on growth at this temperature. Sakacin P production was generally lower in all media at 30 °C compared with the corresponding media at 20 °C, except for the medium with 3% ethanol. No effect on any parameter was observed after adding synthetic inducing peptide (IP-673) at a final concentration of 10 ng ml ¹ to the media containing NaCl or ethanol (data not shown).

Growth and sakacin P production by other producers

Five other sakacin P producers were tested together with Lact. sakei CCUG 42687 for growth and production in DML-B and MRS without pH control in a Bioscreen C. All strains grew and produced bacteriocin in DML-B and MRS at 20 °C ( Table 5). Lactobacillus sakei VB286A grew poorly in DML-B, and specific production (defined for these five strains as sakacin P per O.D. 600 nm) was lower than in the other strains. However, in MRS, Lact. sakei VB286A was the strain with the highest volumetric and specific production of bacteriocin. For all strains, both growth and bacteriocin production were better in a defined medium where the start pH was adjusted by addition of KOH instead of NaOH, and generally, more bacteriocin was produced at 20 °C than at 30 °C (data not shown). Tween-80 was essential for growth and bacteriocin production of all strains. Lactobacillus sakei VB286A had a low requirement for Tween-80 compared with the other strains, and grew and produced sakacin P at a Tween-80 concentration as low as 0·01 g l ¹ (not shown).

Non-producing cells were obtained for all strains except for Lact. sakei VB286A, as described by Eijsink et al. (1996), by diluting culture grown at 30 °C 10⁶-fold. Production could be restored by the addition of synthetic IP-673. There were no differences in growth between producing and non-producing cultures at 30 °C. Non-producing cells were not obtained at 20 °C, even by 10⁹-fold dilution of the culture.

FIGURES

Fig. 1  Production of sakacin P for Lactobacillus sakei CCUG 42687 grown at pH 6·3 (a) and without pH co...

Fig. 2  Effect of Tween-80 on maximum sakacin P production () and maximum dry mass () of Lactobacillus ...

 
Table 1   Strains used in this study

DISCUSSION

All the tested Lact. sakei strains produced sakacin P in DML-B ( Table 5). Hence, there was no component present only in complex media which was essential for production. Production of bacteriocins in defined media has rarely been reported and this is the first report describing the production of a class II bacteriocin by a Lactobacillus species in a completely defined medium, DML-B. Many bacteriocins are likely to be produced in defined media but in order to obtain appreciable amounts, the composition of such media may require some optimization. Using a heterologous expression system in Lact. sakei Lb790 for production of the class II bacteriocins pediocin PA-1, piscicolin 61, sakacin A and sakacin P ( Axelsson et al. 1998), similar amounts of these bacteriocins were obtained in DML-B compared with MRS (Møretrø and Rosshaug, unpublished data). Thus, DML-B should be a suitable medium for the study of bacteriocin production in Lact. sakei and, possibly, other lactobacilli as well.

The specific production of sakacin P in DML-B was highest at low temperatures. This has previously been reported for sakacin P (bavaricin A) in complex medium ( Larsen et al. 1993). Other bacteriocins have also been reported to have optima for production at sub-optimal temperatures for growth ( Krier et al. 1998; Lejeune et al. 1998). The low production of sakacin P at 30 °C was not increased by addition of synthetic inducing peptide. Therefore, it was unlikely that the low production of sakacin P observed at this temperature was caused by low production of the inducing peptide. De Vuyst et al. (1996) suggested that production of bacteriocins is increased under unfavourable growth conditions, and that low growth rates increase production. Raising the temperature to 30 °C, and addition of trace metals, increased the growth rate but reduced the production of sakacin P, thus supporting this hypothesis. However, ethanol and sodium chloride had a negative effect on both growth rate and sakacin P production, showing that growth rate per se is not controlling production. It could be speculated that reduced growth rate, due to an increased energy demand (e.g. salt stress), may have a negative effect on sakacin P production, while reduced growth rate due to reduced rate of enzymatic reactions may be positive, as this could result in increased pools of essential metabolites. The fact that amino acids appeared to be a limiting factor in DML-B for sakacin P production at 30 °C, but not at 20 °C, could support this hypothesis. At high growth rates (30 °C), the amounts of amino acids available for bacteriocin synthesis could have diminished. The low production at high temperatures could also partly be explained by increased degradation/ inactivation of the bacteriocin at high temperatures, as reported by Leroy & De Vuyst (1999) for sakacin K.

The slower growth and sakacin P production observed when NaOH was used instead of KOH for pH adjustment could indicate that the cells were stressed by sodium ions, possibly the same effect as that seen upon addition of NaCl. The medium pH adjustment by NaOH, and the use of NaOH in some stock solutions, resulted in 7-8 g l ¹ of Na⁺ in the final DML medium, the same Na⁺ concentration as in 2% NaCl. Addition of inducing peptide restored some of the enterocin A and B activity in cultures where NaCl or ethanol was added ( Nilsen et al. 1998). In the present study, addition of inducing peptide could not reverse the effects on sakacin P production of NaCl or ethanol. The fact that the addition of synthetic inducing peptide had no effect on producing cultures shows that the induction of sakacin P production is an 'all or nothing' process, as suggested by Eijsink et al. (1996). This differs from the induction of enterocin A and B production, which is dependent on the dose of inducing peptide ( Nilsen et al. 1998).

Tded ( Nilsen et al. 1998). In the present study, addition of inducing peptide could not reverse the effects on sakacin P production of NaCl or ethanol. The fact that the addition of synthetic inducing peptide had no effect on producing cultures shows that the induction of sakacin P production is an 'all or nothing' process, as suggested by Eijsink et al. (1996). This differs from the induction of enterocin A and B production, which is dependent on the dose of inducing peptide ( Nilsen et al. 1998). .

The increase in sakacin P activity stopped when growth stopped, corresponding to the finding that mRNA transcripts of the sakacin P genes are present until growth stops ( Brurberg et al. 1997). The fate of bacteriocin activity in the post-exponential phases varies between different bacteriocins. For some bacteriocins, the activity decreases slightly ( Parente et al. 1997) whereas for others, there is an almost complete loss of activity ( Lejeune et al. 1998) or activity is stable ( Nilsen et al. 1998). The concentration of sakacin P decreased in the stationary phase, and a faster decrease was observed at controlled pH 6·3 than at uncontrolled pH ( Fig. 1a,b). High pH has been shown to promote the rate of decrease in concentration for other bacteriocins as well ( Kaiser & Montville 1993; Parente et al. 1997; Leroy & De Vuyst 1999). Adsorption to cells has been shown for many bacteriocins ( Yang et al. 1992; Parente & Ricciardi 1994) and has been cited as a major cause for the decrease in bacteriocin concentration in the late stages of growth and in the stationary phase ( Leroy & De Vuyst 1999). It is shown here that the decrease in sakacin P continued after the cells were removed ( Table 3) and in addition, that no sakacin P activity could be recovered after the cells had been subjected to a treatment previously shown to release other bacteriocins from cells ( Yang et al. 1992). This suggests that for sakacin P, the inactivation phase is probably not due to bacteriocin adsorption to cells. The significant decrease in concentration of sakacin P during heat treatment could indicate that some chemical reactions, e.g. oxidations, may take place. Worobo et al. (1994) also reported instability of carnobacteriocins at high pH and high temperatures.

No proteolytic activity on casein could be detected in the supernatant fluid, but as this is a relatively insensitive test, proteolytic degradation cannot be completely ruled out. After the initial decrease in activity, a heat-treated sample appeared to be stable. This could indicate that proteolytic degradation is a factor in the inactivation phase. Although it has been suggested that the presence of Tween-80 prevents aggregation of bacteriocin molecules in some cases ( Nissen-Meyer et al. 1992; Huot et al. 1996), aggregation of sakacin P molecules as another possible factor involved in the inactivation phase cannot be disregarded. When a sample was collected during exponential growth at pH 6·3 and the supernatant fluid was further incubated, a decrease in activity was observed (not shown). This could indicate that the factor(s) responsible for the inactivation phase also had a negative effect during actual growth, leading to lower net production at pH 6·3 compared with growth without pH control.

It is not clear what limited the growth of Lact. sakei CCUG 42687 in DML-B. Only marginally higher amounts of cell dry mass were obtained using controlled pH 6·3 compared with uncontrolled pH ( Table 2). In complex media with a surplus of all the medium components and controlled pH, accumulation of lactic acid will eventually stop growth. Normally, this will occur at higher concentrations of lactic acid than the concentration present in DML-B cultures when growth ceased. However, DML-B has a high osmolarity due to the high number of low molecular compounds, and it is possible that at this high osmolarity lactic acid can be inhibitory for growth at lower concentrations than in complex media.

The effects of low temperature, different Tween-80 concentrations and addition of KOH vs NaOH found for Lact. sakei CCUG 42687 were essentially all confirmed with other Lact. sakei strains producing sakacin P, indicating that these are general effects on the production of sakacin P in defined medium. There was a twofold variation within the group of six strains with respect to the production level of sakacin P ( Table 5). Preliminary data (A. Holck, L. Kröckel, F.K. Vogensen and K. Harmark, personal communication) suggest that all the sakacin P producers contain the same chromosomal gene cluster sakacin P (spp.) as strains Lb674 and LTH673, which have been thoroughly investigated ( Hühne et al. 1996; Brurberg et al. 1997). Variations between strains are therefore probably due to host factors other than the primary bacteriocin-related genes. Similar strain variations have been reported for other bacteriocins ( Yang & Ray 1994).

CONCLUDING REMARKS

The specific production of sakacin P in the defined medium, DML-B, reached levels that were equal to or higher than production in an optimized complex medium. However, as production seemed to be correlated with growth and higher cell dry mass was obtained in complex medium, higher volumetric production was obtained in the latter type of medium. The specific production of sakacin P was notably higher at temperatures lower than those required for optimal growth, leading also to higher volumetric production. Apart from Tween-80 and the level of amino acids (at 30 °C), no specific components in the medium were found to have a positive effect on sakacin P production. Further optimization of sakacin P production using these strains probably requires changes at the genetic level (e.g. gene dosage, strong promoters), with the aim of increasing specific production. Such studies are now being carried out in this laboratory.

This work was supported by grant no. 107897/120 from the Norwegian Research Council. The authors wish to thank Beate F. Hagen for the measurement of proteolytic activity, Tone Katla for the supply of purified sakacin P and Vincent G.H. Eijsink for the supply of synthetic IP-673. Lactobacillus sakei MI401, Lact. sakei VB286A, Lact. sakei LTH673 and Lact sakei CCUG 42687, 27 and Lb674 were kindly provided by Finn K. Vogensen, John Coventry, Ingolf F. Nes and Lothar Kröckel, respectively.

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