Abstracts. T. Moretro

A new, completely defined medium for meat lactobacilli

T. Møretrø, B. F. Hagen and L. Axelsson

ABSTRACT

A new defined medium (DML) for meat lactobacilli is presented. This succinate buffered medium with as few as 34 components supported good growth for 61 strains of typical meat lactobacilli, both isolated from meat and used as meat starters. For many of the strains tested, cell densities in DML were similar to those achieved in MRS medium. The high buffer capacity of DML and the balanced amino acid content allowed growth to continue to higher densities than eight other defined media. DML is compared with a previously published defined medium for Lactobacillus sake and shown to support higher cell densities of all strains tested. DML will allow a better determination of the nutritional requirements of different strains of meat lactobacilli as all strains can be tested under the same conditions, and will also be suitable for the study of growth kinetics and production of different metabolites.

Lactobacillus species are widely distributed in meat products, both as a part of the naturally occurring microflora (Frazier & Westhoff 1988) and as deliberately added starter cultures (Hammes & Knauf 1994). In meat they impart valuable properties such as acidification by production of lactic acid, inhibition of the competing flora by production of hydrogen peroxide, lactic acid and/or bacteriocins (De Vuyst & Vandamme 1994) and production of flavour precursors (Dainty & Blom 1995). It would be of value to isolate and purify the compounds imparting these properties for possible application in foods. To this end, it is necessary to study the physiological conditions most suited for their production. For examination of nutritional requirements a defined medium must be used as such a medium (i) provides reproducibility of chemical composition, (ii) avoids an unnecessary excess of nutrients, allowing the determination of nutrient limitation, (iii) meets the experimentally determined nutritional requirements of several strains and (iv) supports growth at a reasonably high rate (Foucaud et al. 1997).

Lactobacilli are extremely fastidious organisms with numerous growth requirements. They are usually grown in a complex medium such as MRS (De Man et al. 1960). However, several defined media are published for lactobacilli (Dunn et al. 1947; Koser & Thomas 1955; Morishita et al. 1974; Ledesma et al. 1977; McFeeters & Chen 1986; Montel & Labadie 1986; Grobben et al. 1995; Lauret et al. 1996); all are phosphate-buffered media. We found that they were not supporting good growth of meat lactobacilli, mainly because they did not fulfil the amino acid requirement of the lactobacilli and/or had too low a buffer capacity. In the development of the medium, strains of Lactobacillus sake and Lact. curvatus, which dominate among the lactobacilli isolated from meat (Champomier et al. 1987; Schillinger & Lücke 1987; Hugas et al. 1993), and Lact. pentosus and Lact. plantarum strains which are often used as starter cultures in the meat industry (Hammes & Knauf 1994), were used. Starting from a commonly used defined medium for lactobacilli (Ledesma et al. 1977), a general defined medium (DML), which was also shown to support good growth of the typical meat lactobacilli in general, was developed for these strains of Lactobacillus.

MATERIALS AND METHODS

Organisms

The following six Lactobacillus strains were used for development of the defined medium: Lact. curvatus NCFB 2739 (National Collection of Food Bacteria, Reading, UK), Lact. pentosus NCFB 363, Lact. plantarum NCFB 1752, Lact. plantarum NC8(Aukrust & Blom 1992), Lact. sake NCFB 2714 and Lact. sake Lb 16 (Holck et al. 1994). To test growth of various meat isolates on the developed defined medium and to compare it with other media for lactobacilli, 41 strains of Lact. sake and 17 strains of Lact. curvatus were cultivated at 30 °C on DML, MCD and MRS (Oxoid). All strains were isolated from meat products at MATFORSK and identified by DNA probe technology (Nissen & Dainty 1995). In addition, Lact. alimentarius ATCC 29643 (American Type Culture Collection, Rockville, MD, USA), Lact. farciminis ATCC 29644 and three Lactobacillus starter cultures used in the meat industry, FloraCarn L-2 (Chr. Hansen, Hørsholm, Denmark), Biocarna Ferment (Wiesby Biofermentation GmbH, Niebüll, Germany) and Müller 77 (Rudolf Müller & Co. GmbH, Pohlheim, Germany), as well as Pediococcus acidilactici PAC1·0 (Gonzalez & Kunka 1987), were tested under the same conditions. All strains were stored at - 80 °C in MRS medium supplemented with glycerol (18%).

Media

The defined media were prepared from stock solutions of chemicals of analytical grade stored at 4-6 °C, except for cysteine HCl which was added directly to the medium (Table 1). The stock solutions of glutamic acid, glutamine, guanine HCl, succinic acid and xanthine were stored at room temperature to prevent precipitation. pH and volume of the final medium were adjusted and medium was sterilized by filtration (0·22 m) into sterile bottles, and used within 2 d. Water used for medium preparation was purified by the MilliQ system (Millipore Incorporation). Development of an optimal minimal medium was based on a phosphate-buffered medium (DM) described by Ledesma et al. (1977) containing 17 amino acids, nine vitamins, six bases, three mineral salts, acetate, citrate, thioglycollate, Tween-80 and glucose. To avoid precipitation problems at high concentrations of phosphate, succinate, a reduced dicarboxylic acid which cannot be metabolized by lactobacilli, was used as buffer. Growth was also examined on MRS medium, which was sterilized by autoclaving (20 min at 115 °C).

Bioscreen C cultivation

For determination of nutritional requirements and optimal concentration of medium components for growth, cells were cultivated in Honeycomb microplates (Labsystems, Helsinki, Finland) in wells with 400 l medium at a start pH of 6·50 ± 0·05, at 30 °C, in a Bioscreen C (Labsystems), a microplate reader measuring growth kinetically. Growth was monitored by optical density (O.D.) at 600 nm every 10 min, after 20 s agitation. According to the manual, O.D. values of Bioscreen C are linear up to a value of 2·0 (Anonymous 1995). The high frequency of measurements permitted an accurate determination of the maximal growth rates (max). Cells used for inoculum were grown in defined medium and washed twice with medium without the components to be tested before inoculation of plates (1% v/v). All conditions were examined in triplicate. In some cultures, glucose strips (Diabur-Test 5000; Boeringer Mannheim) were used at the end of growth to determine if all glucose had been metabolized.

The following terms are used to determine the relationship between medium components and growth as determined by the single omission technique (Lauret et al. 1996): essential, no growth occurred without the component; not essential, growth occurred without the component; and inhibitory, addition of the component reduced maxOD600, max or both.

Lactobacillus sake is known to have the most fastidious nutritional requirements of all the lactobacilli (Lauret et al. 1996). Hence, a medium supporting growth of Lact. sake strains will permit growth of all other lactobacilli and therefore, only the requirements for these strains were completely elucidated. For Lact. sake, the effects of all non-essential components were determined by multiple omission and these components were divided into: stimulating, addition of the component increased the maximum optical density at 600 nm (maxOD600), maximum growth rate (max) or both; unnecessary, no effect on maxOD600 or max if the component was removed.

Fermentor cultivation

Lactobacillus plantarum NCFB 1752 and Lact. sake NCFB 2714 were grown in fermentors (2 l) at 30 °C on DM1 (Table 1) with 20 mmol l¹ succinate as buffer. The pH was monitored and maintained at 6·0 by the addition of NaOH. The fermentors were inoculated (1% v/v) with an overnight culture cultivated on the same medium. Optical density at 600 nm was measured with a Shimadzu UV160 A spectrophotometer. Concentration of glucose and products in the fermentation broth was determined in supernatant fluids (passed through 0·2 m filters) by HPLC as described by Øyaas et al. (1995). To determine the concentration of amino acids in the fermentation broth, 0·8 ml supernatant fluid (filtered as above) was analysed according to Skjerdal et al. (1996) with one modification, that the buffer used was 0·02 mmol l¹ Na-acetate pH 5·9.

RESULTS

Choice of buffer and buffer concentration

MES, potassium phosphate and succinate were tested for use as buffer. The use of phosphate resulted in precipitation of the medium and hence, this buffer was not suitable. Similarly, MES buffer was found to have an inadequate buffer capacity as the pH in the medium at the end of fermentation was as low as 3·9. Succinate, however, supported good growth and was selected for further studies. When different concentrations of succinate were tested, 100 mmol l¹ succinate resulted in higher maxOD600 compared with 50 and 70 mmol l¹ in cultures with Lact. sake NCFB 2714 and Lact. curvatus NCFB 2739 (data not shown). Succinate concentrations of 150 or 200 mmol l¹ resulted in a longer lag period before growth occurred. All batch experiments were performed with 100 mmol l¹ succinate as buffer.

Determination of nutritional requirements

No growth of Lact. sake Lb16 was obtained in DM with 17 amino acids. Therefore, aspartic acid, glutamine and glycine were added to the DM which resulted in growth of all strains. The concentration of arginine and tyrosine in DM was low compared with the other amino acids, resulting in poor growth. To compensate for this, 10 times higher concentrations of arginine and tyrosine were used in further optimization of the medium.

The nutritional requirements varied considerably between strains and are presented in Tables 2 and 3. Isoleucine, leucine and valine were essential for growth of all strains. Formation of cell clusters in medium without citrate as described previously (Ledesma et al. 1977) was not observed by phase contrast microscopy. Addition of acetate (1%) reduced max of Lact. sake NCFB 2714 and maxOD600 of Lact. curvatus NCFB 2739, but increased maxOD600 of Lact. sake NCFB 2714, Lact. pentosus NCFB 363 and Lact. plantarum NC8. When the three bases cytidine, 2-deoxyguanosine and thymidine were replaced by xanthine, maxOD600 of Lact. pentosus NCFB 363 decreased slightly. However, an increase in the growth of Lact. sake Lb 16 was observed. No strains were affected when guanine was removed from the medium. All strains needed folic acid, nicotinic acid, pantothenic acid and pyridoxal HCl for optimal growth.

Determination of a minimal medium (DM1)

To develop a minimal medium, components neither essential nor stimulating for growth of any strain as determined by the single omission technique were removed simultaneously. No growth was observed when only these components were added. However, acetate, citrate, ferrous sulphate, thioglycollate, glutamic acid, aspartic acid, guanine HCl, vitamin B12 and p-aminobenzoic acid could be removed simultaneously, and the three bases cytidine, 2-deoxyguanosine and thymidine could be replaced with xanthine without reducing maxOD600 or max of any strain by more than 10%, resulting in a minimal medium with 34 components (DM1). The composition of DM1 and growth rates of the different strains on DM1 are presented in Tables 1 and 4, respectively.

HPLC analyses revealed that 97·5% of the products from both the fermentations of Lact. sake NCFB 2714 and Lact. plantarum NCFB 1752 were lactic acid. They also confirmed that succinate was not metabolized during growth (data not shown).

Development of a balanced minimal medium (DML)

Experiments were performed to adjust the concentration of components in DM1 to obtain a more balanced medium in which the concentrations reflected the actual need of each component to a greater extent. Amino acid analysis of fermentation broth of Lact. plantarum NCFB 1752 and Lact. sake NCFB 2714 showed an excess of alanine, methionine, histidine and serine and low concentrations of asparagine, lysine, glycine, tryptophane, valine and glutamine. Reduction of the concentrations of vitamins by 20% reduced the maxOD600 of Lact. plantarum NC8 slightly while the other strains could sustain a reduction of 40% or more without growth being reduced. No effect on growth was observed when the concentration of bases was reduced by 50%. MaxOD600 of the Lact. sake strains increased when the phosphate concentration was increased to 1 g l¹. Growth of Lact. sake NCFB 2714 was slightly inhibited by 0·11% Tween-80; the other strain needed 0·055% Tween-80 for good growth. Higher concentrations of Tween-80 did not improve growth. Reduction of glucose concentration from 2 to 1% did not affect growth but a further reduction to 0·8% slightly reduced the maxOD600 of the Lact. plantarum strains. From these observations, the concentrations of components in DM1 were adjusted, resulting in a new balanced minimal medium, DML, for which the composition is given in Table 1.

Growth characteristics in DML

No differences in morphology could be observed by phase contrast microscopy between cells grown in DML and MRS. The max and maxOD600 on MRS, MCD, DM1 and DML are shown in Table 4. All six strains obtained 1·5-2·5 times the maxOD600 on DML, compared to growth on MCD, a defined medium described for Lact. sake (Lauret et al. 1996). Experiments revealed that the different growth in the two media was due to lower buffer capacity of MCD and different concentrations of amino acids and bases between the media (data not shown). In DML, the pH of all strains was measured to 4·7 after all glucose was metabolized, except for Lact. sake Lb 16 where not all glucose was metabolized and end pH was 5·0. In MCD, the end pH values were between 3·4 and 3·8, except 4·8 for Lact. sake Lb 16.

Growth of meat isolates in DML

As shown in Table 5, DML supported good growth for the isolates of Lact. sake, Lact. curvatus and the starter culture strains. Lactobacillus alimentarius ATCC 29643 and Lact. farciminis ATCC 29644 grew slowly and obtained low maxOD600 in both MCD and DML. Pediococcus acidilactici PAC1·0 grew poorly in DML after a 20 h lag.

TABLES

Table 1 Composition of DM1 and DML

Table 2 Amino acid requirements for growth in DM

Table 3 Requirements of vitamins, bases and salts for growth in DM

Table 4 Maximum growth rates (h¹) and maxOD600 (Bioscreen values) in different media at 30 °C

Table 5 Growth characteristics of 61 lactobacilli of meat origin in MRS, MCD and DML at 30 °C

DISCUSSION

A buffer to be used in a defined medium must (i) have a high buffering capacity in the relevant pH area, (ii) not be metabolized, (iii) not give precipitation of medium components and (iv) be relatively cheap. Succinic acid fulfils all of these criteria, being a reduced dicarboxylic acid with pKa_{1
=} 4·21 and pKa_{2 =} 5·64. Most published media for lactobacilli contain acetate but in our medium, the response to this compound was ambiguous, as some strains were inhibited and others were stimulated by the compound. Removing acetate completely still resulted in good growth of all strains. We hypothesize that the positive effect of acetate on maxOD600 of three of the strains is a buffer effect, and that the compound is necessary in phosphate-buffered media because of the low buffering capacity of these media.

The six strains tested could be divided into two groups based on different requirements of growth: Lact. curvatus NCFB 2739, Lact. sake Lb 16 and Lact. sake NCFB 2714 formed a group with complex growth requirements; and Lact. pentosus NCFB 363, Lact. plantarum NCFB 1752 and Lact. plantarum NC8 formed a group with more simple requirements. This grouping reflects the phylogenetic relationship between the species (Collins et al. 1991).

No requirement for glutamic acid was observed for any of the six lactobacilli tested. This is contradictory to the findings for 28 oral lactobacilli by Koser & Thomas (1955) and for 24 Lact. plantarum strains by Ruiz-Barba & Jimenéz-Damp;;acute;az (1994) who found that glutamic acid was essential for growth of these strains. However, those experiments were performed in medium without glutamine. We found that for the Lact. sake strains, glutamate could be replaced by glutamine but not vice versa, indicating that glutamine is essential for growth and that glutamate is unnecessary if glutamine is present. All amino acids except aspartic and glutamic acid were necessary for rapid growth of Lact. sake Lb 16. Good growth of Lact. sake NCFB 2714 was also obtained when alanine was omitted together with aspartic and glutamic acid. When cysteine was omitted in addition to aspartic and glutamic acid, lower growth was observed. When any of the other amino acids were omitted, only poor growth of the Lact. sake strains was obtained. Lauret et al. (1996), also in accordance with our results, found that only aspartic and glutamic acid could be omitted to sustain good growth of all nine strains of Lact. sake tested.

All strains tested had an absolute requirement for the branched amino acids, isoleucine, leucine and valine, indicating that mutations in the genes coding for common enzymes catalysing the synthesis of these amino acids have occurred (Morishita et al. 1981; Gottschalk 1986). These results correspond well with findings for Lact. sake by Montel & Labadie (1986) and Lauret et al. (1996) and for Lact. plantarum by Ruiz-Barba & Jiménez-Damp;;acute;az (1994). However, Ledesma et al. (1977) and Koser & Thomas (1955) found that while leucine and valine were essential for Lact. plantarum, isoleucine was only stimulatory.

The information obtained regarding consumption of amino acids of one strain could not be directly used to adjust the concentration of amino acids in a common medium because the relative consumption of amino acids varied between strains. A common medium must have an excess of all amino acids as different strains need different amounts of the various amino acids.

As described for Lactococcus lactis by Cocaign-Bousquet et al. (1995), the removal of ammonium salt from the medium did not affect the growth negatively. In fact ammonium chloride was slightly inhibitory to the Lact. plantarum strains and Lact. sake NCFB 2714. This illustrates that the amino acid and nucleotide content of the medium satisfies the nitrogen requirements for biomass synthesis.

The requirement of riboflavin and biotin separated the six strains into the two groups described earlier (see above). Riboflavin was essential for growth, but biotin had no effect on growth of Lact. curvatus NCFB 2739 and the Lact. sake strains, while the Lact. plantarum strains and Lact. pentosus NCFB 363 had no requirement for riboflavin but were stimulated by biotin. Lactobacillus plantarum was earlier reported not to require pyridoxal or folic acid (Rogosa et al. 1961), in contrast to the results presented here. Lactobacillus sake had no requirement for p-aminobenzoic acid, but thiamin stimulated growth (10-15%). This is in contrast to results previously described by Lauret et al. (1996) but in agreement with results reported by others (Rogosa et al. 1961). The differences observed in the growth requirements of the same species at different laboratories could be due to differences in the test medium. Screening of various strains for growth requirements must be performed on the same medium.

Lactobacillus plantarum had the ability to grow without bases and thus to synthezise purines and pyrimidines. However, increased growth was observed when cells were grown with one base, no matter which. Lactobacillus sake needed both purines and pyrimidines for growth, in agreement with results previously reported (Lauret et al. 1996).

Tween-80 had a different effect on the Lact. sake strains, stimulating Lact. sake Lb 16 and inhibiting Lact. sake NCFB 2714. An inhibitory effect of 0·11% Tween-80 on Lact. sake has not previously been reported.

DML did not support good growth of Lact. alimentarius ATCC 29643 and Lact. farciminis ATCC 29644. These strains are both isolated from meat (Kandler & Weiss 1986) and define a distinct group as determined by 16S rRNA sequencing (Schleifer & Ludwig 1995). Although DML did not support good growth of these lactobacilli, it supported good growth of the lactobacilli that are most commonly associated with meat.

The new, completely defined medium for meat lactobacilli (DML) described here contains only 34 components, making it less complex than previously described media as well as supporting better growth. This indicates that the medium will be most suitable for physiological studies of lactobacilli commonly associated with meat, and perhaps many other lactobacilli as well.

ACKNOWLEDGEMENTS

This work was supported by grant no. 107897/120 from the Norwegian Research Council and in part by AAIR project no. 94-1517. The fermentations and the HPLC analyses were performed at SINTEF Applied Chemistry, Trondheim, Norway. The authors thank Ivar Storrø at SINTEF for helpful discussions, the staff at SINTEF for technical assistance and especially Kathinka Q. Lystad for performing the HPLC analyses. They also wish to thank Inger Line Hamre and Hege Lande for excellent technical assistance. Lactobacillus sake Lb 16 was kindly supplied by Lothar Kröckel. The meat isolates of Lact. sake and Lact. curvatus were kindly supplied by Hilde Nissen, MATFORSK.

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