INTRODUCTION

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The thermophilic lactic acid bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are important in the dairy industry, since they are used in the fermentation of milk to form yogurt. Several strains which are capable of forming exopolysaccharides (EPS), which give a higher viscosity and a thicker texture to the product, have been isolated and analyzed (1-3, 9, 16, 24, 26). Since the addition of stabilizers of animal or plant origin to natural yogurts is prohibited in France and The Netherlands and since there is a growing popularity for food products without additions, the utilization of EPS-producing lactic acid bacteria has gained more popularity.

For optimal growth, lactic acid bacteria require very complex media like milk, whey ultrafiltrate, or complex synthetic media such as MRS broth (6) and M17 broth (25). However, for the investigation of the physiological background of EPS production in lactic acid bacteria, a chemically defined medium is required, and the exact nutritional requirements of the lactic acid bacteria remain unknown when complex media are used. A chemically defined medium containing a carbohydrate source, mineral salts, amino acids, vitamins, and nucleic acid bases is therefore more suitable to investigate the influence of nutrients on the growth, the metabolic pathways, and the synthesis of EPS in these bacteria. Recently, a simple synthetic growth medium for some lactic acid bacteria was formulated. These bacteria belong to the genera Lactococcus (5, 13), Streptococcus (21), and Leuconostoc (8). However, for L. delbrueckii subsp. bulgaricus, the minimal requirements are still unknown and the effect of medium composition other than the carbohydrate source on EPS production has not yet been elucidated. A synthetic culture medium in which several lactobacilli were able to grow has been formulated (15), but the growth requirements of L. delbrueckii subsp. bulgaricus were only partially investigated.

The use of chemically defined media is particularly important when the quantitative and qualitative production of EPS by lactic acid bacteria and the regulation of EPS synthesis are being studied. Previously, we used a chemically defined medium which contained a carbohydrate source, mineral salts, amino acids, nucleic acid bases and vitamins (10). In the present work we have simplified and optimized this medium for growth and EPS production by using the technique of the omission of a single medium component, which is generally used to investigate the requirements for medium components (22). By using this technique, we formulated a simplified chemically defined medium in which L. delbrueckii subsp. bulgaricus NCFB 2772 was able to produce increased levels of EPS. Previously, it has been possible to study only the effect of the carbohydrate source. The strain metabolized only four carbohydrates, glucose, lactose, fructose, and mannose, and the production of EPS was dependent on the carbohydrate source. The strain produced considerably larger amounts of EPS when grown on glucose or lactose than when grown on fructose to equal cell densities. Compared with growth on the other carbohydrate sources, growth on mannose led to much lower levels of growth and EPS production (10, 11).

A defined medium is also very useful in the investigation of the composition of the EPS produced. It was found that complex media like MRS strongly interfere with the isolation procedure of macromolecules like EPS (9) and that the exact amount and composition of the EPS produced are unclear when these complex media are used. In the chemically defined medium used in our previous work, L. delbrueckii subsp. bulgaricus NCFB 2772 grown on glucose produced two EPS fractions concurrently and in almost equal amounts; these two fractions differed in molecular weight and composition of the EPS repeating unit. The high-molecular-weight fraction (molecular weight, 1.7 × 10⁶) contained glucose, galactose, and rhamnose at a ratio of 1:5:1, and the low-molecular-weight fraction (molecular weight, 4 × 10⁴) was composed of glucose, galactose, and rhamnose at a ratio of 1:11:0.4. When fructose was used as the sole carbohydrate source, a low-molecular-weight EPS fraction was the major product (12). It is possible that not only the carbohydrate source but also other medium components affect the composition of the EPS.

In this work, the effect of the omission of medium components on the growth of and EPS production by L. delbrueckii subsp. bulgaricus NCFB 2772 was investigated and a simplified defined medium, in which the strain produced a considerably larger amount of EPS, was formulated.

	MATERIALS AND METHODS

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Bacterial strain and culture medium. L. delbrueckii subsp. bulgaricus NCFB 2772 was obtained from the National Collection of Food Bacteria (Reading, United Kingdom). Cultures were stored at 80°C in MRS medium (6) plus 15% glycerol until required and were reactivated in MRS medium at 37°C and initial pH 6.0 for 16 h. Growth experiments were performed in a chemically defined medium in which growth of and EPS production by the strain were shown previously (10); this medium contained 7.4 mM KH₂PO₄, 14.1 mM Na₂HPO₄, 4.3 mM citric acid, 82.3 mM sodium acetate, 18.7 mM NH₄Cl, 0.8 mM MgSO₄ · 7H₂O, 0.3 mM MnCl₂ · 4H₂O, 1 ml of Tween 80 per liter, 0.07 mM adenine, 0.07 mM guanine, 0.09 mM uracil, 0.07 mM xanthine, 1.1 mM L-alanine, 0.5 mM L-arginine, 0.7 mM L-asparagine, 0.8 mM L-aspartic acid, 2.5 mM L-cysteine, 0.7 mM L-glutamine, 0.7 mM L-glutamic acid, 1.3 mM glycine, 0.6 mM L-histidine, 0.8 mM L-isoleucine, 0.8 mM L-leucine, 0.6 mM L-lysine hydrochloride, 0.7 mM L-methionine, 0.6 mM L-phenylalanine, 0.9 mM L-proline, 1.0 mM L-serine, 0.8 mM L-threonine, 0.5 mM L-tryptophan, 0.6 mM L-tyrosine, 0.9 mM L-valine, 1 ml of a trace element solution (81.1 mM HCl, 0.8 mM CoCl₂ · 6H₂O, 0.01 mM CuCl₂ · 2H₂O, 7.5 mM FeCl₂ · 4H₂O, 0.1 mM H₃BO₃, 0.2 mM Na₂MoO₄ · 2H₂O, 0.1 mM NiCl₂, 0.5 mM ZnCl₂) per liter, and 1 ml of a vitamin solution (0.7 mM p-aminobenzoic acid, 0.08 mM biotin, 0.1 mM folic acid, 0.24 mM lipoic acid, 1.6 mM nicotinic acid, 0.4 mM calcium pantothenate, 2.1 mM pyridoxamine, 0.3 mM pyridoxine, 0.27 mM riboflavin, 0.6 mM thiamine, 0.07 mM vitamin B₁₂) per liter. Glucose was used as the carbohydrate source at an initial concentration of 111 mM unless otherwise stated. The defined medium was sterilized by being passed through a 0.2-µm-pore-size sterile filter (Schleicher & Schuell, Dassel, Germany, or Gelman Sciences, Ann Arbor, Mich.).

Growth, EPS isolation, and EPS characterization. Batch cultivations were performed in unshaken, nitrogen-flushed sealed bottles (volume, 115 ml) containing 50 ml of defined medium at 37°C and initial pH 6.0, unless otherwise stated. Growth was monitored after 48 h by measurement of the optical density at 600 nm (OD₆₀₀). EPS were isolated as described previously (10), the total carbohydrate content of the isolated EPS was measured by the phenol-sulfuric acid method of Dubois et al. (7), and the sugar composition of the EPS was determined after hydrolysis by high-performance liquid chromatography (10). The molecular weight of the EPS was measured by high-performance size exclusion chromatography as described previously (12).

Growth experiments to evaluate nutritional requirements. To identify the nutritional requirements of L. delbrueckii subsp. bulgaricus NCFB 2772, the strain was grown in batch cultures with defined medium and the technique of omission of one of the medium components was used. Before being used for growth experiments, cells grown in defined medium were centrifuged (15,000 × g for 10 min at 4°C), washed twice in 20 mM phosphate buffer (pH 6.0), resuspended in the same buffer, and finally added to medium from which one or more of the medium components had been omitted. In these test media, the strain was subcultured three times in succession by adding 0.5 ml of a culture incubated for 48 h to 50 ml of fresh medium. After this, the OD₆₀₀ of the third subcultures were compared with the OD₆₀₀ of a positive control without nutrient omissions. These experiments were performed in triplicate.

Simplified defined medium for growth and EPS production. Based on the results of the analyses of the nutritional requirements of L. delbrueckii subsp. bulgaricus NCFB 2772, a simplified medium containing all the components necessary for growth and EPS production by the strain was synthesized. Growth and EPS production were investigated in a pH-controlled batch culture, using a glass fermentor (Applikon, Schiedam, The Netherlands) with a working volume of 1.5 liters at 40°C, pH 6.1 ± 0.1, 50 rpm, and on N₂ atmosphere. Glucose was used as the carbohydrate source at an initial concentration of 166 mM. Growth and EPS production by L. delbrueckii subsp. bulgaricus in the simplified medium were compared with growth and EPS production under the same conditions in the complete medium.

	RESULTS

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Gas requirement. The growth of L. delbrueckii subsp. bulgaricus NCFB 2772 was not significantly affected by the composition of the gas phase. Growth and EPS production under a nitrogen atmosphere, an N₂-CO₂ atmosphere with different ratios, and air were comparable. Growth was lower in the presence of 100% O₂ in shaken sealed bottles (volume, 115 ml) with 25 ml of medium in a horizontally shaking water bath at 37°C, resulting in a final OD₆₀₀ of 0.9; however, glucose fermentation pattern and EPS production related to growth were not affected.

Nutritional requirements for growth. It appeared that most amino acids were essential for the growth of L. delbrueckii subsp. bulgaricus NCFB 2772. The single omission of aspartic acid, glutamic acid, or glycine affected growth only slightly, whereas the omission of asparagine, glutamine, or threonine resulted in a stronger reduction of growth (Table 1). No growth was observed when any of the other amino acids was omitted from the growth medium. The strain did not grow when only the essential amino acids were present. When a growth medium without aspartic acid, glutamic acid, and glycine was used, almost no growth was observed after 48 h but the OD₆₀₀ of the culture increased to 0.9 after 96 h. Omissions of single or multiple amino acids did not affect the amount of EPS relative to the cell density or the sugar composition of the EPS (data not shown). The same was observed when guanine or xanthine was omitted from the growth medium. On the other hand, the omission of uracil prevented growth completely and the omission of adenine resulted in a reduction of the OD₆₀₀ and EPS production by approximately 50%.

Enhanced EPS production by multiple-vitamin omission. A single omission of the nonessential vitamins did not change the specific EPS production, but when L. delbrueckii subsp. bulgaricus was grown in a medium containing no vitamins except riboflavin, calcium pantothenate, and nicotinic acid, the OD₆₀₀ of the cultures was much lower after 48 h whereas the specific EPS production increased significantly (Table 2). Omission of single vitamins did not change the monomeric sugar composition of the EPS. Omission of multiple vitamins resulted in EPS with a slightly lower content of galactose monomer.

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View larger version (17K): [in this window] [in a new window]   FIG. 1.   Growth of and EPS production by L. delbrueckii subsp. bulgaricus in pH-controlled batch cultures at 40°C and pH 6.0 ± 0.1 in complete defined medium (A) and simplified medium (B). Each value represents the mean of duplicate measurements and varied from the mean by not more than 10%. , OD600; , EPS production.

	DISCUSSION

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L. delbrueckii subsp. bulgaricus NCFB 2772 produces EPS when grown in a defined medium supplemented with a carbohydrate source. The amount of the EPS produced was thought to be affected only by the growth temperature or the carbohydrate source, and it was believed that EPS production was strictly coupled to growth (10, 11). In this work, the nutritional requirements of the strain were investigated to determine whether other nutrients can affect the growth of and EPS production by the strain.

Use of the technique of single-component omission gave a clear indication of the nutrient requirement for the growth of L. delbrueckii subsp. bulgaricus NCFB 2772. Nevertheless, the strain did not grow if all the components which were individually not required were omitted. For instance, a single omission of either aspartic acid, glutamic acid, or glycine resulted in good growth of the culture, whereas growth was poor when these three amino acids were all absent. Since nitrogen metabolism in this strain is not well understood yet, it is difficult to predict the combined effect of amino acids in the growth medium. Compared to other lactic acid bacteria such as Lactobacillus plantarum (23), Lactococcus lactis (13), and Streptococcus thermophilus (21), L. delbrueckii subsp. bulgaricus NCFB 2772 required more amino acids. In contrast to the findings of Ledesma et al. (15), who proposed that a requirement for glutamic acid, valine, and leucine is a taxonomic criterion for the genus Lactobacillus, L. delbrueckii subsp. bulgaricus NCFB 2772 grew well in the absence of glutamic acid. The omission of single or multiple amino acids had no effect on the production of EPS relative to cell growth. Regarding the vitamin requirements, it appeared that only nicotinic acid, calcium pantothenate, and riboflavin were essential for growth. Ledesma et al. (15) found that L. delbrueckii subsp. bulgaricus ATCC 9224 required nicotinic acid and calcium pantothenate for growth, since these vitamins are involved in coenzyme biosynthesis by lactobacilli. Riboflavin, a component of flavin coenzymes, appears to be essential for the growth of lactic acid bacteria (5).

Multiple omission of all nonessential vitamins reduced the growth of the strain but, surprisingly and in contrast to the results obtained for the amino acids, affected the production of EPS strongly. For the first time, it was observed that EPS production in a lactic acid bacterium was affected by a growth factor other than the carbohydrate source, temperature, or pH and that the regulation of the EPS production beyond the stationary growth phase was influenced by the medium composition. Up to now, nothing was known about the relationship between the vitamin requirement and EPS production in lactic acid bacteria. Cerning et al. (4) and Kojic et al. (14) found that L. casei CG11 produces EPS when grown in a basal minimal medium (19) containing only folic acid, nicotinic acid, calcium pantothenate, pyridoxine, and riboflavin. The EPS production by L. casei CG11 continued beyond the stationary growth phase. L. delbrueckii subsp. bulgaricus CRL 420 also produced some EPS after growth had ceased (16), but this organism was grown in a complex medium, so that the influence of vitamins on the EPS production by this strain is unknown. We demonstrated that only the total amount of EPS produced was affected by the omission of multiple vitamins and that the ratio of the high-molecular-weight fraction and the low-molecular-weight fraction of the EPS was not affected, as was found when fructose instead of glucose was used as the carbohydrate source (12). This means that the omission of multiple vitamins affected the production of both the high-molecular-weight fraction and the low-molecular-weight fraction, in contrast to the carbohydrate source, whose effect was mainly on the production of the high-molecular-weight EPS fraction (12).

It was found that EPS production under controlled pH was significantly higher than in acidified batch cultures (Table 2; Fig. 1). This was also observed in our previous work (10), and it was found that L. casei also produced considerably more EPS when grown under constant pH than without pH control (20). Higher EPS production as a result of maintaining the pH of the culture medium at a constant value was also reported for Xanthomonas campestris (18) and Pseudomonas sp. strain EPS-5028 (17).

In conclusion, a simplified defined medium was formulated in which L. delbrueckii subsp. bulgaricus NCFB 2772 grew less well than in the complete defined medium but produced about twice as much EPS and in which the EPS production continued strongly after cell growth had ceased. The single-omission technique appeared to be indirectly successful. By using this technique, the nutritional requirements were found and omission of multiple vitamins resulted in enhanced EPS production, although it is unclear which factor is responsible for this enhancement. More investigations on the physiological effects of vitamins on the production of EPS will be necessary for a better understanding of the mechanisms of EPS production.