J. T. M. Wouters

Identification, Cloning, and Characterization of a Lactococcus lactis Branched-Chain α-Keto Acid Decarboxylase Involved in Flavor Formation

ABSTRACT The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain α-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3′ terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain α-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.

Enhanced flavour formation by combination of selected lactococci from industrial and artisanal origin with focus on completion of a metabolic pathway.

E.H.E. AYAD, A. VERHEUL, W.J.M. ENGELS, J.T.M. WOUTERS AND G. SMIT. 2001. Combinations of lactococcal strains from various origins with divers properties were developed as new starters for new dairy products. Flavour formation by such tailor‐made cultures was studied. In some cases, a strongly enhanced flavour was observed. For instance, the combination of B1157 and SK110 strains in milk resulted in a very strong chocolate‐like flavour. B1157 produces only a moderate chocolate‐like flavour, whereas SK110 alone fails to produce this flavour. Headspace gas chromatography results corroborate the organoleptic evaluations. High levels of branched‐chain aldehydes were found when B1157 and SK110 were grown together. The enzyme activities involved in this pathway were studied; both strains contain transaminase activity. Although B1157 had a very high amino acid decarboxylating activity, its release of amino acids from milk protein was limited. SK110 was strongly limited in decarboxylating activity, although this strain is very active in proteolysis. By combining these strains, the substrates released by SK110 can directly be used by the other strain, resulting in the completion of the whole flavour‐formation pathway. This opens new avenues for the preparation of tailor‐made cultures.

Persistence of the pBR 322 plasmid in Escherichia coli K 12 grown in chemostat cultures

Populations of a Escherichia coli K 12 strain, containing the vector plasmid p BR 322, were grown in chemostat culture under glucose- and phosphatelimited conditions. Resistance to tetracycline and ampicillin were lost after prolonged cultivation, resulting in the production of apparent plasmid-free populations which were more competitive than the original population. This competitiveness between plasmid-free and plasmid-containing populations was greatest in environments where the nutrient restriction was severe. Also during sequential subcultivation in batch cultures loss of plasmid was observed.

Pyrolysis Mass Spectrometry of Whole Cells, Cell Walls and Isolated Cell Wall Polymers of Bacillus subtilis var. niger WM

SUMMARY: Bacillus subtilis var. niger WM was grown in continuous culture under phosphate-limited and under magnesium-limited conditions. Whole cells, cell walls and the isolated wall polymers peptidoglycan, teichoic acid and teichuronic acid were analysed by Curie-point pyrolysis mass spectrometry. Characteristic ion peaks for the wall polymers were established and facilitated the interpretation of the mass pyrograms of walls and whole cells. The mass pyrograms of magnesium-limited cells showed the characteristic peaks for protein, peptidoglycan and teichoic acid. Phosphate-limited cells showed peaks characteristic of teichuronic acid instead of teichoic acid.

Influence of nutrient limitation and growth rate on the outer membrane proteins of Klebsiella aerogenes NCTC 418

Four major proteins with molecular weights of 78 000, 37 000, 34 000 and 20 000 were present in the envelope of Klebsiella aerogenes when cultured at a high specific growth rate. However, at lower growth rates, the protein content and composition of the envelope depended on the imposed nutrient limitation. Under potassium-, carbon-, sulphur- and phosphorus-limited conditions, derepression of synthesis of limitation-specific proteins was observed, their apparent molecular weights being 90 000, 48 000, 41 000 and 36 000, respectively. Nitrogen-limited cells had no additional proteins. For a particular limiting nutrient, expression of the limitation-specific proteins was independent of the chemical or physical form in which the nutrient was supplied. Under potassium or sulphur limitation the specific proteins were present maximally at the lowest imposed growth rate, whereas under carbon limitation a maximum expression of these proteins was found at moderate growth rates. It is concluded that limitation-specific proteins which are associated with the outer membrane function in the uptake of limiting nutrients or, possibly, limitation-releasing compounds.

Expression and loss of the pBR322 plasmid in Klebsiella aerogenes NCTC 418, grown in chemostat culture

Klebsiella aerogenes harbouring the plasmid pBR322 was grown in continuous culture at various growth rates under glucose, phosphate or ammonia limitation. With tetracycline in the medium, the maximum culture β-lactamase activity was found at the higher growth rates. When tetracycline was absent, loss of resistance to the drug occurred. Concomitant with the occurrence of drug-sensitive cells, the culture β-lactamase activity decreased. At the higher growth rates the enzyme activity decreased at a slightly higher rate than did the resistance to tetracycline. From this it was concluded that the β-lactamase activity per mg cellular dry weight of the drug-resistant fraction of the population was still decreasing during the appearance of drug-sensitive cells. At the higher growth rates, this decrease was independent of the nutrient that was growth-limiting.

Formation and Regeneration of Geotrichum candidum Protoplasts

Summary: The formation of protoplasts from Geotrichum candidum, by means of a lytic enzyme complex of Streptomyces satsumaensis, was considerably stimulated by adding thiols, including dithiothreitol, 2-mercaptoethanol and cysteamine, to the incubation medium. Treatment of the mycelium with these compounds before incubation with lytic enzymealso enhanced protoplast formation, dithiothreitol giving the most rapid release of protoplasts. The same stimulating effect could be obtained with a proteolytic enzyme. These results suggest the presence of proteinaceous material in the outer layers of the hyphal wall. Geotrichum candidum protoplasts regenerated in liquid as well as solid media if a suitable osmotic stabilizer was present. Up to 20% of the protoplasts could grow into new mycelium.

The effect of tweens on the lipolytic activity ofGeotrichum candidum

Geotrichum candidum produces an extracellular lipase in different media provided that the pH does not decrease too much during growth. The enzyme has a particular substrate specificity; only esters of unsaturated fatty acids which are insoluble in water, are hydrolysed. Although the lipase fails to hydrolyse tweens, the latter stimulate its production but at the same time tweens and other surface-active compounds inhibit the lipolytic action of the enzyme.

Loss of phage resistance encoded by plasmid pSK112 in chemostat cultures of Lactococcus lactis ssp. cremoris SK110.

In cultures of L. lactis ssp. cremoris SK110, phage SK11G-resistant through the presence of pSK112, phage-sensitive variants segregated spontaneously that lacked the plasmid. In overnight batch culture these comprised up to 1% of the total population. Upon prolonged incubation in chemostat culture, a further loss of resistance was observed after a lag period. At high growth rates (0.7 h-1) this period amounted to approximately 35 generations, whereas cultures grown at rates of 0.4 and 0.1 h-1 remained resistant for 55 and 70 generations, respectively. At average-to-high growth rate, characteristics of the partially mixed populations that evolved were comparable to those of pure cultures of L. lactis ssp. cremoris SK110. However, in the culture fluid of the mixed populations that occurred at growth rate 0.1 h-1, higher acetate and formate concentrations were found than in the fluid of pure cultures of L. lactis ssp. cremoris SK110. This indicated that the former metabolized lactose more efficiently. Competition experiments between the resistant strain and a cured, sensitive derivative, L. lactis ssp. cremoris SK112, gave stable mixed populations. It is concluded that at average-to-high growth rates, loss of resistance from cultures of L. lactis ssp. cremoris SK110 had occurred due to instability of the plasmid and not to a competitive disadvantage of the resistant strain towards emerging sensitive variants.

Differentiation of Phage Sensitive and Phage Resistant Streptococcus Cremoris Strains by Pyrolysis Mass Spectrometry and Discriminant Analysis of the Cell Walls

Cell wall preparations of variants from four different Streptococcus cremoris strains R1, 4, 10 and 40 were investigated by classical microbiological techniques and by pyrolysis mass spectrometry combined with multivariate analysis. Discriminant analytical data correlated highly with plaque forming ability, pointing to chemical differences between the cell walls of phage sensitive and phage resistant variants.