Diacetylactis Strains Research Articles

Cloning, sequencing, and expression of the pyruvate carboxylase gene in Lactococcus lactis subsp. lactis C2.

A functional pyc gene was isolated from Lactococcus lactis subsp. lactis C2 and was found to complement a Pyc defect in L. lactis KB4. The deduced lactococcal Pyc protein was highly homologous to Pyc sequences of other bacteria. The pyc gene was also detected in Lactococcus lactis subsp. cremoris and L. lactis subsp. lactis bv. diacetylactis strains.

Effect of α-acetolactate decarboxylase inactivation on α-acetolactate and diacetyl production by Lactococcus lactis subsp. lactis biovar diacetylactis

Strains of Lactococcus lactis subsp. lactis biovar diacetylactis deficient in α-acetolactate decarboxylase produce α-acetolactate. This unstable compound is a precursor of acetoin and an aromatic compound, diacetyl. Following random mutagenesis of strain CNRZ 483, α-acetolactate decarboxylase-negative mutant 483 M1 was selected. When grown in milk, its growth and acidification characteristics were similar to those of the parental strain. In anaerobic conditions, the parental strain produced 2.10 mM acetoin and less than 0.05 mM diacetyl. The mutant accumulated up to 2.11 mM α-acetolactate, which spontaneously degraded to acetoin and diacetyl. After 24 h of culture, the α-acetolactate concentration was only 0.49 mM and the acetoin and diacetyl concentrations reached 1.50 mM and 0.26 mM, respectively. Diacetyl production by both strains increased in aerobic conditions, as well as when citrate was added. In contrast to cultures of the parental strain, however, diacetyl and acetoin concentrations in mutant cultures continued to increase without reaching a plateau. The results also showed that diacetyl production by wild type L. lactis subsp. lactis biovar diacetylactis strains cannot be explained uniquely by the spontaneous decarboxylation of the α-acetolactate produced in the culture medium.

Production of fermented milk using a malty compound-producing strain of Lactococcus lactis subsp. lactis biovar. diacetylactis, isolated from Zimbabwean naturally fermented milk

Malty compound-producing Lactococcus lactis subsp. lactis biovar. diacetylactis strain INF-DM1, originally isolated from naturally fermented milk in Zimbabwe was used to prepare fermented milk from ordinary milk, milk enriched with 2.5% (w/v) skimmed milk powder and by 2.5% (w/v) increase in dry matter by ultrafiltration. Inoculated milk was incubated at 22, 30 and 37°C. Analyses were made after 0, 9, 18 and 24 h incubation and also after 24 h incubation followed by storage for one week at 4°C. Samples were analysed for volatile compounds including malty compounds and for organic acids, pH and log cfu/g. All samples were also judged for sensory attributes. Products made from enriched milks showed increased viscosity which was most marked in ultrafiltrated milk incubated at 30 and 37°C. The levels of certain compounds (lactic acid, citrate and diacetyl) were significantly affected by milk type. Incubation temperature had a significant effect on starter growth rate and the rate of production and amount of the malty compounds, lactate, diacetyl, ethanol, acetoin and acetaldehyde. 3-Methyl butanal concentrations were above the taste threshold level of 0.06 ppm in almost all products, including stored products. Although initial growth rate was fastest at 37°C, an uncoupling of acid production and growth was observed after 9 h incubation, suggesting that this is above the optimum temperature for this strain. In addition, products incubated at 37°C showed a tendency to whey separation, indicating that this temperature is also too high to give optimum product quality. All products attained good scores in sensory analysis provided that fermentation was complete. Variation in the levels of malty compounds during the fermentation had no significant effect on the sensory score for total flavour.

Development and Use of a Screening Procedure for Production of (alpha)-Acetolactate by Lactococcus lactis subsp. lactis biovar diacetylactis Strains

A method was developed to screen and isolate mutagenized Lactococcus lactis subsp. lactis biovar diacetylactis strains accumulating (alpha)-acetolactate. This compound is accumulated by (alpha)-acetolactate decarboxylase-deficient strains and undergoes spontaneous degradation into diacetyl on agar plates. The diacetyl produced is detected by a colorimetric reaction yielding a red halo around the colonies.

Flavour development in aseptic cheese curd slurries prepared with single-strain starter bacteria in the presence and absence of adjuncts

Summary - Eight strains of Lactococcus starter bacteria were tested under microbiologically controlled conditions for their ability to pro duce Cheddar cheese flavour, For this purpose, aseptic Cheddar chee se curds were prepared with each of the organisms, and the resulting curds slurried with sterile salt and water, and stored at 30 C for accelerated f1avour development. Distinct though weak Cheddar cheese flavour was produced by the Lactococcus lactis subsp cremoris strain SK Il, but not by strains ASCC457, ASCC50, ASCC 109, ASCC660, ASCC360, or the L lactis subsp lactis strain ASCC381, or the L lactis subsp lactis biovar diacetylactis strain ASCC250. Different starter strains produced different degrees of bitterness, with strains ASCC457, 109 and 660 producing the most bitter slurries. Strains SKII, ASCC50, ASCC360, and ASCC250 produced only slight bitterness in slurries ripened for 15 days, Free fatty acid concentrations in the slurries differed depending on the starter strain used, implying that different starter strains have different lipase activity. Cheese flavour development was slow in the slurries containing only starter bacteria. In contrast, slurries made with the strain SKII in the presence of the adjuncts Lactobacillus helveticus ASCC395, Brevibacterium linens CSCC750 or Lactobacillus casei subsp casei NCDOl51 underwent rapid f1avour development, giving distinct and strong chee se (but not Cheddar) flavours for each adjunct. These differences in flavour, however, cou Id not be explained in terms of differences in the non-sulphur volatile composition of the slurries determined by static headspace analysis.

Conductimetric Analyses of Bacteriophage Infection of Two Groups of Bacteria in DL-Lactococcal Starter Cultures

Two conductimetric methods were used for simple and rapid detection of bacteriophages against the two major groups of bacteria in DL-lactococcal, mixed-strain starter cultures. Bacteriophages against the acid-producing bacteria Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris in a starter were analyzed in a Malthus® instrument using reconstituted skim milk as the medium. The presence of phages against Lactococcus lactis ssp. lactis biovar. diacetylactis strains in a starter could be analyzed by measurement of CO2 production with an indirect conductance method in the Rabit® instrument. For both methods, the presence of phages against bacteria in a starter increased the detection time. At 30°C and .1% inoculum, a very sensitive assay was obtained that detected phages against acid-producing strains in a starter within 3 to 4h. The presence of 40 to 1000 infectious phages was sufficient for detection with a mixed-strain starter culture. Bacteriophages against CO2- producing bacteria were detected after 7 to 9h with a .1% inoculum at 30°C. Bacteriophage infection of single strains of bacteria reduced total change in conductance. For pure strains of an acid- or a CO2-producing strain, the detection limits were 1 and 100 pfu, respectively.

Identification and characterization of the alpha-acetolactate synthase gene from Lactococcus lactis subsp. lactis biovar diacetylactis

The conversion of 3-13C-labelled pyruvate in an acetoin-producing clone from a Lactococcus lactis subsp. lactis biovar diacetylactis strain DSM 20384 plasmid bank in Escherichia coli was studied by 13C nuclear magnetic resonance analysis. The results showed that alpha-acetolactate was the first metabolic product formed from pyruvate, whereas acetoin appeared at a much slower rate and reached only low concentrations. This alpha-acetolactate production shows that the cells express the gene for alpha-acetolactate synthase (als). Nucleotide sequence analysis identified an open reading frame encoding a protein of 554 amino acids. The deduced amino acid sequence exhibits extensive similarities to those of known alpha-acetolactate synthases from both prokaryotes and eukaryotes. The als gene is expressed on a monocistronic transcriptional unit, which is transcribed from a promoter located just upstream of the coding region.

The stability of the lactose and citrate plasmids in Lactococcus lactis subsp. lactis biovar. diacetylactis

The ability of Lactococcus lactis subsp. lactis biovar. diacetylactis strain C17 to ferment lactose and citrate are stable functions when cells are continuously cultivated under carbon limitation with lactose and citrate present in the medium. Switching the carbohydrate source to glucose rapidly resulted in a population which could no longer ferment lactose. The presence of citrate had no effect on the rapidity of the loss of ability to utilize lactose. The ability of strain C17 to utilize citrate was unaffected by cultivation for more than two weeks on citrate-free medium, containing either lactose or glucose as fermentable carbohydrate. These results confirm the lack of stability of the lactose plasmid and demonstrate for the first time that the citrate plasmid in L. lactis subsp. lactis biovar. diacetylactis is remarkably stable.

The stability of the lactose and citrate plasmids inLactococcus lactissubsp.lactisbiovar.diacetylactis

The ability of Lactococcus lactis subsp. lactis biovar. diacetylactis strain C17 to ferment lactose and citrate are stable functions when cells are continuously cultivated under carbon limitation with lactose and citrate present in the medium. Switching the carbohydrate source to glucose rapidly resulted in a population which could no longer ferment lactose. The presence of citrate had no effect on the rapidity of the loss of ability to utilize lactose. The ability of strain C17 to utilize citrate was unaffected by cultivation for more than two weeks on citrate-free medium, containing either lactose or glucose as fermentable carbohydrate. These results confirm the lack of stability of the lactose plasmid and demonstrate for the first time that the citrate plasmid in L. lactis subsp. lactis biovar. diacetylactis is remarkably stable.

Development and application of oligonucleotide probes for identification of Lactococcus lactis subsp. cremoris

Lactococcus lactis subsp. cremoris is of considerable interest to the dairy industry, which relies upon the few available strains for the manufacture of cheddar cheese free of fermented and fruity flavors. The subspecies cremoris differs from related subspecies by the lack of a few phenotypic traits. Our purpose was to identify unique rRNA sequences that could be used to discriminate L. lactis subsp. cremoris from related subspecies. The 16S rRNAs from 13 Lactococcus strains were partially sequenced by using reverse transcriptase to identify domains unique to L. lactis subsp. cremoris. All five strains of the subspecies cremoris had a unique base sequence in a hypervariable region located 70 to 100 bases from the 5' terminus. In this region, all L. lactis subsp. lactis biovar diacetylactis strains examined had a sequence identical to that of L. lactis subsp. lactis 7962, which was different from other strains of the subspecies lactis by only one nucleotide at position 90 (Escherichia coli 16S rRNA structural model) (J. Brosius, J. L. Palmer, J. P. Kennedy, and H. F. Noller, Proc. Natl. Acad. Sci. USA 75:4801-4805, 1978). Oligonucleotide probes specific for the genus Lactococcus (212RLa) and for the subspecies cremoris (68RCa) were synthesized and evaluated by hybridization to known rRNAs as well as fixed whole cells. Efficient and specific hybridization to the genus-specific probe was observed for the 13 Lactococcus strains tested. No hybridization was seen with the control species. All five strains of the subspecies cremoris hybridized to the subspecies-specific probe.

Partial characterization and comparison of cell wall proteinases from 5 strains of Streptococcus lactis

Cell wall proteinases from 5 strains of Streptococcus lactis including one S. lactis subsp. diacetylactis were compared and partially characterized. They shared sorne common enzymatic features: only one activity optimum at 30-40°C, at pH 5.5-6 on casein and 4-4.5 on haemoglobin. Crude cell wall extracts from the S. lactis strains were fully inhibited by DFP, but this inhibition was only partial for the S. lactis subsp. diacetylactis strain. Electrophoregrams of as!, {3 and K-casein hydrolysates obtained with the cell-wall extracts of these strains were quite similar for each protein substrate. {3-caseinwas preferentially broken down; as! and K-caseins were hydrolysed to a lesser extent.

Occurrence of ?-acetolactate decarboxylases among lactic acid bacteria and their utilization for maturation of beer

Acetolactate decarboxylase activity has been detected among three genera, nine species and 263 strains of lactic acid bacteria tested in the course of a screening for acetolactate decarboxylases amenable for use in brewing as maturation aid. Streptococcus diacetylactis strain FD-64-D was found to generate a decarboxylase exhibiting a satisfactory activity and an excellent stability at the pH prevailing in beer and wort. This decarboxylase could not be solubilized but enzymatically active, freeze-dried cells were effective for satisfactory flavour maturation of beer although difficulties were encountered during attempts to remove the applied cell material by filtration of the beer. Lactobacillus casei DSM 2547 was likewise found to produce a decarboxylase exhibiting a satisfactory activity and stability at the low pH of beer and which, in addition, was readily solubilized. A method has been developed for pilot scale production of preparations of this decarboxylase suitable for use in brewing.

Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing.

The production and regeneration of bacterial protoplasts promoted the loss of three different plasmid-specified traits in Streptococcus lactis subsp. diacetylactis strains. The loss of five different plasmids, including small multicopy molecules, was readily detected in Streptococcus lactis 712 by screening lysates of random protoplast regenerants on agarose gels. In this strain sequential rounds of protoplast regeneration were used to produce a plasmid-free strain and derivatives carrying only single molecules from the plasmid complement. During these experiments a 33-megadalton plasmid, pLP712, was found to encode genes for lactose and protein utilization. Only this plasmid was required for normal growth and acid production in milk; the remaining four plasmids appeared to be cryptic. Lactose-defective derivatives of a strain carrying only pLP712 were readily isolated. Although these derivatives included instances of plasmid loss, deletions of pLP712 were frequently found. Many different deleted derivatives of pLP712, including some in which the lactose or protein utilization determinant or both were lost, were isolated. The molecular instability of pLP712 largely accounted for previous observations of plasmid complements in S. lactis 712 after lactose determinant curing or transfer by conjugation and transduction. Curing of cryptic molecules from multiple plasmid complements by protoplast regeneration may prove to be generally valuable in lactic streptococci and other gram-positive species.

Conjugal transfer of genetic information in group N streptococci.

Streptococcus lactis strains ML3 and C(2)O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM(4) were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac(-)) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac(+) Str(r)) recombinants were found when the Lac(+) Str(s) donor was mixed with Lac(-) Str(r) LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM(4), nor was reversion responsible for the high number of Lac(+) Str(r) recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C(2)O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM(4). In S. lactis C2 x LM0230 matings, the Str(r) marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.