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Mechanism of the citrate transporters in carbohydrate and citrate cometabolism in Lactococcus and Leuconostoc species.

Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178-2185, 1996). Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis. The cloned gene was expressed in a Lactococcus lactis Cit- strain, and the gene product was functionally characterized in membrane vesicles. Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange. These properties are essential for generation of a membrane potential under physiological conditions and show that the Leuconostoc CitP retains its properties when it is embedded in the cytoplasmic membrane of Lactococcus lactis. Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP of Lactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citrate degradation in Lactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism in Lactococcus lactis is a proton motive force-generating process.

Amino Acid Sequence↗

Changes in galactose and lactic acid content of sweet whey during storage.

Whey is often stored or transported for a period of time prior to processing. During this time period, galactose and lactic acid concentrations may accumulate, reducing the quality of spray-dried whey powders in regard to stickiness and agglomeration. This study surveyed industry samples of Cheddar and mozzarella cheese whey streams to determine how galactose and lactic acid concentrations changed with storage at appropriate (4 degrees C) and abuse (37.8 degrees C) temperatures. Samples stored at 4 degrees C did not exhibit significant increases in levels of lactic acid or galactose. Mozzarella whey accumulated the greatest amount of galactose and lactic acid with storage at 37.8 degrees C. Whey samples derived from cheese made from single strains of starter culture were also evaluated to determine each culture's contribution to galactose and lactic acid production. Starter cultures evaluated included Streptococcus salivarius ssp. thermophilus. Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. cremoris, and Lactococcus lactis ssp. lactis. Whey derived from L. helveticus accumulated a significantly greater amount of lactic acid upon storage at 37.8 degrees C as compared with the other cultures. Galactose accumulation was significantly decreased in whey from L. lactis ssp. lactis stored at 37.8 degrees C in comparison with the other cultures. Results from this study indicate that proper storage conditions (4 degrees C) for whey prevent accumulation of galactose and lactic acid while the extent of accumulation during storage at 37.8 degrees C varies depending on the culture(s) used in cheese production.

Cheese↗

Microbiological analysis and starter culture growth in retentates.

Pasteurized skim milk was concentrated by UF to 2-, 4-, and 5-fold. The retentates were evaluated for microbiological quality, heat treatments to inactivate microorganisms, and lactic acid bacterial starter culture activity. Aerobic mesophilic bacterial counts in raw milk decreased from an initial 1.4 x 10(6) to 3.9 x 10(2) cfu/ml after pasteurization. During UF, counts increased from 3.9 x 10(2) cfu/ml UF, counts increased from 3.9 x 10(2) cfu/ml in pasteurized milk to 1.4 x 10(3), 1.4 x 10(4), and 1.8 x 10(4) cfu/ml in 2-, 4- and 5-fold retentates, respectively. Psychrotrophic bacterial counts decreased from 9.9 x 10(5) cfu/ml in raw milk to 3.7 x 10(1) cfu/ml in pasteurized milk and gradually increased to 1.0 x 10(2), 2.5 x 10(2), and 1.4 x 10(3) cfu/ml in 2-, 4-, and 5-fold retentates, respectively. Thermophilic bacterial counts remained less than 10 cfu/ml in all samples. Skim milk and retentates inoculated with five starter cultures at 1% failed to decrease the pH below 4.6 in (2-, 4- and 5-fold). The 4- and 5-fold retentates inoculated with Lactococcus lactis spp. cremoris or Lactococcus lactis spp. lactis cultures were partially coagulated with pH greater than 5.6. In general, the pH of retentates remained higher than that of skim milk. Clotting of uninoculated samples was observed, and a spore-forming contaminant, tentatively characterized as Bacillus cereus and capable of clotting milk at a pH greater than 6, was isolated from the clotted samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Factors affecting calcium lactate and liquid expulsion defects in Cheddar cheese.

This paper summarizes the results of 2 studies designed to investigate the influence of several manufacturing and curing treatments on the appearance of Cheddar cheese defects. Specifically, 2 defects, calcium lactate crystal formation and the expulsion of free liquid (weeping) were monitored in Cheddar cheese. Both studies were conducted at a commercial cheese manufacturing facility that produces Cheddar in 18.14-kg (40-lb) blocks. In the first study we monitored cheese calcium, both total and soluble during manufacture and early curing. In the second study we measured cheese pH from 3 d through 8 mo, as well as some factors that are influenced by cheese pH. Early cheese pH (3 d to 7 d) patterns were used to select vats of cheese for retail packaging. Mild Cheddar packaged at 30 d postmanufacture and sharp Cheddar packaged at 8 mo postmanufacture from the same vats were monitored for the incidence and severity of the defects. Our results indicated that factors measured in early stages of manufacture and curing (less than 7 d) such as cheese pH at mill, lactic acid concentration, nonprotein nitrogen, and calcium (total and soluble) in cheese did not correlate with the appearance of either calcium lactate or expulsion of free liquid in packaged cheeses. Factors including pH, lactic acid concentrations, and soluble calcium measured during curing (greater than 7 d) of cheese were found to be statistically significant in the development of defects and appeared to be associated with use of specific starter culture groups. In the study, 5 different starter culture groups, each consisting of a 4-strain blend of Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were used to manufacture the cheeses. Cheese manufactured with one particular culture group showed no incidence of calcium lactate crystal formation or weeping during curing and shelf-life of cheeses in this study. This starter group also generated the least amount of pH change in cheese during the first month of curing. From these results we conclude that starter culture group, more than any other factor measured, played an important role in the development of calcium lactate and liquid expulsion defects in Cheddar cheese. Starter culture group appeared to strongly influence cheese pH, lactic acid, and soluble calcium concentrations during curing and storage.

Calcium↗

Monitoring the chemical and textural changes during ripening of Iranian White cheese made with different concentrations of starter.

The effect of the concentration of starter inoculated to milk on the composition, free tyrosine-tryptophan content, microstructure, opacity, and fracture stress of Iranian White cheese (IWC) was studied during 50 d of ripening in brine. Three treatments of cheese were made using 1-fold (IWC1S), 2-fold (IWC2S), and 4-fold (IWC4S) concentrations of a direct-to-vat mesophilic mixed culture containing Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis as starter. As ripening progressed, moisture and protein contents of the treatments continuously decreased, whereas their total ash, salt, and salt in moisture contents increased. Fat content and pH of cheeses remained stable during ripening. The pH of cheese milk at the time of renneting, which decreased by increasing the concentration of starter (6.57, 6.49, and 6.29 for IWC1S, IWC2S, and IWC4S, respectively), significantly affected most of the chemical characteristics and opacity of cheese. Lower pH values at renneting decreased moisture and ash contents, whereas cheese protein content increased. The concentration of free tyrosine-tryptophan in curd increased at first 29 d but decreased between d 29 and 49 of aging. The changes observed in cheese whiteness followed the changes in moisture content of the treatments. As the concentration of starter inoculated to milk increased, the value of fracture stress at a given ripening time significantly decreased, leading to a less resistant body against applied stress. A similar trend was also observed for fracture strain during cheese ripening. The micrographs taken by scanning electron microscopy provided a meaningful explanation for decrease in the value of fracture stress. As the cheese ripening progressed or the concentration of starter increased, the surface area occupied by the protein fraction in cheese microstructure decreased, leading the way to lower the force-bearing component in cheese texture.

Animals↗

The contribution of lactococcal starter proteinases to proteolysis in cheddar cheese.

The contribution of the lactococcal proteinase to proteolysis and flavor development in Cheddar cheese was investigated using the starter strains Lactococcus lactis ssp. lactis UC317, its proteinase-negative derivative FH041, and variants of UC317 modified in proteinase production, location, and specificity. Lactococcus lactis ssp. lactis FH041 was transformed by electroporation with plasmids pCI3601, pCI3602, or pNZ521. Plasmids pCI3601 and pCI3602 harbor the cloned proteinase genes of L. lactis ssp. lactis UC317 on a high copy number vector and, as such, encode an increased concentration of cell wall-associated and secreted enzymes, respectively. Plasmid pNZ521 contains the cloned proteinase genes from Lactococcus lactis ssp. cremoris SK11. Assessment of proteolysis and flavor development in Cheddar cheese made with these strains revealed that starter proteinases are required for the accumulation of small peptides and free amino acids in Cheddar cheese. Proteolysis was not enhanced by an approximately threefold increase in concentration of the lactococcal proteinase. The strain in which the proteinase remained attached to the cell wall appeared to contribute more to proteolysis than the strain that secreted the enzyme. Water-soluble peptides unique to Lactococcus lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 were detected by PAGE and HPLC, respectively. Sensory evaluation showed that the flavors of all cheeses made with proteinase-positive starters were similar, but cheeses made with proteinase-negative starters lacked flavor.

Amino Acids↗

Molecular properties of Streptococcus thermophilus plasmid pER35 encoding a restriction modification system.

Bacteriophage attack on lactic fermentation bacteria (LFB) is costly to the dairy industry because it results in product loss. One mechanism used by LFB to protect themselves from bacteriophage attack is restriction of foreign DNA. Three plasmids, pER16, pER35, and pER36, from three different strains of the thermotolerant dairy fermentation bacterium Streptococcus thermophilus were sequenced. One of these plasmids, pER35, isolated from S. thermophilus ST135, encoded a type IC restriction-modification (R-M) system very similar to those encoded on plasmids pIL2614 in Lactococcus lactis subsp. lactis and pND861 in Lactococcus lactis biovar diacetylactis. The high degree of identity between the R-M systems encoded on pER35, pIL2614, and pND861 indicated the potential for horizontal transfer of these genes between different species of lactic fermentation bacteria. Similar to the functional R-M system encoded on pIL2614 that protects the mesophilic L. lactis subsp. lactis against phage attack, the R-M system on pER35 most likely functions in the same role in S. thermophilus ST135. The plasmid pER16 was found to encode the specificity subunit of the R-M system, but not the R or M subunits. In addition, all three plasmids encoded proteins that are present on other S. thermophilus plasmids, including a protein for rolling-circle replication (RepA) and a low-molecular-weight stress protein (Hsp). The presence of a complete R-M system encoded on a plasmid in S. thermophilus, a species that often lacks plasmids, is novel and may be beneficial for protecting S. thermophilus from bacteriophage attack under dairy fermentation conditions.

Amino Acid Sequence↗

Characterization of dominant microbiota of a Ghanaian fermented milk product, nyarmie, by culture- and nonculture-based methods.

AIMS: To characterize the predominant micro-organisms in a Ghanaian traditional fermented dairy product, nyarmie, made from cows' milk, using both culture- and nonculture-based methods. METHODS AND RESULTS: Samples of nyarmie were analysed from three production sites in Accra, by determining the counts on selective culture media. The microbial diversity occurring in nyarmie was also evaluated by 16S/18S ribosomal DNA PCR amplification and denaturing gradient gel electrophoresis. Results showed that nyarmie contained lactococci and lactobacilli in the range of 10(8) and 10(10) CFU ml(-1), respectively, and yeasts at around 10(7) CFU ml(-1). The pH ranged between 3.49 and 4.25. The predominant lactic acid bacteria (LAB) in nyarmie were Leuconostocmesenteroides ssp. mesenteroides, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lact.helveticus, Lact. delbrueckii ssp. lactis and Lactococcus lactis, while Saccharomyces cerevisiae was the predominant yeast species. Lactobacillus delbrueckii ssp. delbrueckii was not detected by cultivation but its predominance was revealed by PCR-DGGE analysis. CONCLUSIONS: The flora in products from different producers varied in the LAB composition present and may result in variations in product quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Development and use of starter cultures for nyarmie may be beneficial in improving the consistency of product quality.

Animals↗

The behavior of selected microorganisms during the manufacture of high moisture Jack cheeses from ultrafiltered milk.

Whole milk was pasteurized and concentrated two times by ultrafiltration. Starter cultures, Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were propagated in either reconstituted skim milk, two times UF retentate, or UF permeate, or a direct vat system was used for the starter culture. The cheese milk was simultaneously inoculated with starter culture and Pseudomonas fragi 4973, Staphylococcus aureus 196E, and Salmonella typhimurium var. Hillfarm. Control whole milk, UF control milk, inoculated whole milk, and inoculated UF milk were made into Monterey Jack cheese using traditional procedures. The process of cheese manufacture was followed by determination of pH, titratable acidity, and microbial population levels. The cheeses were stored for 6 mo and analyzed every month for percentage solids and microbial population levels. Generally, numbers of contaminant microbes increased at a similar rate during manufacture in all cheeses. During the 6-mo ripening period, bacterial starter culture population levels remained high, psychrotrophs declined slowly, Staphylococcus levels remained stable, and Salmonella populations decreased. No Staphylococcus enterotoxin was detected by reverse passive latex agglutination assay.

Animals↗

Isolation, screening and characterization of bacteriocin-producing lactic acid bacteria isolated from traditional fermented food.

100 lactic acid bacterial strains isolated from traditional fermented foods (yoghurt, milk cream, sour dough and milk) were screened for bacteriocin production. Twenty six strains producing a nisin-like bacteriocin were selected. Most of these isolates gave only a narrow inhibitory spectrum, although one showed a broad inhibitory spectrum against the indicator strains tested, this strain was determined as Lactococcus lactis. The influence of several parameters on the fermentative production of nisin by Lactococcus lactis was studied. Production of nisin was optimal at 30 degrees C and in the pH range 5.5-6.3. The effect of different sulphur and nitrogen sources on Lactococcus lactis growth and nisin production was studied. Magnesium sulfate and manganese sulfate were found to be the best sulphur sources while triammonium citrate was the best inorganic nitrogen source and meat extract, peptone and yeast extract were the best organic nitrogen source for nisin production.

Bacteria↗

Identification of a cold shock gene in lactic acid bacteria and the effect of cold shock on cryotolerance.

When Lactic Acid Bacterial cultures were frozen at -20 degrees C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10 degrees C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25-37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested.

Amino Acid Sequence↗

Cloning and molecular analysis of promoter-like sequences isolated from the chromosomal DNA of Lactobacillus acidophilus ATCC 4356.

Promoter-like sequences from the chromosomal DNA of thermophilic strain Lactobacillus acidophilus ATCC 4356 were cloned. Analysis of the three DNA fragments showing promoter activity, designated P3, P6, and P15, were performed in Lactobacillus reuteri, Lactococcus lactis, and E. coli. The reporter cat-86 gene was expressed in all three bacterial species under control of the fragments P3 and P6. Fragment P15 showed promoter activity only in Lactobacillus reuteri and E. coli but not in Lactococcus lactis. The three host-specific transcriptional start points (TSPs) were used when transcription of the cat-86 gene was controlled by fragment P3 in Lactobacillus reuteri, E. coli, and Lactococcus lactis. Similarly, fragment P15 initiated transcription of the cat-86 gene at two distinctive sites in Lactobacillus reuteri and E. coli. Only within fragment P6, a common TSP was used in Lactobacillus reuteri and E. coli, but different from that used in Lactococcus lactis. Each TSP was preceded by the putative -35 and -10 hexamers. Computer analysis of the fragment P3 sequence revealed the existence of divergent promoter-like sequence (P3rev) located on the complementary DNA strand. Fragments P6 and P15 were also functional in Lactobacillus acidophilus ATCC 4356 from which chromosomal DNA they were originally cloned.

Amino Acid Sequence↗

Evidence of a relationship between autolysis of starter bacteria and lipolysis in cheddar cheese during ripening.

Cell viability, autolysis and lipolysis were studied in Cheddar cheese made using Lactococcus lactis subsp. cremoris AM2 or Lactococcus lactis subsp. cremoris HP. Cheddar cheese was made in triplicate over a 3 month period and ripened for 238 days at 8 degrees C. Cell viability in cheese was lower for AM2 (a non-bitter strain) than for strain HP (a bitter strain). Autolysis, monitored by the level of the intracellular marker enzyme, lactate dehydrogenase (EC 1.1.1.27) in cheese 'juice' extracted by hydraulic pressure, was much greater in the cheese made using AM2 than that made with HP. Lipolysis was determined by the increase during ripening of individual free fatty acids (FFA) from butyric (C4:0) to linolenic acid (C18:3) measured using a high performance liquid chromatographic technique. Levels of individual FFA from butyric (C4:0) to linolenic (C18:3) acids increased significantly (P<0.05) during ripening in cheeses made with either starter culture. Palmitic (C16:0) and oleic (C18:1) acids were the most abundant FFA throughout ripening in all cheeses. Levels of caprylic (C8:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids were significantly higher (P<0.05) in cheeses manufactured with Lc. lactis subsp. cremoris AM2 than in cheeses manufactured with Lc. lactis subsp. cremoris HP. Differences in levels of lipolysis between strains was not due to differences in the specific lipolytic or esterolytic activities in cell free extracts of the strains as measured by activity on triolein (lipase) and p-nitrophenylbutyrate (esterase) substrates. Therefore, evidence is provided for a relationship between the extent of starter cell autolysis and the level of lipolysis during Cheddar cheese ripening.

Bacteriolysis↗

Conservation of the major cold shock protein in lactic acid bacteria.

Primers designed from consensus regions of the major cold shock gene of different bacterial species were used in PCR amplification of Lactic Acid Bacteria (LAB). An appropriately-sized PCR product was obtained from Lactococcus lactis subsp. lactis LL43-1 and MG1363; Lactococcus lactis subsp. cremoris LC10-1, LC11-1, and LC12-1; Streptococcus thermophilus ST1-1; Enterococcus faecalis EF1-1; Lactobacillus acidophilus LA1-1; Lactobacillus helveticus LH1-1; Pediococcus pentosaceus PP1-1; and Bifidobacterium animalis BA1-1. The PCR products were cloned and sequenced. The deduced amino acid sequences displayed high sequence similarity with the major cold shock proteins of Escherichia coli and Bacillus subtilis and the human Y-box factor. The amino acid residues of the cold shock domain implicated in nucleic acid binding in several unrelated species were also highly conserved in the LAB strains. It is possible, therefore, that this protein in LAB may also act as a transcriptional enhancer to other cold shock genes and/or act as an RNA chaperone unwinding tightly folded RNA molecules.

Amino Acid Sequence↗

Impact of autolytic, proteolytic, and nisin-producing adjunct cultures on biochemical and textural properties of cheddar cheese.

The effect of incorporating a highly autolytic strain (Lactobacillus delbrueckii subsp. bulgaricus UL12) a proteolytic strain (Lactobacillus casei subsp. casei L2A), or a nisin Z-producing strain (Lactococcus lactis, subsp. lactis biovar diacetylactis UL719) into Cheddar cheese starter culture (Lactococcus lactis KB and Lactococcus cremoris KB) on physicochemical and rheological properties of the resultant cheeses was examined. Cheeses were ripened at 7 degrees C and analyzed over a 6-mo period for viable lactococcal and lactobacilli counts, pH, titratable acidity (TA), lipolysis, proteolysis, and textural characteristics. The combination of the nisin-producing strain and autolytic adjuncts significantly increased the production of water-soluble nitrogen, free amino acids, and free fatty acids. The effect of Lc. diacetylactis UL719 alone or of Lb. casei L2A on water-soluble nitrogen and free amino acid contents were also significant, whereas their effect on free fatty acids was not. Viable counts of Lb. bulgaricus UL12 were significantly reduced in the presence of Lc. diacetylactis UL719. Lactobacilli-containing cheeses showed significantly lower values for hardness, fracturability, and springiness. It could be concluded that the addition of Lb. bulgaricus UL12 together with a nisin-producing strain produces a greater increase in cheese proteolysis and an improvement in Cheddar cheese texture.

Cheese↗

Cloning and expression of the plasmid encoded beta-D-galactosidase gene from a Lactobacillus plantarum strain of dairy origin.

The beta-galactosidase (beta-Gal) gene from Lactobacillus plantarum C3.8 was cloned and expressed in Lactococcus lactis and Escherichia coli. Hybridization experiments indicated that the gene is located on a plasmid and is present in other strains of Lactobacillus plantarum. Its sequence is very similar to a Leuconostoc lactis beta-Gal gene. Expression of the gene, both in Lactobacillus plantarum and in Lactococcus lactis, was four-fold higher in cells growth in lactose compared to those grown in glucose. The presence of the beta-Gal gene in Lactococcus lactis allowed this bacterium to be efficient in clotting milk.

Amino Acid Sequence↗

[Study of the genetic stability of expression plasmid vector pMG36e in host bacteria].

OBJECTIVE: To study the genetic stability of expression plasmid vector pMG36e in Escherichia coli JM109 and Lactococcus lactis MG1363/36e and observe the effect of erythomycin for its stability. METHODS: We used classical method to determine the genetic stable rates of pMG36e in E. coli JM109 and Lactococcus lactis MG1363/36e with or without erythomycin selective pressure. RESULTS: When E. coli JM109 were continuously incubated to 120 generations, its pMG36e genetic stable rates were 100% and 96% respectively with or without erythomycin selective pressure. When Lactococcus lactis MG1363/36e were continuously incubated to 100 generations, its pMG36e genetic stable rates were the same 98%, with or without erythomycin selective pressure. CONCLUSION: pMG36e had good genetic stability in this two hosts. Erythromycin selective pressure had no significant effect for the genetic stability of pMG36e.

Culture Media↗

Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.

A transferable dual-plasmid inducible gene expression system for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible gene expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible system can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.

Gene Expression Regulation, Bacterial↗