Characterization of amino acid transport in the dairy strain Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1273

AIMS: To identify and characterize amino acid transport in Leuconostoc mesenteroides. METHODS AND RESULTS: The transport of labelled amino acids was measured in whole cells of Leuc. mesenteroides CNRZ 1273. Systems were operative under physiological conditions of growth, energy dependent and differed from peptide transport. Some of the systems were shared by several amino acids. Kinetic analysis indicated the presence of three transport systems with very high (VH), high (H) and low affinity (H) for the 11 amino acids studied. The K(t) values (micromol l(-1)) ranged from 0.088 to 0.815 (VH), 6-390 (H) and 320-4500 (L) and the V(max) values [nmol s(-1) (g dry weight)(-1)] from 0.015 to 0.8 (VH), 15-95 (H) and 90-470 (L). CONCLUSIONS: The study showed the presence of three transport systems in Leuc. mesenteroides for all amino acids tested, some of them being shared by several amino acids. SIGNIFICANCE AND IMPACT OF THE STUDY. The findings are discussed with reference to the growth of Leuc. mesenteroides in milk as pure or in mixed-strain culture with Lactococcus lactis.     

Development of a miniaturized selective counting strategy of lactic acid bacteria for evaluation of mixed starter in a model cassava fermentation

A miniaturized most probable number (MPN) method for the selective enumeration of three bacteria species ( Lactobacillus plantarum A6, Leuconostoc mesenteroides and Lactococcus lactis ) is described. This selective count method, based on specific consumption of carbon substrate and resistance to antibiotics, was used for the quantitative assessment of the three bacteria during mixed cultures in a model cassava fermentation. A typical microbial succession pattern was observed: (i) Lactococcus lactis and Leuc. mesenteroides dominated during the first hours of fermentation as their growth was very rapid ; (ii) from hour 12, Lactobacillus plantarum replaced the two latter strains and Lactococcus lactis disappeared gradually, followed by Leuc. mesenteroides . The growth rates of each strain appeared to be independent of the others, while acidification rates increased strongly in mixed cultures compared with pure cultures. No positive interactions resulting from the amylolytic character of Lactobacillus plantarum A6, and no negative interactions resulting from the Nis⁺ property of Lactococcus lactis , were revealed between the three strains under the model conditions used.     

Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA

Of 215 leuconostocs isolated from field grass, natural whey cultures and water-buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA-PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains.     

Molecular and physiological characterization of dominant bacterial populations in traditional mozzarella cheese processing

The development of the dominant bacterial populations during traditional Mozzarella cheese production was investigated using physiological analyses and molecular techniques for strain typing and taxonomic identification. Analysis of RAPD fingerprints revealed that the dominant bacterial community was composed of 25 different biotypes, and the sequence analysis of 16S rDNA demonstrated that the isolated strains belonged to Leuconostoc mesenteroides subsp. mesenteroides , Leuc. lactis , Streptococcus thermophilus , Strep. bovis , Strep. uberis, Lactococcus lactis subsp. lactis , L. garviae, Carnobacterium divergens , C. piscicola, Aerococcus viridans , Staphylococcus carnosus, Staph. epidermidis , Enterococcus faecalis , Ent. sulphureus and Enterococcus spp. The bacterial populations were characterized for their physiological properties. Two strains, belonging to Strep. thermophilus and L. lactis subsp. lactis , were the most acidifying; the L. lactis subsp. lactis strain was also proteolytic and eight strains were positive to citrate fermentation. Moreover, the molecular techniques allowed the identification of potential pathogens in a non-ripened cheese produced from raw milk.     

Identification and characterization of an oligopeptide transport system in Leuconostoc mesenteroides subsp. mesenteroides CNRZ 1463

AIMS: To identify and characterize an oligopeptide transport system in Leuconostoc mesenteroides CNRZ 1473. METHODS AND RESULTS: The uptake of a model substrate was monitored by determining intracellular concentrations of the corresponding amino acids by means of reversed-phase HPLC analysis. The oligopeptide transport system is specific for peptides containing at least four amino acid residues and operative under physiological conditions of growth. It is expressed maximally in the presence of oligopeptides, enhanced in the presence of Mg2+ or Ca2+ ions, and driven by ATP or a related energy-rich phosphorylated intermediate. CONCLUSIONS: The study showed evidence for and characterized the oligopeptide transport system of Leuc. mesenteroides for the first time. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the growth of Leuc. mesenteroides in mixed-strain cultures for the dairy industry.     

Utilization of dipeptides by Lactococcus lactis ssp. cremoris

Different strains of Lactococcus lactis ssp. cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis. These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis. Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine. After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated. This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently. The specific growth rate of L. lactis ssp. cremoris E8 depends upon the amino acid source in the medium. No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L. lactis ssp. cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides.     

Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of alpha-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced alpha-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

Characterization and distribution of lactic acid bacteria in cassava fermentation during fufu production

One hundred and thirty four lactic acid bacterial strains isolated during the 96-h period of cassava fermentation for fufu production were identified. The spectrum and proportion of the strains include Lactobacillus plantarum , 81%; Leuconostoc mesenteroides , 16%; Lact. cellobiosus , 15%; Lact. brevis , 9%; Lact, coprophilus , 5%; Lact. lactis , 4%; Leuc. lactis , 3% and Lact. bulgaricus , 1%. The isolates were characterized into strains. The succession among the lactic isolates was established. Lactobacillus plantarum was identified as the most dominant lactic acid bacterial strain involved in the fermentation.     

Novel paired starter culture system for sauerkraut, consisting of a nisin-resistant Leuconostoc mesenteroides strain and a nisin-producing Lactococcus lactis strain.

Nisin-resistant Leuconostoc mesenteroides NCK293 and nisin-producing Lactococcus lactis subsp. lactis NCK401 were evaluated separately and in combination for growth and nisin production in a model sauerkraut fermentation. Strains were genetically marked and selectively enumerated by using antibiotic-containing media. The growth and survival of L. mesenteroides were similar in the presence and absence of Lactococcus lactis subsp. lactis. The growth of Lactococcus lactis subsp. lactis was not inhibited, although the maximum cell density was reduced and the population decline was more pronounced in the presence of L. mesenteroides. Nisin was detected within 24 h, and levels were relatively constant over the 12-day test period. The maximum cell populations and nisin level achieved could be altered by changing the initial cell ratios of L. mesenteroides and lactococcus lactis subsp. lactis. Isogenic nisin-producing and nisin-negative Lactococcus lactis subsp. lactis derivatives were used in combination with nisin-resistant L. mesenteroides to demonstrate that nisin levels produced in mixed culture were sufficient to retard the onset of the growth of nisin-sensitive, homofermentative Lactobacillus plantarum ATCC 14917.     

Novel paired starter culture system for sauerkraut, consisting of a nisin-resistant Leuconostoc mesenteroides strain and a nisin-producing Lactococcus lactis strain.

Nisin-resistant Leuconostoc mesenteroides NCK293 and nisin-producing Lactococcus lactis subsp. lactis NCK401 were evaluated separately and in combination for growth and nisin production in a model sauerkraut fermentation. Strains were genetically marked and selectively enumerated by using antibiotic-containing media. The growth and survival of L. mesenteroides were similar in the presence and absence of Lactococcus lactis subsp. lactis. The growth of Lactococcus lactis subsp. lactis was not inhibited, although the maximum cell density was reduced and the population decline was more pronounced in the presence of L. mesenteroides. Nisin was detected within 24 h, and levels were relatively constant over the 12-day test period. The maximum cell populations and nisin level achieved could be altered by changing the initial cell ratios of L. mesenteroides and lactococcus lactis subsp. lactis. Isogenic nisin-producing and nisin-negative Lactococcus lactis subsp. lactis derivatives were used in combination with nisin-resistant L. mesenteroides to demonstrate that nisin levels produced in mixed culture were sufficient to retard the onset of the growth of nisin-sensitive, homofermentative Lactobacillus plantarum ATCC 14917.     

Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk.

The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt+) and -negative (Prt-) strains of Lactococcus lactis. The Prt- strains showed monophasic exponential growth, while the Prt+ strains grew in two phases. The first growth phases of the Prt+ and Prt- strains were in same, and no hydrolysis of casein was observed. Also, the levels of consumption of amino acids and peptides in the Prt+ and Prt- strains were similar. At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth. The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth. Oligopeptide transport-deficient strains (Opp-) of L. lactis were unable to utilize oligopeptides and grew poorly in milk. However, a di- and tripeptide transport-deficient strain (DtpT-) grew exactly like the wild type (Opp+ Dtpt+) did. These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase. In the second phase of growth of Prt+ strains, milk proteins are hydrolyzed to peptides by the proteinase. Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E.R.S. Kunji, A. Hagting, C.J. De Vries, V. Juillard, A.J. Haandrikman, B. Poolman, and W.N. Konings, J. Biol. Chem. 270:1569-1574, 1995).(ABSTRACT TRUNCATED AT 250 WORDS)     

Media and process parameters affecting the growth, strain ratios and specific acidifying activities of a mixed lactic starter containing aroma-producing and probiotic strains

AIMS: The effects of medium-composition and fermentation parameters on the properties of mixed mesophilic starters were studied. The starter was composed of Lactococcus lactis ssp. lactis (L. lactis), Lactococcus lactis ssp. cremoris (L. cremoris), Lactobacillus rhamnosus (Lact. rhamnosus) and Leuconostoc mesenteroides ssp. cremoris (Leuc. cremoris). METHODS AND RESULTS: The media used were reconstituted skim milk (RSM), and whey-based media with either citrate or phosphate buffers. The fermentation parameters were incubation temperature (22 degrees C or 32 degrees C), no pH control, and pH control in pH zones of either pH 6.0-5.8 or pH 6.0-5.2. The starter properties were strain ratio, specific acidifying activity (SAA), total population, residual carbohydrates and organic acids produced. The growth of L. lactis was favoured under pH control in whey-based media. High concentrations of Lact. rhamnosus were favoured in whey-based media prepared at 32 degrees C. The highest contents of Leuc. cremoris were obtained in starters prepared in RSM at 22 degrees C without pH control. Starters prepared under pH control gave the highest populations and made it possible for significantly lower inoculation rates (IR) to be used to carry out subsequent milk fermentations. However, the SAA of starters prepared under pH control were lower than the SAA of starters grown without any pH control. CONCLUSIONS: None of the conditions enabled the strain ratio at inoculation to be maintained. The data show that it is possible to prepare a mesophilic starter that has a significant probiotic Lact. rhamnosus content; this starter could be used in the preparation of probiotic-containing cheeses or in Leuc. cremoris for aroma production in fermented milks. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on what should be expected with respect to strain ratios and IR if cheesemakers decide to shift their aroma-producing starter production method from the traditional 'milk-based without pH control' method to whey-based media used with pH-zone control strategies.     

Relationship between utilization of proline and proline-containing peptides and growth of Lactococcus lactis.

Proline, which is the most abundant residue in beta-casein, stimulates growth of Lactococcus lactis in a proline-requiring strain (Lactococcus lactis subsp. cremoris Wg2) and in a proline-prototrophic strain (Lactococcus lactis subsp. lactis ML3). Both strains lack a proline-specific uptake system, and free proline can enter the cell only by passive diffusion across the cytoplasmic membrane. On the other hand, lactococci can actively take up proline-containing peptides via the lactococcal di- and tripeptide transport system, and these peptides are the major source of proline. Consequently, lactococcal growth on amino acid-based media is highly stimulated by the addition of proline-containing di- and tripeptides. Growth of L. lactis subsp. lactis ML3 on chemically defined media supplemented with casein does not appear proline limited. Addition of dipeptides (including proline-containing peptides) severely inhibits growth on a casein-containing medium, which indicates that the specific growth rate is determined by the balanced supply of different di- or tripeptides which compete for the same di- and tripeptide transport system.     

Extracellular material from Leuconostoc mesenteroides subsp. dextranicum mediates the aggregation of lactococcal cells: implications for the isolation of single strains

Cultures of Lactococcus lactis subsp. cremoris , originally derived from mixed cheese starter cultures, were assessed as pure following single colony selection and subculturing, yet nevertheless gave rise, under stress conditions, to an isolate with the ability to ferment citrate. The isolate was characterized with respect to its citrate enzymology and lactose fermentation and was identified as Leuconostoc mesenteroides subsp. dextranicum and assigned the strain number 663. The extracellular material (ECM) from Leuc. mesenteroides subsp. dextranicum 663 was characterized and found to contain carbohydrate, protein and phosphate (30.9, 50.7 and 18.4%, by weight, respectively). Glucose was the most prominent sugar (39% by weight of total carbohydrate) with mannose, galactose and rhamnose being the other major monosaccharides. The ECM protein resolved into a large number of bands on SDS-polyacrylamide gel electrophoresis, the most prominent having molecular masses of 40 and 49 kDa. The ECM from Leuc. mesenteroides subsp. dextranicum 663 caused aggregation of suspensions of lactococcal cells and may facilitate intergeneric interactions and/or co-culture during cheese starter strain isolation.     

Random amplified polymorphic DNA analysis for differentiation of Leuconostoc mesenteroides subspecies isolated from Tenerife cheese

AIMS: In the present study, a RAPD-PCR fingerprinting method was developed to assign Tenerife cheese Leuconostoc mesenteroides strains to its three subspecies (mesenteroides, cremoris and dextranicum). METHODS AND RESULTS: Arbitrarily primed-PCR gave different DNA banding patterns for each type strain of Leuc. mesenteroides subspecies consisting in three major and intense bands of: 1800, 1600 and 1150 bp for subspecies mesenteroides 1800, 1150 and approximately 350 bp for subspecies cremoris; and 1800, 1600 and 1500 bp for subspecies dextranicum. DNA fingerprints of Tenerife cheese Leuc. mesenteroides subspecies were coincident to that of their respective type strain. RAPD profiles were reproducible with DNA template obtained by two different extraction methods. CONCLUSIONS: Tenerife cheese Leuc. mesenteroides strains were rapidly and unequivocally assigned to one of the subspecies by comparing their RAPD-PCR fingerprints with those displayed by type strains used as standards. This technique can be applied to complement preliminary identification of Leuc. mesenteroides to the species level by other molecular methods such as protein fingerprinting. SIGNIFICANCE AND IMPACT OF THE STUDY: RAPD-PCR allows reliable, reproducible and rapid molecular differentiation of Tenerife cheese Leuc. mesenteroides subspecies with no need to use time-consuming and often ambiguous biochemical tests.     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

Behavior of psychrotrophic lactic acid bacteria isolated from spoiling cooked meat products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10 degrees C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10 degrees C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4 degrees C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10(8) CFU/g at 10 degrees C after 7 to 12 days.     

Charged casein-derived oligopeptides competitively inhibit the transport of a reporter oligopeptide by Lactococcus lactis

AIM: To study the effect of casein-derived peptides, accumulated during growth of Lactococcus lactis in milk, on its oligopeptide transport (Opp) function. METHODS AND RESULTS: This effect was estimated by analysing the ability of casein-derived peptides to compete for the transport of a reporter peptide by whole L. lactis cells. The transport of the reported peptide was monitored by determining the intracellular concentrations of the corresponding amino acids by means of reverse-phase high-performance liquid chromatography (HPLC). Uptake of the reporter peptide was competitively inhibited by casein-derived peptides. The competition was only because of charged casein-derived peptides, including anionic peptides. The design of specific pure peptides made it possible to evidence for a positive (or negative) influence exerted by the positively (or negatively) charged side chain of the N-terminal amino acid on the competition. CONCLUSIONS: Charged casein-derived peptides impaired the oligopeptide transport function of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate an inhibition of Opp when too many peptides are produced by the proteinase. Peptide transport by Opp therefore represents a bottleneck for increasing the growth rate of L. lactis in milk.     

Isolation and identification of cultivable lactic acid bacteria in traditional yak milk products of Gansu Province in China

Various traditional fermented yak milk and raw milk foods could be considered as an abundant resource for obtaining novel lactic acid bacteria (LAB) with unique properties. Eighty-eight samples of yak milk products were collected from Gansu Province in China. Three hundred and nineteen strains of LAB isolated from these samples were identified by phenotypic methods, 16S rRNA gene sequence analysis and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technology. Among the isolates, one hundred and sixty-four isolates (51.41% of the total) were classified under Lactobacilli, and one hundred and fifty-five (48.59%) belonged to cocci. All the isolates were classified to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella) and twenty-one species. Lactobacillus helveticus (87 strains), Leuconostoc mesenteroides subsp. mesenteroides (49 strains), Streptococcus thermophilus (39 strains), Lactobacillus casei (31 strains) and Lactococcus lactis subsp. lactis (19 strains) were considered as the predominant populations in the yak milk products. The results showed that there were abundant genus and species LAB existing in yak milk products in Gansu Province in China. The obtained LAB pure cultures may be a valuable source for further starter selection.     

Biodiversity of lactic acid bacteria in Moroccan soft white cheese (Jben)

The bacterial diversity occurring in traditional Moroccan soft white cheese, produced in eight different regions in Morocco, was studied. A total of 164 lactic acid bacteria were isolated, purified and identified by whole-cell protein fingerprinting and rep-PCR genomic fingerprinting. The majority of the strains belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Enterococcus. Sixteen species were identified: Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, Lactococcus garvieae, Lactococcus raffinolactis, Leuconostoc pseudomesenteroides, Leuconostoc mesenteroides, Leuconostoc citreum, Eterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus saccharominimus and Streptococcus sp.