Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Cloning and Characterization of a Novel <Emphasis Type="Italic">tuf</Emphasis> Promoter from <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> IL1403

Genetic engineering of lactic acid bacteria (LAB) requires a reliable gene expression system. Especially, a stable promoter is an important genetic element to induce gene expression in such a system. We report on a novel tuf promoter (Ptuf) of Lactococcus lactis subsp. lactis IL1403 that was screened and selected through analysis of previously published microarray data. Ptuf activity was examined and compared with three other known lactococcal promoters (PdnaJ, PpfkA, and Pusp45) using different bacteria as expression hosts. Each promoter was, respectively, fused to the promoterless and modified bmpB gene as a reporter, and we estimated promoter activity through BmpB expression. All promoters were active in IL1403, and Ptuf activity was strongest among them. The activity of each promoter differed by host bacteria (Lactobacillus plantarum Lb25, Lactobacillus reuteri ATCC23272, and Escherichia coli Top10F’). Ptuf had the highest activity in IL1403 when growth reached late log phase. The activity of each promoter correlated with the expression of each cognate gene in the microarray data (R ² = 0.7186, P = 0.06968). This study revealed that novel food-grade promoters such as IL1403 Ptuf can be selected from microarray data for food-grade microorganisms and Ptuf can be used to develop a reliable gene expression system in L. lactis.     

Enhancement of Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.     

Enhanced production of lactic acid by an adapted strain of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactobacillus delbrueckii subsp. lactis strains were developed having increased activity, by gradually acclimatizing the bacteria to acidic conditions over repeated batch culture. Cells from one batch culture were used as the inoculum for the subsequent batch culture and thereby an adapted strain of Lactobacillus was obtained showing improved lactic acid productivity, cell growth and total glucose utilization. Furthermore, the acclimatized cells used significantly less nitrogen for a given level of lactic acid production, which is significant from an industrial point of view. The developed procedure decreases fermentation time and nutrient use, leading to reduced operation costs, while providing a lactic acid yield superior to previously reported methods.     

Optimized Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">ureB</Emphasis> Gene in the <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Nisin-Controlled Gene Expression (NICE) System and Experimental Study of Its Immunoreactivity

In the development of an oral vaccine against Helicobacter pylori, H. pylori urease subunit B (UreB) was expressed in a food-grade delivery vehicle, Lactococcus lactis NZ3900. The ureB gene (Genbank accession no. FJ436980) was amplified by polymerase chain reaction (PCR) from MEL-Hp27. The PCR-amplified ureB gene was cloned in the E. coli–L. lactis shuttle vector pNZ8110 and transformed into E. coli MC1061. After the transformant had been identified, the recombinant plasmid was purified and electrotransformed into L. lactis NZ3900. The conditions of UreB expression in the L. lactis transformant were optimized by orthogonal experiment. The maltose binding protein (MBP)-UreB fusion protein expressed by TB₁/pMAL-c2X-ureB was used to cultivate mice polyclonal anti-UreB serum after purification by the amylose prepacked column. The Western blot method was adopted to confirm whether the UreB expressed by L. lactis transformant had immunoreactivity. The optimized conditions for UreB expression were as follows. Nisin 40 ng/ml was added to the medium when the recombinant grew to OD₆₀₀≈0.30–0.40 and the induction time lasted 5 h. As a result, the maximum yield of UreB was 27.26 μg/mL of medium, and the maximum percentage of UreB in cell extracts of the L. lactis transformant reached its peak at 20.19%. Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity. All these results make an appealing case for construction of the food-grade vaccine for H. pylori.     

Characterization of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> isolated from surface waters

A group of nine presumptive enterococci was isolated on enterococcal selective media Slanetz-Bartley agar and/or kanamycin-esculin-azide agar during a screening of Enterococcus spp. in surface waters. All strains formed a homogeneous cluster separated from all enterococcal species using rep-PCR fingerprinting with the (GTG)(5) primer but they matched fingerprints revealed by Lactococcus lactis subsp. lactis representatives. Further identification using extensive biotyping and automated ribotyping with EcoRI (RiboPrinter(R) microbial characterization system) confirmed all strains as L. lactis subsp. lactis in full correspondence with the (GTG)(5)-PCR. We demonstrated that L. lactis subsp. lactis strains occur in different surface waters and can be confused with enterococci due to their positive growth on selective enterococcal media as well as positive results in tests commonly used for identification of the genus Enterococcus (esculin hydrolysis, acetoin and pyrrolidonyl arylamidase production, growth at 10 degrees C and in 6.5% NaCl). The (GTG)(5)-PCR fingerprinting was revealed as a reliable and fast method for the identification of L. lactis subsp lactis while automated ribotyping with EcoRI proved to be a good tool for intrasubspecies typing purposes.     

Nisin-controlled extracellular production of apidaecin in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Apidaecins are heat-stable, nonhelical antibacterial peptides isolated from lymph fluid of the honeybee (Apis mellifera). These peptides are active against a wide range of gram-negative bacteria and they are the most prominent components of the honeybee humoral defense against microbial invasion. In the present study, one isoform of apidaecin, apidaecin Ho, was expressed extracellularly in the food-grade bacterium Lactococcus lactis. Results showed that expression driven by the lactococcal nisA promoter and Usp45 signal peptide resulted in efficient secretion of apidaecin in L. lactis subsp. cremoris NZ9000. Recombinant apidaecin was purified by gel filtration and semipreparative RP-HPLC, and about 10 mg active recombinant apidaecin was obtained from 1,000 ml culture. This is the first report on the nisin-controlled extracellular production of active apidaecin in L. lacits. The expression and delivery of apidaecin in the food-grade L. lactis may provide a clue to facilitate the widespread application of apidaecin in the control and prevention of gram-negative bacteria infections of human and animals.     

High efficiency electrotransformation of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> spp. <Emphasis Type="Italic">lactis</Emphasis>cells pretreated with lithium acetate and dithiothreitol

Background  

A goal for the food industry has always been to improve strains of Lactococcus lactis and stabilize beneficial traits. Genetic engineering is used extensively for manipulating this lactic acid bacterium, while electropolation is the most widely used technique for introducing foreign DNA into cells. The efficiency of electrotransformation depends on the level of electropermealization and pretreatment with chemicals which alter cell wall permeability, resulting in improved transformation efficiencies is rather common practice in bacteria as in yeasts and fungi. In the present study, treatment with lithium acetate (LiAc) and dithiothreitol (DTT) in various combinations was applied to L. lactis spp. lactis cells of the early-log phase prior to electroporation with plasmid pTRKH3 (a 7.8 kb shuttle vector, suitable for cloning into L. lactis). Two strains of L. lactis spp. lactis were used, L. lactis spp. lactis LM0230 and ATCC 11454. To the best of our knowledge these agents have never been used before with L. lactis or other bacteria.     

Fermentation performance of an exopolysaccharide-producing strain of<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis> subsp.<Emphasis Type="Italic"> bulgaricus</Emphasis>

The formation of exopolysaccharide (EPS) and extracellular metabolites was studied in a strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2483), grown under batch culture conditions in a semi-defined medium incorporating lactose and casein hydrolysate. Performance parameters were derived from the fermentation data, and kinetic models were applied in order to describe the production of EPS, extracellular metabolites, and biomass produced. Lactose was split intracellularly, with the resultant galactose being exported from the cell, and the glucose being metabolised further to EPS and lactic acid. Production of EPS, lactate, and galactose was closely growth-associated and followed a pattern of primary kinetics. A marginally lower galactose level relative to the modelled levels throughout most of the time course of the fermentation suggests that not all galactose is exported from the cell, and that a low level of flux to other metabolites, such as EPS, might exist.     

Elicitation of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> with dead cells of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in a bioreactor increases production of undecylprodigiosin

Since microorganisms normally co-exist with other species in nature, they have developed complex metabolic and physiological responses as a result of such interspecies interactions. We utilized some of these interactions by introducing heat-killed cells of Bacillus subtilis and Staphylococcus aureus to Streptomyces coelicolor cultures and, as a result, stimulated undecylprodigiosin production. Undecylprodigiosin is not only an antibiotic; it has also been attributed with antitumor activities, but, in a defined medium, pure cultures of S. coelicolor produced only low concentrations. Elicitation with B. subtilis increased the maximum undecylprodigiosin concentration by threefold and S. aureus by fivefold compared with the pure culture of S. coelicolor. Growth and glucose consumption of elicited S. coelicolor, however, remained similar to those observed in the pure culture. Furthermore, another positive outcome of the elicitation with both B. subtilis and S. aureus was the earlier onset of undecylprodigiosin production by 24 h compared with the pure culture of S. coelicolor. This is the first time that such a phenomenon has been seen in 2L bioreactors. Our work supports the use of biotic elicitation in order to enhance the production of secondary metabolites for industrial-scale applications.     

GC-MS based metabolomics analysis reveals the effects of different agitation speeds on the level of proteinogenic amino acids in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">cremoris</Emphasis> MG1363

Lactococcus lactis subsp. cremoris MG1363 is an opportunistic lactic acid bacterium (LAB) that has emerged as one of the most promising candidate cell factories. The availability of genome-level information and U.S. Federal Drug administration’s designation of ‘generally recognized as safe’ (GRAS) are two of the more important key factors for its wide-ranging applications in numerous biotechnological processes. Several studies have shown that various physiological conditions, such as temperature, salinity and pH, can influence the physiological growth of L. lactis; agitation, in particular, can increase the production of amino acids and fermentation by-products. However, the effect of different agitation speeds on the growth of L. lactis’ has rarely been examined. In the study reported here, we used a gas chromatography–mass spectrometry-based metabolomics approach to investigate the effects of different agitation speeds on the production of proteinogenic amino acids (PAAs) by L. lactis MG1363. Lactococcus lactis MG1363 was grown under four different agitation speeds (50, 100, 150 and 200 rpm) at a constant temperature of 30 °C, and the differences in the specific growth rate and levels of PAAs were determined. Approximately 15 PAAs with concentrations ranging from 0 to 50 mmol/L were detected under all conditions. Partial least squares discriminant analysis (PLS-DA) revealed a distinct difference when L. lactis was incubated at 100 and 150 rpm. Heatmap analysis showed that the levels of pyruvate-, glutamate- and aspartate-based amino acids were varied under the different agitation conditions. The time-series analysis showed an increment of lysine when L. lactis’ cells were cultured with shaking at 50, 100 and 200 rpm. Taken together, these results highlight the changes in the levels of PAAs in L. lactis cells in response to agitation. In addition, the collected dataset will be useful for optimization of ¹³C-labeling based experiments in L. lactis.     

Identification and Characteristics of Nisin Z-Producing <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> Isolated from Kimchi

We isolated bacteriocin-producing Lactococcus lactis subsp. lactis from Kimchi. The bacteriocin inhibited strains of Clostridium perfringens, C. difficile, Listeria monocytogenes, vancomycin-resistant Enterococcus, and one out of four methicillin-resistant Staphylococcus aureus strains, as well as some closely related lactic acid bacteria. In tricine-SDS-PAGE, the bacteriocin migrated with an apparent molecular weight of about 4 kDa to the same location as nisin A and crude nisin Z. The gene encoding this bacteriocin was found to be identical to that of nisin Z with direct PCR sequence methods. The inhibitory activity was stable against heat and pH, but it was lost at 100°C for 1 h and at 121°C for 15 min. The bacteriocin was inactivated by proteolytic enzymes, but was not affected by lysozyme, lipase, catalase, or β-glucosidase. There were some differences in characteristics from those of nisins described previously. Received: 21 June 2002 / Accepted: 22 July 2002     

Molecular genetic and physiological differentiation of <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis> and <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>: Analysis of strains from the all-Russian collection of microorganisms (VKM)

Molecular genetic identification of 52 Kluyveromyces strains from VKM, mainly of dairy origin, was carried out. Restriction analysis of 5.8S-ITS rDNA fragments was used to differentiate between Kl. lactis var. lactis, Kl. lactis var. drosophilarum (European population of “krassilnikovii”), and Kl. marxianus. Kl. lactis was shown to differ from Kl. marxianus in its ability to assimilate α-glucosides: maltose, melezitose, and α-methyl-glucoside.     

Food-Grade Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> UreB Subunit in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its Immunoreactivity

Helicobacter pylori is the principal cause of chronic active gastritis, peptic ulcer, and gastric cancer. To develop an oral vaccine against H. pylori infection, we had expressed the H. pylori ureB gene (Genbank accession no. FJ436980) in nisin-controlled expression vectors using Lactococcus lactis NZ3900 as host. The ureB gene was amplified by PCR from a H.pylori strain MEL-Hp27. Then the ureB gene was fused translationally downstream of the nisin-inducible promoter nisA in a L. lactis plasmid pNZ8149. Lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade expression system employing L. lactis NZ3900. The conditions of UreB expression in this system were optimized by orthogonal experiment. The optimized conditions have been determined as follows: induction of expression was carried out at the cells density of OD₆₀₀ ≈ 0.4 with 25 ng/ml nisin, and harvest after 5 h. The maximum percentage of recombinant UreB was estimated to be 7% of total soluble cellular proteins and the yield was 12.9 μg/ml. Western blot demonstrated that the UreB protein was expressed in the L. lactis transformant and had favorable immunoreactivity. These results indicated that the lactococci-derived vaccines could be promising candidates as alternative vaccine strategies for preventing H. pylori infection.     

Use of <Emphasis Type="Italic">Quercus ilex</Emphasis> subsp. <Emphasis Type="Italic">ballota</Emphasis> phenological and pollen-production data for interpreting <Emphasis Type="Italic">Quercus</Emphasis> pollen curves

Although aerobiological data are often used in phenological research as an indicator of flowering, airborne pollen concentrations are influenced by a number of factors that could affect pollen curves. This paper reports on a study of various aspects of reproductive biology in Q. ilex subsp. ballota, together with environmental factors influencing pollen release and transport, with a view of achieving reliable interpretation of Quercus pollen curves in Ourense (NW Spain). Aerobiological data were recorded from 2002 to 2004 at two sites in the province of Ourense. From 1st February to the end of the flowering period, phenological observations were carried out on 19 trees from the Q. ilex subsp. ballota population found in the Ourense area. Pollen production was calculated for the same trees. The chilling and heating requirements for triggering development were also calculated. The mean flowering period lasted 11-15 days. Reduced pollen output per catkin and, especially, a reduced number of catkins per tree in 2003 and 2004, prompted a marked decline in overall pollen production. Major differences observed in Q. ilex subsp. ballota pollen curves were attributed to the considerable influence both of weather conditions during pollination and pollen production. In years with high pollen production and weather conditions favouring pollen release, Q. ilex subsp. ballota contributed almost 10% to the total Quercus pollen curve. Around 20% of the pollen trapped was captured before or after flowering periods.     

The <Emphasis Type="Italic">Lactococcus lactis</Emphasis> FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of <Emphasis Type="Italic">Escherichia coli</Emphasis> and the FabH protein of <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.     

A novel chimeric prophage vB_LdeS-phiJB from commercial <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis>

Prophage vB_LdeS-phiJB (phiJB) was induced by mitomycin C and UV radiation from the Lactobacillus delbrueckii subsp. bulgaricus SDMCC050201 isolated from a Chinese yoghurt sample. It has an isometric head and a non-contractile tail with 36,969 bp linear double-stranded DNA genome, which is classified into the group a of Lb. delbrueckii phages. The genome of phiJB is highly modular with functionally related genes clustered together. Unexpectedly, there is no similarity of its DNA replication module to any phages that have been reported, while it consists of open-reading frames homologous to the proteins of Lactobacillus strains. Comparative genomic analysis indicated that its late gene clusters, integration/lysogeny modules and DNA replication module derived from different evolutionary ancestors and integrated into a chimera. Our results revealed a novel chimeric phage of commercial Lb. delbrueckii and will broaden the knowledge of phage diversity in the dairy industry.     

Establishment of taxonomic status of new prospective bacteriocin-synthesizing <Emphasis Type="Italic">Lactococci</Emphasis> strains of various origins

The taxonomic status of new prospective bacteriocin-synthesizing strains of mesophilic lactococci isolated from raw milk and milk products from different regions of Russia and also of strain F-119, obtained by protoplast fusion of two related strains with low bacteriocin-synthesizing activity, was established by classical methods of identification. The values of antibiotic activity displayed by the strains toward a test microorganism Bacillus coagulans were up to 4650 IU/ml, which is significantly higher than in natural lactococci strains. In spite of some differences in morphology, ability to ferment carbohydrates, requirements for nutrients, and antibiotic suspectability, the strains were identified as Lactococcus lactis subsp. lactis. The new strains differed from the classic nisin-producing strain L. lactis subsp. lactis MGU by a remarkably broad spectrum of bactericidal and fungicidal activity. Study of 16S rRNA gene sequences of new natural strains, fusants F-119 and another one obtained earlier, F-116, and their parental strains in comparison with reference strains confirmed the new strains’ taxonomic status as Lactococcus lactis subsp. lactis. The nucleotide sequences of 16S rRNA genes were deposited with GenBank under accession numbers EF100777-EF114305.     

Identification and characterization of dominant lactic acid bacteria isolated from traditional fermented milk <Emphasis Type="Italic">Dahi</Emphasis> in Bangladesh

We isolated a total of 266 strains of lactic acid bacteria (LAB) from 28 dahi samples that were collected from different areas in Bangladesh. The isolated strains were identified on basis of their morphological, physiological and biochemical characteristics, the lactic acid isomer produced, the ability to ferment sugars and 16S rDNA analysis. Among the isolates, the cocci (73%) were dominant over the rods (27%). The distribution of the isolates by genus was as follows: Streptococcus (50%), Lactobacillus (27%), Enterococcus (9%), Leuconostoc (5%), Lactococcus (5%) and Pediococcus (4%). In this study, S. bovis was the most predominant species as this species represents 47.0% of the total isolates in dahi. The other species we isolated were identified as Lb. fermentum, Lb. delbrueckii ssp. bulgaricus, Lb. delbrueckii ssp. lactis, Lb. sp., Ec. faecium, S. thermophilus, Leuc. mesenteroides ssp. mesenteroides, Leuc. mesenteroides ssp. dextranicum, Lc. lactis ssp. lactis, Lc. raffinolactis and P. pentosaceus.