Sake Strains Research Articles

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His(-), Met(-), Lys(-), Trp(-), Leu(-), Arg(-), and Ura(-) auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.

PURIFICATION AND MECHANISTIC ACTION OF A BACTERIOCIN PRODUCED BY A BRAZILIAN SAUSAGE ISOLATE, LACTOBACILLUS SAKE 2a

ABSTRACT Bacteriocin 2a is produced by a Lactobacillus sake strain isolated from Brazilian sausage. This bacteriocin was purified 72,000 fold by acid extraction from L. sake 2a, followed by cation exchange and reverse phase chromatography. Bacteriocin 2a dissipated the proton motive force in Listeria monocytogenes and reduced the intracellular ATP with no detectable increase in extracellular ATP. Bacteriocin 2a also mediated a concentration‐dependent efflux of carboxyfluorescein from liposomes made from L. monocytogenes lipids. These data indicate that bacteriocin 2a forms pores in the membrane of target cells.

Inhibition of Listeria monocytogenes by a bacteriocinogenic Lactobacillus sake strain in modified atmosphere-packaged Brazilian sausage

Lactobacillus sake 2a is a bacteriocinogenic strain isolated from “lingüiça frescal”, a Brazilian sausage. The combined effect of modified-atmosphere (MA) packaging (100% CO 2 and 50% CO 2/50% N 2) and addition of L. sake 2a on inhibition of growth of Listeria monocytogenes was evaluated in “lingüiça” stored at 6 °C. By the end of the first week, the inhibition of L. monocytogenes due to MA was significant ( P⩽0.05) while the presence of L. sake 2a did not influence significantly the growth of the pathogen. After 14 days, a reduction of 1.3–1.4 log in counts of L. monocytogenes was observed in samples containing L. sake 2a only or MA packaged only, while a reduction of 3.5 log was detected in those submitted to both treatments. Results indicate that inhibition of L. monocytogenes in “lingüiça frescal” by the bacteriocinogenic L. sake 2a is enhanced by the packaging of the product in MA.

Antioxidative properties of Lactobacillus sake upon exposure to elevated oxygen concentrations

The ability of bacteria to overcome oxidative stress is related to the levels and types of antioxidative mechanisms which they possess. In this study, the antioxidative properties in Lactobacillus sake strains from different food origins were determined at low temperature (8°C) and upon exposure to oxygen levels between 20 and 90% O 2. The L. sake strains tested grew well at 8°C and in the presence of 20% O 2, however, most of the strains could not grow at O 2 levels as high as 50 and/or 90%. Cell-free extracts of all strains possessed certain levels of hydroxyl radical scavenging, metal chelating and reducing capacities essential for growth of cells at ambient O 2. At elevated O 2 concentrations, a high H 2O 2 splitting capacity and low specific rates of H 2O 2 production were demonstrated in the O 2-insensitive strain L. sake NCFB 2813, which could grow at elevated O 2 conditions. Although H 2O 2 was generated in the O 2-sensitive L. sake DSM 6333 at levels which were not directly toxic to the cells (<0.2 mM), we can conclude that its removal is essential for cell protection at elevated O 2 conditions.

Superoxide dismutase plays an important role in the survival of Lactobacillus sake upon exposure to elevated oxygen.

In this study, the responses of two Lactobacillus sake strains to elevated oxygen concentrations at 8 degrees C were investigated. L. sake DSM 6333 (L. sake(sens)), unlike L. sake NCFB 2813 (L. sake(ins)), showed a low growth rate in the presence of 90% O(2) and a rapid loss in viability shortly after entry into stationary phase. The steady-state cytosolic superoxide radical (O(2)(-)) concentration in L. sake(sens) was 0.134 microM and in the oxygen-insensitive mutant LSUV4 it was 0.013 microM. The nine- to ten-fold decrease in the rate of O(2)(-) elimination in L. sake(sens) indicates the significance of the O(2)(-)-scavenging system in protecting against elevated O(2). The superoxide dismutase (SOD) activity was 10- to 20-fold higher in L. sake(ins) than in L. sake(sens), depending on the growth phase. An oxygen-insensitive mutant of L. sake(sens), designated as strain LSUV4, had a ten-fold higher SOD activity than the wild-type strain, which likely restored its oxygen tolerance. Damage to proteins in L. sake(sens) was evidenced by the increased protein carbonyl content and reduced activities of the [Fe-S]-cluster-containing enzymes fumarase and fumarate reductase. This study forms a physiological basis for understanding the significance of elevated oxygen stress as an additional method for inhibition of microbial growth in relation to food preservation.

Viability, Efficacy, and Storage Stability of Freeze-Dried Biocontrol Agent Candida sake Using Different Protective and Rehydration Media

Viability, efficacy against Penicillium expansum on Golden Delicious apples, and storage stability of freeze-dried Candida sake strain CPA-1 were studied. The effect of several protective agents and rehydration media was investigated in the freeze drying of C. sake. Skimmed milk at 10% concentration was a good rehydration medium for all protectants tested. In general, good viability results were obtained when the same solution was used as a protectant and as a rehydration medium. The best survival was obtained when C. sake cells were protected with 10% lactose + 10% skimmed milk and rehydrated with skimmed milk (85% viability). The potential for biocontrol of the best freeze-dried treatments against P. expansum on apples was compared with that of fresh cells. Freeze-dried treatments at 1 × 107 CFU/ml reduced the incidence of decay by 45 to 66%. The best biocontrol effect was obtained with cells that had been freeze dried using 10% lactose + 10% skimmed milk as a protectant and 1% peptone as a rehydration medium, with a 66% reduction in rot incidence. However, in all treatments, the efficacy of freeze-dried cells was significantly lower than fresh cells. The stability of freeze-dried samples decreased during storage and was influenced by storage temperature. In the best treatment, storage of C. sake cells for 60 days at 4°C resulted in final concentrations of 2.5 × 108 CFU/ml, which was a 10-fold reduction in relation to the initial starting concentration of cells prior to freeze drying.

Purification and characterization of an intracellular beta-glucosidase from a Candida sake strain isolated from fruit juices.

A yeast strain isolated in the laboratory from fruit juices was studied and classified as Candida sake. The strain produces an intracellular beta-glucosidase when grown with cellobiose as the carbon source. The enzyme was purified by ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular beta-glucosidase, estimated by gel filtration, was 240 kDa. The tetrameric structure of the beta-glucosidase was determined following treatment of the purified enzyme with sodium dodecyl sulfate. The enzyme exhibited optimum activity at 52 degrees C and pH 4.25 with citrate-phosphate buffer. The enzyme was active against soluble glycosides with the (1-->4)-beta configuration, and from Lineweaver Burk plots, a Km value of 6.9 mmol/L was found for p-nitrophenyl-beta-D-glucopyranoside. The beta-glucosidase was found to be tolerant to glucose inhibition with a Ki value of 0.2 mol/L.

Growth Control of Listeria monocytogenes on Cold-Smoked Salmon Using a Competitive Lactic Acid Bacteria Flora

A Lactobacillus sake strain LKE5 and four strains of Carnobacterium piscicola were evaluated as biopreservation cultures to control the growth of Listeria monocytogenes on vacuum-packed, cold-smoked salmon stored at 5°C. All five strains were antilisterial as live cultures in an agar diffusion assay. Cell-free supernatants of two strains of C. piscicola and L. sake LKE5 were also antilisterial because of the production of bacteriocins. The presence of high cell numbers of strains of C. piscicola had no influence on the sensory quality of cold-smoked salmon stored at 5°C, but L. sake LKE5 caused strong sulfurous offflavors and was rejected as a culture for biopreservation of cold-smoked salmon. A bacteriocin-producing strain of C. piscicola (A9b) initially caused a 7-day lag phase of L. monocytogenes, followed by a reduction in numbers of L. monocytogenes from 103 CFU/ml to below 10 CFU/ml after 32 days of incubation, coinciding with the detection of antilisterial compounds. The presence of a nonbacteriocin-producing strain of C. piscicola (A10a) prevented the growth of L. monocytogenes during the 32-day incubation. The growth of L. monocytogenes was strongly repressed on cold-smoked salmon in the presence of C. piscicola A9b and A10a, respectively. The initial cell numbers of L. monocytogenes that were found on Oxford plates incubated at 25°C reached low maximum cell counts of 104 and 2 × 103 after 14 and 20 days of storage in mixed culture with C. piscicola A9b and A10a.

A system for heterologous expression of bacteriocins in Lactobacillus sake.

A system for efficient heterologous expression of class II bacteriocins is described that is based on introducing two plasmids in a bacteriocin-negative Lactobacillus sake strain. The first plasmid (pSAK20) contains the genes necessary for transcriptional activation of the Sakacin A promoter as well as export and processing of bacteriocin precursors. The second plasmid (a pLPV111 derivative) contains the structural and immunity genes for the bacteriocin of interest fused to the sakacin A promoter. Using this system, various bacteriocins were produced at levels equal to or higher than those obtained with the corresponding wild-type producer strains.

A system for heterologous expression of bacteriocins in Lactobacillus sake

A system for efficient heterologous expression of class II bacteriocins is described that is based on introducing two plasmids in a bacteriocin-negative Lactobacillus sake strain. The first plasmid (pSAK20) contains the genes necessary for transcriptional activation of the Sakacin A promoter as well as export and processing of bacteriocin precursors. The second plasmid (a pLPV111 derivative) contains the structural and immunity genes for the bacteriocin of interest fused to the sakacin A promoter. Using this system, various bacteriocins were produced at levels equal to or higher than those obtained with the corresponding wild-type producer strains.

Inhibition of Listeria monocytogenes in a pork product by a Lactobacillus sake strain

A Lactobacillus sake strain ( L. sake 2a), isolated from Brazilian sausage (`lingüiça'), possessed antilisterial activity. The inhibitory activity observed in culture media was due to a proteinaceous compound, as demonstrated by the sensitivity to proteolytic enzymes. Inhibition due to hydrogen peroxide, organic acids and lytic bacteriophages was ruled out. The inhibition of Listeria monocytogenes by L. sake 2a was also demonstrated in laboratory-prepared `lingüiça'. After four weeks at 8°C, counts of L. monocytogenes in the pork product co-inoculated with L. sake 2a were about 6 log 10 lower than those in control samples containing only L. monocytogenes. These results suggest that L. sake 2a strain produces a bacteriocin-like substance that can be used to inhibit the growth of L. monocytogenes on Brazilian sausage.

Characterization of Lactobacillus sake isolates from dry-cured sausages by restriction fragment length polymorphism analysis of the 16S rRNA gene.

Lactobacillus sake strains originally isolated from dry-fermented sausages were characterized by phenotypic and genotypic methods, including DNA-DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates of Lact. sake showing a characteristic phenotype (melibiose-positive, maltose- and arabinose-negative) were identified by DNA-DNA hybridization. Subsequently, RFLP studies using EcoRI and HindIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, EcoRI-digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When HindIII-digested DNA was hybridized with the cDNA probe, strain-specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when EcoRI and HindIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake.

Candida sake: a relevant species in the context of HIV-associated oropharyngeal candidosis?

Candida sake is routinely identified in the oral cavity of patients infected with the human immunodeficiency virus (HIV) using the commercial identification system ATB 32 C. To establish the prevalence of C. sake and to evaluate this designation repeatedly found using the ATB 32 C system, 94 HIV-infected patients were investigated for the presence of oral candidosis based on clinical and microbiological grounds. A total of 186 Candida isolates from 62 patients were obtained. Using the assimilation assay, C. sake was suspected in 49 isolates, but only seven strains were positively identified according to ATB 32 C. With respect to antifungal susceptibility and clinical parameters the 49 strains did not differ markedly from the other strains. Only antifungal susceptibility to amphotericin B, ketoconazole, and flucytosine was increased in C. sake strains when the positively and equivocally identified strains by ATB 32 C were taken together. In addition, amplifying genomic DNA with primers T3B and AP3, C. sake could not be identified in four strains and in one strain, respectively. Therefore biochemical identification of C. sake seems to be misleading and clinical relevance may be lacking.

Ropy slime-producing Lactobacillus sake strains possess a strong competitive ability against a commercial biopreservative

Aseptically handled Frankfurters were treated with a commercial Lactobacillus alimentarius biopreservative and inoculated with different cell concentrations of four ropy slime-producing Lactobacillus sake strains. The packages were vacuum sealed and kept at 6°C for 28 days, after which the production of ropy slime was evaluated. The inoculation test was controlled by sealing the different control packages containing either aseptically manufactured sausages without any bacterial inoculation, packages containing biopreservative only or packages inoculated only with the four different ropy slime-producing strains. Authenticity of the biopreservative strain after the cold storage period was ascertained by performing EcoRI restriction endonuclease analysis of 30 randomly selected isolates originating from the biopreservative control packages. All patterns were identical to the pattern of the original L. alimentarius biopreservative strain. The biopreservative was found to be ineffective against the four ropy slime-producing L. sake strains. The strongest slime producers inoculated with approximately 1 colony forming units (CFU)/cm2 could compete efficiently with the L. alimentarius inoculated at a level of 107 CFU/cm2 on sausage surfaces. This commercial biopreservative failed to occupy the vital niche of the four ropy slime-producing L. sake strains leading to spoilage in almost all packages.

Lactobacillus curvatus subsp. curvatus subsp. nov. and Lactobacillus curvatus subsp. melibiosus subsp. nov. and Lactobacillus sake subsp. sake subsp. nov. and Lactobacillus sake subsp. carnosus subsp. nov., new subspecies of Lactobacillus curvatus Abo-Elnaga and Kandler 1965 and Lactobacillus sake Katagiri, Kitahara, and Fukami 1934 (Klein et al. 1996, emended descriptions), respectively.

Lactobacillus curvatus and Lactobacillus sake are each genetically homogeneous species, as indicated by the high levels of DNA homology (> or = 76%) exhibited by strains of these taxa. However, the results of a numerical analysis of total soluble cell protein patterns and biochemical test data revealed that there are two phenotypic subgroups within L, curvatus and two phenotypic subgroups within L. sake. The overall randomly amplified polymorphic DNA (RAPD)-PCR band patterns obtained for the majority of L. curvatus strains corresponded well to the pattern obtained for the type strain of L. curvatus (strain DSM 20019). However, six strains of L. curvatus had different, but similar, RAPD-PCR profiles and grouped in a separate genetic cluster, which was linked to one of the clusters of L. sake strains. On the basis of these results, differences in biochemical and physiological characteristics, and total soluble cell protein profiles, we describe the subspecies L. curvatus subsp. curvatus subsp. nov. and L. curvatus subsp. melibiosus subsp. nov. for L. curvatus Abo-Elnaga and Kandler 1965 (Klein et al. 1996, emended description). Strains of L. sake grouped in two RAPD-PCR clusters, which was consistent with previous reports of phenotypic heterogeneity. Strains of Lactobacillus bavaricus, including type strain LMG 9844, clustered with the type strain of L. sake (strain NCFB 2714), indicating that these organisms belong to the same genetic group. We propose that strains of L. sake Katagiri, Kitahara, and Fukami 1934 (Klein et al. 1996, emended description) should be reclassified as members of L. sake subsp. sake subsp. nov. and L. sake subsp. carnosus subsp. nov. Strains of L. bavaricus are reclassified as members of L. sake subsp. sake, and the name L. bavaricus Stetter and Stetter 1980 is rejected.

Technological and sensorial evaluation of Lactobacillus strains as starter cultures in fermented sausages

The performance of several lactobacilli strains isolated from naturally fermented sausages as starter cultures was evaluated. Microbiological, physical and chemical changes, in addition to sensorial aspects were studied. From the 12 different strains tested, 10 were capable of leading the fermentation in every batch throughout the process. The monitoring of the inoculated strains was easily carried out by plasmid profiles and by checking the pH rate drop of the sausages, which was slower for the non-inoculated lots. Treatment using natural fermentation resulted in a product where hydrogen sulphide odours, which could be related to the higher content of Enterobacteriaceae throughout the ripening process, diminished its overall acceptability. The lots seeded with different L. sake strains were found to be low in acid in sensory evaluation, correlating with a low lactic acid content. In contrast, the L. plantarum lot gave rise to an overacidified product related to having the highest amount of lactic acid at the end of the process. As a general rule lactobacilli strains isolated from meat origins are good candidates as starter cultures in the manufacture of dry-fermented sausages and produce satisfactory products depending on the specific strains used more than on the species.

Characterization of Lactobacillus sake strains associating with production of ropy slime by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) patterns

Randomly amplified polymorphic DNA (RAPD) patterns and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of ropy slime-producing Lactobacillus sake strains. The two most revealing commercially available primers (OPJ 12 and OPJ 16, Operon Inc. Alameda, USA) and two rare-cutting enzymes ( AvrII and SmaI) were chosen from a pretested lot for the typing of 69 ropy slime-producing strains, 7 non-ropy isolates and 4 non-ropy reference strains. Both RAPD and PFGE patterns confirmed the group division established in previous studies and provided new information concerning ropy slime-producing strains. PFGE patterns were found to have the greatest discriminatory power, revealing the genetic variation of the main group of ropy slime-producing L. sake strains and distinguishing all non-ropy strains from slime-producers.

RRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime

The rRNA gene restriction patterns (ribotypes) of 69 ropy slime producing Lactobacillus sake strains isolated mainly from vacuum-packaged meat products of ten meat plants were determined. Ribotypes were compared to the corresponding patterns of non-ropy L. sake strains, and also to other species of the genus Lactobacillus, Carnobacterium and Weissella associated with meat products. Ropy slime-producing L. sake strains were divided into four characteristic groups corresponding to the phenotypic carbohydrate grouping. No association between certain ribotypes and individual plants was detected. Ribotyping was unable to distinguish slime producing and non-ropy strains of L. sake group sharing the same carbohydrate pattern. Otherwise, ribotyping distinguished the ropy slime producing strains from the non-ropy L. Sake reference strains and all L. sake strains from other species of the genus Lactobacillus, Carnobacterium and Weissella. These results suggest that ribotyping is a suitable method for detection and surveillance of the contamination of ropy slime-producing L. sake strains but the patterns alone cannot be used as markers of slime production capability.

Control of beef spoilage by a sulfide-producing Lactobacillus sake strain with bacteriocinogenic Leuconostoc gelidum UAL187 during anaerobic storage at 2 degrees C.

Chill-stored, vacuum-packaged beef inoculated with sulfide-producing Lactobacillus sake 1218 developed a distinct sulfide odor within 3 weeks of storage at 2 degrees C, at which time the bacteria had reached maximum numbers of 10(6) CFU cm(-2). Coinoculation of the meat with the wild-type, bacteriocinogenic (Bac+) strain of Leuconostoc gelidum UAL187 delayed the spoilage by L. sake 1218 for up to 8 weeks of storage. Coinoculation of meat samples with an isogenic, slowly growing Bac+ variant, UAL187-22, or with the Bac- variant UAL187-13 did not delay the onset of spoilage by L. sake 1218. The study showed that spoilage of chill-stored, vacuum-packaged beef by a susceptible target organism could be dramatically delayed by the Bac+ wild-type strain of L. gelidum UAL187. Inoculation with L. sake 1218 can be used as a model system to determine the efficacy of biopreservation of vacuum-packaged meats.

Intergeneric and intrageneric conjugal transfer of plasmids pAMβ1, pIL205 and pIP501 in Lactobacillus sake

Transfer of the broad host range conjugative plasmids pAM β 1, pIP501 and its derivative pIL205 into Lactobacillus sake was studied. Frequencies up to 5 × 10 −6 transconjugants per recipient cell were found from Lactococcus lactis strain IL1403 to various strains of L. sake. The transfer takes place by a conjugation-like process on solid surface agar. Intrageneric transfer between L. sake strains was also observed, although at lower frequencies (10 −7 transconjugants per recipient cell). The ability to transfer these plasmids into L. sake strains which are either refractory to transformation or poorly transformable provides a method of genetic transfer, thus opening the way for cloning strategies developed in other Gram-positive bacteria.