Molecular characterization of Lactobacillus curvatus and Lact. sake isolated from sauerkraut and their application in sausage fermentations

Lactobacillus curvatus and Lact. sake are best adapted to meat fermentations and dominate the flora during the whole process. In fermenting sauerkraut, Leuconostoc mesenteroides subsp. mesenteroides is the major organism only during the early phase. In this environment Lact. curvatus and Lact. sake provide up to 50% of the microbial flora especially of the later phase, depending on the process conditions. Strains of Lact. curvatus and Lact. sake isolated from fermenting sauerkraut were identified by hybridization with species specific 23S rRNA-targeted oligonucleotide probes and further characterized. In 59 of 72 strains, plasmid DNA was detected. Small cryptic plasmids of 20 strains were found to be homologous with pLc2, a 2·6 kb plasmid from Lact. curvatus LTH683, which was originally isolated from meat. The ability to compete was investigated in fermenting sausages of two strains each of Lact. curvatus and Lact. sake isolated from sauerkraut. One strain each of Lact. curvatus and Lact sake was found to outnumber the meat-borne flora and govern the process.     

Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages

AIM: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. CONCLUSIONS: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. SIGNIFICANCE AND IMPACT OF THE STUDY: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.     

Rapid GC analysis of cellular fatty acids for characterizing Lactobacillus sake and Lact. curvatus strains of meat origin

A rapid procedure based on the gas chromatographic analysis of cellular fatty acids was used to differentiate between strains of Lactobacillus sake and Lact. curvatus isolated from dry salami. All strains had very similar fatty acid profiles except four of them which lacked C19 cycl acid, but neither this feature nor other differences in single fatty acid contents could be successfully correlated with the biochemical discrimination of Lact. sake from Lact. curvatus . When, however, strains were compared on the basis of the total content of fatty acids with 18 carbon atoms divided by that with 16 carbon atoms, a very good correlation with strain characterization by classical methods was achieved. It was concluded that selected cellular fatty acid ratios might be useful for characterizing phylogenetically related strains of lactic acid bacteria.     

Conventional taxonomy of lactobacilli surviving radurization of meat

All of the 113 catalase-negative, Gram-positive, rod-shaped strains isolated from radurized minced beef (5 kGy) were homofermentative, non-thermophilic, and belonged to the sub-genus Streptobacterium. The majority of the strains (100) were identified as Lactobacillus sake. These were divided into four sub-groups based on their sugar fermentation pattern: group IA1 (melibiose (+), maltose (-), amygdalin (-), 76 strains); group IA2 (melibiose (+), maltose (-), amygdalin (+), 14 strains); group IB1 (melibiose (+), maltose (+), amygdalin (+), four strains); group IB2 (melibiose (+), maltose (+), amygdalin (-), six strains). Of the remaining strains, two produced L(+)-lactic acid and were identified as L. farciminis, three were identified as L. curvatus and eight showed characteristics of both L. sake and L. curvatus and were designated 'L. sake/curvatus.' With one exception, all strains were aciduric and relatively insensitive to the chemical preservatives tested. Most L. sake strains produced significant amounts of H2O2. Electron microscopy confirmed a possible relationship between the thickness of the cells and radiation resistance. The problems and limitations of this type of taxonomic study and possible reasons for the predominance of L. sake species in radurized meat are discussed.     

Electrotransformation of meat lactobacilli. Effect of several parameters on their efficiency of transformation

Intact cells of several lactobacilli isolated from Spanish dry fermented sausages ( Lactobacillus curvatus, Lact. sake, Lact. plantarum and Lact. bavaricus ) were transformed by electroporation. With pNZ12 as a vector, transformation efficiencies of 2.4 times 10⁵, 3.8 times 10³ and 8.8 times 10² transformants μg^-1 DNA were observed for Lact. curvatus CTC435, Lact. sake CTC335 and Lact. bavaricus CTC232, respectively.
Effects of variation in experimental parameters on transformation efficiency were evaluated. Strains, vectors and buffers were the determinant parameters. The growth phase of the culture, cell concentration, voltage, use of cell wall weakening agents and the purity of the vector influenced the transformation efficiency in most strains.     

Numerical taxonomy and identification of lactic acid bacteria from spoiled, vacuum-packaged vienna sausages

Sixty-one lactic acid bacteria from spoiled vacuum-packaged vienna sausages and 15 reference strains were tested for 72 phenotypic characteristics. An identification key and a computer data base, both specific for lactic acid bacteria from meat sources, were used for identification and the results were compared. There was a high correlation (86.9%) between the two procedures in the identification of strains to genus level. However, only a 54.8% correlation was obtained in identifying strains to the species level. With numerical taxonomy ( S _sm matching coefficient with average linkage clustering) 60 strains were recovered in six clusters at the 89% similarity level. While most Leuconostoc strains clustered separately from the Lactobacillus strains, the identity of many leuconostocs was not clarified. The presence of a heterogeneous cluster containing typical and 'atypical' strains of the Lactobacillus saké/curvatus group and a separate homogeneous Lact. curvatus cluster was noted. Closer examination of the data suggested that the 'atypical' lactobacilli were all strains of Lact. saké.     

Enzyme production by lactobacilli and the potential link with infective endocarditis

H.J. OAKEY, D.W.S. HARTY AND K.W. KNOX. 1995. Fifty-six strains of lactobacilli were examined for the production of glycosidases and proteases (arylamidases) that could be associated with the ability to grow in vivo and/or be a factor in the pathogenesis of endocarditis. The strains were from seven species, with an emphasis on Lactobacillus rhamnosus and Lact. paracasei subsp. paracasei , both of which have been associated with endocarditis and provided 12 of the 13 strains isolated from cases of the disease. Other species were Lact. acidophilus, Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris.
Commonly expressed glycosidase activities were α-D-galactosidase and β- N -acetyl-D-glucosaminidase followed by β-D-glucosidase and α-L-fucosidase. The combined production of β- N -acetyl-D-glucosaminidase and α-D-galactosidase was a feature of the endocarditis isolates. In contrast, β-D-galactosidase was produced by very few of the strains within species implicated in endocarditis but most of the strains of Lact. salivarius, Lact. fermentum and Lact. oris.
The most commonly produced arylamidases active against substrates employed for testing human blood clotting cascade were activated protein C(Ca)-like, activated factor X(Xa)-like and Hageman factor-like followed by kallikrein-like and chymotrypsin-like enzymes. Kallikrein-like enzyme activity was shown more frequently by strains from species commonly isolated from cases of endocarditis ( Lact. rhamnosus and Lact. paracasei subsp. paracasei ) than from other oral species ( Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris ).
The data indicate that some lactobacilli can produce enzymes that would enable the breakdown of human glycoproteins and the synthesis and lysis of human fibrin clots, characteristics which aid the colonization and survival of bacteria infecting an endocarditis vegetation.     

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.     

Conventional taxonomy of lactobacilli surviving radurization of meat

All of the 113 catalasc-negative, Gram-positive, rod-shaped strains isolated from radurized minced beef (5 kGy) were homofermentative, non-thermophilic, and belonged to the sub-genus Streptobacterium. The majority of the strains (100) were identified as Lactobacillus sake. These were divided into four sub-groups based on their sugar fermentation pattern: group IA₁ (melibiose (+), maltose (—), amygdalin (—), 76 strains); group IA₂ (melibiose (+), maltose (—), amygdalin (+), 14 strains); group IB₁ (melibiose (+), maltose (+), amygdalin (+), four strains); group IB₂ (melibiose (+), maltose (+), amygdalin (—), six strains). Of the remaining strains, two produced L(+)-lactic acid and were identified as L. farciminis , three were identified as L. curvatus and eight showed characteristics of both 'L. sake and L. curvatus and were designated 'L. sake/curvatus.' With one exception, all strains were aciduric and relatively insensitive to the chemical preservatives tested. Most L. sake strains produced significant amounts of H₂O₂. Electron microscopy confirmed a possible relationship between the thickness of the cells and radiation resistance. The problems and limitations of this type of taxonomic study and possible reasons for the predominance of L. sake species in radurized meat are discussed.     

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 Eco RI and Hin dIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, Eco RI-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 Hin dIII-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 Eco RI and Hin dIII 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 .     

Taxonomic characterization of Lactobacillus delbrueckii subsp. bulgaricus isolates from a Cameroonian zebu's fermented raw milk

Atypical thermophilic homofermentative lactobacilli were isolated as one of the major microflora from Pindidam, an indigenous Cameroonian zebu's fermented raw milk. On the basis of their physiological characteristics, obtained isolates constituted a homogeneous group belonging to the species Lactobacillus delbrueckii. Nevertheless, their carbohydrate fermentation pattern was unusual and their identification to one of two subspecies Lact. delbrueckii subsp. bulgaricus (Lact. d. bulgaricus ) or Lact. delbrueckii subsp. lactis (Lact. d. lactis ) was rendered difficult. DNA-DNA hybridization confirmed that they belonged to the species Lact. delbrueckii and the electrophoretic mobility of the D-(—)-lactate dehydrogenase (LDH) furthermore precisely assigned them to Lact. d. bulgaricus. Whole-cell protein profiles were only able to weakly discriminate between these wild isolates and type strains of the species Lact. delbrueckii. The isolates from Pindidam were well adapted to their specific environmental conditions and demonstrated both high growth rates and ability to support elevated acidic levels in fermented milk.     

A polyphasic taxonomic study of Chryseobacterium strains isolated from dairy sources

A polyphasic taxonomic study, employing protein electrophoresis (SDS-PAGE), gas chromatographic analysis of cellular fatty acids (FAME), mol% G+C determination and DNA-DNA hybridizations, was undertaken on 103 dairy isolates shown to belong to Chryseobacterium. Reference strains of the Chryseobacterium species, CDC group IIb and Embedobacter brevis were included. SDS-PAGE analysis yielded good differentiation between the investigated species. About half of the strains could be clustered into nine major groups while the other half occupied a separate position. With FAME analysis no clear differentiation of the Chryseobacterium species (except C. meningosepticum) and SDS-PAGE groups could be achieved. FAME analysis, however, gave good differentiation between the Chryseobacterium and Empedobacter strains. The mol% G+C of the isolates tested, ranged between 36.4 and 39.0. The combination of SDS-PAGE and DNA-DNA hybridization identified a large group of dairy isolates as C. indologenes, one isolate as C. gleum and two new genotypic groups, comprising five and 15 dairy isolates respectively, emerged from the polyphasic study. Another large part of strains have a separate or uncertain position in Chryseobacterium and remained classified as Chryseobacterium species CDC group IIb.     

The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

Lactose metabolism in Lactobacillus curvatus and Lactobacillus sake

Abstract The lactose metabolism was investigated in five strains of Lactobacillus curvatus and 14 strains of L. sake isolated from meat or meat-derived products. Strains with the ability to ferment lactose were found in both species. They exhibited either phospho-β-galactosidase (P-β-gal) or β-galactosidase (β-gal) activity, or both. P-β-gal activity of L. curvatus and L. sake was induced and detected only in the presence of lactose or galactose. Furthermore, catabolite repression by glucose was demonstrated. The immunological properties of the P-β-gal enzymes of these organisms resemble those of Lactococcus lactis . Several strains of L. sake but none of L. curvatus exhibited β-gal activity which was constitutive. In hybridisation experiments, the β-gal genes of L. sake and L. casei ATCC393 showed over 60% DNA-homology. The presence of β-gal genes in L. sake was demonstrated in both β-gal-producing and non-producing strains. This observations is consistent with a genetic potential of lactic acid bacteria exceeding their physiological capabilities.     

Antimicrobial activity of lactic acid bacteria isolated from sour doughs: purification and characterization of bavaricin A, a bacteriocin produced by Lactobacillus bavaricus MI401

Three hundred and thirty-five lactic acid bacteria were isolated from sour doughs and screened for antagonistic activity. Of these 145 showed activity against one or several of the indicator strains used in the screening. The antimicrobial activity of 18 isolates were due to a proteinaceous compound. These 18 isolates belonged to three different Lactobacillus species: Lactobacillus bavaricus, Lactobacillus curvatus and Lactobacillus plantarum. The spectrum of antimicrobial activity for the three species suggested that the inhibitory components were different. The inhibitory compound from Lact. bavaricus MI401 was chosen for further study. The proteinaceous nature, antimicrobial activity against closely-related species, heat resistance and sensitivity to alkaline treatment strongly indicated that this substance was a bacteriocin, which we designated bavaricin A. The bacteriocin was purified to homogeneity by ammonium sulphate precipitation, ion exchange, hydrophobic interaction and reverse-phase chromatography. The purification resulted in 193000-fold increase in specific activity. SDS-PAGE of bavaricin A showed a molecular weight of 3500—4000 Da. By amino acid sequencing 41 amino acids were determined. Bavaricin A had a bactericidal mode of action and inhibited nine out of 10 Listeria monocytogenes. Lactobacillus bavaricus MI401 produced bavaricin A at temperatures from 4°C to 30°C. The production of active bavaracin A was inhibited at increasing sodium chloride concentration. In the presence of 3% sodium chloride at 4°C no active bavaricin A could be detected. Nitrite (100 ppm) did not affect the production of active bavaricin A.     

Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans

Lactobacillus pentosus TV35b, isolated from the posterior fornix secretions of the vagina of a prenatal patient, produced a bacteriocin-like peptide (pentocin TV35b), which is inhibitory to Clostridium sporogenes, Cl. tyrobutyricum, Lact. curvatus, Lact. fermentum, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp. and Candida albicans. The mechanism of activity of pentocin TV35b is bactericidal, as shown by a decrease in the viable cell numbers of Lact. sake from approximately 4 x 108 to less than 10 cfu ml - 1 over a period of 4 h. Pentocin TV35b added to the growth medium of C. albicans stimulated the formation of pseudohyphae during the first 36 h, followed by a slight repression in cell growth. Production of pentocin TV35b was at its maximum towards the end of the logarithmic growth phase of strain TV35b. The peptide was purified by ammonium sulphate precipitation, followed by SP-Sepharose cation exchange chromatography. The molecular size of pentocin TV35b was estimated to be between 2.35 and 3.4 kDa, according to tricine-SDS PAGE. However, results obtained by electrospray ionization mass spectroscopy indicated that the peptide is 3930.2 Da in size. Amino acid analysis performed by using the Pico-Tag(R) method and a Nova-Pak C18 HPLC column indicated that pentocin TV35b consists of 33 amino acids with a total mass of 3929.63 Da. Pentocin TV35b is inactivated when treated with papain and Proteinase K, but remains active after incubation at pH 1-10 for 2 h at 25 degrees C, and when heat-treated for 30 min at 100 degrees C.     

The microbial association of Greek taverna sausage stored at 4 and 10 degrees C in air, vacuum or 100% carbon dioxide, and its spoilage potential

Strains of the Lactobacillus sakei/curvatus group, mainly non-slime-producing Lact. sakei, dominated the microbial flora of industrially manufactured taverna sausage, a traditional Greek cooked meat, stored at 4 degrees C and 10 degrees C in air, vacuum and 100% CO2. Atypical, arginine-positive and melibiose-negative strains of this group were isolated. The isolation frequency of Lact. sakei/curvatus from sausages stored anaerobically was as high as 92-96%, while other meat spoilage organisms were practically absent. Conversely, in air-stored sausages, leuconostocs, mainly Leuconostoc mesenteroides ssp. mesenteroides, had a considerable presence (14-21%), whereas Brochothrix thermosphacta, pseudomonads and Micrococcaceae grew, but failed to increase above 10(5) cfu g(-1) in all samples during storage. Only yeasts were able to compete against LAB and reached almost 10(7) cfu g(-1) after 30 d of aerobic storage at 10 degrees C. The great dominance (> 10(8) cfu g(-1)) of LAB caused a progressive decrease of pH and an increase of the concentration of L-lactate, D-lactate and acetate in all sausage packs. The growth of LAB and its associated chemical changes were more pronounced at 10 degrees C than 4 degrees C. At both storage temperatures, L-lactate and acetate increased more rapidly and to a higher concentration aerobically, unlike D-lactate, which formed in higher amounts anaerobically. Storage in air was the worst packaging method, resulting in greening and unpleasant off-odours associated with the high acetate content of the sausages. Carbon dioxide had no significant effect on extending shelf-life. The factors affecting the natural selection of Lact. sakei/curvatus in taverna sausage are discussed. Moreover, it was attempted to correlate the metabolic activity of this group with the physicochemical changes and the spoilage phenomena occurring in taverna sausage under the different storage conditions.     

Diversity of ethanol-tolerant lactobacilli isolated from Douro fortified wine: clustering and identification by numerical analysis of electrophoretic protein profiles

Eighty-two ethanol-tolerant (≥ 15% v/v) isolates of Lactobacillus were obtained from Douro fortified wines and associated winery equipment. The diversity and specific identity of these isolates was studied with computer-assisted comparison of the whole-cell, SDS-solubilized protein profiles (as generated by SDS-PAGE); with this method four species were identified. The majority of isolates (89%) were identified as Lact. hilgardii , and three differentiable electrophoretic groups of this species were evident. The identification was confirmed by DNA-DNA dot-blot hybridization studies. Also identified were one strain each of Lact. fructivorans, Lact. collinoides and Lact. mali.     

Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide

AIMS: The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. METHODS AND RESULTS: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. RESULTS AND CONCLUSIONS: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.     

Radiation resistance of lactobacilli isolated from radurized meat relative to growth and environment.

Of 113 lactobacilli isolated from radurized (5 kGy) minced meat, 7 Lactobacillus sake strains, 1 L. curvatus strain, and 1 L. farciminis strain were used for radiation resistance studies in a semisynthetic substrate (i.e., modified MRS broth). Five reference Lactobacillus spp., one Staphylococcus aureus strain, and one Salmonella typhimurium strain were used for comparative purposes. All L. sake isolates exhibited the phenomenon of being more resistant to gamma-irradiation in the exponential (log) phase than in the stationary phase of their growth cycles by a factor of 28%. Four references strains also exhibited this phenomenon, with L. sake (DSM 20017) showing a 68% increase in resistance in the log phase over the stationary phase. This phenomenon was not common to all bacteria tested and is not common to all strains with high radiation resistance. Four L. sake isolates and three reference strains were used in radiation sensitivity testing in a natural food system (i.e., meat). The bacteria were irradiated in minced meat and packaged under four different conditions (air, vacuum, CO2, and N2). Organisms exhibited the highest death rate (lowest D10 values [doses required to reduce the logarithm of the bacterial population by 1] ) under CO2 packaging conditions, but resistance to irradiation was increased under N2. The D10 values of the isolates were generally greater than those of the reference strains. The D10 values were also higher (approximately two times) in meat than in semisynthetic growth medium.