Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products.

A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp. in dairy products and vegetables. Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc. A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leuconostoc mesenteroides subsp. dextranicum, 16.9% were Leuconostoc mesenteroides subsp. paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.     

Fungi in flour and refrigerated dough products

Summary Fungal counts of 19 flour samples and 11 wheat samples from 11 flour mills of the Kansas-Nebraska and Pacific Northwest wheat-growing areas were made over a period of 2 years. In addition, 50 spoiled and 22 fresh samples of refrigerated dough products were examined for their fungal content. Methods for obtaining counts of fungi from flour and refrigerated dough were improved through the use of tetracycline as a bacterial inhibitor. Fungal counts ranged from 85 to 8,100 per gram in flour and from 90 to 1,400 in wheat. Generally, mold counts were higher in the flour than in the wheat. Judging from earlier reports, improved handling of wheat and better milling practices are effecting a gradual reduction in fungal counts in commercial flours. More than 500 fungi were isolated from the flours and doughs and were identified. Except for a few Fungi Imperfecti and Mucorales, the majority of the species from the flours belong in the generaAspergillus andPenicillium. Not only do they belong to these genera, but to specific groups in each. In the genusAspergillus, representatives of theA. candidus, A. glaucus, A. flavus-oryzae, andA. versicolor groups were the only ones present. InPenicillium the series commonly found wereP. cyclopium, P. citrinum, andP. urticae, and the remaining species were scattered among various series. Fungal counts of both fresh and spoiled dough samples were comparatively low. Essentially, the fungal flora of the doughs was a reflection of the flour microflora. Spoilage of the dough products did not appear to be of fungal origin.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.     

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.     

Microbiological analysis of rock cod (Sebastes spp.) stored under elevated carbon dioxide atmospheres

The numbers and types of microorganisms on fresh rock cod fillets and fillets stored in air or in a modified atmosphere (MA; 80% CO(2), 20% air) at 4 degrees C were compared. Samples were analyzed after 0, 7, 14, and 21 days of storage. The isolation plates were incubated aerobically, anaerobically, or under MA at 4, 20, or 35 degrees C. After 7 days of storage in air, the fillets were obviously spoiled and had a 3- to 4-log cycle increase in microbial counts. Plate counts increased more slowly on MA-stored fillets. After 21 days, the counts on the latter had increased only 2 log cycles, and the fillets did not seem spoiled. The microbial flora changed greatly during MA storage. Only Lactobacillus spp. (70%) and an Aeromonas sp.-like isolate (30%) were found on plates incubated aerobically at 4 and 20 degrees C, and only Lactobacillus spp. was found on plates incubated aerobically and anaerobically at 35 and at 20 degrees C under MA. Isolation plates incubated at 20 degrees C in air gave the highest counts in the shortest incubation time and the greatest diversity of bacterial types recovered. No Vibrio parahaemolyticus, Staphylococcus aureus, or Clostridium botulinum type E were isolated from the fresh or MA-stored fillets.     

Characterization of lactic acid bacteria isolated from seafood

Lactic acid bacteria were isolated from various samples of seafood: fresh pollock, brine shrimp, gravad fish, vacuum-packed seafood (surimi, smoked tuna, salted cod), and fish stored under 100% CO₂ at 5°C (smoked tuna, fresh and salted cod, salmon). Eighty-six independent isolates were obtained and were grouped according to cell morphology, presence or absence of diaminopimelic acid in the cell wall, and lactate configuration. Fifty-four isolates were identified as belonging to the genus Lactococcus and most of them exhibited DNA homologies with L. lactis subsp. lactis. Four strains were identified as Lactobacillus plantarum , eight strains as genus Leuconostoc and 16 belonged to the genus Carnobacterium. One facultative heterofermentative Lactobacillus and three other isolates were not identified. Of the strains 47% showed similar patterns of carbohydrate fermentations especially among strains belonging to the genera Lactococcus and Carnobacterium. Most of the strains (64%) grew at 5°C, in salted media and in fish extract medium without added sugar. Carnobacterium piscicola and Carn. divergens were the only reference strains able to grow in the same conditions as well as psychrotroph strains isolated from seafood. A numerical analysis could not be used because of the divergent properties of isolates of the same genus and strong similarities between different genera.     

Enumeration of thermophilic Bacillus species in composts and identification with a Random Amplification Polymorphic DNA (RAPD) protocol

The thermophilic microbial flora of general garden and domestic wastes composts, derived from thermogenic, post-thermogenic and maturation phases, was analysed using spore and total plate counts in combination with an optimised RAPD protocol. A total of 459 isolates were recovered obtained at 55 degrees C, and another 56 at 70 degrees C using tryptic soy-starch agar plates, with near-equal numbers being derived from total plate counts or spore preparations. The isolates were obtained from 11 compost samples and were assigned to eighteen different RAPD fingerprint types, with 76.1% of these ultimately being positively assigned by their RAPD profiles to just 2 species including Bacillus thermodenitrificans and B. licheniformis. Viable cell numbers ranged from 1.4 to 150 x 10(6) colony forming units per gram compost (wet weight), with the highest two counts being from 2 week and 4 week old compost samples with temperatures of 70 degrees C and 55 degrees C, respectively. B. thermodenitrificans was a dominant isolate (representing more than 50% of isolates from total plate counts) in 7 of the 11 individual compost total plate count samples between 30 degrees C to 73 degrees C, and accounted for 68.9% of all isolates overall. Another relatively common Bacillus species that was identified with RAPDs in significant numbers included B. licheniformis (7.2% of all isolates and dominant isolate in 1 sample). Three other relatively common RAPD profiles could not be identified by comparison with known species in a RAPD profile database but were tentatively identified using 16S rDNA sequence comparisons. These were B. sporothermodurans (4.9% of all isolates and dominant in 1 sample), B. thermosphaericus (7.4% and dominant in 1 sample) and Terrabacter tumescens (5.0%). Overall, based on the vegetative and spore count results and the subsequent RAPD-based identification, the data strongly support a significant role for B. thermodenitrificans in the composting process, and casts doubt on the notion that B. stearothermophilus sensu strictu (DSMZ 22) is a prominent member within compost ecology.     

Microbiological Analysis of Rock Cod (Sebastes spp.) Stored Under Elevated Carbon Dioxide Atmospheres

The numbers and types of microorganisms on fresh rock cod fillets and fillets stored in air or in a modified atmosphere (MA; 80% CO₂, 20% air) at 4°C were compared. Samples were analyzed after 0, 7, 14, and 21 days of storage. The isolation plates were incubated aerobically, anaerobically, or under MA at 4, 20, or 35°C. After 7 days of storage in air, the fillets were obviously spoiled and had a 3- to 4-log cycle increase in microbial counts. Plate counts increased more slowly on MA-stored fillets. After 21 days, the counts on the latter had increased only 2 log cycles, and the fillets did not seem spoiled. The microbial flora changed greatly during MA storage. Only Lactobacillus spp. (70%) and an Aeromonas sp.-like isolate (30%) were found on plates incubated aerobically at 4 and 20°C, and only Lactobacillus spp. was found on plates incubated aerobically and anaerobically at 35 and at 20°C under MA. Isolation plates incubated at 20°C in air gave the highest counts in the shortest incubation time and the greatest diversity of bacterial types recovered. No Vibrio parahaemolyticus, Staphylococcus aureus, or Clostridium botulinum type E were isolated from the fresh or MA-stored fillets.     

Characterization of micrococcaceae isolated from salt used for Spanish dry-cured ham

The microbial flora of salt used in the production of Spanish dry-cured ham was studied. The results indicated that Micrococcaceae constituted the predominant flora. Identification of the 369 isolates belonging to the family Micrococcaceae revealed that 60% belonged to genus Staphylococcus, 25% to Micrococcus, 6% to Kocuria, 5.4% to Dermacoccus and 0.5% to Stomatococcus. The species most often isolated was Staph. xylosus (28.9%), followed by M. lylae (21.4%), Staph. equorum (18.55%) and D. nishinomiyaensis (5.4%). The results indicate that the salt during salting process of dry-cured hams offers an ecosystem suitable for the survival of the staphylococci and micrococci.     

Polyphasic study of the spatial distribution of microorganisms in Mexican pozol, a fermented maize dough, demonstrates the need for cultivation-independent methods to investigate traditional fermentations

The distribution of microorganisms in pozol balls, a fermented maize dough, was investigated by a polyphasic approach in which we used both culture-dependent and culture-independent methods, including microbial enumeration, fermentation product analysis, quantification of microbial taxa with 16S rRNA-targeted oligonucleotide probes, determination of microbial fingerprints by denaturing gradient gel electrophoresis (DGGE), and 16S ribosomal DNA gene sequencing. Our results demonstrate that DGGE fingerprinting and rRNA quantification should allow workers to precisely and rapidly characterize the microbial assemblage in a spontaneous lactic acid fermented food. Lactic acid bacteria (LAB) accounted for 90 to 97% of the total active microflora; no streptococci were isolated, although members of the genus Streptococcus accounted for 25 to 50% of the microflora. Lactobacillus plantarum and Lactobacillus fermentum, together with members of the genera Leuconostoc and Weissella, were the other dominant organisms. The overall activity was more important at the periphery of a ball, where eucaryotes, enterobacteria, and bacterial exopolysacharide producers developed. Our results also showed that the metabolism of heterofermentative LAB was influenced in situ by the distribution of the LAB in the pozol ball, whereas homolactic fermentation was controlled primarily by sugar limitation. We propose that starch is first degraded by amylases from LAB and that the resulting sugars, together with the lactate produced, allow a secondary flora to develop in the presence of oxygen. Our results strongly suggest that cultivation-independent methods should be used to study traditional fermented foods.     

Factors Influencing the Occurrence of High Numbers of Iodine-Resistant Bacteria in Iodinated Swimming Pools

It has been shown that, although iodinated swimming-pool waters are usually free from coliform bacteria and enterococci, the total counts frequently become relatively high. Pseudomonas alcaligenes and Alcaligenes faecalis have been shown to account for most of these high counts. It was of interest, therefore, to compare the microbial flora of four alternately chlorinated and iodinated swimming pools. By means of the membrane filter method and suitable selective media, examinations were made for total viable counts, coliform bacteria, enterococci, staphylococci, Streptococcus salivarius, and P. aeruginosa. Colonies also were picked from membrane filters incubated on standard plate count agar and identified. The results showed that, although viable counts were significantly higher during the iodinated periods, the specific types of bacteria determined were either fewer than or the same as in chlorinated periods. During chlorination, the predominant microbial flora consisted of staphylococci and members of the genus Bacillus. During iodination, however, the P. alcaligenes-A. faecalis group accounted for 92 to 99% of the microbial flora. The accumulation of high numbers of these bacteria was shown to be due to their iodine resistance and their ability to grow rapidly in pool water in the absence of free iodine.     

Identification of lactic acid bacteria isolated from Portuguese wines and musts by SDS-PAGE

Thirty-two strains were isolated from spoiled port wines, from musts and from various styles of young, Northeastern Portuguese red table wines that had undergone spontaneous malolactic fermentation. Comparison of their SDS-PAGE whole-cell protein patterns with an SDS-PAGE database of lactic acid bacteria indicated that the isolates were members of the species Leuconostoc oenos or Lactobacillus paracasei subsp. paracasei. The latter were found in low acidity table wines and in port wine. The isolation of Lactobacillus paracasei strains from wines indicates the importance of using known strains for wine deacidification because spontaneous malolactic fermentation of table wines can occur from an indigenous flora, adapted to the particular composition of the wine.     

Lactic acid bacteria associated with vacuum-packed cooked meat product spoilage: population analysis by rDNA-based methods

AIM: To determine the lactic acid bacteria (LAB) implicated in bloating spoilage of vacuum-packed and refrigerated meat products. METHODS AND RESULTS: A total of 18 samples corresponding to four types of meat products, with and without spoilage symptoms, were studied. In all, 387 colonies growing on de Man, Rogosa and Sharpe, yeast glucose lactose peptone and trypticase soy yeast extract plates were identified by internal spacer region (ISR), ISR-restriction fragment length polymorphism and rapid amplified ribosomal DNA restriction analysis profiles as Lactobacillus (37%), Leuconostoc (43%), Carnobacterium (11%), Enterococcus (4%) and Lactococcus (2%). Leuconostoc mesenteroides dominated the microbial population of spoiled products and was always present at the moment bloating occurred. Lactobacillus sakei, Lactobacillus plantarum and Lactobacillus curvatus were found in decreasing order of abundance. The analysis of two meat products, 'morcilla' and 'fiambre de magro adobado' obtained from production lines revealed a common succession pattern in LAB populations in both products and showed that Leuc. mesenteroides became the main species during storage, despite being below the detection level of culture methods after packing. CONCLUSIONS: Our results pointed to Leuc. mesenteroides as the main species responsible for bloating spoilage in vacuum-packed meat products. SIGNIFICANCE AND IMPACT OF THE STUDY: Prevention of bloating spoilage in vacuum-packed cooked meat products requires the sensitive detection of Leuc. mesenteroides (i.e. by PCR).     

Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments.

Strict anaerobic culture techniques were used to quantitatively and qualitatively evaluate the anaerobic heterotrophic bacteria present at the sediment-water interface of hyperutrophic Wintergreen Lake (Augusta, Mich.). Anaerobic plate counts remained constant from March through December, 1973, ranging from 2.4 X 10(6) to 5.7 X 10(6) organisms/g (dry weight) of sediment. The isolatable bacteria represented a small percentage of the total microbial community, which was shown by direct microscopic counts to be 2.0 X 10' organisms/g (dry weight) of sediment during June and July. Bacteria of the genus Clostridium dominated the isolates obtained, accounting for 71.8% of the 960 isolates examined. A single species, Clostridium bifermentens, comprised 47.7% of the total. Additional bacterial groups and the percentage in which they were isolated included: Streptococcus sp. (10.8%), unidentified curved rods (9.5%y, gram-positive nonsporing rods (5.6%), and motile gram-negative rods (1.9%). Temperature growth studies demonstrated the ability of all the isolates to grow at in situ sediment temperatures. Gas-liqid radiochromatography was used to determine the soluble metabolic end products from [U-14C]glucose and a U-14C-labeled amino acid mixture by representative sedimentary clostridial isolates and by natural sediment microbial communities. At in situ temperatures the natural sediment microflora produced soluble fermentative end products characteristic of those elaborated by the clostridial isolates tested. These results are considered strong presumptive evidence that clostridia are actively metabolizing in the sediments of Wintergreen Lake.     

新疆伊犁地区原牛乳中乳酸菌的多样性分析

【目的】对新疆伊犁地区原牛奶中乳酸细菌的遗传多样性进行分析。【方法】采用菌落培养、Rep-PCR(Repetitive genomic fingerprinting)指纹图谱和16S r RNA基因序列分析相结合的方法研究牛乳内乳酸菌的遗传多样性。【结果】从5份原牛乳中分离出乳酸菌29株,基因序列分析和系统进化分析显示29株乳酸菌隶属于5个属,分别为:Lactococcus、Lactobacillus、Leuconostoc、Pediococcus和Enterococcus。优势属为Leuconostoc(27.6%),其次为Lactococcus(24.0%)。【结论】新疆伊犁地区原牛乳中乳酸菌多样性丰富,为开发新疆地区益生乳酸菌提供了丰富的活性资源。     

Characterization and Antibacterial Activity Against Helicobacter pylori of Lactic Acid Bacteria Isolated from Thai Fermented Rice Noodle

Probiotics and Antimicrobial Proteins - A total of 32 lactic acid bacteria (LAB) were isolated from Khanom-jeen, a Thai traditional fermented rice noodle. They belonged to the genus Leuconostoc...     

Psychrotrophic bacterial flora of raw ewes' milk, with particular reference to gram negative rods

The microbial flora of 141 samples of raw ewes' milk was determined, before and after storage for 72 h at 4 degrees and 7 degrees C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated (ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

Psychrotrophic bacterial flora of raw ewes'milk, with particular reference to Gram negative rods

The microbial flora of 141 samples of raw ewes'milk was determined, before and after storage for 72 h at 4° and 7°C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated ( ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

An ecological study of lactic acid bacteria from Almagro eggplant fermentation brines

AIM: Identification of the predominant lactic acid bacteria (LAB) involved in spontaneous fermentations of Almagro eggplants, and evaluation of the biodiversity by molecular typing. METHODS AND RESULTS: Almagro eggplant fermentations in three factories (A, B and C) enjoying Protected Designation of Origin (PDO) status were monitored by chemical and microbiological analysis of brines. LAB isolates from brines were identified by phenotypic analysis and by species-specific PCR reactions and typed by randomly amplified polymorphic DNA (RAPD)-PCR. All isolates from factories A and C belonged to the genus Lactobacillus (Lact.), whereas isolates from factory B belonged to Lactobacillus (50%), Leuconostoc (Ln.) (25%) and Lactococcus (Lc.) (25%); 1.9% of this microbiota was considered cosmopolitan. The genera Leuconostoc and Lactococcus and the species Lact. acidophilus and Lact. paracasei had never previously been reported in Almagro eggplant fermentations. CONCLUSION: Considerable differences in the composition of the lactic acid microbiota participating in the Almagro eggplant fermentations exist. Brine NaCl concentration has a notable influence both in number and in the species participating. SIGNIFICANCE AND IMPACT OF THE STUDY: The original aspect of this work consists of an ecological study of the LAB taking part in spontaneous Almagro eggplant fermentations from different factories. Participation of Leuconostoc and Lactococcus species and of Lact. acidophilus and Lact. paracasei, which had never before been described for this pickle, and the evidence that a lactic fermentation does not always take place, were the most relevant results.     

Diversity of bacteria associated with Bursaphelenchus xylophilus and other nematodes isolated from Pinus pinaster trees with pine wilt disease

The pinewood nematode (PWN), Bursaphelenchus xylophilus, has been thought to be the only causal agent of pine wilt disease (PWD), however, since bacteria have been suggested to play a role in PWD, it is important to know the diversity of the microbial community associated to it. This study aimed to assess the microbial community associated with B. xylophilus and with other nematodes isolated from pine trees, Pinus pinaster, with PWD from three different affected forest areas in Portugal. One hundred and twenty three bacteria strains were isolated from PWN and other nematodes collected from 14 P. pinaster. The bacteria strains were identified by comparative analysis of the 16S rRNA gene partial sequence. All except one gram-positive strain (Actinobacteria) belonged to the gram-negative Beta and Gammaproteobacteria. Most isolates belonged to the genus Pseudomonas, Burkholderia and to the family Enterobacteriaceae. Species isolated in higher percentage were Pseudomonas lutea, Yersinia intermedia and Burkholderia tuberum. The major bacterial population associated to the nematodes differed according to the forest area and none of the isolated bacterial species was found in all different forest areas. For each of the sampled areas, 60 to 100% of the isolates produced siderophores and at least 40% produced lipases. The ability to produce siderophores and lipases by most isolates enables these bacteria to have a role in plant physiological response. This research showed a high diversity of the microbial community associated with B. xylophilus and other nematodes isolated from P. pinaster with PWD.     

Effect of γ Irradiation on the Microflora of Freshwater Fish: III. Spoilage Patterns and Extension of Refrigerated Storage Life of Yellow Perch Fillets Irradiated to 0.1 and 0.2 Megarad1

Maximal shelf life was determined and microbial flora were compared for irradiated (0.1 and 0.2 Mrad) and nonirradiated yellow perch fillets stored at 1 C. Shelf life was estimated by organoleptic determinations. Microbiological studies included determination of the effects of irradiation on the total aerobic microbial population, lag phase, and rate of growth. Genera of organisms isolated from fillets through the course of microbial spoilage were identified, and the proteolytic activity of the organisms was determined. Plate counts for fish prior to irradiation showed the presence of approximately 10(6) organisms per g of sample. Irradiation to 0.1 and 0.2 Mrad produced 1.4 and 3 logarithm reductions of the initial count, respectively. Irradiation to 0.1 and 0.2 Mrad approximately doubled the product's shelf life. Organisms initially isolated from the nonirradiated fillets, in order of decreasing number, consisted of Flavobacterium, Micrococcus-Sarcina, Achromobacter-Alcaligenes-Mima, Pseudomonas, Microbacterium, Vibrio, Bacillus, Corynebacterium, Lactobacillus, Brevibacterium, and Aeromonas. By the 6th and 9th days of fillet storage, Pseudomonas and the Achromobacter group were the predominant organisms. All members of the genus Flavobacterium, but not all members of the genus Pseudomonas, were proteolytically active on raw fish juice-agar and skim milk-agar media. The Achromobacter group was found to be nonproteolytic on both media. Residual flora of fillets irradiated to 0.1 and 0.2 Mrad consisted of the Achromobacter group, Lactobacillus, Micrococcus-Sarcina, and Bacillus. Their sequence in predominance, however, varied with dose level. Not all proteolytic bacteria in the fillets were eliminated by 0.1 and 0.2 Mrad; proteolytic Micrococcus-Sarcina survived these treatments.