Detection of the bacteriocin propionicin PLG-1 with polyvalent anti-PLG-1 antiserum.

Polyclonal antibodies against the bacteriocin propionicin PLG-1 were produced in rabbits at high titer (256,000 to 512,000, as determined by indirect enzyme-linked immunosorbent assay [ELISA]). Anti-PLG-1 antiserum neutralized the antimicrobial activity of PLG-1 preparations in a well diffusion assay. Cross-reacting protein was detected using an indirect ELISA of the culture supernatant from a fed-batch fermentation of the producer strain Propionibacterium thoenii P127 within the first 24 h of incubation, but bacteriocin activity was not detected in the same culture until 217 h of incubation. Culture supernatants from 156 strains of classical dairy propionibacteria were tested by indirect ELISA at 5 and 12 days of incubation for production of cross-reacting protein and by well diffusion assay for bacteriocin activity. Cross-reacting protein was detected in 52 strains: all of the tested strains of P. thoenii, most of the strains of Propionibacterium jensenii, and a minority of the Propionibacterium acidipropionici and Propionibacterium freudenreichii strains. Of these 52 strains, only 4 strains of P. thoenii showed bacteriocin activity in a well diffusion assay. Eight bacteriocin-negative mutants of strain P127 were negative in both ELISA and well diffusion assays. Western blot analysis showed that three protein bands bound anti-PLG-1 antibodies in culture supernatants: a 9.1-kDa band that is assumed to be the PLG-1 monomer and 16.2- and 27.5-kDa bands that may be precursors, multimers, or complexes of PLG-1.

Reduction of Escherichia coli O157:H7 counts on whole fresh apples by treatment with sanitizers.

The objectives of this study were to determine if washing of whole apples with solutions of three different sanitizers (peroxyacetic acid, chlorine dioxide, or a chlorine-phosphate buffer solution) could reduce a contaminating nonpathogenic Escherichia coli O157:H7 population by 5 logs and at what sanitizer concentration and wash time such a reduction could be achieved. Sanitizers were tested at 1, 2, 4, 8, and 16 times the manufacturer's recommended concentration at wash times of 5, 10, and 15 min. Whole, sound Braeburn apples were inoculated with approximately 1 x 108 or 7 x 106 CFU per apple, stored for 24 h, then washed with sterile water (control) or with sanitizers for the prescribed time. Recovered bacteria were enumerated on trypticase soy agar. Washing with water alone reduced the recoverable population by almost 2 logs from the starting population; this can be attributed to physical removal of organisms from the apple surface. No sanitizer, when used at the recommended concentration, reduced the recovered E. coli population by 5 logs under the test conditions. The most effective sanitizer, peroxyacetic acid, achieved a 5-log reduction when used at 2.1 to 14 times its recommended concentration, depending on the length of the wash time. The chlorine-phosphate buffer solution reduced the population by 5 logs when used at 3 to 15 times its recommended concentration, depending on wash time. At no concentration or wash time tested did chlorine dioxide achieve the 5-log reduction.

Growth and oil production of Apiotrichum curvatum in tomato juice.

The lipid-accumulating yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D) grew in shake flask culture in freshly prepared tomato juice or tomato pulp; growth was improved when the medium was supplemented with sucrose and a basal salts mixture. Under controlled conditions in the fermentor, biomass and oil production reached a maximum at 96 h, compared with 130 h in shake flasks. Biomass production increased with the addition of 0.3% (wt/vol) urea to the juice, but decreased with the addition of 0.5% (wt/vol) yeast extract or Casamino Acids. The total amount of lipid produced and the percent of intracellular oil per cell dry weight were greatest in juice supplemented with 0.3% urea.

Quality of commercial shelf-stable soymilk products.

Four brands of shelf-stable soymilks (three lots each) were evaluated for their quality. All samples were tested for trypsin inhibitor activity, microbial load (coliforms, aerobic mesophiles, and psychrotrophs), solids content, pH, color, and viscosity. Storage stability at 5 degrees C of the opened original containers was evaluated by microbiological analyses. Physical properties were consistent among three lots of the same brand, but varied among brands. Trypsin inhibitor activity ranged from 3.43 to 10.35 mg/g dried soymilk. The pH values of the soymilks varied between 6.41 and 7.34. Viscosity was measured between 16.5 and 26.4 mPa. Solids content varied considerably among soymilks, from 6.88 to 12.37% (wt/vol). Freshly opened commercial soymilk contained fewer than one microorganism per ml in all microbial counts. During storage at 5 degrees C, microbial counts increased sharply after 2 to 3 weeks. A single microorganism predominated in each stored sample. Shelf life (defined as the time during which aerobic mesophile counts remained below 10(6) CFU/ml) varied from 3 to 4 weeks. The appearance and pH of soymilks remained unchanged even after 4 weeks of storage. Five types of microorganisms were found in stored soymilk samples; none of them produced acid.

Response of cultures of Propionibacterium to acid and low pH: tolerance and inhibition.

Seventeen Propionibacterium strains were tested for acid production and final pH achieved on glucose, fructose, or maltose as the primary carbon source. On average, strains of Propionibacterium acidipropionici produced more acid and reached lower final pH values than did strains of the other species. Three strains of P. acidipropionici, one Propionibacterium jensenii, and two Propionibacterium thoenii strains were tested further for the ability to survive and/or grow at low pH with lactic, hydrochloric, or propionic acid as acidulant. The organic acids were more inhibitory than hydrochloric acid; propionic acid was most inhibitory. In all cases, the P. jensenii and P. thoenii strains initiated growth and survived at lower pH values than did the p. acidipropionici stains. The ability to produce large amounts of acid or achieve low final pH values did not coincide with the ability to initiate growth or survive in low-pH conditions. Strains could not initiate growth below pH 5.0, but cultures started at neutral pH reached final pH values of less than 4.4. At neutral pH, strains could grow in the presence of increased lactate concentrations (up to 180 mM) or propionate concentrations (150 mM) that were inhibitory at acid pH. attempts to isolate variants able to initiate growth below pH 5.0 were unsuccessful.

Propionic acid production by extractive fermentation. I. Solvent considerations.

Solvent selection for extractive fermentation for propionic acid was conducted with three systems: Alamine 304-1 (trilaurylamine) in 2-octanol, 1-dodecanol, and Witcohol 85 NF (oleyl alcohol). Among them, the solvent containing 2-octanol exhibited the highest partition coefficient in acid extraction, but it was also toxic to propionibacteria. The most solvent-resistant strain among five strains of the microorganism was selected. Solvent toxicity was eliminated via two strategies: entrapment of dissolved toxic solvent in the culture growth medium with vegetable oils such as corn, olive, or soybean oils; or replacement of the toxic 2-octanol with nontoxic Witcohol 85 NF. The complete recovery of acids from the Alamine 304-1/Witcohol 85 NF was also realized with vacuum distillation.

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production by Propionibacterium acidipropionici.

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor with Propionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 milligrams propionic acid and 12.8 milligrams acetic acid were obtained in about 146 h in laboratory medium with 79 milligrams glucose added over five feeding periods. The highest concentration of propionic acid, 45 milligrams, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 milligrams propionic acid and 11 milligrams acetic acid were produced in 108 h from 59.4 milligrams total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30-37 milligrams propionic acid and 13 milligrams acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.

Broth recycle in a yeast fermentation.

Fermentation is a water-intensive process requiring treatment of large amounts of effluent broth. It is desirable to increase the ratio of product produced to the volume of effluent by minimizing the discharge of effluent from the fermentation process. A study of recycling spent fermentation process. A study of recycling spent fermentation broth for the subsequent fermentation was carried out with Apiotrichum curvatum an oleaginous yeast, as the working culture. Spent broth from a defined medium was recycled t replace as much as 75% of the water and salts for subsequent batches and this was repeated for seven sequential batches without affecting cell mass and lipid production. A 64% vlume reduction of wastewater was achieved in this manner. However, when using whey permeate as the medium, lipid production dropped after three consecutive recycle operations at 50% recycle, and after two consecutive recycle operations at 75% and 100% recycle. Accumulation of ions in the broth appeared to be responsible for the inhibition. An ion exchange step was able to eliminate the ion buildup and restore fermentation performance. (c) 1994 John Wiley & Sons, Inc.

Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici.

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 x 10(9) cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%-70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semide-fined medium, dilution rates up to 0.31 h-1 could be used, which gave higher volumetric productivities (0.96 g l-1 h-1 for propionic acid and 0.26 g l-1 h-1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.

Inhibition of psychrotrophic organisms by propionicin PLG-1, a bacteriocin produced by Propionibacterium thoenii.

Propionibacterium thoenii strain P127, which produces the bacteriocin propionicin PLG-1, was grown in a skim milk medium and produced bacteriocin in that medium. No bacteriocin activity was detected in skim milk medium in which strain P127-1, a bacteriocin-negative variant of strain P127, had been grown. Five psychrotrophic spoilage or pathogenic organisms (one strain each of Listeria monocytogenes, Pseudomonas fluorescens, Vibrio parahaemolyticus, Yersinia enterocolitica, and one strain of Corynebacterium sp.) were incubated for 24 h in laboratory medium, nonfermented skim milk, and skim milk that had been fermented by strain P127 or P127-1. Strains were inhibited only in the skim milk fermented by strain P127, as evidenced by loss in numbers of viable cells after 24 h at 10 degrees C and less growth than in other media after 24 h at optimal growth temperatures. Growth of selected strains was delayed or slowed during prolonged incubation (21 d) at 10 degrees C. Propionicin PLG-1 shows promise as a preservative for food products.

Isolation and purification of propionicin PLG-1, a bacteriocin produced by a strain of Propionibacterium thoenii.

Production of propionicin PLG-1 by Propionibacterium thoenii P127 was pH dependent, with maximal activity detected in supernatants of cultures grown at pH 7.0 Propionicin PLG-1 was purified by ion-exchange chromatography and isoelectric focusing. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of propionicin PLG-1 purified through isoelectric focusing resolved a protein band with a molecular weight of 10,000. Propionicin PLG-1 was bactericidal to sensitive cells, demonstrating single-hit kinetics. The producing strain harbored a single plasmid (pLG1) with an approximate size of 250 kb. Preliminary data indicate that both propionicin PLG-1 and immunity to the bacteriocin are encoded on the chromosome. Exposure of strain P127 to acriflavine or to N-methyl-N'-nitro-N-nitrosoguanidine yielded isolates that no longer produced bacteriocin activity and isolates that were cured of the plasmid. However, loss of bacteriocin production was not correlated with loss of the plasmid. Isolates cured of the plasmid were phenotypically identical to plasmid-bearing cells in fermentation patterns, pigment production, and growth characteristics.

Propionic Acid Production by a Propionic Acid-Tolerant Strain of Propionibacterium acidipropionici in Batch and Semicontinuous Fermentation.

A propionic acid-tolerant derivative of Propionibacterium acidipropionici P9 was obtained by serially transferring strain P9 through broth that contained increasing amounts of propionic acid. After 1 year of repeated transfers, a strain (designed P200910) capable of growth at higher propionic acid concentrations than P9 was obtained. An increase in the proportion of cellular straight-chain fatty acids and uncoupling of propionic acid production from growth were observed for strain P200910. Growth rate, sugar utilization, and acid production were monitored during batch and semicontinuous fermentations of semidefined medium and during batch fermentation of whey permeate for both strain P200910 and strain P9. The highest propionic acid concentration (47 g/liter) was produced by P200910 in a semicontinuous fermentation. Strain P200910 produced a higher ratio of propionic acid to acetic acid, utilized sugar more efficiently, and produced more propionic acid per gram of biomass than did its parent in all fermentations.

Evidence of peroxisomes and peroxisomal enzyme activities in the oleaginous yeast Apiotrichum curvatum.

The presence of peroxisomes and peroxisomal enzyme activities were investigated in the oleaginous yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D.) Catalase, a marker enzyme for peroxisomes, was measured in cell-free extracts prepared by sonication. The nature of the carbon and nitrogen sources in the growth medium greatly affected catalase activity. Cells grown on corn oil had high specific activity of catalase, but those grown on glucose, sucrose, or maltose had low specific activity. High specific activity of catalase was measured in cultures grown on media that supported poor growth (with soluble starch as carbon source or with methylamine, urea, or asparagine as nitrogen source). Peroxisomes from cells grown on corn oil were separated from other subcellular fractions in a discontinuous sucrose gradient. Major peaks of activity of fatty acid beta-oxidation and of two key enzymes in the glyoxylate cycle were found in fractions containing peroxisomes, but not in fractions corresponding to the mitochondria. Peroxisomal beta-oxidation showed equivalent activity with palmitoyl CoA or n-octanoyl CoA as substrate. Mitochondria did not seem to contain NAD-linked glutamate dehydrogenase. Peroxisomes with a homogeneous matrix and core surrounded by a single-layer membrane were observed with an electron microscope in cells grown on corn oil, but not in those grown on glucose. Staining with 3,3'-diaminobenzidine revealed that catalase activity was located in peroxisomes. Peroxisomes in this oleaginous yeast play important roles in lipid metabolism.

Partial Purification and Characterization of a Bacteriocin Produced by Propionibacterium thoenii.

A partially purified bacteriocin produced by Propionibacterium thoenii designated propionicin PLG-1 was found to be active against closely related species and exhibited a broad spectrum of activity against other microorganisms. Propionicin PLG-1 was found to be heat labile, sensitive to several proteolytic enzymes, and stable at pH 3 to 9. Propionicin PLG-1 was isolated from solid medium, partially purified by ammonium sulfate precipitation, and purified further by gel filtration. Gel filtration experiments revealed that bacteriocin PLG-1 was present as two different protein aggregates with apparent molecular weights of more than 150,000 and approximately 10,000. Resolution of these protein aggregates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein common to both with an apparent molecular weight of 10,000.

Lipid metabolism and cell composition of the oleaginous yeast Apiotrichum curvatum grown at different carbon to nitrogen ratios.

Apiotrichum curvatum ATCC 20509, an oleaginous yeast that can accumulate up to 60% of its cellular dry weight as intracellular lipid when grown with excess carbon, was grown in nitrogen-limited, balanced, and lactose-free medium with asparagine as nitrogen source and lactose as carbon source. Biomass and lipid accumulation were measured, cell composition was analyzed, and catalase activity was followed as marker enzyme for peroxisomes. The organism accumulated 54% of its dry weight as total cellular lipid when grown under nitrogen limitation and accumulated only 20-25% of its dry weight as lipid when grown in balanced medium. When starved for carbon, cells utilized endogenous lipid and carbohydrate as carbon and energy sources; the intracellular contents of lipid and carbohydrate decreased by 31 and 26%, respectively. Intracellular carbohydrates also seemed to be used as intermediates for lipid accumulation and lipid turnover. Catalase activity was strongly induced (over 10-fold increase in specific activity) when cells metabolized endogenous lipid. The lipid content of cells was inversely related to catalase activity and to intracellular protein or total nitrogen content. Lipid content showed no correlation with intracellular carbohydrate content.

Characterization of Propionibacterium plasmids.

Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.

Isolation and characterization of mutants of Propionibacterium strains.

Procedures were developed to isolate and characterize mutants of strains of dairy propionibacteria. These procedures included the construction of minimal defined media to support growth of the strains, optimization of conditions of exposure of the strains to nitrosoguanidine, and identification of the phenotypes of the mutants that were generated. The minimal defined medium contained inorganic salts, adenosine, three vitamins, and sodium lactate as the carbon source, with cysteine, methionine, or cysteine plus methionine added as required by some strains. For mutagenesis, cells were exposed to either 100, 200, or 1000 micrograms/ml nitrosoguanidine, depending on the sensitivity of the strain, for 60 min at 35 degrees C. At least nine stable mutants were isolated and characterized for each of the five strains under study. The most frequent mutations generated were requirements for arginine, histidine, methionine, and uracil and alteration in pigment production.

Microbiological Quality of Commercial Tofu.

A study was done to investigate the microbiological quality of commercial tofu available in local retail outlets. A sampling method was first developed to obtain accurate and representative microbial counts of individual pieces of tofu. Plate count determination of total aerobic organisms, psychrotrophs, coliforms, sporeformers, yeasts and molds, and staphylococci were made on 60 tofu samples (representing three lots each of four different brands) obtained within 24 h after delivery to the retail store. In addition, for two brands that provided manufacturer's pull dates, the same microbial counts were obtained for samples stored in the laboratory at 10°C until the pull date. Of the tofu sampled immediately after purchase, 83% of the lots tested had total counts greater than 106 colony-forming units (CFU)/g and psychrotrophic counts greater than 104 CFU/g. In addition, 67% of the lots tested had confirmed coliform counts greater than 103 CFU/g. Very low levels (less than 10 CFU/g) of all other microbial groups tested for were found in the majority of lots. Samples held until the manufacturer's pull date contained higher total and psychrotrophic counts but lower or stable counts of other organisms compared with samples tested immediately after purchase. To improve the microbiological quality of tofu, processors need to reduce initial loads by improving sanitation and processing techniques, and retailers should provide more consistent and colder refrigerated storage.

Chromosomal mapping of a gene affecting enterotoxin A production in Staphylococcus aureus.

In a previous study, transformation demonstrated that a gene governing enterotoxin A production (entA+) in Staphylococcus aureus strain S-6 was located on the chromosome between the purB110 and ilv-129 markers; in contrast, the entA+ gene of strain FRI-196E was shown not to be located in the same position. In the current study, 54 enterotoxin A-producing strains of S. aureus were examined to locate the entA+ gene. Conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 were used as recipients for chromosomal mapping. Of the 54 strains tested, 23 were found to contain the entA+ gene at the original locus between the purB110 and ilv-129 markers. Twenty-seven strains could not be analyzed either because their DNA was genetically ineffective in transforming strain 8325 (23 strains), or Pur+ Ilv+ transformants could not be recovered (four strains). Four other strains contained an entA+ gene that could not be located in any of the chromosomal linkage groups. A new insertion site for Tn551 was located within the hla+ gene involved in alpha-toxin production. It eliminated alpha-toxin production and was used to separate the entA+ gene from the hla+ marker in the purB110-ilv-129 region. This segment of the chromosome is shown to consist of the purB110, entA+, hla+, and ilv-129 markers in that order.