Detection of Pediococcus spp. in brewing yeast by a rapid immunoassay.

A membrane immunofluorescent-antibody test was developed to detect diacetyl-producing Pediococcus contaminants in brewery pitching yeast (yeast [Saccharomyces cerevisiae] slurry collected for reinoculation). Centrifugations at 11 and 5,100 x g separate yeast cells from bacteria and concentrate the bacteria, respectively. Pelleted bacteria resuspended and trapped on a black membrane filter are reacted with monoclonal antibodies specific for cell surface antigens and then with fluorescein-conjugated indicator antibodies. Whether pitching yeast is contaminated with pediococci at 0.001% is determined in less than 4 h. The sensitivity of the assay is 2 orders of magnitude below the Pediococcus detection limit of direct microscopy.     

Interactions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity.

The natural variation in the susceptibilities of gram-positive bacteria towards the bacteriocins nisin and pediocin PA-1 is considerable. This study addresses the factors associated with this variability for closely related lactic acid bacteria. We compared two sets of nonbacteriocinogenic strains for which the MICs of nisin and pediocin PA-1 differed 100- to 1,000-fold: Lactobacillus sake DSM20017 and L. sake DSM20497 and Pediococcus dextrinicus and Pediococcus pentosaccus. Strikingly, the bacteriocin-sensitive and -insensitive strains showed a similar concentration-dependent dissipation of their membrane potential (delta psi) after exposure to these bacteriocins. The bacteriocin-induced dissipation of delta psi below the MICs for the insensitive strains did not coincide with a reduction of intracellular ATP pools and glycolytic rates. This was not observed with the sensitive strains. Analysis of membrane lipid properties revealed minor differences in the phospho- and glycolipid compositions of both sets of strains. The interactions of the bacteriocins with strain-specific lipids were not significantly different in a lipid monolayer assay. Further lipid analysis revealed higher in situ membrane fluidity of the bacteriocin-sensitive Pediococcus strain compared with that for the insensitive strain, but the opposite was found for the L. sake strains. Our results provide evidence that the association of bacteriocins with the cell membrane and their subsequent insertion take place in a similar way for cells that have a high or a low natural tolerance towards bacteriocins. For insensitive strains, overall membrane constitution rather than mere membrane fluidity may preclude the formation of pores with sufficient diameters and lifetimes to ultimately cause cell death.     

Bacterial surface antigen-specific monoclonal antibodies used to detect beer spoilage pediococci.

Fourteen monoclonal antibodies (Mabs) were isolated that react with surface antigens of Pediococcus beer spoilage organisms, including P. damnosus, P. pentosaceous, P. acidilactici, and unspeciated isolates. Immunoblotting, enzyme immunoassays (EIAs) of protease- and neuraminidase-treated surface antigen extracts, carbohydrate competition EIAs, and cardiolipin EIAs were used to characterize the bacterial antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment or surface antigen extraction of washed bacteria. In most cases, the Mabs bind to Pediococcus surface antigens that appear to be covalently bound cell wall polymers resistant to alteration or removal from the bacterial surface. These bacterial surface antigen reactive Mabs show good potential for rapid, sensitive, and specific immunoassay detection of Pediococcus beer spoilage organisms.     

Fermentation of millet to produce kamu a Nigerian starch-cake food

Microorganisms present in the unfermented grains of millet at the Initial stage of steeping and after sieving at the initial stage of souring for the preparation of kamu were moulds (Aspergillus versicolor, Penicillim nigricans and Rhizopus stolonifer), bacteria (Pediococcus pentosaceus and Lactobaclllus plantarum) and a yeast (Saccharomyces cerevisiae). Only the bacteria and yeast persisted to the end of the steeping period. These, together with another yeast, Candida krusel, brought about the final souring of kamu.     

Beta-1,3-glucanase from Delftia tsuruhatensis strain MV01 and its potential application in vinification

During vinification microbial activities can spoil wine quality. As the wine-related lactic acid bacterium Pediococcus parvulus is able to produce slimes consisting of a β-1,3-glucan, must and wine filtration can be difficult or impossible. In addition, the metabolic activities of several wild-type yeasts can also negatively affect wine quality. Therefore, there is a need for measures to degrade the exopolysaccharide from Pediococcus parvulus and to inhibit the growth of certain yeasts. We examined an extracellular β-1,3-glucanase from Delftia tsuruhatensis strain MV01 with regard to its ability to hydrolyze both polymers, the β-1,3-glucan from Pediococcus and that from yeast cell walls. The 29-kDa glycolytic enzyme was purified to homogeneity. It exhibited an optimal activity at 50°C and pH 4.0. The sequencing of the N terminus revealed significant similarities to β-1,3-glucanases from different bacteria. In addition, the investigations indicated that this hydrolytic enzyme is still active under wine-relevant parameters such as elevated ethanol, sulfite, and phenol concentrations as well as at low pH values. Therefore, the characterized enzyme seems to be a useful tool to prevent slime production and undesirable yeast growth during vinification.     

Distribution of Bacterial Contaminants in a South African Lager Brewery

Bacteria isolated from contaminated pitching yeast, fermenting wort and beer samples from a South African lager brewery over a one-year period were tentatively identified by an improved, rapid diagnostic procedure as pediococci (41%), homofermentative lactobacilli (5%), heterofermentative lactobacilli (9%), Acetobacter spp. (7%), Gluconobacter spp. (13%) and Hafnia protea (25%). Pediococci and lactobacilli dominated samples taken from fermentation, storage and 'bright' beer tanks but were absent from pitched wort samples, from collection vessels and the single pitching yeast sample investigated. Acetic acid bacteria and H. protea were widely distributed in collection vessel, fermentation and storage tank samples, and H. protea was isolated from recycled pitching yeast.     

Production of 21% (v/v) ethanol by fermentation of very high gravity (VHG) wheat mashes

Summary Very high gravity wheat mashes containing 300 g or more sugares per liter were prepared by enzymatic hydrolysis of starch and fermented with a commercial preparation of active dry yeast. The active dry yeast used in this study was a blend of several strains ofSaccharomyces cerevisiae. The fermentation was carried out at 20°C at different pitching rates (inoculation levels) with and without the addition of yeast extract as nutrient supplement. At a pitching rate of 76 million cells per g of mash an ethanol yield of 20.4% (v/v) was obtained. To achieve this yeast extract must be added to the wheat mash as nutrient supplement. When the pitching rate was raised to 750 million cells per g of mash, the ethanol yield increased to 21.5% (v/v) and no nutrient supplement was required. The efficiency of conversion of sugar to ethanol was 97.6% at the highest pitching rate. This declined slightly with decreasing pitching rate. A high proportion of yeast cells lost viability at high pitching rates. It is suggested that nutrients released from yeast cells that lost viability and lysed, contributed to the high yield of ethanol in the absence of any added nutrients.     

Purification,kinetic and functional characterization of membrane bound dipeptidyl peptidase-III from NCDC 252: a probiotic lactic acid bacteria

Molecular Biology Reports - Pediococcus acidilactici is a probiotic lactic acid bacteria possessing studied in-vitro probiotic properties. Study of membrane proteins is crucial in developing...     

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.     

Impact of pitching rate on yeast fermentation performance and beer flavour

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e. higher inoculum size). However, the impact of the pitching rate on crucial fermentation and beer quality parameters has never been assessed systematically. In this study, five pitching rates were applied to lab-scale fermentations to investigate its impact on the yeast physiology and beer quality. The fermentation rate increased significantly and the net yeast growth was lowered with increasing pitching rate, without affecting significantly the viability and the vitality of the yeast population. The build-up of unsaturated fatty acids in the initial phase of the fermentation was repressed when higher yeast concentrations were pitched. The expression levels of the genes HSP104 and HSP12 and the concentration of trehalose were higher with increased pitching rates, suggesting a moderate exposure to stress in case of higher cell concentrations. The influence of pitching rate on aroma compound production was rather limited, with the exception of total diacetyl levels, which strongly increased with the pitching rate. These results demonstrate that most aspects of the yeast physiology and flavour balance are not significantly or negatively affected when the pitching rate is changed. However, further research is needed to fully optimise the conditions for brewing beer with high cell density populations.     

Population diversity of yeasts and lactic acid bacteria in pig feed fermented with whey, wet wheat distillers' grains, or water at different temperatures

The diversity of populations of yeast and lactic acid bacteria (LAB) in pig feeds fermented at 10, 15, or 20 degrees C was characterized by rRNA gene sequencing of isolates. The feeds consisted of a cereal grain mix blended with wet wheat distillers' grains (WWDG feed), whey (W feed), or tap water (WAT feed). Fermentation proceeded for 5 days without disturbance, followed by 14 days of daily simulated feed outtakes, in which 80% of the contents were replaced with fresh feed mixtures. In WWDG feed, Pichia galeiformis became the dominant yeast species, independent of the fermentation temperature and feed change. The LAB population was dominated by Pediococcus pentosaceus at the start of the fermentation period. After 3 days, the Lactobacillus plantarum population started to increase in feeds at all temperatures. The diversity of LAB increased after the addition of fresh feed components. In W feed, Kluyveromyces marxianus dominated, but after the feed change, the population diversity increased. With increasing fermentation temperatures, there was a shift toward Pichia membranifaciens as the dominant species. L. plantarum was the most prevalent LAB in W feed. The WAT feed had a diverse microbial flora, and the yeast population changed throughout the whole fermentation period. Pichia anomala was the most prevalent yeast species, with increasing occurrence at higher fermentation temperatures. Pediococcus pentosaceus was the most prevalent LAB, but after the feed change, L. plantarum started to proliferate. The present study demonstrates that the species composition in fermented pig feed may vary considerably, even if viable cell counts indicate stable microbial populations.     

Lipidome profiling of Saccharomyces cerevisiae reveals pitching rate-dependent fermentative performance

A high cell density strategy has been used in bioethanol production to shorten the fermentation period. To reveal the molecular basis of fermentative behavior in high cell density, the profiling of the phospholipids and sterols of Saccharomyces cerevisiae during fermentation at five different pitching rates (1, 5, 10, 20, and 40 g/L) was investigated. Using LC/ESI/MSⁿ technology, 148 phospholipid species were detected, of which 91 species were quantified, and using the gas chromatography–time-of-flight mass spectrometry procedure, a total of 11 sterols were quantified. Phospholipid samples from different pitching rates were discriminated into three groups using principal component analysis (1, 5 g/L, and the others). The main changes in the lipid profile of yeast cells with higher pitching rates were as follows: (a) the relative contents of phosphatidylglycerol and phosphatidylserine were higher while phosphatidylinositol was lower compared with lower pitching rates, (b) the saturated and the relatively shorter fatty acyl chains of phospholipids decreased, and (c) the content of ergosterol was higher. These findings suggested a regulation of the property of the membrane at the situation of high cell density and a possible approach of self-protection of the yeast cells against the high density stresses.     

Lactic Acid Bacteria and Yeasts as Means of Decreasing Wine Acidity

Two hundred monocultures of lactic acid bacteria and 30 associations of yeasts and lactic acid bacteria have been studied. A stable association was developed which was capable of decreasing wine acidity. The association contained two species of bacteria, Leuconostoc oenosand Pediococcus pentosaceus, and the yeast Saccharomyces cerevisiae. The physiology of the microorganisms was studied, and their effects on the chemical composition of wines were determined.     

Changes in membrane fatty acid composition of Pediococcus sp. strain NRRL B-2354 in response to growth conditions and its effect on thermal resistance.

Membrane fatty acid composition and thermal resistance (D value) of Pediococcus sp. were determined for mid-exponential-phase (ME) and stationary-phase (ST) cells grown in tryptic soy broth (TSB) and tryptone-glucose-yeast extract (TGY) at 28 and 37 degrees C. As the cells entered the stationary phase of growth, the unsaturated fatty acid, C18:1 n11c, produced during the exponential phase of growth was converted to its cyclic form, C19:0 Delta9c. This shift in membrane fatty acid composition was accompanied by an increase in the D values of this bacterium. Data from this study suggest that the membrane fatty acid composition of Pediococcus sp. is dependent on the growth conditions and that membrane fatty acid composition plays a critical role in thermal resistance. Thermal inactivation curves of Pediococcus sp. cells grown in TGY at 28 degrees C indicated the presence of a cell population that is heterogeneous in thermal resistance. The growth of this bacterium in TGY at 37 degrees C and in TSB at 28 and 37 degrees C resulted in cell populations that were uniform in thermal resistance with a lag time for thermal inactivation. Thermal inactivation curves of ME and ST cultures were similar. The data presented here suggest that the cell population's uniformity of thermal inactivation is independent of the growth phase of the culture.     

A medium designed for monitoring pitching yeast contamination in beer using a conductimetric technique

Nitrogen assimilation is the most readily utilized source of conductance changes when pitching yeast is grown in a glucose-based medium. A simple growth medium comprising yeast nitrogen base, in which nitrogen is supplied as ammonium sulphate, and glucose gave good growth but little change in conductance. Inclusion of a succinate buffer in the medium to remove protons liberated as a result of nitrogen uptake produced a large decrease in conductance and detection times that correlated well with enumeration of yeast by plate counting. The medium will allow more rapid and automated detection of pitching yeast survival in pasteurized beer although individual calibration for each beer type will be necessary.     

Isolation and characterization of Pediococcus halophilus from salted anchovies (Engraulis anchoita)

The presence of bacteria in salted anchovies during and at the end of the curing process was investigated. Attempts to isolate bacteria under aerobic or anaerobic conditions led to the isolation of only bacteria of the genus Pediococcus which were identified as Pediococcus halophilus. The isolates correspond to a rather heterogeneous group in which some of the members differ in some biochemical tests from the types described in the literature.     

Imaging of Lactobacillus brevis single cells and microcolonies without a microscope by an ultrasensitive chemiluminescent enzyme immunoassay with a photon-counting television camera.

An ultrasensitive chemiluminescent enzyme immunoassay (CLEIA) was developed for the rapid detection and quantification of Lactobacillus brevis contaminants in beer and pitching yeast (Saccharomyces cerevisiae slurry collected for reinoculation). L. brevis cells trapped on a 47-mm nucleopore membrane (0.4-micron pore size) were reacted with a peroxidase-labelled Lactobacillus group E antibody and then subjected to an enhanced CLEIA analysis with 4-iodophenol as the enhancer. The combination of a nucleopore membrane with low background characteristics that enables the antigen-antibody reaction to proceed through the pores of the membrane and a labelled antibody prepared by the maleimide hinge method with minimal nonspecific binding characteristics was essential to minimize background in the detection of single cells. An ultrahigh sensitive charge-coupled device (CCD) camera equipped with a fiber optics image intensifier permitted the imaging of single cells. A clear correlation existed between the number of luminescent spots observed and the plate count [y (CLEIA) = 0.990x (plate count) + 15.9, where n = 7, r = 0.993, and P < 0.001]. Microscopic observation confirmed that the luminescent spots were produced by single cells. This assay could be used to detect approximately 20 L. brevis cells in 633 ml of beer within 4 h. Our ultrasensitive CLEIA could also be used to detect microcolonies approximately 20 microns in diameter which had formed on a membrane after 15 to 18 h of incubation. This method, which we called the microcolony immunoluminescence (MIL) method, increased the signal-to-noise ratio dramatically. The MIL method could be used to detect a 10(0) level of L. brevis contamination in 633 ml of beer and a 1/10(8) level of L. brevis contamination in pitching yeast within 1 day (15 to 18 h to form microcolonies and 2 h for CLEIA).     

Isolation and characterization of Pediococcus halophilus from salted anchovies (Engraulis anchoita).

The presence of bacteria in salted anchovies during and at the end of the curing process was investigated. Attempts to isolate bacteria under aerobic or anaerobic conditions led to the isolation of only bacteria of the genus Pediococcus which were identified as Pediococcus halophilus. The isolates correspond to a rather heterogeneous group in which some of the members differ in some biochemical tests from the types described in the literature.     

Pediocin PA-1, a bacteriocin from Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells.

Pediocin PA-1 is a bacteriocin which is produced by Pediococcus acidilactici PAC1.0. We demonstrate that pediocin PA-1 kills sensitive Pediococcus cells and acts on the cytoplasmic membrane. In contrast to its lack of impact on immune cells, pediocin PA-1 dissipates the transmembrane electrical potential and inhibits amino acid transport in sensitive cells. Pediocin interferes with the uptake of amino acids by cytoplasmic membrane vesicles derived from sensitive cells, while it is less effective with membranes derived from immune cells. In liposomes fused with membrane vesicles derived from both sensitive and immune cells, pediocin PA-1 elicits an efflux of small ions and, at higher concentrations, an efflux of molecules having molecular weights of up to 9,400. Our data suggest that pediocin PA-1 functions in a voltage-independent manner but requires a specific protein in the target membrane.