Phil Bremer

Ecological Behavior of Lactobacillus reuteri 100-23 Is Affected by Mutation of the luxS Gene

ABSTRACT The luxS gene of Lactobacillus reuteri 100-23C was amplified by PCR, cloned, and then sequenced. To define a physiological and ecological role for the luxS gene in L. reuteri 100-23C, a luxS mutant was constructed by insertional mutagenesis. The luxS mutant did not produce autoinducers AI-2 or AI-3. Complementation of the luxS mutation by a plasmid construct containing luxS restored AI-2 and AI-3 synthesis. In vitro experiments revealed that neither the growth rate, nor the cell yield, nor cell survival in the stationary phase were compromised in the luxS mutant relative to the wild type and complemented mutant. The ATP content of exponentially growing cells of the luxS mutant was, however, 65% of that of wild-type cells. Biofilms formed by the luxS mutant on plastic surfaces in a bioreactor were thicker than those formed by the wild type. Biofilm thickness was not restored to wild-type values by the addition of purified AI-2 to the culture medium. In vivo experiments, conducted with ex-Lactobacillus-free mice, showed that biofilms formed by the mutant strain on the epithelial surface of the forestomach were approximately twice as thick as those formed by the wild type. The ecological performance of the luxS mutant, when in competition with L. reuteri strain 100-93 in the mouse cecum, was reduced compared to that of a xylA mutant of 100-23C. These results demonstrate that LuxS influences important ecological attributes of L. reuteri 100-23C, the consequences of which are niche specific.

Proteolysis produced within biofilms of bacterial isolates from raw milk tankers

In this study, six bacterial isolates that produced thermo-resistant enzymes isolated from the internal surfaces of raw milk tankers were evaluated for their ability to produce proteolysis within either single culture biofilms or co-culture biofilms. Biofilms were formed in an in vitro model system that simulated the upper internal surface of a raw milk tanker during a typical summers day of milk collection in New Zealand. The bacterial isolates were further evaluated for their ability to form biofilms at 25, 30 and 37°C. Mutual and competitive effects were observed in some of the co-culture biofilms, with all isolates being able to form biofilms in either single culture or co-culture at the three temperatures. The proteolysis was also evaluated in both biofilms and corresponding planktonic cultures. The proteolysis per cell decreased as the temperature of incubation (20-37°C) increased. Furthermore, mutualistic interactions in terms of proteolysis were observed when cultures were grown as co-culture biofilms. This is the first study to show that proteolytic enzymes can be produced in biofilms on the internal surfaces of raw milk tankers. This has important implications for the cleaning and the temperature control of raw milk transport tankers.

Salt modulates bacterial hydrophobicity and charge properties influencing adhesion of Pseudomonas aeruginosa (PA01) in aqueous suspensions.

The influence on cell hydrophobicity of differential extension with ionic strength of lipopolysaccharide molecules (LPS), which exist as charged and uncharged polymers at the surface of the gram-negative bacterium Pseudomonas aeruginosa (PA01), has been investigated. Attenuated total reflection infrared (ATR-IR) spectral absorptions from a single layer of cells adsorbed to ZnSe increased in intensity with increasing NaCl concentration up to 0.1 mol L(-1). Dynamic contact angle measurements (Wilhelmy plate tensiometry) made with a ZnSe plate having an adsorbed cell layer and the adherence of the cells to hexadecane suggest that PA01 cells were most hydrophobic in contact with 0.1 mol L(-1) NaCl solutions. These data indicate a charge screening induced compression of the charged LPS polymers decreasing the cell-surface approach distance and increasing the cell hydrophobicity due to the greater surface predominance of the uncharged LPS polymers. Interestingly, adsorbed cell layers in 0.3 mol L(-1) NaCl had a lower IR absorption intensity, and PA01 cells suspended in 0.3 mol L(-1) were found to be more hydrophilic, indicating that other factors influence the cell-surface approach distance and hydrophobicity. The examination of cell electrophoretic mobility variation with NaCl concentration suggests that the compression of charged polysaccharides increases the polysaccharide charge density and may also reduce the flow of liquid through the polysaccharide layer affecting the effective potential at the interface, the cell hydrophobicity, and the cell-surface approach distance.

Mechanisms of Cation Exchange by Pseudomonas aeruginosa PAO1 and PAO1 wbpL, a Strain with a Truncated Lipopolysaccharide

ABSTRACT The ability of bacterial cells to sequester cations is well recognized, despite the fact that the specific binding sites and mechanistic details of the process are not well understood. To address these questions, the cation-exchange behavior of Pseudomonas aeruginosa PAO1 cells with a truncated lipopolysaccharide (LPS) (PAO1 wbpL) and cells further modified by growth in a magnesium-deficient medium (PAO1 wbpL − Mg2+) were compared with that of wild-type P. aeruginosa PAO1 cells. P. aeruginosa PAO1 cells had a negative surface charge (zeta potential) between pH 11 and 2.2, due to carboxylate groups present in the B-band LPS. The net charge on PAO1 wbpL cells was increasingly positive below pH 3.5, due to the influence of NH3+ groups in the core LPS. The zeta potentials of these cells were also measured in Na+, Ca2+, and La3+ electrolytes. Cells in the La3+ electrolyte had a positive zeta potential at all pH values tested. Growing P. aeruginosa PAO1 wbpL in magnesium-deficient medium (PAO1 wbpL − Mg2+) resulted in an increase in its zeta potential in the pH range from 3.0 to 6.5. In cation-exchange experiments carried out at neutral pH with either P. aeruginosa PAO1 or PAO1 wbpL, the concentration of bound Ca2+ was found to decrease as the pH was reduced from 7.0 to 3.5. At pH 3.5, the bound Mg2+ concentration decreased sharply, revealing the activity of surface sites for cation exchange and their pH dependence. Infrared spectroscopy of attached biofilms suggested that carboxylate and phosphomonoester functional groups within the core LPS are involved in cation exchange.

Biogenic amines and potential histamine - Forming bacteria in Rihaakuru (a cooked fish paste).

The biogenic amine concentration in Rihaakuru (a fish paste) (n=28), obtained from different parts of the Maldives (North, South, and Central), was determined by HPLC. Ten biogenic amines were detected; agmatine, not detected (ND) - 161ppm; cadaverine, ND - 387ppm; histamine, ND - 5487ppm; putrescine, ND - 290ppm; phenylethylamine, ND - 23ppm; serotonin, ND - 91ppm; spermine, ND - 329ppm; spermidine, ND - 79ppm; tryptamine, ND - <5ppm; and tyramine, ND - 50ppm. Nine biogenic amines were found in 3 samples, 8 in 10 samples, 7 in 6 samples, 6 in 3 samples, 4 in 5 samples, and 1 was found in 1 sample. Histamine was detected at levels that are regarded as a risk to human health. Fourteen isolates were selected from two randomly selected samples out of the 28 samples of Rihaakuru and screened for histamine production. Twelve of the 14 isolates produced histamine, with the highest histamine producers being Bacillus massiliensis Nai5 (6.65ppm) and Bacillus polyfermenticus (5.58ppm); while Bacillus malacitensis produced the least (<0.5ppm).

Effect of Season on the Sensory Quality of Sea Urchin (Evechinus chloroticus) Roe

The influence of season, gender, and reproductive maturity on the sensory properties (appearance, odor, taste, flavor, texture/mouth feel, and after taste) of roe from sea urchins (Evechinus chloroticus) was investigated. A trained sensory panel applied descriptive analysis, with a comprehensive sensory vocabulary of 35 descriptors to analyze sea urchin roe obtained from samples collected during the austral autumn, winter, summer, and spring, over a 2-y period. Sensory differences between genders were less pronounced in autumn (March to May) and winter (June to August) when more nutritive cells were present in the roe, and more pronounced over spring (September to November) and summer (December to February) months as the gametogenic cells matured. Roe from female sea urchins were commonly associated with sulfur odor, bitter taste, and metallic flavor, while roe from the male sea urchins were associated with sweet taste. During autumn, female roe were closest in sensory quality to male roe (least bitter and most sweet compared to other seasons), while there were little differences in sweetness and bitterness of male roe over the 4 seasons. Despite the relative size of the roe being lowest in autumn, it appeared to be the optimum season to harvest E. chloroticus sea urchins to obtain high-quality roe.

Lipolysis within single culture and co-culture biofilms of dairy origin

Bacteria in raw milk can produce heat-stable lipases, which survive pasteurisation and subsequently reduce the shelf life of dairy products because of their ability to break down the milk fat and increase rancidity. In this study, four bacteria, originating from the surfaces of raw milk transport tankers, and a known lipase-producing bacterium were evaluated for their ability to produce lipolysis in planktonic and biofilm cultures. Lipolysis was determined using two separate assays that measured hydrolysis of the ester p-nitrophenol palmitate (pnpp) and the lipid tributyrin. The hydrolysis of pnpp per CFU within biofilms and planktonic cultures ranged from 0.01 to 8.35 and 0.01 to 0.07 nU/CFU respectively. The amount of butyric acid released from hydrolysis of tributyrin per CFU within biofilms and planktonic cultures ranged from 0.1 to 1110.3 and 0.1 to 0.3 ng/CFU, respectively. The hydrolysis of pnpp and tributyrin per CFU within biofilms was at least 10 times higher compared with the corresponding planktonic cultures. This is the first study to show that lipolysis occurs within biofilms of bacteria that were originally isolated from the surfaces of raw milk tankers. This is relevant to the dairy industry, highlighting the importance of eliminating biofilms on milk tanker surfaces as a source of heat-stable lipases.

In Vitro and In Vivo Flavor Release from Intact and Fresh-Cut Apple in Relation with Genetic, Textural, and Physicochemical Parameters

UNLABELLED Flavor release from 6 commercial apple cultivars (Fuji, Granny Smith, Golden Delicious, Jonagold, Morgen Dallago, and Red Delicious) under static conditions (intact or fresh-cut samples) and during consumption of fresh-cut samples (nosespace) was determined by proton transfer reaction mass spectrometry. Textural (firmness, fracturability, flesh elasticity, and rupture) and physicochemical (pH, acidity, and water content) properties of the apples were also measured. Static headspace analysis of intact fruits revealed Fuji and Granny Smith apples had the lowest concentration for all measured flavor compounds (esters, aldehydes, alcohols, and terpenes), whereas Red Delicious apples had the highest. Fresh-cut samples generally showed a significant increase in total volatile compounds with acetaldehyde being most abundant. However, compared to intact fruits, cut Golden and Red Delicious apples had a lower intensity for ester related peaks. Five parameters were extracted from the nosespace data of peaks related to esters (m/z 43, 61), acetaldehyde (m/z 45), and ethanol (m/z 47): 2 associated with mastication (duration of mastication-t(con); time required for first swallowing event-t(swal)), and 3 related with in-nose volatile compound concentration (area under the curve-AUC; maximum intensity-I(max); time for achieving I(max)-t(max)). Three different behaviors were identified in the nosespace data: a) firm samples with low AUC and t(swal) values (Granny Smith, Fuji), b) mealy samples with high AUC, I(max), t(swal) values, and low t(con) (Morgen Dallago, Golden Delicious), and c) firm samples with high AUC and I(max) values (Red Delicious). Strengths and limitations of the methodology are discussed. PRACTICAL APPLICATION Volatile compounds play a fundamental role in the perceived quality of food. Using apple cultivars, this research showed that in vivo proton transfer reaction mass spectrometry (PTR-MS) could be used to determine the relationship between the release of volatile flavor compounds and the physicochemical parameters of a real food matrix. This finding suggests that in vivo PTR-MS coupled with traditional physicochemical measurements could be used to yield information on flavor release from a wide range of food matrices and help in the development of strategies to enhance food flavor and quality.

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra

Aims:  The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials.

Adhesive Secretions of Live Mussels Observed in Situ by Attenuated Total Reflection–Infrared Spectroscopy

The chemical species involved in the adhesion of blue mussels (Mytilus galloprovincialis) and greenshell mussels (Perna canaliculus) to surfaces has been investigated using in situ attenuated total reflection infrared (ATR-IR) spectroscopy. Mussel spat ranging in size from 0.5 to 25 mm were placed in a flow cell containing a ZnSe multiple internal reflection prism and supplied with temperature-controlled seawater. Distinctively different absorption spectra were obtained when the mussels were predominantly moving across the surface or forming permanent bonds. With limited movement, the absorption spectrum was characteristic of protein with peaks near 1647 cm−1 (amide I), 1543 cm−1 (amide II), and 1235 cm−1 (amide III). When the mussels were observed to be moving across the surface of the ATR-IR crystal there was a strong broad absorption maximum around 1200–900 cm−1 (carbohydrate polymers), presumably due to the secretion of a weakly acidic mucopolysaccharide. Distinct differences in the spectra obtained from the adhesive secretions of blue or greenshell mussels were not observed. The data presented is the first reported use of IR spectroscopy to obtain in situ, real-time, chemical data on the interactions between invertebrates and substrates immersed in sea water.