The inhibitory effect of natural microflora of food on growth of Listeria monocytogenes in enrichment broths

The aims of this study were to (i) compare the inhibitory effects of the natural microflora of different foods on the growth of Listeria monocytogenes during enrichment in selective and non-selective broths; (ii) to isolate and identify components of the microflora of the most inhibitory food; and (iii) to determine which of these components was most inhibitory to growth of L. monocytogenes in co-culture studies. Growth of an antibioticresistant marker strain of L. monocytogenes was examined during enrichment of a range of different foods in Tryptone Soya Broth (TSB), Half Fraser Broth (HFB) and Oxoid Novel Enrichment (ONE) Broth. Inhibition of L. monocytogenes was greatest in the presence of minced beef, salami and soft cheese and least with prepared fresh salad and chicken pâté. For any particular food the numbers of L. monocytogenes present after 24 h enrichment in different broths increased in the order: TSB, HFB and ONE Broth. Numbers of L. monocytogenes recovered after enrichment in TSB were inversely related to the initial aerobic plate count (APC) in the food but with only a moderate coefficient of determination (R2) of 0.51 implying that microbial numbers and the composition of the microflora both influenced the degree of inhibition of L. monocytogenes. In HFB and ONE Broth the relationship between APC and final L. monocytogenes counts was weaker. The microflora of TSB after 24 h enrichment of minced beef consisted of lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas spp., Enterobacteriaceae, and enterococci. In co-culture studies of L. monocytogenes with different components of the microflora in TSB, the lactic acid bacteria were the most inhibitory followed by the Enterobacteriaceae. The least inhibitory organisms were Pseudomonas sp., enterococci and B. thermosphacta. In HFB and ONE Broth the growth of Gram-negative organisms was inhibited but lactic acid bacteria still reached high numbers after 24 h. A more detailed study of the growth of low numbers of L. monocytogenes during enrichment of minced beef in TSB revealed that growth of L. monocytogenes ceased at a cell concentration of about 102 cfu/ml when lactic acid bacteria entered stationary phase. However in ONE Broth growth of lactic acid bacteria was slower than in TSB with a longer lag time allowing L. monocytogenes to achieve much higher numbers before lactic acid bacteria reached stationary phase. This work has identified the relative inhibitory effects of different components of a natural food microflora and shown that the ability of low numbers of L. monocytogenes to achieve high cell concentrations is highly dependent on the extent to which enrichment media are able to inhibit or delay growth of the more effective competitors.

Bryozoan populations reflect nutrient enrichment and productivity gradients in rivers

1. The hypothesis that nutrient enrichment will affect bryozoan abundance was tested using two complementary investigations; a field-based method determining bryozoan abundance in 20 rivers of different nutrient concentrations by deploying statoblast (dormant propagule) traps and an experimental laboratory microcosm study measuring bryozoan growth and mortality. These two methods confirmed independently that increased nutrient concentrations in water promote increases in the biomass of freshwater bryozoans. 2. Statoblasts of the genus Plumatella were recorded in all rivers, regardless of nutrient concentrations, demonstrating that freshwater bryozoans are widespread. Concentrations of Plumatella statoblasts were high in rivers with high nutrient concentrations relative to those with low to moderate nutrient concentrations. Regression analyses indicated that phosphorus concentrations, in particular, significantly influenced statoblast concentrations. 3. Concentrations of Lophopus crystallinus statoblasts were also higher in sites characterised by high nutrient concentrations. Logistic regression analysis revealed that the presence of L. crystallinus statoblasts was significantly associated with decreasing altitude and increasing phosphorus concentrations. This apparently rare species was found in nine rivers (out of 20), seven of which were new sites for L. crystallinus. 4. Growth rates of Fredericella sultana in laboratory microcosms increased with increasing nutrient concentration and high mortality rates were associated with low nutrient concentrations. 5. Our results indicate that bryozoans respond to increasing nutrient concentrations by increased growth, resulting in higher biomasses in enriched waters. We also found that an important component of bryozoan diets can derive from food items lacking chlorophyll a. Finally, bryozoans may be used as independent proxies for inferring trophic conditions, a feature that may be especially valuable in reconstructing historical environments by assessing the abundance of statoblasts in sediment cores.

Using a runway paradigm to assess the relative strength of rats' motivations for enrichment objects

Laboratory animals should be provided with enrichment objects in their cages; however, it is first necessary to test whether the proposed enrichment objects provide benefits that increase the animals’ welfare. The two main paradigms currently used to assess proposed enrichment objects are the choice test, which is limited to determining relative frequency of choice, and consumer demand studies, which can indicate the strength of a preference but are complex to design. Here, we propose a third methodology: a runway paradigm, which can be used to assess the strength of an animal’s motivation for enrichment objects, is simpler to use than consumer demand studies, and is faster to complete than typical choice tests. Time spent with objects in a standard choice test was used to rank several enrichment objects in order to compare with the ranking found in our runway paradigm. The rats ran significantly more times, ran faster, and interacted longer with objects with which they had previously spent the most time. It was concluded that this simple methodology is suitable for measuring rats’ motivation to reach enrichment objects. This can be used to assess the preference for different types of enrichment objects or to measure reward system processes.

A further study of the recovery and purification of surfactin from fermentation broth by membrane filtration

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, a two-step membrane filtration process was developed using a lab scale tangential flow filtration (TFF) unit with 10 kDa MWCO regenerated cellulose (RC) and polyethersulfone (PES)membranes at three different transmembrane pressure (TMP) of 1.5 bar, 2.0 bar and 2.5 bar. Two modes of filtrations were studied, with and without cleaning of membranes prior to UF-2. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effectively retained by the membrane in UF-2. Flux of permeates, rejection coefficient (R) of surfactin and proteinwere measured during the filtrations. Overall the three different TMPs applied have no significant effect in the filtrations and PES is the more suitable membrane to selectively separate surfactin from fermentation broth, achieving high recovery and level of purity. In addition this two-step UF process is scalable for larger volume of samples without affecting the original functionality of surfactin, although membranes permeability can be affected due to exposure to methanolic solution used in UF-2.

n-3 fatty acid enrichment of edible tissue of poultry: A review

There is clear evidence of the nutritional benefits of consuming long-chain n-3 PUFA, which are found predominantly in oily fish. However, oily fish consumption, particularly in the United Kingdom, is declining, as is the consumption of all meats with the exception of poultry, which has increased in consumption by 73% in the last 30 yr. This pattern, if less marked, is reflected throughout Europe, and therefore one means of increasing long-chain n-3 PUFA consumption would be to increase the long-chain n-3 PUFA content in the edible tissues of poultry. This review considers the feasibility of doing this, concentrating particularly on chickens and turkeys. It begins by summarizing the benefits to human health of consuming greater quantities of n-3 FA and the sources of n-3 PUFA in the human diet. The literature on altering the FA composition of poultry meat is then reviewed, and the factors affecting the incorporation of n-3 PUFA into edible tissues of poultry are investigated. The concentration of alpha-linolenic acid (ALA) in the edible tissues of poultry is readily increased by increasing the concentration of ALA in the birds' diet (particularly meat with skin, and dark meat to a greater extent than white meat). The concentration of EPA in both white and dark meat is also increased when the birds' diet is supplemented with EPA, although supplementing the diet with the precursor (ALA) does not result in a noticeable increase in EPA content in the edible tissues. Although supplementing the birds' diets with relatively high concentrations of DHA does result in an increased concentration of DHA in the tissues, the relationship between dietary and tissue concentrations of DHA is much weaker than that observed with ALA and EPA. The impact that altering the FA composition of edible poultry tissue may have on the organoleptic and storage qualities of poultry products is also considered.

Effect of species and genotype on the efficiency of enrichment of poultry meat with n-3 polyunsaturated fatty acids

The effect of poultry species (broiler or turkey) and genotype (Wrolstad or BUT T8 turkeys and Ross 308 or Cobb 500 broilers) on the efficiency with which dietary longchain n-3 PUFA were incorporated into poultry meat was determined. Broilers and turkeys of both genotypes were fed one of six diets varying in FA composition (two replicates per genotype x diet interaction). Diets contained 50 g/kg added oil, which was either blended vegetable oil (control), or partially replaced with linseed oil (20 or 40 g/kg diet), fish oil (20 or 40 g/kg diet), or a mixture of the two (20 g linseed oil and 20 g fish oil/kg diet). Feeds and samples of skinless breast and thigh meat were analyzed for FA. Wrolstad dark meat was slightly more responsive than BUT T8 (P = 0.046) to increased dietary 18:3 concentrations (slopes of 0.570 and 0.465, respectively). The Ross 308 was also slightly more responsive than the Cobb 500 (P= 0.002) in this parameter (slopes of 0.557 and 0.449). There were no other significant differences between the genotypes. There was some evidence (based on the estimates of the slopes and their associated standard errors) that white turkey meat was more responsive than white chicken meat to 20:5 (slopes of 0.504 and 0.289 for turkeys and broilers, respectively). There was no relationship between dietary 18:3 n-3 content and meat 20:5 and 22:6 contents. If birds do convert 18:3 to higher FA, these acids are not then deposited in the edible tissues.

Bryozoan populations reflect nutrient enrichment and productivity gradients in rivers

P>1. The hypothesis that nutrient enrichment will affect bryozoan abundance was tested using two complementary investigations; a field-based method determining bryozoan abundance in 20 rivers of different nutrient concentrations by deploying statoblast (dormant propagule) traps and an experimental laboratory microcosm study measuring bryozoan growth and mortality. These two methods confirmed independently that increased nutrient concentrations in water promote increases in the biomass of freshwater bryozoans. 2. Statoblasts of the genus Plumatella were recorded in all rivers, regardless of nutrient concentrations, demonstrating that freshwater bryozoans are widespread. Concentrations of Plumatella statoblasts were high in rivers with high nutrient concentrations relative to those with low to moderate nutrient concentrations. Regression analyses indicated that phosphorus concentrations, in particular, significantly influenced statoblast concentrations. 3. Concentrations of Lophopus crystallinus statoblasts were also higher in sites characterised by high nutrient concentrations. Logistic regression analysis revealed that the presence of L. crystallinus statoblasts was significantly associated with decreasing altitude and increasing phosphorus concentrations. This apparently rare species was found in nine rivers (out of 20), seven of which were new sites for L. crystallinus. 4. Growth rates of Fredericella sultana in laboratory microcosms increased with increasing nutrient concentration and high mortality rates were associated with low nutrient concentrations. 5. Our results indicate that bryozoans respond to increasing nutrient concentrations by increased growth, resulting in higher biomasses in enriched waters. We also found that an important component of bryozoan diets can derive from food items lacking chlorophyll a. Finally, bryozoans may be used as independent proxies for inferring trophic conditions, a feature that may be especially valuable in reconstructing historical environments by assessing the abundance of statoblasts in sediment cores.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Effects of enrichment of refined olive oil with phenolic compounds from olive leaves

The possibility of preparing olive oil, with the same nutritional value and stability characteristics found in virgin olive oil, by the enrichment of refined olive oil with olive leaf polyphenols was studied. To obtain antioxidant phenols similar to those found in virgin olive oil, these components were extracted from the leaves of several olive cultivars from the Northern region of Portugal, namely, Carrasca, Ripa, Negruche, Cordovil, Verdeal, Madural, and Bical cultivars, under several conditions. The concentration of a leaf extract required for addition to refined olive oil to obtain the same stability as virgin olive oil was determined. The extract from 1 kg of leaves was sufficient to fortify 50-320 L of refined olive oil to a similar stability as a virgin olive oil sample depending on the metal concentration of the oil, cultivar, and time of the year when the leaves were picked.

Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media

Aims: To make a preliminary assessment of the incidence of Salmonella in Egyptian dairy products, and to investigate the effectiveness of various protocols for the detection of the pathogen in these products. Methods and Results: Samples of milk and related dairy products were randomly collected from local markets and examined for the presence of Salmonella. While most samples were free of the organism, isolates of Salmonella enterica subsp. enterica serovar Typhimurium PT 8 could be recovered from 'matared' cream specimens. These isolates were susceptible to antibiotics usually used to challenge infections caused by Salmonella. A combination of buffered peptone water, Muller-Kauffman tetrathionate broth, and brilliant green phenol red agar gave the best results for the detection of the pathogen. Selenite-cystine broth and Hektoen enteric agar were ineffective as an enrichment and a plating medium, respectively, in the isolation of Salmonella. A modified identification strategy that reduces the burden of serological testing of presumptive isolates is proposed. Conclusions, Significance and Impact of the Study: 'Matared' cream could be a vehicle for transmitting Salmonella. Using the above combination of media, beside the suggested modified confirmatory procedure, should increase the effectiveness and ease of the detection of Salmonella in milk and dairy products.

Apolipoprotein E enrichment of immuno-separated chylomicron and chylomicron remnants following saturated fatty acids

Aim: We examined the effect of meat fatty acids on lipid and apolipoprotein concentrations of very low density lipoprotein (VLDL) and chylomicron/chylomicron remnants in lipid fractions with a Svedberg flotation rate (S-f) 60-400 and S-f 20-60. Methods and results: Six healthy middle-aged men received in random order mixed meals enriched with saturated (SFA), polyunsaturated (PUFA) or monounsaturated (MUFA) fatty acids on 3 occasions. VLDL and chylomicron/chylomicron remnants in the lipid fractions were separated by immunoaffinity chromatography against apo B-100. In the S-f 60-400 chylomicron/chylomicron remnants, triacylglycerol and cholesterol concentrations were significantly tower following PUFA compared with SFA and MUFA (P <= 0.05). Apolipoprotein (apo) E responses were significantly higher after SFA in chylomicron/chylomicron remnants and VLDL compared with PUFA and MUFA (P < 0.007). However, apo B responses (particle number) were higher following MUFA than SFA (P = 0.039 for chylomicron/chylomicron remnants). Composition of the chylomicron/chylomicron remnants (expressed per particle) revealed differences in their triacylglycerol and apo E contents; in the Sf 60-400 fraction, SFA-rich chylomicron/chylomicron remnants contained significantly more triacylglycerol than MUFA (P = 0.028), more apo E than PUFA- and MUFA-rich particles (P < 0.05) and in the S-f 20-60 fraction, more apo E than MUFA (P = 0.009). Conclusion: There are specific differences in the composition of chylomicron/ chylomicron remnants formed after saturated compared with unsaturated fatty acid-rich meals which could determine their metabolic fate in the circulation and subsequent atherogenicity. (C) 2005 Elsevier B.V. All rights reserved.

Greater enrichment of triacylglycerol-rich lipoproteins with apolipoproteins E and C-III after meals rich in saturated fatty acids than after meals rich in unsaturated fatty acids

Background: Although there is considerable interest in the postprandial events involved in the absorption of dietary fats and the subsequent metabolism of diet-derived triacylglycerol-rich lipoproteins, little is known about the effects of meal fatty acids on the composition of these particles. Objective: We examined the effect of meal fatty acids on the lipid and apolipoprotein contents of triacylglycerol-rich lipoproteins. Design: Ten normolipidemic men received in random order a mixed meal containing 50 L, of a mixture of palm oil and cocoa butter [rich in saturated fatty acids (SFAs)], safflower oil [n-6 polyunsaturated fatty acids (PUFAs)]. or olive oil [monounsaturated fatty acids (MUFAs)] on 3 occasions. Fasting and postprandial apolipoproteins B-48. B-100, E. C-II, and C-III and lipids (triacylglycerol and cholesterol) were measured in plasma fractions with Svedberg flotation rates (S-f) >400 S-f 60-400, and S-f 20 - 60. Results: Calculation of the composition of the triacylglycerol-rich lipoproteins (expressed per mole of apolipoprotein B) showed notable differences in the lipid and apolipoprotein contents of the SFA-enriched particles in the S-f > 400 and S-f 60-400 fractions. After the SFA meal, triacylglycerol-rich lipoproteins in these fractions showed significantly greater amounts of triacylglycerol and of apolipoproteins C-II (Sf 60-400 fraction only), C-III, and E than were found after the MUFA meal (P < 0.02) and more cholesterol, apolipoprotein C-III (Sf > 400 fraction only), and apolipoprotein E than after the PUFA meal (P < 0.02). Conclusions: Differences in the composition of S-f > 400 and S-f 60-400 triacylglycerol-rich lipoproteins formed after saturated compared with unsaturated fatty acid-rich meals may explain differences in the metabolic handling of dietary fats.

Recovery and purification of surfactin from fermentation broth by a two-step ultrafiltration process

Surfactin is a bacterial lipopeptide produced by Bacillus subtilis and it is a powerful surfactant, having also antiviral, antibacterial and antitumor properties. The recovery and purification of surfactin from complex fermentation broths is a major obstacle to its commercialization; therefore, two-step membrane filtration processes were evaluated using centrifugal and stirred cell devices while the mechanisms of separation were investigated by particle size and surface charge measurements. In a first step of ultrafiltration (UF-1), surfactin was retained effectively by membranes at above its critical micelle concentration (CMC); subsequently in UF-2, the retentate micelles were disrupted by addition of 50% (v/v) methanol solution to allow recovery of surfactin in the permeate. Main protein contaminants were effective]), retained by the membrane in UF-2. Ultrafiltration was carried out either using centrifugal devices with 30 and 10 kDa MWCO regenerated cellulose membranes, or a stirred cell device with 10 kDa MWCO polyethersulfone (PES) and regenerated cellulose (RC) membranes. Total rejection of surfactin was consistently observed in UF-1, while in UF-2 PES membranes had the lowest rejection coefficient of 0.08 +/- 0.04. It was found that disruption of surfactin micelles, aggregation of protein contaminants and electrostatic interactions in UF-2 can further improve the selectivity of the membrane based purification technique. (C) 2007 Elsevier B.V. All rights reserved.

Comparison between pre-enrichment in single- or double-strength buffered peptone water for recovery of Salmonella enterica serovar Typhimurium DT104 from acidic marinade sauces containing spices.

The ability of the standard pre-enrichment procedure in buffered peptone water (BPW) to recover Salmonella Typhimurium from acidic marinade sauces containing spices was tested by inoculating marinade sauces with known numbers of an antibiotic-resistant marker strain of Salmonella Typhimurium DT104 prior to pre-enrichment. Viable numbers of salmonellae present in BPW after 24h incubation depended on the inoculum level. If initial cell numbers were low (below 103 cfu per 250 ml BPW) final cell concentrations were also low and, in some cases, no growth occurred. The problem was overcome by use of double-strength BPW that neutralised the acidity and allowed good recovery from otherwise inhibitory marinade sauces.

Comparison of algal and fish sources on the oxidative stability of poultry meat and its enrichment with omega-3 polyunsaturated fatty acids

Human consumption of long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) is below recommendations, and enriching chicken meat (by incorporating LC n-3 PUFA into broiler diets) is a viable means of increasing consumption. Fish oil is the most common LC n-3 PUFA supplement used but is unsustainable and reduces the oxidative stability of the meat. The objective of this experiment was to compare fresh fish oil (FFO) with fish oil encapsulated (EFO) in a gelatin matrix (to maintain its oxidative stability) and algal biomass at a low (LAG, 11), medium (MAG, 22), or high (HAG, 33 g/kg of diet) level of inclusion. The C22:6n-3 contents of the FFO, EFO, and MAG diets were equal. A control (CON) diet using blended vegetable oil was also made. As-hatched 1-d-old Ross 308 broilers (144) were reared (21 d) on a common starter diet then allocated to treatment pens (4 pens per treatment, 6 birds per pen) and fed treatment diets for 21 d before being slaughtered. Breast and leg meat was analyzed (per pen) for fatty acids, and cooked samples (2 pens per treatment) were analyzed for volatile aldehydes. Concentrations (mg/100 g of meat) of C20:5n-3, C22:5n-3, and C22:6n-3 were (respectively) CON: 4, 15, 24; FFO: 31, 46, 129; EFO: 18, 27, 122; LAG: 9, 19, 111; MAG: 6, 16, 147; and HAG: 9, 14, 187 (SEM: 2.4, 3.6, 13.1) in breast meat and CON: 4, 12, 9; FFO: 58, 56, 132; EFO: 63, 49, 153; LAG: 13, 14, 101; MAG: 11, 15, 102; HAG: 37, 37, 203 (SEM: 7.8, 6.7, 14.4) in leg meat. Cooked EFO and HAG leg meat was more oxidized (5.2 mg of hexanal/kg of meat) than the other meats (mean 2.2 mg/kg, SEM 0.63). It is concluded that algal biomass is as effective as fish oil at enriching broiler diets with C22:6 LC n-3 PUFA, and at equal C22:6n-3 contents, there is no significant difference between these 2 supplements on the oxidative stability of the meat that is produced.