Pretreatment of different food rest materials for bioconversion into fungal lipid-rich biomass.

Food rest materials have the potential to be used as media components in various types of fermentations. Oleaginous filamentous fungi can utilize those components and generate a high-value lipid-rich biomass, which could be further used for animal and human use. One of the main limitations in this process is the pretreatment of food rest materials, needed to provide homogenization, sterilization and solubilization. In this study, two pretreatment processes-steam explosion and enzymatic hydrolysis-were evaluated for potato and animal protein-rich food rest materials. The pretreated food rest materials were used for the production of fungal lipid-rich biomass in submerged fermentation by the oleaginous fungus Mucor circinelloides. Cultivation media based on malt extract broth and glucose were used as controls of growth and lipid production, respectively. It was observed that media based on food rest materials can support growth and lipid production in M. circinelloides to a similar extent as the control media. More specifically, the use of potato hydrolysate combined with chicken auto-hydrolysate resulted in a higher fungal total biomass weight than using malt extract broth. When the same C/N ratio was used for glucose and rest materials-based media, similar lipid content was obtained or even higher using the latter media.

Predicting the concentration of verotoxin-producing Escherichia coli bacteria during processing and storage of fermented raw-meat sausages.

A model to predict the population density of verotoxigenic Escherichia coli (VTEC) throughout the elaboration and storage of fermented raw-meat sausages (FRMS) was developed. Probabilistic and kinetic measurement data sets collected from publicly available resources were completed with new measurements when required and used to quantify the dependence of VTEC growth and inactivation on the temperature, pH, water activity (aw), and concentration of lactic acid. Predictions were compared with observations in VTEC-contaminated FRMS manufactured in a pilot plant. Slight differences in the reduction of VTEC were predicted according to the fermentation temperature, 24 or 34°C, with greater inactivation at the highest temperature. The greatest reduction was observed during storage at high temperatures. A population decrease greater than 6 decimal logarithmic units was observed after 66 days of storage at 25°C, while a reduction of only ca. 1 logarithmic unit was detected at 12°C. The performance of our model and other modeling approaches was evaluated throughout the processing of dry and semidry FRMS. The greatest inactivation of VTEC was predicted in dry FRMS with long drying periods, while the smallest reduction was predicted in semidry FMRS with short drying periods. The model is implemented in a computing tool, E. coli SafeFerment (EcSF), freely available from http://www.ifr.ac.uk/safety/EcoliSafeFerment. EcSF integrates growth, probability of growth, and thermal and nonthermal inactivation models to predict the VTEC concentration throughout FRMS manufacturing and storage under constant or fluctuating environmental conditions.

Effect of starter culture inoculation on feed hygiene and microbial population development in fermented pig feed composed of a cereal grain mix with wet wheat distillers' grain.

AIMS: Investigating the influence of an added starter culture on the properties of fermented liquid pig feed. METHODS AND RESULTS: Diets of cereal grain blended with wet wheat distillers' grain that were either not inoculated (WWDG), inoculated with a silage starter culture at start (WWDGsc1) or at start and at each backslopping (replacement of 80% the content with fresh mixture, simulating feed outtake, WWDGsc5) were fermented for 5 days, followed by 5 days of daily backslopping. Numbers of undesirable micro-organisms (enterobacteria, moulds) were reduced in all fermentations; particularly enterobacteria in the starter culture inoculated diets. Lactobacillus plantarum present in the starter culture became dominant in diets WWDGsc1 and WWDGsc5. However, Lactobacillus panis that was dominating WWDG was also abundant in WWDGsc1 and WWDGsc5. Yeast populations were not influenced by the starter culture, with Pichia fermentans dominating all fermentations. All diets had similar chemical characteristics with the exception of a significant increase of all tested organic acids in WWDGsc5. CONCLUSIONS: The addition of a starter culture influences the bacterial population in fermented liquid feed, but there is also a strong impact of the flora already present in the feed ingredients. The yeast population is not influenced by adding a lactic acid bacteria (LAB) starter culture. A consortium of LAB and yeast strains adapted to the fermentation should be used as starter culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that it is possible to influence the current unpredictable and spontaneous process of feed fermentation when appropriate starter cultures are used. For this purpose, LAB and yeasts with desirable characteristics should be isolated.

Secreted enteric antimicrobial activity localises to the mucus surface layer.

OBJECTIVES: The intestinal mucosa is constantly exposed to a dense and highly dynamic microbial flora and challenged by a variety of enteropathogenic bacteria. Antibacterial protection is provided in part by Paneth cell-derived antibacterial peptides such as the alpha-defensins. The mechanism of peptide-mediated antibacterial control and its functional importance for gut homeostasis has recently been appreciated in patients with Crohn's ileitis. In the present study, the spatial distribution of antimicrobial peptides was analysed within the small intestinal anatomical compartments such as the intestinal crypts, the overlaying mucus and the luminal content. METHODS: Preparations from the different intestinal locations as well as whole mouse small intestine were extracted and separated by reversed-phase high-performance liquid chromatography. Antibacterial activity was determined in extracts, and the presence of antimicrobial peptides/proteins was confirmed by N-terminal sequencing, mass spectrometry analysis and immunodetection. RESULTS: The secreted antibacterial activity was largely confined to the layer of mucus, whereas only minute amounts of activity were noted in the luminal content. The extractable activity originating from either crypt/mucus/lumen compartments respectively (given as a percentage) was for Listeria monocytogenes, 48 (4)/44 (4)/8 (8); Enterococcus faecalis, 44 (10)/49 (3)/7 (7); Bacterium megaterium, 56 (4)/42 (3)/2 (1); Streptococcus pyogenes, 48 (4)/46 (3)/6 (6); Escherichia coli, 46 (4)/47 (3)/7 (7); and Salmonella enterica sv. Typhimurium, 38 (3)/43 (7)/19 (10). A spectrum of antimicrobial peptides was identified in isolated mucus, which exhibited strong and contact-dependent antibacterial activity against both commensal and pathogenic bacteria. CONCLUSION: These findings show that secreted antimicrobial peptides are retained by the surface-overlaying mucus and thereby provide a combined physical and antibacterial barrier to prevent bacterial attachment and invasion. This distribution facilitates high local peptide concentration on vulnerable mucosal surfaces, while still allowing the presence of an enteric microbiota.

Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides.

AIMS: To test seven selected putative signal peptides from Lactobacillus plantarum WCFS1 in terms of their ability to drive secretion of two model proteins in Lact. plantarum, and to compare the functionality of these signal peptides with that of well-known heterologous signal peptides (Usp45, M6). METHODS AND RESULTS: Signal peptide functionality was assessed using a series of modular derivatives of the pSIP vectors for peptide pheromone-controlled high-level gene expression in lactobacilli. Several of the constructs with homologous signal peptides yielded similar or higher reporter protein activities than constructs with heterologous signal peptides. Two of the homologous signal peptides (Lp_0373 and Lp_0600) appeared as especially promising candidates for directing secretion, as they were among the best performing with both reporter proteins. CONCLUSIONS: We have identified homologous signal peptides for high-level secretion of heterologous proteins in Lact. plantarum. With the model proteins, some of these performed better than commonly used heterologous signal peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: The homologous signal peptides tested out, in this study, could be useful in food-grade systems for secretion of interesting proteins in Lact. plantarum. The constructed modular secretion vectors are easily accessible for rapid signal peptide screening.

Early changes in rectal nitric oxide and mucosal inflammatory mediators in Crohn's colitis in response to infliximab treatment.

BACKGROUND: Treatment with tumor necrosis factor-alpha monoclonal antibody (infliximab) reduces clinical activity and intestinal inflammation in Crohn's disease. AIM: To study the time-course of the effects of infliximab with reference to mucosal cytokine and inducible nitric oxide synthase expression. METHODS: Thirty-two patients with Crohn's disease were treated with single dose infliximab (5 mg/kg). Disease activity was assessed days 1, 3, 7 and 28 using Harvey-Bradshaw index. Rectal nitric oxide levels were determined and rectal biopsies collected before treatment, 1 h after infusion and on days 3, 7 and 28. Immunohistochemical staining against inducible nitric oxide synthase, tumor necrosis factor-alpha, interleukin-1beta and interferon-gamma were performed. RESULTS: Clinical response was seen in 14 patients with down-regulation of global immunohistochemistry expression, reaching nadir day 3. Rectal nitric oxide was increased at baseline (3578 +/- 1199 parts per billion, ppb) compared with controls (89 +/- 13 ppb) (P < 0.001). In patients with clinical response, rectal nitric oxide decreased from 3926 +/- 1687 ppb to 1050 +/- 428 ppb day 28 (P < 0.05). CONCLUSIONS: Down-regulation of mucosal inflammatory mediators occurs after infliximab. Rectal nitric oxide levels parallel down-regulation of inducible nitric oxide synthase, tumor necrosis factor-alpha, interleukin-1beta and interferon-gamma and may serve as a quantitative biomarker of intestinal inflammation.

Alteration in human defensin-5 expression following gastric bypass surgery.

BACKGROUND: Roux-en-Y gastric bypass surgery provides a novel human model to investigate small bowel mucosal innate immunity, in which there is loss of gastric acid-mediated protection against orally-acquired microorganisms. AIM: To study changes in jejunal mucosal immunoreactivity of human defensin (HD)-5, an antimicrobial peptide normally produced by Paneth cells. METHODS: Mucosal samples were obtained from 18 female patients (24-54 years), from the same segment of jejunum during and after gastric bypass surgery. Samples were used for bacterial culture and immunohistochemistry using anti-HD-5 antibody. The number of immunoreactive cells per crypt and villus were determined and expressed as mean (SD). RESULTS: No bacteria were cultured from any of the perioperative jejunal samples but colonies of bacteria normally present in the pharynx were identified during culture of all postoperative jejunal biopsy specimens (1->100 colonies). Paneth cell numbers per crypt were unchanged after gastric bypass (4.16 (0.71) vs 4.24 (0.78)). However, following surgery, there was an increase in HD-5-positive intermediate cells per crypt (0.25 (0.41) vs 1.12 (0.66), p<0.01), HD-5 staining enterocytes per crypt (0.03 (0.09) vs 1.38 (1.10), p<0.01), HD-5 staining material in the crypt lumen (crypt lumens: 5.0% (10.9%) vs 68.1% (27.9%), p<0.01) and HD-5 immunoreactivity coating the luminal surface of villus enterocytes (villi sampled: 15.0% (31.0%) vs 67.5% (42.0%), p<0.01). CONCLUSIONS: Bacteria normally resident in the pharynx were present in the proximal jejunal mucosa following Roux-en-Y gastric bypass surgery. After gastric bypass, there was increased secretion of HD-5 and an increase in HD-5 expressing intermediate cells and enterocytes in the crypt. The increase in HD-5 expression in the jejunal mucosa following gastric bypass surgery is likely to be secondary to exposure to orally-acquired microorganisms.

A fluorescence method for the visualization of inflammatory cells in intestinal mucosa.

Treatment of paraffin or cryostat sections from rat, rabbit and man, with fluorescein isothiocyanate (FITC) in buffer (1 microgram/l), resulted in visualization of highly fluorescent leukocytes with a granular staining in light microscopy. FITC staining of paraffin-embedded colonic tissue was compared with three other staining methods, i.e., (1) FITC-conjugated anti-neutrophil antibodies, (2) endogenous peroxidase with 3,3-diaminobenzidine, and (3) FITC-conjugated Lotus tetragonolobus lectin. Good correlations between FITC staining and the three other methods were found (r:0.97, 0.98 and 0.98; P less than 0.001). Inhibition of receptors for the FITC conjugates prior staining, by preincubation with unconjugated anti-neutrophil antibodies or L-fucose, abolished the staining with antibodies and lectin. This study shows that staining with unlabeled FITC represents a quick and convenient method for the estimation of phagocytic cells (polymorphonuclear cells and macrophages) in histological specimens.

Statistical aspects of cell motility determinations with a modified chemotaxis assembly for multiwell filter assays.

A multiple chamber assembly for determination of neutrophil migration consisting of disposable lower plastic chambers and an easily cleaned and inert upper chamber made of stainless steel was constructed and used in experimental work. Using results of chemotaxis determinations in the assembly and a statistical model the consequences of various ways to present cell migration data are discussed. With any method for multiple measurements of chemotaxis of cells from a given sample, there are occasional extreme results ('outliers') deviating considerably from other determinations. We show that the medians of chemotaxis determinations performed in triplicate are less influenced by such outliers than the mean values. The risk of an outlier affecting the median value was in our assembly about 0.2%, compared with a 10% chance of influencing the mean value.

Immune complex induced experimental colitis: beneficial effect of sulphasalazine in rabbits.

Experimental colitis was induced in rabbits by exposing the colon mucosa to 1% formalin followed by i.v. injections of soluble immune complexes made with antigen in excess. The animals were preimmunized with Escherichia coli O14:K7:H--inducing antibodies cross-reactive to intestinal epithelium. Animals with this colitis were divided in two groups. One group was treated with sulphasalazine and the other was given vehicle only. Sulphasalazine was administered daily at 125.5 mumol (50 mg) per kg body weight. The administration was started at the same day as the colitis was initiated. At Day 6, 13 and 30 following induction of colitis, biopsies were sampled and histologically evaluated. Inflammation was assessed by scores for inflammatory cells, crypt distortion, decreased crypt number and presence of crypt abscesses, thus corresponding to the picture seen in humans. A statistically significant lower score of inflammation was seen on Day 6 and 13 (P less than 0.01) and on Day 30 (P less than 0.05) following induction of colitis in animals treated with sulphasalazine.

Experimental colitis induced by dextran sulphate sodium in mice: beneficial effects of sulphasalazine and olsalazine.

BACKGROUND: Animal models of inflammatory bowel disease are artificial and more or less representative of human disease. However, the dextran sulphate sodium (DSS) induced intestinal inflammation model has recently been shown to fulfil some pathological criteria for an adequate experimental model. AIM: To determine whether this form of experimental intestinal inflammation responds to established therapy used for human inflammatory bowel disease. METHODS: DSS was used to induce intestinal inflammation in conventional Balb/c mice and athymic nu/nu CD-1(BR) mice, and the well-documented 5-aminosalicylic acid (5-ASA) based anticolitis drugs sulphasalazine (SASP) and olsalazine (OLZ) were used to study therapeutic effects. Parameters which have been shown to reflect DSS-induced intestinal inflammation (body weight, colon length, spleen weight, diarrhoea, and rectal bleeding) were measured in the Balb/c mice. RESULTS: Significant amelioration was seen on these parameters after different treatment protocols. Survival in nu/nu CD-1 mice was studied, and after 16 days a death rate of 50% was noted in the DSS group. SASP (100 mg/kg/day) and OLZ (50 mg/kg/day) significantly prolonged the survival to 29 and 38 days, respectively. SASP and OLZ showed a dose-dependent effect in the range between 10 and 100 mg/kg/day, doses closely corresponding to those used in humans. CONCLUSIONS: SASP and OLZ are able to ameliorate the DSS-induced intestinal inflammation. The dose-response patterns suggested that the active therapeutic moiety for the two drugs appears to be mainly the liberated 5-ASA molecule.

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750.

Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus. Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated M(r) from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 (dvn750) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M(r) = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.

A system for heterologous expression of bacteriocins in Lactobacillus sake.

A system for efficient heterologous expression of class II bacteriocins is described that is based on introducing two plasmids in a bacteriocin-negative Lactobacillus sake strain. The first plasmid (pSAK20) contains the genes necessary for transcriptional activation of the Sakacin A promoter as well as export and processing of bacteriocin precursors. The second plasmid (a pLPV111 derivative) contains the structural and immunity genes for the bacteriocin of interest fused to the sakacin A promoter. Using this system, various bacteriocins were produced at levels equal to or higher than those obtained with the corresponding wild-type producer strains.

Purification and cloning of sakacin 674, a bacteriocin from Lactobacillus sake Lb674.

Sakacin 674, a bacteriocin produced by Lactobacillus sake Lb764 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and sequential ion exchange, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence of sakacin 674 was determined by Edman degradation. The bacteriocin consisted of 43 amino acid residues and had a calculated molecular mass of 4436.6 Da, which is in good agreement with the molecular mass of 4437.2 as determined by mass spectrometry. The structural gene encoding sakacin 674 (sakR) was located on the chromosome. This gene was cloned and sequenced. It encoded a primary translation product of 61 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin 674. Sakacin 674 resembled other known bacteriocins and was very similar to sakacin P.

A model assay to demonstrate how intrinsic factors affect diffusion of bacteriocins.

A rapid and simple method to elucidate how intrinsic factors in a given food product affect bacteriocin diffusion and efficacy is described. The basic idea of this assay is to help predict which bacteriocin or other inhibitory substance to select for a given product, where increased security towards specific microorganisms is wanted. In an agar plate model system the effect of five factors (number of indicator cells, pH and concentration of NaCl, agar and soy oil) on the diffusion of the bacteriocins sakacin A, sakacin P, pediocin PA-1, piscicolin 61 and nisin was studied. An experimental design permitting simultaneous evaluation of the effect of all factors was used. The results indicated that each bacteriocin has a characteristic intrinsic factor effect profile. However, pH and load of indicator cells affect all the bacteriocins.

Helix pomatia a hemagglutinin, a surface marker for bovine T-lymphocytes.

Bovine peripheral blood lymphocytes (PBL) were isolated from blood collected from 6 cattle. After treatment with neuraminidase, 40 or 60% of the cells were shown to combine with Helix Pomatia A hemagglutinin (HP) depending whether a direct or indirect fluorescence technique was used. About 20% of the cells were Ig-bearing. With double staining fluorescence technique, it was shown that cells attaching to HP were not Ig-bearing and the reverse. With the aid of HP, covalently bound to Sepharose, Ig-bearing cells could be separated from cell populations attaching to HP. The fraction of cells forming rosettes with sheep erythrocytes was proportional to that of HP attaching cells both before and after fractionation on the HP column. It is therefore concluded that HP is a marker for bovine T-cells, and that this lectin may be used to separate B-cells from T-cells.

Accelerated production of dry fermented sausage.

The scope of this paper is to review work connected with accelerated ripening of dry fermented sausages by addition of proteolytic enzymes. An overview of the following topics is given: practical sausage experiments with addition of various proteinases of bacterial origin, including data from sensory, biochemical and gc/ms analyses; biochemical and genetic characterization of the enzyme shown to be most useful in these experiments, the serine proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151; experiments to transform starter cultures with the genes for production of this proteinase and proposals for future work in this field.

Cleaning of conveyor belt materials using ultrasound in a thin layer of water.

Cleaning of conveyor belts in the food industry is imperative for preventing the buildup of microorganisms that can contaminate food. New technologies for decreasing water and energy consumption of cleaning systems are desired. Ultrasound can be used for cleaning a wide range of materials. Most commonly, baths containing fairly large amounts of water are used. One possibility to reduce water consumption is to use ultrasonic cavitation in a thin water film on a flat surface, like a conveyor belt. In order to test this possibility, a model system was set up, consisting of an ultrasound transducer/probe with a 70-mm-diameter flat bottom, operating at 19.8 kHz, and contaminated conveyor belt materials in the form of coupons covered with a thin layer of water or water with detergent. Ultrasound was then applied on the water surface at different power levels (from 46 to 260 W), exposure times (10 and 20 s), and distances (2 to 20 mm). The model was used to test two different belt materials with various contamination types, such as biofilms formed by bacteria in carbohydrate- or protein-fat-based soils, dried microorganisms (bacteria, yeasts, and mold spores), and allergens. Ultrasound treatment increased the reduction of bacteria and yeast by 1 to 2 log CFU under the most favorable conditions compared with water or water-detergent controls. The effect was dependent on the type of belt material, the power applied, the exposure time, and the distance between the probe and the belt coupon. Generally, dried microorganisms were more easily removed than biofilms. The effect on mold spores was variable and appeared to be species and material dependent. Spiked allergens were also efficiently removed by using ultrasound. The results in this study pave the way for new cleaning designs for flat conveyor belts, with possibilities for savings of water, detergent, and energy consumption.

Effects of post-processing treatments on sensory quality and Shiga toxigenic Escherichia coli reductions in dry-fermented sausages.

The effects of post-processing treatments on sensory quality and reduction of Shiga toxigenic Escherichia coli (STEC) in three formulations of two types of dry-fermented sausage (DFS; salami and morr) were evaluated. Tested interventions provided only marginal changes in sensory preference and characteristics. Total STEC reductions in heat treated DFS (32°C, 6days or 43°C, 24h) were from 3.5 to >5.5 log from production start. Storing of sausages (20°C, 1month) gave >1 log additional STEC reduction. Freezing and thawing of sausages in combination with storage (4°C, 1month) gave an additional 0.7 to 3.0 log reduction in STEC. Overall >5.5 log STEC reductions were obtained after storage and freezing/thawing of DFS with increased levels of glucose and salt. This study suggests that combined formulation optimisation and post-process strategies should be applicable for implementation in DFS production to obtain DFS with enhanced microbial safety and high sensory acceptance and quality.

Heterogeneity in resistance to food-related stresses and biofilm formation ability among verocytotoxigenic Escherichia coli strains.

This study assessed the resistance of ten verocytotoxigenic Escherichia coli (VTEC) isolates of commonly encountered serogroups/-types and two non-pathogenic E. coli strains to various food-related stresses (acid, alkaline, heat and high hydrostatic pressure treatments) and their biofilm formation ability. In addition, the global changes in the cellular composition in response to the exposure to these adverse environments were monitored by Fourier Transform Infrared (FT-IR) spectroscopy for two of the strains. Large inter-strain variations in stress resistance were observed. The most tolerant strains belonged to serogroup O157 which included both the O157:H7 type strain EDL933 and a representative isolate of the sorbitol fermenting O157:H- VTEC clone (strain MF3582). Strain C-600, a non-pathogenic laboratory strain, was sensitive to multiple stresses. Although wide variation in biofilm-forming ability was observed among VTEC isolates, no consistent relationships between biofilm-forming ability and capacity to withstand stress exposures were found. Analysis of the allelic status of the rpoS gene, involved in the general stress response of stationary-phase cells, allowed detection of loss-of-function mutations for two strains, E218/02 and MF2411, both of them showing as common features a high sensitivity to alkaline and heat treatments and a poor ability to form mature biofilms. Evidences found in this study confirm rpoS as a highly mutable gene in nature, and suggest its relevance not only for the mount of an active stress response but also for the establishment of mature biofilm communities. Our findings contribute to increase the knowledge on the resistance of VTEC to environmental stresses commonly encountered in the food chain, which can lead to improved strategies for preventing VTEC infections.