Food waste from restaurant sector - Characterization for biorefinery approach.

The aim of this study is the analysis of food waste (FW) composition from local catering services to assess potential biorefinery development. Moisture content of different FW samples showed that 27-47% (w/w) was organic material. Main components were lipids (25.7-33.2, w/w), starch (16.2-29.4%, w/w) and proteins (23.5-18.3%, w/w) on a dry basis. A metal profile with Na and Mg as main components, followed by trace elements, i.e. Zn or Fe, was also found in food waste samples. Statistical tests in combination with principal component analysis provides an efficient methodology to establish specific composition variations between FW from different catering services, while relating them to FW typology. The combination of chemical characterization with statistical study constitutes a promising decision-making tool for FW processing and valorization. The innovative methodology presented in this study provides systematic evaluation of FW composition and variability to allow selection of the most appropriate valorization paths.

Isolation, identification and screening of yeasts towards their ability to assimilate biodiesel-derived crude glycerol: microbial production of polyols, endopolysaccharides and lipid.

AIMS: To assess the ability of various newly isolated or belonging in official collections yeast strains to convert biodiesel-derived glycerol (Gly) into added-value compounds. METHODS AND RESULTS: Ten newly isolated yeast strains belonging to Debaryomyces sp., Naganishia uzbekistanensis, Rhodotorula sp. and Yarrowia lipolytica, isolated from fishes, metabolized Gly under nitrogen limitation. The aim of the study was to identify potential newly isolated microbial candidates that could produce single-cell oil (SCO), endopolysaccharides and polyols when these micro-organisms were grown on biodiesel-derived Gly. As controls producing SCO and endopolysaccharides were the strains Rhodotorula glutinis NRRL YB-252 and Cryptococcus curvatus NRRL Y-1511. At initial Gly (Gly0 ) ≈40 g l-1 , most strains presented remarkable dry cell weight (DCW) production, whereas Y. lipolytica and Debaryomyces sp. produced non-negligible quantities of mannitol and arabitol (Ara). Five strains were further cultivated at increasing Gly0 concentrations. Rhodotorula glutinis NRRL YB-252 produced 7·2 g l-1 of lipid (lipid in DCW value ≈38% w/w), whereas Debaryomyces sp. FMCC Y69 in batch-bioreactor experiment with Gly0 ≈80 g l-1 , produced 30-33 g l-1 of DCW and ~30 g l-1 of Ara. At shake-flasks with Gly0 ≈125 g l-1 , Ara of ~48 g l-1 (conversion yield of polyol on Gly consumed ≈0·62 g g-1 ) was achieved. Cellular lipids of all yeasts contained in variable concentrations oleic, palmitic, stearic and linoleic acids. CONCLUSIONS: Newly isolated, food-derived and non-previously studied yeast isolates converted biodiesel-derived Gly into several added-value metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternative ways of crude Gly valorization through yeast fermentations were provided and added-value compounds were synthesized.

Lipid production and characterization by Mortierella (Umbelopsis) isabellina cultivated on lignocellulosic sugars.

AIMS: To study and characterize the lipids produced by Mortierella (Umbelopsis) isabellina, during its growth on mixtures of glucose and xylose. METHODS AND RESULTS: Glucose and xylose were utilized as carbon sources, solely or in blends, under nitrogen-limited conditions, in batch-flask trials (initial sugars at 80 g l-1 ). Significant lipid production (maximum lipid 17·8 g l-1 ; lipid in DCW 61·0% w/w; lipid on glucose consumed 0·23 g g-1 ) occurred on glucose employed solely, while xylose concentration in the growth medium was conversely correlated with lipid accumulation. With increasing xylose concentrations into the blend, lipid storage decreased while xylitol in significant concentrations (up to 24 g l-1 ) was produced. Irrespective of the sugar blend employed, significant quantities of endopolysaccharides were detected in the first growth steps (in the presence of nitrogen into the medium or barely after its disappearance) while lipids were stored thereafter. Neutral lipids, mainly composed of triacylglycerols, were the main microbial lipid fraction. Phospholipids were quantified both through fractionation and subsequent gravimetric determination and also through determination of phosphorus, and it seemed that the second method was more accurate. Phospholipids were mainly composed of phosphatidylcholine and another nonidentified compound presumably being phosphatidyldimethylethanolamine. CONCLUSIONS: Mortierella isabellina is suitable to convert lignocellulosic sugars into lipids. SIGNIFICANCE AND IMPACT OF THE STUDY: Differentiations between metabolism on xylose and glucose were reported. Moreover, this is one of the first reports indicating extensive analysis of microbial lipids produced by M. isabellina.

Detection of intracellular IFN-γ and IL-4 cytokines in CD4+ and CD8+ T cells in the peripheral blood of dogs naturally infected with Leishmania infantum.

Canine leishmaniosis (CanL) is a systemic zoonotic disease the clinical manifestations of which can range from self-healing cutaneous lesions to disseminated visceral disease. Effective activation of cellular immunity is the cornerstone of resistance against Leishmania infantum in infected dogs. The aim of this cross-sectional, controlled study was the intracellular detection of interleukin 4 (IL-4) and interferon-γ (IFN-γ) in CD4+ and CD8+ lymphocytes in the peripheral blood of 40 dogs naturally infected with L. infantum by applying flow cytometry. The percentage of CD4+IL-4+ and CD8+IL-4+ lymphocytes (with or without immunostimulation) was low in the clinically healthy and subclinically infected dogs in contrast to clinically affected ones. In the same groups of dogs, the percentage of CD4+IFN-γ+ and CD8+IFN-γ+ T cells in their resting phase and following specific immunostimulation with Leishmania soluble antigen (LSA) was also low. CD4+IL-4+ and CD8+IL-4+ T cell percentage was higher in sick compared to clinically healthy and subclinically infected dogs, after immunostimulation. The corresponding figure of CD8+IL-4+ cells in sick dogs after LSA immunostimulation was also increased thus underlining the important role these cells may play in humoral immunity and perhaps the progression of CanL.

Biorefining of by-product streams from sunflower-based biodiesel production plants for integrated synthesis of microbial oil and value-added co-products.

This study focuses on the valorisation of crude glycerol and sunflower meal (SFM) from conventional biodiesel production plants for the separation of value-added co-products (antioxidant-rich extracts and protein isolate) and for enhancing biodiesel production through microbial oil synthesis. Microbial oil production was evaluated using three oleaginous yeast strains (Rhodosporidium toruloides, Lipomyces starkeyi and Cryptococcus curvatus) cultivated on crude glycerol and nutrient-rich hydrolysates derived from either whole SFM or SFM fractions that remained after separation of value-added co-products. Fed-batch bioreactor cultures with R. toruloides led to the production of 37.4gL(-1) of total dry weight with a microbial oil content of 51.3% (ww(-1)) when a biorefinery concept based on SFM fractionation was employed. The estimated biodiesel properties conformed with the limits set by the EN 14214 and ASTM D 6751 standards. The estimated cold filter plugging point (7.3-8.6°C) of the lipids produced by R. toruloides is closer to that of biodiesel derived from palm oil.

Cutaneous immune mechanisms in canine leishmaniosis due to Leishmania infantum.

Canine leishmaniosis (CanL) caused by the parasite Leishmania infantum is a systemic disease with variable clinical signs. The disease is endemic in the Mediterranean countries and dogs are the main domestic reservoir of the parasite. The quite complicated immune response against the parasite is crucial for the evolution of CanL infection with the skin playing a major role in its immunopathogenesis. After the inoculation of Leishmania promastigotes into the dermis by sand fly bites, complement factors, Langerhan's cells, neutrophils, fibroblasts and keratinocytes are involved in the activation of the innate arm of the skin immune system, with the macrophages and dendritic cells to play a major key role. The effective activation of cellular immunity is the cornerstone of dog's resistance against the parasite. Promastigotes reaching the dermis are engulfed, processed and transferred by APCs to draining lymph nodes to stimulate naïve T-cells for proliferation and differentiation into armed effector T-cells. Th1 cells activate the infected macrophages to kill Leishmania, whereas Th2 cells divert the immune response to humoral immunity and down regulation of cellular immunity with Th1 cell anergy. Inhibition of co-stimulatory molecules expression by infected macrophages contributes to T-cell anergy. In canine subclinical infections cutaneous lymphocytic infiltrate and parasites are absent, as opposed to dogs with clinical leishmaniosis. CD8+ cells constitute a significant population of cellular immunity in CanL since they outnumber CD4+ cells in the dermis, producing IFN-γ in sub clinically infected dogs and high levels of IL-4 in dogs with clinical leishmaniosis. Numerous B-lymphocytes have been shown to heavily infiltrate the dermis at least in exfoliative dermatitis in CanL. A mixed Th1/Th2 cytokine profile has been found in the dermis of naturally infected with L. infantum dogs. In the skin of dogs with clinical leishmaniosis, where plasma cells outnumber T lymphocytes in the dermal infiltrate, there is an overproduction of IL-4, IL-13 and TNF-α leading to Th2-biased humoral immune response. The issue of humoral immunity polarization in CanL remains controversial. Much still needs to be learned about other mechanisms underlying the complex interaction between the skin immune system and the parasite.

Lipid production by yeasts growing on biodiesel-derived crude glycerol: strain selection and impact of substrate concentration on the fermentation efficiency.

AIMS: To screen yeasts in relation to the potential to produce single cell oil (SCO) from biodiesel-derived glycerol and to enhance SCO production in Lipomyces starkeyi and Rhodosporidium toruloides yeasts. METHODS AND RESULTS: Yarrowia lipolytica, Cryptococcus curvatus, R. toruloides and L. starkeyi were grown in nitrogen-limited flask cultures. Yarrowia lipolytica strains produced citric acid and mannitol. Lipomyces starkeyi DSM 70296 and R. toruloides NRRL Y-27012 showed potential for SCO production, and were cultivated at increasing the initial glycerol concentrations with the initial nitrogen concentration remaining constant. Significant biomass and SCO production were reported even in cultures with high initial glycerol concentrations (i.e. 180 g l(-1) ). Lipid quantities of c. 12 g l(-1) (lipid in dry cell weight 35-40%) were obtained for both L. starkeyi and R. toruloides, quite high values compared with literature values for oleaginous micro-organisms growing on glycerol. However, these strains exhibited different kinetic profiles in the synthesis of intracellular polysaccharides. Lipomyces starkeyi produced a significant quantity of polysaccharides (c. 7 g l(-1) ). The yeast lipids contained mainly oleic and palmitic and to a lesser extent linoleic and stearic acids. CONCLUSIONS: Lipomyces starkeyi and R. toruloides are potential SCO producers from crude glycerol. SIGNIFICANCE AND IMPACT OF THE STUDY: Very scarce numbers of reports have indicated the production of SCO by L. starkeyi and R. toruloides growing on glycerol. We report here that these yeasts are able efficiently to convert raw glycerol into SCO, while L. starkeyi also synthesizes intracellular polysaccharides in marked quantities.

Emergence and maintenance of multidrug-resistant Escherichia coli of canine origin harbouring a blaCMY-2-IncI1/ST65 plasmid and topoisomerase mutations.

OBJECTIVES: To characterize the mechanisms implicated in fluoroquinolone (FQ) and expanded-spectrum cephalosporin (ESC) resistance in three clinical and seven faecal multidrug-resistant (MDR; resistant to at least three antimicrobial classes) Escherichia coli isolates from a dog with atopic dermatitis, also suffering from recurrent otitis, that had already been exposed to prolonged antimicrobial treatment and colonized for a long period. METHODS: MICs of FQs, ESCs and other antimicrobials were determined by the broth microdilution method. Phenotypic tests (efflux pump inhibition and combination disc tests) and isoelectric focusing were combined with genotypic analyses [PCRs, sequencing, conjugation, S1 nuclease PFGE, PCR-based replicon typing, plasmid multilocus sequence typing (pMLST) and PCR mapping] to characterize the molecular basis of FQ and ESC resistance. Isolates were further characterized by MLST and PFGE. RESULTS: Three otitis and five faecal isolates with enrofloxacin MICs of 32 to >128 mg/L displayed the GyrA:S83L+D87N/ParC:E62K/ParE:G545D pattern harbouring novel ParC and ParE substitutions, whereas the two remaining faecal isolates were susceptible or borderline resistant single-step mutants (GyrA:S83L pattern) and carried qnrS1. Efflux pump overexpression also contributed to FQ resistance and the MDR phenotype. The three otitis and five faecal isolates also exhibited cefoxitin/ceftazidime MICs of 32-64 mg/L and harboured blaCMY-2, adjusted to ISEcp1, on an IncI1/ST65 conjugative plasmid, previously described in Salmonella Heidelberg from poultry. Interestingly, all isolates shared an identical MLST type (ST212), with the otitis isolates showing indistinguishable patterns with the high-level resistant faecal E. coli isolates. CONCLUSIONS: The long-term maintenance of FQ- and ESC-resistant clones harbouring topoisomerase mutations and a blaCMY-2-IncI1/ST65 plasmid in canine commensal flora after prolonged antimicrobial use may contribute to the dissemination of multidrug resistance.

Pathologic mechanisms underlying the clinical findings in canine leishmaniasis due to Leishmania infantum/chagasi.

In dogs with symptomatic or asymptomatic leishmaniasis, Leishmania infantum appears to induce a mixed Th1/Th2 immune response that in the sick dog may eventually result in tissue damage via different pathomechanisms, notably granulomatous inflammation (eg, nodular dermatitis, osteomyelitis), immune complex deposition (eg, glomerulonephritis), and/or autoantibody production (eg, polymyositis). This is a compensatory but detrimental mechanism generated mainly because of the insufficient killing capacity of macrophages against the parasite in the susceptible dog. Clinical disease is typically exemplified as exfoliative and/or ulcerative dermatitis, with or without nasodigital hyperkeratosis and onychogryphosis, glomerulonephritis, atrophic myositis of masticatory muscles, anterior uveitis, keratoconjunctivitis sicca, epistaxis, and/or polyarthritis, appearing alone or in various combinations. The pathogenesis of these clinical conditions has recently been highlighted, to a greater or lesser extent. The usually subclinical conditions expressed as chronic colitis, chronic hepatitis, vasculitis, myocarditis, osteomyelitis, orchiepididymitis, and meningoencephalomyelitis, though uncommon, are of pathologic importance from a differential point of view. The leading cause of death among canine leishmaniasis patients is chronic proteinuric nephritis that may progress to end-stage kidney disease, nephrotic syndrome, and/or systemic hypertension. However, even the asymptomatic proteinuria, when profuse, may be a serious problem because it predisposes to arterial thromboembolism and eventually contributes to the deterioration of the body condition.

Basal cell carcinoma in a dog with chronic solar dermatitis.

A seven-year-old, entire male, American Staffordshire bull terrier was diagnosed with chronic solar dermatitis and basal cell carcinoma, based on physical examination, cutaneous cytology and histopathology. Immunohistochemistry revealed that the tumour cells did not express p53. To the authors' knowledge this is the first reported case of canine basal cell carcinoma developing as a complication of chronic solar dermatitis.

Evaluating glucose and xylose as cosubstrates for lipid accumulation and γ-linolenic acid biosynthesis of Thamnidium elegans.

AIMS: To study the biotechnological production of lipids containing rich amounts of the medically and nutritionally important γ-linolenic acid (GLA), during cultivation of the Zygomycetes Thamnidium elegans, on mixtures of glucose and xylose, abundant sugars of lignocellulosic biomass. METHODS AND RESULTS: Glucose and xylose were utilized as carbon sources, solely or in mixtures, under nitrogen-limited conditions, in batch-flask or bioreactor cultures. On glucose, T. elegans produced 31.9 g l(-1) of biomass containing 15.0 g l(-1) lipid with significantly high GLA content (1014 mg l(-1)). Xylose was proved to be an adequate substrate for growth and lipid production. Additionally, xylitol secretion occurred when xylose was utilized as carbon source, solely or in mixtures with glucose. Batch-bioreactor trials on glucose yielded satisfactory lipid production, with rapid substrate consumption rates. Analysis of intracellular lipids showed that the highest GLA content was observed in early stationary growth phase, while the phospholipid fraction was the most unsaturated fraction of T. elegans. CONCLUSIONS: Thamnidium elegans represents a promising fungus for the successful valorization of sugar-based lignocellulosic residues into microbial lipids of high nutritional and pharmaceutical interest. SIGNIFICANCE AND IMPACT OF THE STUDY: Xylitol production and cultivation in bioreactor trials is reported for the first time for T. elegans, while cultivation on xylose-based media resulted in high GLA production by this fungus.

Evaluation of by-products from the biodiesel industry as fermentation feedstock for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Cupriavidus necator.

Utilization of by-products from oilseed-based biodiesel production (crude glycerol, rapeseed meal hydrolysates) for microbial polyhydroxyalkanoate (PHA) production could lead to the replacement of expensive carbon sources, nutrient supplements and precursors for co-polymer production. Batch fermentations in shake flasks with varying amounts of free amino nitrogen led to the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) with a 2.8-8% 3HV content. Fed-batch fermentations in shake flasks led to the production of 10.9g/L P(3HB-co-3HV) and a 55.6% P(3HB-co-3HV) content. NaCl concentrations between 2 and 6g/L gradually became inhibitory to bacterial growth and PHA formation, whereas in the case of K(2)SO(4), the inhibitory effect was observed only at concentrations higher than 20g/L. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and nuclear magnetic resonance ((13)C NMR) demonstrated that the incorporation of 3HV into the obtained P(3HB-co-3HV) lowered glass transition temperature, crystallinity and melting point as compared to polyhydroxybutyrate. Integrating PHA production in existing oilseed-based biodiesel plants could enhance the viability and sustainability of this first generation biorefinery.

Serum acute phase proteins as clinical phase indicators and outcome predictors in naturally occurring canine monocytic ehrlichiosis.

BACKGROUND: Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, is an important tick-borne disease of global importance. Currently, limited information is available on the diagnostic and prognostic value of acute phase proteins (APPs) in dogs naturally infected with E. canis. HYPOTHESIS: APPs may be useful indicators of the clinical phase of CME and predictive of the clinical outcome (death or survival). ANIMALS: Fifty-six dogs naturally infected with E. canis and 7 clinically healthy control dogs. METHODS: C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp), and albumin concentrations determined on admission were retrospectively compared among 27 dogs with nonmyelosuppressive CME, 29 dogs with myelosuppressive CME and 7 healthy dogs. Diagnosis of CME was based on clinical and clinicopathological findings, seropositivity to E. canis, polymerase chain reaction amplification of E. canis-specific 16S rDNA, microscopic observation of Ehrlichia sp. morulae in blood monocytes or some combination of these. RESULTS: Mean concentrations of CRP, SAA, and Hp were significantly higher in the myelosuppressed dogs compared with the other groups, but no significant differences were found in the concentration of albumin. Survival analysis of the affected animals indicated that APP concentrations were not associated with clinical outcome; the latter was strongly associated with pancytopenia (odds ratio for death 22.7) and neutropenia (odds ratio for death 7.7). CONCLUSIONS AND CLINICAL IMPORTANCE: CRP, SAA, and Hp serum concentrations on admission are useful indicators of the clinical phase of CME, but are not useful predictors of clinical outcome.

Application of novel starter cultures for sourdough bread production.

Sourdough application has been extensively increased in the last years due to the consumers demand for food consumption without the addition of chemical preservatives. Several starter cultures have been applied in sourdough bread making targeting the increase of bread self-life and the improvement of sensorial character. More specific, Lactobacillus acidophilus and Lactobacillus sakei as single and mixed cultures were used for sourdough bread making. Various sourdough breads were produced with the addition of sourdough perviously prepared with 10% w/w L. acidophilus, 10% w/w L. sakei and 5% w/w L. acidophilus and 5% w/w L. sakei at the same time. Various chemical parameters were determined such as lactic acid, total titratable acidity and pH. The results revealed that the produced sourdough bread made with sourdough containing the mixed culture was preserved for more days (12 days) than all the other breads produced in the frame of this study, since it contained lactic acid in higher concentrations. The respective total titratable acidity varied between 10.5 and 11 ml NaOH N/10. The same sourdough bread had a firmer texture, better aroma, flavor and overall quality compared to other sourdough breads examined in this study, as shown by sensory evaluation tests and results obtained through SPME GC-MS analysis, which revealed significant differences among the different bread types.

Leukoencephalopathy associated with parvovirus infection in Cretan hound puppies.

Leukoencephalopathies in dogs encompass presumably inherited conditions such as leukodystrophies, hypomyelination or spongiform degeneration, but other causes, such as virus infections and toxic or nutritional factors, might also play a contributory role. In this report, we provide evidence of parvovirus infection and replication in the brains of five 6-week-old Cretan hound puppies suffering from a puppy shaker syndrome and leukoencephalopathy. Although these puppies belonged to two different litters, they were closely related, tracing back two generations to the same sire. Histologically, a mild to moderate lymphohistiocytic meningitis, with focal lymphohistiocytic leukoencephalitis in two animals, and a mild to moderate vacuolation with myelin loss, mainly in the white matter of the cerebellum was detected. Vacuolation was also found in the corpus callosum, fimbria hippocampi, mesencephalon, capsula interna, basal ganglia, and hypothalamus. By immunohistology and in situ hybridization, either parvoviral antigen, DNA, mRNA, or replicative intermediate DNA were detected in the cerebellum, hippocampus, periventricular areas, corpus callosum, cerebral cortex, medulla oblongata, and spinal cord. Parvovirus antigen, DNA, and mRNA were present in cells of the outer granular layer of the cerebellum and in periventricular cells, most likely representing spongioblasts, glial cells, neurons, endothelial cells, occasional macrophages, and ependymal cells. Sequencing revealed canine parvovirus type 2 stretches. Thus, an association of parvovirus infection with the leukoencephalopathy seems likely, possibly facilitated by a genetic predisposition due to the mode of inbreeding in this particular dog breed.

Absence of myelofibrosis in dogs with myelosuppression induced by Ehrlichia canis infection.

Bone marrow (BM) pathology was assessed in 10 dogs with Ehrlichia canis-induced aplastic pancytopenia. BM core biopsy sections were stained with haematoxylin and eosin and with haematoxylin/van Gieson and Gordon and Sweets' reticulin stain for the detection of collagen and reticulin fibres, respectively. Iron stores were assessed by Perls' Prussian blue staining. There was no significant deposition of collagen or reticulin in any sample, but in seven dogs the BM was depleted of haemosiderin. These findings suggest that myelofibrosis does not play a significant role in the development of BM failure in canine monocytic ehrlichiosis and that iron deficiency may exacerbate the anaemia in the myelosuppressive phase of the disease.

Thermally-dried immobilized kefir on casein as starter culture in dried whey cheese production.

The aim of the present study was to evaluate the use of thermally-dried immobilized kefir on casein as a starter culture for protein-enriched dried whey cheese. For comparison reasons, dried whey cheese with thermally-dried free kefir culture and with no starter culture were also produced. The effect of the nature of the culture, the ripening temperature and the ripening process on quality characteristics of the whey cheese was studied. The association of microbial groups during cheese maturation suggested repression of spoilage and protection from pathogens due to the thermally-dried kefir, as counts of coliforms, enterobacteria and staphylococci were significantly reduced in cheeses produced using thermally-dried kefir starter cultures. The effect of the starter culture on production of volatile compounds responsible for cheese flavor was also studied using the SPME GC/MS technique. Thermally-dried immobilized kefir starter culture resulted in an improved profile of aroma-related compounds. The preliminary sensory evaluation ascertained the soft, fine taste and the overall improved quality of cheese produced with the thermally-dried immobilized kefir. The potential of protein-based thermally-dried starter cultures in dairy products is finally highlighted and assessed.