Uso de antibióticos convencionais e antimicrobianos a base de lúpulo no controle da infecção bacteriana em fermentação alcoólica

The aim of this work was to compare the efficiency of conventional antibiotics in relation to hop-based antimicrobials, in industrial-scale bioethanol production. The comparison was made by calculating the lactic acid bacteria population reduction in two consecutive fermentation cycles. To conduct the experiment, it was used five treatments (three conventional antibiotics: Kamoran WP, Corstan and Alcapen 1030, and two hop-based antimicrobials: BetaBio and IsoStab). The samples were collected in the fermentation vat. In order to quantify the initial lactic acid bacteria population, a sample was collected at the end of the fermentation process (wine) before the treatment with antibiotics or antimicrobials, and to determine the final population, another sample was collected at the end of the fermentation process (wine) after the treatment with antibiotics or antimicrobials. The experiment was completely randomized and the statistical analysis was performed through analysis of variance (ANOVA) for data processed using the equation y’ = . After the data transformation, the Levene's test was applied to verify data adherence to normal distribution, and the averages were compared through Tukey’s test at 5% probability. The results showed that the hop-based antimicrobials (IsoStab and BetaBio) can be used to substitute the conventional antibiotics (Kamoran, Alcapen and Corstan), since there was no statistical difference between the treatments.

Desenvolvimento de um embutido cárneo fermentado, com teores reduzidos de gordura e sais de cura, através da utilização de culturas probióticas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Imunonutrição de frangos de corte alimentados com ácidos orgânicos em alternativa aos quimioterápicos

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Growth potential of Salmonella spp. and Listeria monocytogenes in nine types of ready-to-eat vegetables stored at variable temperature conditions during shelf-life

Growth potential (delta) is defined as the difference between the population of a microorganism at the end of shelf-life of specific food and its initial population. The determination of 6 of Salmonella and Listeria monocytogenes in RTE vegetables can be very useful to determine likely threats to food safety. However, little is known on the behavior of these microorganisms in several RTE vegetables. Therefore, the aim of this study was to determine the delta of both pathogens in nine different types of RTE vegetables (escarole, collard green, spinach, watercress, arugula, grated carrot, green salad, and mix for yakisoba) stored at refrigeration (7 degrees C) and abuse temperature (15 degrees C). The population of aerobic microorganisms and lactic acid bacteria, including those showing antimicrobial activity has been also determined. Results indicated that L monocytogenes was able to grow (delta >= 0.5 log(10)) in more storage conditions and vegetables than Salmonella. Both microorganisms were inhibited in carrots, although a more pronounced effect has been observed against L monocytogenes. The highest 5 values were obtained when the RTE vegetables were stored 15 degrees C/6 days in collard greens (delta=3.3) and arugula (delta=3.2) (L monocytogenes) and arugula (delta=4.1) and escarole (delta=2.8) (Salmonella). In most vegetables and storage conditions studied, the counts of total aerobic microorganisms raised significantly independent of the temperature of storage (p<0.05). Counts of lactic acid bacteria were higher in vegetables partially or fully stored at abuse temperature with recovery of isolates showing antimicrobial activity. In conclusion, the results of this study show that Salmonella and L monocytogenes may grow and reach high populations in RTE vegetables depending on storage conditions and the definition of effective intervention strategies are needed to control their growth in these products. (C) 2012 Elsevier B.V. All rights reserved.

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese

Aims To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity. Methods and Results Forty-three bacteriocinogenic enterococci isolates were identified by 16s rDNA, fingerprinted by RAPD-PCR analysis and tested by PCR for the presence of genes for lantibiotics (lanM, lanB and lanC) and enterocins (entA, entB, entP, entL50AB and entAS48) and by phenotypical methods for bacteriocin production and inhibitory spectrum. Also, the virulence of the isolates was evaluated by PCR for genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for gelatinase, lipase, DNAse and a- and beta-haemolysis. Most isolates (93.0%) harboured at least one lantibiotic or enterocin gene and were positive for several tested virulence genes, mainly asa1 (100%), gelE (93.0%) and efaA (83.7%). 53.5% of the isolates presented beta-haemolysis. Conclusions Enterococcus spp. isolates presented an interesting potential application for food preservation because of bacteriocin production; however, virulence-related genes were identified in all RAPD profiles. Significance and Impact of the Study The study demonstrated the contradictory characteristics of the tested Enterococcus isolates: they presented a good potential for application in food biopreservation but contained several virulence factors.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Sardinian goat's milk as source of bacteriocinogenic potential protective cultures

Goat breeding in Sardinia constitutes an important source of income for farming and shepherding activities. In this study 170 LAB strains were isolated from Sardinian goat's milk and tested for bacteriocins production against several food-borne pathogenic microorganisms. Four isolates (SD1, SD2, SD3 and SD4) were selected for their effective inhibition on Listeria monocytogenes. The strains were classified as members of Enterococcus genus, according to their biochemical and physiological characteristics, and then genetically identified as Enterococcus faecium. In MRS broth at 37 degrees C, bacteriocins SD1 and SD2 were produced at much higher levels (51200 AU/ml) compared to bacteriocin SD3 (3200 AU/ml) and bacteriocin SD4 (800 AU/ml). Their peptides were inactivated by proteolytic enzymes, but not when treated with alpha-amylase, catalase and lipase. The four bacteriocins remained stable at pH from 2.0 to 12.0, after exposure to 100 degrees C for 120 min and were not affected by the presence of surfactants and salts (N-Laourylsarcosine, NaCl, SDS, Triton X-100, Tween 20, Tween 80 and urea). Their molecular size was determined to be approximately 5 kDa by tricine-SDS-PAGE. Since the strains exhibited a strong antimicrobial activity against 21 L monocytogenes strains and 6 Salmonella spp. isolates, they should be considered as potential bio-preservatives cultures for fermented food productions. Moreover, due to their technological features, the four strains could be taken in account for using as adjunct NSLAB (non-starter lactic acid bacteria) rather than as starter culture. (C) 2011 Elsevier Ltd. All rights reserved.

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 degrees C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 degrees C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese. (c) 2012 Elsevier Ltd. All rights reserved.

Evaluation of relative biological efficiency of additives in sugarcane ensiling

The objective of this study was to evaluate the effects of adding alkalis on the fermentative pattern, aerobic stability and nutritive value of the sugarcane silage. A completely randomized design with 6 additives in two concentrations (1 or 2%), plus a control group, totalizing 13 treatments [(6x2)+1] with four replications, was used. The additives were sodium hydroxide (NaOH), limestone (CaCO3), urea (CO(NH2)(2)), sodium bicarbonate (NaHCO3), quicklime (CaO) and hydrated lime (Ca(OH)(2)). The material was ensiled in 52 laboratory silos using plastic buckets with 12 L of capacity. Silos were opened 60 days after ensiling, when organic acids concentration, aerobic stability and chemical composition were determined. The Relative Biological Efficiency (RBE) was calculated by the slope ratio method, using the data obtained from ratio between desirable and undesirable silage products, according to the equation: D/U ratio = [lactic/(ethanol + acetic + butyric)]. All additives affected dry matter, crude protein, acid detergent fiber, neutral detergent fiber contents and buffering capacity. Except for urea and quicklime, all additives increased the in vitro dry matter digestibility. In general, these additives altered the fermentative pattern of sugarcane silage, inhibiting alcoholic fermentation and improving lactic acid production. The additive that showed the best RBE in relation to sodium hydroxide (100%) was limestone (89.4%). The RBE values of urea, sodium bicarbonate and hydrated lime were 49.2%, 47.7% and 34.3%, respectively.

Constitutive models for biodegradable thermoplastic ropes for ligament repair

The concept behind a biodegradable ligament device is to temporarily replace the biomechanical functions of the ruptured ligament, while it progressively regenerates its capacities. However, there is a lack of methods to predict the mechanical behaviour evolution of the biodegradable devices during degradation, which is an important aspect of the project. In this work, a hyper elastic constitutive model will be used to predict the mechanical behaviour of a biodegradable rope made of aliphatic polyesters. A numerical approach using ABAQUS is presented, where the material parameters of the model proposal are automatically updated in correspondence to the degradation time, by means of a script in PYTHON. In this method we also use a User Material subroutine (UMAT) to apply a failure criterion base on the strength that decreases according to a first order differential equation. The parameterization of the material model proposal for different degradation times were achieved by fitting the theoretical curves with the experimental data of tensile tests on fibres. To model all the rope behaviour we had considered one step of homogenisation considering the fibres architectures in an elementary volume. (C) 2012 Elsevier Ltd. All rights reserved.

Cytoplasmic and extracellular expression of pharmaceutical-grade mycobacterial 65-kDa heat shock protein in Lactococcus lactis

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U. S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.

Organic milk improves Bifidobacterium lactis counts and bioactive fatty acids contents in fermented milk

In functional dairy products, polyunsaturated fatty acids such as, conjugated linoleic acid (CLA) and alpha-linolenic acid (ALA) have been highlighted for their benefits related to prevention of some chronic diseases. In order to study the effect of type of milk (conventional vs. organic, characterized by a specific fatty acid composition), Bifidobacterium animalis subsp. lactis (BB12, B94, BL04 and HN019) counts, acidification activity and chemical composition (pH, lactose, lactic acid contents and fatty acids profile) were investigated before fermentation and after 24 h of products stored at 4 degrees C. Organic and conventional milk influenced acidification performance and bacteria counts, which was strain-dependent. Higher counts of BB12 were observed in organic milk, whereas superior counts of BL04 were found in conventional milk. Organic fermented milk showed lower levels in saturated fatty acids (FA) and higher in monounsaturated FA contents. Similarly, among bioactive FA, organic fermented milks have higher amounts of trans vaccenic acid (TVA-C18:1t), conjugated linoleic acid (CLA) and slightly higher contents of alpha-linoleic acid (ALA). (C) 2012 Elsevier Ltd. All rights reserved.

In Vitro Evaluation of the Probiotic Potential of Bacteriocin Producer Lactobacillus sakei 1

Lactobacillus sakei 1 is a food isolate that produces a heat-stable antimicrobial peptide (sakacin 1, a class ha bacteriocin) inhibitory to the opportunistic pathogen Listeria monocytogenes. Bacterial isolates with antimicrobial activity may be useful for food biopreservation and also for developing probiotics. To evaluate the probiotic potential of L. sakei I, it was tested for (i) in vitro gastric resistance (with synthetic gastric juice adjusted to pH 2.0, 2.5, or 3.0); (ii) survival and bacteriocin production in the presence of bile salts and commercial prebiotics (inulin and oligofructose); (iii) adhesion to Caco-2 cells; and (iv) effect on the adhesion of L. monocytogenes to Caco-2 cells and invasion of these cells by the organism. The results showed that L. sakei I survival in gastric environment varied according to pH, with the maximum survival achieved at pH 3.0, despite a 4-log reduction of the population after 3 h. Regarding the bile salt tolerance and influence of prebiotics, it was observed that L. sakei 1 survival rates were similar (P > 0.05) for all de Man Rogosa Shame (MRS) broth formulations when tests were done after 4 h of incubation. However, after incubation for 24 h, the survival of L. sakei 1 in MRS broth was reduced by 1.8 log (P < 0.001), when glucose was replaced by either inulin or oligofructose (without Oxgall). L. sakei 1 was unable to deconjugate bile salts, and there was a significant decrease (1.4 log) of the L. sakei 1 population in regular MRS broth plus Oxgall (P < 0.05). In spite of this, tolerance levels of L. sakei 1 to bile salts were similar in regular MRS broth and in MRS broth with oligofructose. Lower bacteriocin production was observed in MRS broth when inulin (3,200 AU/ml) or oligofructose (2,400 AU/ml) was used instead of glucose (6,400 AU/ml). L. sakei I adhered to Caco-2 cells, and its cell-free pH-neutralized supernatant containing sakacin I led to a significant reduction of in vitro listerial invasion of human intestinal Caco-2 cells.

Effect of inulin on the growth and metabolism of a probiotic strain of Lactobacillus rhamnosus in co-culture with Streptococcus thermophilus

Metabolic studies are very important to improve quality of functional dairy products. For this purpose, the behaviors of pure cultures of Streptococcus thermophilus (St) and Lactobacillus rhamnosus (Lr) as well a co-culture of them (St-Lr) were investigated during skim milk fermentation, and the inulin effect as prebiotic was assessed. Lr was able to metabolize 6 g/100 g more galactose than St and St-Lr. Final lactic acid production by Lr was higher (9.8 g/L) compared to St (9.1 g/L) and St-Lr (9.1 g/L). Acetic acid concentration varied from 0.8 g/L (St-Lr) to 1.5 g/L (Lr) and that of ethanol from only 0.2 g/L (St-Lr) to 0.4 g/L (Lr), which suggests the occurrence in Lr of a NADH oxidase activity and citrate co-metabolization via pyruvate, both dissipating a part of the reducing power. Diacetyl and acetoin accumulated at the highest levels (18.4 and 0.8 mg/L, respectively) with St-Lr, which suggests possible synergistic interactions between these microorganisms as well as the Lr capability of co-metabolizing citrate in the presence of lactose. Inulin stimulated both biomass growth and levels of all end-products, as the likely result of fructose release from its partial hydrolysis and subsequent metabolization as an additional carbon and energy source. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Co-metabolic models of Streptococcus thermophilus in co-culture with Lactobacillus bulgaricus or Lactobacillus acidophilus

To shed light on the interactions occurring in fermented milks when using co-cultures of Streptococcus thermophilus with Lactobacillus bulgaricus (StLb) or Lactobacillus acidophilus (StLa), a new co-metabolic model was proposed and checked either in the presence of Inulin as a prebiotic or not. For this purpose, the experimental data of concentrations of substrates and fermented products were utilized in balances of carbon, reduction degree and ATP. S. thermophilus exhibited always quicker growth compared to the other two microorganisms, while the percentage of lactose fermented to lactic acid, that of galactose metabolized, and the levels of diacetyl and acetoin formed strongly depended on the type of co-culture and the presence of inulin. The StLb co-culture led to higher acetoin and lower diacetyl levels compared to StLa, probably because of more reducing conditions or limited acetoin dehydrogenation. Inulin addition to StLa suppressed acetoin accumulation and hindered that of diacetyl, suggesting catabolite repression of alpha-acetolactate synthase expression in S. thermophilus. Both co-cultures showed the highest ATP requirements for biomass growth and maintenance at the beginning of fermentation, consistently with the high energy demand of enzyme induction during lag phase. Inulin reduced these requirements making biomass synthesis and maintenance less energy-consuming. Only a fraction of galactose was released from lactose, consistently with the galactose-positive phenotype of most dairy strains. The galactose fraction metabolized without inulin was about twice that in its presence, which suggests inhibition of the galactose transport system of S. thermophilus by fructose released from partial inulin hydrolysis. (C) 2012 Elsevier B.V. All rights reserved.