Evaluation of different selective media for enumeration of probiotic micro-organisms in combination with yogurt starter cultures in fermented milk

The aim of this study was to assess selective plating methodologies for the enumeration and identification of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium animalis ssp. lactis in fermented milks. Seven agar media (MRS with added sorbitol, clindamycin or vancomycin, acidified MRS, RCA with added aniline blue and dicloxacilin, M17 and ST) were evaluated. The results showed that RCA dicloxacilin agar was suitable for the selective enumeration of B. animalis ssp. lactis in fermented milk. Either MRS (acidified) or M17 agar could be used for enumeration of L. delbrueckii ssp. bulgaricus and S. thermophilus, respectively. MRS media containing antibiotics were effective for the enumeration of the probiotic organisms (L. rhamnosus and L. acidophilus) inoculated in fermented milks.

Dried Tomato-Flavored Probiotic Cream Cheese with Lactobacillus paracasei

A dried tomato-flavored probiotic cream cheese (P) containing Lactobacillus paracasei Lpc-37 was developed for the purpose of this study. The same product, but without probiotic addition (C) was used as control. Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were used as lactic starter cultures. Chemical composition analyses and sensory tests were performed on days 1 and 7, respectively. Titratable acidity, pH value and L. paracasei population were determined every 7 d during the refrigerated storage (21 d) of the cream cheeses. The experiment and analyses were performed in triplicate, using standard methods. Probiotic population remained greater than 10(7) CFU/g throughout the storage period, thereby characterizing the product as potentially probiotic. Cream cheeses C and P did not differ on the sensory tests, both obtaining good overall acceptance by the consumers, of which 82.6% stated that they certainly or probably would buy the product.

Oxygen supply in Bacillus thuringiensis fermentations: bringing new insights on their impact on sporulation and delta-endotoxin production

The growth kinetics, sporulation, and toxicity of Bacillus thuringiensis var. israelensis were evaluated through the analysis of batch cultures with different dissolved oxygen (DO) profiles. Firstly, DO was maintained constant at 5%, 20%, or 50% throughout fermentation in order to identify the most suitable one to improve the main process parameters. Higher biomass concentration, cell productivity, and cell yield based on glucose were obtained with 50% DO. The higher aeration level also resulted in higher spore counts and markedly improved the toxic activity of the fermentation broth, which was 9-fold greater than that obtained with 5% DO (LC50 of 39 and 329 mg/L, respectively). Subsequently, using a two-stage oxygen supply strategy, DO was kept at 50% during the vegetative and transition phases until the maximum cell concentration was achieved. Then, DO was changed to 0%, 5%, 20%, or 100% throughout sporulation and cell lysis phases. The interruption of oxygen supply strongly reduced the spore production and thoroughly repressed the toxin synthesis. On the contrary, when DO was raised to 100% of saturation, toxic activity increased approximately four times (LC50 of 8.2 mg/L) in comparison with the mean values reached with lower DO levels, even though spore counts were lower than that from the 50% DO assay. When pure oxygen was used instead of normal air, it was possible to obtain 70% of the total biomass concentration achieved in the air assays; however, cultures did not sporulate and the toxin synthesis was consequently suppressed.

Influence of milk type and addition of passion fruit peel powder on fermentation kinetics, texture profile and bacterial viability in probiotic yoghurts

The effect of the addition of passion fruit peel powder (PFPP) on the fermentation kinetics and texture parameters, post-acidification and bacteria counts of probiotic yoghurts made with two milk types were evaluated during 28 days of storage at 4 degrees C. Milks were fermented by Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (CY340), and one strain of probiotic bacteria: Lactobacillus acidophilus (L10 and NCFM), Bifidobacterium animalis subsp. lactis (8104 and HN019). The addition of PFPP reduced significantly fermentation time of skim milk co-fermented by the strains L10, NCFM and HN019. At the end of 28-day shelf-life, counts of B. lactis Bl04 were about 1 Log CFU mL(-1) higher in whole yoghurt fermented with PFPP regarding its control but, in general, the addition of PFPP had less influence on counts than the milk type itself. The titratable acidity in yoghurts with PFPP was significantly higher than in their respective controls, and in skim yoghurts higher than in the whole ones. The PFPP increased firmness, consistency (except for the NCFM strain of L acidophilus) and cohesiveness of all skim yoghurts. The results point out the suitability of using passion fruit by-product in the formulation of both skim and whole probiotic yoghurts. (C) 2012 Elsevier Ltd. All rights reserved.

Probiotic yogurts manufactured with increased glucose oxidase levels: Postacidification, proteolytic patterns, survival of probiotic microorganisms, production of organic acid and aroma compounds

We investigated the effect of increased glucose oxidase concentration as a technological option to decrease oxidative stress during the processing of probiotic yogurts. Probiotic yogurts were produced with increased concentrations of glucose oxidase (0, 250, 500, 750, or 1,000 mg/kg) and submitted to physicochemical and microbiological analysis at 1, 15, and 30 d of refrigerated storage. Higher concentrations of glucose oxidase (750 and 1,000 mg/kg) and a longer storage time were found to have an influence on the characteristics of the probiotic yogurt, contributing to more extensive post-acidification, an increase in the dissolved oxygen level, and higher proteolysis. In addition, increased production of aroma compounds (diacetyl and acetaldehyde) and organic acids (mainly lactic acid) and a decrease in the probiotic bacteria count were reported. The use of glucose oxidase was a feasible option to minimize oxidative stress in probiotic yogurts. However, supplementation with excessive amounts of the enzyme may be ineffective, because insufficient substrate (glucose) is present for its action. Consumer tests should be performed to evaluate changes in the sensory attributes of the probiotic yogurts with increased supplementation of glucose oxidase. In addition, packaging systems with different permeability to oxygen should be evaluated.

Evaluation of gamma-radiation on oolong tea odor volatiles

The aim of this study was to evaluate the gamma radiation effects on odor volatiles in oolong tea at doses of 0, 5, 10, 15 and 20 kGy. The volatile organic compounds were extracted by hydrodistillation and analyzed by GC/MS. The irradiation has a large influence on oolong tea odor profile, once it was identified 40% of new compounds after this process, the 5 kGy and 20 kGy were the doses that degraded more volatiles found naturally in this kind of tea and the dose of 10 kGy was the dose that formed more new compounds. Statistical difference was found between the 5 kGy and 15 kGy volatile profiles, however the sensorial analysis showed that the irradiation at dose up 20 kGy did not interfere on consumer perception. (C) 2011 Elsevier Ltd. All rights reserved.

X-ray diffraction and theoretical investigation of the Gedunin crystal structure

The Gedunin compound (C28H34O6) is a natural product extracted from Trichilia pallida that has shown a wide activity. The crystallographic structure shows two conformers in the asymmetric unit, which differ in a rotation of the furan group. To understand this molecular arrangement, the density functional calculations. Molecular Electrostatic Potential (MEP) and thermodynamic function calculation have been performed at the B3LYP/6-311++g(d,p) level. Both conformers were optimized and the agreement with the experimental structure was very good, making possible further theoretical analysis of the structure. The inter-conversion between two conformers depends on the energy barrier. This process is studied in the vacuum and shows two transition states with a low energetic barrier for a potential energy curve scanning rigid around furan group: 4.37 kcal/mol and 16.52 kcal/mol. As the first transition state has a notably lower energetic barrier, the preferred inter-conversion pathway between the conformers involves the first rather than the second transition state. Understanding this transition state in detail led us to perform its optimization, showing an energetic barrier around 3.66 kcal/mol. The negative free energy and low enthalpy confirm that the process is spontaneous and exothermic. The results show that this requirement makes the existence of the two conformers in the asymmetric unit possible. The structure of molecules in the asymmetric unit is better understood when the MEP is used on the interaction between molecules. For Gedunin, both molecules have shown MEP with well-defined regions, and this behavior contributes to the observed link between molecules and for the negative regions complementing positive regions of another molecule. (C) 2011 Elsevier B.V. All rights reserved.

Novos táxons e notas sobre Cerambycidae (Coleoptera) da Costa Rica

Nova espécie descrita: Eupogonius intonsus sp. nov. de Guanacaste. Nova subespécie descrita: Eburia lanigera costaricensis subsp. nov. de Guanacaste. Novos registros: Cymatonycha meridionalis Martins & Galileo, 1995 (Puntarenas) e Nesozineus triviale Galileo & Martins, 1996 (Guanacaste). É comentada a variabilidade no colorido elitral de Eburodacrys havanensis Chevrolat, 1862.

Potencial do flavedo (epicarpo) de Citrus aurantifolia cv. Tahiti no controle do bolor verde e da antracnose em citros

O Brasil é considerado o maior produtor de citros e o maior exportador de suco de laranja. Doenças de pós-colheita representam uma grande perda para a citricultura, sendo que para a exportação de frutos são rígidas as exigências com relação a isenção de resíduos químicos nos mesmos. Patógenos de importância em pós-colheita de citros incluem o Penicillium digitatum, agente causal do bolor-verde e o Colletotrichum gloeosporioides, agente causal da antracnose. Dada a importância econômica que representam estas doenças dos frutos cítricos, tanto em termos de comprometimento da qualidade e dificuldade de controle, a busca de alternativas adicionais que possam viabilizar a capacidade produtiva e garantir a obtenção de frutos com excelentes padrões de qualidade torna-se imprescindível. Portanto, estudou-se os efeitos dos extratos aquosos do flavedo de Citrus aurantifolia var. Tahiti, Lentinula edodes, Agaricus subrufescens (syn. Agaricus brasiliensis), albedo de Citrus sinensis var. Valência e do ácido jasmônico no controle póscolheita do bolor verde e da antracnose e na indução de resistência em frutos de laranjeira Valência (Citrus sinensis). Foi possível observar que o extrato aquoso do flavedo (C. aurantifolia) apresentou efeito inibitório sobre os patógenos, quando tratados em pós-colheita, em função da redução dos sintomas e esporulação. Porém, os extratos de albedo (C. sinensis), L. edodes, A. subrufescens e o ácido jasmônico não apresentaram efeitos sobre P. digitatum e C. gloeosporioides.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Período de incubação de Guignardia citricarpa em diferentes estádios fenológicos de frutos de laranjeira 'Valência'

A mancha preta dos citros (MPC), causada pelo fungo Guignardia citricarpa, produz lesões em frutos, os quais ficam depreciados para o mercado interno e os restringem para a exportação. O grande período de suscetibilidade dos frutos cítricos, em adição ao fato de G. citricarpa causar infecções latentes, dificulta o entendimento sobre o período de incubação da doença. O objetivo do trabalho foi determinar o período de incubação da MPC inoculando frutos de laranjeira 'Valência' em diferentes estádios fenológicos. Para a inoculação foram empregadas suspensões de conídios de G. citricarpa (10³, 10(4), 10(5) e 10(6) conídios mL-1) em diferentes diâmetros dos frutos (1,5; 2,0; 2,5; 3,0; 5,0 e 7,0 cm). O período de incubação da MPC para os diferentes diâmetros dos frutos inoculados apresentou uma relação polinomial negativa. Em frutos com até 3 cm de diâmetro o período de incubação médio foi superior a 200 dias, enquanto que em frutos com diâmetros superiores a 5 cm o período de incubação médio foi inferior a 84 dias. A MPC apresenta período de incubação variável dependente do estádio fenológico em que os frutos são infectados. A concentração de conídios de G. citricarpa, na infecção, não interfere no período de incubação da doença.

Sistema inmune inespecífico en dorada (Sparus aurata): activación del sistema interferón

Programa de doctorado en Sanidad Animal y Seguridad Alimentaria

Rapporti fra fattori ambientali e proteine di parete in Bifidobacterium

The normal gut microbiota has several important functions in host physiology and metabolism, and plays a key role in health and disease. Bifidobacteria, which are indigenous components of gastrointestinal microbiota, may play an important role in maintaining the well-being of the host although its precise function is very difficult to study. Its physiological and biochemical activities are controlled by many factors, particularly diet and environment. Adherence and colonization capacity are considered as contributing factors for immune modulation, pathogen exclusion, and enhanced contact with the mucosa. In this way, bifidobacteria would fortify the microbiota that forms an integral part of the mucosal barrier and colonization resistance against pathogens. Bifidobacteria are not only subjected to stressful conditions in industrial processes, but also in nature, where the ability to respond quickly to stress is essential for survival. Bifidobacteria, like other microorganisms, have evolved sensing systems for/and defences against stress that allow them to withstand harsh conditions and sudden environmental changes. Bacterial stress responses rely on the coordinated expression of genes that alter various cellular processes and structures (e.g. DNA metabolism, housekeeping genes, cell-wall proteins, membrane composition) and act in concert to improve bacterial stress tolerance. The integration of these stress responses is accomplished by regulatory networks that allow the cell to react rapidly to various and sometimes complex environmental changes. This work examined the effect of important stressful conditions, such as changing pH and osmolarity, on the biosynthesis of cell wall proteins in B. pseudolongum subsp. globosum. These environmental factors all influence heavily the expression of BIFOP (BIFidobacterial Outer Proteins) in the cell-wall and can have an impact in the interaction with host. Also evidence has been collected linking the low concentration of sugar in the culture medium with the presence or absence of extracromosomal DNA.

Cross-Talk tra Bifidobacterium e intestino umano: impatto sulle attività health promoting

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important healthpromoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. The first health-promoting activities studied in these job was the oxalate-degrading activity. Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyper absorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis, reducing the risk of kidney stone development. In this study, the oxalate-degrading activity of 14 bifidobacterial strains was measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-CoA decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of B. animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unravelling the presence of other two independently transcribed open reading frames, potentially responsible for B. animalis subsp. lactis ability to degrade oxalate. Transcriptional analysis, using real-time quantitative reverse transcription PCR, revealed that these genes were highly induced in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Acidic conditions were also a prerequisite for a significant oxalate degradation rate, which dramatically increased in oxalate pre-adapted cells, as demonstrated in fermentation experiments with different pH-controlled batch cultures. These findings provide new insights in the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, in the second part of the job, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified six putative plasminogen-binding proteins in the cell wall fraction of three strain of Bifidobacterium. The data suggest that plasminogen binding to Bifidobactrium is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction. In these job w studied a new approach based on to MALDI-TOF MS to measure the interaction between entire bacterial cells and host molecular target. MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.

Variazione della capacità riproduttiva del corallo Leptopsammia pruvoti (Scleractinia, Dendrophylliidae) lungo un gradiente latitudinale di temperatura

La temperatura influenza molti dei processi fisiologici degli organismi marini e, considerato che la riproduzione dei coralli sembrerebbe essere sensibile agli stress, è necessario comprendere come questa possa reagire ai cambiamenti climatici globali per riuscire a prevedere le future risposte delle popolazioni. Leptopsammia pruvoti (Scleractinia, Dendrophylliidae) è un corallo solitario non zooxantellato presente in Mediterraneo e lungo le coste Atlantiche dal Portogallo alla Gran Bretagna meridionale, dalla superficie fino a 70 metri di profondità.. È un organismo gonocorico con fecondazione interna. In questo lavoro di tesi sono stati analizzati gli aspetti della gametogenesi di L. pruvoti a diverse latitudini per ottenere risultati preliminari riguardanti le possibili correlazioni tra attività riproduttiva e parametri ambientali (temperatura e irradianza). Tale studio si colloca all’interno del progetto europeo sul riscaldamento globale e biologia dei coralli FP7–IDEAS-ERC “Corals and Global Warming: The Mediterranean versus the Red Sea” (CoralWarm). I risultati presentati in questo lavoro sono relativi a cinque popolazioni di L. pruvoti (Genova, Calafuria, Palinuro, Scilla e Pantelleria) disposte lungo un gradiente latitudinale di temperatura e irradianza nel versante occidentale della penisola italiana. I campioni sono stati raccolti mediante campionamenti mensili effettuati tramite immersioni subacquee. Su ogni campione sono state effettuate misurazioni biometriche e analisi cito-istometriche. Nelle popolazioni analizzate i parametri riproduttivi (fecondità, abbondanza, indice gonadico, dimensione) sono stati messi in relazione con la temperatura di fondo (DT, °C) e l’irradianza solare (W/m2) peculiari di ogni sito. L. pruvoti ha mostrato una sessualità gonocorica in tutti i siti considerati. In tutte le popolazioni, la presenza di due stock di ovociti e la distribuzione degli stadi di maturazione degli spermiari durante le fasi di attività riproduttiva, nell’arco dell’anno, hanno permesso di definire un periodo di reclutamento e uno di maturità gonadica, suggerendo che tali eventi siano influenzati dai cambiamenti stagionali della temperatura dell’acqua e del fotoperiodo. Nel periodo di reclutamento, la fecondità presentava una debole correlazione positiva con i parametri ambientali considerati. Al momento della maturità gonadica gli ovociti erano di dimensioni maggiori e meno numerosi rispetto al periodo precedente, nelle popolazioni con una maggiore temperatura e irradianza. Una possibile interpretazione potrebbe essere l’esistenza di un processo di fusione degli ovociti nel periodo di maturità gonadica che sembrerebbe più marcato nelle popolazioni più calde e maggiormente irradiate. La spermatogenesi, diversamente, non ha delineato finora un pattern chiaramente interpretabile. Con i dati attualmente in nostro possesso, non possiamo tuttavia escludere che le differenze riscontrate nelle dimensioni di ovociti e spermiari lungo il gradiente latitudinale considerato siano dovute a un lieve sfasamento nel raggiungimento della maturità gonadica, pur mantenendo la medesima stagionalità nel ciclo riproduttivo delle diverse popolazioni. Ulteriori studi saranno necessari per poter meglio comprendere i complessi meccanismi che regolano e controllano i processi riproduttivi al variare dei parametri ambientali. In questo modo sarà possibile considerare i risulti ottenuti nel contesto dei cambiamenti climatici globali.