Fast screening of Bifidobacterium longum sublethal stress conditions in a novel two-stage continuous culture strategy.

A central issue in the application of probiotics as food additives is their fastidious production and their sensitivity to many environmental stresses. The importance of inducible cell-protective mechanisms triggered by application of sublethal stresses for survival under stress conditions has been demonstrated. Continuous cultures could be a suitable and more efficient method to test stress factors on one culture instead of several repeated batch cultures. In this study, the application of a two-stage continuous culture of Bifidobacterium longum NCC2705 was investigated. The first reactor was operated under fixed conditions at 37 °C and pH 6.0 and used to produce cells with controlled physiology, mimicking cells in the late exponential growth phase. Stress pretreatment combinations of pH (6.0, 5.0 and 4.0), temperature (37, 45 and 47 °C) and NaCl (0, 5 and 10%) were tested in the second reactor. Of all tested combinations, only those of pH 4.0 significantly decreased cell viability in the second reactor compared to control conditions (37 °C, pH 6.0, 0% NaCl) and, therefore, could not be considered as sublethal stresses. Pretreatments with 5 or 10% NaCl had a negative effect on cell viability after gastric lethal stress. A significant improvement in cell resistance to heat lethal stress (56 °C, 5 min) was observed for cells pretreated at 47 °C. In contrast, heat pretreatment negatively affected cell viability after freeze drying and osmotic lethal stresses. The two-stage continuous culture allowed for efficient screening of several stress pretreatments during the same experiment with up to four different conditions tested per day. Optimal sublethal stress conditions can also be applied for producing cells with traditional batch cultures.

Transcriptome analysis and physiology of Bifidobacterium longum NCC2705 cells under continuous culture conditions.

A central issue in the use of probiotics in food and food supplements is their sensitivity to many environmental stress factors. The resistance of probiotic cells to lethal stress can be improved by application of homologous or heterologous sub-lethal stress during culture. This screening procedure is generally performed using batch cultures. Continuous cultures could be a suitable and more efficient method to test different stress factors on one culture instead of repeating several batch cultures. However, before testing stresses using continuous cultures, the physiological stability of continuously produced cells over a considered time period must be first evaluated. A continuous culture of Bifidobacterium longum NCC2705 was maintained for 211 h at a dilution rate of 0.1 per h, mimicking a deceleration growth phase culture. Stable viable cell counts were measured over the culture period, decreasing only moderately from 8.8 to 8.6 log10 cfu/ml. A slight shift in metabolite production, characterized by increased lactate and decreased acetate, formate and ethanol concentrations was observed. Susceptibilities to antibiotics and stress conditions were stable (cefotaxim, ampicillin, ceftazidime) or moderately affected (simulated gastric juices, heat, bile salts, tetracycline, chloramphenicol, penicillin, vancomycin and neomycin) over culturing time. Comparison of gene transcription profiles between samples collected after 31 h of continuous culture and samples collected after 134 and 211 h revealed only limited changes in expression of 1.0 and 3.8% of total genes, respectively. Based on these results, we propose that continuous culture can be used to produce bacterial cells with stable physiological properties suitable for fast and efficient screening of sub-lethal stress conditions.

Development of a rapid screening protocol for selection of strains resistant to spray drying and storage in dry powder.

An efficient screening method for selection of Bifidobacterium longum strains resistant to spray drying and storage was developed based on randomly amplified polymorphic DNA (RAPD) for identification of the best survivors in mixed strains bacterial preparations. Three different primers were used to generate RAPD profiles of 22 B. longum strains. All strains were distinguished according to their RAPD profiles except for the strain NCC2705 and its H(2)O(2) resistant derivative variant. The 22 strains were grouped in 3 batches of 7, 7 and 8 strains and subjected to spray drying and storage at 30 and 37 °C under anaerobic conditions. Batch survival rates after spray drying reached 17.1±4.4%. Strains showing the highest prevalence and/or resistance to storage at 37 °C were selected from individual batches for subsequent spray drying and storage testing. After 67 days of storage, NCC572 was identified as the dominant strain in powder. The stability of strain NCC572 was confirmed by performing single spray drying and storage tests. Out of 22 B. longum strains, a robust strain was identified by combining RAPD with a simultaneous screening test for survival under spray drying and storage. The method allowed a fast screening of B. longum strains in mixture for resistance to spray drying and storage compared to traditional screening procedures carried out with individual strains, in the same conditions. This approach could be applied to other stress conditions.

Transcriptional down-regulation by abscisic acid of pathogenesis-related beta-1,3-glucanase genes in tobacco cell cultures.

Class I isoforms of beta-1,3-glucanases (betaGLU I) and chitinases (CHN I) are antifungal, vacuolar proteins implicated in plant defense. Tobacco (Nicotiana tabacum L.) betaGLU I and CHN I usually exhibit tightly coordinated developmental, hormonal, and pathogenesis-related regulation. Both enzymes are induced in cultured cells and tissues of cultivar Havana 425 tobacco by ethylene and are down-regulated by combinations of the growth hormones auxin and cytokinin. We report a novel pattern of betaGLU I and CHN I regulation in cultivar Havana 425 tobacco pith-cell suspensions and cultured leaf explants. Abscisic acid (ABA) at a concentration of 10 micron markedly inhibited the induction of betaGLU I but not of CHN I. RNA-blot hybridization and immunoblot analysis showed that only class I isoforms of betaGLU and CHN are induced in cell culture and that ABA inhibits steady-state betaGLU I mRNA accumulation. Comparable inhibition of beta-glucuronidase expression by ABA was observed for cells transformed with a tobacco betaGLU I gene promoter/beta-glucuronidase reporter gene fusion. Taken together, the results strongly suggest that ABA down-regulates transcription of betaGLU I genes. This raises the possibility that some of the ABA effects on plant-defense responses might involve betaGLU I.

[Nifedipine and isometric exercise. Evaluation of the pressure response in hypertensive patients]. / Nifedipina e sforzo isometrico. Valutazione della risposta pressoria negli ipertesi.

Because of isometric efforts are a normal component of the usual life, we studied 35 patients suffering from mild and moderate essential hypertension, by means of an isometric exercise test, using the "handgrip method". The purpose of the experimental work was the evaluation of pressure changes at the top of an isometric stress in basal conditions and the demonstration of the protective effect against pressure increase of nifedipine, acutely administered (20 mg sublingual). The results largely confirmed the usefulness of nifedipine in the treatment of hypertension at rest and at the top of an isometric exercise. Moreover, the pressure increase during the same effort after nifedipine administration is smaller than during a therapy with others antihypertensive drugs.

[Carcinoid heart disease. Report of a case with triple valvular lesion (author's transl)]. / Cardiopatia da carcinoide. Presentazione di un caso con triplice lesione valvolare.

A case of carcinoid syndrome, stemming from a tumor of the large intestine with hepatic metastases, is reported. Clinical features included cardiac disease with triple valvular lesion: tricuspid insufficiency with stenosis, pulmonary artery stenosis and mitral insufficiency. More recent views about the pathogenesis of the cardiac involvement in the carcinoid syndrome are reported, and the cardiac therapy is discussed.