Probiotic Strawberry Yogurts: Microbiological,Chemical and Sensory Properties

This study was performed to determine the viability of Lactobacillus acidophilus and Bifidobacterium bifidum in yogurt made with strawberry marmalade (SM) and to examine the quality properties of probiotic yogurt. Acidity, pH, bacterial counts and sensory analysis of the yogurt samples were investigated on days 1, 3, 5, 7, 10 and 14 during storage at 4 °C. The survival rate of L. acidophilus was greater than that of B. bifidum. The viability of L. acidophilus decreased during the storage period, but B. bifidum numbers remained stable during the storage period. The highest L. acidophilus count (7.20 log cfu/g) was found in L. acidophilus + B. bifidum SM yogurt on day 1. The highest B. bifidum count (6.13 log cfu/g) was detected in yogurt containing L. acidophilus + B. bifidum SM yogurt on day 7. Yeast and mould counts of all yogurts increased during the storage period. Coliform bacteria and Staphylococcus aureus were not detected in the yogurt samples. The highest overall acceptance sensory score was observed in yogurts containing L. acidophilus. Considering the sensory and probiotic characteristics of all yogurt samples, this study suggested that strawberry yogurt with a suitable 5–7 day storage period can be produced with single L. acidophilus addition or single B. bifidum addition.     

Long-term survival and resistance of submerged pseudomonad cultures in the exopolymer mass

An issue on the cellular forms that ensure survival of pseudomonads is important due to wide occurrence of these bacteria in the environment and their role for clinical microbiology. The present work demonstrates the high survival potential of Pseudomonas aurantiaca and P. аeruginosa in the mass of exopolymers produced by cells. Exopolymer formation occurred only during incubation of the post-stationary phase cultures of P. aurantiaca (at 4°C) and P. aeruginosa (at 4 and 20°C). After storage for 1.5–12 months, the number of colony-forming units in the exopolymer was 30 to 68% of the viable cell titer in stationary-phase cultures. Antibiotic-tolerant persister cells that were revealed in the exopolymer cultures after treatment with ciprofloxacin (2.5–100 μg/mL) were more resistant to the antibiotic than persisters in suspension cultures, with the threshold doses of 25 and 2.5 μg/mL, respectively. The cells embedded in the exopolymer were found to be more resistant to 5-min heating at 60–70°C than the vegetative cells of suspension cultures, which did not survive such heat treatment conditions. Electron microscopic investigation revealed morphological heterogeneity of exopolymer-embedded pseudomonads, including the presence of the cells similar to cystlike dormant forms. The populations developing on solid media inoculated with the exopolymer mass with cells were found to contain 1.5 to 2 orders of magnitude more persisters tolerant to high ciprofloxacin doses (25 μg/mL for P. aurantiaca and 100 μg/mL for P. aeruginosa) than the populations developing after inoculation with second-transfer vegetative cells of the cells of planktonic cultures. The results obtained improve our understanding of pseudomonad survival in the environment.     

Molecular genetics of type 1 diabetes mellitus: Achievements and future trends

Type 1 diabetes mellitus (T1DM) is a widespread severe disease that results from autoimmune destruction of β cells in Langerhans islets of the pancreas. To date, several loci involved in T1DM have been reliably identified using various approaches: the MHC locus, VNTR within the 5′-nontranscibed region of the insulin gene (INS), CTLA4 (T-cell surface receptor), PTPN22, PTPN2 (T-cell tyrosine phosphatases), IL2 (interleukin 2, IL-2), IL2RA (IL-2 receptor α chain), KIAA0350 (unknown function), and IFIH1 (receptor for double-stranded DNA generated in virus infections). Functional analysis of their protein products confirmed the hypothesis that T1DM is underlain by deregulation of the mechanisms of immune tolerance and, on the other hand, a destructive immune response against the body’s own proteins after virus infection or some other immune stress. Thus the protein products of MHC, INS, PTPN22, and PTPN2 are involved in the intrathymic formation of the T-cell repertoire, responsible for immune defense of the body. On the other hand, nonspecific activation of T cells, which starts autoimmune destruction of pancreatic β cells, is most likely associated with the protein products of CTLA4, IL2, IL2RA, and, possibly, PTPN22 and PTPN2. Apart from the genes with unknown functions, the only exception is IFIH1, but its association with T1DM confirms that certain virus infections can activate autoreactive T cells and lead to T1DM.     

Antirestriction and antimodification activities of T7 Ocr: Effects of amino acid substitutions in the interface

The T7 antirestriction protein Ocr, encoded by 0.3 (ocr), specifically inhibits ATP-dependent type I restriction-modification systems. T7 0.3 (ocr) was cloned in pUC18. Ocr inhibited both restriction and modification activities of the type I restriction-modification system (EcoKI) in Escherichia coli K12. The Ocr F53D A57E mutant was obtained and proved to inhibit only restriction activity of EcoKI. The 0.3 (ocr) and Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P _ltetO-1 promoter, strongly controlled by the TetR repressor. The bioluminescence intensity and luciferase content varied up to 5000-fold in E. coli K12 MG1655Z1 tetR⁺ (pZE21-luxCDABE) cells, depending on the environmental concentration of the inductor anhydrotetracycline. The antirestriction activity of Ocr and Ocr F53D A57E was studied as a function of their concentration in the cell. The dissociation constant K _d, characterizing the binding with EcoKI, differed 1000-fold between Ocr and Ocr F53D A57E (10^?10 M versus 10^?7 M).     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Photosynthetic activity and components of the electron transport chain in the aerobic bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>-containing bacterium <Emphasis Type="Italic">Roseinatronobacter thiooxidans</Emphasis>

Bioenergetics of the aerobic bacteriochlorophyll a-containing (BCl a) bacterium (ABC bacterium) Roseinatronobacter thiooxidans is a combination of photosynthesis, oxygen respiration, and oxidation of sulfur compounds under alkaliphilic conditions. The photosynthetic activity of Rna. thiooxidans cells was established by the photoinhibition of cell respiration and reversible photobleaching discoloration of the BCl a of reaction centers (RC), connected by the chain of electron transfer with cytochrome c ₅₅₁ oxidation. The species under study, like many purple bacteria and some of the known ABC bacteria, possesses a light-harvesting pigment-protein (LHI) complex with the average number of 30 molecules of antenna BCl a per one photosynthetic RC. Under microaerobic growth conditions, the cells contained bc ₁ complex and two terminal oxidases: cbb ₃-cytochrome oxidase and the alternative cytochrome oxidase of the a ₃ type. Besides, Rna. thiooxidans was shown to have several different soluble low- and high-potential cytochromes c, probably associated with the ability of utilizing sulfur compounds as additional electron donors.     

Intrapopulation heterogeneity of the fluorescence parameters of the marine plankton alga <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis> at various nitrogen levels

Fluorescence of the marine alga Thalassiosira weissflogii (Grunow) Fryxell et Hasle with open (F _o ) and closed (F _m ) reaction centers of photosystem 2 (PS 2) and its relative variable fluorescence (F _v/F _m ) were measured at various levels of inorganic nitrogen. A significant heterogeneity of the population in terms of these parameters was revealed. Some cells within the population were more sensitive to nitrogen deficiency, and their photosynthetic apparatus was disrupted to a greater extent. The cells within a population also differed in terms of their ability to recover after incubation at low nitrogen levels. Enhancement of nitrogen deficiency resulted in an increase in the variability of the F _o and F _v/F _m values of the cells. Fluorescence variability decreased at a less pronounced deficiency. Fluorescence variability should be taken into consideration in the studies concerning responses of algae to changes in nutrient contents.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Cytochromes <Emphasis Type="Italic">c</Emphasis> of the anaerobic methacrylate reducer <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> AM-1

Cytochromes c were found in the cells of the bacterium Geobacter sulfurreducens AM-1 grown on acetate and methacrylate. The periplasmic extract of G. sulfurreducens AM-1 contained about 88% of the total content of cytochromes c of intact cells. The analysis of cytochromes c from the native cells of G. sulfurreducens AM-1, from the periplasmic extract and from the cells treated by an alkaline solution showed the presence of nine proteins containing heme c. The molecular masses of cytochromes c from G. sulfurreducens AM-1 were 12.5, 15.5, 25.7, 29.5, 34.7, 41.7, 50.1, 63.1, and 67.6 kDa; localization of each cytochrome c was determined. Three heme-containing proteins (15.5 kDa, 25.7 kDa, and 29.5 kDa with the most intensive staining) were present mainly in the periplasm of the bacterium. The other two (50.1 and 67.6 kDa) were supposedly localized in the cell membrane. Cytochromes c with the molecular masses of 12.5, 15.5, and 67.6 kDa are considered as possible components of the methacrylate redox system of G. sulfurreducens AM-1.     

Dormant forms of <Emphasis Type="Italic">Micrococcus luteus</Emphasis> and <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> not platable on standard media

The colony-forming ability of long (3–9 months) incubated cystlike resting cells (CRC) of the nonspore-forming gram-positive bacteria Micrococcus luteus and Arthrobacter globiformis was studied in this work. The preservation of the CRC proliferative potential as assayed by plating on standard LB agar was shown to depend on the conditions of the formation of the dormant cells. In aged post-stationary cultures of micrococci and arthrobacters grown under carbon and phosphorus limitation the number of colony-forming units (CFU/ml) of CRC decreased in the course of 3–9 month incubation to the level of 10⁶–10⁷ CFU/ml. However, M. luteus CRC obtained under carbon and nitrogen limitation and A. globiformis CRC obtained under nitrogen limitation and starvation completely lost their ability to form colonies on standard solid medium after 4–6 months of incubation and turned into a ‘non-culturable’ (non-platable) state. In this case, the ratio of live cells in the population of M. luteus and A. globiformis ‘non-culturable’ CRCs (determined by the Live/Dead staining test) was 10–44% of the total cell number. To study the possible preservation of proliferative potential in non-platable CRCs, various methods of their reactivation were applied. Although preincubation of CRC suspensions in a buffer solution of 0.1 M K₂HPO₄ (pH 7.4) or in the presence of lysozyme (1 or 10 μg/ml) resulted in increased numbers of live cells (determined by the Live/Dead test) or in disruption of the cell conglomerates, it did not increase considerably the CFU titer on LB medium. Variations in the medium composition, such as addition of sodium pyruvate as an antioxidant or dilution of the medium, promoted the formation of macrocolonies by a small portion of nonplateable CRC of M. luteus (50?80 CFU/ml), whereas the number of the cells capable of microcolony formation (mCFU) was 1.8–6.8 × 10⁵ mCFU/ml, exceeding the CFU titers by four orders of magnitude. The application of semisolid agar and the most probable number (MPN) method was the most efficient for determination of the mCFU titer, and an almost complete reversion of ‘non-culturable’ micrococcal CRCs to microcolony formation was observed (up to 2.3 × 10⁷ mCFU/ml). The usefulness of diluted complete media for the restoration of the colony-forming ability of the dormant forms was confirmed in experiments with ‘nonculturable’ CRCs of A. globiformis. The development of special procedures and methods for determining actively proliferating cells not detected by ordinary methods is of great importance for advanced monitoring studies.     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

Thermotolerance and place memory in adult <Emphasis Type="Italic">Drosophila</Emphasis> are independent of natural variation at the <Emphasis Type="Italic">foraging</Emphasis> locus

Natural polymorphisms at the foraging (for) gene influence several behaviors. However, it is seldom clear how different for alleles could be selected. In one case, Drosophila with the rover allele (for ^r ) have higher locomotor activity in the presence of food than animals with the sitter allele (for ^s ), suggesting a complementary feeding strategy. There are, in addition, differences between for ^r and for ^s Drosophila in some tests of short-term memory (for ^r animals generally perform at higher levels) and thermotolerance (for ^s larvae are more resistant to the effects of high-temperature). We asked whether there could be a direct compensating advantages in adult for ^s flies that could maintain the natural for variants. First, are adult for ^s flies more thermotolerant? Second, do for ^r flies have a higher short-term place memory? Third, as an alternative, might for ^s flies have higher place memory? Our results do not confirm these possibilities. Thus, a thermotolerance advantage of for ^s flies does not compensate for a potential for ^r short-term memory advantage; for ^r flies do not have a universal advantage in short-term memory; and for ^s flies do not have an advantage in place memory that could compensate for for ^r advantages in other learning contexts.     

Patterns of growth and oxidation of natural pyrites by the representatives of acidophilic chemolithotrophic microorganisms

The patterns of the growth and oxidation of different types of natural pyrites were studied for the three microbial species adapted to these substrates and belonging to phylogenetically remote groups: gram-negative bacterium Acidithiobacillus ferrooxidans, gram-positive bacterium Sulfobacillus thermotolerans, and the archaeon Ferroplasma acidiphilum. For both A. ferrooxidans strains, TFV-1 and TFBk, pyrite 4 appeared to be the most difficult to oxidize and grow; pyrite 5 was oxidized by both strains at an average rate, and pyrite 3 was the most readily oxidized. On each of the three pyrites, growth and oxidation by TFBk were more active than by TFV-1. The effectiveness of the adaptation of S. thermotolerans Kr1^T was low compared to the A. ferrooxidans strains; however, the adapted strain Kr1^T showed the highest growth rate on pyrite 3 among all the cultures studied. No adaptation of strain Kr1^T to pyrite 5 was observed; the rates of growth and pyrite oxidation in the third transfer were lower than in the first transfer. The strain F. acidiphilum Y^T was not adapted to pyrites 3 and 5; the rates of growth and pyrite oxidation were the same in the first five transfers. The strains of three species of the microorganisms studied, A. ferrooxidans, S. thermotolerans, and F. acidiphilum, grew on pyrite 3 (holetype (p) conductivity) and oxidized it better than pyrite 5 (mixed-type (n-p) conductivity). The most readily oxidized were the pyrites with a density of 5.6–5.7 g/cm³ and high resistance values (ln R = 8.8). The pyrite oxidation rate did not depend on the type of conductivity. Changes in the chromosomal DNA structure were revealed in strain TFBk on adaptation to pyrites 3 and 4 and in the TFV-1 plasmid profile on adaptation to pyrite 3. Correlation between genetic variability and adaptive capabilities was shown for A. ferrooxidans. No changes in the chromosomal DNA structure were found in S. thermotolerans Kr1^T and F. acidiphilum Y^T on adaptation to pyrites 3 and 5. Plasmids were absent in the cells of these cultures.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Embryonal effects of <Emphasis Type="Italic">t</Emphasis>-haplotypes in mice

The effects of t-haplotypes on embryonic morphology in house mouse Mus musculus were described. Lethal mutations, t-haplotypes, in homozygotes induce abnormal embryogenesis and zygotic death at different developmental stages, which depends on the time of their action in ontogeny. Death commonly occurs in the first semester of pregnancy from the morula to the mature embryo stage (day 9–10), and the embryogenetic abnormalities and their timing were Specific for each t-haplotype. Such mutations were analyzed to identify the gene products (proteins) affecting the nervous system development. The t-complex proved to contain tandem repeats coding for regulatory factors modulating the expression of specific structural genes in mouse neurons.     

Effect of a corrinoid on <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> DNA synthesis

Methanosarcina barkeri is capable of synthesizing large amounts of corrinoids, compounds of the vitamin B₁₂ group, although not cobalamin. In the present work, exogenous cobalamin was demonstrated to upregulate DNA synthesis in M. barkeri cell suspensions incubated under air. The effect is similar to the one in Propionibacterium freudenreichii cells, though less pronounced. The growth of the archaeon under anaerobic conditions was shown to be suppressed by cobalamin and 5,6-dimethylbenzimidazole. The data obtained suggest the presence of a corrinoid-dependent ribonucleotide reductase in the archaeal cells which provides for deoxyribose precursors for DNA biosynthesis independently of the presence of molecular oxygen in the medium. Growth suppression under anoxic conditions by cobalamin and 5,6-dimethylbenzimidazole may be due to a decrease in the concentration of factor III, a polyfunctional corrinoid dominating in M. barkeri cells.     

<Emphasis Type="Italic">Malassezia</Emphasis> spp. in Acoustic Meatus of Bats (<Emphasis Type="Italic">Molossus molossus</Emphasis>) of the Amazon Region,Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, “Rondônia”, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.