Two-generation diet-induced obesity model producing mice with increased amount of body fat in early adulthood.

The aim of our study was to develop a model producing obese mice in early adulthood (4-6 weeks) based on their over-nutrition during fetal and early postnatal development. The fertilized dams of the parental generation were fed the standard diet supplemented with high-energy nutritional product Ensure Plus during gestation and lactation. Delivered weanlings were then fed with standard or supplemented diet and assessed for body fat deposits using EchoMRI at the time of early and late adulthood. Maternal over-feeding during the period before weaning had the most significant effect on obesity development in the filial generation. In weanlings, significantly higher body fat deposits and average body weight were recorded. Later, further significant increase in percentage of body fat in both male and female mice was observed. Withdrawal of the Ensure Plus supplement caused a decrease in the percentage of body fat in part of the filial generation. In offspring fed the standard diet, higher fat deposits persisted till the time of late adulthood. We conclude that this diet-induced obesity model might be used in exploration of the effects of elevated body fat on physiological functions of various organ systems during juvenile and early adulthood periods of life of a human being.

Expression of adrenergic receptors in bovine and rabbit oocytes and preimplantation embryos.

Catecholamines play an important role in embryogenesis, and data obtained in the rodent model indicate that they can act even during the preimplantation period of development. Using RT-PCR with specific oligonucleotide primers distinguishing among all members of the adrenergic receptor family, we examined expression of adrenergic receptors in bovine and rabbit oocytes, morulas and blastocysts. We found several profiles of adrenoceptor mRNA expression. Transcripts for some receptor subtypes (bovine alpha 2 receptors, rabbit α2A, α2C, ß1 and ß2 receptors) were detected at all examined stages, which suggests receptor expression throughout (or at most stages) the preimplantation developmental period. Expression in oocytes but not at later stages was found in only one adrenoceptor subtype (rabbit α1B). In contrast, mRNA for several adrenoceptors was found in embryos but not in oocytes (bovine beta adrenoceptors and rabbit α1A). Nucleotide sequences of our PCR products amplified in rabbit oocytes, and preimplantation embryos represent the first published mRNA sequences (partial sequences coding at least one transmembrane region) of rabbit α2C, ß1 and ß2 adrenoceptors. Our results suggest that the expression of adrenergic receptors can be a general feature of mammalian oocytes and preimplantation embryos. On the other hand, comparison of three mammalian species (cattle, rabbit and mouse) revealed possible interspecies differences in the expression of particular adrenoceptor subtypes. Our results support the opinion that stress mediators can act directly in cells of preimplantation embryos.

Effects of borneol and thymoquinone on TNBS-induced colitis in mice.

Components of plant essential oils have been reported to have health benefit properties, including antioxidative, anti-tumour, antimicrobial, anti-stress, and immunomodulative activities. We examined the anti-inflammatory effects of thymoquinone, the active ingredient in the volatile oil of Nigella sativa seeds, and borneol, the active component of Salvia officinalis essential oil, on TNBS-induced colitis in mice. Thymoquinone was added to the commercial diet at a concentration of 0.05 % and borneol at two concentrations (0.09% and 0.18%) and fed to ICR mice 5 days before induction of TNBS colitis. Seven days after TNBS administration the mice were killed and macroscopic and histological scores were evaluated. Cytokine mRNA expression in colonic tissue was assessed using quantitative realtime RT-PCR. We did not detect any significant changes in macroscopic and histological scores between experimental and control groups, but we observed a significant decrease in proinflammatory cytokine (IL-1beta and IL-6) mRNA expression in colon tissue in the 0.09% and 0.18% borneol-treated groups of mice in comparison to the control group. Surprisingly, we were not able to confirm anti-inflammatory effects of thymoquinone in TNBS colitis. In conclusion, our data show that borneol is able to significantly suppress proinflammatory cytokine mRNA expression in colonic inflammation, although no significant morphological changes are visible.

Expression of serotonin receptors in mouse oocytes and preimplantation embryos.

Serotonin receptors have been found in several reproductive organs as well as in the central nervous system. Serotonin-binding sites have been demonstrated in duck ovarian follicles and the testis, hamster ovaries, human granulosa cells and mouse placenta. Local production of serotonin by the rat ovary, oviduct, uterus and testis has also been reported. We analyzed the expression of three types of serotonin receptors: 5-HT1B, 5-HT2C and 5-HT1D by reverse transcription-polymerase chain reaction in mouse unfertilized oocytes and preimplantation embryos from zygotes to the blastocyst stage in vivo. Transcripts for 5-HT1B and 5-HT2C serotonin receptors were detected neither in unfertilized oocytes nor at any stages of in vivo developing preimplantation embryos. Serotonin 5-HT1D receptor mRNA was present in unfertilized oocytes, zygotes, 2-cell embryos, compacted morulae and in vivo produced expanded blatocysts. The expression of the mRNA 5-HT1D serotonin receptor was also detected in blastocysts cultured in vitro. When added to the culture medium, specific serotonin 5-HT1D agonist sumatriptan (1 microM) significantly inhibited the development of mouse embryos cultured in vitro. Demonstration of the expression of 5-HT1D serotonin receptor in mouse oocytes and preimplantation embryos supports the idea of a functional serotonin (5-HT1D) receptor in early mammalian development.

Effect of enterocin CCM 4231 on Listeria monocytogenes in Saint-Paulin cheese.

The bacteriocin production by Enterococcus faecium strain in cheese milk and cheese was demonstrated. Purified enterocin CCM 4231 exhibited an anti-listerial effect during Saint-Paulin cheese manufacture. During cheese production the strain grew to a final concentration of 10.1 +/- 0.01 log CFU per mL per g in cheese. Then only a slight decrease of the cell concentration was noticed during ripening and was almost stable for 8 weeks. No significant differences in pH were observed between the experimental and reference cheeses. Bacteriocin production during cheese manufacture was detected only in milk samples and curd, reaching a level of 100 AU/mL. After addition of purified enterocin CCM 4231 (concentration 3200 AU/mL) into the experimental cheese, the initial concentration of 6.7 +/- 0.06 log CFU per mL of Listeria monocytogenes Ohio was reduced up to 1.9 +/- 0.01 log CFU per mL per g. After 6 weeks and at the end of the experiment the difference of surviving cells of L. monocytogenes Ohio in ECH was only one or 0.7 log cycle compared to the control cheese. Although enterocin CCM 4231 partially inhibited L. monocytogenes in Saint-Paulin cheese manufacture, an inhibitory effect of enterocin added was shown in 1-week cheese; however, it was not possible to detect bacteriocin activity by the agar spot test. The traditional fermentation and ripening process was not disturbed, resulting in acceptable end-products, including sensory aspects.

Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on "bryndza", a traditional Slovak dairy product from sheep milk.

"Bryndza" is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in "bryndza" was investigated with the aim to reduce the contaminant agents. "Bryndza" was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, "bryndza" was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 degrees C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in "bryndza" before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10(3) cfu/ml/g, Staphylococcus aureus (10(2) cfu/ml/g) and enterococci (10(4) cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10(2) cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10(3) cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in "bryndza". However, bacteriocin activity was not determined by laboratory analyses.

Use of enterocin CCM 4231 to control Listeria monocytogenes in experimentally contaminated dry fermented Hornád salami.

The effectiveness of enterocin CCM 4231 in controlling Listeria monocytogenes contamination in dry fermented Hornád salami was examined. Three independent salami treatments were conducted under pilot plant and laboratory conditions. Salamis were produced according to standard technological parameters and stages with ripening for 3 weeks. The reference samples consisted of the meat mixture without either L. monocytogenes or bacteriocin addition. The control sample (CS) consisted of the meat mixture with 1% of L. monocytogenes inoculum (10(8) cfu ml(-1)) added; while the experimental sample (ES) consisted of the same mixture with enterocin CCM 4231 (12800 AU g(-1)) added. Sampling was done on the first day of the experiment, before and after bacteriocin addition for ES, on the second day and after 1, 2 and 3 weeks. The enterocin addition resulted in the reduction of L. monocytogenes by 1.67 log cycle in the ES when compared to the CS immediately after addition of the bacteriocin. Although on the second day, the growth of L. monocytogenes in ES reached 3.38 cfu g(-1) (log 10), a difference of 1.72 log was found between the ES and the CS. After 1 week of ripening, the L. monocytogenes count in the CS reached 10(7) cfu g(-1); while in the ES the count was 10(4) cfu g(-1), a difference which was maintained after 2 and 3 weeks of ripening. However, bacteriocin activity in the ES could not be detected analytically. The meat mixture used did not contain Listeria.

The use of enterocin CCM 4231 in soy milk to control the growth of Listeria monocytogenes and Staphylococcus aureus.

The inhibitory effect of enterocin CCM 4231 (concentration 3200 AU ml-1) was used to control the growth of Listeria monocytogenes Ohio and Staphylococcus aureus in soy milk. The growth and bacteriocin (enterocin) production of producer strain CCM 4231 in soy milk was also checked. Bacteriocin production by CCM 4231 strain in soy milk was first detected after 2 h from the beginning of cultivation (100 AU ml-1). The stationary phase for CCM 4231 was reached after 6 h reaching 10.38 cfu ml-1 (log10) with a slight increase up to 24 h (10.43 cfu ml-1, log10), and the maximum bacteriocin production in soy milk (200 AU ml-1) was noted after 8 h of the beginning of cultivation with stability up to 24 h. The addition of enterocin CCM 4231 at 3200 AU ml-1 to a growing indicator strain, L. monocytogenes Ohio, in soy milk resulted in inhibition for 24 h. The high inhibitory effect of enterocin was found after 1 h and 2 h of its addition (in 5 h-6 h of cultivation), the difference between the experimental and the control samples (ES, CS) being 4.96 log cycles at 5 h and 5.15 log cycles at 6 h. Staphylococcus aureus was not fully inhibited, although a difference of 3.55 log cycles was found when ES and CS were compared at the end of cultivation (24 h). The pH was not influenced by enterocin addition. The inhibitory effect of enterocin CCM 4231 against L. monocytogenes Ohio in soy milk was probably bacteriocidal; while Staph. aureus was influenced bacteriostatically. In general, the observed inhibitory activity confirmed the possibility for further application of bacteriocins in food environments as the protective agents. Of course, legislation problems must be solved.

Anti-staphylococcal effect of enterocin in Sunar and yogurt.

Enterocin was used to control the growth of Staphylococcus aureus strains SA1 and Oxford 209P in Sunar (milk nourishment for suckling babies) and during the yogurt-making process. Reduction by three orders of magnitude was noted in the growth of SA1 strain in Sunar milk nourishment between the enterocin-containing (ES) and the control samples (CS) at 1-d cultivation. An inhibitory effect of enterocin was observed when surviving of SA1 cells were checked 6 h after the start of cultivation (2 h after enterocin application; enterocin was applied after 4 h). Decrease in the count of Oxford 209P strain in yogurt was detected in ES after 1 d of storage in comparison with CS (10(3) and 10(0) CFU mL-1 g-1). Thus a decrease by three orders of magnitude was found between ES and CS at the time mentioned. On the other hand, no bacteriocin activity was detected in ES after 1 d. Activity was detected only immediately after enterocin addition to ES (400 AU/mL) as well as after 1 and 3 1/2 h (200 AU/mL). Although the slight regrowth of the indicator was obtained up to 1 week of yogurt storage, the difference between ES and CS persisted. The lowest pH of the final yogurt product was noted in the reference yogurt sample but differences among the pH values of yogurt samples were not significant.

Antimicrobial effect of enterocin CCM 4231 in the cattle slurry environment.

The antagonistic effect of enterocin CCM 4231 towards enterococci, staphylococci, Escherichia coli, listeriae and pseudomonads in the cattle slurry environment was assessed during periods of 1 and 2 weeks. The maximum decrease in the viable cells of enterococci and staphylococci (5.39 to 1.1 log CFU ml-1, and 4.3 to 2.3 log CFU ml-1, respectively) was detected on the second day after enterocin CCM 4231 addition to cattle slurry. E. coli cells, listeriae and pseudomonads decreased insignificantly. After 1 week, enterococci were completely inhibited. Staphylococci were suppressed by reaching a 1.8 log CFU ml-1 difference between the experimental and the control samples. A stable suppressive effect of enterocin CCM 4231 on the growth of listerial cells became significant with 2.59 log CFU ml-1 between the experimental and the control samples in the second week of bacteriocin addition. This was demonstrated in an experiment with enterocin addition to slurry which was sterilized and then inoculated with Listeria monocytogenes Ohio culture. Further possibilities of using bacteriocins for the treatment of animal waste are discussed.

Occurrence of bacteriocin production among environmental enterococci.

The occurrence of enteroccoci in cattle dung water from the basins of 25 cattle farms of 15 northeastern Slovakia districts was screened as well as bacteriocin production among selective enterococcal isolates. The average total count of enterococci detected reached 5.0 x 10(3) cfu ml-1. Enterococcus faecium was the predominant species (25%) followed by Ent. casseliflavus (19.2%), Ent. faecalis (9.6%), Ent. avium and Ent. durans (1.9%). The antagonistic activity of isolates showed a mainly antilisterial effect. Enterococcus faecalis V24 strain produced a heat stable, largely hydrophobic antimicrobial substance with best production in the range pH 4 to 7 and with a strong inhibitory effect even against Gram-negative bacteria.

Inhibition effect of enterocin CCM 4231 in the rumen fluid environment.

Enterocin CCM 4231 is a bacteriocin with a broad antimicrobial spectrum produced by the ruminal strain Enterococcus faecium CCM 4231. Its inhibitory effect towards enterococci, Ent. faecium EF 26/42, staphylococci, Streptococcus bovis AO 24/85 and Escherichia coli, as well as towards Listeria monocytogenes OHIO strain, in the rumen fluid environment was studied during culture at 37 and 30 degrees C for 24 h and 20 days. Enterocin CCM 4231 was added to the samples at a concentration of 3200 AU ml-1. The best inhibitory effect was noted against enterococci at both cultivation temperatures. A decrease in total cell count from 10(8) cfu ml-1/or 10(4) cfu ml-1 and from 10(5) cfu ml-1 to 10(1) cfu ml-1 was detected. Addition of enterocin to the rumen fluid also inhibited staphylococci (from 10(5) cfu ml-1 to 10(4) cfu ml-1 and/or 10(3) cfu ml-1). Gram-negative E. coli cells were inhibited at both cultivation temperatures (decrease from 10(6) cfu ml-1 to 10(1) cfu ml-1 at 37 degrees C, and from 10(7) cfu ml-1 to 10(5) cfu ml-1 at 30 degrees C). Enterococcus faecium EF 26/42 and Streptococcus bovis AO 24/85, the strains growing in the rumen fluid, were the most sensitive to the addition of enterocin during the first 24 h of fermentation (decrease from 10(10) cfu ml-1 and 10(8) cfu ml-1 to 10(6) cfu ml-1 and 10(4) cfu ml-1). An antilisterial effect of the bacteriocin was also confirmed. Further application of bacteriocin in ruminal ecology was indicated.

A bacteriocin-mediated antagonism by Enterococcus faecium BC25 against ruminal Streptococcus bovis.

A bacteriocin-like activity produced by Enterococcus faecium BC25, isolated from the the rumen of a cow, was partially purified and characterized. The active substance was prepared by ammonium sulfate and chloroform/methanol precipitation of culture supernatant. The bacteriocin was a protein of molecular mass 17.5 kDa. Activity was inactivated by trypsin and proteinase K. The bacteriocin BC25 inhibited growth of amylolytic ruminal strains of Streptococcus bovis, including S. bovis AO 24/85. Results showed that bacteriocine substance BC25 has a bacteriostatic effect when present in concentrations exceeding 125 AU/ml. Agar overlays and batch culture growth experiments proved that E. faecium BC25 was producing bacteriocin that inhibited the growth of S. bovis.