Comparison of the acidifying activity of Lactococcus lactis subsp. lactis strains isolated from goat's milk and Valdeteja cheese

AIMS: This work was carried out to study the acid production by Lactococcus lactis subsp. lactis strains isolated from goat's milk and goat cheese (Valdeteja variety) in order to select a suitable starter culture for industrial goat cheese manufacturing. METHODS AND RESULTS: The titrable acidity of 45 Lactococcus lactis subsp. lactis strains isolated from a home-made batch of Valdeteja cheese with excellent sensory characteristics was measured over a period of 18 h. The strains were divided into two groups depending on the acid production rate: 20 fast acid producer (F) strains and 25 slow acid producer (S) strains. The kinetic parameters (lag phase, maximum acid production rate and value of upper asymptote curve) of the acid production curves for F and S strains were significantly (P < 0.001) different. CONCLUSIONS: Significant (P < 0.001) differences between titrable acidity of F and S strains were observed after the second hour of incubation. SIGNIFICANCE AND IMPACT OF THE STUDY: An F strain acetoin producer (Lactococcus lactis subsp. lactis 470Ch2) was selected as autochthonous starter culture for industrial Valdeteja goat cheese manufacturing.     

Genome shuffling of Lactobacillus plantarum C88 improves adhesion

Genome shuffling is an important method for rapid improvement in microbial strains for desired phenotypes. In this study, ultraviolet irradiation and nitrosoguanidine were used as mutagens to enhance the adhesion of the wild-type Lactobacillus plantarum C88. Four strains with better property were screened after mutagenesis to develop a library of parent strains for three rounds of genome shuffling. Fusants F3-1, F3-2, F3-3, and F3-4 were screened as the improved strains. The in vivo and in vitro tests results indicated that the population after three rounds of genome shuffling exhibited improved adhesive property. Random Amplified Polymorphic DNA results showed significant differences between the parent strain and recombinant strains at DNA level. These results suggest that the adhesive property of L. plantarum C88 can be significantly improved by genome shuffling. Improvement in the adhesive property of bacterial cells by genome shuffling enhances the colonization of probiotic strains which further benefits to exist probiotic function.     

酿酒酵母和粟酒裂殖酵母属间融合和融合子特性

酿酒酵母(Saccharomyces cerevisiae Hansen)PW218和粟酒裂殖酵母(Schizosaccharomyces pombe Lindn)PW232的原生质体用20mmol/L CaCl_2和30％PEG(MW6000)处理进行属间融合,获得了10多株融合子,融合率为0.65～1.96×10~(-5)。对F_2和F_(10)两株融合子进行了葡萄糖、木糖及葡萄糖和木糖混合液的摇瓶实验结果表明F_(10)融合子利用葡萄糖、木糖及两种糖混合液产乙醇的能力大大高于两亲株。F_2融合子对木糖以及葡萄糖和木糖混合液的发酵能力亦较两亲株高,其中利用木糖产乙醇的量分别比PW218和PW232提高1.38倍和2.65倍。     

大肠杆菌F18菌毛及其亚型的PCR鉴定

F18菌毛是产肠毒素大肠杆菌 (ETEC)与产vero细胞毒素大肠杆菌 (VTEC)的重要致病因子 ,可介导细菌对小肠细胞的黏附 ,并具有F18ab和F18ac 2个抗原亚型。根据已发表的F18ab菌毛A亚单位 (FedA ab)的基因 (fedA ab)设计 3条引物 ,建立了 2种聚合酶链式反应 (PCR)扩增方法。通过对F18ab 大肠杆菌、F18ac 大肠杆菌、K88 大肠杆菌、K99 大肠杆菌、987P 大肠杆菌、F4 1 大肠杆菌的试验 ,结果表明所建立的PCR方法可特异性鉴定F18 大肠杆菌并区别其亚型F18ab与F18ac     

Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage

This study assessed potential probiotic Lactobacillus strains isolated from the feces of breast-fed infants and from Taiwanese pickled cabbage for their possible use in probiotic fermented foods by evaluating their (i) in vitro adhesive ability, resistance to biotic stress, resistance to pathogenic bacteria, and production of β-galactosidase; (ii) milk technological properties; and (iii) in vivo adhesive ability, intestinal survival and microbial changes during and after treatment. Five Lactobacillus isolates identified as Lactobacillus reuteri F03, Lactobacillus paracasei F08, Lactobacillus rhamnosus F14, Lactobacillus plantarum C06, and Lactobacillus acidophilus C11 that showed resistance to gastric juice and bile salts were selected for further evaluation of their probiotic properties. All the strains demonstrated the ability to adhere to Caco-2 cells, particularly, strain L. plantarum C06 and L. reuteri F03 showed satisfactory abilities, which were similar to that of the reference strain L. rhamnosus GG. The strains L. paracasei F08 and L. acidophilus C11 had the highest β-galactosidase activity. Most of the strains were resistant to aminoglycosides and vancomycin but sensitive to ampicillin, erythromycin, and penicillin. All the 5 strains elicited antibacterial activity against both Gram-positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and -negative (Escherichia coli and Salmonella enterica) pathogens. Moreover, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 could grow rapidly in milk without nutrient supplementation and reached 10? cfu/mL after 24 h of fermentation at 37 °C. The viable cell counts of the 3 strains remained above 10? cfu/mL after 21 d of storage at 4 °C. In the animal feeding trial, the number of intestinal lactobacilli increased significantly after administration of milk fermented with the 3 strains, and the counts of fecal coliforms and Clostridium perfringens were markedly reduced. Lactobacillus strains could also survive in the ileal intestinal tissue of the treated rats. Technologically interesting Lactobacillus isolates may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

Curing and induction of the Fels 1 and Fels 2 prophages in the Ames mutagen tester strains of Salmonella typhimurium

A method is described for curing the Ames Salmonella mutagen tester strains of their Fels 1 and Fels 2 prophages with the aid of the antitumor drug daunorubicin. Non-lysogenic derivatives corresponding to TA100 and TA1535 were isolated and designated TAQ100 and TAQ1535 respectively. In addition, the Fels 1 monolysogens TAQ100F1 and TAQ1535F1, as well as the Fels 2 monolysogens TAQ100F2 and TAQ1535F2, were obtained. Finally, strains corresponding to TA98 and TA1538 cured of Fels 2, but retaining a cryptic Fels 1 (F1d) prophage were isolated and designated TAQ98F1d and TAQ1538F1d respectively. The various cured derivatives were identified by colony hybridization with ³²P-labeled probes of Fels 1 and Fels 2 DNA. Southern blot hybridizations confirmed that phage-specific Fels DNA sequences were missing from the cured strains. The Fels 2-cured strains were resistant to Fels 2, but Fels 1 grew, albeit poorly, on the Fels 1-cured strains. Strains TAQ100F1, TAQ1535F1, TAQ100F2 and TAQ1535F2 were used in prophage induction assays, in the presence of rat-liver extract where necessary. Daunorubicin, bleomycin, mitomycin C, aflatoxin B1, 2-amino-dipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-2) were found to induce Fels 1 and/or Fels 2 in at least one of these strains. The induction of the Fels prophages in the TAQ monolysogens may provide a useful complement to the Ames test for the detection of DNA-damaging agents and potential carcinogens.     

Epicoccum nigrum for biocontrol agents in vitro of plant fungal pathogens

Epicoccum nigrum strains were evaluated in vitro as potential biological agents for control of the growth of Fusarium avenaceum, F. graminearum, F. oxysporum and Botrytis cinerea. Individual biotic effects of five strains of E. nigrum on the various fungi were determined using the biotic series method elaborated for fungi, on potato dextrose agar (PDA - Difco) medium. Our results show that E. nigrum strains limited the growth of all isolates of Fusarium spp., but not of those of Botrytis cinerea.     

Interspecific differences in cryoresistance of lichen symbiotic algae of genus Trebouxia assessed by cell viability and chlorophyll fluorescence

Unicellular algae of genus Trebouxia are the most frequent symbiotic photobionts found in lichen species adapted to extreme environments. When lichenised, they cope well with freezing temperature of polar regions, high-mountains environments and were successfully tested in open-space experiments. Trebouxia sp. is considered potential model species for exobiological experiments. The aim of this paper is to evaluate cryoresistence of Trebouxia sp. when isolated from lichen thalli and cultivated on media. In our study, six algal strains were exposed to repeated freezing/thawing cycles. The strains of Trebouxia sp. (freshly isolated from lichen Lasallia pustulata), Trebouxia erici, Trebouxia asymmetrica, Trebouxia glomerata, Trebouxia irregularis, and Trebouxia jamesii from culture collection were cooled from 25 to -40 °C at two different rates. The strains were also shock frozen in liquid nitrogen. After repeated treatment, the strains were inoculated and cultivated on a BBM agar for 7 days. Then, cell viability was assessed as relative share of living cells. Potential quantum yield of photochemical reactions in PS II (F(V)/F(M)), and effective quantum yield of photochemical reactions in PS II (Φ(PSII)) were measured. While the slow cooling rate (0.5 °C min(-1)) did not cause any change in viability, F(V)/F(M), and Φ(PSII), the fast cooling rate (6.0 °C min(-1)) caused species-specific decrease in all parameters. The most pronounced interspecific differences in cryoresistance were found after shock freezing and consequent cultivation. While T. asymmetrica and T. jamesii exhibited low viability of living cells (18.9% and 34.7%) and full suppression of photosynthetic processes, the other strains had viability over 60%, and unaffected values of F(V)/F(M), and Φ(PSII). This indicated a high degree of cryoresistance of T. glomerata, T. erici, T. irregularis and Trebouxia sp. strains. These strains could be used for detailed investigation of underlying physiological mechanisms and as models for astrobiological tests taken in the Earth facilities.     

Characterization of Lethal Zygosis Associated with Conjugation in Escherichia coli K-12

When F(-) cells are mixed with an excess of Hfr cells there is a lethal event which results in a decrease in the number of F(-) survivors. We have described and discussed the parameters affecting this phenomenon of lethal zygosis, and these include the cultural conditions of both donor and recipient cells prior to mixing and the use of aeration throughout the period of the experiment. The absence of lethal zygosis with filtrates and supernatant fluids from donors suggests a dependence on direct cell-cell contact as found in conjugation. The phenomenon, which is normally observed in liquid media, also occurs on solid media, and use of these two methods has allowed examination of strains of different mating types. Whereas most Hfr strains capable of producing normal yields of recombinants showed killing activity, no F(+) and only one F' donor produced lethal zygosis. Only F(-) strains were sensitive to this phenomenon. The relationship between lethal zygosis and the various stages of conjugation is discussed.     

Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles

Fusarium species are globally distributed filamentous ascomycete fungi that are frequently reported as plant pathogens and opportunistic human pathogens, leading to yield loss of crops, mycotoxin contamination of food and feed products as well as damage to human and livestock. Human infections of Fusarium spp. are difficult to treat due to broad antifungal resistance by members of this genus. Their role as disease-causing agents in crops and humans suggests a need for antifungal resistance profiles as well as a simple, rapid, and cost effective identification method. Fusarium strains were isolated from food and clinical samples. High-resolution melting curve (HRM) analysis was performed using specific primers targeting internal transcribed spacer (ITS) region, followed with evaluation of specificity and sensitivity. The antifungal susceptibility of four Fusarium species was studied using the Sensititre YeastOne method. HRM analysis revealed reproducible, unimodal melting profiles specific to each of the four Fusarium strains, while no amplification of the negative controls. The minimum detection limits were 100–120 copies based on a 2 µl volume of template. Clear susceptibility differences were observed against antifungal agents by different Fusarium isolates, with amphotericin B and voriconazole displayed strongest antifungal effects to all the tested strains. We developed a simple, rapid, and low-cost qPCR-HRM method for identification of four Fusarium spp. (F. oxysporum, F. lateritium, F. fujikuroi, and F. solani). The antifungal susceptibility profiles supplied antifungal information of foodborne and clinical Fusarium spp. and provided guidance for clinical treatment of human infections.

     

Two inbred strains of mice with high and low mammary tumor potentials established from the same basal stock of Swiss albino (SHN and SLN): history of mammary tumorigenesis and reproduction after the 30th generation of full-sib mating.

We established SHN and SLN strains of mice with a high and a low mammary tumor potentials from the same basal stock of Swiss albino. The selection history of females (mothers) for subsequent generations, mammary tumorigenesis and reproduction up to recent generations are presented and compared with the results up to F30 published in 1979. The changes with generation of each parameter varied in both strains except the conditions of female (mother) selection for subsequent generations, which were generally constant. However, the fluctuations in parameters up to F30 became generally smaller with advancing generations. Based on the fluctuations from generation to generation of mammary tumorigenic and reproductive parameters, an interdependency between mammary tumor potential and reproductivity is suggested in SLN, but less so in SHN. The pup growth rate was increased and associated with selection for mammary tumorigenesis in SHN; this parameter is usually used as an indicator of mother lactational performance in mice. The significance of selection for mammary tumor potential in association with reproductivity was further discussed.     

The type subtyping of meningococci by means of whole-cell immunoenzyme analysis]

In this work the method of the whole-cell enzyme immunoassay, used for the serotype-subtyping of meningococci by means of specific monoclonal antibodies, is described. High specificity of the method, the simplicity of the assay procedure and evaluation of its results, as well as the availability of this method for practical use, have been demonstrated. The results of this investigation confirm the importance of the evaluation of type-subtype appurtenance of reference and laboratory strains used in experiments. Study of 72 meningococcal strains obtained from patients has revealed their polyclonal character in respect of their type-subtype signs.     

The genetic profile and molecular diagnosis of thiophanate-methyl resistant strains of Fusarium asiaticum in Japan

Fusarium asiaticum strains resistant to thiophanate-methyl were detected in four prefectures in Japan though their proportion in the total population was low in all instances. The F167Y or F200Y mutation in the β2-tubulin gene (FGSG06611.3) was detected in thiophanate-methyl resistant (TMR) strains. A PCR-based diagnostic method based on these mutations was developed and applied for all 17 TMR strains that have been detected so far in Japan. Three and 14 TMR strains were the F167Y and F200Y mutation types, respectively. Analysis by 11 variable number of tandem repeat markers showed that TMR strains from the same site had an identical genotype, while TMR strains from different sites were dissimilar. This result indicates that TMR strains did not spread clonally to the different sites.     

Genotype-specific environmental impact on the variance of blood values in inbred and F1 hybrid mice

Mice are important models for biomedical research because of the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Inbred mouse strains as well as F₁ hybrid mice are routinely used as genetically defined animal models; however, only a few studies investigated the variance of phenotypic parameters in inbred versus F₁ hybrid mice and the potential interference of the genetic background with different housing conditions. Thus, we analyzed the ranges of clinical chemical and hematologic parameters in C3H and C57BL/6 inbred mice and their reciprocal F₁ hybrids (B6C3F1, C3B6F1) in two different mouse facilities. Two thirds of the blood parameters examined in the same strain differed between the facilities for both the inbred strains and the F₁ hybrid lines. The relation of the values between inbred and F₁ hybrid mice was also affected by the facility. The variance of blood parameters in F₁ hybrid mice compared with their parental inbred strains was inconsistent in one facility but generally smaller in the other facility. A subsequent study of F₁ hybrid animals derived from the parental strains C3H and BALB/c, which was done in the latter housing unit, detected no general difference in the variance of blood parameters between F₁ hybrid and inbred mice. Our study clearly demonstrates the possibility of major interactions between genotype and environment regarding the variance of clinical chemical and hematologic parameters.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

Comparative dose-survival curves of representative Clostridium botulinum type F spores with type A and B spores.

Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with dose-response data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters.     

Statistical Analysis of Chemical Release Rates from Soils

Two statistical methods for determining the precision of best-fit model parameters generated from chemical rate of release data are discussed. One method uses the likelihood theory to estimate marginal confidence intervals and joint confidence regions of the release model parameters. The other method uses Monte Carlo simulation to estimate statistical inferences for the release model parameters. Both methods were applied to a set of rate of release data that was generated using a field soil. The results of this evaluation indicate that the precision of F (the fraction of a chemical in a soil that is released quickly) is greater than the precision of k₁ (the rate constant describing fast release), which is greater than the precision of k₂ (the rate constant describing slow release). This occurs because more data are taken during the time period described by F and k₁ than during the time period described by F and k₂. In general, estimates of F will be relatively precise when the ratio of k₁ to k₂ is large, estimates of k₁ for soil/chemical matrices with a high F will be relatively precise, and estimates of k₂ for soil/chemical matrices with a low F will be relatively precise, provided that sufficient time is allowed for full release.     

Characteristics of the aortic elastic network and related phenotypes in seven inbred rat strains

Extracellular matrix (ECM) molecules such as elastin and collagen provide mechanical support to the vessel wall and are essential for vascular function. Evidence that genetic factors influence aortic ECM composition and organization was concluded from our previous studies showing that the inbred Brown Norway (BN) rat differs significantly from the outbred Long-Evans (LE) and the inbred LOU rat with respect to both thoracic aortic elastin content and internal elastic lamina (IEL) rupture in the abdominal aorta and iliac arteries. Here, we measured aortic elastin and collagen contents as well as factors that may modulate these parameters [insulin growth factor (IGF)-I, transforming growth factor (TGF)-beta(1), and matrix metalloproteinase (MMP)-2] in seven inbred rat strains, including BN and LOU. We also investigated whether IEL ruptures occur in strains other than BN. We showed that LOU, LE, BN, and Fischer 344 (F344) rats were significantly different for aortic elastin content and elastin-to-collagen ratio, whereas LE, Lewis, WAG, and Wistar-Furth (WF) were similar for these parameters. BN and F344 had the lowest values. BN was the only strain to present numerous IEL ruptures, whereas F344, LE, and WF presented a few and the other strains presented none. In addition, IGF-I and TGF-beta(1) levels in the plasma and aorta differed significantly between strains, suggesting genetic control of their production. Because inbred rat strains provide interesting models for quantitative trait locus analysis, our results concerning elastin, collagen, IEL ruptures, and cytokines may provide a basis for the search for candidate genes involved in the control of these phenotypes.     

Long-term preservation of freeze-dried mouse spermatozoa

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.     

Biological and mathematical description of the growth pattern of Fusarium oxysporum in a sterilized soil

Abstract The population dynamics of 4 F. oxysporum strains incubated in sterilized soil have been studied for 35 days. Enumeration of fungal propagules was carried out by the soil dilution plate technique. The qualitative variations of the populations were viewed by epifluorescent microscopy after staining the fungal propagules in soil with Calcofluor. A logistic model was used to describe population dynamic parameters. The predominant mode of growth of F. oxysporum in sterilized soil involved abundant production of conidia during the exponential phase. The increase of the propagules resulting from mycelial development during the lag phase was not taken into account by the logistic model. By determining the density of the population at the end of the lag phase, growth parameters of different F. oxysporum strains have been identified allowing the saprophytic competence of each strain to be characterized.