甘蓝花药培养胚状体诱导形成影响因子研究

用甘蓝F1、F2和自交系S33个世代6种基因型材料进行甘蓝花药培养诱导胚状体形成影响因子研究。结果显示:(1)高浓度蔗糖对甘蓝胚状体形成具有显著的诱导作用,6%蔗糖浓度是甘蓝花药培养的最适浓度,其胚状体的诱导率最高达12.2%;(2)材料基因型是影响花药培养的主要因素,F2和F1代材料胚状体诱导效果好,且胚状体诱导率F2代(F2P192和F2P194)18.9%比F1代(F1S17和F1S13)17.1%较高,但差异不显著,自交系S3代材料很难诱导出胚状体;(3)B5培养基比MS培养基更适合甘蓝花药胚状体的诱导培养。结果表明,甘蓝F2代是其花药诱导培养胚状体的最佳基因型材料,B5 2.0 mg/L 2,4-D 2.0 mg/L KT 6%Suc是甘蓝花药诱导培养胚状体的最适培养基。     

Purification and Characterization of Spirosomes in Lactobacillus brevis

Spirosomes, very fine spiral particles, were isolated from a protoplastlysate of Lactobacillus brevis ATCC 8287 by differential centrifugation and purified further by potassium tartrate density gradient centrifugation. The purified spirosome preparation showed a maximum peak around 275 nm on the ultraviolet absorption spectrum and it consisted of about 94.5% protein. The buoyant density in CsCl of the spirosomes was 1.320 g/cm³. The spirosomes were composed mainly of a single protein (spirosin) with an apparent molecular weight of about 95,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The protein of the spirosomes was found to be composed predominantly of neutral amino acids accompanied by approximately equal amounts of acidic and basic amino acids. The spirosomes showed one antigenic determinant in the immunodiffusion test. The spirosomes were readily degraded by the action of proteolytic enzymes and lost their antigenicity, but they were not affected by treatment with either deoxyribonuclease or ribonuclease. The spiral structure of the spirosome was also found to be disintegrated by treatment with 1 m guanidine hydrochloride, 4 m urea or 0.1% SDS, but not by the action of deoxycholate, non-ionic detergents or mercaptoethanol, as observed in the electron microscope.     

Growth Response of Lactobacillus brevis to Aeration and Organic Catalysts

Under stationary and anaerobic conditions, greater cell yields of Lactobacillus brevis were obtained from autoclaved than from filter-sterilized glucose media. Fructose, tentatively identified as a product generated by the heating process, served as an excellent catalyst for inducing growth. The addition of micromolar quantities of pentoses or potential pentose precursors to the filter-sterilized medium was equally effective in stimulating growth. These organic catalysts were not essential for growth under aerobic conditions. Upon agitation, similar cell yields were obtained from the autoclaved and filter-sterilized media. The micromolar quantities of lactic acid produced per micromole of carbohydrate fermented appeared to be similar under aerobic and static conditions of incubation. The final concentration of acetic acid increased as the result of agitation. This increase in volatile acidity was accompanied by a significant decrease in ethyl alcohol production. The cell yield was increased nearly 50% under aerobic conditions.     

荷斯坦奶牛红毛性状的基因检测及红毛形成机制的探讨

荷斯坦奶牛黑素皮质素受体1(MC1R)基因310G缺失突变产生e等位基因,导致红白花毛色。实验利用PCR-RFLP和DNA测序技术建立了荷斯坦奶牛红毛性状的基因检测方法,并在4个荷斯坦奶牛全同胞家系中得到了证实,可见其可用于鉴定携带e等位基因的种公牛,以指导奶牛育种。通过分析黑素皮质素(MC)受体蛋白家族序列,找到了MC1R蛋白结构与功能的区域(TM3、TM6、TM7和EL3)。利用VHMPT软件预测MC1R突变蛋白的结构,结果显示其丢失了TM3、TM6、TM7和EL3区,也失去了与α-促黑素(α-MSH)结合的区域,最终导致红毛的产生。     

Localization and Characterization of alpha-Glucosidase Activity in Lactobacillus brevis

Lactobacillus brevis is found together with the yeast Brettanomyces lambicus during the overattenuation process in spontaneously fermented lambic beer. An isolated L. brevis strain has been shown to produce an alpha-glucosidase with many similarities to the glucosidase earlier found in B. lambicus. The enzyme was purified by ammonium sulfate precipitation, gel (Sephadex G-150 and Ultrogel AcA-44) filtration, and ion-exchange chromatography (DEAE-Sephadex A-50). The molecular weights of the enzyme, as determined by gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were about 50,000 and 60,000, respectively. Optimum catalytic activity was obtained at 40 degrees C and pH 6.0. The enzyme showed a decrease of hydrolysis with an increase in the degree of polymerization of the substrate. The K(m) values for p-nitrophenyl-alpha-d-glucopyranoside, maltose, and maltotriose were 0.51, 3.0, and 5.2 mM, respectively. There was lack of inhibition by 0.15 mM acarbose and 0.5 M turanose, but the enzyme was inhibited by Tris (K(i) value of 25 mM). The alpha-glucosidase of L. brevis together with the enzyme of B. lambicus seems to be a key factor in the overattenuation of lambic beer, although the involvement of other lactic acid bacteria (pediococci) cannot be excluded.     

The two-step biotransformation of monosodium glutamate to GABA by Lactobacillus brevis growing and resting cells

Gama-aminobutyric acid (GABA) is a natural functional amino acid. In the current study, Lactobacillus brevis TCCC13007, a high GABA-producing strain, was isolated from naturally pickled Chinese vegetables. A two-step cellular bioconversion process was established using L. brevis TCCC13007 for the production of GABA. First, L. brevis cells were grown anaerobically in 7% monosodium glutamate (MSG)-containing medium at an initial pH of 6.0 and a controlled pH of 4.6 for 16 to 66 h; approximately 38 g L(-1) of GABA was obtained after 66 h of fermentation at a conversion rate of 98.6%. In the second stage of the process, about 7.6 g L(-1) of GABA was produced three more times at a conversion rate of 92.2% using the same batch of resting cells in the substrate-containing buffer under optimized conditions. Thus, the total GABA yield reached 61 g L(-1). A model system for the biotransformation of MSG to GABA was established using L. brevis TCCC13007 resting cells. The reaction rates were found to follow the classic Michaelis-Menten equation at low substrate concentrations (<80 mM). Kinetic analysis of the biotransformation revealed that L. brevis TCCC13007 resting cells produced GABA similar to that produced by purified glutamate decarboxylase from L. brevis.     

Production of brevicin 286 by Lactobacillus brevis VB286 and partial characterization

Brevicin 286 was produced by Lactobacillus brevis VB286 isolated from vacuum-packaged meat and was partially purified by ammonium sulphate precipitation, gel filtration and dialysis. The bacteriocin was susceptible to proteolytic enzymes, stable to heating at 100°C particularly under acidic pH conditions, and showed a relatively narrow spectrum of activity with notable activity against Listeria sp. Production of brevicin 286 was optimal during exponential growth at 20°C. Higher rates of cell growth occurred between 30 and 37°C but with little or no expression of brevicin 286. A food-grade formulation consisting of 4% yeast extract and 1% glucose was found to be adequate for optimal brevicin 286 production and the bacteriocin-containing culture supernate was successfully spray dried with full recovery of antibacterial activity in the resultant powder.     

Aggregation of Lactobacillus brevis associated with decrease in pH by glucose fermentation

ABSTRACT

Some Lactobacillus brevis strains were found to aggregate upon the addition of glucose, which resulted in glucose fermentation and pH decrease. Surface layer proteins (Slp) that represented the outermost layer of the bacteria decreased under these low pH conditions, probably because of the partial detachment of Slp from the cell surface triggered by the acidic environment. Similar observations of decreased Slp and aggregation were observed under the culture conditions, confirming that L. brevis aggregation was due to the partial Slp detachment under the acidic conditions of glucose fermentation. Such Slp detachment might affect the electrostatic nature of L. brevis cells by initiating the formation of irregular charge across the L. brevis cell surface, thereby leading to aggregation. These observations would be useful for elucidating the aggregation mechanism of lactic acid bacteria, which was considered to be involved in the probiotic effect of the bacteria.     

Coexpression of pyruvate decarboxylase and alcohol dehydrogenase genes in Lactobacillus brevis

Lactobacillus brevis ATCC367 was engineered to express pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) genes in order to increase ethanol fermentation from biomass-derived residues. First, a Gram-positive Sarcina ventriculi PDC gene (Svpdc) was introduced into L. brevis ATCC 367 to obtain L. brevis bbc03. The SvPDC was detected by immunoblot using an SvPDC oligo peptide antiserum, but no increased ethanol was detected in L. brevis bbc03. Then, an ADH gene from L. brevis (Bradh) was cloned behind the Svpdc gene that generated a pdc/adh-coupled ethanol cassette pBBC04. The pBBC04 restored anaerobic growth and conferred ethanol production of Escheirichia coli NZN111 (a fermentative defective strain incapable of growing anaerobically). Approximately 58 kDa (SvPDC) and 28 kDa (BrADH) recombinant proteins were observed in L. brevis bbc04. These results indicated that the Gram-positive ethanol production genes can be expressed in L. brevis using a Gram-positive promoter and pTRKH2 shuttle vector. This work provides evidence that expressing Gram-positive ethanol genes in pentose utilizing L. brevis will further aid manipulation of this microbe toward biomass to ethanol production.     

Proposed Mechanism for HII Phase Induction by Gramicidin in Model Membranes and Its Relation to Channel Formation

A model is proposed for the molecular mechanism of H_II phase induction by gramicidin in model membranes. The model describes the sequence of events that occurs upon hydration of a mixed lipid/gramicidin film, relating them to gramicidin channel formation and to relevant literature on gramicidin and lipid structure.     

RNA-seq-based transcriptomic analysis of AHL-induced biofilm and pyocyanin inhibition in Pseudomonas aeruginosa by Lactobacillus brevis

International Microbiology - Quorum sensing (QS) regulates hundreds of genes in Pseudomonas aeruginosa, and many of which encode extracellular virulence factors. Lactobacillus as a probiotic has...     

Rapid identification of the three major species of dairy obligate heterofermenters Lactobacillus brevis, Lactobacillus fermentum and Lactobacillus parabuchneri by species-specific duplex PCR

In this study, the biodiversity of 154 strains of lactic acid bacteria, including 112 dairy product isolates presumptively identified as obligately heterofermentative lactobacilli (OHL) by classical microbiological tests, as well as 23 OHL-type strains, was investigated by PCR-based methods and gene sequencing. Using these techniques, 51% of the cheese isolates were actually identified as OHL. The non-OHL isolates were identified to the Leuconostoc, Lactobacillus, Weisella, Pediococcus or Streptococcus genera. Among the OHL cheese isolates, five species were directly identified including three of the most frequently isolated species -Lactobacillus fermentum, Lactobacillus parabuchneri and Lactobacillus brevis- and two rarely isolated species - Lactobacillus diolivorans and Lactobacillus reuteri. A sixth group made up of two dairy isolates was also identified and according to 16S rRNA gene and intergenic spacer region (ISR) sequencing data corresponded to Lactobacillus sp. and may constitute a new species. Species-specific primers were designed for the rapid and reliable detection of the three most frequently isolated species by species-specific duplex PCR.     

Induction of microcycle sporulation in Bacillus brevis spp. AG4 by amethopterin

Addition of amethopterin to medium before inoculation inhibited DNA synthesis and induced microcycle sporogenesis in Bacillus brevis spp. AG4. Synthesis of RNA and proteins occurred at a considerably reduced rate.Abbreviations TVC total viable counts - HSC heat stable counts - CDGS chemically defined medium for growth and sporulation - TCA trichloro acetic acid     

Relationship between Endogenous Factor and Decrease in Color Formation of Low Quality Pork

To clarify the reason for the decrease in CFR (color forming ratio) of pale, soft and exudative (PSE) porcine muscle, the effect of simulation of the PSE conditions on cooked cured meat color was investigated in fractionated components from porcine skeletal muscle, 24 hr postmortem. Although the solubility of total proteins and heme pigments markedly decreased in sarcoplasm by this treatment, no decline in CFR of the sarcoplasm could be observed when compared to the controls. A similar tendency was observed in both high- and low-molecular weight fractions of sarcoplasm. On the other hand, myofibrils simulated the PSE conditions in the presence of myoglobin significantly decreased CFR, and the extent of this decline was nearly consistent with the case of whole muscle. The reasons for the decrease in CFR of PSE muscle are discussed.     

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis

AIMS: Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. METHODS AND RESULTS: A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. CONCLUSION: The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.     

Increase in Viscosity and Gel Formation of Fruit Juice by Purified Pectinesterase

The pectinesterase produced by Sclerotinia libertiana was purified by the column chromatography employing cation exchange resin Amberlite CG 50 and CM cellulose. The specific activity per mg protein was increased 266-fold. The homogeneity of the enzyme preparation was confirmed by ultracentrifugal analysis and starch zone electrophoresis. The purified pectinestrase was found to remarkably increase the viscosity of pectin solution and fruit juice, and, in a pectin solution of appropriate concentration (about 0.5~1 %) and some fruit juices, it finally formed the cosity thus increased and gel formed by the enzyme action did not change for a long time.