Effect of culture medium composition on Trichoderma reesei’s morphology and cellulase production

The objective of this study was to determine how fungal morphology influences the volumetric cellulase productivity of Trichoderma reesei cultured in four media with lactose and lactobionic acid as fed-batch in a 7 L stirred tank bioreactor. The use of a cellulose–yeast extract culture medium yielded the highest enzyme production with a volumetric enzyme activity of 69.8 U L⁻¹ h⁻¹, and a maximum fungal biomass of 14.7 g L⁻¹. These findings were associated with the following morphological characteristics of the fungus: total mycelia was 98% of total mean projected area, mean hyphae length of 10 mm, mean hyphae volume of 45.1 mm³, mean hyphae diameter of 7.9 μm, number of branches 9, and number of tips per hypha 29. A positive correlation was found between the total mycelia, the number of tips and the volumetric enzyme productivity, indicating the weight of these variables on the enzyme productivity.     

Effect of preculturing conditions on growth of Lactobacillus rhamnosus on medium containing glucose and citrate

Lactobacillus rhamnosus can metabolize citrate through a citrate inducible transport system. The growth curves of L. rhamnosus on medium containing glucose and citrate was found to be highly dependent on preculturing conditions. It exhibited diauxic growth when precultured on glucose, but demonstrated simultaneous consumption when cultured on citrate. The maximum specific growth rate for cells growing on glucose + citrate was 0.38 h-1, which was higher than the growth rate on individual substrates (0.28 h-1). Simultaneous consumption also yielded higher net flavour compounds, diacetyl and acetoin. Flux analysis indicated that L. rhamnosus requires oxygen for balancing excess NADH through NADH oxidase. The flux analysis provided insights into the metabolic network of L. rhamnosus.     

The antimicrobial properties of different strains of Lactobacillus spp. isolated from kefir

The characteristics of 58 strains of Lactobacillus spp. isolated from kefir were studied. These strains were tested for adherence to human enterocyte-like Caco-2 cells, resistance to acidic pH and bile acid, antimicrobial activities against enteropathogenic bacteria and inhibition of Salmonella typhimurium attachment to Caco-2 cells. The best probiotic properties were observed in L. acidophilus CYC 10051 and L. kefiranofaciens CYC 10058. L. kefiranofaciens CYC 10058 produced an exopolysaccharide, which revealed that it was closely related to kefiran, a polysaccharide with antitumoral properties. This is the first in vitro study about the antimicrobial characteristics of the Lactobacillus population of kefir.     

Identification and differentiation of Lactobacillus, Streptococcus and Bifidobacterium species in fermented milk products with bifidobacteria

The aim of this study was to identify and discriminate bacteria contained in commercial fermented milks with bifidobacteria by the use of amplified ribosomal DNA restriction analysis (ARDRA) and randomly amplified polymorphic DNA (RAPD) techniques. ARDRA of the 16S rDNA gene and RAPD were performed on 13 Lactobacillus strains, 13 Streptococcus and 13 Bifidobacterium strains isolated from commercial fermented milk. Lactobacillus delbrueckii, Streptococcus thermophilus and Bifidobacterium animalis isolates were identified by genus- and species-PCR and also, they were differentiated at genus and species level by ARDRA using MwoI restriction enzyme. The ARDRA technique allowed for the discrimination among these three related genus with the use of only one restriction enzyme, since distinctive profiles were obtained for each genus. Therefore it can be a simple, rapid and useful method for routine identification. Also, RAPD technique allowed the discrimination of all bacteria contained in dairy products, at genus- and strain-level by the performance of one PCR reaction.     

Chitosan-mediated changes in cell wall composition, morphology and ultrastructure in two wood-inhabiting fungi

The effect of chitosan on cell wall deposition was investigated in the two wood-inhabiting fungal species Trichoderma harzianum (CBS 597.91) and Sphaeropsis sapinea (NZFS 2725). The study used three independent analytical techniques to quantify chitin in the fungal mycelium. A colorimetric method for the detection of d-glucosamine was compared with two gas chromatography–mass spectroscopy (GC-MS) methods employing alditol acetates analysis and pyrolysis. The latter used a stable-isotope-labelled internal standard, d₃-N-acetyl glucosamine. At least in the case of S. sapinea, the study provided evidence of an increase in the chitin content in the mycelium due to chitosan treatment, indicating that chitosan treatment affected cell wall deposition. Electron microscopy techniques showed alteration in surface morphology and cell wall texture due to chitosan treatment. The implications of these results are discussed with a view to analysing possible mechanisms for growth inhibitory effects of chitosan on fungal hyphae.     

The ultrastructure of the yolk nucleus during early cleavage of Nassarius reticulatus L. (Gastropoda,Prosobranchia)

Summary In all blastomeres of Nassarius from 4- to 16-cell stage yolk nuclei occur. Most of them are spherical bodies, lying juxtanuclearly between the nucleus and the apical plasmalemma. Strangely they are not ultrastructurally uniform but fall into two categories (Fig. 5): Type I is a massive spherical accumulation of mitochondria embedded in a dense intermitochondrial substance, which appears to contain both granules and filaments. Type II is a ball of radially arranged small Golgi stacks clustered around a centre of Golgi vesicles and other organelles embedded in a ground cytoplasm structurally similar to the intermitochondrial substance of type I. The function of both types of yolk nuclei is unknown. These segmentation yolk nuclei have nothing to do with yolk synthesis any more. On the other hand there are no indications that yolk nuclei occurrence is correlated with the break-down of yolk because neither lipid droplets nor protein yolk granules are observed in or beside the yolk nuclei.We wish to thank Mrs. Chr. Mehlis for her valuable technical assistance as well as Professor J. Bergérard for the excellent working conditions on the Station biologique at Roscoff (France).     

Seasonal changes of the ultrastructure of the pars tuberalis of the hypophysis of Rana temporaria

Summary In the pars tuberalis of the hypophysis of Rana temporaria, which shows the ultrastructural characteristics of a polypeptide hormone secreting endocrine gland, seasonal changes of the ultrastructure are described. In accordance with the literature, these seasonal changes of ultrastructure are interpreted as the morphological expression of seasonal changes of endocrine activity of the pars tuberalis.     

Diversity of Lactobacillus and Bifidobacterium in feces of herbivores, omnivores and carnivores

The Lactobacillus and Bifidobacterium population in the feces of 26 animals (16 species) were studied by culture-dependent and culture-independent techniques. Lactobacilli were detected from a few herbivores, all carnivores and some omnivores. Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus vaginalis and Lactobacillus ingluviei were the most dominant lactobacilli in carnivores. These species were, however, not predominant in herbivores and omnivores. Lactobacillus brevis, Lactobacillus casei, Lactobacillus parabuchneri, Lactobacillus plantarum, Lactobacillus sakei, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, usually present in raw plant material, were present in omnivores but not in carnivores. Bifidobacteria were detected in only four herbivores and two omnivores. Bifidobacterium pseudolongum was the only Bifidobacterium species detected in herbivores. Bifidobacteria detected in the two omnivores are phylogenetically not closely related to known species and are possible novel species in the genus.     

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.     

The ultrastructure of the byssal apparatus of Mytilus galloprovincialis

The ultrastructure of the byssus of Mytilus galloprovincialis was analysed by transmission electron microscopy in thin sections of either embedded or frozen samples. All parts of the byssus (stem core laminae, stem outer laminae, threads proximal and distal parts) appear to be formed by the same basic filamentous components organized in different ways at the submicroscopic level and embedded in a variable quantity of matrix. The filaments appear to consist of a central electron-lucent zone (3 nm in diameter), surrounded by an electron-dense rim (total diameter 7 nm). The matrix has a granular or microfilamentous structure. The stem and the threads differ greatly in their submicroscopic organization, but their basic constituents (filaments and matrix) are similar. Peculiar filamentous banded elements (FBE) were found mainly in the stem outer laminae. A relation between the ultrastructure and mechanical properties of the different parts of the byssus was established. The presence of collagen is discussed; since no morphological evidence of any of the known forms of collagen organization was revealed by electron microscopy, it is suggested that byssus collagen may be localized in the matrix and in the FBE.     

High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure

High-pressure freezing of chemically untreated nodules of soybean (Glycine max (L.) Merr.), in sharp contrast to chemical fixation and prefixation, appears to preserve the ultrastructure close to the native state. This is supported by the observation that the peribacteroid membrane of high-pressure-frozen samples is tightly wrapped around the bacteroids, a finding that is fully consistent with the current views on the physiology of oxygen and metabolite transport between plant cytosol and bacteroids. In soybean root nodules, the plant tissue and the enclosed bacteria are so dissimilar that conventional aldehyde-fixation procedures are unable to preserve the overall native ultrastructure. This was demonstrated by high-pressure freezing of nodules that had been pre-fixed in glutaraldehyde at various buffer molalities: no buffer strength tested preserved all ultrastructural aspects that could be seen after high-pressure freezing of chemically untreated nodules.     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Premetamorphic changes in the ultrastructure of Calliphora fat cells

Summary In the fat body of Calliphora a drastic reorganization of the cell structure, which is related to premetamorphic functional changes, can be observed during the 3rd larval stage of development. These changes include an accumulation of protein storage granules, a decrease in the relative volume of the mitochondria and the surface area of endoplasmic reticulum membrane, and the disappearance of vacuoles. Absolute values obtained for organelle areas and membrane perimeters confirm the assumption that large numbers of organelles are eliminated by autophagic activity prior to the pupation period. A temporary restoration of the endoplasmic reticulum is found immediately before pupariation. Large variations in organelle density were found between individual fat cells and between the nuclear, mid- and peripheral regions of the cytoplasm.     

On the ultrastructure of polypeptide hormone-producing cells in the gut of the ascidian,Ciona intestinalis L. and the Bivalve,Mytilus edulis L.

Summary Ultrastructural evidence has been found for the presence of polypeptide hormone-producing cells in the gut of Ciona intestinalis L. and Mytilus edulis L. which do not appear to have been described before. Due to their localization and ultrastructural characteristics, it is suggested that the cells in Mytilus edulis probably produce an insulin-like substance and that some of these cells in Ciona intestinalis may produce 5-HT (5-Hydroxytryptamine). In each species only one granulated cell type can be observed. The granules, which are electron dense and membrane bound, also show a halo. The average diameter of the granules is 100–200 nm for Ciona and 200–400 nm for Mytilus.I thank Mr. G. Bargsten, M.A., Dept. of Marine Zoology, University of Kiel, for the supply of the animals     

Evidence of metal binding activities of pentadecapeptide from Panax ginseng

A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg²⁺. Mobility was also affected in the presence of La³⁺, Mn²⁺, Ca²⁺ and Zn²⁺ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca²⁺-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg²⁺ and Mn²⁺ ions.     

Use of SDS-PAGE of cell-wall proteins for rapid differentiation of Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus

Cell-wall protein profiles of different strains of Lactobacillus helveticus and L. delbrueckii subsp. lactis isolated from regional cheeses were studied by SDS-PAGE. The patterns were highly reproducible and the presence of numerous bands with molecular weight ranging from 14 to 160 kDa allowed L. delbrueckii subsp. lactis to be differentiated from L. helveticus. The method is a reliable and rapid way to identify thermophilic lactobacilli.     

Development and Application of Random DIG-Labelled Probes Based on Total Genomic Lactobacillus DNA

Summary Nine Lactobacillus-specific and non-isotopically (digoxygenin) labelled probes were developed on the basis of Lactobacillus total chromosomal DNA. Their specificity and applicability for Lactobacillus discrimination was proven by DNA–DNA hybridization to reference strains from the American Type Culture Collection (ATCC). The DNA probes were divided into three groups depending on the ability to hybridize to DNA from the same and/or from a group of related Lactobacillus strains. They were assayed in the species-specific detection of vaginal strains from the genus Lactobacillus. Six DNA probes were successfully applied for characterization of 21 newly isolated vaginal Lactobacilli. The species affliation of some isolates was determined. The developed DNA probes were evaluated for usage as a qualitative hybridization test for detection of Lactobacillus species in mixed cultures, obtained directly from vaginal samples without strain isolation.     

Fatty acid composition of Simonsiella strains

Gas-liquid chromatography of methyl esters of bound fatty acids extracted from the cells of 48 Simonsiella strains showed that these aerobic, gliding, multicellular-filamentous bacteria have fatty acid profiles of the pattern considered typical of Gramnegative eubacteria. All strains contained predominantly tetradecanoic acid (29.5%), 9-hexadecenoic acid (22.2%), an unidentified acid with an equivalent chain length of approximately 20 carbon atoms (15.8%), and dodecanoic acid (11.4%).Discriminant analysis of the mean relative percentages of 12 fatty acids correctly assigned 94% of the strains to groups based on their source of origin (i.e., the oral cavities of sheep, cat, human or dog); the relative amounts of only 3 of the fatty acids (9-octadecenoic acid, hexadecanoic acid, and tetradecanoic acid) provided most of this discrimination.     

Effect of bacteriocinogenic Lactobacillus spp. on the shelf life of fufu, a traditional fermented cassava product

Quantitative determination of antimicrobial compounds produced by the Lactobacillus strains under test was carried out. L. plantarum F1 produced the highest quantity of lactic acid (16.4 g/l) while the lowest amount (0.3 g/l) was produced by L. jensenii F9. All the test organisms produced hydrogen peroxide, with L. brevis OG1 having the highest yield of 0.037 g/l. Diacetyl was also produced by all the organisms, with L. plantarum F1 and L. brevis OG1 having the highest yield of 1.7 g/l, while the lowest producer was L. jensenii F9 (0.86 g/l). Determination of bacteriocin activity was carried out. L. plantarum F1 exhibited 6400 AU/ml bacteriocin activity, while L. brevis OG1 had the lowest activity of 3200 AU/ml using E. coli NCTC10418 as indicator organism. However, L. fermentum F5 and L. jensenii F9 did not produce any detectable bacteriocin. The pH value in the culture supernatant of L. plantarum F1 reached 3.1 within 48 h of incubation, while that of L. jensenii F9 was 5.2. Fufu was prepared using both bacteriocin-producing (BP) L. plantarum F1 and L. brevis OG1, and non-bacteriocin producing L. fermentum F5 and L. jensenii F9. No viable cells of Salmonella typhimurium ATCC13311 and Shigella flexneri AP23498 were detected after 12 h in the cassava products fermented with mixed starter culture of L. plantarum F1 and L. brevis OG1. The rate of survival of enteropathogens in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was much lower when compared to cassava fermented with mixed starter culture of L. fermentum F5 and L. jensenii F9. At 12 h, the viable count of E. coli NCTC10418 in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was 1.1 log₁₀ c.f.u./g whereas in cassava fermented with mixed starter cultures of L. fermentum F5 and L. jensenii F9, 8.5 log₁₀ c.f.u./g was obtained The study revealed that fufu produced with BP mixed starter cultures had a better shelf life and kept for 13 days before spoilage occurred, relative to 5 days observed for fufu produced using non-bacteriocin-producing starter cultures, and 6 days for the traditional fermented fufu.     

Mixed-acid fermentation and polysaccharide production by Lactobacillus helveticus in milk cultures

Lactobacillus helveticus grown in milk with pH control at 6.2 had a slower growth rate (=0.27 h^–1) and produced less exopolysaccharide (49 mg l^–1) but increased lactic acid production (425 mM) compared to cultures without pH control (=0.5 h^–1, 380 mg exopolysaccharide l^–1, and 210 mM lactate), respectively. Both cultures displayed a mixed-acid fermentation with formation of acetate, which is linked not only to citrate metabolism, but also to alternative pathways from pyruvate.