Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants

AIMS: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. METHODS AND RESULTS: Wheat and two corn silages were prepared in 0.25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10(6) CFU g(-1). Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. CONCLUSIONS: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental micro-organisms in the silage or in the rumen.     

Time course of lipolytic activity and lipid peroxidation after whole-body gamma irradiation of rats

The content of fluorescing products of lipid peroxidation (LFP) and hormone-stimulated lipolytic activity were determined in rat epididymal adipose tissue during a 29-day interval after whole-body gamma irradiation. An increase in LFP was accompanied by a decrease in lipolytic activity. It is suggested that these effects are interrelated and that the decrease in lipolysis in irradiated, semi fasting rats is an additional deteriorating factor leading to death in some animals.     

Recent advances and applications of the lipolytic activity of Carica papaya latex

The lipolytic enzymes of Carica papaya have been the subjects of a growing research activity in the last years due to the structural characteristics, substrate specificity over lipases from different sources and the wide availability from the large worldwide figures of papaya agro-waste. This review attempts to present an inclusive discussion of the recent advances of these lipolytic enzymes starting from findings related to their structure and mechanism and later examining the most employed pretreatments and the general optimized parameters for its use. Furthermore, individual sections comprising applications in glycerolipid modification, catalysis for the synthesis of non-glycerolipid products and the kinetic resolution of racemic compounds are discussed. The attractive features of CPL cited in this literature survey serve as an invitation for other research groups interested in the development of new and sustainable biochemical technologies.     

Ricin RCA60: evidence of its phospholipase activity.

The present work documents, on a qualitative and quantitative basis, the lipolytic activity of ricin protein RCA60 on glycerophospholipids. RCA60 demonstrates a low level of hydrolysis towards radioactive dipalmitoyl-glycerophosphatidylcholine. This observation was confirmed on a better substrate, palmitoyl-oleoyl-glycerophosphatidylcholine, after analysis of the reaction products by thin-layer and gas chromatography. A comparable hydrolytic activity was observed when palmitoyl-oleoyl-glycerophosphatidylethanolamine was used as substrate. The nature of the hydrolysis products supports the conclusion that RCA60 demonstrates phospholipase A1 and A2 activities as well as a lysophospholipase activity of A1 and A2 type. The insensitivity of this lipolytic activity towards calcium ions and the presence of the already described consensus sequence of lipases, Gly-Xaa-Ser-Xaa-Gly, in the primary sequence of the B-chain of RCA60 support the idea that the lipolytic activity of RCA60 is more related to the lipase family than to the phospholipases A. We hypothesize that such activity contributes to the mechanism which underlies the expression of the cytotoxicity of RCA60.     

EXTRACELLULAR LIPOLYTIC ENZYME ACTIVITY OF A NEWLY ISOLATED DEBARYOMYCES HANSENII

A strain isolated from waste of a milk products plant and exhibited extracellular lipolytic activity was identified as Debaryomyces hansenii by 5.8S rRNA and 28S rRNA gene sequence analyses. Lipolytic activity was assayed spectrophotometrically by using p-nitrophenylpalmitate. Higher specific lipolytic activities were obtained in the presence of tristearin (0.68 U/mg prot), oleic acid (0.56 U/mg prot), and soybean oil (0.36 U/mg prot) than other triglycerides, fatty acids, and vegetable oils considered as carbon sources. Cheese whey appeared to be a good alternative to lipidic substances for lipolytic activity. Among various organic and inorganic nitrogen sources, soy flour was found to attain the lipolytic activity similar to that provided by universal yeast medium components. This work is the first report on the discussion of lipolytic activity enhancement by D. hansenii through modulating the cultivation medium. It also proposes low cost medium nutrients that could be of industrial value and could serve as basal nutrients for further optimization studies on the lipase production by D. hansenii.     

Effect of sodium taurodeoxycholate, CaCl2 and albumin on the action of pancreatic lipase on droplets of trioleoylglycerol and the release of lipolytic products into aqueous media

1. Effects of various substances on the activity of pancreatic lipase and on the release of lipolytic products into aqueous media were studied with droplets of trioleoylglycerol suspended from a membrane filter at the top of a flow-through chamber. The droplets were perifused for 7 min with a commercial preparation of pancreatic lipase in 0.15 M NaCl solution at pH 6.5 and then perifused for 60 min with lipase-free media, either 0.15 M NaCl at pH 6.5 or basal medium at pH 7.4 (70 mM sodium barbital) containing different additives. 2. About 6% of the trioleoylglycerol in droplets was hydrolyzed during the perifusion with lipase. Another 15% was hydrolyzed in 30 min, but none thereafter, when the droplets were perifused with 0.15 M NaCl alone. The rate of hydrolysis was doubled and prolonged when droplets were perifused with basal medium at pH 7.4. Lipolytic products formed at pH 7.4 were 62% oleic acid, 20% monooleoylglycerol and 18% dioleoylglycerol, yet only 4% of the lipolytic products were released into the perifusate. 3. Sodium taurodeoxycholate (TDC) (17 mM ) added to basal medium increased 18 x the amount of lipolytic products released into the perifusate but increased lipolysis only 13%. The molar ratio of oleic acid to monooleoylglycerol in the perifusate was 5.7 during the first 30 min and 4.0 during the last 30 min. 4. Ca2+ (3.3 mM) added to basal medium increased lipolysis 87% but did not affect the amount (4%) of lipolytic products released into the perifusing medium. 5. TDC and Ca2+ added to basal medium produced the largest increase in lipolysis, with 59% of trioleoylglycerol hydrolyzed in 15 min and 91% in 60 min. The amount of lipolytic products released into the perifusing medium, however, was not increased above that released into medium containing TDC alone. 6. Serum albumin (0.6 mM) and Ca2+ added to basal medium increased 14 x the amount of lipolytic products released into the perifusate without affecting the basal lipolytic rate. Albumin, however, suppressed by 40% the stimulatory effect of Ca2+ on pancreatic lipase activity.     

Autolytic phenotype of Lactococcus lactis strains isolated from traditional Tunisian dairy products

AIMS: To evaluate the autolytic properties of Lactococcus lactis strains isolated from artisan Tunisian dairy products, their peptidoglycan hydrolase content and their activity spectrum. METHODS AND RESULTS: The autolytic phenotype of Lactococcus strains was evaluated under starvation conditions in potassium phosphate buffer. The results obtained highlighted a high degree of diversity among the strains analysed, allowing the identification of high and low autolytic Lactococcus lactis strains. Peptidoglycan hydrolase content was evaluated by renaturing SDS-PAGE using cells of Micrococcus lysodeikticus as a target for the enzymatic activity. A major activity band migrating at about 45 kDa was observed. The lytic activity, evaluated in the presence of different chemicals, was retained in 8% NaCl, 15 mmol l(-1) CaCl2, and in a pH range between 5 and 9.5. The substrate specificity of peptidoglycan hydrolase from Lactococcus strains was evaluated in renaturing SDS-PAGE incorporating cells of different bacterial species. The major autolysin of Lactococcus lactis was active against cells of Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Listeria monocytogenes. CONCLUSIONS: Autolytic activity is widely distributed in Lactococcus lactis and the rate of autolysis is strain-dependent. The major peptidoglycan hydrolase showed a wide spectrum of activity against several lactic acid bacteria and bacterial species involved in food-related infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The autolytic phenotype of Lactococcus lactis strains isolated from Tunisian artisan dairy products has been determined, and the data obtained should allow the selection of strains of technological interest in the cheese-ripening process.     

Identification of novel lipolytic genes and gene families by screening of metagenomic libraries derived from soil samples of the German Biodiversity Exploratories

Microbial metagenomes derived from soils are rich sources for the discovery of novel genes and biocatalysts. Fourteen environmental plasmid and seven fosmid libraries obtained from 10 German forest soils (A horizons) and six grassland soils (A and B horizons) were screened for genes conferring lipolytic activity. The libraries comprised approximately 29.3 Gb of cloned soil DNA. Partial activity-based screening of the constructed libraries resulted in the identification of 37 unique lipolytic clones. The amino acid sequences of the 37 corresponding lipolytic gene products shared 29-90% identity to other lipolytic enzymes, which were mainly uncharacterized or derived from uncultured microorganisms. Multiple sequence alignments and phylogenetic tree analysis revealed that 35 of the predicted proteins were new members of known families of lipolytic enzymes. The remaining two gene products represent two putatively new families. In addition, sequence analysis indicated that two genes encode true lipases, whereas the other genes encode esterases. The determination of substrate specificity and chain-length selectivity using different triacylglycerides and p-nitrophenyl esters of fatty acids as substrates supported the classification of the esterases.     

A bacterial population study of commercialized wastewater inoculants

AIMS: To assess the bacterial diversity and safety of wastewater inoculants, which are commercially available products used to improve the aerobic digestion processes of the domestic waste compost in the septic tank. METHODS AND RESULTS: Eighteen wastewater inoculants were analysed on nonselective and selective media and the cultivable bacteria were identified. In all wastewater inoculants, the number of CFUs were between 10(4) and 10(7) g(-1) powder on nonselective media and Bacillus was the predominant cultivable genus. Culture-independent molecular methods such as sequencing of 16S rRNA clone libraries and denaturating gradient gel electrophoresis demonstrated the high prevalence of interfering chloroplast 16S rRNA from plant material and the presence of Bacillus spp. Only after selective enrichments and cultivation, the presence of one pathogenic strain (Klebsiella pneumoniae subsp. pneumoniae) and one opportunistic strain of (Enterobacter cloacae) bacteria were detected in six different products. CONCLUSION: The predominant cultivable species of the wastewater inoculants were Bacillus spp. and after enrichment six products were found to contain opportunistic or pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of opportunistic pathogenic strains in the inoculants might represent a risk for immunocompromised, the elderly or children. A clear labelling should therefore be displayed on the product.     

Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion

The technique of freeze fracture was used to visualize triglyceride (TG) hydrolysis and the production of lipolytic products (LPs) in vitro and in vivo in the presence of bile salts (BS). Three systems were investigated: pure lipolytic products (oleic acid and monoolein) in the presence of a pure bile salt (taurodeoxycholate (TDC)), lipolytic products produced from TG by pancreatic lipase in the presence of a variety of bile salts, and lipolytic products produced in the intestine of the killifish, Fundulus heteroclitus, after fat feeding. In vitro, lamellae (4-5 nm thick with 0-8-nm water spacings) appeared on the surface of TG droplets in all preparations with LP/BS molar ratios of 1.5 or greater and spherical vesicles (diameter range, 20-130 nm) were produced from these lamellae. With model killifish bile (taurocholate-cholate 1:1) at LP/BS ratios between 1.5 and 4, homogeneous vesicles or particles (mean diameter, 23.8 nm) were produced by lipase at pH 6.9. In vivo, lamellar product phases also occurred after fat feeding. The smallest visible LP/BS structures by freeze fracture electron microscopy were approximately 20 nm globular particles. Large disc-shaped micelles either were not present or were below the resolution limit of the replica (approximately 10 nm). The dominant aggregated lipolytic product phase was composed of multiple layers of rough-textured lamellae. No evidence of cubic structure was seen. These results show that lamellar and vesicular lipolytic product phases can be intermediates in intestinal fat digestion. However, no evidence for the direct endocytotic absorption of these product phases by the intestinal microvillus membrane was found.     

The entomopathogen Metarhizium anisopliae can modulate the secretion of lipolytic enzymes in response to different substrates including components of arthropod cuticle

The filamentous fungus Metarhizium anisopliae is a well-characterized, arthropod pathogen used in the biological control of arthropod pests. Studies on the regulation of enzymes related to host infection such as proteases and chitinases have been reported but little is known about regulation of lipolytic enzymes in this fungus. Here we present the effects of different carbon sources such as components of the arthropod cuticle on the secretion of lipolytic enzymes by M. anisopliae. Differences in the induction of lipolytic activity were observed between the several carbon sources tested. Higher activities of lipase or lipase/esterase were found in culture media containing the arthropod integument components chitin and cholesteryl stearate. Several bands of lipolytic activity were also detected in zymograms, thus suggesting an important set of lipolytic enzymes secreted by the fungus. These results show that the fungus can modulate the secretion of lipolytic activity in response to host integument components, thus reinforcing the potential role of these enzymes during M. anisopliae infection.     

The survival of silage inoculant lactic acid bacteria in rumen fluid

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.     

Exploitation of genetically modified inoculants for industrial ecology applications

The major growth seen in the biotechnology industry in recent decades has largely been driven by the exploitation of genetic engineering techniques. The initial benefits have been predominantly in the biomedical area, with products such as vaccines and hormones that have received broad public approval. In the environmental biotechnology and industrial ecology sectors, biotechnology has the potential to make significant advances through the use of genetically modified (GM) microbial inoculants that can reduce agri-chemical usage or remediate polluted environments. Although many GM inoculants have been developed and tested under laboratory conditions, commercial exploitation has lagged behind. Here, we review scientific and regulatory requirements that must be satisfied as part of that exploitation process. Particular attention is paid to new European Union (EU) regulations (Directives) that govern the testing and release of genetically modified organisms and microbial plant protection inoculants in the EU. With regard to the release of GM inoculants, the impact of the inoculant and the fate of modified genes are important concerns. Long term monitoring of release sites is necessary to address these issues. Data are reported from the monitoring of a site 6 years after release of GM Sinorhizobium meliloti strains. It was found that despite the absence of a host plant, the GM strains persisted in the soil for at least 6 years. Horizontal transfer and microevolution of a GM plasmid between S. meliloti strains was also observed. These data illustrate the importance of assessing the long-term persistence of GM inoculants. This revised version was published online in August 2006 with corrections to the Cover Date.     

Lipolytic fermentations of stickwater by Geotrichum candidum and Candida lipolytica.

Stickwater, a by-product of the fish meal and oil industry, is an aqueous suspension of fish proteins, lipids, and other materials, and also contains soluble nonprotein nitrogen but not carbohydrate. It is usually partially evaporated by heat to a marketable form called "fish solubles," which is sold with an acid preservative as an animal feed supplement. However, fish solubles are only used to a limited extent in feeds, because the lipids of solubles (averages 11%) are relatively prone to oxidative rancidity development. An investigation was undertaken to digest and/or stabilize lipids in stickwater by lipolytic fermentations and, at the same time, to attempt to increase the protein content as single cell protein. Strains of the yeast Candida lipolytica and the yeast like mold Geotrichum candidum were employed for these investigations. Stickwater fermentations were performed in a laboratory bench top fermentor. Respirometric studies of lipid metabolic activity and microbial observations were periodically performed during these fermentations. Rapid microbial growth and metabolic activity were observed in well aerated cultures. Fermented products were evaluated for chemical composition. Lipid residues were characterized by thin-layer chromatographic procedures. There was evidence of abundant microbial growth, increased lipolytic activity, and decreased lipid content. However, evidence was lacking to show that the protein content of stickwater was actually increased.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Lipase Production and Activity as a Function of Incubation Time, pH and Temperature of Four Lipolytic Micro-organisms

S ummary . The lipolytic activity in supernatant fractions of cultures of Saccharomycopsis lipolytica, Micrococcus caseolyticus, Bacillus licheniformis , and a Staphylococcus sp. was studied. Nutrient broth with and without emulsified olive oil was used as substrate. Optimal pH values and temperatures for the lipase produced by the 4 different micro-organisms were determined. The lipolytic activity generally reached a maximum after incubation for 2–6 days. The subsequent decrease in the lipolytic activity was associated with a high proteolytic activity only for Micrococcus caseolyticus . The lipolytic activity was decreased by the presence of olive oil in the medium. Determination of the lipolytic activity after a certain time of incubation, the maximal lipolytic activity and a time-integrated lipolytic activity are compared as estimators for the potential hydrolytic capacity of micro-organisms.     

Synergistic effect of vespa amino acid mixture on lipolysis in rat adipocytes

The effect of Vespa amino acid mixture (VAAM) on the release of lipolytic products was examined in isolated rat adipocytes. Concentrations of 112.5 to 225 ppm of VAAM showed significantly greater release of non-esterified fatty acids (NEFA) and glycerol than the same concentrations of casein amino acid mixture (CAAM). The integrated relative release of NEFA and glycerol was lower in response to individual administration of amino acids comprising VAAM than to VAAM itself. Further, amino acids mixtures deficient in a single amino acid comprising VAAM showed significantly lower release of lipolytic products than VAAM. These data suggest that the synergistic effect of VAAM on the release of lipolytic products is a function of concurrent exposure to the unique composition of amino acids found in VAAM as compared to the effect of exposure to the same individual un-mixed amino acids or to a mixture lacking one of the amino acids comprising VAAM.     

Microbial inoculants with higher capacity to colonize soils improved wheat drought tolerance

Microbial inoculants have gained increasing attention worldwide as an eco-friendly solution for improving agriculture productivity. Several studies have demonstrated their potential benefits, such as enhanced resistance to drought, salinity, and pathogens. However, the beneficial impacts of inoculants remain inconsistent. This variability is attributed to limited knowledge of the mechanisms by which microbial inoculants affect crop growth and a lack of ecological characteristics of these inoculants that limit our ability to predict their beneficial effects. The first important step is believed to be the evaluation of the inoculant's ability to colonize new habitats (soils and plant roots), which could provide crops with beneficial functions and improve the consistency and efficiency of the inoculants. In this study, we aimed to investigate the impact of three microbial inoculants (two bacterial: P1 and P2, and one fungal: P3) on the growth and stress responses of three wheat varieties in two different soil types under drought conditions. Furthermore, we investigated the impact of microbial inoculants on soil microbial communities. Plant biomass and traits were measured, and high-throughput sequencing was used to characterize bulk and rhizosphere soil microbiomes after exposure to drought stress. Under drought conditions, plant shoot weight significantly increased (11.37%) under P1 treatments compared to uninoculated controls. In addition, total nitrogen enzyme activity increased significantly under P1 in sandy soil but not in clay soil. Importantly, network analyses revealed that P1, consisting of Bacillus paralicheniformis and Bacillus subtilis, emerged as the keystone taxa in sandy soil. Conversely, P2 and P3 failed to establish as keystone taxa, which may explain their insignificant impact on wheat performance under drought conditions. In conclusion, our study emphasizes the importance of effective colonization by microbial inoculants in promoting crop growth under drought conditions. Our findings support the development of microbial inoculants that robustly colonize plant roots for improved agricultural productivity.     

Adrenergic lipolysis in human fat cells: properties and physiological role of alpha-adrenergic receptors (author's transl)

Lipolytic activity of human isolated fat cells from different fat deposits was studied. The purpose of the present investigations was to determine the epinephrine responsiveness, with regard to alpha- and beta-adrenergic receptor site activity, of omental and subcutaneous adipocytes (abdominal or from the lateral part of the thigh). Adipocytes were obtained from normal subjects or from obese subjects on iso- or hypocaloric diets. The lipolytic effect of epinephrine varied according to the fat deposits, while the beta-lipolytic effect of isoproterenol was more stable (Fig. 1). We explored the possible involvement of adrenergic alpha-receptors, in order to explain these results. The potentiating action of phentolamine on epinephrine-induced lipolysis, and the antilipolytic effect of alpha-agonists on basal or theophylline--induced lipolysis, were found to be a good indication of alpha-adrenergic activity. The alpha-adrenergic antilipolytic effect was most prominent in adipose tissue from the lateral part of the thigh, and less noticeable in omental adipocytes. In conclusion, the inability of epinephrine to induce lipolysis, and the epinephrine-induced inhibition of lipolysis observed when the basal rate of FFA release was spontaneously increased in subcutaneous fat-cells of the thigh, could be explained by an increased alpha adrenergic responsiveness (Fig. 2). Moreover, various alpha-adrenergic agonists (phenylephrine, noradrenaline and adrenaline) showed a clear inhibiting effect on theophylline-stimulated adipocytes from the thigh. The pharmacological study of the antilipolytic effect of epinephrine on theophylline-induced lipolysis showed that the inhibition was linked to a specific stimulation of the alpha-receptors of the subcutaneous adipocytes (Fig. 4). From the different sets of experiments, it is shown that the modifications in the lipolytic effect of epinephrine on adipocytes of different areas could be explained by the occurrence of a variable alpha-adrenergic effect initiated by catecholamine. Furthermore, theophylline stimulation of lipolysis provides an accurate system to investigate the alpha-inhibiting effect of catecholamines. Our study was completed by the investigation of the lipolytic activity of subcutaneous fat cells from obese subjects submitted to a hypocaloric diet (800-1 000 Cal/day). An increased alpha-inhibitory effect of epinephrine was shown on the increased basal lipolytic activity observed in the fat cells of obese subjects on a hypocaloric diet (Fig. 5); a similar effect was observed when these adipocytes were stimulated by theophylline. To conclude, these investigations allow the alpha-adrenergic effect to be considered as a regulator mechanism of the in vitro lipolytic activity in human adipose tissue, since the antilipolytic effect is operative whenever the basal rate of lipolysis is increased (spontaneously, after caloric restriction, or with a lipolytic agent such as theophylline).     

Effect of 2-deoxy-D-glucose on lipolytic processes in blood and adipose tissue of rat

The effect of 2-deoxy-D-glucose on lipolytic processes in the blood and adipose tissue was studied. Rats treated with this antimetabolite showed a significant increase in serum glucose, FFA and glycerol level, as well as in the lipid mobilizing activity. On the other hand, the lipolytic activity of rat serum decreased when compared to control group. From these results it may be concluded that during hypothermia induced by administration of 2-deoxy-D-glucose intracellular, but not intravascular, lipolysis is enhanced.