Biomedical subjects

Leo Eberl

Publications and source records attributed to Leo Eberl.

At least 37 records · Page 2Linked to original sources

PubMed2005

Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis patients. P. aeruginosa colonizes the lungs by forming biofilm microcolonies throughout the lung. Quorum sensing (QS) renders the biofilm bacteria highly tolerant to otherwise lethal doses of antibiotics, and protects against the bactericidal activity of polymorphonuclear leukocytes (PMNs). It has been previously demonstrated that QS is inhibited by garlic extract. In this study, the synergistic effects of garlic and tobramycin, and PMNs activities have been evaluated. P. aeruginosa was grown in vitro in continuous-culture once-through flow chambers with and without garlic extract. The garlic-treated biofilms were susceptible to both tobramycin and PMN grazing. Furthermore, the PMNs showed an increase in respiratory burst activation, when incubated with the garlic-treated biofilm. Garlic extract was administered as treatment for a mouse pulmonary infection model. Mice were treated with garlic extract or placebo for 7 days, with the initial 2 days being prophylactic before P. aeruginosa was instilled in the left lung of the mice. Bacteriology, mortality, histopathology and cytokine production were used as indicators. The garlic treatment initially provoked a higher degree of inflammation, and significantly improved clearing of the infecting bacteria. The results indicate that a QS-inhibitory extract of garlic renders P. aeruginosa sensitive to tobramycin, respiratory burst and phagocytosis by PMNs, as well as leading to an improved outcome of pulmonary infections.

Animals↗

PubMed2005

Quorum sensing: the power of cooperation in the world of Pseudomonas.

Work over the past few years has provided evidence that quorum sensing is a generic regulatory mechanism that allows bacteria to launch a unified, coordinated response in a population density-dependent manner to accomplish tasks which would be difficult, if not impossible, to achieve for a single bacterial cell. Quorum sensing systems are widespread among pseudomonads and the one of the human opportunistic pathogen Pseudomonas aeruginosa belongs to the most extensively studied cell-to-cell communication systems. In this organism, quorum sensing is highly complex and is made up of two interlinked N-acyl homoserine lactone (AHL)-dependent regulatory circuits, which are further modulated by a non-AHL-related signal molecule and numerous regulators acting both at the transcriptional and post-transcriptional level. This genetic complexity may be one of the key elements responsible for the tremendous environmental versatility of P. aeruginosa. Work of the past few years showed that quorum sensing is essential for the expression of a battery of virulence factors as well as for biofilm formation in P. aeruginosa and thus represents an attractive target for the design of novel drugs for the treatment of P. aeruginosa infections. Furthermore, the cell-to-cell communication ability was also demonstrated in a number of additional pseudomonads.

4-Butyrolactone↗

PubMed2005

The rhizosphere as a reservoir for opportunistic human pathogenic bacteria.

During the last years, the number of human infections caused by opportunistic pathogens has increased dramatically. One natural reservoir of opportunistic pathogens is the rhizosphere, the zone around roots that is influenced by the plant. Due to a high content of nutrients, this habitat is a 'microbial hot-spot', where bacterial abundances including those with strong antagonistic traits are enhanced. Various bacterial genera, including Burkholderia, Enterobacter, Herbaspirillum, Ochrobactrum, Pseudomonas, Ralstonia, Staphylococcus and Stenotrophomonas, contain root-associated strains that can encounter bivalent interactions with both plant and human hosts. Mechanisms responsible for colonization of the rhizosphere and antagonistic activity against plant pathogens are similar to those responsible for colonization of human organs and tissues, and pathogenicity. Multiple resistances against antibiotics are not only found with clinical strains but also with strains isolated from the rhizosphere. High competition, the occurrence of diverse antibiotics in the rhizosphere, and enhanced horizontal gene transfer rates in this microenvironment appear to contribute to the high levels of natural resistances. While opportunistic bacteria from the rhizosphere have some properties in common, each of these emerging pathogens has its own features, which are discussed in detail for Burkholderia, Ochrobactrum and Stenotrophomonas.

Bacteria↗

PubMed2005

Screening for quorum-sensing inhibitors (QSI) by use of a novel genetic system, the QSI selector.

With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Furthermore, it has become apparent that the bacterial life style also contributes significantly to this problem. Bacteria living in the biofilm mode of growth tolerate conventional antimicrobial treatments. The discovery that many bacteria use quorum-sensing (QS) systems to coordinate virulence and biofilm development has pointed out a new, promising target for antimicrobial drugs. We constructed a collection of screening systems, QS inhibitor (QSI) selectors, which enabled us to identify a number of novel QSIs among natural and synthetic compound libraries. The two most active were garlic extract and 4-nitro-pyridine-N-oxide (4-NPO). GeneChip-based transcriptome analysis revealed that garlic extract and 4-NPO had specificity for QS-controlled virulence genes in Pseudomonas aeruginosa. These two QSIs also significantly reduced P. aeruginosa biofilm tolerance to tobramycin treatment as well as virulence in a Caenorhabditis elegans pathogenesis model.

4-Butyrolactone↗

PubMed2005

Biofilm formation, extracellular polysaccharide production, and cell-to-cell signaling in various Enterobacter sakazakii strains: aspects promoting environmental persistence.

Enterobacter sakazakii is considered an opportunistic pathogen and has been implicated in food-associated cases of meningitis or enteritis, especially in neonates and infants. The organism has been detected in various types of food and in food production units, but so far only powdered infant formula has been linked to outbreaks of disease. Survival and persistence in such environments requires the ability to adapt to high osmotic potentials and/or dry conditions. Fifty-six E. sakazakii strains were evaluated for several features important for persistence and survival: (i) biofilm formation and the putative production of cellulose as one of the components of the extracellular matrix, (ii) adherence to hydrophilic and hydrophobic surfaces, (iii) the production of extracellular polysaccharides, and (iv) the ability of E. sakazakii to produce cell-to-cell signaling molecules. Pellicle and flock formation was observed in 21 of the strains grown in Luria-Bertani broth and in 44 of the strains grown in brain heart infusion broth. Calcofluor-stained fibrils, observed microscopically in every (fragile or rigid) pellicle, suggested the presence of cellulose as an extracellular compound in this type of biofilm. Twelve isolates did not form any pellicle or flocks under either condition. Twenty-three of the isolates exhibited the potential to adhere to glass surfaces in shaken cultures, and 33 strains showed biofilm formation at the air-solid interface of polyvinyl chloride microtiter wells. Sixteen isolates adhered to both surfaces. Twenty-four of the isolates tested produced a milky, viscous mass, considered as extracellular polysaccharide. High-performance liquid chromatography analysis of the polysaccharide revealed the presence of glucose, galactose, fucose, and glucuronic acid. Thin-layer chromatography analyses performed on ethyl acetate extracts of cell-free supernatants of the 56 strains indicated the presence of two different types of acylated homoserine lactones (3-oxo-C6-HSL and 3-oxo-C8-HSL). These findings illustrate the ability of E. sakazakii to produce cell-to-cell signaling molecules.

Bacterial Adhesion↗

PubMed2004

Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates.

Two strains, Pseudomonas aeruginosa TB10839 and TB121838, which belong to the TB clonal lineage, have been isolated from sputa of cystic fibrosis patients. Despite the fact that the strains are closely related, their pathogenic potential differs dramatically: while strain TB10839 is capable of proliferating in polymorphonuclear granulocytes, strain TB121838 is not. Comparative two-dimensional polyacrylamide gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry was employed to map the extracellular, intracellular, and surface sub-proteomes of TB10839 and TB121838 and to identify differentially expressed proteins. About 4% of all detected protein spots were differentially expressed between both strains including absent or present spots and spots with a more than 2-fold changed intensity. This percentage reflects a relatively high degree of intraclonal variability. Many of the protein spots in TB10839 that were missing or expressed at lower levels in TB121838 were identified as quorum-sensing regulated virulence factors. It might be speculated that the increased expression of these proteins contributes to pathogenic competence of TB10839.

Clone Cells↗

PubMed2004

Pseudomonas aeruginosa and Burkholderia cepacia in cystic fibrosis: genome evolution, interactions and adaptation.

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and are the major pathogens in cystic fibrosis (CF). The two bacteria not only inhabit the same environmental niches but can also form mixed biofilms in the lungs of CF patients. Hence, it appears very likely that the two organisms are capable of interacting with each other. Work of the past few years has shown that both bacteria utilize quorum-sensing systems, which rely on N-acyl-homoserine lactone signal molecules, to control the expression of virulence factors and biofilm development. Most importantly, evidence has been presented that these signal molecules also serve as a universal language for communication between the two organisms. Moreover, analyses of the diversity in P. aeruginosa revealed the presence of genome islands that contain genes that are highly homologous to genes identified in strains of Burkholderia sp. This finding suggests that there is a frequent exchange of genetic material between the two organisms.

4-Butyrolactone↗

PubMed2004

An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum).

The fish pathogen Vibrio anguillarum produces quorum sensing signal molecules, N-acyl homoserine lactones (AHLs), which in several Gram-negative human and plant pathogenic bacteria regulate virulence factors. Expression of these factors can be blocked using specific quorum-sensing inhibitors (QSIs). The purpose of this study was to investigate the effect of a QSI, furanone C-30, on mortality of rainbow trout during challenge with V. anguillarum. Addition of 0.01 or 0.1 microM furanone C-30 to rainbow trout infected by cohabitation caused a significant reduction in accumulated mortality from 80-100% in challenge controls to 4-40% in treated groups. Furanone C-30 had no effect in an immersion challenge system, probably due to a very high water exchange and a rapid dilution of furanone C-30. Growth and survival of V. anguillarum were not affected by the concentrations of furanone C-30 used in the challenge experiments, thus avoiding selection for resistance. To elucidate the mechanism of disease control by furanone C-30, we determined its effect on the bacterial proteome, motility, and respiration. No effects were seen of furanone C-30 in any of these experiments. Although no cytotoxic effect on HeLa cells were observed, exposure to 1 microM (or higher) concentrations of furanone C-30 had detrimental effects on the rainbow trout. Our results indicate that QSIs can be used in non-antibiotic based control of fish diseases. However, they also underline the need for development of novel, less toxic QSI compounds and the need for understanding the exact mechanism(s) of action.

4-Butyrolactone↗

PubMed2004

Expression of Pseudomonas aeruginosa exoS is controlled by quorum sensing and RpoS.

In Pseudomonas aeruginosa, virulence determinants and biofilm formation are coordinated via a hierarchical quorum sensing cascade, which involves the transcriptional regulators LasR and RhlR and their cognate homoserine lactone activators C12-HSL [N-(3-oxododecanoyl)-L-homoserine lactone] and c4-hsl (n-butanoyl-L-homoserine lactone), which are produced by LasI and RhlI, respectively. The exoenzyme S regulon of P. aeruginosa, comprises genes for a type III secretion system and for four anti-host effector proteins (ExoS, T, U and Y), which are translocated into host cells. It is a reasonable assumption that this ExoS regulon should be downregulated in the biofilm growth state and thus should also be under the regulatory control of the Las/Rhl system. Therefore, an exoS'-gfp reporter construct was used, and the influence of the Las and Rhl quorum sensing systems and the effect of the stationary-phase sigma factor RpoS on regulation of the exoS gene was examined. Evidence is provided for downregulation of exoS during biofilm formation of P. aeruginosa PAO1. The rhlI mutant PDO100 and rhlR mutant PDO111, but not the lasI mutant PDO-JP1, showed approximately twofold upregulation of the exoS'-gfp reporter in comparison to PAO1. Upregulation of exoS'-gfp in the PDO100 mutant could be repressed to normal level by adding C4-HSL autoinducer, indicating a negative regulatory effect of RhlR/C4-HSL on exoS expression. As RhlR/C4-HSL is also involved in regulation of RpoS, the P. aeruginosa rpoS mutant SS24 was examined and the exoS'-gfp reporter was found to be fivefold upregulated in comparison to PAO1. For the first time evidence is reported for a regulatory cascade linking RhlR/RhlI and RpoS with the expression of the anti-host effector ExoS, part of the exoenzyme S regulon. Moreover, these data suggest that the exoenzyme S regulon may be downregulated in P. aeruginosa biofilms.

4-Butyrolactone↗

PubMed2004

Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa.

Pathogenesis of Pseudomonas aeruginosa is controlled to a major extent by the two quorum-sensing systems las and rhl. The previously uncharacterized gene PA2591 was identified as a major virulence regulator, vqsR, in the quorum-sensing hierarchy. vqsR is a member of the LuxR family and possesses a las box in its upstream region. Transposon inactivation of vqsR abrogated the production of N-acylhomoserine lactones and the secretion of exoproducts and diminished bacterial virulence for Caenorhabditis elegans. Cytotoxicity towards macrophages was not affected. vqsR mRNA was expressed more strongly in the presence of human serum and oxidative stress than under standard growth conditions. High-density oligonucleotide microarrays were used to compare the global expression profile of a wild-type strain and a vqsR mutant. One-hundred-and-fifty-one and 113 genes were significantly differentially expressed in the presence of H(2)O(2) and human serum, respectively. The disruption of vqsR repressed the expression of genes that are known to be promoted by quorum sensing and activated the expression of genes that are known to be repressed by quorum sensing. Moreover, the vqsR mutant harboured less mRNA transcript for the production of siderophores and membrane-bound elements of antibiotic resistance. The protein encoded by PA2591 regulates several traits of pathogenicity; hence, the name vqsR ('virulence and quorum-sensing regulator') was assigned to PA2591.

4-Butyrolactone↗

PubMed2004

Impact of violacein-producing bacteria on survival and feeding of bacterivorous nanoflagellates.

We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.

Animals↗

PubMed2004

Identification of a novel virulence factor in Burkholderia cenocepacia H111 required for efficient slow killing of Caenorhabditis elegans.

Burkholderia cenocepacia H111, which was isolated from a cystic fibrosis patient, employs a quorum-sensing (QS) system, encoded by cep, to control the expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are expressed only when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. While the wild-type strain effectively kills the nematode Caenorhabditis elegans, the pathogenicity of mutants with defective quorum sensing is attenuated. To date, very little is known about the cep-regulated virulence factors required for nematode killing. Here we report the identification of a cep-regulated gene, whose predicted amino acid sequence is highly similar to the QS-regulated protein AidA of the plant pathogen Ralstonia solanacearum. By use of polyclonal antibodies directed against AidA, it is demonstrated that the protein is expressed in the late-exponential phase and accumulates during growth arrest. We show that B. cenocepacia H111 AidA is essential for slow killing of C. elegans but has little effect on fast killing, suggesting that the protein plays a role in the accumulation of the strain in the nematode gut. Thus, AidA appears to be required for establishing an infection-like process rather than acting as a toxin. Furthermore, evidence is provided that AidA is produced not only by B. cenocepacia but also by many other strains of the Burkholderia cepacia complex.

Amino Acid Sequence↗

PubMed2003

Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors.

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

Animals↗

PubMed2003

Analysis of N-acyl-L-homoserine lactones produced by Burkholderia cepacia with partial filling micellar electrokinetic chromatography--electrospray ionization-ion trap mass spectrometry.

A method for the analysis of N-acyl-L-homoserine lactones (AHLs) with micellar electrokinetic chromatography coupled to electrospray ionization-ion trap mass spectrometry, combining the flexibility of capillary electrophoresis with the unmatched structural information provided by mass spectrometry is presented. Different surfactants were evaluated, with sodium dodecyl sulfate (SDS) yielding the best results considering sensitivity and flexibility. We examined the interaction of AHLs with the SDS micelles at different analysis conditions and applied the optimized method to the analysis of a real bacterial sample. Two AHLs from Burkholderia cepacia colonizing the rhizosphere of traditional Indian rice cultivars could be unambiguously determined in an ethyl acetate extract with high resolution flexibility.

4-Butyrolactone↗

PubMed2003

Analysis of the quorum-sensing regulon of the opportunistic pathogen Burkholderia cepacia H111 by proteomics.

Burkholderia cepacia H111, an important pathogen for persons suffering from cystic fibrosis, employs a quorum-sensing (QS) system, cep, to control expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are only expressed when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. In this study, we compared the protein pattern of the intracellular, extracellular, and surface protein fractions of an AHL-deficient cepI mutant with the one of the parent strain H111 by means of two-dimensional gel electrophoresis (2-DE). Our analysis showed that 55 proteins out of 985 detected spots were differentially expressed; these are expected to represent QS-controlled gene products. Addition of the respective signal molecules to the growth medium of the cep mutant fully restored the wild-type protein expression profile. In total about 5% of the B. cepacia proteome was downregulated and 1% upregulated in the cepI mutant, indicating that quorum sensing represents a global regulatory system. Nineteen proteins were identified with high confidence by N-terminal sequence analysis.

Burkholderia cepacia↗

PubMed2003

Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics.

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N-acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem.

4-Butyrolactone↗

PubMed2003

Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system.

Burkholderia cepacia H111, which was isolated from a cystic fibrosis patient, effectively kills the nematode Caenorhabditis elegans. Depending on the medium used for growth of the bacterium two different killing modes were observed. On high-osmolarity medium the nematodes became paralysed and died within 24 h. Using filter assays we provide evidence that this killing mode involves the production of an extracellular toxin. On nematode growth medium killing occurs over the course of 2-3 days and involves the accumulation of bacteria in the intestinal lumen of C. elegans. We demonstrate that the cep quorum-sensing system of H111 is required for efficient killing of C. elegans under both killing conditions. Using the C. elegans phm-2 mutant that has a non-functional grinder evidence is provided that the cep system is required to enter the intestinal lumen but is dispensable for the colonization of the gut. Furthermore, we demonstrate that the type II secretion machinery is not essential for nematode killing.

Animals↗

PubMed2003

Quorum-sensing-directed protein expression in Serratia proteamaculans B5a.

N-Acyl-L-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities, thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore, chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.

4-Butyrolactone↗