Increase In Antimicrobial Activity Research Articles

Isolation and assessment of potential probiotic microorganisms from modified indigenous foods preparation

Objective: The objective of present study is to isolate potential probiotic microorganisms from modified indigenous foods and assessment of its antimicrobial activity against some selected pathogenic strains including E. coli, E. faecilis, K. pneumonia, S. dysentrae, S.epidermidis, S. saprophyticus, S. pyogenes and S. aureus. These isolated microorganisms were further characterized using microscopic and biochemical methods. Method: Six different set of probiotic indigenous foods were prepared by Bulgur Wheat and Corn grits. Each probiotic isolate was grown in Man Rogasa Sharp (MRS) broth for 24 h at 37°C in order to prepare Cell Free Supernatant (CFS) The CFS obtained from isolated strain was tested for antimicrobial activity by well diffusion method. Results: The study finally led us to isolate creamy white, circular, convex elevated and smooth rough textured colonies by using MRS agar containing bromocresol purple. The isolates were Gram positive, rod shaped, catalase negative and shows increase in antimicrobial activity (zone of inhibition) of isolates CFS against E.coli (22.3±1.2), E. faecalis (23±0.8), S. pyogenes (24.3±0.4), S. saprophyticus (18 ± 0.8), S. aureus (16.6±0.4), S.epidermidis (17±0), K. pneumonae (18±0.8), S. dysentrae (17±0.8), which is more than processed probiotic CFS. It was observed that isolates demonstrated good capacity to resist bile salts by showing growth under exposure to 0.3% bile salts and able to grow in pH 2 as well as pH 3. Conclusion: These results suggest that these isolates could successfully transit the human stomach and may be capable of reaching the intestinal environment and functioning effectively there.

Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus–Burkholderia symbiosis

The majority of insects possess symbiotic bacteria. Since symbiont titers can affect host phenotypes of biological importance, host insects are expected to evolve some mechanisms for regulating symbiont population. Here we report that, in the Riptortus–Burkholderia gut symbiosis, titers of the beneficial symbiont transiently decrease at the pre-molt stages in host development. This molting-associated suppression of the symbiont population is coincident with the increase of antimicrobial activity in the symbiotic midgut, which is observed in both symbiotic and aposymbiotic insects. Two genes, pyrrhocoricin-like antimicrobial peptide and c-type lysozyme, exhibit significantly increased expression in the symbiotic midgut at the pre-molt stages. These results suggest that the molting-associated up-regulation of antimicrobial activity in the symbiotic midgut represents a physiological mechanism of the host insect to regulate symbiosis, which is presumably for defending molting insects against injury and infection and/or for allocating symbiont-derived energy and resources to host molting.

Expedient Synthesis of SMAMPs via Click Chemistry.

A novel series of synthetic mimics of antimicrobial peptides (SMAMPs) containing triazole linkers were assembled using click chemistry. While only moderately active in buffer alone, an increase in antimicrobial activity against Staphylococcus aureus and Escherichia coli was observed when these SMAMPs were administered in the presence of mouse serum. One compound had minimum inhibitory concentrations (MICs) of 0.39 μg/mL and 6.25 μg/mL, respectively, and an HC50 of 693 μg/mL. These values compared favorably to peptide-based antimicrobials. A correlation between the net positive charge and SMAMP antimicrobial activity was observed. The triazole linker, an amide surrogate, was found to provide better antimicrobial activity against both S. aureus and E. coli when compared to other analogues.

Effects of hyperbaric oxygen treatment on antimicrobial function and apoptosis of differentiated HL-60 (neutrophil-like) cells

AimsNeutrophil apoptosis is important in the resolution of inflammation in chronic wounds. Hyperbaric oxygen (HBO) therapy, an intermittent inhalation of 100% oxygen at greater than atmospheric pressure, appears to be an effective treatment for chronic wounds. The aim was to use HL-60 cells differentiated using all-trans retinoic acid (ATRA) (neutrophil-like cells) to test the hypothesis that an HBO-induced increase in antimicrobial activity might lead to an increase in apoptosis, thereby contributing to neutrophil clearance from chronic wounds. Main methodsATRA differentiated HL-60 cells, an in vitro neutrophil model, were used to test the effects of normoxia, hypoxia (5% O2), hyperoxia (95% O2), hyperbaric normoxia (pressure) (8.8% O2 at 2.4 ATA) and HBO (97.9% O2 at 2.4 ATA) on antimicrobial function [NBT staining, superoxide and H2O2 production, and phagocytosis activity] and apoptosis (caspase 3/7 activity and morphological changes observed using SYBR Safe staining). Key findingsA single 90min HBO exposure caused an increase in the respiratory burst activity of neutrophil-like cells post exposure. Phagocytosis of Staphylococcus aureus was also increased. HBO pre-treatment had a pro-apoptotic effect, increasing caspase 3/7 activity and causing morphological changes associated with apoptosis. SignificanceThe potential detrimental effect of enhanced antimicrobial activity induced by HBO may be offset by enhanced apoptosis. Both hyperoxia and pressure alone seemed to contribute to the HBO-induced increases in antimicrobial activity and apoptosis, although there was no consistent pattern. These data contribute to explaining the effectiveness of HBO in the treatment of chronic wounds.

Phytochemical and Antimicrobial Evaluation of Aqueous and Organic Extracts of Calotropis procera Ait Leaf and Latex

The aqueous and ethanol extract of Calotropis procera leaf and latex were investigated. The leaves and latex from the plant were tested for antimicrobial activities. The bioactive constituents extracted from the leaf and latex were tested against pathogenic organisms ( Eschericia coli , Salmonella typhi , Bacillius subtilis , Candida albicans , Aspergillus niger ) using the Agar well diffusion method. The ethanolic latex extract showed significant activity against all the test organisms. The results revealed that ethanol is a more effective extractive solvent for antimicrobial activity of leaf and latex of C . procera . The ethanol extract of the latex gave the widest zone of inhibition (21 mm) against B . subtilis . All the extracts inhibit the growth of all the organisms except B . subtilis of which the aqueous extract has no effect. The Minimum Inhibitory Concentration (MIC) for the latex extract was between 3 and 7.5 mg/ml for bacteria, and 5.0 to 7.0 mg/ ml for fungi. For the leaf extract the MIC for bacteria was between 5.0 and 10.5 mg/ml and 11 and 15 mg/ml for fungi. The results also showed an increase in antimicrobial activity with increase in temperature. This study therefore revealed that C . procera latex extract demonstrated strong and better inhibitory activity on the test organisms than the leaf extract. These findings therefore provide an explanation for the traditional medicinal use of C . procera extracts.

Simple screening method to identify toxic/non-toxic ionic liquids: Agar diffusion test adaptation

A wide range of ionic liquids (ILs), containing a diverse set of cations, anions and alkyl chain lengths, was screened for their antimicrobial activity toward four microorganisms, Escherichia coli CCT-0355, Staphylococcus aureus ATCC-6533, Fusarium sp. LM03 and Candida albicans ATCC-76645. For that purpose an adaptation of the Agar Diffusion test was validated and successfully applied as a rapid screen method to identify toxic ILs, avoiding the use of more complex and expensive techniques. The effects of the cation alkyl chain length were studied, being observed both the “alkyl side chain” effect (increase in antimicrobial activity with the elongation of the alkyl chain) and “cut-off” effect (beyond a given chain length, the toxicity cannot be increased any further). Imidazolium-based ILs have in general, negative effects on the growth of these microorganisms dependent on the anion and alkyl chain length (growth inhibition halo from 1.98±0.04mm for [C2mim]Cl to 39.53±0.81mm for [C10mim]Cl). On the opposite, the phosphonium-based ILs do not seem to have negative effects for the longest alkyl chains (growth inhibition halos between 0.00±0.00 and 7.30±0.42mm). It was also observed that the alkyl chain, cation family, and anion moiety all have significant effects on the antimicrobial activity these effects being well correlated with the lipophilicity of the ILs tested. The results also show that the microorganisms responses to the diverse ILs tested are dependent on their morphologic differences.

Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33

With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC(50) = 16-64μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.

Synthesis, spectral characterization, DNA interaction studies, anthelmintic and antimicrobial activity of transition metal complexes with 3-(2- hydroxybenzylideneamino)-2-methylquinazolin-4(3H)-one and 1,10- phenanthroline

Mixed ligand complexes of cobalt(II) (1), copper(II) (2) and oxovanadium(IV) (3) with 3- (2-hydroxy benzylideneamino)-2-methylquinazolin-4(3H)-one and 1,10-phenanthroline have been synthesized and characterized by elemental analyses, IR, electronic, 1 H-NMR, mass spectra, molar conductance and thermal studies. The synthesized compounds were screened for their in vitro antimicrobial activity. The results show a significant increase in antimicrobial activity of the complexes compared to ligand. Antihelmintic activity of the compounds has been tested on earthworms and the enhanced activity was observed upon complexation. In addition, DNA binding and DNA cleavage studies for the newly prepared compounds were also studied. These studies indicate that the DNA binding and cleavage efficacy were increased in the complexes relative to the parental ligand.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Influence of gamma radiation on the antimicrobial activity of crude extracts of Anacardium occidentale L., Anacardiaceae, rich in tannins

Anacardium occidentale L. Anacardiaceae, known as cashew, commonly found in northeastern of Brazil, has high levels of secondary metabolites, particularly tannins, used as raw material for herbal medicines. An efficient alternative to decontaminate plant products is the total sterilization or reduction of the initial microbial count, the process of gamma irradiation with 60Co. The objective of this study was to analyze the antimicrobial activity of crude extracts of bark and leaves of A. occidentale, based on the quantification of total phenols and tannins, before and after exposure to gamma radiation from 60Co. The extracts were obtained in the laboratory by cold maceration in ethanol, filtered and dryness. They were divided into non-irradiated control group (0 kGy) and irradiated: groups exposed to gamma radiation at doses of 5, 7.5 and 10 kGy. The total phenols was obtained by the Folin-Ciocalteau method and tannins, by the precipitation of casein. The antimicrobial potential activities of these extracts were also evaluated. The results showed that gamma radiation doses employed in this study did not influence statistically the percentage of total phenols and tannins in the bark extracts, at levels ranging from 5.73±0.14 and 5.20±0.14, respectively. The levels of metabolites in the leaves were statistically (p<0.05) influenced by radiation, observed average total phenols between 3.13±0.04 (0 kGy) and 3.50±0.08 (10 kGy), and tannin between 2.47±0.06 (0 kGy) and 2.93±0.04 (10 kGy). The extracts of bark and leaves were active against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Enterococcus faecalis, Mycobacterium smegmatis, Candida albicans. Gamma radiation caused an increase in antimicrobial activity of extracts against Staphylococcus aureus (Gram positive), with average inhibition zones for shells: 14.33±058 (0 kGy) and 22.33±0.58 (10 kGy), and leaves: 11.33±0.58 (0 kGy) and 19.00±1.00 (10 kGy). Exposure to radiation caused changes in physical and chemical constituents of phenolic extracts of leaves of cashew, increasing levels of tannins.

Antimicrobial Effects of Hypochlorite onEscherichia coliin Water and Selected Vegetables

In this study, the antimicrobial effects of hypochlorite (HOCl) on Escherichia coli in tap water were investigated. The effects of 0.1% thyme oil and 100 mg/L HOCl on E. coli on vegetables (lettuce, parsley leafs, and red pepper) were also studied. E. coli was reduced by 2.54, 3.33, 3.93, 4.87, and 5.57 log colony forming units (cfu)/mL with 0.25, 0.5, 1.0, 10, and 50 mg/mL HOCl, respectively. There was an increase of more than 30% in the inactivation of E. coli with 10 degrees C rise in temperature, a remarkable increase in antimicrobial activity at pH 5.0 was also observed with 5.62 log cfu/mL reductions in 30 sec, as well as marked neutralization of the effect in the presence of 0.1% peptone in water was noted. Biphasic kinetics in the inactivation curves of E. coli was observed. HOCl, thyme oil, and their mixture reduced the number of E. coli between 1.23 and 3.75 log cfu/mL after 5-min exposure on vegetables. The degree of E. coli inactivation depends on concentration of residual chlorine, suspending medium, type of vegetables, and the use of thyme essential oil.

Synthesis and antimicrobial activities of 3- O-alkyl analogues of (+)-catechin: Improvement of stability and proposed action mechanism

We report here the synthesis and biological properties of 3- O-alkyl analogues of (+)-catechin ( 5), which itself is one of the major natural polyphenols found in green tea and has several physiological activities. Starting from 5, a series of 3- O-alkyl-(+)-catechin derivatives were investigated as potent antimicrobial agents. The presence of an alkyl chain rather than acyl on 3- O- showed an increase in antimicrobial activity which may be due to stability in standard culture condition. The most promising compound is 8e, 3- O-decyl analogue, with the MIC of 0.5–2, 32–128 and 2–4 μg/mL against Gram-positive bacteria, Gram-negative bacteria and human pathogenic fungi, respectively. Regarding action mechanism, the antimicrobial activity is possibly due to the lipophilicity and disrupting ability of the analogues to the liposome membrane.

Salmonella gallinarum와 Staphylococcus epidermidis 균주에 대한 한약재의 항균 활성

The present study was conducted to investigate the antimicrobial activities of extracts from approximately 40 different traditional Korean medicinal herbs against S. gallinarum and S. epidermidis. The extracts from Schizandra chinensis Baill., Melia azedarach Linne, Caesalpinia sappan Linne. and Rhus javanica Linne. exhibited high antimicrobial activities against S. gallinarum, whereas the extracts from Melia azedarach Linne and Rhus javanica Linne. exhibited high antimicrobial growth for S. epidermidis. Minimum inhibitory concentrations (MIC) of Melia azedarach Linne, Caesalpinia sappan Linne. and Rhus javanica Linne. for S. gallinarum were 1.2 mg/mL, whereas MIC of exracts from Rhus javanica Linne. extract for S. epidermidis were 0.6 mg/mL. Heat treatment of the extracts from Schizandra chinensis Baill. and Rhus javanica Linne. caused a significant reduction in antimicrobial activities against S. gallinarum. but didn't affect antimicrobial activities against S. edidermidis. Alkaline treatment of the extracts from Schizandra chinensis Baill. caused a significant reduction in antimicrobial activities against S. gallinarum, while similar treatment of the extracts from Rhus javanica Linne. caused a significant increase in antimicrobial activities against S. edider- midis. Since extracts from Rhus javanica Linne. and Caesalpinia sappan Linne. exhibited the highest antimicrobial activities, these extracts at the concentrations of 100, 300 or 500 ppm were added and then bacterial growth-inhibiting activities for S. gallinarum and S. epidermidis by these two extracts were further examined. Optical density at 620 nm (OD620) after 24 hours incubation in the absence of Rhus javanica Linne. extract ranged from 0.30 to 0.45 compared with OD620 value ranging from 0.06 to 0.18 in the presence of 100, 300 or 500 ppm of the extract, indicating that growth of all bacteria was significantly inhibited within 24 hours by the addition of at least 100 ppm of Rhus javanica Linne extract. Value of OD620 after 24 hours incubation in the absence of Caesalpinia sappan Linne. extract ranged from 0.30 to 0.55 compared with OD620 value ranging from 0.05 to 0.15 in the presence of 300 or 500 ppm of the extract, indicating that growth of all bacteria was also significantly inhibited within 24 hours by the addition of at least 300 ppm of Caesalpinia sappan Linne. extract. In conclusion, these findings suggest that extracts from Rhus javanica Linne. and Caesalpinia sappan Linne. may play important roles in antimicrobial activities against S. gallinarum and S. epidermidis.

Directed alteration of a novel bovine β-defensin to improve antimicrobial efficacy against methicillin -resistant Staphylococcus aureus (MRSA)

β-Defensin antimicrobial peptides are critical components of the innate immune response in many species and may be useful against pathogens that have acquired resistance to standard antibiotic therapies. We analysed a panel of recently discovered bovine β-defensins in order to identify sites that may have particular functional importance against antibiotic-resistant bacteria. Modifications were introduced to increase charge at positively selected (PS) sites, to make charge-neutral changes at PS sites, to increase hydrophobicity and to confer a hydrophilic C-terminal. Whilst all four peptide modifications increased antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) compared with the native form of bovine β-defensin 123 ( P = 0.02), conferring the hydrophilic tail caused the most significant increase, with a 50% lethal dose (LD 50) of 3.91 μg/mL. The peptide with increased charge at PS sites showed the most significant increase in antimicrobial activity against a non-resistant strain of S. aureus ( P = 0.02). Therefore, informed modifications of the amino acid sequence can significantly affect the specificity and antimicrobial activity of a peptide.

Lipid interactions of acylated tryptophan‐methylated lactoferricin peptides by solid‐state NMR

Lactoferricin (LfB) is a 25-residue innate immunity peptide released by pepsin from the N-terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR-NH2) retains antimicrobial activity and is thought to constitute the "antimicrobial active-site" (Tomita, Acta Paediatr Jpn. 1994; 36: 585-91). Here we report on N-acylation of 1-Me-Trp5-LfB6, Cn-RRWQ[1-Me-W]R-NH2, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp5) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn-RRWQ[1-Me-W]R-NH2 (deuterated 1-Me-Trp5 underlined), were monitored by solid state 31P NMR and 2H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn-RRWQ[&1-Me-W]R-NH2 peptides added at a 1:25 peptide to lipid ratio. 2H-NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The 2H NMR quadrupolar splittings suggest that the 1-Me-Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. 31P-NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0-RRWQ[1-Me-W]R-NH2, resulted in a 3-4 fold increase in antimicrobial activity against E. coli. N-acylation with a C12 fatty acid enhanced activity almost 90 fold.

Antibacterial Activity of Dextran-Conjugated Lysozyme against Escherichia coli and Staphylococcus aureus in Cheese Curd

The purposes of this research were to glycosylate lysozyme with dextran under Maillard reaction conditions and assess the antimicrobial characteristics of the lysozyme-dextran conjugate in a culture medium and cheese curd. Solutions containing lysozyme and dextran were incubated at 60°C and at 79% relative humidity. Gel permeation chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis were used to follow the glycosylation process. Under optimum conditions 3.7 mol of dextran were coupled to 1 mol of lysozyme. Lytic activity of the conjugate against the cell wall of Micrococcus luteus was about 62% of that of native lysozyme. Evaluation of the lysozyme-dextran conjugate against test microorganisms (Staphylococcus aureus and Escherichia coli) in culture media indicated a progressive increase in antimicrobial activity, with an increase in enzyme-conjugate concentration. The lysozyme-dextran conjugate was also effective against E. coli in a natural food system, as it reduced the bacterial count by 3 log in cheese curd after 40 days of storage. Unlike E. coli, the antimicrobial action of lysozyme against S. aureus was not improved by conjugation with dextran in both in vitro and in vivo tests. Antimicrobial activity of the lysozyme-dextran conjugate against gram-negative bacteria is probably related to the remaining lytic activity as well as the excellent surfactant properties of the lysozyme-dextran conjugate. These results might increase the applicability of lysozyme as a natural antimicrobial ingredient in different food products.

Enzyme-Assisted Processing Increases Antimicrobial and Antioxidant Activity of Bilberry

The effects of nine cell wall-degrading enzymes on the antimicrobial and antioxidant activities of bilberry were studied. Antimicrobial activity was measured using the human pathogens Salmonella enterica sv. Typhimurium and Staphylococcus aureus as test strains. Enzyme treatments liberated phenolics from the cell wall matrix, which clearly increased the antimicrobial activity of berry juices, press cakes, and berry mashes on the basis of plate counts. Antibacterial effects were stronger against Salmonella than against Staphylococcus bacteria. In general, the increase in activity measured as colony-forming units per milliliter was 3-5 logarithmic units against Salmonella and 1-2 units against Staphylococcus bacteria. Increase in antimicrobial activity was observed only in acidic conditions, which is also the natural environment in various berry products, such as juices. The activity profile of the pectinase preparation affected the chemistry of the phenolics due to the presence of deglycosylating activities in some preparations. The difference in phenolic profiles was reflected in the antimicrobial effects. Bilberry mashes treated with Pectinex Ultra SP-L, Pectinex 3 XL, and Pectinex BE XXL were most efficient against Salmonella bacteria, whereas mashes treated with Pectinex Smash, Pectinex BE 3-L, and Biopectinase CCM showed the strongest antimicrobial activity against Staphylococcus bacteria. Due to the liberation of phenolics from the cell wall matrix the antioxidant activity measured as radical scavenging activity was also increased on average about 30% by the enzymatic treatments. The highest increase in phenolic compounds was about 40%. Highest increases in anthocyanins and in antioxidant activity were observed in berry mash treated with Pectinex Smash XXL enzyme, and the lowest increase was observed after treatment with Pectinex BE 3-L. Enzyme-assisted processing is traditionally used to improve berry and fruit juice yields. However, enzymatic treatments also have an impact on the functional properties of the products. The increased liberation of phenolics from the cell wall matrix can prolong the shelf life of berry products by limiting the growth of contaminants during processing or storage. The increased amount of phenolic compounds may also have a positive effect on gut well-being.

P1114 Increase of antimicrobial activity of β-lactam antibiotics in combination with sodium dioctylsulfosuccinate

P1114 Increase of antimicrobial activity of β-lactam antibiotics in combination with sodium dioctylsulfosuccinate

Kallikrein‐mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin

The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL-37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL-37 were identified in normal skin. Through the use of selective inhibitors, SELDI-TOF-MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5-deficient mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.

Synthesis of Ermanin, 5,7-Dihydroxy-3,4'-dimethoxyflavone from Kaempferol, 3,5,7,4'-Tetrahydroxyflavone with Two O-Methyltransferases Expressed in E. coli

Enzymatic modifications of natural compounds have been drawn attention because they provide regioselectivity and chiral selectivity, which are hardly achievable with chemical synthesis. Using a mobilized enzyme or a whole cell containing a particular gene delivers useful tools for modification of natural compounds. In addition, using a whole cell, known as biotransformation, has advantage of saving expensive cofactors. Flavonoids and alkaloids are secondary metabolites produced mainly in plants. Their biological activities have an impact on human health so that they serve as target molecules to develop new drugs. One of common modification reactions found in these two groups of compounds is O-methylation due to free hydroxyl groups. Various Omethylation reactions have been reported and a number of genes (O-methyltransferases: OMTs) that medicated Omethylation have been cloned and characterized. Some of OMTs that use flavonoids as substrates are highly specific and thus they could be used for the regioselective modification of flavonoids. O-methylation of flavonoids resulted in reduction of chemical reactivity and increase of antimicrobial activity. So far, O-methylated flavonoids have been mainly isolated from plants, which could be a limitation for biological assays. Kaempferol (3,5,7,4'-tetrahydroxyflavone) is one of commonly found flavonoids in nature. Even though several biological activities of kaempferol have been established, its dimethoxy form, ermanin (5,7-dihydroxy-3,4'dimethoxyflavone) was known to have several activities including antiviral, anti-inflammatory, cytotoxic, and antibacterial activity. Lists of its biological activity might be extended if large amount of ermanin is supplied. Previously, we cloned and characterized two OMT genes, SOMT-2(soybean O-methyltransferase-2) and ROMT-9 (rice O-methyltrasnferase-9). The transgenic E. coli expressing SOMT-2 transferred a methyl group to 4'-hydroxyl group of flavonoids. ROMT-9 expressed in E. coli showed different regioselectivity depending on the availability of 3'hydroxyl groups. It transferred a methyl group to 3'hydroxyl group if flavonoids have 3'-hydroxyl group. But, when 3'-hydroxyl group is not present, it methylated a 3hydroxyl group. Here we report the biological synthesis of ermanin from kaempferol with two OMTs expressed in E. coli. ROMT-9 and SOMT-2 were cloned in one expression vector and both proteins were induced from E. coli containing both ROMT-9 and SOMT-2 by adding IPTG at 100 μM. In addition, ROMT-9 and SOMT-2 were induced separately from E. coli containing either ROMT-9 or SOMT2. After 4 hr induction, the cells from each culture were harvested and resuspended in LB containing ampicillin (50 μg/mL). 100 μM of kaempferol (3,5,7,4'-tetrahydroxyflavone) was added. The mixture was further incubated for 15 hrs at 28 C. Analysis of culture filtrates from three reactions using high performance liquid chromatography (HPLC) revealed that kaempferol was converted into a new product which had different HPLC retention time from kaempferol itself. ROMT-9 produced a new peak at 15.2 min (Fig. 1B) and SOMT-2 produced a new peak at 19.9 min