Further aspects on the hemolytic activity of the antibiotic lipopeptide iturin A

The bacterial lipopeptide iturin A is able to cause hemolysis of human erythrocytes in a dose-dependent manner. Hemolysis takes place at iturin concentrations below its critical micellar concentration. Relative kinetics determinations clearly show that K(+) leakage occurs prior to hemoglobin release. Furthermore, hemolysis can be prevented by addition to the outer solution of osmotic protectants of appropriate size. Altogether these results indicate that iturin A-induced hemolysis follows a colloid-osmotic mechanism, with the formation of a membrane pore of average diameter 32 A. Iturin A is capable of inducing leakage of an aqueous fluorescent probe trapped in human erythrocyte ghosts, but not in large unilamellar liposomes made of various lipid compositions. The different permeabilizing effects of iturin A on model and biological membranes are discussed on the light of the presented results.     

Interactions of antibiotics of the iturin group with human erythrocytes

The peptidolipid antibiotics, iturin A and bacillomycin L have a disrupting effect on erythrocyte membrane leading to a simultaneous release of K+ ions and hemoglobin. The formation of ghosts is accompanied by a partial solubilisation of lipid components. Binding experiments with radioactive antibiotics show that about 7 X 10(7) molecules of iturin A and 4 X 10(7) molecules of bacillomycin L are bound to one erythrocyte at the concentration giving 100% hemolysis. This concentration is reduced by about 20% after treatment of erythrocytes by phospholipase A2. Scatchard plots show that the affinity for erythrocyte membrane is higher with bacillomycin L than with iturin A. This difference is probably correlated to the differences in their peptide moieties. The substitution of tyrosyl residue leads to a ten-fold increase of the concentrations giving 100% hemolysis, probably due to a low distribution coefficient of derivatives between the membrane and the solvent. This result and the humped Scatchard curves obtained with both antibiotics may be related to the self-association of the compounds in aqueous solutions.     

Methylation of the antifungal lipopeptide iturin A modifies its interaction with lipids

Iturin A, extracted from the culture media of Bacillus subtilis, is an antifungal lipopeptide, the peptide cycle of which includes a D-Tyr residue in position 2. The antibiotic strength of iturin A is related to a change in the permeability of the membrane cells which leads to a leakage of K+ from the intracellular medium. Methylation of the D-Tyr residue dramatically decreases the biological activity of iturin A. Using the intrinsic fluorescence of D-Tyr we have shown that both iturin A and O-methyl-tyrosine iturin A enter the lipid membranes. When dimyristoylphosphatidylcholine vesicles contain iturin A we observe a change in the order degree of the lipid phase and an increase in the transition temperature. The methylated derivative has no effect. Two model membranes have been used to study the permeability changes induced by iturin A and O-methyltyrosine iturin A. Studying ionic permeability we have found that the conductance of a planar lipid membrane increases very much less when the lipopeptide is methylated. On the other hand, the release of carboxyfluorescein trapped in lipid vesicles is less upon addition of O-methyltyrosine-iturin A. We conclude that the Tyr residue of the peptide cycle plays a role in determining the interactions of iturin A with lipid membrane.     

Action of peptidolipidic antibiotics of the iturin group on erythrocytes: Effect of some lipids on hemolysis

Iturin A, bacillomycin L and bacillomycin L dimethyl ester have a strong lytic activity upon human erythrocytes while iturin C is totally inactive. The hemolytic action of the antibiotics is inhibited by free cholesterol as well as by cholesterol included in mixed liposomes of phosphatidylcholine-cholesterol and to a lesser extent by phosphatidylcholine liposomes. This inhibition is the result of an interaction between the antibiotic and added lipids which diminishes the concentration of free antibiotic available to lyse erythrocytes. The inhibitory effect of liposomes on hemolysis demonstrates the affinity of the antibiotic for artificial membranes, especially those containing cholesterol.     

Mechanism of polymer-induced hemolysis: nanosized pore formation and osmotic lysis

Hemolysis induced by antimicrobial polymers was examined to gain an understanding of the mechanism of polymer toxicity to human cells. A series of cationic amphiphilic methacrylate random copolymers containing primary ammonium groups as the cationic functionality and either butyl or methyl groups as hydrophobic side chains have been prepared by radical copolymerization. Polymers with 0-47 mol % methyl groups in the side chains, relative to the total number of monomeric units, showed antimicrobial activity but no hemolysis. The polymers with 65 mol % methyl groups or 27 mol % butyl groups displayed both antimicrobial and hemolytic activity. These polymers induced leakage of the fluorescent dye calcein trapped in human red blood cells (RBCs), exhibiting the same dose-response curves as for hemoglobin leakage. The percentage of disappeared RBCs after hemolysis increased in direct proportion to the hemolysis percentage, indicating complete release of hemoglobin from fractions of RBCs (all-or-none leakage) rather than partial release from all cells (graded leakage). An osmoprotection assay using poly(ethylene glycol)s (PEGs) as osmolytes indicated that the PEGs with MW > 600 provided protection against hemolysis while low molecular weight PEGs and sucrose had no significant effect on the hemolytic activity of polymers. Accordingly, we propose the mechanism of polymer-induced hemolysis is that the polymers produce nanosized pores in the cell membranes of RBCs, causing an influx of small solutes into the cells and leading to colloid-osmotic lysis.     

Antagonistic Mechanism of Iturin A and Plipastatin A from Bacillus amyloliquefaciens S76-3 from Wheat Spikes against Fusarium graminearum

Controlling toxigenic Fusarium graminearum (FG) is challenging. A bacterial strain (S76-3, identified as Bacillus amyloliquefaciens) that was isolated from diseased wheat spikes in the field displayed strong antifungal activity against FG. Reverse-phase high performance liquid chromatography and electrospray ionization mass spectrometry analyses revealed that S76-3 produced three classes of cyclic lipopeptides including iturin, plipastatin and surfactin. Each class consisted of several different molecules. The iturin and plipastatin fractions strongly inhibited FG; the surfactin fractions did not. The most abundant compound that had antagonistic activity from the iturin fraction was iturin A (m/z 1043.35); the most abundant active compound from the plipastatin fraction was plipastatin A (m/z 1463.90). These compounds were analyzed with collision-induced dissociation mass spectrometry. The two purified compounds displayed strong fungicidal activity, completely killing conidial spores at the minimal inhibitory concentration range of 50 µg/ml (iturin A) and 100 µg/ml (plipastatin A). Optical and fluorescence microscopy analyses revealed severe morphological changes in conidia and substantial distortions in FG hyphae treated with iturin A or plipastatin A. Iturin A caused leakage and/or inactivation of FG cellular contents and plipastatin A caused vacuolation. Time-lapse imaging of dynamic antagonistic processes illustrated that iturin A caused distortion and conglobation along hyphae and inhibited branch formation and growth, while plipastatin A caused conglobation in young hyphae and branch tips. Transmission electron microscopy analyses demonstrated that the cell walls of conidia and hyphae of iturin A and plipastatin A treated FG had large gaps and that their plasma membranes were severely damaged and separated from cell walls.     

Hemolysis of human erythrocytes induced by melamine-cyanurate complex

Melamine is a widely-used chemical in industries. In recent years, melamine has been found to be involved in outbreaks of renal injury in infants and animals. Pathological studies indicated that the melamine-induced acute renal failure was related to the concurrence of melamine and other triazine analogs such as cyanuric acid. In the present study, human erythrocytes were used as an in vitro model to explore the cytotoxicity of melamine and its complex with cyanuric acid. The results demonstrated that mixing melamine and cyanuric acid resulted in the formation of insoluble particles and that the insoluble melamine-cyanurate complex induced membrane damages of human erythrocytes. The membrane damages included hemolysis, K⁺ leakage, alterations in cell shape and membrane fragility, and inhibition of enzymatic activity. By contrast, either melamine or cyanuric acid alone had no effect on erythrocyte membranes. The results of this study may provide a fresh insight into the melamine toxicology.     

Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH₄Cl, urea, and ammonium citrate); and metal ions (Zn²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ca²⁺, and K⁺). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg²⁺ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg²⁺. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.     

Comparing Methods for Identifying Bacillus Strains Capable of Producing the Antifungal Lipopeptide Iturin A

Lipopeptides represent a unique class of bioactive microbial secondary metabolites, and iturin A shows attractive antibiotic properties among them. This study compares three methods, such as yeast/fungal growth inhibition assay, quantitative high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR) for identifying a number of Bacillus species that produce iturin A. We examined the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD and lpa-14 amplification. Twenty standard strains and 120 field-collected Bacillus spp. isolates were tested in this study. Four B. subtilis and one B. circulan strains from ATCC, and B. amyloliquefaciens B128, a known iturin A producer, exhibited positive results. Of the 120 field-collected isolates, 42 strains were positive. The potential of producing iturin A by these PCR-positive strains were then confirmed by conventional methods such as fungal growth inhibition assay and HPLC analysis. The consistency between results of PCR, HPLC, and fungal growth inhibition assay suggests that the PCR method could be used as an alternative tool for fast screening of iturin A-producing Bacillus strains from the environment. This is the first report of detecting iturin A production from B. circulans.     

Aggregational behavior of aqueous dispersions of the antifungal lipopeptide iturin A

Iturin A, a lipopeptide isolated from Bacillus subtilis, possesses a strong antifungal activity, and has been devoted to a great deal of attention. Since iturin is an amphiphilic compound with a great propensity to self-associate in solution as well as inside the membrane, the question arises to whether its aggregational behavior is dependent on the concentration of the lipopeptide. In order to test this, the ability of iturin suspensions to encapsulate water-soluble molecules has been examined. Iturin was dispersed at different concentrations above its critical micellar concentration, in a buffer containing the water-soluble dye 5,6-carboxyfluorescein. For iturin A micelles, a Stokes radius of 1.3 nm and an aggregational number of 7 was obtained. The results shown in this work clearly demonstrate that iturin dispersions in water, at concentrations of 0.7, 1.4 and 3 mM, i.e. far above the critical micellar concentration (40 microM), are capable of encapsulating carboxyfluorescein, probably by adopting a type of aggregate different from the micelle. Negative-staining electron microscopy shows the presence of vesicles with an average size of 150 nm. By using (14)C-iturin, it is shown that, at 3 mM concentration, 40 % of the iturin molecules adopt this vesicular state. It is proposed that iturin molecules form a fully interdigitated bilayer, where each hydrocarbon tail span the entire hydrocarbon width of the bilayer, resulting in multilamellar vesicles capable of encapsulating an aqueous compartment. The possible implications of these results to the membrane destabilizing effect of iturin A, are discussed according to the dynamic cone-shape of the iturin molecule.     

Cloning, sequencing, and characterization of the iturin A operon

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser-->L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn-->L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.     

Iturin A-like lipopeptides from Bacillus subtilis trigger apoptosis,paraptosis, and autophagy in Caco-2 cells

This study revealed that iturin A-like lipopeptides produced by Bacillus subtillis induced both paraptosis and apoptosis in heterogeneous human epithelial colorectal adenocarcinoma (Caco-2) cells. Autophagy was simultaneously induced in Caco-2 cells treated with iturin A-like lipopeptides at the early stage and inhibited at the later stage. A western blot analysis showed that the lipopeptides induced apoptosis in Caco-2 cells via a mitochondrial-dependent pathway, as indicated by upregulated expression of the apoptotic genes bax and bad and downregulated expression of the antiapoptotic gene bcl-2. The induction of paraptosis in Caco-2 cells was indicated by the occurrence of many cytoplasmic vacuoles accompanied by endoplasmic reticulum (ER) dilatation and mitochondrial swelling and dysfunction. ER stress also occurred with significant increases in reactive oxygen species and Ca²⁺ levels in cells. Autophagy was detected by a transmission electron microscopy analysis and by upregulated expression of LC3-II and downregulated expression of LC3-I. The inhibition of autophagy at the later stage was shown by upregulated expression of p62. This study revealed the capability of iturin A-like B. subtilis lipopeptides to simultaneously execute antitumor potential via multiple pathways.     

Hemolysis of human red blood cells by riboflavin-Cu(II) system

The photodynamic action of riboflavin is generally considered to involve the generation of reactive oxygen species, whose production is enhanced when Cu(II) is present in the reaction. In the present study we report that photoactivated riboflavin causes K(+) loss from fresh human red blood cells (RBC) in a time dependent manner. Addition of Cu(II) further enhances the K(+) loss and also leads to significant hemolysis. Riboflavin in a 2:1 stoichiometry with Cu(II) leads to maximum K(+) loss and up to 45% hemolysis. Bathocuproine, a specific Cu(I)-sequestering agent, when present in the reaction, inhibits the hemolysis completely. Free radical scavengers like superoxide dismutase, potassium iodide and mannitol inhibited the hemolysis up to 55% or more. However, thiourea was the most effective scavenger showing 90% inhibition. These results suggest that K(+) leakage and hemolysis of human RBC are basically free radical mediated reactions.     

The influence of process parameters in production of lipopeptide iturin A using aerated packed bed bioreactors in solid-state fermentation

The strain Bacillus iso 1 co-produces the lipopeptide iturin A and biopolymer poly-γ-glutamic acid (γ-PGA) in solid-state fermentation of substrate consisting of soybean meal, wheat bran with rice husks as an inert support. The effects of pressure drop, oxygen consumption, medium permeability and temperature profile were studied in an aerated packed bed bioreactor to produce iturin A, diameter of which was 50 mm and bed height 300 mm. The highest concentrations of iturin A and γ-PGA were 5.58 and 3.58 g/kg-dry substrate, respectively, at 0.4 L/min after 96 h of fermentation. The low oxygen uptake rates, being 23.34 and 22.56 mg O₂/kg-dry solid substrate for each air flow rate tested generated 5.75 W/kg-dry substrate that increased the fermentation temperature at 3.7 °C. The highest pressure drop was 561 Pa/m at 0.8 L/min in 24 h. This is the highest concentration of iturin A produced to date in an aerated packed bed bioreactor in solid-state fermentation. The results can be useful to design strategies to scale-up process of iturin A in aerated packed bed bioreactors. Low concentration of γ-PGA affected seriously pressure drop, decreasing the viability of the process due to generation of huge pressure gradients with volumetric air flow rates. Also, the low oxygenation favored the iturin A production due to the reduction of free void by γ-PGA production, and finally, the low oxygen consumption generated low metabolic heat. The results show that it must control the pressure gradients to scale-up the process of iturin A production.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.     

Role of the interaction between puerarin and the erythrocyte membrane in puerarin-induced hemolysis

Adverse drug reactions (ADR), especially intravenous hemolysis, have largely limited the application of puerarin injections in clinics. This study investigated the underlying mechanisms of puerarin-induced hemolysis. Our results show that puerarin induced concentration-dependent and time-dependent hemolysis when human erythrocytes were incubated in saline solution with more than 2 mM puerarin for over 2 h. However, incubation in PBS or addition of 1 mM of lidocaine to the saline solution completely abolished the hemolysis. Providing materials that could start ATP synthesis did not reverse the hemolysis, and puerarin did not affect Na⁺–K⁺–ATPase activity. In addition, puerarin (0.1–2 mM) did not cause calcium influx or exhibited pro-oxidant activity in erythrocytes. Puerarin exhibited different influences on the membrane microviscosity of erythrocytes in saline and PBS. Moreover, 1 mM lidocaine inhibited 8 mM puerarin-induced reduction of membrane microviscosity in saline solution. SDS–PAGE analysis of membrane proteins revealed that 2 mM puerarin treatment induced the appearance of several new protein bands but attenuated the expression of protein bands 2.1, 3, 4.1, 4.2 and 5. These results suggest that high concentrations of puerarin-induced hemolysis were associated with the changes of membrane lipids and of the composition of erythrocytes membrane proteins but not with ATP depletion, pro-oxidation and calcium influx. These changes could be related to the intercalation of amphiphilic puerarin at high concentration into the erythrocyte membrane in certain media, resulting in membrane disorganization and, eventually, cytolysis. Hence, in clinics, determining the optimal dose of puerarin is critical to avoid overdosing and ADR.     

The unconventional adverse effects of fungal pretreatment on iturin A fermentation by Bacillus amyloliquefaciens CX-20

Fungal pretreatment is the most common strategy for improving the conversion of rapeseed meal (RSM) into value-added microbial products. It was demonstrated that Bacillus amyloliquefaciens CX-20 could directly use RSM as the sole source of all nutrients except the carbon source for iturin A fermentation with high productivity. However, whether fungal pretreatment has an impact on iturin A production is still unknown. In this study, the effects of fungal pretreatment and direct bio-utilization of RSM for iturin A fermentation were comparatively analysed through screening suitable fungal species, and evaluating the relationships between iturin A production and the composition of solid fermented RSM and liquid hydrolysates. Three main unconventional adverse effects were identified. (1) Solid-state fermentation by fungi resulted in a decrease of the total nitrogen for B. amyloliquefaciens CX-20 growth and metabolism, which caused nitrogen waste from RSM. (2) The released free ammonium nitrogen in liquid hydrolysates by fungal pretreatment led to the reduction of iturin A. (3) The insoluble precipitates of hydrolysates, which were mostly ignored and wasted in previous studies, were found to have beneficial effects on producing iturin A. In conclusion, our study verifies the unconventional adverse effects of fungal pretreatment on iturin A production by B. amyloliquefaciens CX-20 compared with direct bio-utilization of RSM.     

Molecular and Biochemical Approaches for Characterization of Antifungal Trait of a Potent Biocontrol Agent Bacillus subtilis RP24

Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma–Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4′-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.     

Optimization of Antifungal Effect of Surfactin and Iturin to <Emphasis Type="Italic">Penicillium notatum</Emphasis> in Syrup of Peach by RSM

In the paper, the sensitivity of Penicillium notatum to surfactin and iturin was determined, and the optimization of the antifungal of surfactin and iturin to Penicillium notatum in syrup of peach by a response surface methodology (RSM) was researched. Results demonstrated that Penicillium notatum was sensitive to them, whose minimal inhibitory concentration (MIC) was 62.5 and 31.25 μg ml⁻¹ respectively. In the optimization experiment, when the temperature was 2.37°C, the action time was 25.21 h, and the concentration (surfactin/iturin weight ratio 1:1) was 40.26 μg ml⁻¹, Penicillium notatum could be sterilized by 5 orders of magnitude. All the results in the experiment indicated surfactin and iturin could kill remarkably Penicillium notatum in syrup of peach.