International Journal of Biological Macromolecules 66 (2014) 7–14

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Synthesis of biocontrol macromolecules by derivative of chitosan with surfactin and antifungal evaluation Bo Yuan, Pei-Yuan Xu, Yue-Ji Zhang, Pei-Pei Wang, Hong Yu, Ji-Hong Jiang ∗ The Key Laboratory of Biotechnology for Medicinal Plants of Jiangsu Province, Jiangsu Normal University, Xuzhou, Jiangsu 221116, PR China

a r t i c l e

i n f o

Article history: Received 28 December 2013 Received in revised form 29 January 2014 Accepted 7 February 2014 Available online 13 February 2014 Keywords: Synthesis Biological macromolecules Anti-sapstain fungi

a b s t r a c t A derivative of chitosan was prepared with chitosan and ␤-cyclodextrins, which was synthesized by the immobilization reaction, as a carrier to adsorb surfactin produced from Bacillus amyloliquefaciens and got biological macromolecules. The antifungal activity against three sapstain fungi by a combination of macromolecules was tested. The results showed that the macromolecules inhibited the mycelium growth of sapstain fungi Lasiodiplodia rubropurpurea, L. crassispora, and L. theobromae by about 73.22%, 76.72%, and 70.22%, respectively. The macromolecules were relatively thermally stable with more than 50% of the antifungal activity even after being held at 121 ◦ C for 30 min. Meanwhile, the activity of the macromolecules remained more than 55% at a pH value ranging from 4 to 12. The macromolecules were resistant to hydrolysis by most protein-denaturing detergents and other enzymes. The results indicated the macromolecules might provide an alternative bioresource for the bio-control of sapstain. © 2014 Published by Elsevier B.V.

1. Introduction Plant diseases can cause death or other delirious effects on plant leading to great economic losses. Particularly, these impacts are caused by pathogenic fungi. Chemical fungicide has been considered to be one of the most effective tools that could control pathogenic diseases. However, the frequent and long-term application of traditional fungicides has caused serious hazards such as environmental pollution and threats to human health. This has resulted in an increasing awareness of the environmental impacts of the chemicals used for plant protection and preservation purposes among the industrial sector and the general public [1]. Sapstain is a major issue for timber producers as well as pulp and paper manufacturers. Recently it was observed that, fungi colonization and disfigurement of freshly fell materials prior to drying can result in significant economic losses [2]. Although the sapstain fungi cause little or no significant damage to the structure elements of the timber, they have a detrimental effect on the esthetic value of the wood due to the colonization by their pigmented mycelia. At present, chemical control remains the main measure to reduce the wood discoloration [3]; however, it may pose significant risks to environment and public health [1]. Therefore, it is highly critical to discover new antifungal alternatives for the sapstain.

∗ Corresponding author. Tel.: +86 516 83403515; fax: +86 516 83403515. E-mail address: [email protected] (J.-H. Jiang). http://dx.doi.org/10.1016/j.ijbiomac.2014.02.011 0141-8130/© 2014 Published by Elsevier B.V.

Chitosan is a cheap and naturally occurring biosorbent, obtained from the deacetylation of chitin extracts from crustaceans. Partial deacetylation of chitin results in the production of chitosan, which is a polysaccharide composed of ␤-(1,4)-2-acetamido-2deoxyglucopyranose and 2-amino-2-deoxyglucopyranose units, a biodegradable high molecular weight cationic polysaccharide with excellent film-forming ability [4]. The chitosan has been extensively studied in the adsorption of metals and dyes [5]. The antifungal activity of chitosan has also been reported in several studies, both in vitro and in vivo, although chitosan activity against fungi has been shown to be less efficient as compared to its activity against bacteria [4]. The inhibitory efficiency of chitosan has been related to its properties such as deacetylation degree (DD) and molecular weight (Mw). In others works, researchers have reported that the level of fungal inhibition is highly dependent on chitosan concentration, indicating that chitosan performance is related to its application of an appropriate rate. Bacillus strains produce various kinds of bioactive lipopetides that is applied in diverse areas for biocontrol. Among these, surfactin, iturin and fengycin are the principal lipopeptides produced by B. subtilis. Surfactin is considered to be one of the most potent biosurfactant and exhibit antiviral properties. Iturin and fengycin are among the active antifungal compounds [6,7]. In addition, surfactin is able to induce systemic resistance in plants and could be used in near future as a biocontrol agent for plant diseases [8]. Surfactin is a bacterial lipopeptide produced biologically by several strains of B. subtilis [9,10]. Surfactin is an extraordinarily powerful biosurfactant that is known to decrease the surface tension of

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water. It exerts a detergent-like action on biological membranes [11], and is clearly distinguishable with its exceptional emulsifying, foaming, antiviral and anti-mycoplasma activities [12]. Surfactin has potential applications in fields of plant disease biocontrol [8] and biomedicine [13]. Moreover, lipopeptide can be widely used in the food and cosmetic industries and for enhanced oil recovery for the bioremediation of oil-contaminated sites [14–17]. Both B. amyloliquefaciens and B. subtilis have been used as lipopeptide producers and there have been many attempts to increase lipopeptide production in these two organisms. Bio-control for wood discoloration is an important research area for our group and in this study we report the antifungal activity of extracts from an endophytic bacterium, designated strain B. amyloliquefaciens CGMCC 5569 isolated from Chinese medicinal Ginkgo biloba collected from Xuzhou, China. Both the filtrate and the ethyl acetate extract of strain CGMCC 5569 showed growth inhibition activity against the sapstain fungi L. rubropurpurea, L. crassispora, and L. theobromae obviously. The extract is a mixture of series of fengycin, surfactin and bacillomycin as identified by LC–MS [1]. Other researches have reported that the chitosan has antifungal activity, however, there are only a few reports describing a multicompound drug and the study of chitosan on loaded antifungal drugs. In this study the endophytic B. amyloliquefaciens CGMCC 5569 was used and we evaluated the activity for the surfactin loaded by chitosan. The polymeride showed a strong growth inhibition activity against the sapstain fungi. The effects were significantly better than surfactin itself and other chemical fungicides. The advantages and strong anti-sapstain fungi activity indicated that the polymeride might provide an alternative bioresource for the bio-control of sapstain. 2. Methods 2.1. Microorganism Strain B. amyloliquefaciens CGMCC 5569 was isolated from ginkgo (G. biloba) collected from Xuzhou, China. Two or three segments were randomly cut from each stem, washed in running water and sterilized successively with 75% ethanol and 40% formaldehyde for 3 min each. The segments were washed again using sterile distilled water and cut into 1 cm long sections using a small knife and placed on potato sucrose agar (PSA: potato, 200 g; sucrose, 20 g; agar, 15 g; distilled water, 1 L). The same medium without agar (PS) was used as a liquid medium. After incubation for 1–2 days at 30 ◦ C, bacteria appeared on the plates and were isolated individually as single colonies on PSA. The strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) and maintained in LB medium (Lysogeny Broth Medium). The sapstain strain of L. theobromae was provided by the Forest Microbial Resources of China (CFCC). The stain of L. rubropurpurea and L. crassispora were kindly provided by Professor Gui-hua Zhao and maintained on PDA medium (Potato Dextrose Agar medium). Chitosan (degree of deacetylation (DD) 95%, average-molecular weight (Mw) 230 kDa), was purchased from Qingdao Baicheng Biochemical Corp. Cyclodextrin (CD) was obtained from Tianjin Damao Chemical Co., Ltd. 2.2. Surfactin preparation The fermentation was performed in LB medium at 30 ◦ C, 100 rpm for 72 h. The seed culture (12 L) was centrifuged at 4000 rpm at room temperature for 5 min. The cell-free supernatant was extracted exhaustively five times with ethyl acetate (filtrate:ethyl acetate = 1:2 vol/vol). The solvent was removed by using a rotary vacuum evaporator R-206 D (SENCO, Shanghai,

China) under reduced pressure to yield a brown viscous tarry residue (5.2 g) and stored at 4 ◦ C. Surfactin was extracted in accordance with the Hsieh method [18] with minor modifications. Cell suspension was centrifuged at 10,000 rpm for 5 min to prepare the cell-free supernatant (CFS). The CFS was acidified with 1 N HCl to pH 2 and left overnight at 4 ◦ C. The produced off-white to buff cake in the centrifuge tubes was dried in a hot-air oven at 70 ◦ C. The dried materials were transferred to 50 mL methylene chloride contained in a 250-mL conical flask and left covered overnight at room temperature with intermittent shaking. The organic extract was filtered and the residue on the filter paper was re-extracted with another 50 mL fresh methylene chloride and refiltered for a second time. The pooled organic phase was evaporated under vacuum (Buchi, Germany) at 40 ◦ C. The residue obtained was characterized as such or after dissolving in 5 mM Tris–HCl buffer, pH 8.5 (the crude surfactin solution). 2.3. Preparation of ˇ-CD-chitosan (CS-ˇ-CD) 3% (w/v) chitosan powder was completely dissolved in 1% (v/v) acetic acid. This solution was extruded through a syringe needle (27 G) into a coagulant bath consisting of 1 N sodium hydroxide solution containing 26% (v/v) ethanol under stirring condition to form spherical gels. The solution was allowed to stand for 3 h and the spherical gels were removed by filtration and rinsed with deionized (DI) water until neutrality. Finally, the beads were dried at room temperature for more than 24 h for further application. The method of covalent binding was used for immobilization of ␤-CD to chitosan beads. In this process, 1,6-hexamethylene diisocyanate (HMDI) acts as a spacer between the ␤-CD and chitosan. One gram of chitosan beads was placed in 25 mL of a mixture of toluene and HMDI containing 5% (v/v) of HMDI. The mixture was magnetically stirred at room temperature after adding few drops of stannous 2ethylhexanoate that catalyzed the immobilization reaction. After stirring for 40 min, the supernatant was discarded and the chitosan beads were dried using nitrogen gas. The chitosan beads with bound HMDI were then placed in 25 mL of DMF solution containing 2% (w/v) ␤-CD. The mixture was magnetically stirred for 1 h at room temperature after adding few drops of stannous 2-ethylhexanoate. The supernatant was decanted and the CS-␤-CD beads were washed several times with DI water followed by ethanol and finally with DI water before freeze drying. 2.4. Scanning electron microscopy (SEM) This allows the identification of the appearance, morphology and size of the structures originated as a result of the film formation process. Microstructural analysis of the surface and section of the dry films were carried out using SEM technique in a Hitachi S-3400N (Japan). The samples were cut from films and mounted in copper stubs. To allow the observation, samples were gold coated (15 nm) and observed using an accelerating voltage of 10 kV. 2.5. Adsorption of surfactin The adsorption of surfactin was carried out at 30 ◦ C in 20 mL at pH 7 with ultrapure water in a 50 mL glass recipient under 160 rpm agitation. 600 mg of nonwoven membrane was added to each flask in the form of 0.5 cm × 0.5 cm square pieces and completely wetted with a small volume of water before adding surfactin. The initial surfactin concentration was 200 mg/L. The surfactin samples were immersed in the chitosan solution (pH was to be studied in the following experiment) for 1 min, followed by washing in deionized water and drying at 115 ◦ C, in a procedure similar to those employed in the LbL technique [19]. The samples were filtered through 0.2-␮m cellulose filters before analysis

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and the residual surfactin was quantified using HPLC (Aglient 1260 system, USA) with water:ACN:TFA mixture as eluent and a C18 column (5 ␮m, 250 mm × 4.6 mm, VYDAC 218 TP, Hesperia, CA, USA) as described in Gancel [20]. As surfactin occurs in different molar masses depending on the length of the fatty acid chain, the total quantity of surfactin was calculated from the total surface area of HPLC peaks with a surfactin standard. The amount of surfactin encapsulated (embedded rate) in the SFC–CD–CS was calculated by measuring the difference between the total amount of surfactin added in the SFC–CD–CS preparation buffer and the amount of non-entrapped surfactin remaining in the aqueous suspension after the coacervation process. For this purpose, SFC–CD–CS suspension was centrifuged in a series 35–55–85% (w/v) sucrose gradients as described above (1000 rpm for 20 min). The supernatant was analyzed for surfactin concentration, which accounted for the non-entrapped surfactin, using the method account. The embedded rate was calculated as follows:



embedded rate(%) =

w0 − w × 100 w0

Here, w is the non-entrapped surfactin and w0 is the total surfactin concentration. Three replicates of each test were carried out and the results were averaged. And then, we designed the difference method and calculated the embedded rate between difference concentration of CD (0.5, 1.0, 2.0, 3.0, and 4.0%), CS (1.0, 2.0, 3.0, 4.0, and 5.0%), even difference response time (the response time for the surfactin and CS–CD) and the pH (the CD and CS concentrations were determined). 2.6. Antifungal assay and comparison with various chemicals Antifungal assay was evaluated in vitro by mycelium growth rate test method described by Guo [21]. Chitosan samples were dissolved in 0.5% (w/v) acetic acid at an original concentration of 1% (w/v). The solution was mixed with sterile molten potato dextrose agar (PDA) to obtain final concentrations of 100 mg/L, 200 mg/L and 400 mg/L, respectively. The antifungal index was calculated with the following equation:



antifungal index(%) =

Db − Dt × 100 Db

Here, Dt is the colony diameter in the test plate and Db is the colony diameter in the blank control. Three replicates of each test were carried out and the results were averaged. The Scheffe method was used to evaluate the differences in antifungal index in the tests. Results with p < 0.05 were considered statistically significant. Different concentrations of chitosan (95% DD; 100, 200, and 400 mg/L), CD–CS (100, 200, and 400 mg/L) and SFC–CD–CS (100, 200, and 400 mg/L) were used in the antifungal assay. The polyoxin and triadimefon were used in same concentration as positive control. 2.7. pH and thermal stability The drug-loading SFC–CD–CS was adjusted to various pH values in the range from 1.0 to 14.0 using 2 M HCl or 2 M NaOH and then maintained at 4 ◦ C for 24 h. Antifungal activity was assayed after the samples were readjusted to pH 7.0. A similar procedure was used to assess the effect of temperature on antifungal activity of the drug-loading SFC–CD–CS. The SFC–CD–CS was held at temperatures of 25, 80, 100, and 121 ◦ C, respectively, for 30 min, and was tested for their antifungal activity after being cooled to room temperature following the process of Section 2.6 (200 mg/L). The relative remaining activity was measured by comparison with that of the samples held at pH 7.0 and room temperature. The data

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are presented as the mean values of the three replications ± SEM (standard error of the mean). 2.8. Susceptibility to denaturation by enzymes The sensitivity of antifungal metabolites (SFC–CD–CS) to denaturation by enzymes was tested using the following commercial enzyme preparations: ␣-chymotrypsin (pH 7.5), trypsin (pH 8.1), pepsin (pH 2.1), subtilopeptidase A (pH 7.5), carboxypeptidase A (pH 7.5), proteinase K (pH 8.5), lysozyme (pH 6.1), a-amylase (pH 6.1), dextranase (pH 6.1) and lipase A (pH 7.0). All enzymes were purchased from Sigma Chemical Co. and were dissolved in distilled water at a concentration of 1 mg/mL, except carboxypeptidase A which was used at a concentration of 20 ␮L/mL water. The pH of distilled water was adjusted to those values indicated for optimum activity of each enzyme. SFC–CD–CS solution were added to enzyme solutions at a concentration of 20 mg SFC–CD–CS solution per mL enzyme solution and incubated at 37 ◦ C for 2 h. Then, the mixture of SFC–CD–CS solution and enzyme was autoclaved for 15 min to inactivate enzymes before assaying for residual antifungal activity against mycelial growth as described previously (Section 2.6). 2.9. Susceptibility to protein-denaturing detergents Susceptibility to denaturation by protein-denaturing detergents was studied by treating the metabolites with nonionic (Triton X100, Tween-20, Tween-80, Nonidet P-40), anionic (sodium dodecyl sulphate, deoxycholic acid), cationic (hexadecyltrimethyl ammonium bromide) and dipolar ionic (N-hexadecyl-N,N-dimethyl3-ammonio-1-propanesulphonate) detergents. Detergents were obtained from Guoyao Chemical Co., which was added to antifungal metabolites at concentrations of 0.1 mL or 0.01 g of detergent per mL antifungal solutions containing 20 mg dried metabolites mL−1 distilled water. These preparations were incubated at 30 ◦ C for 6 h, sterilized at 121 ◦ C for 15 min and qualitatively assayed for antifungal activity against mycelial growth of L. theobromae, L. rubropurpurea and L. crassispora under the process described in Section 2.6. An agar diffusion assay was used as previously described. Detergents added to distilled water were used as control treatments. 2.10. Statistical analysis All experiments were carried out in triplicate, and average values with standard deviation errors were reported. Mean separation and significance were analyzed using the SPSS software package (SPSS 12, Chicago). 3. Results and discussion 3.1. Immobilization of ˇ-cyclodextrin and characterization 1,6-Hexamethylene diisocyanate (HMDI) is generally utilized as a strong cross-linker of amino or hydroxyl groups since it possesses two isocyanate groups (HN C O). The hypothetical illustration of ␤-CD immobilized to chitosan using HMDI as a cross-linker is shown in Scheme 1. Under the suitable conditions (pH < 6), the hydroxyl groups of chitosan reacts with an isocyanate to form a urethane product ( NH CO O ) due to the transfer of proton from hydroxyl to nitrogen atom of isocyanate. In addition, isocyanate also reacts with hydroxyl groups of ␤-CD to form a product identical to urethane. It is assumed that the cross-linking of the hydroxyl groups of chitosan with HMDI results in chitosan–HMDI complex, which then binds with the hydroxyl groups of ␤-CD to form CS–CD. HMDI cannot bind to amino groups of chitosan due to the lower

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Scheme 1. Hypothetical illustration of ␤-CD immobilized to chitosan using HMDI as a cross-linker.

affinity for amino group as compared to hydroxyl groups under low pH value. Fig. 1 shows the surface structure of commercial chitosan (Fig. 1(a)), crosslinked chitosans (Fig. 1(b)). As shown in Fig. 1, the chitosan surfaces are smooth, but the crosslinked chitosans have large pore structures which are clearly visible. These pores could provide sites or location for the surfactin. 3.2. The embedded rate on difference craft The embedded rate for the surfactin was found to be increasing with the concentration of ␤-CD and chitosan initially. However, it started decreasing when the ␤-CD and chitosan reached a certain concentration (␤-CD was 3.0% and chitosan was 4.0%). The embedded rate reached the high value under these conditions (about 60.12 ± 1.55) (Table 1). The embedded rate was decreasing for ␤-CD and chitosan concentrations higher than the value. Fig. 2 shows the effect on embedded rate on different pH and response time. This figure has a double X layer, the blue line (––) show the difference in pH. The embedded rate reached the highest when the pH was maintained at 4. On the other side, the embedded rate was almost zero at highly acidic (pH = 1) and weak alkaline range (pH = 8), even at neutral range. This was mainly because of the dissolution characteristic of the chitosan. Acid environment fitted with the chitosan due to the amino group within the structure of chitosan. Chitosan is a weak base polysaccharide, having an average group density of 0.837 per disaccharide unit [22], and insoluble at neutral and alkaline pH values. With a large number of NH2 and OH in the chitosan, the solubility of the chitosan could be improved. The NH2 and OH could interact with hydrone and produce the hydration in the acidic aqueous solution. In such a context, the chitosan will expand gradually and the shape changes with the degree of hydration. The hydration could affect the adsorption of the surfactin. Meanwhile, the viscosity would be reduced in the

Fig. 2. The effect of the time and pH on the surfactin embedded rate. , and  indicates that the embedded rate for difference time and pH, respectively.

low pH range because of the change in shape from chain to globular that might improve the adsorption area. On the other side, the viscosity will increase with the pH value. For chitosan (a weak polybase), the opposite scenario the case as the ionization of amine groups decreased greatly when the solution pH increased above 6.0 (around the pKa of chitosan 6.3) [23], and at pH higher than 7.5 usually less than 10% of amine groups were ionized. It could affect the adsorption of the surfactin because of the structure itself. The adsorption itself on the response of carboxyl of the surfactin and amino groups of the chitosan. The less the amino groups, the

Fig. 1. SEM of chitosan (a) and crosslinked chitosan (b).

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Table 1 The embedded rate of surfactin by different ␤-CD and CS (%). ␤-CD/%

Chitosan/% (acetic acid) 1.0

0.50 1.00 2.00 3.00 4.00

17.09 21.29 23.76 26.34 20.16

2.0 ± ± ± ± ±

0.22 0.68 1.33 0.27 0.57

22.09 32.68 41.23 47.28 40.86

3.0 ± ± ± ± ±

0.65 1.02 0.66 0.36 1.22

worse the adsorption. On the basis of this result, hereafter, all the experiments in this paper were carried out at pH 4.0. The active line (——) represents the effect of the absorption time for the CS–CD binding on the drug’s embedded rate for surfactin. Obviously, the maximum embedded rate was obtained in 6.5 h. This figure indicated that the absorption of surfactin consisted of two rapid phases: a primary rapid phase and a secondary slow phase. At the first 2 h, the surfactin removal was observed in the rapid phase, from 3 h to 6 h, the adsorption was slow. It reached equilibrium after 6 h and the corresponding embedded rate was 64.68%. The primary rapid phase indicated that the rapid uptake could be due to the concentration gradient of the surfactin and more active sites at the beginning of the adsorption process. During the second phase of the surfactin uptake, the active sites of the CS–CD beads were occupied by the surfactin that reduced the space available for adsorption [24]. Some researchers have reported that the adsorption behavior reduced in later stage because of the repulsive forces between the solute molecules of the solid and bulk phases, rendering it difficult for the remaining vacant surface sites to occupy [25]. 3.3. Antifungal activity of original chitosan, CS–CD, and SFC–CD–CS (chitosan load surfactin) Sapstain can cause a detrimental effect on the esthetic value of the wood due to the colonization by their pigmented mycelia. Generally, L. rubropurpurea, L. crassispora, and L. theobromae were considered to be the pathogenic fungi for the sapstain [1]. It has

36.22 47.28 51.42 59.72 55.57

4.0 ± ± ± ± ±

0.44 0.57 0.55 0.72 0.54

42.32 51.36 57.27 60.12 54.31

5.0 ± ± ± ± ±

0.92 0.69 0.55 1.55 0.88

38.24 41.44 48.43 52.12 41.39

± ± ± ± ±

0.51 0.99 0.55 0.87 0.22

been shown that three types of chitosan have broad-spectrum antifungal activity against a variety of sapstain pathogenic fungi (Table 2). The inhibitory effects of different concentrations of acetic acid aqueous solution on three fungi corresponding to the investigated concentrations of sample were evaluated. At low concentrations, no higher than 100 mg/L (corresponding to 200 mg/L, the concentration of tested sample), acetic acid aqueous solution clearly had no inhibitory effects on three fungi. Whereas at the concentration of 200 mg/L, acetic acid would inhibit the growth of L. rubropurpurea, L. crassispora, and L. theobromae, with effects of 10.80%, 12.45%, and 11.71%, respectively. As shown in Table 2, the SFC–CD–CS exhibited higher antifungal activity against L. rubropurpurea, L. crassispora, and L. theobromae than CS–CD and original chitosan. For L. crassispora and L. theobromae, the antifungal activity was enhanced visibly at lower concentrations. For example, the inhibitory effect of SFC–CD–CS on L. crassispora was about 41.7% at 100 mg/L, while that of original chitosan only reached 18.2 and the CD–CS reached 19.4%, respectively. On the other side, the inhibitory effect for SFC–CD–CS was higher than polyoxin (21.72%) and triadimefon (35.55%). The antifungal activity against three kinds of sapstain fungi was more pronounced at higher concentrations. The inhibitory effects on three kinds of sapstain fungi for SFC–CD–CS ranged from 70.22% to 76.22% at 400 mg/mL, whereas that original chitosan only ranged from 34.66% to 37.82% and the CD–CS ranged from 48.71% to 52.72%. Futhermore, it was worthwhile to note that the antifungal activity of SFC–CD–CS against L. rubropurpurea, L. crassispora, and L. theobromae was comparable to that of the positive control used

Table 2 Antifungal activity of original chitosan, CD-CS, and SFC–CD–CS at different concentrations. Samples

Concentrations (mg/L)

Inhibitory effect (%) L. rubropurpurea

L. crassispora

L. theobromae

Acetic acid

100 200 400

0 ± 0.62 0.62 ± 1.98 10.80 ± 1.02

0.0 ± 2.69 2.71 ± 2.74 12.45 ± 2.04

0.27 ± 1.55 2.95 ± 2.88 11.71 ± 1.65

Original chitosan

100 200 400

9.21 ± 0.97 21.95 ± 1.26 37.82 ± 1.57

18.20 ± 1.88 25.98 ± 1.76 34.66 ± 2.06

12.63 ± 1.28 24.60 ± 1.97 36.97 ± 1.22

CD-CS

100 200 400

11.26 ± 1.20 33.99 ± 1.69 51.22 ± 1.16

19.40 ± 1.97 35.25 ± 1.88 52.72 ± 1.67

11.24 ± 1.67 29.27 ± 1.99 48.71 ± 2.15

SFC–CD–CS

100 200 400

39.22 ± 1.69 61.77 ± 1.09 73.22 ± 1.77

41.70 ± 2.37 65.77 ± 1.25 76.72 ± 3.33

37.63 ± 2.39 57.68 ± 1.77 70.22 ± 2.61

Surfactin

100 200 400

27.44 ± 1.67 36.74 ± 1.98 52.67 ± 1.77

32.77 ± 1.66 46.06 ± 1.17 50.67 ± 2.07

22.15 ± 1.57 42.73 ± 2.04 55.66 ± 1.77

Polyoxin

100 200 400

19.33 ± 2.87 31.22 ± 1.96 45.79 ± 2.02

21.72 ± 1.94 42.35 ± 2.03 55.70 ± 1.47

12.77 ± 2.11 38.87 ± 3.11 51.66 ± 2.74

Triadimefon

100 200 400

27.97 ± 1.98 41.88 ± 1.22 61.22 ± 1.91

35.55 ± 1.98 55.72 ± 1.33 63.71 ± 1.98

16.77 ± 2.63 37.54 ± 2.11 50.71 ± 2.07

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(Triadimefon and polyoxin). They have higher effects and better friendly than these chemical drugs on the basis of the characteristics such as non-toxic, easily degradable, and so on. Chitosan is a biopolymer of natural origin, not toxic for human and warm-blooded animals, and could be used in medicines, health products, food industry and cosmetics [26]. The antifungal activity of chitosan is believed to occur from the interaction between the cationic chain and the negatively charged residues of macromolecules exposed on the fungal cell surface, leading to leakage of intracellular electrolytes and other constituents [27]. But the inhibitory effect of the chitosan was not obviously on the sapstain fungi (Table 2). Surfactin is an extraordinarily powerful biosurfactant that is known to decrease the surface tension of water; it exerts a detergent-like action on biological membranes, and is distinguishable by its exceptional emulsifying, foaming, antiviral and anti-mycoplasma activities [28]. However, surfactin is not widely available because of its high production cost, which results primarily from high recovery expenses [29]. Bio-absorption may be a selective process to solve the problem of recovery of surfactin and the low inhibitory effect of the chitosan. The SFC–CD–CS which embellish with ␤-CD could offer more sites (Table 1) for the surfactin and also increase the activities (Table 2). We suggest application of chemically modified chitosan together with surfactin to improve the efficacy of the inoculant as a biocontrol agent for the wood sapstain. The results above make it evident that the chitosan (with ␤CD)/surfactin combination is more effective in controlling sapstain fungi than chitosan or surfactin alone. This may well be due to the antifungal activities of both chitosan (with ␤-CD) and surfactin. However, results also indicate that the concentration of SFC–CD–CS used could stimulate three kinds of sapstain fungi growth. When the sapstain fungi were grown in the presence of SFC–CD–CS, their growth was reduced or delayed. It is well documented that B. amyloliquefaciens CGMCC 5569 [1] possess innate resistance to fungicides and other antifungal benign molecules. Once established, B. amyloliquefaciens CGMCC 5569 grows fast and rapidly captures the substrate. Furthermore, Bacillus spp. are known to have several antagonistic mechanisms including production of lipopeptide that contribute to their success as surfactin and so on. The current study confirmed our previous work where a greater inhibition of sapstain fungi with the production of B. amyloliquefaciens CGMCC 5569 was observed. Previous studies have shown that the activity depended on the lipopeptide, including surfactin and others [1]. In the present work, modificatory chitosan (SFC–CD–CS) was produced by ␤-cyclodextrins with chitosan. The improved activity of SFC–CD–CS compared to chitosan because of the chitosan adsorbed with the antifungal reagent. In addition to the applications, chitosan has been demonstrated to be useful in regulating the release of bioactive agents [30]. More remarkable, those researches were all about the drugs for human and animals, however, reagents for the control the pathogenic fungi were seldom mentioned. To the best of our knowledge, it was the first report that chitosan adsorbed the surfactin and the macromolecules could exhibit strong activities against three sapstain fungi. 3.4. The stability for SFC–CD–CS Results of pH and thermal stability shown in Fig. 3 demonstrated that the antifungal activity of the SFC–CD–CS against L. rubropurpurea, L, crassispora, and L. theobromae. The SFC–CD–CS was relatively thermostable with more than 50% of its activity being retained even after the sample was held at 121 ◦ C for 30 min (Fig. 3a). There was no difference between amounts of inhibitory effect by heat stability (>80 ◦ C). The SFC–CD–CS was susceptible for the pH value lower than or higher than 13, and the

Table 3 Inhibitory of mycelial growth (inhibitory effects) of sapstain fungi by SFC–CD–CSa treated with different enzymes. Enzyme

␣-Chymotrypsin Trypsin Pepsin Subtilopeptidase A Carboxypeptidase A Proteinase K Lysozyme ␣-Amylase Dextranase Lipase A

Inhibitory effects/% L. rubropurpurea

L. crassispora

L. theobromae

55.52 62.04 7.83 54.57 55.12 63.54 48.58 46.98 47.52 50.25

59.24 64.98 11.27 58.11 60.05 66.98 52.68 51.02 53.66 56.87

52.65 58.67 6.18 53.04 55.29 60.12 44.25 36.68 42.22 49.22

Each value is expressed as mean ± SD (n = 3). Differences were considered to be statistically significant if p < 0.05. a The sample concentration were all 200 mg/L.

antifungal inhibitory effect significantly reduced under these conditions. However, the activity remained more than 55% when the SFC–CD–CS was exposed to conditions in the pH ranging from 4 to 12 (Fig. 3b). The results of sensitivity to enzymes and protein-denaturing detergents are shown in Table 3. None of the enzymes used in this study had an effect on inhibitory effects for three kinds of sapstain fungi. SFC–CD–CS treated with enzymes inhibited sapstain fungi by 7.83–66.98%. On comparing with the effect on pH shown in Fig. 3b, none of the enzymes except the pepsin inhibited the activity of SFC–CD–CS against the sapstain fungi while SFC–CD–CS treated with enzymes for 2 h. The pH value of pepsin was 2.1, the pH environment might affect the chitosan and surfactin. Meanwhile, the inhibitory effect of SFC–CD–CS was the lowest when the pH was lower than 3 (Fig. 3b). Detergent controls such as sodium dodecyl sulphate, hexadecyltrimethyl ammonium bromide and N-hexadecyl-N, N-demethyl-3-ammonio-1-propanesulphonate inhibited the mycelial growth of sapstain fungi (Table 4). The effect of these detergents on the stability of anti-sapstain fungi activity was observed when the SFC–CD–CS was treated with Triton X-100, Tween-20 or deoxycholic acid. On the other hand, Nonidet P-40 and Triton X-100 completely destroyed the anti-sapstain fungi activity. Tween-80 did not significantly affect the activity, however, Tween-20 was slightly destructive, reducing the inhibitory activity by almost 40% (comparative with the inhibitory effects in Table 2). Table 4 Inhibitory of mycelial growth (inhibitory effects) of sapstain fungi by SFC–CD–CSa treated with different protein-denaturing detergents. Detergentc

Inhibitory effects/% L. rubropurpurea

Triton X-100 Tween-20 Tween-80 Nonidet P-40 Sodium dodecyl sulphate Deoxycholic acid Citrimide Palmityl sulfobetaine

b

0.89/1.77 0.16/26.87 0.09/59.57 0.07/0.16 0.66/63.24 1.88/62.18 0.87/63.77 1.72/64.87

L. crassispora

L. theobromae

1.72/2.59 0.25/27.58 0.61/63.77 0.11/3.85 0.71/65.75 1.76/64.74 1.69/65.71 0.63/67.08

0.66/0.74 0.77/21.77 0.36/56.75 0.19/7.58 0.76/59.11 1.02/57.98 0.66/56.39 0.47/59.51

Each value is expressed as mean ± SD (n = 3). Differences were considered to be statistically significant if p < 0.05. a The samples concentration were all 200 mg/L. b a/b means that [no SFC–CD–CS/with SFC–CD–CS]. c Triton X-100: t-Octylphenoxypolyethoxyethanol; Tween-20 = polyoxyethylene sorbitan monolaurate; Tween-80 = polyoxyethylene sorbitan monooleate; Citrimide = hexadecyltrimethylammonium bromide; Palmityl sulfobetaine = Nhexadecyl-N,N-dimethyl-3-ammonio-1-propanesulphonate.

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Fig. 3. Effect of temperature (a) and pH (b) on the antifungal activity of the SFC–CD–CS. , 䊉, and  indicate that the antifungal activity of the SFC–CD–CS against L. rubropurpurea, L. crassispora, and L. theobromae, respectively. The filled, clear, and shade bars represent the antifungal activity of the SFC–CD–CS against above three kinds of fungi, respectively.

In this study, the biological macromolecules which contained modified chitosan and surfactin could be used as a potential biocontrol agent applied to control the sapstain. This finding provides more information about the possible application of chitosan in controlling pathogenic fungi or plant diseases. It is an important industrial application for chitosan and its modified form, in addition to known activities, such as control cholesterol [31], antibacterial activities [32], and the heavy metals adsorption [33]. The effect on the anti-sapstain fungi might be provided by chitosan and surfactin, however, the mechanism of the biological macromolecules against the sapstain fungi is still unknown. So the mechanism of the biological macromolecules might be known from the future study. Furthermore, the efficiency of in vivo disease control provided should be confirmed in field trials. 4. Conclusion Biological macromolecules (CD–CS) were produced by both chitosan and ␤-cyclodextrins. As a bicontrol agent carrier, the macromolecules adsorbed the surfactin under an adaptive pH and time (pH = 4, adsorption time was 6 h, Fig. 2). High antifungal activities against three sapstain fungi L. theobromae, L. rubropurpurea, and L. crassispora were observed. The SFC–CD–CS clearly exhibit the higher activity than chitosan and surfactin itself. Meanwhile, the SFC–CD–CS is heat-stable and operate at mild pH rage. Furthermore, little of the enzymes used in this study had no effect on the anti-sapstain fungi activity, however, some protein-denaturing detergents could damage the activity of the SFC–CD–CS. The use of SFC–CD–CS presents an attractive feature because chitosan is obtained from natural raw sources, an environment friendly material of low cost. Despite the fact that SFC–CD–CS show good performance, further studies should be devoted to transfer the process to industrial scale. Also, regeneration studies need to be performed in more extent to recover the adsorbent – surfactin, enhancing the economic feasibility of the process. Acknowledgements This work has been financially supported by the grants from the Chinese National Science Foundation (31170605, 31370646), the Program of Natural Science Foundation of the Jiangsu Higher Education Institutions of China (13KJD350001), the Program of Natural

Science Foundation of the Jiangsu Normal University (12XLB04), the Science and Technology Plan Project of Xuzhou (XF13C036), the Program of National Student Innovation Training (12SSJCXZD03), the Scientific Research Foundation for Doctorate Teacher of Xuzhou Normal University (10XLR05). References [1] B. Yuan, Z. Wang, S. Qin, G.H. Zhao, L.H. Wei, J.H. Jiang, Bioresource Technology 114 (2012) 536–541. [2] A. Bruce, D. Stewar, S. Verrall, R.E. Wheatley, International Biodeterioration and Biodegradation 51 (2003) 101–108. [3] D.Q. Yang, Canada Journal of Microbiology 51 (2005) 1–6. [4] G.J. Tsai, Z.Y. Wu, W.H. Su, Journal of Food Protection 63 (2000) 747–752. [5] S. Babel, T. Kurniawan, Journal of Hazardous Materials 97 (2003) 219–243. [6] P. Jacques, in: G. Soberon-Chavez (Ed.), Biosurfactants. Microbiology Monographs 20, Springer-Verlag, Berlin Heidelberg, 2011, pp. 57–91. [7] F. Semeh, D. Krasimir, G. Frederique, V. Peggy, J. Philippe, N. Iordan, Bioresource Technology 126 (2012) 1–6. [8] M. Ongena, P. Jacques, Trends in Microbiology 16 (2008) 115–125. [9] K. Arima, A. Kakinuma, G. Tamura, Biochemical and Biophysical Research Communications 31 (1968) 488–494. [10] N. Behary, A. Perwulez, C. Campagne, D. Lecouturier, P. Dhulster, A.S. Mamede, Colloids and Surfaces B 90 (2012) 137–143. [11] C. Carrillo, J.A. Teruel, F.J. Aranda, A. Ortiz, Biochimica et Biophysica Acta: Biomembranes 1611 (2003) 91–97. [12] F. Peypoux, J.M. Bonmatin, J. Wallach, Applied Microbiology and Biotechnology 51 (1999) 553–563. [13] M. Kowall, J. Vater, B. Kluge, T. Stein, P. Franke, D. Ziessow, Journal of Colloid and Interface Science 204 (1998) 1–8. [14] C.N. Mulligan, R.N. Yong, B.F. Gibbs, Journal of Hazardous Materials 85 (2001) 111–125. [15] K.D. Schaller, S.L. Fox, D.F. Bruhn, K.S. Noah, G.A. Bala, Applied Biochemistry and Biotechnology 115 (2004) 827–836. [16] X.M. Bie, Z.X. Lu, F.X. Lu, X.X. Zeng, World Journal of Microbiology and Biotechnology 21 (2005) 925–928. [17] M. Kanlayavattanakul, N. Lourith, Journal of Cosmetic Science 32 (2010) 1–8. [18] F.C. Hsieh, M.C. Li, T.C. Lin, S.S. Kao, Current Microbiology 49 (2004) 186–191. [19] F.L. Leite, L.G. Paterno, C.E. Borato, P.S.P. Herrmann, O.N. Oliveira, L.H.C. Mattoso, Polymer 46 (26) (2005) 12503–12510. [20] F. Gancel, L. Montastruc, L. Tao, Z. Ling, Process Biochemistry 44 (2009) 975–978. [21] Z.Y. Guo, R. Chen, R.E. Xing, S. Liu, H.H. Yu, P.B. Wang, C.P. Li, P.C. Li, Carbohydrate Research 341 (2006) 351–354. [22] F.L. Mi, H.W. Sung, S.S. Shyu, Polymer 44 (2003) 6521–6530. [23] X.Z. Shu, K.J. Zhu, W.H. Song, International Journal of Pharmaceutics 5 (2001) 19–28. [24] Y. Sag, Y. Aktay, Process Biochemistry 36 (2000) 157–173. [25] R. Saravanane, T. Sundararajan, R. Sivamurthyreddy, Indian Journal of Environmental Health 44 (2002) 78–81. [26] P. Joeke, H.S. Luc, L.W. Gerrie, T.D. Ever, H.N. Els, Biological Control 48 (2009) 301–309.

14

B. Yuan et al. / International Journal of Biological Macromolecules 66 (2014) 7–14

[27] O.P. Rafael, T. Mirelle, C.C.G. Teresa, L.D.B. Vanildo, C.T. João, J.T. Marcio, A.O.T. Vera, Microbiology Research 168 (2013) 50–55. [28] J.F. Zhao, Y.H. Li, C. Zhang, Z.Y. Yao, L. Zhang, X.M. Bie, F.X. Lu, Z.X. Lu, Journal of Industrial Microbiology Biotechnology 39 (2012) 889–896. [29] M.S. Yeh, Y.H. Wei, J.S. Chang, Biotechnology Progress 21 (2005) 1329–1334. [30] J.P. Yoon, M.L. Yong, Y.L. Ju, J.S. Yang, P.C. Chong, J.L. Seung, Journal of Controlled Release 67 (2000) 385–394.

[31] A.O. Enayat, F.A. Hanan, A.N. Somaia, Regulatory Toxicology and Pharmacology 62 (1) (2012) 29–40. [32] Z.S. Cai, Y.M. Sun, X.M. Zhu, L.L. Zhao, G.Y. Yue, Polymer Bulletin 70 (3) (2013) 1085–1096. [33] F.C. Wu, R.L. Tseng, R.S. Juang, Journal of Environmental Management 91 (4) (2010) 798–806.