Purification and Biochemical Characterization of a Pathogenesis Related Endochitinase Arachis hypogaea.
Plants protect themselves by producing phytoalexins and pathogenesis related (PR) proteins in response to different physical and biological stresses as well as against pathological attack. In the present study, chitinase, a PR protein, from peanut seeds was purified to homogeneity by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-75 column and ion exchange chromatography on Q-Sepharose column. Chitinase was identified as monomeric protein with molecular mass of 30 kDa by SDS-PAGE. Chitinase activity was also evaluated on chitin agar plates showing distinct zones. The optimum pH and temperature of purified chitinase activity were found to be 5.2 and 35C, respectively. The enzyme was stable within broad pH range of 3.6 to 7.6 and up to 55C temperature.
Amongst the various substrates, acid swollen chitin was found to be the best substrate for chitinase when used at the concentration of 1% exhibiting its high specificity in catalyzing glycosidic bonds between N-acetylglucosamine residues. Chitinase activity of the purified enzyme was inhibited by Hg2+ and Ag2+ at 1 mM concentration and enhanced by Ca2+, Mg2+. The increase in the presence of 2-mercaptomethanol demonstrates the existence of sulfhydryl groups in enzyme active site. The enzyme exhibited a strong inhibitory action towards a variety of fungal species including Fusarium oxysporum, Trichoderma reesei, Aspergillus flavus and A. niger. Purified chitinase showed fungicidal properties against A. flavus on white bread and therefore, could be applied in bakery products to inhibit the formation and growth of fungal colonies.
Keywords: Antifungal activity; Chitinase; Characterization; Purification; Aspergillus flavus
Plants respond to pathogenic attack, wounding, exposure to heavy metals, salinity, cold and ultraviolet rays through different defense mechanisms. These defense mechanisms include: strengthening of the cell wall through lignin, suberin and callose deposition as well as by the synthesis of phytoalexins which are toxic to bacteria and fungi by producing Pathogenesis-Related (PR) proteins like chitinases, b-1,3-glucanases, and thaumatin-like proteins (Bowles, 1990).
Chitinases (EC 126.96.36.199) belong to families 18 and 19 of the glycoside hydrolases (Henrissat, 1991). They catalyze the hydrolysis of the b-1, 4-linkages between N-acetylglucosamine residues of chitin. Chitin is the second most abundant polymer in nature after cellulose (Tharanathan and Kittur, 2003). Chitinases have seven classes (I-VII) (Neuhaus, 1999). However, most chitinases present in leguminous plants belong to class I-IV, because information about other three classes is not known yet (Wang et al., 2009). Class I chitinases are basic and consist of N-terminal cysteine rich chitin binding domain. Class II chitinases are acidic and lack the chitin-binding domain but however are similar to class I chitinases. Class III chitinases have no similarity with class I or class II chitinases. Class IV chitinases are also not similar with class I chitinases but it is smaller in size due to four deletions (Shinshi et al., 1990).
Depending on cleavage pattern, chitinolytic enzymes are divided into two categories that are: N-acetylglucosaminidases and Chitinases. N-acetylglucosaminidases (EC 188.8.131.52.2) cause the release of terminal, non-reducing N-acetylglucosamine (GlcNAc) residues from chitin. They have the highest affinity for the dimer N, N-diacetylchitobiose (GlcNAc)2 and convert it to two monomers. Chitinases are further divided into endochitinases and exochitinases. Endochitinases degrade chitin from any point along the polymer chain forming random-sized length products, while exochitinases cleave (GlcNAc)2 from the non-reducing end of the chain (Seidl, 2008).
To date, different types of chitinases have been purified and characterized from different plants like sugar beet (El Sayed et al., 2000), soya bean (Gijzen et al., 2001), pineapple (Taira et al., 2005), pomegranate (Yang et al., 2011), and persimmon (Zhang et al., 2013).
Plant chitinases and glucanases often act together to increase antifungal activity by inhibiting the growth of many pathogenic fungi (Busam et al., 1997). Antifungal effect of chitinases have been extensively studied in many plants including mung bean (Ye and Ng, 2005), lima bean (Wang et al., 2008), cranberry beans (Wang et al., 2009), tea leaves (Nisha et al., 2011) and fava beans (Wang et al., 2012). Different types of cell wall degrading chitinases and glucanases have been successfully transferred to crops such as silver birch (Pappinen et al., 2002) and grapevine (Yamamoto et al., 2000). The objective of the present investigation as the purification and characterization of a chitinase from peanut seeds and its antifungal activity towards some pathogenic fungi.
Materials and Methods
100 grams of peanut seeds (Arachis hypogaea L), purchased from a local market, were ground to powder and soaked in 300 mL of 60 mM acetic acid for one hour on ice. The mixture was centrifuged (SiGMA(r) Laborzentrifugen) at 8,000 rpm for 30 min. pH of the supernatant was adjusted to 7.6 and placed at 4C for overnight and again centrifuged at 8,000 rpm for 30 min. The supernatant was designated as crude extract for further investigations.
Chitinase Activity Assay
Chitinase activity was checked by using 1% acid swollen chitin (0.5 g chitin soaked in 50 mM sodium acetate buffer, pH 5.2 containing 0.02% sodium azide for 2 h). The reaction mixture was prepared by adding 0.5 mL acid swollen chitin and 0.5 mL suitably diluted enzyme in a test tube following incubation at 35C for 30 min. The mixture was centrifuged at 4,000 rpm for 10 min. The reducing sugars released in supernatant were measured by DNS method (Miller, 1959). Blank contained 0.5 ml of 50 mM sodium acetate buffer (pH 5.2) instead of enzyme. One unit of enzyme activity was defined as the amount of enzyme that released 1 umol N-acetyl-D-glucosamine from acid swollen chitin per minute under the standard assay conditions.
Zymogram for Chitinase Activity
15% polyacrylamide gel electrophoresis was carried out under native condition by using the procedure described by Davis (1964). 0.5% acid swollen chitin was used as substrate for chitinase in the zymogram technique. The gel was stained with 0.1% Congo red dye for 30 min following destaining in 2M NaCl solution.
Total Protein Estimation
The protein concentration was estimated by using Bradford method with bovine serum albumin (Sigma Chemicals Co., USA) as the standard (Bradford, 1976).
Purification of Chitinase
Ammonium sulfate precipitation: The crude chitinase extract was adjusted to 80% saturation with ammonium sulfate. The precipitate was harvested by centrifugation at 8,000 rpm for 20 min. Pellet was suspended in 50 mM sodium acetate buffer (pH 5.2) and stored at 4C. The chitinase activity and protein concentration of the solution was determined as described above.
Gel Filtration Chromatography
Ammonium sulfate precipitated sample was dialyzed against 50 mM sodium acetate buffer (pH 5.2) at 4C overnight with four changes at three hours interval. Dialyzed sample was lyophilized and loaded on Sephadex G-75 (Pharmacia Fine Chemicals, Sweden) column (0.25 x 30 cm) that was pre-equilibrated with the 50 mM sodium acetate buffer (pH 5.2). The protein elution was done with the same buffer at a flow rate of 1 mL/min. Total 60 fractions (2 mL each) were collected. Chitinase activity and protein concentration of each fraction was determined.
Fractions displaying chitinase activity were pooled and subjected to ultrafiltration at 4C in an Amicon ultrafiltration cell fitted with 10,000 MW cutoff membrane (PTGC 043 Millipore Co., USA) under 50 pounds per square inch nitrogen pressure. The concentrated enzyme sample was fractionated on Q-Sepharose (Sigma Chemical Co. USA) column (1.6 x 10 cm) equilibrated with Tris-HCl buffer (pH 8.5) at natural flow rate. The bound proteins were eluted by a linear gradient of 0 to 1.5M NaCl in the same buffer. 1 mL fractions were collected by using a fraction collector Frac-100 (Pharmacia Fine Chemicals) and assayed for protein and chitinase activity.
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Homogeneity and molecular mass of the purified chitinase was estimated by (SDS-PAGE) (BIO-RAD Laboratories Inc., USA) performed by the method described by Laemmli (1970). Samples were mixed 1: 4 with sample buffer and boiled for 10 min. Gel containing standard protein size markers (Invitrogen) and samples after electrophoresis, was stained in 0.1% (w/v) Coomassie brilliant blue R-250 (Sigma), 30% (v/v) methanol and 10% (v/v) glacial acetic acid solution and destained in a solution containing 30% (v/v) methanol and 10% glacial acetic acid in distilled water. The molecular mass of purified antifungal protein was determined by comparison of its electrophoretic mobility with those of unstained protein ladder.
Characterization of Purified Chitinase
Effect of pH on activity and stability: The effect of pH on the activity of purified chitinase was estimated by maintaining the pH of assay mixtures using 1% acid swollen chitin as a substrate. Mcllvaine buffer was used for pH range 2.8 to 7.6 and Clark and Lubs buffer for pH range 8.4 to 10.
pH stability of purified chitinase was determined by incubating enzyme in buffers of different pH ranging 2.8 to 10 at room temperature for one hour without substrate. The residual activity was then determined under the standard assay conditions.
Effect of Temperature on Activity and Stability
The optimal temperature for purified chitinase was determined at different temperatures in the range of 20C to 80C by incubating a known amount of chitinase activity with 1% acid swollen chitin as a substrate in optimized buffer.
Temperature stability profile for purified chitinase was obtained by preincubating enzyme in the buffer of optimized pH at various temperatures ranging 10 to 80C for one hour prior to the addition of substrate and determining the residual activity under standard assay conditions.
The substrate specificity of the purified chitinase was investigated by incubating the enzyme with different concentrations (0.5-1.5%) of acid swollen chitin, colloidal chitin, chitotriose, glycol chitin, carboxymethyl cellulose (CMC), and chitibiose as substrate in 50 mM sodium acetate buffer (pH 5.2). Acid swollen chitin was taken as control to measure the relative activity for each substrate.
Activation and Inhibition Studies
The enzyme was pre incubated with a 1 mM concentration of different metals (MgCl2, CaCl2, CuSO4, MnCl2, AgNO3, HgCl2 and b-mercaptoethanol) at room temperature. The residual chitinase activity was measured under the standard assay conditions after one hour.
Chitinase Diffusion Assay
Well plate method was used to make wells (4 mm diameters) on chitin agar plates containing 4% agar and 1% acid swollen chitin in 50 mM acetate buffer (pH 5.2). Each well was loaded with different amounts of chitinase activity (1-3 U) while control well contained only 50 mM sodium acetate buffer (pH 5.2). The plates were incubated at 37C for 24 h. Zones of chitinase activity were observed after staining and destaining with 0.1% congo red dye and 2M NaCl, respectively.
Antifungal Activity of Chitinase
The mycelium of A. fumigatus, A. flavus, F. oxysporum, T. reesei were inoculated separately in the middle of the petri plates containing 2% malt extract agar media and incubated at 35C. After 2 days when colony diameter was 3-4 cm, sterile filter paper discs (0.5 cm in diameter) soaked in chitinase solution containing different concentrations (30, 60, 90 ug) were placed on plates at a distance of about 0.5 cm away from the edge of the growing colonies. The discs soaked in buffer without chitinase served as control. The plates were further incubated at 35C until the mycelial growth encircled the peripheral disc containing the control and had produced crescents of inhibition around the discs loaded with chitinase antifungal protein (Wong and Ng, 2005).
Chitinase as Food Preservative
Antifungal potential of chitinase against bread mold was evaluated with fresh bread pieces according to the method described by Hans et al. (2010). Fresh white bread obtained from local market was sliced approximately into 30 mm pieces. The fungal spores were spread on the bread and chitinase solution applied on the bread samples. The bread pieces without enzyme were used as controls. The pieces packed in plastic bags were incubated at 25C up to seven days.
Purification of the Chitinase
The summary of purification of chitinase is presented in Table 1. Ammonium sulfate (80%) precipitated sample after dialysis and lyophilization was subjected to Sephadex G-75 column that was pre-equilibrated with 50 mM sodium acetate buffer (pH 5.2). Chitinase activity was separated as a single peak as shown in Fig. 1. Active fractions were pooled and further purified by Q-Sepharose column chromatography. Bound proteins were eluted with a gradient of NaCl (0 to 1.5M) in Tris - HCl buffer of pH 8.5 (Fig. 2). The purified enzyme preparation had a specific activity of 101 U/mg of protein, while the final percentage recovery was 23.8.
Chitinase catalyzes the cleavage of chitin as it diffuses through the gel, leaving a light, unstaine d circular zone around the well, because the congo red dye binds only to undigested chitin. A plate clearing assay for chitinases (Zarei et al., 2012) was used to evaluate the activity of purified chitinase producing distinct zones of chitin hydrolysis as shown in Fig. 3. This chitin plate clearing test seems to be semi-quantitative since diameter of the hydrolysis zone increased with increasing enzyme concentration.
Table 1: Purification scheme of chitinase from Arachis hypogaea L. seeds
Purification Steps###Total activity (U)###Total protein (mg)###Specific activity (U/mg)###Purification fold###Yield (%)
Ammonium sulphate precipitation###305.6###32.5###9.4###1.1###80.4
After Gel Filtration Chromatography###205###6.0###34.1###4.0###53.9
After Ion Exchange Chromatography###90.5###0.9###101###12.02###23.8
Characterization of Purified Chitinase
Molecular weight determination: The molecular weight of purified chitinase was appraised to be 30 kDa on SDS-PAGE as shown in Fig. 4A. Chitinase activity was further confirmed under native conditions in 15% (w/v) polyacrylamide gel containing 0.5% acid swollen chitin as substrate following staining with congo red. Chitinolytically active enzyme was visualized as clear band on red background of the gel as shown in Fig. 4B.
Effect of pH and Temperature on Activity and Stability of Purified Chitinase
Purified chitinase was found to be the most active at pH 5.2 as shown in Fig. 5. Purified chitinase was stable in the broad pH range from 3.6 to 7.6 as shown in Fig. 5. Enzyme retained 99% of its activity at pH 4.4 and about 55% at pH 9.2. The purified enzyme was optimally active at 35C as shown in Fig. 6.
Substrate Specificity of Purified Chitinase
Polymeric substrates having glycosidic bonds were used as substrates for determination of substrate specificity of purified enzyme. Acid swollen chitin was found to be the best substrate at 1% concentration but significant activity was also obtained against colloidal chitin as shown in Fig. 7.
However, glycol chitin and chitotriose were found to be poor substrates demonstrating reduced approach of enzyme to the substrate. Activity of chitinase was very low against carboxymethyl cellulose and chitibiose even when enzyme concentration was increased up to 1.5%.
Inhibition and Activation Studies
The effect of some potential metal ions on chitinase activity was investigated in an assay system containing 1 mM of each chemical. As shown in Fig. 8, the chitinase activity was strongly inhibited by Ag+ and almost completely by Hg2+. During present studies, the presence of Mg+2 and Ca+2 enhanced 30 and 15% chitinase activity, respectively when used at a concentration of 1 mM in the reaction mixtures.
Antifungal Activity of Chitinase
Antifungal assays were performed using major phytopathogenic fungi. The purified chitinase showed antifungal activity by forming a crecent at concentrations 30, 60, and 90 ug against A. fumigatus, T. reesei, A. flavus, and F. oxysporum as shown in Fig. 9.
Chitinase as Food Preservative
Antifungal potential of chitinase was assessed as a food preservative by applying purified enzyme on bread infected with A. flavus as white bread is mainly vulnerable by fungal pathogens, particularly the species of genus Penicillum, Aspergillus, Fusarium, and Rhizopus (Lavermicocca et al., 2003; Guynot et al., 2005). The fungal spores and antifungal protein when used in different combinations at a concentration of 60 ug of purified chitinase produced maximal inhibitory effect on the growth of A. flavus (Fig. 10).
Chitinases are very important enzymes that are distributed in bacteria, fungi, plants and animals. These are produced under normal conditions as well as a result of different environmental and developmental factors in plants. Due to compact structure, enzymes are stable at extreme conditions of pH and temperature. Chitinases are also resistant to proteolytic degradation and remain soluble and stable under such conditions retaining their antifungal activities and surviving ability during food processing (Gorjanovic, 2009).
Enzyme purification is essential for understanding the mode of action and substrate specificity of an enzyme. Product inhibition studies and calculation of Michaelis-Menten constants can be carried out with purified. Many other workers have reported multistep procedure for purification of chitinase. Ye and Ng (2005) purified a chitinase after three chromatographic steps (CM-Sephadex C-50, POROS HS and Sephadex G-75 column chromatography) from mung beans to 3.9 folds and Taira et al. (2005) purified chitinase from pineapple leaf after four steps such as chitin affinity column chromatography, Butyl-Toyopearl 650M column chromatography, Resource Q column chromatography, HPLC on Phenyl superpose column.
A plate clearing assay was used to evaluate the activity of purified chitinase producing distinct zones of chitin hydrolysis as shown in Fig. 3. Chitinase catalyzes the cleavage of chitin as it diffuses through the gel, leaving a light, unstained circular zone around the well, because the congo red dye binds only to undigested chitin. During current trials, chitin plate clearing test seemed to be semi-quantitative since diameter of the hydrolysis zone increased with increasing enzyme concentration. Plate assay method was also performed by Zarei et al. (2012) to examine the active fungal strains producing chitinases.
Chitinases of plant origin are generally endochitinases and possess molecular masses in the range of 25 to 35 kDa (Sahai and Manocha, 1993). Similar molecular weight chitinases are reported by Patel et al. (2010), Yang and Gong (2002), Ye and Ng (2005) from Ipomoea carnea latex (30 kDa), Hydrangea macrophylla (30 kDa) and Phaseolus mungo seeds (30.8 kDa), respectively. However, low molecular weight chitinase were also purified from Vicia faba seeds (25.5kDa) (Wang et al., 2012) and P. limensis seeds (28.6 kDa) (Wang et al., 2008). Many researchers have reported higher molecular weight of chitinase contrary to present studies i.e., Beta vulgaris (64 kDa) (El sayed et al., 2000), leaf rachises of Cycas (40 kDa) (Taira et al., 2009), Astragalus membranaceus seed (35 kDa) (Kopparapu et al., 2011) and Camellia sinensis leaves (34 kDa) (Nisha et al., 2011).
pH is one of the major factors affecting tertiary and quaternary structure of proteins and enzymes. Since enzymes are amphoteric in nature, therefore possess both acidic and basic amino acids. Based on dissociation constants and pH of the environment, these varying charges on amino acids affect the overall charge along with their peripheral distribution thus influencing the activity, stability and solubility of enzymes (Chaplin and Bucke, 1990). Chitinase purified during present studies showed optimum activity at 5.2, which is in close conformity to the findings of Wang et al. (2008) and Santos et al. (2004) who purified a chitinase from lima bean and Adenanthera pavonina seeds showing maximum activity at pH 5.4 and 5.5, respectively. While chitinase obtained from Oryza sativa possessed pH optimum of 5.5-6.5 (Xayphakatsa et al., 2008). On the contrary, purified chitinases, reported by Bansode and Bjekal (2005) and Kuzu et al. (2012) exhibited maximum activity at pH 9.
Since purified chitinase showed pH optima in the acidic range, therefore this enzyme could possibly be exploited for control of fungal plant pathogens (Moore et al., 2004). Some chitinases are reported displaying even broader pH range. Patel et al. (2010) purified a chitinase from Ipomoea carnea latex that was stable from pH 4.0 to 9.5, while chitinase purified by Kopparapu et al. (2011) had been shown to maintain its activity in the pH range of 3.0-9.0.
Temperature is one of the critical constraints that regulate the enzyme activity. The resistance of enzymes to thermal denaturation is due to the presence of hydrophobic interactions, hydrogen bonding and ionic stabilization in polypeptide chains (Voordouw et al., 1976). During contemporary trials, 35C was found to be optimal temperature for chitinase, which is in accordance with the results reported by Molano et al. (1979) from wheat grains. Research papers are also present on chitinases that possess high optimum temperatures like P. mungo seed chitinase (40-50C) (Ye and Ng, 2005), Oryza sativa chitinase (35 to 40C) (Xayphakatsa et al., 2008), Ipomoea carnea latex (50C) (Patel et al., 2010), Vicia faba seed chitinase (50C) (Wang et al., 2012) and A. membranaceus seed chitinase (50C) (Kopparapu et al., 2011).
Thermostable chitinase could be useful for the chitin industry and biotechnological applications; therefore, the thermostability of purified chitinase was examined at various temperatures. Such studies demonstrated that activity of purified chitinase was preserved up to 55C when incubated for one hour (Fig. 6). Earlier reports have also revealed comparable thermal stability of chitinases i.e., peanut seeds below 50C (Wang et al., 2008). However, Patel et al. (2010) have reported even high thermally stable peanut chitinase (up to 80C) from Ipomoea carnea. Wide-ranging pH and temperature stability is criterion of enzymes for their industrial applications as many biotechnological processes occur at high temperature and at acidic or basic pH values (Antranikian, 2009).
Chitinase is specific in hydrolyzing the compounds that contain glycosidic linkage. Ikeda et al. (2009) have also reported that amongst different insoluble substrates used for substrate specificity studies, colloidal chitin was more effectively digested by chitinase purified from Pennahia argentatus.
Generally, the presence of various chemicals results in inhibition or activation of enzymes. Rouvinen et al. (1990) suggested that enzyme inhibition might be due to the binding of metal ions to sulfhydryl groups of cysteine residues playing a significant role in maintaining the integral active site structure of enzymes. Xia et al. (2001) have described the different prospects of Hg2+ impact on enzyme. The ions can bind the sulfhydryl or carboxyl groups of amino acids or interacts with tryptophan residues as such. Furthermore, in other cases metals can lead to the dearth or substitution of indispensable ions in metalloproteins (Van Assche and Clijsters, 1990). Kim et al. (2003) have reported inhibitory effect of Ag+ and Hg2+ on chitinase similar to present studies, purified from Streptomyces sp. M-20 when used at 1 mM concentration. The mechanism by which metal ions enhance the reaction is through their binding to the substrates so as to orient them properly for enzymatic reaction.
Metal ions may facilitate the reaction through the charge shielding of bound substrates (Villafrance, 1992).
Interestingly, in contrast to our studies, Ca2+ and Mg2+ partially inhibited Serratia marcescens chitinase while Mn2+ enhanced enzyme activity at 1 mM concentration (Zarei et al., 2011). Five chitinases purified from different microorganisms, Bansode and Bjekal (2005) reported that Ca2+ remarkably increased the chitinase activity as compared to other metal ions used. This study also reported that enzyme activity of purified chitinase was enhanced (21%) in the presence of 2-mercaptomethanol in the assay mixture suggesting the presence of SH-groups in active site of the enzyme. These findings were further established when purified enzyme was found to be inhibited in the presence of divalent ions such as HgCl2 and such metal cations enzyme inhibition revealed the presence of at least one Cys residue in the enzyme active site (Rouvinen et al., (1990). Present results also agree with earlier findings reported previously by Kim et al. (2003) and Ueno et al. (1990).
Fungi are widely distributed in every ecological unit. They have ability to inhabit plants, human and animals as their substrates. The previous studies revealed that fungi are the major threat to immune-compromised patients and their infection lead to the death of the patient. However, these infections can be suppressed by the development of antifungal that have the ability to prevent such infections (Shoham and Levitz, 2005). Chitinases occupy a distinct place in agricultural biotechnology because their lytic activity inhibit fungal development by degrading chitin and glucan components of cell wall and have proven their potential as antifungal agents (Punja and Zhang, 1993; Jung et al., 2005). Antifungal effect of different plant chitinases has been studied. Kirubakaran and Sakthivel (2007) purified a 35 kDa chitinase exhibiting antifungal activity against B. cinerea, P. theae, B. oryzae, Alternaria sp., C. lunata and R. solani.
A 28 kDa chitinase purified from maize seeds had the potential to inhibit the growth of T. reesei, A. solani and F. oxysporum (Huynh et al., 1992). Ye and Ng (2005) isolated chitinase from mung beans with antifungal activity against F. oxysporum, M. arachidicola, P. aphanidermatum and S. rolfsii.
Besides the advancement in scientific knowledge, food industry is still facing unsolved food spoilage problems due to incursion of molds and spores. The use of conventional chemical preservatives or other physical procedures in food items have their own inherent disadvantages. Hence to evade chemical preservation methods, natural plant extracts could be promising alternative; therefore, to explore new plants materials with antifungal properties have become an interesting research area in food industry (Irkin and Korukluoglu, 2007). The cell wall of the fungi contains mostly chitin along with other polysaccharides and chitinase enzymes break down chitin of fungal cell wall by hydrolyzing its ss-(1, 4) glycosidic linkages, therefore directly prevent germination and growth of molds and fungi. Hans et al. (2010) recounted that antifungal activity of oat (Avena sativa) seed extracts exhibited its great potential to prevent and repress the spore germination of P. roqueforti, when applied on rye bread.
Moreover Fig leaves extracts exhibiting both antifungal and antibacterial activities against detrimental microbes in foods have been reported previously (Hu et al., 2007; Balestra et al., 2009; Oliveira et al., 2009; Aref et al., 2010).
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