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Screening of Inulinolytic Potentialities of some Fungi Isolated from Egyptian Soil



Noura El-Ahmady El-Naggar, E.A. Metwally, A.B. El-Tanash and A.A. Sherief
 
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ABSTRACT

Forty five soil samples from different sites of non-cultivated soil, salt marshes and rhizosphere of some wild and cultivated soils collected from seven governorates of Egypt were used as local source for isolation of some inulinolytic fungi. Forty six fungal species belonging to three sub-divisions of Zygomycotina, Ascomycotina and Deutromycotina were identified. Eight unknown filamentous fungi characterized by their black reverse color on PDA medium and Czapek agar plates were recorded. Frequency of occurrence indicates that Aspergillus foetidus var. pallidus (frequency 52.2%) was the most dominant followed by A. sclerotiorum (39.1%). All fungi were able to grow on medium containing sugar cane bagasse and Jerusalem artichoke tubers powder (1:1). No significant correlation observed between the fungal growth, liberated soluble protein and inulinase activities. Inulinase activity indicate that Aspergillus foetidus var. pallidus (564.71±1.22 Ugds-1), A. sclerotiorum (534.78±1.37 Ugds-1), Emericella nidulans (495.73±3.85 Ugds-1) and A. aculeatus (444.37±2.37 Ugds-1) were the most active fungal species able to produce a considerable amount of enzyme activity.

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Noura El-Ahmady El-Naggar, E.A. Metwally, A.B. El-Tanash and A.A. Sherief, 2014. Screening of Inulinolytic Potentialities of some Fungi Isolated from Egyptian Soil. Biotechnology, 13: 152-158.

DOI: 10.3923/biotech.2014.152.158

URL: https://scialert.net/abstract/?doi=biotech.2014.152.158
 
Received: April 12, 2014; Accepted: May 09, 2014; Published: May 06, 2019



INTRODUCTION

Inulin is a widespread naturally occurring polyfructan, occurs as a reserve carbohydrate in bulbs, tubers and roots of many plant families including; Liliaceae, Amaryllidaceae and Asteraceae (Carpita et al., 1989; Chi et al., 2011). It consists of linear chains of β 2, 1-D fructofuranose molecules terminated with a glucose residue at the reducing end (Lopez-Molina et al., 2005). Such inulin sources have received a great interest as they represent relatively inexpensive and abundant substrates for the microbial production of high fructose syrup which has gained importance in food, drink and pharmaceutical industries (Zhao et al., 2010). Fructose syrup has beneficial effects in diabetic patients, increases the iron absorption in children, has high sweetening capacity so it can be used in the diet of obese persons, stimulates calcium absorption in postmenopausal women, stimulates growth of bifidobacteria in large and small intestine, prevents colon cancer (Rocha et al., 2006).

The use of microbial inulinase (β-D-fructan fructanohydrolase) for hydrolysis of inulin containing extracts for the production of high fructose syrup has been reported by Chen et al. (2011a). Microorganisms including, Penicillium, Aspergillus and Kluyveromyces are the most industrial strains that used as inulinase producers (Pandey et al., 1999). Their inulinases are classified into exo-inulinases (EC 3.2.1.80) that split terminal fructose from the non-reducing end and endo-inulinases (EC 3.2.1.7) that hydrolyze the internal β-(2→1)-fructofuranosidic linkages of inulin (Chi et al., 2011). Traditionally, inulinases are produced by submerged fermentation (Gill et al., 2003). Inulinase can also be produced by solid-state fermentation (SSF) (Xiong et al., 2007).

Increasing potential of microbial inulinase applications for the production of ultra-high-fructose syrup, inulo-oligosaccharide, bioethanol, single-cell protein, citric acid, 2, 3 butanediol, lactic acid and sugar alcohols (Yun et al., 1997; Saha, 2006; Ricca et al., 2009; Liu et al., 2010; Lim et al., 2011; Chi et al., 2011) are promoted us to screen for new inulinase-producing microorganisms. In the present study, an attempt try to isolate and identify some inulinase-producing filamentous fungi from the soil and rhizosphere of some plants for further screening of their inulinase producing potentialities by solid state fermentation technique.

MATERIALS AND METHODS

Collection of soil samples: Forty five soil samples were collected from different sites of non-cultivated soil, salt marshes and rhizosphere of wild and cultivated soils were during 2009 from seven governorates of Egypt, Dakahlia, Kafr El-Sheikh, Beheira, Alexandria, Fayoum, Monofeya and South Sinai.

Isolation and purification of inulinolytic fungi: The fungal species were isolated according to method of Warcup (1950). The 25 g of each soil sample was suspended in 225 mL of sterilized distilled water (1:10; 10-1 dilution) and subsequently 10 mL of this suspension were added to 990 mL of sterilized distilled water. Petri-dishes containing inulin agar medium according to Nakamura et al. (1997) plus chloranphenicol (100 mg L-1) were inoculated with 1.0 mL of the 1:1000 diluted soil suspensions. The inoculated plates were incubated at 28±2.0°C for 3 days. Morphological appearances of the plates were observed and fragments of distinct colonies were transferred separately to the same inulin agar medium to obtain pure isolates which were then maintained and stored at 4°C.

Identification of the fungi: Pure isolated fungi were confirmed for identification through Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Egypt, by observing its macroscopic characteristics (colour, texture appearance and diameter of the colonies) and microscopic (microstructures), according to Domsch et al. (1993): For soil fungi, Zycha and Siepmann (1973) for families, genera and species of Mucorales, Baijal and Mehrotra (1980) for genus Cunninghamella, Schipper and Stalpers (1984) for genus Rhizopus, Raper and Fennell (1965, 1973) and Samson (1992) for the genus Aspergillus and its teleomorphs, Pitt (1979) for the genus Penicillium and its teleomorphic states of Eupencillium and Talaromyces, Barron (1977) for genera of Hyphomycetes from soil, Booth (1971) for Fusarium species and Ellis (1971, 1976) for dematiaceous Hyphomycetes.

Basal medium: The basal medium (free of carbon) used in experiments was contained the following (g L-1 of acetate buffer, 0.1 M, pH 4.8): peptone, 5.0, NH4H2PO4, 8.0, (NH4)2HPO4, 4.0, KCl, 0.5, MgSO4.7H2O, 0.5 and FeSO4.7H2O, 0.01 according to Nakamura et al. (1997).

Inoculum preparation: Fungal cultures were sub-cultured on modified agar media containing 1.0% inulin as a sole carbon source, at 30°C for 5.0 days and maintained at 4°C. Induced slant was mixed with 10 mL sterile basal medium for preparing spore suspension. The spore count in the suspension was about 2.0x107 spore mL-1.

Solid state fermentation: Fermentation was carried out in 250 mL Erlenmeyer flask with 0.1 g mixed substrate of sugar cane bagasse and Jerusalem artichoke tubers powder by a ratio of 1:1, mixed with 2.0 mL of freshly prepared basal medium (0.1 M acetate buffer, pH 4.8). The flasks were autoclaved at 121°C at 15 lbs for 20 min after cooling each flask was inoculated under aseptic conditions and distributed carefully with 2.0 mL spore suspension previously prepared (final moisture content was 80%) and incubated for 5.0 days at 30°C under static conditions.

Extraction of inulinase: The fermented substrates were mixed and homogenized well with 50 mL of 0.1 M acetate buffer (pH 4.8). The mixture was shaken thoroughly on a rotary shaker (150 rpm) at room temperature (20±2°C) for 60 min. The mixtures were filtered off through muslin cloth; then centrifuged at 5000xg for 10 min. After centrifugation, the supernatant was collected and stored in a deepfreezer (-20°C) until its use as a crude inulinase (Chen et al., 2011b).

Inulinase assay: The inulinase activity was determined by measuring the reducing sugars released by the hydrolysis of inulin according Smogyi (1952) method. The assay mixture for inulinase containing 0.1 mL of crude enzyme and 0.1 mL of 0.1% (w/v) inulin (freshly prepared in 0.1 M acetate buffer; pH 4.8). The reaction mixture was incubated at 40°C for 30 min, then, liberated reducing sugars were estimated at 700 nm using Spectro UV-Vis RS spectrophotometer. One unit (U) of inulinase was defined as the amount of enzyme that released one μmol of fructose per minute from inulin at 40°C and other assay conditions.

Estimation of soluble protein: The soluble protein concentration was determined by the method of Bradford (1976), by measuring the optical density of the color against blank at 595 nm using spectro UV-Vis RS spectrophotometer. The protein concentration was calculated using μg standard curve of bovine serum albumin (BSA).

Statistical analysis: The analysis of data was done by using the statistical software Graph-Pad Prism 4. Each experiment was repeated three times (minimum) and the data are means of triplicate determinations. Results with probability levels greater than 5% were regarded as non-significant.

RESULTS AND DISCUSSION

The increased potential of inulinase applications (production of high fructose syrups, fructooligosaccharides, ethanol and inulo-oligosaccharides which are extensively used in pharmaceutical and food industry), promoted us to search for new sources of inulinases and hence microorganisms are the best sources for commercial production of inulinases because of their easy cultivation and high yields of the enzyme, our attempt was made to isolate and identify inulinase-producing filamentous fungi from the soil and rhizosphere of some plants and screening their inulinase producing potentialities.

In this study approximately 380 filamentous fungal isolates (Table 1) were isolated by dilution plate method on inulin agar medium, the number of isolates and its biodiversity are restricted by inulin (1.0%) used in isolation medium (sole carbon source). The rhizospheres of inulin rich plants including; Jerusalem artichoke (Helianthus tuberosus L.) (Abuzahran site, Beheira) and Allium cepa, (Sinbillawain; Dakahlia) were the richest soils (24, 22 isolates, respectively) compared with the others. This high fungal occurrence may be attributed to the bioactive materials of root exudates (sugars, organic acids and amines…) which released into the plant rhizosphere during their life cycle which enhance plant growth and fungal populations (Westover et al., 1997) furthermore this may also due to the release of root fragments that represent potential substrates for fungi, being therefore, a way of these microorganisms to obtain inulin as a carbon source. In this connection, De Souza-Motta et al. (2003) reported the distribution of about 50 fungal species from rhizosphere of Asteraceae plant. By contrast, the least number of isolates (3 isolates) were obtained from salt marshes soils of Baltim (Kafr El-Sheikh) and Burj Al Arab (Alexandria), due to high salt concentrations which interfere or prevent the fungal population as well as plant growth. Furthermore, decreasing the number of isolates in the non cultivated salt marshes may be also prohibited by the presence of inulin in isolation plats as sole carbon source.

The isolates were classified and identified as 46 isolates by Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo, Egypt. Results (Table 2) showed 38 fungi were identified to the species level while, 8 species (not completely identified) belonging to deuteromycotina were recorded by their isolation code numbers. The well identified species belonged to three sub-division called Zygomycotina (4 species), Ascomycotina (29 species) and Deutromycotina (5 species). This predominance of Deuteromycetes and Ascomycetes are in agreement with results obtained by Garrett (1976) and De Souza-Motta et al. (2003) where, they isolated about 49 fungal species belonging to Ascomycota, Deuteromycota and Zygomycota from the rhizospheres of sunflower cultivated in field and in greenhouse. On the other hand Santos et al. (1989) reported the predominance of Deuteromycota in sugar cane rhizosphere, by analyzing 142 samples obtained from 22 plantation sites, spread over several regions of the Pernambuco State (Brazil).

The frequency of fungal occurrence showed that Aspergillus foetidus var. pallidus (frequency 52.2%) was the most prevalent followed by Aspergillus sclerotiorum (39.1%), Aspergillus aculeatus (34.8%) and Emericella nidulans (34.8).

Table 1:Occurrence of inulinase producing fungal isolates from soils and rhizosphere of some wild and cultivated crops collected from different sites of seven Egyptian governorates, Dakahlia, Kafr El-Shaikh, Beheira, Alexandria, Fayoum, Monofeya and South Sinai
Image for - Screening of Inulinolytic Potentialities of some Fungi Isolated from Egyptian 
  Soil
N.B: Total isolates (T) = 380, No. of isolates (N) and Percentage: (N/T)x100, Si: El- Sinbillawain, Ma: Mansoura, BO: Bani Obeid, Ba: Baltim, Ha: Hamoul, MA: Minshat Abdullah, Kh: Elkhatatba, RM: Ras Mohamed, protected area, AZ: Abu Zahran, Ga: Ganaklis, BuA: Burj Al Arab

Table 2:List of identified inulinase producing fungi and their frequencies after isolation from soils and rhizosphere of some wild and cultivated crops from different sites of seven Egyptian governorates of El-Dakahlia, Kafr El-Shaikh, El-Beheira, Al-Alexandria, El-Fayoum, El Monofeya and South Sinai
Image for - Screening of Inulinolytic Potentialities of some Fungi Isolated from Egyptian 
  Soil
N.B: Total isolates (T) = 380, Identified isolates (I): 46, Repetition of isolates (R), Frequency (FI): (Repetition/Identified isolates)x100, Frequency (FT): (Repetition/Total isolates)x100

Table 3: Inulinase activities and protein conc. produced by different isolated fungi
Image for - Screening of Inulinolytic Potentialities of some Fungi Isolated from Egyptian 
  Soil

Dominance of Aspergillus species in the rhizosphere of inulin rich plants were reported by Bonciu et al. (2010).

The results (Table 3) showed that, all fungi were able to grow on the selective medium with different levels. However, no significant correlation between the fungal biomass (data not recorded) and inulinase activities in such studied fungi. Aspergillus foetidus var. pallidus (564.71±1.22 Ugds-1), A. sclerotiorum (534.78±1.37 Ugds-1), Emericella nidulans (495.73±3.85 Ugds-1), A. aculeatus (444.37±2.37 Ugds-1), Penicillium wortmannii (304.00±1.22 Ugds-1), A. brevipes (297.51±2.94 Ugds-1), A. pseudo-niger (252.36±1.78 Ugds-1) and A. fumigatus var. ellipticus (220.19±3.86 Ugds-1) were the most active fungal species able to produce a considerable amount of active inulinase. While, moderate or little inulinase activities were detected by some fungi (001.84±0.04-191.12±1.20 Ugds-1). About 10 species were negative results for inulinase productivity under the studied fermentation conditions. These results are in agreement with that obtained by fungi isolated from Asteraceae rhizosphere with some different behavior (De Souza-Motta et al., 2003); this behavior may attribute to the genetical and physiological characteristics among strains of the same fungal species. AbdAl-Aziz et al. (2012) reported that Aspergillus and Penicillium species isolated from rotten Jerusalem artichoke tubers are the most potent inulinase producers.

Among 46 isolated species more than 78% (36 from 46 species) were inulinolytic fungi. The most common genera were Aspergillus, Emericella and Penicillium. In this connection Onodera and Shiomi (1992) and Kumar et al. (2005) reported P. trzebinskii and A. niger as inulinase producers. In the present study, A. foetidus var. pallidus and A. sclerotiorum are recorded as the highest inulinase producer as compared with the other species after 4.0 incubation days. Jing et al. (2003) showed that natural inulin from Jerusalem artichoke tuber powder is believed to be an inducer for inulinase production, it induces much more inulinase over that observed with fructose. Therefore, our results reported that inulinase is an inducible enzyme; it induced by its substrate as reported by Saber and El-Naggar (2009) and El-Hersh et al. (2011). The advantage of using natural inulin degrading fungi isolated from nature habitats over the genetically manipulated technique is the easier adaptation and succession, such isolate can be effectively used for biotransformation of natural inulin to mono-sugars. So, we used both A. foetidus var. pallidus and A. sclerotiorum as inulinase producers for further studies.

CONCLUSION

In this study, approximately 380 filamentous fungal isolates belonging to three sub-divisions, Zygomycotina, Ascomycotina and Deutromycotina were screened for their inulinolytic potentialities, the results revealed that Aspergillus foetidus var. pallidus (564.71±1.22 Ugds-1), A. sclerotiorum (534.78±1.37 Ugds-1), Emericella nidulans (495.73±3.85 Ugds-1) and A. aculeatus (444.37±2.37 Ugds-1) were the most active fungal species able to produce a considerable amount of enzyme activity.

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