Investigation of Secondary Metabolites and its Bioactivity from Sarocladium kiliense SDA20 Using Shrimp Shell Wastes

Utilization of shrimp waste an alternative technology to replace dangerous chemical methods that addresses the future trends in total utilization of shrimp waste for recapture of bioactive compounds. Low molecular weight fungal secondary metabolites produced by a wide variety of fungi. These compounds determined as mycotoxins.1,2 Mycotoxins are a varied group of compounds produced by an extensive range of different fungi, normally after a period of balanced growth.3 Fungi are capable to produce one or more mycotoxin. Therefore, several mycotoxins are often at the same time found in a single product.4 Many organisms produce large numbers of secondary metabolites and the complexity and diversity is sometimes amazing. Secondary metabolites are organic compounds can be used in traditional medicine and in modern times, as pharmaceuticals, flavoring in foods, fragrances in cosmetics, agrochemicals and drinks, etc.5,6 Filamentous fungi can be cultivated beneath in fermentation medium contain nutrients for fungal growth, changing in this medium composition affects the biomass yield and quality as well as metabolites.7 The high cost of synthetic media has interested to use another new fermentation media designed from inexpensive by-products and wastes, which are able to support and fulfill nutritional requirements for microbial growth.8 Substrates used for cultivation of fungi must be easily biodegradable, easily available, cheap, and contain sufficient required micro and macro-nutrients.9 Shrimp aquaculture is an attractive economic activity of great impact and commercial importance.10 The waste generated from the worldwide production and processing of shell-fish and fish scales is a serious problem of growing magnitude. This abundant waste may pose environmental hazard due to the easy deterioration. The use of shrimp shell waste due to their low commercial value and applications of these residues can be used as good substrate by fungi for production of secondary metabolites from cheap and low-cost substrate.11 The waste created from the overall generation and preparing of shellfish and fish scales is difficult issue of developing greatness. Recently, bacterial biofilm considered as one of the major public health problems. This process gives bacteria the ability to maintain their attachment to living and non-living surface and to keep the bacterial cells together by production of a complex exopolymers formed from proteins, polysaccharides and nucleic acids.12-14 Several reports have now clearly confirmed that, biofilm play a significant role in the persistence of pathogenic bacteria.15 Biofilms are consideded as a source of pathogenic bacteria ABSTRACT Introduction: In this study we isolated bioactive compounds using different chromatographic techniques from culture filtrate of Sarocladium kiliense SDA20 grown in fermentation media containing shrimp shell waste as substrate under optimum conditions. Antibacterial and antibiofilm activities of crude extract and purified compounds were evaluated. Methods: The test fungi strain Sarocladium kiliense SDA20 was isolated from Egyptian soil and identified by18 S ribosomal RNA. Optimization conditions were carried out in fermentation media containing shrimp shell waste as sole carbon source, inoculated by 106 spores/ml of Sarocladium kiliense SDA20 at pH 7.0 produce 84.5% of the total toxins. Different chromatographic techniques for ethyl acetate extract of culture filtrate of fungi were used resulting in isolation of pure compounds were elucidated spectroscopically and comparing their data in literature. GC/MS analysis of extract was used for identification of other chemical compounds. Antibacterial and biofilm activity was evaluated using MTT assay. Results: Five compounds for the first time were identified: Cholest-5-en-3-ol (C1), Palmitic acid (C2), Oleic acid (C3), Nicotinamide (C4), Tricin (C5). GC-MS analysis showed the presence of twenty-seven compounds. Antibacterial activity of crude and pure compounds displayed a strong inhibitory activity against Bacillus subtilis. C1, C4, C5 showed moderate activity against Escherichia coli, followed by C2, C3. Meanwhile, Staphylococcus aureus was less susceptible to pure compounds with low activity of C3, C5. The crude extract and pure compounds displayed biofilm inhibition activity against four pathogenic bacterial strains. Conclusion: Shrimp shell wastes are one of the economic bioproducts used for the production of bioactive secondary metabolites from fungi.


INTRODUCTION
Utilization of shrimp waste an alternative technology to replace dangerous chemical methods that addresses the future trends in total utilization of shrimp waste for recapture of bioactive compounds. Low molecular weight fungal secondary metabolites produced by a wide variety of fungi. These compounds determined as mycotoxins. 1,2 Mycotoxins are a varied group of compounds produced by an extensive range of different fungi, normally after a period of balanced growth. 3 Fungi are capable to produce one or more mycotoxin. Therefore, several mycotoxins are often at the same time found in a single product. 4 Many organisms produce large numbers of secondary metabolites and the complexity and diversity is sometimes amazing. Secondary metabolites are organic compounds can be used in traditional medicine and in modern times, as pharmaceuticals, flavoring in foods, fragrances in cosmetics, agrochemicals and drinks, etc. 5,6 Filamentous fungi can be cultivated beneath in fermentation medium contain nutrients for fungal growth, changing in this medium composition affects the biomass yield and quality as well as metabolites. 7 The high cost of synthetic media has interested to use another new fermentation media designed from inexpensive by-products and wastes, which are able to support and fulfill nutritional requirements for microbial growth. 8 Substrates used for cultivation of fungi must be easily biodegradable, easily available, cheap, and contain sufficient required micro and macro-nutrients. 9 Shrimp aquaculture is an attractive economic activity of great impact and commercial importance. 10 The waste generated from the worldwide production and processing of shell-fish and fish scales is a serious problem of growing magnitude. This abundant waste may pose environmental hazard due to the easy deterioration. The use of shrimp shell waste due to their low commercial value and applications of these residues can be used as good substrate by fungi for production of secondary metabolites from cheap and low-cost substrate. 11 The waste created from the overall generation and preparing of shellfish and fish scales is difficult issue of developing greatness. Recently, bacterial biofilm considered as one of the major public health problems. This process gives bacteria the ability to maintain their attachment to living and non-living surface and to keep the bacterial cells together by production of a complex exopolymers formed from proteins, polysaccharides and nucleic acids. [12][13][14] Several reports have now clearly confirmed that, biofilm play a significant role in the persistence of pathogenic bacteria. 15 Biofilms are consideded as a source of pathogenic bacteria that are involved in many infectious diseases. 16,17 Despite several attempts to find drugs that specifically target bacterial biofilms, still there is undeniable need to discover anti-biofilm agents that could inhibit bacterial biofilm formation. The aim of this study was isolation of bioactive compounds using different chromatographic techniques from culture filtrate of Sarocladium kiliense SDA20 accession number (MN636431) grown in fermentation media containing shrimp shell waste as substrate under optimum conditions. Then evaluation the biological activities (antibacterial activity and antibiofilm activity) of the crude extract and the purified compounds.

Microorganisms
The test fungal strain Sarocladium kiliense SDA20 was isolated from the Egyptian soil at El Behira province and identified by18 S ribosomal RNA. Cultures were kept on potato dextrose slants and kept at 4 °C.

DNA extraction
The molecular identification of the selected fungal strain was carried out by sequencing of the 18srRNA gene. The extraction of the genomic DNA was performed using Qiagen DNeasy Mini Kit following the manufacturer's manual.

Sequencing
The amplified products were examined by electrophoresis and sequenced in Macrogen Companies, South Korea. The sequence produced was analyzed by using BLASTN program, to study the similarity and homology of the 18S rRNA gene sequences with the similar existing sequences available at NCBI database.

Substrate used
Shrimp shell waste was collected, washed and dried in oven at 70 • C, then grinded, stored until used.

Effect of different inoculum size on metabolites production
Different inoculum size ranged from (10 4 , 10 6 , 10 8 &10 10 ) spores/ml were examined to determine total secondary metabolites production.

Effect of different initial pH values on metabolites production
Different initial pH values of fermentation media for compounds production were adjusted at (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5& 8.0) using dilute HCl or NaOH solutions. Inoculated medium was then incubated for 7 days.

Instruments
NMR spectra were recorded on a Jeol Ex-500 spectrometer: 500 MHz ( 1 H-NMR), 125 MHz ( 13 C-NMR). UV spectro photometer (Shimadzu UV-240). ESI-MS were recorded on a Waters-Micromass Quattro Premier Triple Quadrupole mass spectrometer. Column chromatography (CC) was carried out on silica gel F254 (Merck) in glass blades. TLC was performed with silica gel 60 GF254 plates (Merck, Darmstadt, Germany), then the plates were visualized using UV light and by spraying with vanillin in H2SO4. The GC-MS was performed using the GC-MS system (Agilent Technologies), equipped with gas chromatograph (7890B) and mass spectrometer detector (5977A). The GC was equipped with HP-5MS column (30 m x 0.25mm internal diameter and 0.25 μm film thickness).

Preparing extract
The culture filtrate (700 g) of Sarocladium kiliense SDA20 was extracted with ethyl acetate till exhaustion. The extract was evaporated under reduced pressure to yield (30 g) of crude extract. A portion of this extract was subjected to column chromatography for compounds isolation, part was kept for GC-MS analysis, and other was stored for biological study.

Isolation of the compounds
The crude extract was subjected to silica gel column chromatography, eluted with petroleum ether and ethyl acetate step-gradient, starting with100% petroleum ether with increasing polarity, in the ratio (100:0, 9:1, 8:2,7:3, 6:4, 4:6, 3:7, 2:8, 1:9, 0:100). The elutes were monitored using TLC, and were viewed under UV light (254 and365 nm) and by spraying with 1% vanillin/ 5% H 2 SO 4 / EtOH reagent, followed by heating at 100°C. The fractions obtained were collected and combined based on TLC profiles, to give five major fractions from which 5 compounds were obtained. Five different fractions were eluted by different concentrations of ethyl acetate in petroleum ether (Table  1) and purified by TLC on silica gel "G" plate using petroleum ether/ ethyl acetate (9:1) as developing system, giving compounds C 1, C 2, C 5, and (8:2) developing system giving compounds C 3, C 4. The compounds were identified with their physical and spectroscopic data and comparing data with reported literature.

Gas chromatography-mass spectrometry analysis (GC-MS)
The GC-MS system (Agilent Technologies) was equipped with gas chromatograph (7890B) and mass spectrometer detector (5977A) at  Central Laboratories Network, National Research Centre, Cairo, Egypt. The GC was equipped with HP-5MS column (30 m x 0.25 mm internal diameter and 0.25 μm film thickness). Analysis was carried out using Helium as the carrier gas at a flow rate of 1 ml/min at a split-less mode, injection volume of 1 μl and the following temperature program: 60°C for 2 min; rising at 5°C /min to 300°C and held for 10 min. The injector and detector were held at 280°C and 300°C, respectively. Mass spectra were obtained by electron ionization voltage (EI) at 70 ev and using a spectral range of m/z 50-550 and solvent delay 3 min. Identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.

Antibacterial activity
To measure the antibacterial activity of the crude and pure compounds. Gram-positive bacteria )Staphylococcus aureus ATCC 9144 and Bacillus subtilis ATCC 29212( and gram-negative bacteria (Pseudomonas areuginosa ATCC 27853 and Escherichia coli ATCC 25922) were used as test organisms and antibacterial tests were performed as in. 18 The tests were performed in 96-well flat polystyrene plates. 10μl of test extracts (final concentration of 250 μg/ml) were added to 80 μl of lysogeny broth (LB broth) followed by addition of 10 μl of bacterial culture suspension (log phase), then the plates were incubated overnight at 37°C. After incubation, the positive antibacterial effect of the tested compound observed as clearance in the wells, while compounds that didn't have an effect on the bacteria, the growth media appeared opaque in wells. The absorbance was measured after about 20 h at OD600 in a Spectrostar Nano Microplate Reader (BMG LABTECH GmbH, Allmendgrun, Germany). The positive control (Bacteria plus distilled water), while, negative control (growth media plus distilled water).

Minimum inhibitory concentration (MIC)
To determine the MIC of the tested compounds that displayed the antibacterial activity, 80 μL of lysogeny broth (LB broth) were mixed with 10 μL of different concentrations of test compounds and followed by addition of 10 μL of a fresh culture (1×106 CFU/mL) of test microorganisms. Micro plates were incubated at 37°C for 24 h. MIC are defined as the lowest concentration of the antimicrobial agent that inhibited the growth of microorganisms. 19 Antibiofilm activity The biofilm inhibitory activity of the ethyl acetate crude extract and pure compounds were measured using 96-well flat polystyrene plates toward and four clinical microbes comprising gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and gram-negative bacteria (Pseudomonas areuginosa and Escherichia coli) according to method of 20 with some modifications. Briefly, each well was filled with 180 µL lysogeny broth (LB broth) then inoculated with 10 µL of pathogenic bacteria followed by addition of 10 µL of samples along with control (without test sample

Collection of samples and isolation of fungal isolate
Soil samples were collected from El Saf Province -Giza Governorate . Soil was taken at 10 cm depth. Soil samples were firstly sieved and then dried for 5 days at 25°C. The samples were kept after drying at 10 °C followed by fungal strain isolation. Serial dilution of soil suspension was prepared up to 10 −6 dilution. Then, 0.1mL of suspension from dilutions 10 −3 to 10 −6 was spread on Potato dextrose agar media plates at 28 ± 2 °C for 6-8 days until the fungal growth colonies was observed. The fungi isolated on culture medium from soil were further purified by using streak method. Routinely, the purified fungal cultures were transferred by streaking every 6 to 8 days onto fresh Potato dextrose agar plates. Kept in refrigerator at 4°C.

Phylogenic identification of fungal isolates
The fungal isolate Sarocladium kiliense SDA20 was identified by sequencing of its ITS region. The obtained sequence was aligned with other identified strains in the Gene bank database using online BLAST tool to determine the similarity score (http://www. blast.ncbi.nlm.nih.gov/Blast). The obtained result confirmed a very close similarity of the gene sequence with 97.45% homology of the isolate SDA20 with Sarocladium kiliense strain. The isolates identified as Sarocladium kiliense SDA20 and submitted in the gene bank under accession number MN636431 The phylogenetic tree was constructed using MEGA X program 21 and neighbour-joining method 22 (Figure 1).

Different inoculum size
The data recorded in (Table 2) showed that increase in inoculums size of S.arocladium kiliense MN636431strain led to increase in the total secondary metabolites required which produce its maximum percentage at 10 6 spores/ml. This results were obesit to 23 who found that 9 ×10 4 spores /ml of A. flavus were suitable for metabolites produced from fish wastes. The fungal counts recorded for metabolites production in the shrimps are considered very high ranging from 1.7 x 104 spores /ml. 24

Different pH values
The results recorded in (Figure 2) reveled that increasing pH values led to increase percentage of total secondary metabolites production till reached 84.5 % at pH 7.0 then activity decreased. This results was coincided with 25 who found that the pH values of fermentation media for marine fungi between 6 and 7.5. The pH must be effected by the varieties of carbon and nitrogen sources also 26 found that the optimum pH of T. thioreducens was at pH 7.0 while T. celericrescens was optimum at pH 5.6-8.3, while this results in contrast with the first obligate T. archae cultured illustrated by 27 had a pH optimum of 4.2-4.8.

Elucidation of the compounds
Five compounds from the ethyl acetate extract of culture filtrate of S. kiliense SDA20 were isolated for the first time as:C1: Cholest-5-en-3-ol, C2:Palmitic acid C3:Oleic acid, C4:Nicotinamide, C5:Tricin (Fig 3).The following compounds were isolated using different chromatographic techniques and identified by spectroscopic methods and comparing data with reported literature (Figure 3).

GC-MS analysis
GC-MS analysis of ethyl acetate extract of culture filtrate showed the presence of twenty-seven phytochemical compounds (Table 3, Figure 4). The identification of compounds was based on the peak area, retention time, molecular formula and comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data. Of the 26 identified compounds, 9-Octadecenoic acid (Z)-,methyl ester had the highest peak area of 22.26% and the lowest was Oleic acid, 3-(octadecyloxy)propyl ester showing 0.44%.

Antibacterial activity
The crude extract of the Sarocladium kiliense SDA20 and the five isolated pure compounds were tested against four pathogenic bacterial strains. Preliminary antibacterial results of the crude extracts showed in ( Figure 5) showed antibacterial activity against S. aureus, Bacillus subtilis and Escherichia coli, while no activity has been detected toward P. areuginosa. The pure compounds (C1:C5) were also tested for their antibacterial activity at concentration of (250 μg/mL). All pure compounds displayed a strong inhibitory activity against Bacillus subtilis. On the other hand, Compounds (C1, C4 and C5) showed moderate antibacterial activity against E. coli, followed by compound (C2 and C3). Meanwhile S. aureus was less susceptible to pure compounds with very low activity of compounds (C3 and C5). Finally, like the crude extract, the pure compound did not display any antibacterial activity against P. areuginosa. Recent investigations have been carried out on natural products from endophytic fungi because of their ability to produce a unique bioactive metabolite, including steroids, alkaloids, flavonoids, benzopyranones, quinonesterpenoids, and others with antifungal, antibacterial, and antiviruses agents 33 , 34 studied the antimicrobial bioactivity of 21 fungal endophytes strains isolated from C. hainanensis Li. Another study conducted by using 35 fungal endophytes were isolated from seven medicinal plants and their antimicrobial activity have been investigated towards a group of pathogenic bacteria. Three fungal strains displayed significant antibacterial activity against human pathogenic bacteria P. aeruginosa, E. coli, S. aureus, and K. pneumonia. 35

Minimum inhibitory concentration (MIC)
The Minimum inhibitory concentration (MIC) of the S. kiliense MN636431biologically active crude extract and pure compounds were determined and the data were presented in (Table 4).

Biofilm inhibitory activity
Using MTT assays, the biofilm inhibition activity of the crude extract and isolated pure compounds were assayed toward four pathogenic bacteria (P. aeruginosa, S. aureus, E. coli and B. subtilis). Biofilm were assessed by crystal violet staining data presented represents mean +/-SD of three independent experiments. Results in (Figure 6) reveled that        preliminary antibiofilm results of the crude extract displayed moderate biofilm inhibition activity against B. subtilis, S. aureus and E. coli with biofilm inhibitory ratio of 58, 45 and 45% respectively, and low biofilm inhibition activity against P. aeruginosa up to 36%. The pure compounds were also evaluated for their ability to eradicate bacterial biofilm formation. As a result, C 1, C 2 reduced the biofilm formation of the strain B. subtilis up to 78 % and 71 % respectively, followed by C 3 (52%) ( Figure 6A). S. aureus biofilm formation was inhibited over 59 % by C 1 and C 2, while C 3 and C 5 have a low inhibition activity ( Figure  6D). In case of E coli, the highest percentage of biofilm inhibition was shown by C1 (59%) and C 3 (53%) followed by C 2, C 5 and C 4 ( Figure 6B). On the other hand, P. aeruginosa strain didn't display any response to isolated pure compounds (C 1: C 5) ( Figure 6C). Recently, many researchers have been studied the relationship between biofilm formation and the persistence of bacterial infections. They reported that, once bacteria form a biofilm, they become more resistant to antibiotic. 15 For this reason, bacterial biofilms are considered as a remarkable source of pathogenic diseases such as osteomyelitis, dental caries, otitis media and nosocomial infections such as urinary tract, lower respiratory tract and surgical site infections. 16,17,36,37 In this context, many trials have been performed to find an effective antibiofilm agents. In the study conducted by 38 the extract of endophytes Chlamydomonas sp. was found to be very potential as antibiofilm agent against P. aeruginosa PA14 and P. aeruginosa ATCC 10145. 38

DISCUSSION
Bioactive compounds were isolated from culture filtrate of Sarocladium kiliense SDA20 grown in fermentation media containing shrimp shell waste as substrate. This media produced the high activity in the mycotoxins production under optimium conditions using inoculum size 10 6 spores/ml of Sarocladium kiliense SDA20 at pH 7.0, as shown in (Table 2) and ( Figure 2). The ethyl acetate extract of culture filtrate of Sarocladium kiliense SDA20 was subjected to silica gel column chromatography, The fractions obtained were examined using TLC and the similar fractions were collected to give five major fractions, from which 5compounds were isolated for the first time, Cholest-5en-3-ol, palmitic acid,oleic acid, nicotinamide and tricin The isolated compounds were identified by spectroscopic methods and comparing data with reported literature. GC-MS analysis of ethyl acetate extract of culture filtrate showed the presence of twenty-seven phytochemical compounds. The identification of compounds was based on comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data. The crude extract of the Sarocladium kiliense SDA20 and the five isolated pure compounds were tested against four pathogenic bacterial strains. The crude extract showed antibacterial activity against Staphylococcus aureus, Bacillus subtilis and Escherichia coli, while no activity has been detected toward Pseudomonas areuginosa. All pure compounds displayed a strong inhibitory activity against Bacillus subtilis. On the other hand, Compounds (C1, C4 and C5) showed moderate antibacterial activity against E. coli, followed by compound (C2 and C3). Meanwhile S. aureus was less susceptible to pure compounds with very low activity of compounds (C3 and C5). Using MTT assays, the biofilm inhibition activity of the crude extract and isolated pure compounds were assayed toward four pathogenic bacteria (P. aeruginosa, S. aureus, E. coli and B. subtilis). Results reveled that the crude extract and isolated pure compounds displayed moderate biofilm inhibition activity against the four pathogenic bacteria.

CONCLUSION
Five compounds were isolated for the first time from the ethyl acetate extract of culture filtrate of S.kiliense MN636431. Also, twenty-seven compounds from GC-MS analysis of the extract were identified. Tested the antibacterial activity and MTT assay of crude and five pure compounds displayed a strong inhibitory antibacterial activity against different bacterial strains and the biofilm inhibition activity. The use of fungal endophytes in drug discovery have many advantages due to their diversity that offers an abundant source of active and novel metabolites. On the other hand, the utilization of fungal endophytes can be the act as an alternative source of bioactive metabolites extracted from plants that lead to eliminating of large quantity of plant material. 39