Crude Mycelial Extracts Research Articles

Elicitation of Secondary Metabolites in Aquilaria malaccensis Lam. Callus Culture by Crude Mycelial Extract of Fusarium solani and Methyl Jasmonate

Agarwood is a resinous wood of great economic value produced by trees from the Thymelaeaceae family in response to stress. The natural formation of agarwood can take decades after exposure to the stressors. Artificial agarwood induction by inoculating the stem with fungi has been successfully demonstrated, but resin accumulation occurs very slowly. Cell suspension and callus cultures may serve as an alternative solution to provide a fast-growing plant material to produce artificial agarwood in a short period. Here, we induced agarwood formation in callus cultures of Aquilaria malaccensis by application of crude mycelial extracts of Fusarium solani strains GSL1 or GSL2, or methyl jasmonate (MeJA). After 20 days of treatment with elicitors, all treated calluses had less dry weight than the control group. The gas chromatography–mass spectrometry analysis identified 33 different secondary metabolites among all samples, four of which were present in all treatments and control, i.e., 1-docosene and 1-octadecene (alkenes), 4-di-tert-buthylphenol (phenolic), and benzenepropanoic acid (fatty acid). The 6-methoxy-2-(4-methoxyphenethyl)-4H-chromene-4-one, a chromone derivative, was only detected in callus elicited with the F. solani strain GSL2 and MeJA. All treated calli produced more fatty acid derivatives than the control group. We conclude that elicitors used in this study can induce the production of agarwood-related chemicals such as chromone and fatty acid in callus culture.

Characterization and Toxicity of Crude Toxins Produced by Cordyceps fumosorosea against Bemisia tabaci (Gennadius) and Aphis craccivora (Koch).

Cordyceps fumosorosea, an insect pathogenic fungus, produces different toxins/secondary metabolites which can act as pest control agents. This study reports the extraction and characterization of crude mycelial extracts of C. fumosorosea isolate SP502 along with their bio-efficacy against Bemisia tabaci and Aphis craccivora. Fourier transform infrared spectroscopy, liquid chromatography, mass spectrometery and nuclear magnetic resonance analysis of C. fumosorosea isolate SP502 extracts showed the presence of five major compounds—Trichodermin, 5-Methylmellein, Brevianamide F, Enniatin and Beauvericin—which all may potentially be involved in insecticidal activity. The HPLC analysis of C. fumosorosea mycelial extracts and Beauvericin standard showed similar chromatographic peaks, with the content of Beauvericin in the crude toxin being calculated as 0.66 mg/ml. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of A. craccivora were 46.35, 54.55, 68.94, and 81.92 µg/mL, respectively. The median lethal concentrations of C. fumosorosea mycelial extracts towards first, second, third and fourth instar nymphs of B. tabaci were 62.67, 72.84, 77.40, and 94.40 µg/mL, respectively. Our results demonstrate that bioactive compounds produced by C. fumosorosea isolate SP502 have insecticidal properties and could, therefore, be developed into biopesticides for the management of B. tabaci and A. craccivora.

Kojic acid repurposing as a pancreatic lipase inhibitor and the optimization of its production from a local Aspergillus oryzae soil isolate

BackgroundObesity and its related diseases are increasing worldwide. One of the best therapeutic strategies for obesity management is through the inhibition of pancreatic lipase (PL) enzyme. So far orlistat is the only FDA approved PL inhibitor, but with unpleasant side effects. New efficacious anti-obesity drugs are needed to achieve a successful reduction in the incidence and prevalence of obesity. Many microbial metabolites have PL inhibitory activity. Screening soil inhabitants for PL inhibitors could help in increasing the available anti-obesity drugs. We aimed to isolate and identify alternative PL inhibitors from soil flora.ResultsWe screened the crude mycelial methanolic extracts of 39 soil samples for PL inhibitory activity by the quantitative lipase colorimetric assay, using the substrate p-nitrophenyl palmitate and orlistat as positive control. AspsarO, a PL inhibitor producer, was isolated from an agricultural field soil in Giza, Egypt. It was identified as Aspergillus oryzae using colony morphology, microscopical characteristics, 18S rDNA sequencing, and molecular phylogeny. Increasing the PL inhibitor activity, in AspsarO cultures, from 25.9 ± 2% to 61.4 ± 1.8% was achieved by optimizing the fermentation process using a Placket–Burman design. The dried 100% methanolic fraction of the AspsarO culture had an IC50 of 7.48 μg/ml compared to 3.72 μg/ml for orlistat. It decreased the percent weight gain, significantly reduced the food intake and serum triglycerides levels in high-fat diet-fed Sprague–Dawley rats. Kojic acid, the active metabolite, was identified using several biological guided chromatographic and 1H and 13C NMR techniques and had an IC50 of 6.62 μg/ml. Docking pattern attributed this effect to the interaction of kojic acid with the key amino acids (Lys80, Trp252, and Asn84) in PL enzyme binding site.ConclusionCombining the results of the induced obesity animal model, in silico molecular docking and the lipase inhibitory assay, suggests that kojic acid can be a new therapeutic option for obesity management. Besides, it can lower serum triglycerides in obese patients.

Optimization conditions, antibacterial and antioxidant activities of clitopilus chalybescens

The aim of this work was to study the effect of media, temperature and pH on the mycelial growth of Clitopilus chalybescens strain MFLUCC 13–0809 collected from Lampang, Thailand. The antioxidant and antibacterial activities from the crude mycelial extracts were evaluated. The study indicated that the optimal medium, pH and temperature were observed on yeast extract agar (YEA) pH 5 to 7, and 20–29˚C, respectively. For antibacterial activities, crude extracts from mycelium slightly inhibited the growth of Gram positive bacteria (Bacillus subtilis, Staphylococcus aureus and Micrococcus luteus), but no activities were observed on Gram negative bacteria (Pseudomonas aeruginosa and Escherichia coli). Antioxidant assay indicated that the crude extract had noticeable scavenging activity on 2,2-diphenyl-1 picrylhydrazyle (DPPH) radical. The results suggest that the crude extract from this mushroom mycelium deem to have a potential for further development on antibacterial and antioxidant applications.

Crude extract of Trichoderma elicits agarwood substances in cell suspensionculture of the tropical tree, Aquilaria malaccensis Lam.

Agarwood is the precious fragrant wood produced by the tropical tree Aquilaria, often after elicitation by wounding or fungal attack. In this study we established a cell suspension culture of A. malaccensis from leaf-derived callus and induced agarwood production in the culture using fungal elicitors. Elicitors were made from crude mycelial extracts of two fungal species from the genera Trichoderma and Lasidiplodia. The elicitors were added to the cell suspension culture, initiated with 2 g of fresh calli, at concentrations ranging from 2 to 10 mg L-1. A light agarwood scent was detected from the suspension culture elicited with 8 mg L-1 Trichoderma extract. To increase scent intensity, cell suspension cultures were initiated from 2 to 8 g of calli and treated with 8 mg L-1 Trichoderma extract. The combination of 8 g of calli inoculum and 8 mg L-1 Trichoderma extract produced the most intense fragrance, one comparable to agarwood scent. The cell culture was harvested, extracted in methanol, and analyzed using GC-MS. Several important agarwood compounds were detected including 8-epi-.gama.-eudesmol, Ai-guaiene, and alloaromadendrene oxide-1. Trichoderma appeared to be a suitable inducer for agarwood production when used at an optimal concentration and in combination with a cell suspension culture of Aquilaria.

Crude bacterial extracts of two new Streptomyces sp. isolates as bio-colorants for textile dyeing

Renewed demand for incorporation of natural dyes (bio-colorants) in textile industry could be met through biotechnological production of bacterial pigments. Two new Streptomyces strains (NP2 and NP4) were isolated for the remarkable ability to produce diffusible deep blue and deep red pigment into fermentation medium. Crude mycelial extracts of both strains were used as bio-colorants in conventional textile dyeing procedures avoiding downstream purification procedures. The yields of bio-colorants obtained in this way were 62 and 84 mg per g of mycelia for Streptomyces sp. NP2 and Streptomyces sp. NP4, respectively. Through nuclear magnetic resonance analysis of crude extracts before and after dyeing procedures, it was shown that both extracts contained prodigiosin-like family of compounds that exhibited different dyeing capabilities towards different textile fibers. Polyamide and acrylic fibers were colored to the deepest shade, polyester and triacetate fibers to a noticeable, but much lower shade depth, while cotton and cellulosic fibers stained weakly. These results confirmed that crude bacterial extracts had the characteristics similar to those of ionic and disperse dyes, which was consistent with the identified polypyrrolic prodigiosin-like structures.

Study on the Antimicrobial Activity of Aspergillus sp Isolated from Justicia adathoda

A number of new bioactive compounds from endophytes have been recognised as potential sources of antimicrobial substances. In the present study, the leaf samples of Justicia adathoda was screened for endophytic fungi. The isolated fungi was identified as Aspergillus sp . based on morphological features. Fungal metabolites of the mycelia and the culture broth were extracted with ethyl acetate. The antimicrobial activity of the mycelia extracts and crude culture broth were evaluated by agar well diffusion method against few representative strains. Crude mycelial extract inhibited all the strains significantly, with a mean strongest zone of inhibition of 12.0±1mm against E.coli . Crude culture broth inhibited the strains of Pseudomonas and Klebsiella alone and had a mean stronger zone of inhibition of 13±1mm and 14±1mm than crude mycelia extract. Further FTIR analysis indicated the nature of the antimicrobial compound.

Study on the antimicrobial activity of Aspergillus sp isolated from Justicia adathoda

A number of new bioactive compounds from endophytes have been recognised as potential sources of antimicrobial substances. In the present study, the leaf samples of Justicia adathoda was screened for endophytic fungi. The isolated fungi was identified as Aspergillus sp. based on morphological features. Fungal metabolites of the mycelia and the culture broth were extracted with ethyl acetate. The antimicrobial activity of the mycelia extracts and crude culture broth were evaluated by agar well diffusion method against few representative strains. Crude mycelial extract inhibited all the strains significantly, with a mean strongest zone of inhibition of 12.0±1mm against E.coli. Crude culture broth inhibited the strains of Pseudomonas and Klebsiella alone and had a mean stronger zone of inhibition of 13±1mm and 14±1mm than crude mycelia extract. Further FTIR analysis indicated the nature of the antimicrobial compound. Keywords: Endophytic fungi, Justicia adathoda, Agar well diffusion, Aspergillus sp.

Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C(16) and C(18) fatty acids at pH approximately 7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.

A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2).

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.

Examination of Alternaria alternata mutagenicity and effects of nitrosylation using the Ames Salmonella test

Molds of the genus Alternaria are common food pathogens responsible for the spoilage of fruits, vegetables, grains, and nuts. Although consumption of Alternaria alternata-contaminated foodstuffs has been implicated in an elevated incidence of esophageal carcinogenesis, the mutagenic potencies of several A. alternata toxins seem unable to account for the levels of activity found using crude mycelial extracts. In this study, the mutagenic effects of nitrosylation were examined with the major Alternaria metabolites Altenuene (ALT), Alternariol (AOH), Alternariol Monomethyl Ether (AME), Altertoxin I (ATX I), Tentoxin (TENT), Tenuazonic Acid (TA), and Radicinin (RAD) using the Ames Salmonella strains TA98 and TA100. In the absence of nitrosylation, ATX I was mutagenic when tested from 1 to 100 microg/plate in TA98 with rat liver S9 for activation, while AOH and ATX I were weakly mutagenic +/- S9 in TA100. Incubation with nitrite generally increased mutagenic potencies with ATX I strongly mutagenic +/- S9 in both TA98 and TA100, while ALT, AOH, AME, and RAD responses were enhanced in TA100 + S9. However, subsequent examination of three extracts made from A. alternata culture broth, acetone-washed mycelia, and the acetone washes showed a different mutagenic response with both broth and acetone washes directly mutagenic in TA98 and TA100 but with a reduced response + S9. The acetone-washed mycelial extract was found to have the lowest mutagenic activity of the three extracts tested. Nitrosylation had little effect on the mutagenicity of any of the extracts. Thus, while nitrosylation increases the mutagenicity of ATX I, and to a lesser extent that of several other Alternaria toxins, the results demonstrate that Alternaria produces a major mutagenic activity with a S. typhimurium response different from that found with the purified toxins. Efforts are currently underway to chemically identify this mutagenic species. Published 2001 Wiley-Liss, Inc.

Identification of alkaloids and polyketides in an Actinomycete by high-performance liquid chromatography with mass spectrometric and UV–Visible detection

Analytical HPLC with UV–Vis diode array detection and positive electrospray HPLC–MS were employed to characterise the secondary metabolites of Actinomycetes spec. strain BA 909 in crude mycelial extracts and preparative HPLC fractions. It was shown that the strain produced two families of antibiotics previously reported in the literature, namely the polyketides leptomycin A ( 1) and B ( 2), kazusamycins A ( 3) and B ( 4) and the alkaloids nybomycin ( 5) and deoxynybomycin ( 6), respectively. Compounds 1– 4 were identified by comparison of their HPLC–UV and HPLC–MS data with authentic materials obtained from the original producer strain, whereas the identity of the nybomycins 5 and 6 was established by comparison of structural data with those of reference compounds. From HPLC–UV–Vis analyses it was established that both groups of metabolites are produced as complex mixtures of congeners. The employment of a single HPLC–UV–Vis method, aided by HPLC–MS of selected fractions, allowed for simultaneous detection of the main metabolites in the crude extracts during fermentation. Known metabolites could be distinguished from tentatively new congeners. For example, the data available from HPLC–UV–Vis and HPLC–MS analyses point towards the presence of an anguinomycin A ( 7) isomer, two new isomers of kazusamycins A and B ( 8 and 9), and a new nybomycin derivative ( 1 0) in addition to the known compounds 1– 6. The co-occurrence of both leptomycin and nybomycin type metabolites in the same Actinomycete strain was observed for the first time.

Activation of ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase) in Streptomyces antibioticus.

The activity of the ATP:GTP 3'-pyrophosphotransferase (guanosine pentaphosphate synthetase I [GPSI]) from Streptomyces antibioticus is stimulated maximally by methanol at 20% (vol/vol) in assay mixtures. Although the enzyme is not activated by ribosomes, its activity is stimulated by tRNA (uncharged or charged) and by synthetic mRNA [e.g., poly(U)]. The level of stimulation is greater in the presence of tRNA and poly(U) together than with either RNA alone. Incubation of GPSI with low levels of trypsin also leads to activation of the enzyme. Analysis of the products of mild trypsin digestion revealed the presence of two intermediates whose M(r)s are identical to those of species produced by incubation of purified GPSI with crude extracts of S. antibioticus mycelium. GPSI can be activated by incubation with crude mycelial extracts, and this activation is partially inhibited by the inclusion of trypsin inhibitor in reaction mixtures.

Protein kinases in the entomopathogenic fungus Metarhizium anisopliae

Cyclic AMP (cAMP)- and Ca2+-dependent protein kinase activities of the fungus Metarhizium anisopliae were sought in extracts of ungerminated conidia, germinating conidia and mycelium, as well as in purified plasma membranes from mycelium. Ungerminated conidia contained a Ca2+/calmodulin-dependent protein kinase capable of phosphorylating multiple endogenous proteins and involved in triggering germination. cAMP-dependent protein kinase activity was not detected in ungerminated conidia in spite of the presence of cAMP in these conidia and the pre-germination synthesis of two cAMP-binding proteins. Most phosphorylation events in crude mycelial extracts were Ca2+-dependent but H-series inhibitors of cAMP-dependent kinases selectively repressed phosphorylation of a 27 kDA protein. Plasma membranes from mycelium contained a Ca2+-independent but H-8-sensitive protein kinase with multiple endogenous substrates for phosphorylation. 8-Azido[32P]cAMP bound selectively to a 52 kDa membrane protein indicative of a single cAMP-binding protein. Plasma membranes contained a phosphatase which rapidly (< 1 min) and selectively dephosphorylated a polypeptide of 15·5 kDa, thus being suited to cause rapid and reversible changes in membrane function. Membranes also contained an adenylate cyclase apparently involved in transmembrane signalling reactions, since mechanical or chemical treatments which stress the fungus caused rapid increases in intracellular levels of cAMP. Reconstitution experiments with a homogenate from a crisp-1 mutant of Neurospora crassa suggested G-protein regulation of Metarhizium plasmalemma adenylate cyclase.

Proteinase inhibitory activity in Penicillium urticae

Intracellular proteinase activity in cell extracts of Penicillium urticae increased twofold during storage. Three proteinase fractions showed different sensitivities to an inhibitory component which was present in the original crude mycelial extract. The inhibitory component proved to be nondialyzable and heat stable, but lost activity during storage of the cell extracts, probably as a result of proteolytic digestion. Following electrophoresis of the two most stable proteinase fractions, 18 individual proteinases were separated. All but 2 of these enzymes were shown to have undergone activation during the storage period.

Novel GTP-binding proteins in plasma membranes of the fungus [formula omitted

We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae . Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding [α- 32P]GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties. Four other proteins (31–38.2 kDa) were similar except that they did not bind [α- 32P]GTP after treatment with sodium dodecyl sulfate. An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products. An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti- ras serum. Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive. Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.

CO oxidoreductase from Streptomyces strain G26 is a molybdenum hydroxylase.

CO oxidoreductase was purified to 95% homogeneity from crude mycelial extracts of Streptomyces G26. The purified preparation has a specific activity of 25.7 units/mg, a 13-fold improvement on crude soluble mycelial extracts. The native enzyme (Mr 282,000) is composed of non-identical subunits of Mr 110,000 and 33,000. It is a molybdenum hydroxylase containing 1.6 mol of FAD, 7.3 mol of Fe, 8.3 mol of acid-labile sulphide and 1.3 mol of Mo per mol of enzyme. Purified CO oxidoreductase catalyses the reduction of benzyl viologen, confirming the previously reported ability of this enzyme to interact with low-potential acceptors. Cytochrome c reduction cannot be accounted for entirely by non-enzymic reduction by superoxide radicals. NAD+ and NADP+ are not reduced, nor is clostridial ferredoxin.

An endogenous inhibitor of folylpolyglutamate synthetase in Neurospora crassa

The met-9 mutant (FGSC 552) of Neurospora crassa was examined for ability to synthesize folylpolyglutamates in vivo and in vitro. After culture in defined media, the concentration of mycelial folates was ca 75 % of that found in the Lindegren A wild type (FGSC 853). The derivatives were principally short-chained folates that supported the growth of Lactobacillus casei without conjugase treatment. The levels of polyglutamyl folates in the mutant were only 6–20% of those present in the wild type under comparable growth conditions. Crude mycelial extracts of met-9 had little folylpolyglutamate synthetase (FPGS) activity but ammonium sulphate fractionation resulted in recoveries in the 150–200% range. A protein fraction precipitating at 30–45% of saturation with ammonium sulphate contained a FPGS inhibitor that was more potent on preincubation. The inhibitor was present in other polyglutamate-deficient mutants and in the wild type. This factor was non-dialysable, thermolabile and inactivated by urea and trypsin treatment. The inhibitor fraction lacked significant protease and conjugase activities. Although several enzymes were not affected by preincubation with this protein fraction, the FPGS activities of E. coli, bovine liver and pea cotyledons were strongly inhibited.

Relationship between two major immunoreactive forms of arginase in Neurospora crassa

Two major immunoreactive proteins of Mr 41,700 and 36,100 have been detected in crude mycelial extracts with polyclonal antibodies raised against arginase purified from Neurospora crassa. The latter corresponded to the protein used to obtain the antibodies. Both polypeptides were either missing or present in very low amounts in mutant strains having little or no detectable arginase activity. The relative proportion of the two species was altered in strains containing the nitrogen catabolite regulatory mutation nit-2. Peptide mapping indicated that the two species are very closely related, but several of the peptides which appeared to be identical by staining reacted differently with the antibodies. Both species were produced by in vitro translation of poly(A)+ mRNA, although the larger species was produced to a much smaller extent than was expected from its abundance in vivo. The results suggest the existence of multiple forms of arginase in N. crassa which differ in their response to nitrogen catabolite regulation.

Large‐scale and pH‐controlled fermentation production of the tremorgenic mycotoxin verruculogen

but, since its discovery and subsequent structural de- termination,’ this compound has been little studied despite its unusual neurotropic properties. It is probably the most potent of the known fungal tremorgens, and indirect evidence suggests that this group of mycotoxins may be involved in the etiology of certain neurologic disorders (staggers) of sheep and Until the present studies, it has only been possible to make a qualitative assessment of verruculogen in toxin-producing cultures and this had clearly hindered the development of an efficient fermentation process. The fluorimetric method herein described allows the quantitative assay of verruculogen in both pure solution and crude mycelial extracts. This assay has facilitated the development of a