Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.     

Optimized conditions for the synthesis of vanillic acid under hypersaline conditions by <Emphasis Type="Italic">Halomonas elongata</Emphasis> DSM 2581<Superscript>T</Superscript> resting cells

During growth on ferulic acid, Halomonas elongata DSM 2581^T was capable of promoting the formation of a significant amount of vanillic acid. The products were confirmed by high-performance liquid chromatography and gas chromatography mass-spectrometry analyses. To enhance the formation of vanillic acid and prevent its degradation, a resting-cell method using Halomonas elongata was developed. The growth state of the culture utilized for biomass production, the concentration of the biomass, the amount of ferulic acid that was treated and the reutilization of the biomass were optimized. The optimal yield of vanillic acid (82%) was obtained after a 10-h reaction using 10 mM ferulic acid and 5 g/l of cell pregrown on ferulic acid and harvested at the end of the exponential phase.     

Efficient release of ferulic acid from sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis>) stems by chemical hydrolysis

An efficient method for release of ferulic acid from sweet potato stems was developed. Ferulic acid along with phenolic compounds were released from stems by acid and alkaline treatments. The base hydrolysis with 0.1 N NaOH yielded the highest quantity of total extracts (471.1 mg/g). The stems released more phenolic compounds when 0.0125∼0.025 N NaOH was employed. Where as ferulic acid release was maximal with 0.05 N H₂SO₄ (0.32 mg/g). Ferulic acid was separated from phenolics by column chromatography. Among the elution solvents, ethyl acetate fractions (80%) contained ferulic acid. Ethyl acetate eluants were further fractionated with n-hexane/ethyl acetate/formic acid (100/50/0.5, v/v/v). All fractions showed ferulic acid and phenolic compounds. Fraction V among them was ascribed to ferulic acid with an yield of 5.41 mg/g of dry sweet potato tissue.     

Ferulic acid may prevent infection of Cicer arietinum by Sclerotium rolfsii

High performance liquid chromatography analysis of different parts of Sclerotium rolfsii-infected and healthy seedlings of chickpea (Cicer arietinum) was carried out to examine the status of phenolic compounds. Three major peaks that appeared consistently were identified as gallic, vanillic and ferulic acids. Gallic acid concentrations were increased in the leaves and stems of infected plants compared to healthy ones. Vanillic acid detected in stems and leaves of healthy seedlings was not detected in infected seedlings. There was a significant increase of ferulic acid in those stem portions located above the infected collar region compared to minimal amounts in the roots of healthy seedlings. In vitro studies of ferulic acid showed significant antifungal activity against S. rolfsii. Complete inhibition of mycelial growth was observed with 1000 g of ferulic acid/ml. Lower concentrations (250, 500 and 750 g/ml) were also inhibitory and colony growth was compact in comparison with the fluffy growth of normal mycelium. Higher amounts of phenolics were found in the stems and leaves of S. rolfsii-infected seedlings in comparison to the healthy ones. A role for ferulic acid in preventing infections by S. rolfsii in the stems and leaves of chickpea plants above the infection zone is therefore feasible.     

Formation of coniferyl alcohol from ferulic acid by the white rot fungus Trametes

The white rot fungus, Trametes sp., was cultivated in a medium containing ferulic acid, glucose and ethanol under aerobic conditions in submerged culture. The ferulic acid was transformed into coniferyl alcohol, coniferylaldehyde, dihydroconiferyl alcohol, vanillic acid, vanillyl alcohol, 2-methoxyhydroquinone and 2-methoxyquinone during 48–120 hr of cultivation. The amount of coniferyl alcohol in the culture reached a maximum after 90 hr with ca 40% of the initial amount of ferulic acid. Cinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, p -coumaric acid and sinapic acid were also transformed into the corresponding alcohols, benzoic acids and benzyl alcohols in the fungus culture.     

Rapid and simple assay for feruloyl and p-coumaroyl esterases

A rapid, simple and sensitive method for detection of ferulic and p-coumaric acids using HPLC has been developed which can be used to determine the respective phenolic acid esterase activities of microorganisms. Prior concentration, purification or derivatization of the samples are not required. As little as 0.5 mg ferulic or p-coumaric acid/I could be detected and estimated in < 1 h. The method is specific for the two phenolic acids sice no interference by other components was observed.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State (UOFS), PO Box 339, Bloemfontein 93000, South Africa     

Enhanced transformation of malachite green by laccase of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the presence of natural phenolic compounds

In this study, we investigated the efficacy of phenolic extract of wheat bran and lignin-related phenolic compounds as natural redox mediators on laccase-mediated transformation of malachite green (MG) using purified laccase from the white-rot fungus Ganoderma lucidum. G. lucidum laccase was able to decolorize 40.7% MG dye (at 25 mg l⁻¹) after 24 h of incubation. Whereas, the addition of phenolic extract of wheat bran enhanced the decolorization significantly (p < 0.001) by two- to threefold than that of purified laccase alone. Among various natural phenolic compounds, acetovanillone, p-coumaric acid, ferulic acid, syringaldehyde, and vanillin were the most efficient mediators, as effective as the synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by HPLC, UV–Vis, and liquid chromatography-mass spectrometry-electrospray ionization analysis revealed that N-demethylation was the key mechanism of decolorization of MG by laccase. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium revealed that treatment with laccase plus natural mediators effectively reduced the growth inhibitory levels of MG than that of untreated one. Among all the tested compounds, syringaldehyde showed the highest enhanced decolorization, as a consequence reduced growth inhibition was observed in syringaldehyde-treated samples. The results of the present study revealed that the natural phenolic compounds could alternatively be used as potential redox mediators for effective laccase-mediated decolorization of MG.     

Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of Bacillus coagulans

A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and ¹H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.     

Enhanced vanillin production from ferulic acid using adsorbent resin

High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g l⁻¹ ferulic acid could be added continually and 19.2 g l⁻¹ vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing.     

Dekkera and Brettanomyces growth and utilisation of hydroxycinnamic acids in synthetic media

Dekkera and Brettanomyces yeast are important spoilage organisms in a number of food and beverage products. Isolates of both genera were cultured in a defined medium and supplemented with hydroxycinnamic acids and vinylphenols to investigate their influence on growth and the formation of ethyl phenol derivatives. The growth rate of Brettanomyces species in the presence of acids was reduced, and no significant conversion to vinyl or ethyl derivatives was observed. The growth rate and substrate utilisation rates of Dekkera anomala and Dekkera bruxellensis yeast differed depending on strain and the acid precursor present. Growth of D. bruxellensis was slowed by the presence of ferulic acid with the addition of 1 mM ferulic acid completely inhibiting growth. This study provides an insight into the spoilage potential of these organisms and possible control strategies involving hydroxycinnamic acids.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.     

Comparative physiological effects of three allelochemicals and two herbicides on <Emphasis Type="Italic">Dactylis glomerata</Emphasis>

Effects of three allelochemicals (2-benzoxazolinone (BOA), p-hydroxybenzoic, and ferulic acid) and two herbicides (linuron and fluometuron) on chlorophyll fluorescence, photosynthesis, accumulation of free proline and polyamines, and total content of soluble proteins were measured to assess herbicidal activity on Dactylis glomerata. The application of chemicals generally reduced Fv/Fm (more than 25 % with p-hydroxybenzoic acid, BOA, or linuron). BOA decreased the quantum efficiency of PSII temporarily, but it did not affect photosynthesis. Polyamine content was very low, while proline content was markedly affected by some of the chemicals, thus producing some osmotic adjustment. Ferulic acid did not cause any physiological effect at all. BOA and p-hydroxybenzoic acid caused significant changes in various physiological traits of Dactylis glomerata, specially interfering with the quantum efficiency of the PSII.     

Ferulic acid in the treatment of post‐diabetes testicular damage: relevance to the down regulation of apoptosis correlates with antioxidant status via modulation of TGF‐β1, IL‐1β and Akt signalling

The aim of this study was to investigate the protective effect of ferulic acid at different doses (50 mg kg^?1 alternative day and 50 mg kg^?1 daily) on the streptozotocin (STZ)‐induced post‐diabetes rat testicular damage. Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg). Rats treated with ferulic acid were given once a day orally for 10 weeks, starting 3 days after STZ injection. Testis tissue and blood samples were collected for investigating biochemical analysis, antioxidant status, sperm parameters, and histopathological, immunohistochemical and apoptotic studies. Treatment with ferulic acid to diabetic rats significantly improved the body weight, testis weight, serum insulin level, serum testosterone level and sperm parameters (viability, motility and count). Histopathological study also revealed that ferulic acid‐treated diabetic rats showed an improved histological appearance. Our data indicated that significant reduction in the activity of apoptosis by using terminal deoxyuridine triphosphate nick end‐labelling and reduced expression of transforming growth factor‐β1 and interleukin‐1β in the testis tissue of ferulic acid‐treated diabetic rats. Conversely, it was also revealed that ferulic acid‐treated diabetic rats markedly enhanced the serine/threonine protein kinase protein expression in the testis tissue. Our result suggests that ferulic acid inhibits testicular damage in diabetic rats by declining oxidative stress. Copyright © 2013 John Wiley & Sons, Ltd.     

Concentrations of lignin and wall-bound ferulic acid after wounding in the phloem of Chamaecyparis obtusa

The changes of lignin and wall-bound ferulic acid induced by wounding were quantitatively and histochemically investigated in the phloem of Chamaecyparis obtusa. Histochemical staining of lignin was first observed in the necrotic region of the phloem 7 days after wounding and developed in 14 days. Increases of the wall-bound ferulic acid and lignin concentrations were detected in the necrotic tissue at 7 and 14 days, respectively. The concentrations continued to increase until 28 days. The lignin concentration of the callus tissue was observed to be lower than that of the healthy tissue at 14 days, and reached a similar level after 28 days. No quantitative changes of lignin and wall-bound ferulic acid were observed in other tissues. The results indicated that lignin synthesis could be maintained after the phloem cells were discolored and seemed to be necrotic. Distribution and timing of the wall-bound ferulic acid and lignin accumulation suggested that the increased wall-bound ferulic acid was involved in the lignin synthesis after wounding.     

Germination,duplication cycle and septum formation are altered by caffeine,caffeic acid and cinnamic acid in <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>

Phytogenous Phenolic and benzene compounds have been described as being responsible for many biological activities including antifungal effects. The effect of caffeine and cinnamic and caffeic acids on a model fungus, Aspergillus nidulans, was investigated at its initial stage of germination. Conidia did not germinate in the presence of cinnamic acid (1 mM). Caffeine and caffeic acid exerted a negative effect on germination, on the nuclear duplication cycle and on first septum formation. The effects of caffeine were dose-dependent; effects of caffeic acid (1 mM) were more intense than those of caffeine (10 mM).     

Hemicellulolytic enzymes of the ligninolytic white-rot fungus Phlebia radiata. Influence of phenolic compounds on the synthesis of hemicellulolytic enzymes

The ligninolytic fungus Phlebia radiata growing in a low-nitrogen medium with wheat bran as the sole carbon source was induced by some lignin monomers, e.g. vanillic, veratric and ferulic acids. In the medium these substances showed a mainly stimulating influence on the hemicellulolytic enzymes activity except for arabinofuranosidase and ferulic acid esterase.     

Synthesized esters of ferulic acid induce release of cytochrome c from rat testes mitochondria

Ferulic acid plays a chemopreventive role in cancer by inducing tumor cells apoptosis. As mitochondria play a key role in the induction of apoptosis in many cells types, here we investigate the mitochondrial permeability transition (MPT) and the release of cytochrome c induced by ferulic acid and its esters in rat testes mitochondria, in TM-3 and MLTC-1 cells. While ferulic acid, but not its esters, induced MPT and cytochrome c release in rat testes isolated mitochondria, in TM-3 cells we found that both ferulic acid and its esters induced cytochrome c release from mitochondria in a dose-dependent manner, suggesting a potential target of these compounds in the induction of cell apoptosis. The apoptosis induced by ferulic acid is therefore associated with the mitochondrial pathway involving cytochrome c release and caspase-3 activation. Cione and Tucci have equally contributed to this article.     

Lipid Accumulation during Canola Seed Germination in Response to Cinnamic Acid Derivatives

The objective of this research was to investigate how ferulic and p-coumaric acids affect lipid and fatty acid composition during canola (Brassica napus L.) seed germination. Data showed that both compounds increased total lipid and fatty acid contents in the cotyledons during germination. The largest accumulation in lipids occurred at 1.0 mM p-coumaric acid with an increase in all unsaturated fatty acids. The results suggest that allelochemicals interfere in canola seed germination by reducing lipid mobilization.     

Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal,P<Subscript>i</Subscript>-fertilised and phytohormone-treated <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by P_i fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with P_i or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of P_i concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher P_i levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Laccase, cellulase and xylanase activities during growth ofPleurotus sajor-caju on sagohampas

Sagohampas, the fibrous pith residue left after starch extraction from sago palm, is abundant at sago-processing factories and can be used as a substrate for the production of laccase by solid substrate fermentation (SSF) withPleurotus sajorcaju, an edible mushroom. The fungus grown onhampas with an adjusted carbon : nitrogen ratio of 35:1, exhibited high laccase activity together with variable cellulase (0.3-2.8 U/g) and xylanase (0.9-10.1 U/g) activity. The maximum amount of laccase produced was approximately 17.7 U/g after 6 days of SSF using 4-week-old inoculum at a density of 10%. With the mature four-week inoculum, laccase activity increased 12-fold compared to that achieved with two-week-old inoculum. The optimum pH and temperature of the crude laccase were 6.0 and 50‡C, respectively. The apparent K_m and V_max values obtained were 0.073 mM and 0.962 U/min, respectively. The maximum laccase activity could be almost doubled after 6 days of fermentation by addition of 0.2 mM vanillin or ferulic acid; the cellulose to lignin ratio increased significantly during the 12 days of SSF, from 2.74 in the control to 3.3, when 0.2 mM of either vanillin or ferulic acid was added to the substrate.