Microbial Degradation And Biotransformation Of Trans-Anethole To Anisaldehyde And Anisic Acid

Illicium verum Hook.f. is a kind of local rich natural flavor in Guangxi. Anise oil is extracted from the fruits, branches and leaves of Illicium verum Hook.f. by steam-distillation extraction, in which trans-anethole content of 80% to 90%.trans-Anethole, a type of propenylbenzene compound, is usually used as starting material to synthesize fragrants in flavor and fragrance industry. Most valuable aromatic compounds are produced as intermediates in the biodegradation pathways of propenylbenzene compounds. Thus, it is likely that microorganisms capable of utilizing trans-anethole will produce high value-added natural biological spices, such as anisaldehyde, anisic acid etc. Therefore,trans-anethole was selected as substrate. Microorganisms that can degrade and transfor trans-anethole to anisaldehyde or anisic acid were isolated. Meanwhile, the factors that affected biotransformation, isolation and identification of intermediate products and trans-anethole biodegradation pathways were studied.1. Establishment of methods for analyzing trans-anethole, anisaldehyde and anisic acid in trans-anethole degradation and transformation systemA thin layer chromatography method (TLC), thin layer chromatography-UV spectrophotometry method (TLC-UV), reversed phase high performance liquid chromatography method (RP-HPLC) and thiobarbituric acid spectrophotometric method (TBA) for the analysis of trans-anethole, anisaldehyde and anisic acid in trans-anethole biodegradation and transformation system were developed. The suitable developing solvent of TLC was petroleum ether (bp.60℃-90℃): chloroform:ethyl acetate:formic acid (V/V/V/V)= 25:10:3:0.2. TLC was found to be simple and rapid for qualitative and semiquantitative analysis of trans-anethole, anisaldehyde and anisic acid. The method was suitable for rapid screening of trans-anethole degrading microorganisms. TLC-UV and RP-HPLC method can be simultaneous quantitative analysis of trans-anethole, anisaldehyde and anisic acid. RP-HPLC method was accurate above TLC-UV method. RP-HPLC conditions were as follow:The separation was performed on Kromasil-100A C18 column (250 mm×4.6 mm×5μm) using V(acetonitrile):V(water):V(acetic acid)=70:30:0.02 as the mobile phase. The flow rate was 0.8 mL·min-1. The detection wave length was 260 nm. The injection volume was 5μL, and the column temperature was room temperature. The method showed good linear relationship, precision and repeatability. In addition, anisaldehyde in transformation liquids could also be detected by TBA method.trans-Anethole and anisic acid don't interfered with the determination of anisaldehyde.2. Isolation and identification of trans-anethole degrading microorganismsIsolation of trans-anethole degrading microorganisms which can grow on high concentration of anise oil from the soil under aniseed tree, the fruits,branches and leaves of fresh aniseed, waste residue of aniseed workshop. Then biotransformation of trans-anethole to anisaldehyde or anisic acid with these strains were carried out by fermentation. Bacteria were isolated from soil under aniseed trees by enrichment culture. Bacterial strain BT-13, which utilizes 1%(V/V) anise oil as the sole source of carbon and energy in modified M9 media, was screened out base on its good capability to biotransform trans-anethole to anisic acid. This strain was identified as Pseudomonas sp. according to visual observation, physiological and biochemical experiments and 16S rDNA sequence analysis. Eighty-seven endophytes were isolated from the fruits,branches and leaves of fresh aniseed. It includes sixty-nine strains of endophytic fungus, eighteen strains of endophytic bacteria. A endophytic bacterium designated BZ-15 was newly isolated based on its ability to degrade trans-anethole. Anisic acid was detected in the culture.This strain was identified as Pseudomonas putida according to its 16S rDNA sequence analysis. A fungal strain ZJ-9 was newly isolated from waste residue in aniseed workshop based on its ability to degrade trans-anethole. A small amount of anisaldehyde was detected in the culture. Strain ZJ-9 was identified as Aspergillus niger according to its morphological characteristics, culture characteristics and "fungal identification manual".3. Biodegradation and biotransformation technology of trans-anethole to anisaldehyde and anisic acidThe biotransformation process of trans-anethole to anisaldehyde by Pseudomonas sp. BT-13 was carried out in aqueous-organic solvents biphasic systems. The effects of some key factors such as organic solvent polarity and content, the component of culture medium, conversion time, temperature, medium capacity, rotate speed of rotary shaker and medium initial pH were investigated on the biotransformation of trans-anethole to anisaldehyde by free cell. The results showed that the suitable organic solvent was ethyl acetate, ethyl acetate content was 10%(V/V), the suitable medium was the modified Martin's medium, medium capacity was 20 mL/150 mL flask, medium initial pH 6.5, temperature 30℃, rotate speed of rotary shaker at 150 r·min-1, conversion time 30 h. On the optimum conditions, the molar generation ratio of anisaldehyde was increased to 12.6% by immobilized cells with calcium alginate compared with 7.7% of free cells. In order to separate trans-anethole and anisaldehyde, trans-anethole and anisaldehyde were extracted from biotransformation liquids by equal volume ethyl acetate at first, then trans-anethole was stripped to water phase via saturation sodium bisulfite solution. Because trans-anethole was almost insoluble in water, this reactive-extraction also resulted in anisaldehyde purification. Finally anisaldehyde was obtained from acidic water phase by ethyl acetate.The optimization of conversion conditons for the biotransformation of trans-anethole to anisic acid by Pseudomonas sp. BT-13 was carried out in modified M9 medium, on which the effects of culture medium components, substrate concentration, shaking speed, temperature and initial medium pH were studied. The results showed that the carbon source concentration of above 1 g·L-1 in modified M9 medium could marketdly inhibit the transformation of trans-anethole. The optimal medium composition was obtained:maltose 0.5 g·L-1,NH4Cl 0.5 g·L-1,FeSO4·7H2O 0.01 g·L-1, MgSO4·7H2O 2.0 g·L-1,NaCl 0.5 g·L-1, Na2HPO4 6.8 g·L-1, KH2PO4 3.0 g·L-1 CaCl2 0.02 g·L-1. Under an optimal condition of trans-anethole concentration of 9.83 g·L-1, shaking speed 200 r·min-1, temperature 30℃, pH 7.0, the cumulative concentration of anisic acid could reach to 3.49 g·L-1 with a molar generation ratio of 34.6%, which increased 92.8% than that under the unoptimized conditions. Anisic acid was proved to be the leading product of this biotransformation, while some other intermediates such as anethole epoxide, anisaldehyde,t-anethole-diol etc could also be detected in the culture. When the biotransformation was processed in 5 L fermentor, the anisic acid of 3.57 g·L-1 was obtained with a molar generation ratio of 36.1%. Crystals of anisic acid was obtained from biotransformation liquid afer acidified, extracted with ethyl acetate, vacuum concentration and crystallization.4. Proposed degradation pathway of trans-anethole by Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9The degradation pathway of trans-anethole in Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9 were studied by analyzing intermediates using HPLC and GC-MS. Some main intermediates were identified by comparison with standard sample or GC-MS spectra analysis. Four intermediates that is anethole epoxide, anethyl diol, anisaldehyde and anisic acid were produced in the degradation of trans-anethole by Pseudomonas sp. BT-13; Five intermediates that is anethole epoxide, anethyl diol, anisyl alcohol, anisaldehyde and anisic acid were produced in the degradation of trans-anethole by Aspergillus niger ZJ-9. Under certain conditions, anisyl alcohol was accumulated. These intermediate products are all high value-added aromatic compounds in the perfume and pharmaceutical industries. According to the generation of intermediates, possible degradation pathways of trans-anethole by Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9 were proposed:First the 1,2-CC double bond in the trans-anethole side chain is epoxidized and anethole epoxide is produced.Then the epoxide is hydrolysed, forming anethyl diol which is subsequently oxidized to anisaldehyde. Anisaldehyde can continue to be oxidized to anisic acid. In Aspergillus niger ZJ-9, anisaldehyde also can continue to be reduced to anisyl alcohol. In two strains,trans-anethole was degraded through anethyl diol pathway, but the accumulation of some intermediate products was different.Peroxidase (POD) and catalase (CAT) activity were detected in intracellular enzyme extracted from Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9. POD and CAT activity of Pseudomonas sp. BT-13 were 122 U·mL-1 and 625 U·mL-1, but POD and CAT activity of Aspergillus niger ZJ-9 were 4.8 U·mL-1 and 42.5 U·mL-1, significantly lower than Pseudomonas sp. BT-13. According to the testing enzymes activity and the accumulation of the intermediate products, there were some differences among the key enzymes and enzyme regulation in biodegradation of trans-anethole by Aspergillus niger ZJ-9 and Pseudomonas sp. BT-13. Anethole epoxide, anisaldehyde, and anisic acid were also detected in the biotransformation liquids of trans-anethole by Pseudomonas sp. BT-13 crude enzyme. Adding amount of H2O2 could promote the biotransformation of trans-anethole to anisic acid. Enzymatic transforming suitable pH was 8.