Microbial Transformation of Monoterpenes: Flavor and Biological Activity

Chapter 16

Microbial Transformation of Monoterpenes: Flavor and Biological Activity 1

2

Hiroyuki Nishimura and Yoshiaki Noma 1

Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Sapporo 005, Japan Faculty of Domestic Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770, Japan

Downloaded by CORNELL UNIV on August 16, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch016

2

Microbial transformation of (-)-carvone in Mentha spicata and 1,8 -cineolein Eucalyptus species was studied from the viewpoint of the flavor and biological activities. (-)-cis-Carveol, one of products biotransformed from (-)-carvone, was transformed by Streptomyces bottropensis to produce a novel compound, (+)-bottrospicatol which exhibits the germination inhibitory activity against plant seeds. Furthermore, 1,8-cineole was transformed by a strain of Aspergillus niger to produce 3-hydroxycineoles. Hydrogenolysis of 3 -hydroxycineolesafforded p-menthane-3,8-diols (cis and trans) which exhibit repellent activity against mosquitoes. The flavor of microbial transformation products was evaluated. There is a growing interest among biochemists in the microbial transformation of natural products in terms of the production of economically useful chemicals. In connection with effective utilization of terpenoids which are major constituents in higher plants, the microbial transformation of (-)-carvone (ca. 70% content) in Mentha spicata oil and 1,8-cineole (40-70% content) in Eucalyptus oils has been investigated from viewpoint of the flavor and biological activities. Growth Conditions of Microorganisms. Microorganisms and growth conditions were as follows. A liquid medium having the following composition (%, w/w): glucose 1%; meat extract 0.5%; polypeptone 0.5%; NaCl 0.3% was prepared in distilled water. Strains of Streptomyces A-5-1, Streptomyces bottropensis and Aspergillus niger isolated from soil were inoculated and cultured under static conditions for 3 days at 30°C. After full growth of the microorganism, (-)-carvone, (-)-cw-carveol and 1,8cineole (0.4 - 1.5 g/1 medium) were individually added into the medium. The microorganism were further cultivated for 7 to 15 days under the same conditions. Chromatography and Identification of Metabolic Products. Each culture broth (12 liters) was extracted with ether (3 liters x 4) and the extract was dried over anhydrous sodium sulfate. The ether extract was analyzed by gas liquid chromatography (GLC): Shimadzu GC-4C equipped with a stainless steel column (3 m x 3 mm i.d.) packed with 10% PEG-20M on 80 - 100 mesh of Celite 545. 0097-6156/96/0637-0173$15.00/0 © 1996 American Chemical Society

Takeoka et al.; Biotechnology for Improved Foods and Flavors ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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BIOTECHNOLOGY FOR IMPROVED FOODS AND FLAVORS


Metabolic products were identified by the interpretation of spectral data which were obtained by using the following instruments. Infrared (IR) and mass spectra were taken on a Hitachi-285 grating spectrometer and a JEOL JMS-D300 spectrometer, respectively. NMR spectra were measured on a JEOL JNM-FX200FT spectrometer (200 MHz) in C D C I 3 with (CH3)4Si as an internal standard. Optical rotations were measured on a JASCO DIP-4 spectrometer. Microbial Transformation of (-)-Carvone (1). Microbial transformation of (-)carvone (1) by bacteria (7 - 3), yeast (4), fungi (5) and photosynthetic microorganism, Euglena species (6) has been investigated. It was shown that (-)-carvone (1) was mainly converted via (+)-dihydrocarvone (2) to either (+)-neodihydrocarveol (4) or (-)dihydrocarveol (5), or via (+)-isodihydrocarvone (3) to either (+)-isodihydrocarveol (6) or (+)-neoisodihydrocarveol (7) (Figure 1). However, in some actinomycetes including a strain of Streptomyces, A-5-1 and a strain of Nocardia, 1-3-11, the major metabolic products of (-)-carvone were (-)-trans-carveo\ (8) and (-)-cw-carveol (9) by 1,2reduction (7) (Figure 2). Microbial Transformation of (-)-cw-Carveol (9). Carveol is known to be converted to carvone (7) or l-/?-menthane-6,9-diol (8,9) by microorganisms. However, (-)-ciscarveol (9) was transformed to produce novel bicyclic monoterpenes (10 in Figure 2) by a strain SY-2-1, which was isolated from soil and identified to be Streptomyces bottropensis (10). The time course for the conversion of (-)-ds-carveol (9) is shown in Figure 3. After ten days, more than 95% of 9 was converted to products a-d. The major product (a in Figure 3, 85% yield) was separated and purified by column chromatography on Si02 and by preparative GLC. The IR spectrum of the unknown compound showed absorptions due to a hydroxy 1 group at 3420 cnr and olefin at 1640 cm . The IR spectrum differed from that of (-)-ds-carveol, the starting material, in terms of the lack of absorption due to the isopropenyl group at 880 cm* . The molecula formula was determined to be C10H16O2 from the high resolution FI-MS spectrum, 168.1139 (M+). The *H-NMR spectrum (JEOL JNM-FX 200FT, 200 MHz) indicated the presence of a methyl group on a tertiary carbon (81.24, 3H, s, H-10), a methyl group on a trisubstituted double bond (81.69 - 1.72, 3H, H-7), a trisubstituted double bond adjacent to methylene (85.23, 1H, broad s, H-2), a methylene carrying a hydroxyl group (83.57 and 3.71, 1H each, d, 7=11.4 Hz, H-9), and a methine between a double bond and an ethereal oxygen (84.05, 1H, broad d, band width=12 Hz, H-6 cf. in (-)-cw-carveol, band width=24 Hz; in (-)-fran.s-carveol, band width=6 Hz). The H NMR spectrum differed from that of (-)-cw-carveol in terms of the lack of any signal due to the exo-methylene protons at about 84.65, the appearance of a pair of 1Hdoublets (H-9) at 83.5 - 3.8, and the high field shift of methyl proton signal to 81.24. In addition, the change of band width of the peak of H-6 suggested some conformational changes. In the *H-NMR spectrum of the monoacetate, which was obtained by acetylation of the unknown compound with acetic anhydride in pyridine, the carbinyl proton signals shifted to lower fields (84.08 and 4.17). This result confirmed the presence of a hydroxymethyl group. Based on the evidence mentioned above, it was concluded that product (a) in Figure 3 was (4R, 6/?)-(+)-6,8-oxidomenth-l-en-9-ol (10 in Figure 2), which is a novel 1

-1

1

1



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Microbial Transformation of Monoterpenes

175

Figure 1. Reductive Transformation of (-)-Carvone (1) by Microorganisms.

compound, named (+)-bottrospicatol. However, the relative configuration at the C-8 substituents of (+)-bottrospicatol has not been determined by IR, mass and NMR spectra (10). Absolute Configuration of (+)-Bottrospicatol (10). To determine the stereochemistry, synthesis of (+)-bottrospicatol and isobottrospicatol (Cs-epimer) was carried out according to Figure 4. (-)-Carvone (1) was oxidized by m-chloroperbenzoic acid in dry ether to give the diastereomixture of 8,9-epoxycarvone (85%), colorless oil; [a]* -26.6° (c=0.094, CHC1 ); EI-MS m/z (70eV) 166 (M+, 1%), 151(3), 109(100), 3

3

1

108(75), 82(31), 54(14), 43(18); IR v^ax cm" 1660 (C=0), 1240, 890, 820 (epoxide); NMR (in CDCI3) 81.33 (3H, s, Ci -methyl), 1.83 (3H, s, C -methyl), 2.60 (2H, s, C9-methylene), 6.70 (1H, m, C=C-H). The resulting epoxyketones were stereoselectively reduced by sodium borohydride to give 8,9-epoxy-cw-carveols and the successive reaction of epoxycarveols with 0.1N sulfuric acid led to the diastereomixture of bottrospicatol (68%). Two isomers (10a and 10b) were separated by SiC>2 column chromatography using ether-hexane as the eluting solvent and obtained in the ratio of 6 to 4. As a result, 10a was indicated to be identical with natural (+)-bottrospicatol 0

7




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(%)

0

2

4

6

8

10

Time (day) Figure 3. Time Course for the Conversion of (-)-Carveol by S. bottropensis, SY-2-1. Reproduced with permission from ref. 10. Copyright 1982 Japan Society for Bioscience, Biotechnology, and Agrochemistry.

Figure 4. Synthesis of (+)-Bottrospicatol (10a) and Its Isomer (10b). Reproduced with permission from ref. 11. Copyright 1983 Japan Society for Bioscience, Biotechnology, and Agrochemistry.


178


concerning its Rf value of TLC and spectral data. Subsequently, the p-bromobenzoyl ester of 10a was prepared and crystallized (Figure 5), mp 75.8 - 76.5°C; [oc]^ +16.1° (c=0.050, CHC1 ); FI-MS m/z 352 (M++2, 100%), 350 (M+ 99); EI-MS m/z 137 (100%), 93 (90), 43 (50); IR vJJJ cnr* 1720 (C=0), 1600 (benzene C=C); NMR (in 6

3

81.32 (3H, s, Cio-Me), 1.71 (3H, dd, 7=3.67 and 2.20 Hz, C -Me), 1.90 (1H, d, 7=10.21 Hz, C -Hexo), 2.22 - 2.39 (4H, m, C3-methylene, C -methyne, C5Hendo), 4.11 (1H, d, 7=4.88 Hz, C -methyne), 4.35 (2H, s, C -methylene), 5.25 (1H, m, C=C-H), 7.58 (4H, d, benzene). The absolute configuration of the (+)-bottrospicatol p-bromobenzoyl ester was confirmed by X-ray crystallography (Figure 6). The crystals are orthorhombic, space group p2\2\2\ with a=7.274(l), 6=33.531(5), c=6.670(2)A, Z=4. The chemical structure of (+)-bottrospicatol (10a) was determined as (4R, 6R, S/?)-(+)-6,8oxidomenth-l-en-9-ol, a colorless oil; [a]^ +63.7° (c=0.056, CHCI3); FI-MS m/z 169 (M++1, 42%), 168 (M+ 100), 133 (33); EI-MS m/z 150 (M+-H 0, 0.1%), 137 (10), CDCI3)

7

5

4


6

9

6

2

94 (69), 93 (60), 79 (15), 77 (13), 43 (100); NMR (in CDCI3) 81.24 (3H, s, Ci -Me), 0

1.71 (3H, d, C -Me), 1.85 (1H, d, 7=10.45 Hz, C -Hexo) 2.20 - 2.35 (4H, m, C methylene, C4-methyne,C5-H£?nufo-Hydroxycineole (17). The crystalline 2-endo-d\coho\ had mp 66.0 22

66.5°C and [a] ±0° (c=0.2, EtOH). IR v ^ cnr*: 3385 (OH), 1130 (C-O-C), 1065 (alcoholic C-O). *H-NMR S ^ ? : 1.10 (3H, s, C H at C-7), 1.20 (3H, s, C H at C9), 1.28 (3H, s, C H at C-10), 2.52 (1H, m, 73^,2=10 Hz, 7gem=13 Hz, hexoA^ Hz, J3exo,5exo=3 Hz attributed to W-conformation, C^-Hexo), 3.72 (1H, ddd, ^2,3^=10 Hz, 7 , 3 ^ = 4 Hz, 7 ,6^=2 Hz, HCOH). C-NMR 8 ^ 3 : 22.2 (t, C5), 24.1 (q, C-7), 25.0 (t, C-6), 28.6 (q, C-10), 29.1 (q, C-9), 34.4 (d, C-4), 34.7 (t, C-3), 71.1 (d, C-2), 72.6 (s, C-l), 73.5 (s, C-8), MS m/z (%): 170 (M+, 7.0), 155 3

3

3

3

13

2

2


180



C(5)

COO)

Figure 6. X-Ray Crystallography of (+)-Bottrospicatol p-Bromobenzoyl Ester. Reproduced with permission from ref. 11. Copyright 1983 Japan Society for Bioscience, Biotechnology, and Agrochemistry.

R«CH3,C H5 , D-C3H 2

7

Figure 7. Preparation of (+)-Bottrospicatol Derivatives.



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NISHIMURA & NOMA

v


Concentration

( ppm)

Concentration

(ppm)

Figure 8. Effects of (+)-Bottrospicatol (10a) and Its Related Compounds on the Germination of Lettuce Seeds.



182


0 10

30

50

100 Concentration (ppm)

200

Figure 9. Effects of (+)-Bottrospicatal on the Germination of Several Plant Seeds.

(0.6), 126 (83.7), 111 (29.0), 108 (98.9), 93 (26.6), 83 (22.5), 71 (86.0), 69 (40.3), 43 (100). (±)-3-emfo-Hydroxycineole (20). A novel 3-e/ufo-alcohol was isolated as colorless 26

1

crystals, mp 55.0 - 55.5°C; [a] ±0° (c=0.1, EtOH). IR v ^ cm" : 3420 (OH), 1130 (C-O-C), 1060 (alcoholic C-O). *H-NMR S ^ : 1.07 (3H, s, C H at C-7), 1.22 (3H, s, C H at C-9), 1.30 (3H, s, C H at C-10), 1.98 - 2.20 (2H, m, exo-proton signal at ca. 2.17 collapsed to d with 7 m=13 Hz on irradiation at 64.46, CH?-COH), 1 3

3

3

3

ge


NISHIMURA & NOMA



16.


183

184


3

4.46 (1H, dd, 73,2**0=10 Hz, / 2««to=4 Hz, HCOH). * C-NMR ^ : 13.9 (t, C5) , 27.1 (q, C-7), 28.3 (q, C-9), 28.9 (q, C-10), 31.0 (t, C-6), 40.3 (d, C-4), 43.0 (t, C-2), 65.2 (d, C-3), 70.9 (s, C-l), 73.3 (s, C-8). MS m/z (%): 170 (M+, 13.5), 155 (4.6), 137 (4.4), 127 (4.9), 126 (2.9), 108 (15.5), 93 (24.5), 87 (28.6), 85 (21.5), 84 (22.8), 71 (13.6), 69 (25.5), 59 (14.5), 43 (100). (±)-3-ex0-Hydroxycineole (21). A novel 3-ex0-alcohol was isolated as colorless oil: [a]^ ±0° (c=0.1, EtOH). IR v ^ cm- : 3420 (OH), 1100 (C-O-C), 1058 3>

4

n

1

x

(alcoholic C-O). H-NMR 8 ^ J

: 1.11 (3H, s, C H at C-10), 1.98 - 2.15 (2H, m,

1 3

3

endo-^vo\on signal at ca. 2.07 collapsed to d with 7 m=13 Hz on irradiation at 64.15, CH -COH), 4.15 (1H, ddd, 73,2^=10 Hz, 7 , ^=6 Hz, 7 ,4=2 Hz, HCOH). C NMR 5 ^ : 21.5 (t, C-5), 26.9 (q, C-7), 30.2 (q, C-9), 30.5 (q, C-10), 30.9 (t, C6) , 40.8 (d, C-4), 43.3 (t, C-2), 70.2 (d, C-3), 70.9 (s, C-l), 73.4 (s, C-8). MS m/z (%): 170 (M+, 2.1), 155 (46.1), 137 (6.3), 127 (6.1), 126 (2.8), 108 (8.7), 93 (30.0), 87 (8.3), 85 (12.3), 84 (6.6), 71 (7.3), 69 (9.4), 59 (14.2), 43 (100). ge

1 3


2

3

2

3

1 3

22

(±)-2-Oxocineole (19). The crystalline ketone had a mp of 47.0 - 48.0°C and [a] ±0° (c=0.09, EtOH). The ketone was identical with an oxidation product of 2-endohydroxycineole (17) by CrOypyridine (27). IR v ^ cm" : 1730 (C=0), 1150 (C-O-C). 1

*H-NMR : 1.15 (3H, s, C H at C-7), 1.24 (3H, s, CH3 at C-9), 1.39 (3H, s, C H at C-10), 2.21 (1H, dd, 7g =20 Hz, 7 ^ , 4 = 2 Hz, C -Hemfo), 2.79 (1H, dt, ^gem=20 Hz, 7 =3 Hz, 7 ^ ^ = 3 Hz, C -Hejco). MS m/z (%): 168 (M+, 2.6), 140 (9.6), 111 (2.5), 83 (4.2), 82'(100), 71 (2.5), 69 (8.9), 67 (9.9), 43 (30.8). 3

3

em

3ejco4

3

5

3

3

3

23

(±)-3-Oxocineole (22). A novel ketone was isolated as a colorless oil; [oc] ±0° D

e

(c=0.15, EtOH). IR v " * cm-*: 1735 (C=0), 1150 (C-O-C). *H-NMR max

: 1.16 1 Mo

(3H, s, C H at C-7), 1.24 (3H, s, CH3 at C-9), 1.32 (3H, s, C H at C-10), 2.24 (1H, d\ 7 em=20 Hz, C2-Hendo), 2.40 (1H, dd, 7 =20 Hz, J2exo,6exo=3 Hz, C2~Hexo). MS m/z (%): 168 ( M , 8.6), 153 (64.5), 140 (2.6), 125 (15.3),' 111 (57.9), 83 (54.1), 82 (62.8), 71 (6.4), 69 (10.6), 67 (12.8), 55 (26.5), 43 (100). Chemical Conversion of Hydroxycineoles to Economically Useful Substances. Temperature-programmed GC analysis (10% PEG-20M on Celite 545, 3 m x 3 mm i.d.) showed in order of increasing retention time: 1,8-cineole (16), 3-oxocineole (22, 80mg), 2-oxocineole (19, 17mg), 2-enrfo-hydroxycineole (17, 877mg), 3-endohydroxycineole (20, 809mg) and 3-^jco-hydroxycineole (21, 15mg). Time course changes in the products of metabolic oxidation of 16 by A. niger are shown in Figure 12. A peak of the substrate (16) on a GC trace disappeared in 7 days. Furthermore, 3-ejt0-hydroxycineole (21) and 3-end0-hydroxycineole (20) were converted to /?-menthane-3,8-c/s-diol (23) and its trans isomer (24), respectively, which had previously been isolated as allelochemicals from Eucalyptus citriodora (22, 23) by catalytic hydrogenolysis (H2/Pt02) in a similar manner to the reaction of 2hydroxycineole to p-menthane-2,8-diol (24). Column chromatography (Si02) of reaction mixtures gave cis and trans diols (Figure 13) in ca. 15% and 12% yields, respectively. These two compounds exhibited repellent activities against mosquitoes, Aedes albopictus and Culex pipiens (25). The repellent effects of ester derivatives from 3

3

g

gem

+


NISHIMURA & NOMA


2-e/ufo-hydroxycineole (17) Downloaded by CORNELL UNIV on August 16, 2016 | http://pubs.acs.org Publication Date: August 13, 1996 | doi: 10.1021/bk-1996-0637.ch016

80-

^1,8-Cineole "O-, 3-e/wfo-hydroxycineole (20) - A - , 3-ew-hydroxycineole (21) 2- and 3-oxocineole (19+22)

60-

40-

20-

Figure 12. Time Course of Major Metabolic Products of 1,8-Cineole by Strain of Aspergillus niger.

7

(±) cis

(±) trans

Figure 13. (±)- p-Menthane-3,8-diols (cis and trans).


186



p-menthane-3,8-diols were compared with commercially available repellent, D E E T (N,N-diethyl-m-toluamide). Especially, the caproyl ester (C6) of cw-diol (23) exhibited much higher activity than D E E T in terms of repellency and repellent durability against mosquitoes. Flavor of Microbial Transformation Products. The flavor of terpenoids produced by microbial transformation was evaluated. (-)-Carvone (1) is well known as a spearmint flavor component. Transformation products (4 - 7) of 1 had peppermint-like flavor although these are slightly different to each other in terms of the odor quality. The metabolites, bottrospicatols (10a and 10b) Streptomyces species did not have a characteristic flavor but quite different activities (Figure 8). (+)-Bottrospicatal (15) which was produced by the oxidative reaction of (+)-bottrospicatol (10a) with CrC>3 in pyridine had a weak spice-flavor (slightly black-pepper like). The ester derivatives (13 in Figure 7) had a weak medicinal flavor. The flavor of acetyl ester (13a) was the strongest of all. On the other hand, 1,8-cineole (16) which is a significant constituent of Eucalyptus species has a characteristic camphoraceous odor. Oxocineoles (19 and 22) still possessed camphoraceous odor. However, hydroxycineoles (17,18, 20, 21) had slightly medicinal odor. Interestingly, /?-menthane-3,8-diols (23, 24) which were produced by catalytic hydrogenolysis of 3-hydroxycineoles (20 and 21) exhibited no odor to humans. /?-Menthane-3,8-diols are fascinating to cosmetic companies since the compounds have very high repellent activity against mosquitoes. Further research will be necessary to elucidate relationships between chemical structure and flavor. Acknowledgments We thank Mr. Atsushi Satoh, Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University for preparation of the manuscript. This work was partly supported by the Ministry of Education of Japan (Grant 6056-0124). Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Noma, Y.; Tatsumi, C. Nippon Nogeikagaku Kaishi. 1973, 47, 705-711. Noma, Y.; Nonomura, S.; Ueda, H.; Tatsumi, C. Agric. Biol. Chem. 1974, 38, 735-740. Noma, Y.; Nonomura, S.; Sakai, H. Agric. Biol. Chem. 1975, 39, 437-441. Noma, Y. Ann. Rep. Stud., Osaka Joshigakuen Junior College 1976, 20, 3347. Noma, Y.; Nonomura, S. Agric. Biol. Chem. 1974, 38, 741-744. Noma, Y.; Asakawa, Y. Phytochemistry 1992, 31, 2009-2011. Noma, Y . Agric. Biol. Chem. 1980, 44, 807-812. Dhavalikar, R.S.; Bhattacharyya, P.K. Ind. J. Biochem. 1966, 3, 144-157. Dhavalikar, R.S.; Rangachari, P.N.; Bhattacharyya, P.K. Ind. J. Biochem. 1966, 3, 158-164. Noma, Y.; Nishimura, H.; Hiramoto, S.; Iwami, M . ; Tatsumi, C. Agric. Biol. Chem. 1982, 46, 2871-2872. Nishimura, H.; Hiramoto, S.; Mizutani, J.; Noma, Y.; Furusaki, A . ; Matsumoto, T. Agric. Biol. Chem. 1983, 47, 2697-2699. Jori, A.; Bianchetti, A.; Prestini, P.E. Biochem. Pharmacol. 1969, 18, 20812086.


16.

13. 14. 15. 16. 17. 18.


19. 20. 21. 22. 23. 24. 25.

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Jori, A.; Bianchetti, A.; Prestini, P.E.; Garattini, S. Eur. J. Pharmacol. 1970, 9, 362-368. Jori, A.; Salle, E.D.; Pescador, R. J. Pharm. Pharmacol. 1972, 24, 464-468. Jori, A.; Briatico, G. Biochem. Pharmacol. 1973, 23, 543-545. Nishimura, H.; Paton, D.M.; Calvin, M . Agric. Biol. Chem. 1980, 44, 24952496. MacRae, I.C.; Alberts, V.; Carman, R.M.; Shaw, I.M. Aust. J. Chem. 1979, 32, 917-922. Nishimura, H.; Noma, Y.; Mizutani, J. Agric. Biol. Chem. 1982, 46, 26012604. Carman, R.M.; MacRae, I.C.; Perkins, M . V . Aust. J. Chem. 1986, 39, 17391746. Miyazawa, M . ; Nakaoka, H.; Hyakumachi, M . ; Kamioka, H . Chemistry Express 1991, 6, 667-670. Ratcliffe, R.; Rodehorst, R. J. Org. Chem. 1970, 35, 4000-4002. Nishimura, H.; Kaku, K.; Nakamura, T.; Fukuzawa Y.; Mizutani, J. Agric. Biol. Chem. 1982, 46, 319-320. Nishimura, H.; Nakamura, T.; Mizutani, J. Phytochemistry 1984, 23, 27772779. Gandini, A.; Bondavalli, R.; Schenone, P.; Bignardi, G. Ann. Chim. (Rome) 1972, 62, 188-191. Nishimura, H.; Mizutani, J. Proceeding of Hokkaido Tokai University Science and Engineering 1989, 2, 57-65.


Microbial Transformation of Monoterpenes: Flavor and Biological Activity

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