Biosci. Biotechnol. Biochem., 68 (11), 2401–2404, 2004 Note Increased Levels of Policosanol and Very Long-Chain Fatty Acids in Potato Pulp Fermented with Rhizopus oryzae

y Renaguli MUSA,1;2 Keita YUNOKI,1;2 Mikio KINOSHITA,1 Yuji ODA,3 and Masao OHNISHI1;2;

1Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan 2The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japan 3Department of Upland Agriculture, National Agricultural Research Center for Hokkaido Region, Memuro, Kasai, Hokkaido 082-0071, Japan

Received May 27, 2004; Accepted August 4, 2004

Significant amounts of policosanol and very long- lecular-weight alcohols isolated from sugar cane, in chain fatty acids (VLFAs) ranging in carbon length which the main component is octacosanol;2) there have from 22 to 30 were found in the lipophilic fraction been many reports on its physiological functions such as obtained from potato pulp fermented with Rhizopus inducing a progressive effect on athletic performance, oryzae. It is believed that these compounds would have resistance to stressors, reduction of cholesterol, and originally been present as suberin-related compounds, inhibition of platelet aggregation.3) Therefore, policosa- but not as wax, in the periderm of potato tubers and nol is separated and utilized as a functional foodstuff on concentrated into potato pulp during the process of an industrial scale. However, VLFAs have also been starch production. Moreover, the policosanol and isolated and purified from sugar cane wax. Although VLFAs extracted from potato pulp with organic sol- VLFAs are structurally related to their corresponding vents were found to have increased after fermentation. policosanol, the pharmacological effects of policosanol have been reported to differ from those exhibited by Key words: functional lipids; very long-chain fatty VLFAs; for example, VLFAs from sugar cane wax have acids; octacosanol; potato pulp; Rhizopus been shown to protect lipoprotein fractions against lipid oryzae peroxidation in lipids as well as in the protein moiety by oral administration to rats.4) Potato pulp is a by-product of potato starch produc- In this present study, the composition and concen- tion, reaching annually more than 100 thousand tons in trations of VLFAs and policosanol in potato pulp Hokkaido, the relevant agricultural district in northern fermented with R. oryzae were determined. We also Japan. Despite its relatively high nutrient value, potato analyzed their components in raw potato pulp and potato pulp is mostly composted and used regionally as an tubers, in order to discuss the possible origin of these organic fertilizer, because its high moisture content compounds and the advantage of lactic acid fermenta- (80%) requires an expensive drying process due to the tion. problem of spoilage if left untreated. However, a Potato pulp (80% moisture content), obtained from a practical technique for lactic acid fermentation of potato starch factory near Obihiro in Hokkaido, was autoclaved pulp has been developed.1) Since the low pH due to at 121 C for 5 min. After cooling to room temperature, lactic acid formation can suppress the microorganisms spores of R. oryzae NBRC 4707 were inoculated at a responsible for spoilage, Rhizopus oryzae would be an final concentration of 105/g of wet potato pulp, and the inoculant for ensiling potato pulp to use as cattle feed mixture was fermented for 7 days at 25 C as previously even under aerobic conditions. In addition, fermented described.5) The fermented potato pulp and its raw potato pulp could be expected to become a functional material were lyophilized, before being extracted three foodstuff, because species of the Rhizopus fungus are times with five volumes of a chloroform–methanol used not only for fermenting local foods in China and solution (2:1, v/v) and then twice with three volumes of Southeast Asia but also in the food industry. water-saturated butanol.6) The combined extracts were In our survey of functional compounds in fermented washed with water, and the chloroform layer was potato pulp, policosanol and very long-chain fatty acids evaporated to dryness to yield total lipids (1.3%). (VLFAs) were found in extracts obtained with organic Moreover, after separating into the edible part and the solvents. Policosanol refers to a mixture of high-mo- periderm of potato tubers (the Konahubuki variety used

y To whom correspondence should be addressed. Fax: +81-155-49-5549; E-mail: [email protected] 2402 R. MUSA et al. for starch production) in proportions of 82% and 18%, Table 1. Fatty Acid Composition (mol %) of Total Lipids in both parts were respectively homogenized in a chloro- Fermented and Raw Potato Pulp, and in the Edible and Periderm of Potato Tubers form–methanol mixture, and total lipids were prepared by the procedure just described. The total lipids obtained Potato pulp Potato tuber Fatty acid were separated into the neutral and polar fractions by Fermented Raw Periderm Edible part silicic acid column chromatography. To separate the 12:0 <0:1 0.3 0.1 <0:1 fraction including free fatty acids and aliphatic alcohols, 14:0 0.6 1.0 0.4 0.2 the neutral lipids were dissolved in a chloroform– 15:0 0.1 1.0 0.3 0.2 methanol solution (2:1, v/v) and extracted twice with 16:0 23.3 26.1 18.6 19.5 hexane. 16:1(7) <0:1 <0:1 0.2 0.1 The lipophilic compounds were treated with meth- 16:1(9) 1.6 2.1 — — 17:0 — — 0.2 0.2 anolic 5% HCl for 3 hr at 95 C. The fatty acid methyl 18:0 10.8 5.3 6.2 6.1 esters and fatty alcohols liberated were then extracted 18:1(9) 34.0 5.5 0.8 0.5 with hexane. The fatty acid methyl esters thus obtained 18:1(11) 0.8 1.2 0.7 0.7 were analyzed by GC and GC–MS. The aliphatic 18:2 7.3 9.3 36.2 47.2 alcohols and free fatty acids were respectively converted 18:3 2.7 — — — 18:3 <0:1 3.5 24.7 22.4 to trimethylsilyl derivatives with N-trimethylsilylimid- 20:0 0.1 1.7 1.2 1.1 azole and a mixture of pyridine, N,O-bis(trimethylsilyl) 21:0 <0:1 <0:1 0.1 0.1 trifluoroacetamide and trimethylchlorosilane, before 22:0 0.8 2.3 1.0 0.5 being analyzed by GC–MS. 23:0 <0:1 0.5 0.6 0.5 GC–MS was performed with a QP-2010 gas chroma- 24:0 1.5 4.5 1.6 0.6 25:0 0.1 0.5 0.3 0.1 tograph-mass spectrometer (Shimadzu Co., Kyoto, 26:0 2.0 5.2 1.2 — Japan). The chromatograph was fitted with a capillary 27:0 0.2 0.5 0.2 — column of ULBON HR-1 (50 m 0:25 mm, GL Sci- 28:0 7.0 17.2 3.0 — ence, Tokyo, Japan). The column was programmed from 29:0 2.4 4.2 0.8 — 80 C to 180 Cat15C/min, then increased to 230 C 30:0 4.7 8.1 1.6 — at 2 C/min, and finally at 4 C/min to 290 C. GC was performed with a Shimadzu 9A gas chromatograph in the same capillary column as that used for GC–MS. Table 2. Fatty Alcohol Composition (mol %) of Total Lipids in Tridecanoic acid and 1-nonadecanol (Tokyo Kasei Fermented and Raw Potato Pulp and in the Periderm of Potato Tubers Kogyo Co., Tokyo, Japan) were used as internal Potato pulp standards for quantitative analyses. The data reported Fatty alcohol Potato periderm are averages from two independent experiments. Fermented Raw The total ion chromatogram by GC–MS of hexane Hexadecanol 2.8 2.9 4.3 extracts of the methanolyzates prepared from total lipids Octadecanol 3.9 5.6 4.8 in the fermented potato pulp showed at least twenty- Icosanol 1.2 2.4 1.2 three fatty acid methyl esters ranging from C12 to C30 Henicosanol 5.1 6.5 5.5 Docosanol 3.4 4.9 5.3 (data not shown). With regard to VLFAs, octacosanoic Tricosanol 2.3 2.3 0.9 (28:0) and triacontanoic acids (30:0) were predominant, Tetracosanol 3.6 3.8 3.1 although the principal fatty acid components were oleic Pentacosanol 1.9 2.4 3.4 [18:1(9)] and palmitic acids (16:0) (Table 1). -Lino- Hexacosanol 9.1 8.6 8.0 lenic acid (18:3) was also detected, which is a typical Heptacosanol 3.2 2.7 3.0 fatty acid originating from R. oryzae.7) A GC analysis of Octacosanol 50.3 46.9 49.2 Nonacosanol 9.9 7.5 7.3 the hexane-soluble matter obtained from the neutral Triacontanol 3.3 3.5 4.0 lipids as TMS derivatives gave some peaks correspond- ing to the VLFAs, indicating that VLFAs were partially present in a free form. When GC profiles of the addition to fatty acid methyl ester peaks, at least thirteen component fatty acids were compared between ferment- saturated higher alcohols with a carbon length from 16 ed and raw potato pulp, oleic acid was much richer in to 30 that were newly detected (Table 2). Among these, the fermented potato pulp than in the raw pulp. In octacosanol amounted to approximately 50% of the contrast, the relative proportions of VLFAs were usually whole; the proportions of the other principal fatty found to be higher in the potato pulp before fermentation alcohols with carbon lengths of 26 and 29 were 9.1% (Table 1). This discrepancy can be attributed to in- and 9.9%, respectively. These aliphatic alcohols were creases of such fatty acids as stearic and oleic acids that also found in raw potato pulp (Table 2). were extracted from the mycelia of R. oryzae grown Table 3 shows the fatty acid and alcohol contents per during fermentation. 100 g of dry potato pulp before and after lactic acid The total ion chromatogram of hexane extracts of the fermentation with R. oryzae. The amount of VLFAs in methanolyzate after TMS ether conversion shows, in the extractive lipophilic compounds was 2.7-fold higher Functional Lipids in Fermented Potato Pulp 2403 Table 3. Concentrations (mg/100 g) of Fatty Acids and Fatty related compounds (metabolic intermediates or degra- Alcohols in Fermented and Raw Potato Pulp dation products), ferulic acid esters of aliphatic alcohols Fatty acids Fatty alcohols Octacosanol ranging in carbon length from 16 to 28 have been Potato pulp detected in the periderm of potato tubers.11) Although Total VLFAs* Total Free Total Free VLFAs have been found in the solubilized monomers Raw 150 81 193 35 98 20 and dimers of potato periderm suberin,10) the form of Fermented 845 221 274 45 146 25 VLFAs and of policosanol, apart from their free form, in *Very long-chain fatty acids with a carbon number from 22 to 30. the lipophilic fraction from potato pulp remains unclear. This will need further investigation. after fermentation, while the total fatty acid amount in Significant amounts of VLFAs and policosanol might fermented potato pulp was 5.6-fold more from 150 mg to also be present in the suberin structure of potato pulp, 845 mg. This notable increase of fatty acid components but suberin is not regarded as a nutrient for humans and in fermented potato pulp would have been caused by cannot be expected to exhibit the physiological effects R. oryzae growing in the potato pulp. However, the total from VLFAs and policosanol, because humans do not amounts of higher alcohols in the raw and fermented have digestive enzymes to depolymerize suberin. Only potato pulp were 193 mg and 274 mg, respectively. VLFAs and policosanol in the lipophilic fraction Thus, the increase in alcohol level was observed after extracted by organic solvents without any degradation lactic acid fermentation; the concentration of total of the suberin structure were analyzed in this study. The octacosanol was also found to have increased from increased levels of both compounds after lactic acid 98 mg to 146 mg by fermentation. Quantitative analyses fermentation would have been due to the action of by GC, after TMS conversion with N-trimethylsilylimi- R. oryzae to break the ester bonds of suberin, by which dazole, of the hexane-soluble matter in the neutral lipid VLFAs and policosanol would have been converted to fraction showed that less than 20% of all alcohols was simple lipophilic compounds including their free form present in the free form (Table 3). with high bioavailability. It thus appears that lactic acid To elucidate the origin of the VLFAs and policosanol fermentation with R. oryzae may have the advantage of in potato pulp, the edible part and periderm of potato enhancing the physiological functionality of potato pulp tubers were also analyzed (Tables 1 and 2). The major as a foodstuff, based on the availability of VLFAs and fatty acid components in the edible parts were linoleic policosanol. (18:2), -linolenic (18:3) and palmitic acids, while VLFAs, especially octacosanoic acid, were almost Acknowledgments absent. Although the three fatty acids just listed were also predominant in the periderm, VLFAs with a carbon This work was supported in part by Special Coordi- length from 22 to 30 were notably detected. However, nation Funds for Promoting Science and Technology aliphatic alcohols, including octacosanol, were found in (Leading Research Utilizing Potential of Regional the periderm but not in the edible part of potato tubers Science and Technology) from the Japanese Ministry (Table 2). It thus became clear that VLFAs and of Education, Culture, Sports, Science and Technology. policosanol in the periderm were concentrated in the potato pulp during the process of starch production. TLC of neutral lipids on silica gel with hexane–diethyl References ether–acetic acid (80:20:1, v/v) did not show any spot corresponding to wax in the periderm of potato tubers or 1) Oda, Y., Saito, K., Yamauchi, H., and Mori, M., Lactic acid fermentation of potato pulp by the fungus Rhizopus in potato pulp (data not shown),8) indicating that VLFAs oryzae. Curr. Microbiol., 45, 1–4 (2002). and policosanol were not present as wax components. 2) Arruzazabala, M. L., Carbajal, D., Mas, R., Garcia, M., Although the fatty acid compositions of the periderm of and Fraga, V., Effects of policosanol on platelet potato tubers and raw potato pulp were significantly aggregation in rats. Thromb. Res., 69, 321–327 (1993). different from each other, there was no notable differ- 3) Taylor, J. C., Rapport, L., and Lockwood, G. B., ence in alcohol composition between the two samples Octacosanol in human health. Nutrition, 19, 192–195 (Tables 1 and 2). This indicates that autoxidation or (2003). enzymatic oxidation and subsequent decomposition of 4) Menendez, R., Mas, R., Amor, A. M., Ledon, N., Perez, such polyunsaturated fatty acids as 18:2 and 18:3 J., Gonzalez, R. M., Rodeiro, I., Zayas, M., and Jimenez, occurred during the process of starch production.9) S., Inhibition of rat lipoprotein lipid peroxidation by the It is known that VLFAs and policosanol are present as oral administration of D003, a mixture of very long- chain saturated fatty acids. Can. J. 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