Deinking of soy bean oil based ink printed paper with lipases and a neutral surfactant

Journal of Biotechnology 67 (1999) 229 – 236

Deinking of soy bean oil based ink printed paper with lipases and a neutral surfactant Anne L. Mørkbak, Peter Degn, Wolfgang Zimmermann * Biotechnology Laboratory, Aalborg Uni6ersity, 9000 Aalborg, Denmark Received 23 March 1998; received in revised form 6 October 1998; accepted 16 October 1998

Abstract A screening of different strains of bacteria for the production of lipases which degrade the soy bean oil based binder component of newsprint ink was performed. Three strains were found to be efficient lipase producers and were selected for further investigations. The pH optimum, temperature stability and the optimum enzyme concentrations for the deinking of recovered paper printed with soy bean oil based ink were determined and compared with a commercially available porcine pancreas lipase preparation. A lipase preparation from Pseudomonas aeruginosa with a pH optimum and temperature stability suitable for an application in a deinking process and with a high deinking efficiency in combination with a neutral surfactant could be identified. The deinking effect of the lipases was caused by a partial degradation of the binder of the soy bean oil based inks thereby releasing the ink particles from the paper. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Deinking; Lipase; Paper; Vegetable oil; Ink

1. Introduction Trends in the printing industry to replace the mineral oil in newsprint inks have begun after the oil crisis in the 1970s. Increasing environmental and qualitative demands have resulted in the development of printing inks based on vegetable oils derived from soy beans, rapeseed, castor, linseed * Corresponding author. Tel.: +45-96-358-468, fax: +4598-14255; e-mail: [email protected].

or canola instead of mineral oil. Newsprint inks based on vegetable oil have the advantage of reduced volatile organic compound emission (Power, 1992). Moreover, these inks are derived from renewable and biodegradable sources which facilitate the treatment of the waste water produced in the deinking process and require less aggressive solvents for their removal from the printing press (Anon., 1992). Previously, the prices of vegetable oil inks exceeded those of mineral oil based inks, but this disadvantage has

0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 8 ) 0 0 1 7 9 - 5

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disappeared with the development of new and more advanced formulations. The ease of removal of ink from the printed paper by chemical or enzymatic treatments is mainly influenced by the ink vehicle used. In newsprint inks, the vehicle consists of a mineral or vegetable oil based binder which together with alkyd resins or other hydrocarbon resins entrap the pigment particles and bind them to the surface of the fibres (Borchardt, 1995). In contrast to non-drying mineral oil based inks which just soak into the paper during drying, vegetable oil based inks are semi-drying since internal cross-links in the binder and between the binder and the paper are formed by the fatty acids of the oil. The cross-linking in vegetable oil based inks could potentially result in a poorer deinking and recycling of the printed products. Different results have been reported on the deinkability of recovered paper printed with this type of ink. It has been shown that the content of soy bean oil does not influence the deinkability over age (Dunn and Cathie, 1990; Fuchs et al., 1991; Rosinski, 1995). However, contrary results have also been reported (Anon., 1993). Neutral deinking is a process based on the use of non-ionic surfactants and performed at neutral pH. In contrast to a conventional deinking at high pH, this deinking method avoids the accumulation of alkali-solubilised contaminants and a yellowing of the fibres (Schmid, 1995). A bleaching step can therefore be omitted resulting in a considerable reduction in the amount of chemicals used (Schmid, 1995). However, the removal of residual visible ink particles is less efficient with this method and requires an additional deinking step such as an enzymatic treatment of the paper (Anderson, 1994; Schmid and Schwinger, 1994). Deinking effects can be evaluated by measuring brightness (whiteness), residual ink areas and dirt counts of handsheets made from the recycled paper. Decreases in dirt counts and residual ink areas and increases in brightness values indicate an efficient deinking. Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) are enzymes catalysing the hydrolysis of acyl glycerols at the interface of oil and water (Jensen et al., 1990). Previous studies have shown

that a treatment of paper printed with soy bean oil based ink (VOI) with enzyme preparations containing cellulases, xylanases and lipases in addition to a neutral surfactant resulted in decreased dirt counts and residual ink areas (Mørkbak and Zimmermann, 1998). The deinking of other paper types was also increased by treatment with lipases and esterases (Fukuda et al., 1990; Sharyo and Sakaguchi, 1990; Sugi and Nakamura 1990; Nakano, 1993). These results could be explained by an enzymatic hydrolysis of the oil-based binder or of the resins in the ink. Furthermore, lipases could have a surfactant effect due to their amphiphilic properties and thereby facilitate the deinking of recovered paper. In this paper we show that a commercial lipase preparation and lipase preparations from different bacterial strains could improve the deinking of VOI in combination with a neutral surfactant.

2. Materials and methods

2.1. Micro-organisms and maintenance The lipase-producing strains Streptomyces coelicolor, Streptomyces li6idans, Streptomyces 6enezuelae, Staphylococcus aureus, Bacillus subtilis, Pseudomonas putida and Pseudomonas aeruginosa (Hou and Johnston, 1992) were provided by the Department of Biochemistry and Molecular Biology, University of Georgia, USA. The thermophilic actinomycete isolates TE 13, 22, 75, 80, 82, 87, 118, 135, 207, 208 and 213 were from the culture collection of the Biotechnology Laboratory, Aalborg University. S. coelicolor, S. li6idans, S. 6enezuelae, S. aureus, B. subtilis, P. putida and P. aeruginosa were grown on agar plates at 37°C containing soy flour (DIFCO, MI, USA), 20.0 g l − 1; mannitol (DIFCO, MI, USA), 20.0 g l − 1 and agar (DIFCO, MI, USA), 18.0 g l − 1 in double distilled water. The actinomycete isolates were grown at 55°C on agar plates containing malt extract (Sigma, MO, USA), 7.5 g l − 1; yeast extract (DIFCO, MI, USA), 3.0 g l − 1; glucose, 3.0 g l − 1 and agar, 20.0 g l − 1 in double distilled water.

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2.2. Culture conditions and enzyme preparation S. coelicolor, S. li6idans, S. 6enezuelae and S. aureus were grown in a medium containing malt extract, 7.5 g l − 1; yeast extract, 3.0 g l − 1 and polyethylene glycol, 50.0 g l − 1 in double distilled water. B. subtilis was grown in a medium containing yeast extract, 5.0 g l − 1; tryptone (DIFCO, MI, USA), 10 g l − 1; NaCl, 5.0 g l − 1 and 1 N NaOH, 1 ml l − 1 in double distilled water. P. putida and P. aeruginosa were grown in a medium containing glucose, 3.0 g l − 1; (NH4)2SO4, 1.0 g l − 1; KH2SO4, 2.0 g l − 1 and MgSO4, 0.05 g l − 1 in double distilled water. The actinomycete isolates were grown in a medium containing malt extract, 7.5 g l − 1; yeast extract, 3.0 g l − 1 and glucose, 3.0 g l − 1 in double distilled water. All media (50 ml in 250 ml Erlenmeyer flasks) were adjusted to pH 7.2, inoculated and incubated in a shaking incubator (New Brunswick Scientific, Edison, NJ, USA) at 250 rpm. S. coelicolor, S. li6idans, S. 6enezuelae, S. aureus, B. subtilis, P. putida and P. aeruginosa were grown at 37°C and the actinomycete isolates were grown at 55°C. These cultures were used to inoculate the culture media for lipase production. S. coelicolor, S. li6idans, S. 6enezuelae, S. aureus, P. putida and P. aeruginosa were grown in 250 ml flasks containing 50 ml medium of the following composition: trace element solution (ZnCl2, 0.04 g l − 1; FeCl.3 6 H2O, 0.20 g l − 1; CuCl.2 2 H2O, 0.01 g l − 1; MnCl.2 4 H2O, 0.01 g l − 1; Na2B4O.710 H2O, 0.01 g l − 1 and (NH4)6Mo7O24 · 4 H2O, 0.01 g l − 1 in double distilled water), 1 ml l − 1; mineral salt solution ((NH4)2SO4, 3.0 g l − 1; NaCl, 1.0 g l − 1; NH4Cl, 10.0 g l − 1; MgSO4 · 7 H2O, 1.0 g l − 1; CaCO3, 0.4 g l − 1 and KH2PO4, 6.0 g l − 1 in double distilled water), 12.5 ml l − 1; yeast extract, 1.0 g l − 1; polyethylene glycol, 50.0 g l − 1 and binder from soy bean oil based ink (Akzo Nobel A/S, Denmark), 10.0 g l − 1 in double distilled water. The actinomycete isolates were grown in a medium containing trace element solution, 1 ml l − 1; mineral salt solution, 12.5 ml l − 1; yeast extract, 1.0 g l − 1 and binder from soy bean oil based ink, 10.0 g l − 1 in double distilled water. B. subtilis was grown in a medium containing K2PO4, 7.0 g l − 1; KH2PO4, 2.0 g l − 1; Na3C6H5O7, 0.5 g l − 1; MgSO4, 0.1 g l − 1;

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(NH4)2SO4, 1.0 g l − 1 and binder from soy bean oil based ink, 10.0 g l − 1 in double distilled water. S. coelicolor, S. li6idans, S. 6enezuelae, S. aureus, B. subtilis, P. putida and P. aeruginosa were grown at 37°C and the actinomycete isolates were grown at 55°C. Samples were taken every 12 h, centrifuged at 10 000 rpm for 10 min and the lipase activity was determined in the supernatant. The three strains (TE 135, TE 208 and P. aeruginosa) producing the highest lipase activity were grown for 2–5 days in 500 ml medium in 2.8 l Fernbach flasks and 2.5-l Erlenmeyer flasks. The culture broths were centrifuged at 10 000 rpm for 30 min, the supernatants were freeze dried, solubilised in 50 ml phosphate buffer 0.05 M (pH 7) and stored at 5°C.

2.3. Lipase assay Lipase activity was determined at 37°C using p-nitrophenyl palmitate as substrate (Erdmann et al., 1992).

2.4. PH optimum The pH optimum of the respective lipase preparations was determined by performing the lipase activity assay using 0.05 M sodium phosphate buffers with pH varying from 6 to 8.

2.5. Temperature stability The temperature stability was determined by incubating the lipase preparations for 1 h at temperatures varying from 40 to 70 °C. After incubation, the lipase assays were performed at the optimum pH for the respective lipase preparations.

2.6. Deinking experiments 1.2 kg of VOI was gradually added to 12 l of tap water in a 25 l laboratory pulper, partially disintegrated, and stored at 5°C. The partially disintegrated paper (1.5 l, 10.0% consistency) was diluted to 7.5% consistency with tap water. All samples were treated with a neutral surfactant (Incopur, Kolb AG, Switzerland, 0.065% based

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on paper dry weight) and NaSiO3 (0.4% based on paper dry weight). After addition of the deinking chemicals, the pH was adjusted by addition of 1 M NaOH or HCl. Deinking with the lipase preparations produced by TE208 and TE135 grown on the soy bean oil based binder (TE208L and TE135L) was performed at 8 while deinking with porcine pancreas lipase (PPL, Sigma, MO, USA) and the lipases produced by P. aeruginosa grown on the soy bean oil based binder (PAL) was performed at 7.5. PPL, TE135L, TE205L and PAL were added in concentrations varying from 10 to 500 nkat g − 1 paper dry weight. Pulping (75 000 revolutions) was performed with a NORAM homogeniser (Lorentzen & Wettre, Sweden) at the optimal temperature for the respective lipase. The optimal temperature was 40°C for PPL and TE135L, 60°C for PAL and 70°C for TE208L. Flotation was carried out for 15 min at 1.0% consistency w v − 1 with an air flow of 10 l min − 1.

2.7. Lipase inhibition PPL (8.0 g l − 1) was incubated for 12 h at 25°C with iodine (0.34 g l − 1) in deionised water to inhibit lipase activity (Gargouri et al., 1997). Fifty nkat of active PPL (0.003 g protein), iodine-inhibited PPL (0.003 g protein) or 0.003 g bovine serum albumin (BSA, Sigma, MO, USA) were added per g paper dry weight. Pulping (750 rpm) was performed in a 25 l laboratory pulper for 20 min at 45 – 50°C. The pulping consistency was 10%.

2.8. Handsheets Handsheets for image analysis and brightness measurement were made according to TAPPI T 218 om-83.

2.9. Deinking e6aluation methods Image analysis of the handsheets made from recycled VOI was performed using a PC, a Hewlett Packard ScanJet 3c scanner and Spec

Scan 2000 software (Apogee Systems, USA). Scanning resolution was 600 dpi, threshold was 140 and the analysed area of handsheets was 324 cm2 for each repeat. Dirt counts and residual ink areas of the handsheets were determined by image analysis. Brightness of the handsheets was measured according to TAPPI Test Methods T 452 om-57.

2.10. Statistics The data were evaluated using a one factor analysis of variance and a Fisher’s PLSD test. All tests were done with a confidence interval of 95%.

2.11. Enzymatic hydrolysis of the binder The degradation of the binder in soy bean oil based ink was analysed by incubating 0.1 g of binder with PPL and PAL (200 nkat g − 1) in 1 ml sodium phosphate buffer 0.05 M (pH 8) for 0, 30, 60 and 180 min at 37 °C and 250 rpm. Incopur (2%) was added for emulgation and 1decanol (0.5 mM) as an internal standard. For the detection of hydrolysed fatty acids, the samples were extracted with chloroform/methanol 1:2 (Yorkowski and Walker, 1969, 1970). The free fatty acids were analysed with a gas chromatograph (Chrompack CP 9002) equipped with a CP 9050 liquid sampler. Samples (1.5 ml) were injected in splitless mode onto a CP-wax-58 (FFAP), 25 m×0.32 mm (i.d.) fused silica capillary column. The injection port temperature was 275°C and purge time was 0.5 min with a gas flow of 70 ml min − 1. The temperature was increased by 35°C min − 1 from 45 to 240°C and this temperature was kept for 130 min. The carrier gas was He and the column flow was held constant at 22 cm s − 1. The FID detector temperature was 275°C. Products with retention times of 29.84, 45.46, 49.10 and 55.12 min were identified as hexadecanoic acid (C16), octadecanoic acid (C18), octa-6-enoic acid (C18:1) and octadeca-9:12-dienoic acid (C18:2), respectively, by comparison of their retention times with those of standards (Sigma, MO, USA).

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g − 1 paper dry weight resulted in the highest brightness increase of the handsheets made from recycled VOI, while treatments with TE135L and TE208L had no effect on the brightness (Fig. 2a). Furthermore, treatment of VOI with PAL in concentrations up to 100 nkat g − 1 paper dry weight had no effect on the brightness, while the application of 200–500 nkat g − 1 paper dry weight of PAL increased the brightness. Decreases in residual ink area and dirt count of the handsheets made from recycled VOI were obtained by treatment of VOI with 50 nkat g − 1 paper dry weight of PPL. Higher enzyme concentrations did not result in further decreases (Fig. 2b and c). Treatment with PAL and TE135L also resulted in a decrease in dirt counts and residual ink areas but required much higher enzyme concentrations. Treatment of VOI with 10 nkat g − 1 paper dry weight of TE208L resulted in a slight decrease in dirt count and residual ink area, while at higher concentrations of this enzyme even a slight increase in dirt count and residual ink area was obtained. The differences in the performance of the lipase preparations in the deinking of VOI could be a result of varying degrees of degradation of the soy bean oil based binder or the resins contained in the ink depending on the type of enzymes used. While PPL showed to be the most efficient enzyme, higher concentrations of PAL and TE135L were necessary to obtain a similar effect. Treatment of VOI with PPL and with higher concen-

3. Results and discussion

3.1. Lipase acti6ity of the bacterial strains Analysis of the bacterial strains showed differences in their ability to grow on media containing soy bean oil based binder and in their lipase production (Table 1). The most efficient lipase producers were P. aeruginosa, TE 135 and TE 208 which were selected for further investigations.

3.2. PH optimum and temperature stability of the lipases The pH optima for both PPL and PAL were close to pH 7.5 whereas the pH optima for TE208L and TE135L were at or above pH 8 (Fig. 1a). Due to hydrolysis of the p-nitrophenyl palmitate substrate, pH values above 8 could not be tested. The temperature stabilities of the lipase preparations varied considerably. PPL and TE135L were completely inactivated at 60°C, whereas PAL and TE208L were stable up to 60 and 70°C, respectively (Fig. 1b).

3.3. Deinking of VOI with lipase preparations The lipase preparations showed different effects on the deinking of VOI which were also depending on the amount of enzymes applied. Treatment of VOI with PPL in concentrations up to 50 nkat

Table 1 Lipase activity in cultures of the bacterial strains grown in 50 ml liquid medium containing soy bean oil based binder (10 g l−1) Bacterial strain

Lipase activitya (U ml−1)

Bacterial strain

Lipase activitya (U ml−1)

S. coelicolor S. li6idans S. 6enezuelae S. aureus B. subtilis P. putida P. aeruginosa TE 13 TE 22

0.04 0.04 0.04 0.04 0.06 0.03 0.90 —b 0.06

TE TE TE TE TE TE TE TE TE

0.00 —b 0.08 0.00 0.02 0.09 —b 0.13 0.00

a b

Highest activity determined after 2–5 days. No growth.

75 80 82 87 118 135 207 208 213

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3.4. Mechanism of deinking of VOI by lipases The mechanism of deinking of VOI by lipases was studied by comparing the deinking effects obtained by treatment with enzyme preparations containing iodine-inhibited PPL, active PPL or BSA. BSA was included to compare the effects of the presence of protein on the deinking of VOI with those of the active and inactive PPL. Com-

Fig. 1. PH optima (a) and temperature stabilities (b) of a porcine pancreas lipase preparation (PPL; "), lipases from P. aeruginosa (PAL; ), lipases from TE135 (TE135L; ), and lipases from TE208 (TE208L; “).

trations of PAL resulted in both increases in brightness and decreases in dirt count and residual ink area, while TE135L and TE208L did not affect brightness. This effect could be due to a liberation of ink particles by the enzyme treatment which are below the size range of particles which can be efficiently removed by flotation. This would result in unchanged brightness values even though larger ink particles were removed. The increase in dirt count and residual ink area observed with high concentrations of TE208L could be due to an excessive degradation of the soy bean oil based binder or the resin contained in the ink resulting in the formation of ink particles outside the size range which can be efficiently removed by flotation.

Fig. 2. Effects of different concentrations of a porcine pancreas lipase preparation (PPL; "), lipases from P. aeruginosa (PAL; ), lipases from TE135 (TE135L; ) and lipases from TE208 (TE208L; “) in combination with a neutral surfactant on the deinking of VOI. Changes in brightness (a), residual ink area (b) and dirt count (c) compared to the neutral surfactant treated control were determined.

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Table 2 Initial formation rates of C16, C18, C18:1, and C18:2 during hydrolysis of soy bean oil based binder by a porcine pancreas lipase preparation (PPL) and lipases from P. aeruginosa (PAL) (200 nkat g−1) Enzyme

C16 (nmol min−1)

C18 (nmol min−1)

C18:1 (nmol min−1)

C18:2 (nmol min−1)

PPL PAL

15.0 5.0

6.0 1.0

15.0 4.0

3.0 1.0

pared to the control which was treated with surfactant only, the addition of active PPL increased the brightness and decreased the residual ink of the handsheets made from recycled VOI significantly. In contrast, treatment with BSA or with the inactive PPL preparation did not result in significant deinking effects. The results show that the catalytic activity of PPL and not its surfactant properties or the presence of protein were responsible for the deinking of VOI. This indicates that a degradation of the binder or the resin in the ink is necessary for obtaining a deinking effect. Treatment of soy bean oil based binder with PPL and PAL resulted in a limited release of fatty acids which could be detected by gas chromatography. After 20 min of incubation, less than 1% of the total amount of fatty acids contained in the binder were released indicating that a limited hydrolysis of the binder is sufficient to obtain a deinking effect. The initial formation rates of the major fatty acids contained in soy bean oil (C18, C18:1, and C18:2) obtained by hydrolysis of the binder with PPL were higher compared to PAL indicating a more efficient degradation of the soy bean oil based binder by PPL (Table 2). This is in accordance with the deinking results obtained showing a more efficient deinking of VOI by treatment with PPL compared to PAL. The differences detected in the performance of lipases in the deinking of VOI show the importance of selecting specific lipases suitable for an enzymatic deinking process. Treatment with PPL resulted in the most efficient deinking at low concentrations. However, the higher temperature stability of PAL and the efficient deinking effects obtained at higher temperatures indicate that PAL would be more suitable for application in an industrial deinking process which is often per-

formed at temperatures above 40°C (Biermann, 1993). Our results indicate that the observed deinking effects of the lipases are due to a partial degradation of the vegetable oil based binder thereby releasing ink particles from the paper.

Acknowledgements This work was supported by a grant from the Danish Research Academy to Anne Louise Mørkbak and from the Danish Research Council to Peter Degn. We are grateful to Kolb AG, Switzerland for providing the surfactant and Akzo Nobel A/S, Denmark for providing paper printed with soy bean oil based ink.

References Anderson, J., 1994. Chemical techniques to improve deinking efficiency. World Paper 3, 176 – 180. Anon, 1992. Possibilities for the use of soy oil in European printing inks Cobufimat N.V. report. Geraadsbergen, Belgium. Anon., 1993. The effect of the vegetable ink printing act on the potential market for vegetable oils, Am. Ink Marker 71, 37 – 40, 42, 55, 76. Biermann, C.J., 1993. Essentials of Pulping and Papermaking. Academic Press, San Diego, CA. Borchardt, J.K., 1995. Ink types: The role of ink in deinking. Prog. Paper Recycl. 5, 81 – 87. Dunn, R. and Cathie, K., 1990. Vegetable oil inks, Pira report 32/PB/88/8. Erdmann, H., Fritsche, K., Kordel, M., Lang, S., Lokotsch, M., Markweg, M., Schneider, M., Syldatk, C, Wagner, F., 1992. Lipases: selected applications in preparative organic chemistry. In: Finn, R.K., et al. (Eds.), Biotechnology Focus, vol. 3. Hanser, Mu¨nchen, Germany, pp. 333 – 358. Fuchs, B., Lindquist, U., Wallstrom, E., 1991. Vegetable oil based newsinks and their printability properties and deinkability. TAGA Conference, Rochester, NY, pp. 433 – 464.

A.L. Mørkbak et al. / Journal of Biotechnology 67 (1999) 229–236

236

Fukuda, S., Hayashi, S., Ochiai, H., Ihizumi, T., Nakamura, T., 1990. Japanese patent 229, 290/90. Gargouri, Y., Ransac, S., Verger, R., 1997. Covalent inhibition of digestive lipases: an in vitro study. Biochim. Biophys. Acta 1344, 6 – 37. Hou, C.T., Johnston, T.M., 1992. Screening of lipase activities with cultures from the agricultural research service culture collection. J. Am. Oil Chem. Soc. 69, 1088–1097. Jensen, R.G., Galluzzo, D.R., Bush, V.J., 1990. Selectivity is an important characteristic of lipases(acyl-glycerol hydrolases). Biocatalysis 3, 307–316. Mørkbak, A.L., Zimmermann, W., 1998. Deinking of mixed office waste, old newspaper and vegetable oil-based ink printed paper using cellulases, xylanases and lipases. Prog. Paper Recycl. 7 (2), 14–21. Nakano, J., 1993. Recent research trends in pulping chemistry. J. Korea Tappi 25, 85–91.

.

Power, J., 1992. Printing with soya inks. New Zealand Printer 9/10, 52. Rosinski, J., 1995. Aging and deinking of soy printed newsprint. Prog. Paper Recycl. 4, 55 – 62. Schmid, H., Schwinger, K., 1994. Mill experience of flotation deinking of mixes in neutral medium. Wochenblatt fu¨r Papierfabrikation 122, 234 – 241. Schmid, H., 1995. Deinkinganlage zur Aufbereitung von Haushaltsammelware im neutralen pH Bereich. Wochenblatt fu¨r Papierfabrikation 14/15, 621 – 626. Sharyo, M., Sakaguchi, H., 1990. Japanese patent 2,160,984. Sugi, T., Nakamura, K., 1990. Japanese patent 249,291/91. Yorkowski, M., Walker, B.L., 1969. Formation of methyl esters by rat musosa in methanol. FEBS Lett. 2, 265 – 266. Yorkowski, M., Walker, B.L., 1970. Lipids of the intestinal mucosa of normal and essential fatty acid deficient rats. Can. J. Physiol. Pharmacol. 48, 631 – 639.