Results

Pressing tests without enzymatic pretreatment In the case of Coriandrum and Calendula direct pressing gave low yields. Investigations of the reasons for this suggested that one reason for the poor pressing behaviour of uncrushed seeds lies in their structure. Conventional pressing tools have been designed for processing of seeds with hard and brittle hulls and a higher ratio of oil-storage cells relative to the volume of hulls. Hard hulls assist in the transmission of forces from the pressing tool into the seed. In the case of Calendula and Coriandrum seeds the oil content is between 16 and 22%, while the hulls are soft and elastic hulls. The proportion of hull material is between 60 and 70% in the case of Calendula and between 30 and 50% for Coriandrum. The lack of a hard shell results in poor and unequal transmission of forces so that the results are poor; seed is transported and compacted and only a small amount of oil is expelled. When pressure is relieved, at the end of press channel, the seed material reabsorbs a significant proportion of the extracted oil. The need to have hard seed components became especially apparent in pressing of milled seed. Seeds milled to varying degree of particle size were examined. No oil was removed as a consequence of reducing the size of the few hard particles in these seeds.

Pressing tests following enzymatic pretreatment Tests with enzyme treated seeds gave oil yields that were slightly higher. This oil was removed from the seed but not actually extracted in a form available for further use. Loss of oil due to aqueous extraction during the enzymatic pretreatment considerably reduced the extracted volume of useable oil, so that conventional processing methods produced better results.

Extensive tests were conducted with whole seeds, varying incubation times for up to 12 hours, using enzyme concentrations of up to 10%. However, even the most extreme enzymes treatment failed to lyse the hulls to the necessary degree. The unfavourable ratio between hull and of oil-storage tissue (seeds) and the thick fibrous structure of hulls made it impossible to lyse hulls of whole seed in a reasonably time under the conditions used. Squeezing or dehulling would be required in order to bring the enzymes into direct contact with the oil-storage tissue. A suitable method for dehulling of Calendula and Coriandrum is not known. Milling of seed cannot increase the efficiency of oil extraction from these seeds.

Enzymes used for the tests were selected on the basis of experiments on rape and canola seeds where positive results are known. The use of these enzymes in the case of uncrushed or milled Calendula seed demonstrated that such results cannot be reproduced for processing of other kinds of seed. Experiments were also carried out in the laboratory in order to verify the enzyme activity under standard conditions. Higher yields were found with Calendula, Crambe and Coriandrum permitted higher oil yields but in conjunction with losses caused by aqueous extraction as seen at the pilot scale.

During pressing tests with Calendula in press increasing temperature led to polmerization followed by plugging of the press. Polymerization reflected the content of linolenic acid (C 18:3) and especially on the content of reactive calendula acid (C 18:3 ~ 8, 10, 12), a conjugated fatty acid with trans and cis double bonds that split and form cross-links with further fatty acid molecules and residues forming larger molecules. It is assumed that other seed constituents, such as proteins, promote this process resulting in plastic bodies reflecting the effects of pressure, friction and the temperature rise resulting from this. In fact, such a mechanism has been used in the past for production of linoleum from linseed oil.

Hexane extraction Various experiments in which levels of oil and residual oil content were determined by hexane extraction (Soxhlet) gave remarkable different results. The reason reflects fact the crushing methods employed. Those used for rape and sunflower seeds are not at all suited for Calendula and Coriandrum, and are suitable for Crambe only with certain restrictions. A higher degree of crushing is required for processing of Calendula and Coriandrum. Furthermore, the material to be extracted has to be checked for uncrushed seeds. If these were removed prior to extraction reproduceable results were obtained.

Summary of processing results

• The enzymatic pretreatment of whole seeds will not result in a higher oil yield during the pressing operation due to the high proportion of hulls
• The enzymatic pretreatment of flaked seeds reduces residual oil content in the pressed cake.

. However, yield of oils extracted by pressing is smaller than in case of conventional pressing since aqueous oil extraction takes place during the enzymatic treatment. This is a result of the need to adopt a working temperature for the enzymes at around 55°C.

Dehulling could increase the relative oil content of the substance to be pressed. However, conventional dehulling methods are not suited in this application since they are designed for seeds with brittle and fragile hulls. A suitable dehulling method has not yet been developed for processing of Calendula and Coriandrum. The problems reflect the elasticity of the fibrous hulls and the branching of hull fibres into the oil-storing tissue.

Pressing of Calendula seed in conventional presses results in a marked rise in working temperature, leading to polymerization calendula acid and deterioration of oil quality. Hence, extraction of Calendula oil with a high content of calendula acid by pressing requires adaptation of the pressing tools to the properties of the seed. Cooling of the press is required to minimize polymerization. Cooled oil presses are available. However, since the viscosity of oil rises as temperature decreases, the flow behaviour deteriorates. Extraction of Calendula oil with a high share of calendula acid is possible under a temperature control regime, with a cold extraction method the most suitable. Problems with Coriandrum are similar, also requiring adaptation of pressing tools to the seed characteristics.

Processing of Crambe by pressing provides no problems. The enzymes has no influence on the fatty acid pattern. Processing of Euphorbia seed by pressing also presents no problems. The enzymes had no effect on the content of vernolic acid. To increase the oil yield it was necessary to press twice or use a press tool with a longer pressure screw.

Conclusions

Pros and cons of the enzymatic process:

Advantages

• Lower residual oil content in press cake compared with the conventional pressing procedure
• no organic solvent is needed therefore no danger of explosion and environmental contamination by organic solvents no solvent contaminated meal
• simplification of the process engineering

Disadvantages

• Costs for the enzymes
• Environmental impact of oil contaminated waste water
• High energy needed for heating during enzymatic treatment
• High energy needed for drying of the enzyme treated seed before pressing
• oil losses due to the aqueous extraction.

This suggests that enzymes and/or enzyme complexes should be developed for cell wall lysis at low temperatures (around 20°C) and be suitable for use in solid state, rather than aqueous phase. This would lower energy needs, decrease oil losses and reduce need for waste water treatment.

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Annual Project Report Summary

Summary

This project aims to determine the effectiveness and conditions of use for the application of enzymes in the recovery of vegetable oils and fats from new and conventional oilseeds without the need to use solvents. In this way it is hoped to get recovery yields of more than 90%. The project deals with new oilseeds which include those containing fatty acids of unusual composition suited for use in oleo chemistry. The oilseeds under investigation are from plants which have been cultivated rather erratically in the past, or from novel crops, cultivation of which is just beginning.

INTRODUCTION

This project aims to develop a complete process engineering concept for the recovery of vegetable oils and fats for industrial purposes with the help of enzymes. The proposed enzymatic process is an alternative to or partial substitute for solvent extraction. Such a recovery process, without solvent extraction, would enable a simplified extraction plant to be used. From the environmental point of view an enzymatic process has a number of advantages in handling, recovery and disposal of solvent, as well as reducing other inherent risks - such as the danger of explosion. The application of the enzymatic extraction of the following new oilseeds was planned: Calendula officinalis, Coriandrum sativum, Crambe abyssinica and Euphorbia lagascae. However, a possible problem arose with Euphorbia. This was being processed at a pilot plant in France when two members of the staff fell ill and one had to be treated as an in patient for a week. The symptoms did not correspond to known diseases and diagnosis was difficult. Hence, the literature was investigated. However, it was only possible to obtain the well known descriptions concerning the toxic effects of this plant. Information concerning safety during the processing of Euphorbia and the symptoms of the effects of the poison on humans in the event of contamination has not apparently been reported. Linked to this was the necessity of a very costly cleaning of the equipment and pilot plant after use. Hence, this area is not being pursued. Results, so far, for the other crops are described below.

OBJECTIVE

The project will determine the required parameters and peripheral conditions for the use of enzymes in the recovery of vegetable oils and fats from new oilseeds without solvent extraction: yields of the recovery of more than 90 % expected: the project deals with new oilseeds which include fatty acids with a special design which are very important for oleo-chemistry. The oilseeds are from plants which have been discontinuous, cultivated for this purpose up to now as well as from plants with only a small level of cultivation. In addition genetically manipulated plants will become more significant for this purpose in the future.

To ensure positive results for this project : a partnership will be arranged between two European participants. The participants are the Frech institution Gerdoc and the German association pilot pflanzenoeltechnologie Magdeburg E.V. (PPM E.V.) The operator of the project UEPNO will be the PPM E.V.

The result of the project will be complete process engineering conception for the enzymatic extraction of vegetable oils and fats from new and conventional oilseeds: the enzymatic extraction will be an important part within the recovery and processing of renewable raw materials. The investigations include the process engineering; the environmental effects as well as the analyses of the intermediate; final and byproducts. The new extraction step will be a "smooth process" from the product's and environmental point of view.

MATERIALS AND METHODS

Problems were experienced in obtaining some of the equipment and measuring devices required, but in general such problems were overcome by hire of equipment. As planned, seeds of Calendula (with an oil content of around 17%) were extracted using conventional methods and also using an enzymatic treatment. The conventional method involved pressing, with a screw press, of whole or milled seeds. Alternatively, milled and unmilled seeds were treated with enzymes.

RESULTS

Conventional extraction
Using the conventional process with whole seeds of Calendula the oil yield was around 50%. Milled seed was also pressed. However, no oil could be extracted. In order to determine the cause, various investigations were carried out, indicating that one reason probably reflected the structure of the seed. Traditional press tools are designed for use with hard and brittle seeds and also for seeds with a high oil content, rather than high hull mass. A hard hull is necessary for successful pressing because it aids the power transfer from the press screw into the seed. Calendula seed has a lower oil content of 15 20%. The hull constitutes 60 70% of the mass and has a soft and elastic structure. This hull structure is unfavourable for pressing because of unequal force transmission into the seed. As a result, within the press, the seed is transported and compressed, thus expressing some of the oil. At the end of the pressing canal, the seed expands and soaks up a large part of the expressed oil. During the milling of Calendula the harder seed component (fibres) is destroyed and so impairs the pressing process and no oil is yielded. Different grades of milled seeds were investigated with the same negative result.

Enzymatic processing
The enzymatic treatment of Calendulawas carried out in a container fitted with an electric mixer. For optimal enzyme treatment, the water/seed ratio was to be determined. With milled seed the best ratio was found to be 6:1. For whole seed this was 7:1. The enzymes used included two cellulases, two pectinases and two proteases. The experiments were carried out first with single enzymes and then with an equi mixture of all enzymes. The enzymes were provided in solution. They were added to the seed/water mixture at varying concentrations in the range of 0.1 1.0% of the quantity of seed. The experiments were carried out at a temperature of 55°C and a pH of between 4 and 4.5. After incubation the seed was dried to 6 10% moisture and pressed. The press cakes were extracted using hexane. The total oil yields were around 96%. Pressing of enzymatic treated milled seeds delivered no oil. The results of pressing of whole and enzymatic treated seeds were the same as those of the traditional processed seeds. Investigations were carried out using varying incubation periods (of up to 12 hours) and enzyme concentrations (up to 2%). However, the results showed no great increase in oil yield with any one combination. To determine the activities of enzymes on the oil containing kernels of Calendula, the seeds were flaked, not milled. The flaked kernels were separated from the hulls and enzymatically treated under standard conditions. Enzyme treated whole seeds and the flaked kernels were extracted using hexane. The oil yields of extracted flaked kernels were 5% lower than the yields of extracted whole seeds. The reason for the difference in yields is due to the cell wall breaking during the enzyme treatment of the flaked kernels, which results in oil being lost into the aqueous enzyme solution. Flaked Calendula seeds without enzymatic treatment were pressed. The oil could be extracted during pressing. Analysis of recovered oils showed that the use of enzymes had no influence on the fatty acids. The oil recovered after enzyme treatment contained more seed fibres than the conventionally recovered oils. These fibres, however, are easy to separate from the oil by filtration. Parallel enzymatic investigations at a laboratory scale carried out with Crambe and Coriandrum enabled oil to be extracted following enzyme treatment without pressing.

CONCLUSIONS

The extraction and crushing methods used for rape and sunflower are not suitable for Calendula. The enzymes used were chosen because of their beneficial effects on processing with rape seed. The processing of the enzyme treated milled and whole Calendula seed indicates that the results from rape seed are not repeatable with all types of oil seeds. Further investigations with the use of enzymatic and conventional processes will be carried out with flaked Calendula.

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