250 The Journal of Supercritical Fluids, 1991,4, 250-253

Degreasing of Protein-Hydrolysate by SC-CO$

F. Friischl* and R. Marr

Institut ftir Thermische Verfahrenstechnik und Umwelttechnik, Technische Universittit Graz, InfSeldgasse 25, A-8010 Graz, Austria

and

M. Nussbaumer

EBEWE Arzneimittel Ges.m.b.H., A-4866 Unterach, Austria

Received May 6, 199 1; accepted in revised form August 28, 199 1

The degreasing of a protein-hydrolysate, produced from a pigs-brain raw material, was investigated. A production process based on the removal of fats by dichloromethane should be replaced by a process using an alternative solvent. Therefore, a supercritical-fluid-extraction using CO2 as a solvent was per- formed. The degreasing was investigated with pure gas, as well as with additional ethanol as entrainer. The extraction using an entrainer was necessary because the maximum design pressure of the plant, which is 325 bar, did not bring expected results.

More than 95% of the initially 2 wt % fatty components could be removed from the raw material. This degree of degreasing was reached by a 6-wt % loading of the carbon dioxide with ethanol. A serious dis- advantage of the process was a great loss of nitrogen-containing proteins and amino acids, which are the required residual components in the product. A loss of about 40% of these components is the reason for the process not being investigated any further. Experiments for the degreasing of a powder raw material, which is the raw material in the hydrolysation step of the current process, brought the same good results for the removal of the fats and no nitrogen losses could be observed. The investigated process still was not changed, because the hydrolysation with degreased powder again generated grease-like components which have to be extracted.

Keywords: supercritical fluid extraction, entrainer, degreasing, carbon dioxide, protein-hydrolysate

INTRODUCTION The production of fats from natural substances is a

broadly investigated field of applications for the use of supercritical gases. The extraction of oil from oil seeds and the fractionation of edible oils are well known. The treatment of animal raw materials is seldom reported. Several kinds of fats are highly miscible with supercritical gases, especially with supercritical carbon dioxide. This can be observed at a high pressure level of about 800 bar. Most fatty components are soluble except the phospho- lipids.‘,2 Their solubility can be enhanced by adding ethanol entrainer to the extraction gas. The degreasing of substances at a lower pressure-level (300 bar) also works

+Paper presented at the 2nd International Symposium on Supercritical Fluids, May 20-22, 1991, Boston, MA, USA.

much better when using this kind of entrainer. For applications in food and pharmaceutical industry,

non-toxic solvents have to be used. In Austria, and also many other countries all over the world, governmental re- strictions are increasingly forcing companies to look for alternative ways of production and other solvents. Therefore, supercritical-fluid extraction processes, which are mostly so-called low-waste technologies, are of in- creasing interest for industrial applications. Carbon diox- ide as an extraction gas is preferred. Solvent-free products can be produced by using a harmless, easily available, and cheap solvent. When entrainers are used in SFE, the ad- vantage of getting pure products without residual solvent is lost, but if the entrainer can easily be removed from the product an alternative process is possible.

0896-8446/91/0404-0250$4.00/O 0 1991 PRA Press

The Journal of Supercritical Fluids, Vol. 4, No. 4, 1991

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s Mod V w -1

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Figure 1. Flowscheme of the process.

This was the start of a research cooperation between an Austrian company and the Institut fur Thermische Verfahrenstechnik und Umwelttechnik to investigate the degreasing of a protein hydrolysate. The raw material, a protein hydrolysate, which contains 2 wt % by methy- lene-chloride extractable fatty components on an average (among them phospholipids, lipoproteins, and their degra- dation products) had to be degreased. A percentage of de- greasing of at least 97% was required and a loss of nitro- gen-containing product substances was to be minimized. In the currently performed degreasing process, a loss of 20% of the nitrogen-containing proteins and amino acids occurred. The loss of these substances in the new process should be less or at most equal to the loss in the current process.

PROCESS Figure 1 shows the flow scheme of the performed

process. At the Institut fur Thermische Verfahrenstechnik und Umwelttechnik in Graz, a plant with this setup has been working since 1983. Two extractors, one with 10 L and one with 500 mL, are available.

The experiments were carried out in the small extrac- tor, with a design pressure of 325 bar at 200 “C. The separator can work at pressures up to 100 bar and 100 ‘C.

The extraction gas, which is stored in the liquid state in the storage vessel V, is compressed to extraction pres- sure and heated to the required temperature. By a metering pump, the liquid modifier is pumped into the gas stream. It is dissolved and this gas-mixture extracts the fatty com- ponents from the raw material in the extractor E. The loaded carbon dioxide, which was used as a solvent, is de- compressed by throttling. The gas, which is partly evap- orated, has to be gasified and is heated until it is in a pure gaseous state. By reducing the pressure and raising the temperature of the extraction gas, the density and therefore the solvent capacity of the CO* is decreased and the ex- tracted substances precipitate in the separator S. The gaseous carbon dioxide is recondensed and collected again in the storage vessel V. The cycle-process can then restart.

Degreasing of Protein-Hydrolysate 251

The first experiments were performed without adding entrainer to the CO*, however, after getting unsatisfactory results this equipment was installed. The metering pump is a HPLC-piston pump. Loadings of the extraction gas of 6-8 g/l00 g gas (6-8 wt %) can be reached.

EXPERIMENTAL First, the initial content of fatty components in the

raw material was determined. An extraction with dichloromethane was carried out for the hydrolysate as well as for the powder raw material. After the extraction, the solvent was evaporated and the weight of the residual extracted components was measured. The same procedure was performed with the extracted material. A very inter- esting fact is the content of nitrogen in the material, which was measured by the Kjeldahl method.

The experiments were performed in a 500-mL extrac- tor filled with 250 mL of raw material. The extractor was also equipped with a demister package because the protein- hydrolysate caused serious foam problems which could be overcome by it. The extraction time was between 120 and 240 min. Several extract samples were collected. The experiments with entrainers were finished after 120 min. After the extraction, the extractor was opened and the degreased protein hydrolysate was collected. After rec- ognizing that a big loss of nitrogen occurred, some of the ethanol samples of the separator were also analyzed by the Kjeldahl method. These samples consisted of two phases - a white precipitate and a clear ethanol solution. It should be determined in which phases the nitrogen con- taining substances were dissolved.

RESULTS In the first experiments, the hydrolysate was de-

greased with pure carbon dioxide. The pressure was in- creased from 200 to 300 bar and the temperature was also increased from 20 to 60 “C. The maximum temperature was limited because of some thermodegradable substances in the product. All experiments were performed with a CO2 flow between 6 and 7 kg/h. Initially the hydrolysate contained 2.04 wt % fatty components and 142 mg/g ni- trogen. Serious foam problems occurred during extrac- tion, which were caused by the protein content of the feed solution. To overcome these problems a demister was in- stalled in the extractor which could hinder the foam from blocking the pressure regulating valves.

Figure 2 shows the results for experiments without ‘entrainer. The separation works better at higher tempera- tures and pressures but a maximum 77% of the fatty components could be separated. Therefore, it was decided to use ethanol entrainer in further experiments.

Between 6 and 6.5 wt % ethanol entrainer was added to the carbon dioxide. Much better results for the degreas- ing were achieved. More than 90% of the fatty compo- nents could be removed easily. At 300 bar and 60 “C, the

252 Frijschl et al. The Journal of Supercritical Fluids, Vol. 4, No. 4, 1991

100 Degreasing I%1

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Figure 2. Degreasing with pure CO,. Figure 5. Influence of ethanol-loading on degreasing.

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Figure 3. Degreasing with ethanol-loaded gas.

75 N-Loss [%I 1 I

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Figure 4. Loss of nitrogen vs. degreasing.

best results were obtained - 96.3% degreasing was reached. These results are plotted in Figure 3.

At this point, the solution to the problem was thought to be very near. But the nitrogen balance showed extraordinary losses during extraction. Even the degreas- ing of the protein hydrolysate with pure CO* caused ni- trogen losses of 28%. In Figure 4, these nitrogen losses

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are plotted against the degree of degreasing and it shows that there is a correlation between the two. There are sev- eral kinds of compounds containing nitrogen - amino acids, proteins, and lipoproteins.

Amino acids are reported to have very low solubility in dense carbon dioxide.1-3 Proteins also have low solu- bilities’ and the lipoproteins should be extracted. Very high amounts of amino acids were found in the ethanol phase of the extract and the precipitated solid phase also had a high nitrogen content.

Experiments showed that amino acids are not soluble in ethanol-loaded gas. Therefore, the other dissolved components (lipoproteins) may cause the entraining effect for the amino acids and some proteins. Another possibil- ity is that the co-extracted water can also act as entrainer for the separated nitrogen-containing substances. In par- ticular, the short-chain proteins, which are formed in the hydrolysation step, might be extracted by the water-en- trained COz.

Figure 5 shows that the ethanol loading of the gas has sotie influence on the degree of degreasing, but the loss of nitrogen also could not be reduced, it correlated again with the results of degreasing. In addition to Figures 2, 3, 4, and 5, the results of the experiments are shown in Table I.

The next experiments were performed with a powder material which is the raw material for the hydrolysation. This powder contains 3 wt % by methylene-chloride ex- tractable fatty components and 166.4 mg/g nitrogen. Two experiments were performed. In the first experiment, 8 1.7% of the fatty components could be removed from the powder and the nitrogen loss was only 3.16%. Using a 5.89-wt % ethanol-loaded CO;! resulted in a 96.8% re- moval of the fats and a nitrogen loss of 2.1%. With this powder, a hydrolysation experiment was performed which showed that several lipoproteins were generated in this re- action and therefore, a second extraction was necessary. These findings resulted in the decision that this kind of SFE process would not be further investigated.

The Journal of Supercritical Fluids, Vol. 4, No. 4, 1991 Degreasing of Protein-Hydrolysate 253

TABLE I Results of the Experiments for Degreasing a Protein-Hydolysate with Pure and Modifier-Loaded CO2

P (bar) T PC> Modifier (wt %) Degreasing (%) Nitrogen-Loss (%)

300 20 0.0 0.00 6.20 200 20 0.0 3.92 10.20 300 40 0.0 28.41 10.31 250 50 0.0 52.05 13.72 250 60 0.0 66.01 26.73 300 60 0.0 77.20 36.53 300 40 6.03 54.40 42.47 300 60 1.05 79.44 55.50 300 60 4.90 86.35 50.09 300 60 2.18 87.45 49.55 300 60 3.40 89.68 56.46 250 60 5.52 92.00 64.37 300 50 5.79 92.01 49.70 300 60 5.71 96.28 48.36

CONCLUSIONS The degreasing of a protein hydrolysate was investi-

gated. The performed process uses supercritical carbon dioxide as a solvent. The raw materials tested were a pro- tein hydrolysate of high viscosity and a powder raw mate- rial which is the feed product for the hydrolysation.

The degreasing yield using the pure gas did not reach the required yield of 98%. Raising the temperature as well as pressure of the extraction led to a better removal of the fatty components. The resulting extraction of 77% can be explained by the fact that phospholipids and lipoproteins are not soluble in pure CO*. The loss of ni- trogen-containing substance is difficult to clarify. It seems to be caused by co-extraction of these components, caused either by the other extracted components (fats) working like an entrainer for nitrogen containing sub- stances or the co-extracted water, which is also a strong entrainer. These losses were completely unexpected based on the known solubilities of these substances.

The extraction with ethanol-loaded CO2 brought very good degreasing results of more than 97%. This was reached at a pressure of 300 bar and a temperature of 60 “C. The use of the ethanol entrainer caused a high coex- traction of nitrogen containing substances of nearly 50%. Two interesting facts occurred. Proteins were extracted

and also high amounts of amino acids were found in the ethanol extract.

The replacing of a methylene-chloride-based degreas- ing process by a supercritical fluid extraction process is not successful because of too high coextraction of valu- able substance from the raffinate. The results for the sep- aration of fatty components are satisfying but the selectiv- ity of extraction is not. An unexpectedly high solubility of several amino acids in the ethanol-loaded extraction gas also caused insufficient specification of the raffinate.

Experiments with a smaller ethanol-loading of the CO2 also could not lower the nitrogen loss. Efforts for the degreasing of a powder raw material were successful. The hydrolysation of the degreased material showed that, once again, lipids were generated. Therefore, the SFE-process will not be performed on a commercial scale.

REFERENCES (1) Stahl, E.; Quirin, K. W.; Gerard, D. Verdichtete Gase

zur Extraktion und Raffination; Springer-Verlag: Berlin/Heidelberg, 1987.

(2) McHugh, M. A.; Krukonis, V. J. Supercritical Fluid Extraction, Butterworths: Boston, 1986.

(3) Stahl, E.; Schilz, W. Chem.-lng.-Tech. 1978, 50, 535.