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Precipitation of food protein using high pressure carbon dioxide
Journal of Food Engineering 79 (2007) 1214–1220 www.elsevier.com/locate/jfoodeng
Precipitation of food protein using high pressure carbon dioxide Nawal Khorshid, Md.M. Hossain, M.M. Farid
*
Department of Chemical and Materials Engineering, University of Auckland, Private Bag 92019, Auckland City, New Zealand Received 15 November 2005; accepted 11 April 2006 Available online 6 May 2006
Abstract High pressure carbon dioxide has been applied to precipitate soy protein. The method is suitable for effective precipitation of soy proteins and prevents local pH overshoot, which usually occurs in case of using mineral acids for the precipitation processes. It was possible to achieve 68.3 wt% of soy protein precipitate using 30 bars of pressurized carbon dioxide, at pH of 5.60 and at constant temperature of 22 ± 1 C. The rate of mixing was kept at 300 rpm in all experiments, as more froth was noticed while using 400 rpm with a slight rise in temperature. The qualitative analysis using RP-HPLC for soy protein precipitated at 30 bar, showed two totally separated peaks; one for soy protein glycinin with molecular weight of 350 kDa and the second peak was for, soy protein b-conglycinin with molecular weight 180 kDa. The precipitate at 40 bar showed only one peak for pure soy protein b-conglycinin, which precipitate later because it has lower molecular weight. This process is considered one of the clean processes leading to highly purified food proteins which need no further treatments to purify the product, to be used in food, non food products and pharmaceutical manufacturing. 2006 Elsevier Ltd. All rights reserved. Keywords: High pressure; Carbon dioxide; Soy protein: HPLC; Absorbance
1. Introduction In protein recovery, precipitation remains to be the important technique for concentration and purification. In terms of production volumes, food proteins make up the greater part of the commercially purified protein. Purification of food proteins is in many cases done by means of isoelectric precipitation. Usually, mineral acids such as sulfuric acid and hydrochloric acid are used in these processes. These must be neutralized afterwards, leaving residual salt both in the protein and the residual solution. The use of volatile electrolytes, such as carbon dioxide, can therefore be a valuable tool. After depressurization, the solution returns to a pH close to neutral as the electrolyte returns to the vapor phase. A second advantage of the use of a volatile electrolyte is that it prevents local overshoot in pH during acidification of the solution. In the conventional *
Corresponding author. Tel.: +64 9 373 7599; fax: +64 9 373 7463. E-mail address: [email protected] (M.M. Farid).
0260-8774/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2006.04.037
processes, problems have been reported to occur due to extreme pH values near the acid injection port, which may result in the reduction of precipitate purity and denaturation of the protein (Salt, Liliford, Dunnill, & Eur, 1982; Aalbersberg et al., 2003). Using a volatile acid, the acid concentration is limited by pressure via the vapor–liquid equilibrium, and hence pH cannot fall below its equilibrium value. Casein is, however, not the only protein produced on a large scale by isoelectric precipitation. The technique is also commonly applied to isolate many plant proteins, among which soy protein is the best known. The precipitation of soy protein isolates differs substantially from the precipitation of casein. It resembles, however, the precipitation of other vegetable and legume proteins. Most legumes contain 20–25% protein, but soy beans typically contain 30–45% protein at 13% moisture and as high as 55% protein (moisture-free basis) have been observed (Hammond, Murphy, & Johnson, 2003). In the following section soy proteins are discussed briefly.
N. Khorshid et al. / Journal of Food Engineering 79 (2007) 1214–1220
1.1. Soybean proteins Soybeans contain a broad range of proteins which may be classified in terms of their biological function (as metabolic and storage proteins), their solubility profiles (as albumin or globulin proteins) or their size, often qualified via sedimentation rate in fractional ultracentrifugation. Soy protein have been separated in the ultracentrifuge and characterized as 2S, 7S, 11S, 15S fractions (S is a sedimentation constant, and larger numbers indicate larger proteins). The common way to classify soy proteins is using the later technique in terms of Svedberg units, S. The smaller the Svedberg number the smaller the protein. The 15S fraction consists of glycinin aggregates or polymers and remains insoluble during extract preparation. The bulk of the 2S fraction, on the other hand, does not precipitate at acidic conditions but remains soluble. The 2S fraction is called whey protein. The 11S and 7S fractions make up the majority of protein in soybeans, and are of most value to the food-processing industry. The 7S fraction of soy protein solution contains a-, b-, sconglycinin. Beta-conglycinin (180 kDa) is the main constituent of this fraction and exists as trimer of subunits. These subunits are rich in aspartate asparagines, glutamate/glutamine, leucine, and arginine but low in sulfur containing amino acids. Alpha-conglycinin contains enzymatic activities characteristic of the 2S fraction (Catsimpoolas, 1969). Tauconglycinin, unlike b-conglycinin, remains soluble in acidic solutions and its sedimentation rate remains unchanged at low-ionic strengths. Low concentrations of glycinin may also be present. The 11S fraction of soy protein isolate consists of glycinin. It is the largest single protein fraction, contributing to about 40% of the total globulin mass (Liu, 1997). Associated with protein in soybeans is phytin, the calcium and magnesium salt of hexaphosphoryl inositol. The pH at which a fractionation is most efficient depends on the protein concentration, the protein composition, and the ionic strength of the solution. Soybean proteins, in particular, are of great interest not only for their very widespread use by food industry but also because of their applications in plastics, adhesives, and fiber production. At present, the largest consumer of soy protein isolate is the paper industry, which uses it as a cobinder in paper coatings, but soybeans have also been used in paper and textile-fiber sizing, in building materials, wallpaper coating foams, and in many types of paints and inks (Johnson & Myers, 1995). In the food industry, the addition of soybean protein products allows for a modification of the water-holding, fat-binding, emulsifying capacity, and gelling properties of the food stuff (Liu, 1997). The advantages in using carbon dioxide as a protein precipitant or volatile electrolyte are: • Carbon dioxide is removed by depressurization leaving the solution clean, need no more processing steps to treat the product, like in case of using mineral acids.
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• Since carbon dioxide is considered as a weak acid, it induces slow acidification in to the solution, which leads to a more controlled pH. It prevents pH overshoot which usually occur when using mineral acids. • Using CO2 for precipitation gives better morphology for the protein particles and higher calcium contents (Tomasula, Grag, & Boswell, 1997). • It is considered as a clean process, since carbon dioxide is nontoxic, nonflammable and economical. • The product is highly purified, can be used for infant’s formula as antiallergenic food, for building muscles for athletes and also for pharmaceutical uses. 1.2. Application of high pressure CO2 in precipitating food proteins It is important for environmental reasons to reduce the consumption of the auxiliary compounds in the industrial processing of protein, such as salts, acids and bases, which cannot be easily recycled. High pressure carbon dioxide has been used as a recyclable auxiliary compound in many processes. Supercritical carbon dioxide is used as a reaction medium, a solvent or anti-solvent (Jarzebski & Malinowski, 1995; Thiering, Hofland, Foster, Witkamp, & van der Wielen, 2001). Carbon dioxide can also be used for its acidifying properties in aqueous solutions as represented in Eq. (1), since CO2 reacts with water to produce carbonic acid which gives hydrogen ions, lowering the pH. Carbon dioxide pressurized to 6 MPa can acidify aqueous solutions to a pH-value down to 3–5, depending on the concentration of buffering components (Hofland, van Es, van der Wielen, & Witkamp, 1999).
ð1Þ Low pressure carbon dioxide has been used for stabilization of milk at pH (6.0–6.2). Milk has buffering properties because its pH does not deviate very much from its normal value of pH = 6.6, upon addition of an acid or a base. This is due to various components in milk such as proteins, fat, and carbohydrates, which absorb acids and bases causing buffering. When a pressurized gas such as carbon dioxide is injected into milk, it causes milk to become an acid solution of pH = 5.4. The buffering properties of milk when carbon dioxide is added vary at different pH values. The difference in pH may occur because the high pressure carbon dioxide causes changes in the structure of proteins in milk to be selectively precipitated and give unique functional properties for food use. This is a potential benefit to processors because it demonstrates that the major milk
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protein, casein, could be selectively extracted with high calcium content, instead of by acid precipitation, which is presently used, followed by treatment with calcium hydroxide to add back the calcium to the casein (Olano & Calvo, 1995). 1.3. Reaction of carbon dioxide with water The reaction of the carbon dioxide with water to form carbonic acid is considerably slower than the dissociation of carbonic acid and other dissociation and association reactions occurring in solution. When the reaction of carbon dioxide and water is approximated as a pseudo-first order equilibrium reaction, the concentrations exhibit exponential curves with a characteristic time tr in (s): tr ¼ 1=ðk 1 þ k 2 Þ:
ð2Þ
The characteristic time is equal to 0.05 s for (k1 = 4.37 · 102 s1 and k2 = 19.2 s1), data of Van Eldik and Palmer (1982), where k1 and k2 are the mass transfer coefficient (m s1). 1.4. Mass transfer of high pressure gases Gas–liquid mass transfer is important as it is often timelimiting in process mechanism. The rate of the gas–liquid mass transfer is characterized by the volumetric mass transfer coefficient kma, the product of mass transfer coefficient km (based on the liquid phase) and the specific interfacial area a. The characteristic time tr for mass transfer is equal to the inverse of the kma tr ¼ 1=k m a
ð3Þ
Numerous investigations have been made to determine and correlate kma for specific media and reactor sizes and geometries. The equations for mass transfer coefficient are empirical and contain the stirring power and the superficial gas velocity of gas flow rate as variables (Mezaki & Ogawa, 2000): n
k 1 a ðP g Þ umg =V 1 ;
ð4Þ
where Pg is the power input by the stirrer in presence of a dispersed gas, V1 is the liquid volume and ug is the superficial gas velocity in the vessel. The value of exponent n reported is usually around 0.4. The exponent of the gas velocity m is usually very small or even zero for non-coalescing systems and 0.5 for coalescing systems. 2. Experimental work 2.1. Materials Defatted soy meal was supplied by Cargill food company (Melbourne, Australia), a side product of one of the oil manufacturing company. Soy meal contains 48 wt% proteins. The carbon dioxide (industrial-grade) was supplied by BOC Gas.
2.2. Feed preparation 2.2.1. Extraction of soy meal proteins Soy protein solution was prepared according to the standard procedure of (Bell & Dunnill, 1982). Defatted soy meal was dispersed in demineralized water to give a final concentration of 10 wt%. Soy protein was extracted with alkaline water at pH 8–9. NaOH (2 M) was added to adjust the solution pH, whilst stirring for 30 min, taking care to avoid foaming. The dispersion was then centrifuged for 2 h at 4100 rpm (Mistral 6000 B.Tech) to remove the insoluble carbohydrates. The supernatant contained soluble carbohydrate, and soluble proteins. The extract (supernatant) was immediately used or stored at 5 C to avoid bacterial degradation. 2.3. Experimental setup A high pressure vessel was constructed in the School of Engineering workshop (University of Auckland) and shown in Fig. 1. The volume of the vessel (1) is 1 L and it is designed for a maximum working pressure of 100 bar. The batch vessel (autoclave extractor) is a cylindrical container made of 304 stainless steel with 84 mm internal diameter, 122 mm external diameter and 180 mm height (1). The 20 mm thick autoclave top cover has 12 screws and a copper gasket to prevent any leak. The top cover has access to a 3.3 mm diameter pipe for CO2 inlet, 6.4 mm diameter pipe for the feed inlet, 3.3 mm diameter opening for the thermocouple probe (14), 20 mm diameter opening for the pH probe (12) and 8 mm diameter opening for the mixer (3). The mixer was a pitched blade 46 mm in diameter (2), mounted at 28 mm from the bottom and driven by a motor (3) ‘‘ Parvalux’’ shunt wound A/C motor, UK. Carbon dioxide pressure was set by a pressure regulator (4) and fed to the vessel at the headspace. Temperature of the vessel was measured by a thermocouple, Farnell Inc., UK. Heating was generated by using a tape (5) around the vessel which was connected to a controller. The high pressure sample vessel (6), connected to the bottom through a piston valve (7) and to the high pressure vessel (1) from its side with a tube of 6 mm diameter (8). This side tube was used to pressurize the sample with CO2 through a cross over valve (9), before taking the sample. The sample was taken under the same pressure of the high pressure vessel (1), in order to keep the pH stable. Otherwise taking the sample under atmospheric pressure will cause the pH to rise suddenly causing the precipitate to dissolve again, and depressurizing the whole vessel (1) before taking the sample might block the outlet line due to freezing of the water in it. This would also increase foaming which is undesirable. Depressurizing the vessel after each run is done using a needle valve at the top (10). A safety valve was connected to the CO2 inlet tube (11) and was set to 60 bars. The pH electrode (12) shown in Fig. 1 was a high pressure electrode
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Depressurizing valve (10) Feed Thermocouple (14)
P
Pressure regulator (4)
T
pH
Needle valve (11) (12) (3)
(9)
CO2 Cylinder
Recorder (13)
Side tube 6 mm dia (8) 151.45 mm
Heating element (5)
High pressure vessel (1)
180.45 mm
Mixer (2) 29 mm
45 mm
84 mm
Piston valve (7)
Sample Vessel (6)
Protein + Slurry
Fig. 1. Experimental set up of the high pressure extraction unit.
(Innovative Sensors, Inc. Anaheim, USA.) provided by Bell Technology, NZ. The pH electrode can be used up to a pressure of 68 bar. The signals from the temperature and pressure transducers were taken to a data logger (Picolog TC08) for online measurements (13). The pH probe was connected to a pH meter (Radiometer Copenhagen a pH M82), which was connected to the data logger with galvanic isolator (signal conditioning software). 2.4. Procedure Soy meal extract or reconstituted milk of 700 ml was fed to the top of the extractor through a 6 mm inside dia. tube via a funnel (gravity feed) in all runs, and allowed thermally to equilibrate for 30 min. CO2 was then introduced into the head space at a controlled rate until the set pressure was reached, then the mixer was set on to 300 rpm to assist in dissolving carbon dioxide in to solution. The head space was not evacuated or vented before the experiment and hence contained, some air. At high pressure >10 bar the effect of the residual air is small. The time to reach the equilibrium at low pressure (10– 20 bar) was noticed to be long (60–70 min) compared to that at high pressure (30–45 min). Equilibrium was identified as the point at which the pH becomes constant or varies within ±.05, indicating that the solution was saturated with carbon dioxide at the specific operating pressure. All runs for soy extracts were carried out at room temperature, 22 ± 1 C, and those for the reconstituted milk, were carried out at, 40 ± 1 C. The heat was supplied by a heating
tape fixed around the vessel. The milk was preheated to around, 38–40 C in a microwave oven just before fed to the reactor. A series of experiments were performed for both soy meal extracts and the reconstituted skim milk in which high pressure CO2 was used as acidifying agent. The pressure was increased at a controlled rate until the set pressure was reached. The temperature, pressure and pH readings were recorded on computer, which was then transferred to an Excel file to draw the variables diagram for each run. 2.5. Sampling procedure After the pH has reached equilibrium at a certain pressure, a sample of the stirred suspension was taken through the base of the vessel. After pressurizing the sample vessel (6) by opening the cross over valve (9) for few minutes, then opening the piston valve (7) to let some of the stirred suspension (around 50 ml) to fill the sample vessel, then close the cross over valve. The needle valve (10) was carefully and slightly opened to depressurize the sample vessel. Depressurizing was done very slowly, to minimize foaming in the system. After the slow depressurization step, the front sample vessel valve was opened to take a sample. Every sample was centrifuged for 30 min at 4100 rpm to separate the precipitated proteins from the suspension. After centrifugation the container was opened gently to prevent the curd from re-suspending into the supernatant. This procedure was subsequently repeated at a higher pressure and the same procedure was followed for all other
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3. Results and discussion 3.1. Quantitative analysis results
35
12
30
10
25
8
20 6
15 10
2
pH
0 0
400
800 Time (second)
0 1600
1200
Fig. 2. Variation of pH during CO2 extraction of soy meal protein. (Pressure = 30 bar, temperature = 22 C).
6.60 6.30 pH
Table 1 for soy samples is showing the proteins precipitated under different pressure of carbon dioxide and at different pHs. The precipitated proteins form soy meal was determined by measuring the proteins in the supernatant solution after the precipitation process for each run. The measurment was on the basis of a simple linear relationship between the UV absorbance and the concentration of the solute (Demchenco, 1986) using UV–vis, spectrophotometer (Perkin–Elmer, USA). The difference in wt% protein in the feed and wt% protein in the supernatant, equals the precipitated proteins.
4
Pressure
5
pH
Pressure (bar)
runs. The supernatant was then decanted into another container to prevent any dissolution of the curd and kept for further analysis. The precipitate was freeze-dried and kept for the qualitative analysis, while the supernatant used for the quantitative analysis.
6.00
3.2. Acidification of soy protein solution with carbon dioxide 5.70
The effect of pressurized carbon dioxide on the acidification of soy protein solutions was studied. Experiments were performed by applying different pressure 10, 20, 30, 40 and 50 bar and then the equilibrium pH was measured at different times. All runs were carried out at a constant ambient temperature of around 22 C. The curves of pH as a function of time all have the same pattern. The pH dropped rapidly from 9 and then levels down to a constant value, as the solution get saturated with CO2. This equilibrium pH would initiate the flocculation, coagulation then precipitation of the neutralized protein particles. Fig. 2 showed a sharp drop in pH from 9.0 to 5.6 as pressure was increased to 30 bar then the pH stabilized around pH 5.5 as the solution became saturated with CO2. Fig. 3 shows the relationship between different pressures of CO2 and pH at ambient temperature and 300 rpm for all soy protein extract experiments. At lower pressures (10– 20 bar), pH is strongly influenced (reduced) by pressure increase, and at higher pressures (30–50 bar) the pH drop is minimal. The sensitivity of pH is low in the pressure range of 30–50 bar. The change in pH is only about 0.05, so operating at pressures higher than 50 bar will have no
5.40 0
10
20 30 40 Pressure (bar)
50
60
Fig. 3. Relation between pH and CO2 pressure for soy protein extracts, at ambient temperature and 300 rpm.
advantage in further decreasing the pH, since the solution is saturated with carbon dioxide. 3.3. Protein recovered High protein recovery is desirable from any stream containing them. To determine the yield, a graph of the wt% of protein precipitated under different carbon dioxide pressures were plotted as a function of pressure for soy protein. Fig. 4 shows that the maximum recovery for soy protein is 68.3 wt% under 30 bar of pressurized carbon dioxide and at pH of 5.6. This pH (5.6) is close to the pI or the isoelectric point of soy protein, which is in the range of 4.7–5.2, (Thiering et al., 2001).
Table 1 Precipitated soy proteins under different CO2 pressure and their pH Pressure bar
Absorbance UV (nm)
Concentration mg/ml diluted
Concentration mg/ml
mg Protein precipitated
% Protein precipitated
pH reading
10 20 30 40 45 50
0.7249 0.6569 0.532 0.644 1.0149 1.1753
0.0497 0.0451 0.0365 0.0442 0.0696 0.0806
8.2880 7.5106 6.0825 7.3631 11.6037 13.4376
10.9120 11.6894 13.1175 11.8369 7.5963 5.7624
56.8332 60.8825 68.3201 61.6507 39.5641 30.0125
6.40 5.65 5.60 5.62 5.54 5.52
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wt% protein recovered
80 60 40 20 0
0
10
20
30
40
50
60
Pressure (bar)
Fig. 4. Effect of CO2 pressure on soy protein recovery at ambient temperature of 22 ± 1 C.
3.4. Effect of acidity on protein recovery To confirm the results discussed in the previous section, the direct effect of increasing acidity (pH) by CO2 pressure, on protein recovery is shown in this section. Fig. 5 shows that the highest recovery for soy protein is achieved at pH around 5.6. This indicates there is no need to increase the acidity further, because the amount of protein recovered was decreasing with further increase in pH. 3.5. Qualitative analysis of precipitated proteins Reversed-phase high-performance liquid chromatography (RP-HPLC), manufactured by Waters 600E and ELSD 800, Alltech (USA), was used to analyze the precipitated proteins qualitatively and to determine the purity of proteins. RP-HPLC is a very powerful analytical technique for peptides and proteins (Aguilar, 2003; Mant & Hodges, 1996). Fig. 6 shows the elution profile of the soy protein extract components of the feed showing three major peaks, the first large peak which eluted at 17.9 min coincides with milk caseinate standard, while the second peak eluted at 21.9 min and the third eluted at 22.8 min. Fig. 7 shows a first small peak eluted at 23.3 min and a second largest peak eluted at 28.5 min, which indicates the precipitate was not a pure protein but possibly a mixture of the two major soy proteins which were glycinin of molecular weight of (350 kDa) with pI range 4.9–5.2 and b-con-
Fig. 6. Elution profile of 10 wt% of soy protein extract components of the feed solution.
wt% protein recovered
80 60 Fig. 7. Elution profile of soy protein precipitated at 30 bar of carbon dioxide.
40 20 0 5.20
5.60
6.00 pH
6.40
Fig. 5. Effect of acidity on soy protein recovery.
6.80
glycinin of molecular weight of (180 kDa) with pI range of 4.7–5.0 (Thiering et al., 2001). Fig. 8 shows the elution profile of the soy protein precipitated using 40 bar of carbon dioxide. It shows one major peak eluted at 23 min, indicating a pure soy protein of b-conglycinin, the lower molecular weight soy protein,
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cates a mixture of two soy proteins. The first peak was the pure soy protein glycinin, which usually precipitate at higher pI (5.62) because of its higher molecular weight (350 kDa). The other soy protein was b-conglycinin, which has lower molecular weight of (180 kDa). The chromatogram showed, a pure soy protein in case of using (40 bar), which is the only pure soy protein with higher molecular weight glycinin soy protein. References
Fig. 8. Elution profile of soy protein precipitated at 40 bar of carbon dioxide.
which normally precipitate at lower pH range of 4.7–5.0 than glycinin soy protein, the higher molecular weight soy protein. From the results shown in Figs. 7 and 8, it is clear that the two types of proteins are formed only at 30 bar. This explains why the protein recovery was higher in case of using 30 bar of CO2 for precipitation of protein and at pH of 5.6. 4. Conclusions High pressure carbon dioxide was applied to recover protein from defatted soymeal. This process was found to produce protein in pure form. The advantage of using pressurized carbon dioxide in precipitating food protein is to achieve the precipitation at higher pH with minimum possible pressure, to save energy and to get high protein recovery and more purified protein than that usually obtained using mineral acids, which needs further treatments. CO2, unlike acids, will separate easily from the solution by depressurizing it leaving the solution clean. Highest recovery of soy protein has been achieved at a pressure of 30 bar of CO2 at ambient temperature of 22 ± 1 C, which correspond to a solution pH in the range of 5.4–5.6. The qualitative analysis of RP-HPLC for soy protein precipitate showed two totally separated peaks, which indi-
Aalbersberg, W. Y., Hamer, R. J., Jasperse, P., de Jong, H. H. J., de Kruik, C. G., Walstra, P., et al. (2003). Industrial proteins in perspective. Progress in Biotechnology, 23. Aguilar, M. I. (2003). HPLC of peptides and proteins, methods and protocols. Methods in Molecular Biology, 251, 9–12. Bell, D. J., & Dunnill, P. (1982). The influence of precipitation reactor configuration on the centrifugal recovery of isoelectric soy protein precipitate. Biotechnology and Bioengineering, 24, 2319–2336. Catsimpoolas, N. (1969). Isolaton of glycinin subunit by isoelectric focusing in ureamercaptoethanol. FEHS, 4, 259–261. Demchenco, A. P. (1986). Ultraviolet spectroscopy of proteins. Berlin Heidelberg, New York, London, Tokyo: Springer-Verlag, p. 94. Hammond, E. G., Murphy, P. A., & Johnson, L. A. (2003). Properties and analysis of soybeans. USA: Elsevier Science Ltd., p. 32. Hofland, G. W., van Es, M., van der Wielen, L. A. M., & Witkamp, G. J. (1999). Isoelectric precipitation of casein using high pressure carbon dioxide. American Chemical Society, 38, 4919–4927. Jarzebski, A. B., & Malinowski, J. J. (1995). Potentials and prospects for application of supercritical fluid technology in bioprocessing. Process Biochemistry, 30, 343–352. Johnson, L. A., & Myers, D. J. (1995). Industrial uses for soybeans. In D. R. Erikson (Ed.), A practical handbook of soybean processing and utilization (pp. 380–427). AOCS Press. Liu, K. (1997). Soybeans; chemistry, technology and utilization. New York: International Thomson Publishing, p. 532. Mant, C. T., & Hodges, R. S. (1996). Analysis of peptides by high performance liquid chromatography. Methods in Enzymology, 271, 3–50. Mezaki, M., Mochizuki, M., & Ogawa, K. (2000). Engineering data on mixing. Amsterdam. The Netherlands: Science, p. 15. Olano, A. L., & Calvo, M. M. (1995). Preservation of raw milk with carbon dioxide. Zeitschrift fur Lebensmittel-Untersuchung und-Forschung, 200, 293–296. Salt, D. J., Liliford, R. B., Dunnill, P., & Eur, J. (1982). Factors influencing protein structure during acid precipitation; a study of soy proteins. Journal of Applied Microbiology and Biotechnology, 14, 114–144. Thiering, R., Hofland, G., Foster, N., Witkamp, G. J., & van der Wielen, L. A. M. (2001). Fractionation of soybean proteins with pressurized carbon dioxide as a volatile electrolyte. Biotechnology and Bioengineering, 73, 1. Tomasula, P. M., Grag, J. C., & Boswell, R. T. (1997). A continuous process for casein production using high pressure carbon dioxide. Journal of Food Engineering, 33, 405–419. Van Eldik, R., & Palmer, D. A. (1982). Effects of pressure on the kinetics of the dehydration of carbonic acid and the hydrolysis of carbon dioxide in aqueous solution. Journal of Solution Chemistry, 11, 339–346.
Precipitation of food protein using high pressure carbon dioxide Nawal Khorshid, Md.M. Hossain, M.M. Farid
*
Department of Chemical and Materials Engineering, University of Auckland, Private Bag 92019, Auckland City, New Zealand Received 15 November 2005; accepted 11 April 2006 Available online 6 May 2006
Abstract High pressure carbon dioxide has been applied to precipitate soy protein. The method is suitable for effective precipitation of soy proteins and prevents local pH overshoot, which usually occurs in case of using mineral acids for the precipitation processes. It was possible to achieve 68.3 wt% of soy protein precipitate using 30 bars of pressurized carbon dioxide, at pH of 5.60 and at constant temperature of 22 ± 1 C. The rate of mixing was kept at 300 rpm in all experiments, as more froth was noticed while using 400 rpm with a slight rise in temperature. The qualitative analysis using RP-HPLC for soy protein precipitated at 30 bar, showed two totally separated peaks; one for soy protein glycinin with molecular weight of 350 kDa and the second peak was for, soy protein b-conglycinin with molecular weight 180 kDa. The precipitate at 40 bar showed only one peak for pure soy protein b-conglycinin, which precipitate later because it has lower molecular weight. This process is considered one of the clean processes leading to highly purified food proteins which need no further treatments to purify the product, to be used in food, non food products and pharmaceutical manufacturing. 2006 Elsevier Ltd. All rights reserved. Keywords: High pressure; Carbon dioxide; Soy protein: HPLC; Absorbance
1. Introduction In protein recovery, precipitation remains to be the important technique for concentration and purification. In terms of production volumes, food proteins make up the greater part of the commercially purified protein. Purification of food proteins is in many cases done by means of isoelectric precipitation. Usually, mineral acids such as sulfuric acid and hydrochloric acid are used in these processes. These must be neutralized afterwards, leaving residual salt both in the protein and the residual solution. The use of volatile electrolytes, such as carbon dioxide, can therefore be a valuable tool. After depressurization, the solution returns to a pH close to neutral as the electrolyte returns to the vapor phase. A second advantage of the use of a volatile electrolyte is that it prevents local overshoot in pH during acidification of the solution. In the conventional *
Corresponding author. Tel.: +64 9 373 7599; fax: +64 9 373 7463. E-mail address: [email protected] (M.M. Farid).
0260-8774/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2006.04.037
processes, problems have been reported to occur due to extreme pH values near the acid injection port, which may result in the reduction of precipitate purity and denaturation of the protein (Salt, Liliford, Dunnill, & Eur, 1982; Aalbersberg et al., 2003). Using a volatile acid, the acid concentration is limited by pressure via the vapor–liquid equilibrium, and hence pH cannot fall below its equilibrium value. Casein is, however, not the only protein produced on a large scale by isoelectric precipitation. The technique is also commonly applied to isolate many plant proteins, among which soy protein is the best known. The precipitation of soy protein isolates differs substantially from the precipitation of casein. It resembles, however, the precipitation of other vegetable and legume proteins. Most legumes contain 20–25% protein, but soy beans typically contain 30–45% protein at 13% moisture and as high as 55% protein (moisture-free basis) have been observed (Hammond, Murphy, & Johnson, 2003). In the following section soy proteins are discussed briefly.
N. Khorshid et al. / Journal of Food Engineering 79 (2007) 1214–1220
1.1. Soybean proteins Soybeans contain a broad range of proteins which may be classified in terms of their biological function (as metabolic and storage proteins), their solubility profiles (as albumin or globulin proteins) or their size, often qualified via sedimentation rate in fractional ultracentrifugation. Soy protein have been separated in the ultracentrifuge and characterized as 2S, 7S, 11S, 15S fractions (S is a sedimentation constant, and larger numbers indicate larger proteins). The common way to classify soy proteins is using the later technique in terms of Svedberg units, S. The smaller the Svedberg number the smaller the protein. The 15S fraction consists of glycinin aggregates or polymers and remains insoluble during extract preparation. The bulk of the 2S fraction, on the other hand, does not precipitate at acidic conditions but remains soluble. The 2S fraction is called whey protein. The 11S and 7S fractions make up the majority of protein in soybeans, and are of most value to the food-processing industry. The 7S fraction of soy protein solution contains a-, b-, sconglycinin. Beta-conglycinin (180 kDa) is the main constituent of this fraction and exists as trimer of subunits. These subunits are rich in aspartate asparagines, glutamate/glutamine, leucine, and arginine but low in sulfur containing amino acids. Alpha-conglycinin contains enzymatic activities characteristic of the 2S fraction (Catsimpoolas, 1969). Tauconglycinin, unlike b-conglycinin, remains soluble in acidic solutions and its sedimentation rate remains unchanged at low-ionic strengths. Low concentrations of glycinin may also be present. The 11S fraction of soy protein isolate consists of glycinin. It is the largest single protein fraction, contributing to about 40% of the total globulin mass (Liu, 1997). Associated with protein in soybeans is phytin, the calcium and magnesium salt of hexaphosphoryl inositol. The pH at which a fractionation is most efficient depends on the protein concentration, the protein composition, and the ionic strength of the solution. Soybean proteins, in particular, are of great interest not only for their very widespread use by food industry but also because of their applications in plastics, adhesives, and fiber production. At present, the largest consumer of soy protein isolate is the paper industry, which uses it as a cobinder in paper coatings, but soybeans have also been used in paper and textile-fiber sizing, in building materials, wallpaper coating foams, and in many types of paints and inks (Johnson & Myers, 1995). In the food industry, the addition of soybean protein products allows for a modification of the water-holding, fat-binding, emulsifying capacity, and gelling properties of the food stuff (Liu, 1997). The advantages in using carbon dioxide as a protein precipitant or volatile electrolyte are: • Carbon dioxide is removed by depressurization leaving the solution clean, need no more processing steps to treat the product, like in case of using mineral acids.
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• Since carbon dioxide is considered as a weak acid, it induces slow acidification in to the solution, which leads to a more controlled pH. It prevents pH overshoot which usually occur when using mineral acids. • Using CO2 for precipitation gives better morphology for the protein particles and higher calcium contents (Tomasula, Grag, & Boswell, 1997). • It is considered as a clean process, since carbon dioxide is nontoxic, nonflammable and economical. • The product is highly purified, can be used for infant’s formula as antiallergenic food, for building muscles for athletes and also for pharmaceutical uses. 1.2. Application of high pressure CO2 in precipitating food proteins It is important for environmental reasons to reduce the consumption of the auxiliary compounds in the industrial processing of protein, such as salts, acids and bases, which cannot be easily recycled. High pressure carbon dioxide has been used as a recyclable auxiliary compound in many processes. Supercritical carbon dioxide is used as a reaction medium, a solvent or anti-solvent (Jarzebski & Malinowski, 1995; Thiering, Hofland, Foster, Witkamp, & van der Wielen, 2001). Carbon dioxide can also be used for its acidifying properties in aqueous solutions as represented in Eq. (1), since CO2 reacts with water to produce carbonic acid which gives hydrogen ions, lowering the pH. Carbon dioxide pressurized to 6 MPa can acidify aqueous solutions to a pH-value down to 3–5, depending on the concentration of buffering components (Hofland, van Es, van der Wielen, & Witkamp, 1999).
ð1Þ Low pressure carbon dioxide has been used for stabilization of milk at pH (6.0–6.2). Milk has buffering properties because its pH does not deviate very much from its normal value of pH = 6.6, upon addition of an acid or a base. This is due to various components in milk such as proteins, fat, and carbohydrates, which absorb acids and bases causing buffering. When a pressurized gas such as carbon dioxide is injected into milk, it causes milk to become an acid solution of pH = 5.4. The buffering properties of milk when carbon dioxide is added vary at different pH values. The difference in pH may occur because the high pressure carbon dioxide causes changes in the structure of proteins in milk to be selectively precipitated and give unique functional properties for food use. This is a potential benefit to processors because it demonstrates that the major milk
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protein, casein, could be selectively extracted with high calcium content, instead of by acid precipitation, which is presently used, followed by treatment with calcium hydroxide to add back the calcium to the casein (Olano & Calvo, 1995). 1.3. Reaction of carbon dioxide with water The reaction of the carbon dioxide with water to form carbonic acid is considerably slower than the dissociation of carbonic acid and other dissociation and association reactions occurring in solution. When the reaction of carbon dioxide and water is approximated as a pseudo-first order equilibrium reaction, the concentrations exhibit exponential curves with a characteristic time tr in (s): tr ¼ 1=ðk 1 þ k 2 Þ:
ð2Þ
The characteristic time is equal to 0.05 s for (k1 = 4.37 · 102 s1 and k2 = 19.2 s1), data of Van Eldik and Palmer (1982), where k1 and k2 are the mass transfer coefficient (m s1). 1.4. Mass transfer of high pressure gases Gas–liquid mass transfer is important as it is often timelimiting in process mechanism. The rate of the gas–liquid mass transfer is characterized by the volumetric mass transfer coefficient kma, the product of mass transfer coefficient km (based on the liquid phase) and the specific interfacial area a. The characteristic time tr for mass transfer is equal to the inverse of the kma tr ¼ 1=k m a
ð3Þ
Numerous investigations have been made to determine and correlate kma for specific media and reactor sizes and geometries. The equations for mass transfer coefficient are empirical and contain the stirring power and the superficial gas velocity of gas flow rate as variables (Mezaki & Ogawa, 2000): n
k 1 a ðP g Þ umg =V 1 ;
ð4Þ
where Pg is the power input by the stirrer in presence of a dispersed gas, V1 is the liquid volume and ug is the superficial gas velocity in the vessel. The value of exponent n reported is usually around 0.4. The exponent of the gas velocity m is usually very small or even zero for non-coalescing systems and 0.5 for coalescing systems. 2. Experimental work 2.1. Materials Defatted soy meal was supplied by Cargill food company (Melbourne, Australia), a side product of one of the oil manufacturing company. Soy meal contains 48 wt% proteins. The carbon dioxide (industrial-grade) was supplied by BOC Gas.
2.2. Feed preparation 2.2.1. Extraction of soy meal proteins Soy protein solution was prepared according to the standard procedure of (Bell & Dunnill, 1982). Defatted soy meal was dispersed in demineralized water to give a final concentration of 10 wt%. Soy protein was extracted with alkaline water at pH 8–9. NaOH (2 M) was added to adjust the solution pH, whilst stirring for 30 min, taking care to avoid foaming. The dispersion was then centrifuged for 2 h at 4100 rpm (Mistral 6000 B.Tech) to remove the insoluble carbohydrates. The supernatant contained soluble carbohydrate, and soluble proteins. The extract (supernatant) was immediately used or stored at 5 C to avoid bacterial degradation. 2.3. Experimental setup A high pressure vessel was constructed in the School of Engineering workshop (University of Auckland) and shown in Fig. 1. The volume of the vessel (1) is 1 L and it is designed for a maximum working pressure of 100 bar. The batch vessel (autoclave extractor) is a cylindrical container made of 304 stainless steel with 84 mm internal diameter, 122 mm external diameter and 180 mm height (1). The 20 mm thick autoclave top cover has 12 screws and a copper gasket to prevent any leak. The top cover has access to a 3.3 mm diameter pipe for CO2 inlet, 6.4 mm diameter pipe for the feed inlet, 3.3 mm diameter opening for the thermocouple probe (14), 20 mm diameter opening for the pH probe (12) and 8 mm diameter opening for the mixer (3). The mixer was a pitched blade 46 mm in diameter (2), mounted at 28 mm from the bottom and driven by a motor (3) ‘‘ Parvalux’’ shunt wound A/C motor, UK. Carbon dioxide pressure was set by a pressure regulator (4) and fed to the vessel at the headspace. Temperature of the vessel was measured by a thermocouple, Farnell Inc., UK. Heating was generated by using a tape (5) around the vessel which was connected to a controller. The high pressure sample vessel (6), connected to the bottom through a piston valve (7) and to the high pressure vessel (1) from its side with a tube of 6 mm diameter (8). This side tube was used to pressurize the sample with CO2 through a cross over valve (9), before taking the sample. The sample was taken under the same pressure of the high pressure vessel (1), in order to keep the pH stable. Otherwise taking the sample under atmospheric pressure will cause the pH to rise suddenly causing the precipitate to dissolve again, and depressurizing the whole vessel (1) before taking the sample might block the outlet line due to freezing of the water in it. This would also increase foaming which is undesirable. Depressurizing the vessel after each run is done using a needle valve at the top (10). A safety valve was connected to the CO2 inlet tube (11) and was set to 60 bars. The pH electrode (12) shown in Fig. 1 was a high pressure electrode
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Depressurizing valve (10) Feed Thermocouple (14)
P
Pressure regulator (4)
T
pH
Needle valve (11) (12) (3)
(9)
CO2 Cylinder
Recorder (13)
Side tube 6 mm dia (8) 151.45 mm
Heating element (5)
High pressure vessel (1)
180.45 mm
Mixer (2) 29 mm
45 mm
84 mm
Piston valve (7)
Sample Vessel (6)
Protein + Slurry
Fig. 1. Experimental set up of the high pressure extraction unit.
(Innovative Sensors, Inc. Anaheim, USA.) provided by Bell Technology, NZ. The pH electrode can be used up to a pressure of 68 bar. The signals from the temperature and pressure transducers were taken to a data logger (Picolog TC08) for online measurements (13). The pH probe was connected to a pH meter (Radiometer Copenhagen a pH M82), which was connected to the data logger with galvanic isolator (signal conditioning software). 2.4. Procedure Soy meal extract or reconstituted milk of 700 ml was fed to the top of the extractor through a 6 mm inside dia. tube via a funnel (gravity feed) in all runs, and allowed thermally to equilibrate for 30 min. CO2 was then introduced into the head space at a controlled rate until the set pressure was reached, then the mixer was set on to 300 rpm to assist in dissolving carbon dioxide in to solution. The head space was not evacuated or vented before the experiment and hence contained, some air. At high pressure >10 bar the effect of the residual air is small. The time to reach the equilibrium at low pressure (10– 20 bar) was noticed to be long (60–70 min) compared to that at high pressure (30–45 min). Equilibrium was identified as the point at which the pH becomes constant or varies within ±.05, indicating that the solution was saturated with carbon dioxide at the specific operating pressure. All runs for soy extracts were carried out at room temperature, 22 ± 1 C, and those for the reconstituted milk, were carried out at, 40 ± 1 C. The heat was supplied by a heating
tape fixed around the vessel. The milk was preheated to around, 38–40 C in a microwave oven just before fed to the reactor. A series of experiments were performed for both soy meal extracts and the reconstituted skim milk in which high pressure CO2 was used as acidifying agent. The pressure was increased at a controlled rate until the set pressure was reached. The temperature, pressure and pH readings were recorded on computer, which was then transferred to an Excel file to draw the variables diagram for each run. 2.5. Sampling procedure After the pH has reached equilibrium at a certain pressure, a sample of the stirred suspension was taken through the base of the vessel. After pressurizing the sample vessel (6) by opening the cross over valve (9) for few minutes, then opening the piston valve (7) to let some of the stirred suspension (around 50 ml) to fill the sample vessel, then close the cross over valve. The needle valve (10) was carefully and slightly opened to depressurize the sample vessel. Depressurizing was done very slowly, to minimize foaming in the system. After the slow depressurization step, the front sample vessel valve was opened to take a sample. Every sample was centrifuged for 30 min at 4100 rpm to separate the precipitated proteins from the suspension. After centrifugation the container was opened gently to prevent the curd from re-suspending into the supernatant. This procedure was subsequently repeated at a higher pressure and the same procedure was followed for all other
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3. Results and discussion 3.1. Quantitative analysis results
35
12
30
10
25
8
20 6
15 10
2
pH
0 0
400
800 Time (second)
0 1600
1200
Fig. 2. Variation of pH during CO2 extraction of soy meal protein. (Pressure = 30 bar, temperature = 22 C).
6.60 6.30 pH
Table 1 for soy samples is showing the proteins precipitated under different pressure of carbon dioxide and at different pHs. The precipitated proteins form soy meal was determined by measuring the proteins in the supernatant solution after the precipitation process for each run. The measurment was on the basis of a simple linear relationship between the UV absorbance and the concentration of the solute (Demchenco, 1986) using UV–vis, spectrophotometer (Perkin–Elmer, USA). The difference in wt% protein in the feed and wt% protein in the supernatant, equals the precipitated proteins.
4
Pressure
5
pH
Pressure (bar)
runs. The supernatant was then decanted into another container to prevent any dissolution of the curd and kept for further analysis. The precipitate was freeze-dried and kept for the qualitative analysis, while the supernatant used for the quantitative analysis.
6.00
3.2. Acidification of soy protein solution with carbon dioxide 5.70
The effect of pressurized carbon dioxide on the acidification of soy protein solutions was studied. Experiments were performed by applying different pressure 10, 20, 30, 40 and 50 bar and then the equilibrium pH was measured at different times. All runs were carried out at a constant ambient temperature of around 22 C. The curves of pH as a function of time all have the same pattern. The pH dropped rapidly from 9 and then levels down to a constant value, as the solution get saturated with CO2. This equilibrium pH would initiate the flocculation, coagulation then precipitation of the neutralized protein particles. Fig. 2 showed a sharp drop in pH from 9.0 to 5.6 as pressure was increased to 30 bar then the pH stabilized around pH 5.5 as the solution became saturated with CO2. Fig. 3 shows the relationship between different pressures of CO2 and pH at ambient temperature and 300 rpm for all soy protein extract experiments. At lower pressures (10– 20 bar), pH is strongly influenced (reduced) by pressure increase, and at higher pressures (30–50 bar) the pH drop is minimal. The sensitivity of pH is low in the pressure range of 30–50 bar. The change in pH is only about 0.05, so operating at pressures higher than 50 bar will have no
5.40 0
10
20 30 40 Pressure (bar)
50
60
Fig. 3. Relation between pH and CO2 pressure for soy protein extracts, at ambient temperature and 300 rpm.
advantage in further decreasing the pH, since the solution is saturated with carbon dioxide. 3.3. Protein recovered High protein recovery is desirable from any stream containing them. To determine the yield, a graph of the wt% of protein precipitated under different carbon dioxide pressures were plotted as a function of pressure for soy protein. Fig. 4 shows that the maximum recovery for soy protein is 68.3 wt% under 30 bar of pressurized carbon dioxide and at pH of 5.6. This pH (5.6) is close to the pI or the isoelectric point of soy protein, which is in the range of 4.7–5.2, (Thiering et al., 2001).
Table 1 Precipitated soy proteins under different CO2 pressure and their pH Pressure bar
Absorbance UV (nm)
Concentration mg/ml diluted
Concentration mg/ml
mg Protein precipitated
% Protein precipitated
pH reading
10 20 30 40 45 50
0.7249 0.6569 0.532 0.644 1.0149 1.1753
0.0497 0.0451 0.0365 0.0442 0.0696 0.0806
8.2880 7.5106 6.0825 7.3631 11.6037 13.4376
10.9120 11.6894 13.1175 11.8369 7.5963 5.7624
56.8332 60.8825 68.3201 61.6507 39.5641 30.0125
6.40 5.65 5.60 5.62 5.54 5.52
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wt% protein recovered
80 60 40 20 0
0
10
20
30
40
50
60
Pressure (bar)
Fig. 4. Effect of CO2 pressure on soy protein recovery at ambient temperature of 22 ± 1 C.
3.4. Effect of acidity on protein recovery To confirm the results discussed in the previous section, the direct effect of increasing acidity (pH) by CO2 pressure, on protein recovery is shown in this section. Fig. 5 shows that the highest recovery for soy protein is achieved at pH around 5.6. This indicates there is no need to increase the acidity further, because the amount of protein recovered was decreasing with further increase in pH. 3.5. Qualitative analysis of precipitated proteins Reversed-phase high-performance liquid chromatography (RP-HPLC), manufactured by Waters 600E and ELSD 800, Alltech (USA), was used to analyze the precipitated proteins qualitatively and to determine the purity of proteins. RP-HPLC is a very powerful analytical technique for peptides and proteins (Aguilar, 2003; Mant & Hodges, 1996). Fig. 6 shows the elution profile of the soy protein extract components of the feed showing three major peaks, the first large peak which eluted at 17.9 min coincides with milk caseinate standard, while the second peak eluted at 21.9 min and the third eluted at 22.8 min. Fig. 7 shows a first small peak eluted at 23.3 min and a second largest peak eluted at 28.5 min, which indicates the precipitate was not a pure protein but possibly a mixture of the two major soy proteins which were glycinin of molecular weight of (350 kDa) with pI range 4.9–5.2 and b-con-
Fig. 6. Elution profile of 10 wt% of soy protein extract components of the feed solution.
wt% protein recovered
80 60 Fig. 7. Elution profile of soy protein precipitated at 30 bar of carbon dioxide.
40 20 0 5.20
5.60
6.00 pH
6.40
Fig. 5. Effect of acidity on soy protein recovery.
6.80
glycinin of molecular weight of (180 kDa) with pI range of 4.7–5.0 (Thiering et al., 2001). Fig. 8 shows the elution profile of the soy protein precipitated using 40 bar of carbon dioxide. It shows one major peak eluted at 23 min, indicating a pure soy protein of b-conglycinin, the lower molecular weight soy protein,
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cates a mixture of two soy proteins. The first peak was the pure soy protein glycinin, which usually precipitate at higher pI (5.62) because of its higher molecular weight (350 kDa). The other soy protein was b-conglycinin, which has lower molecular weight of (180 kDa). The chromatogram showed, a pure soy protein in case of using (40 bar), which is the only pure soy protein with higher molecular weight glycinin soy protein. References
Fig. 8. Elution profile of soy protein precipitated at 40 bar of carbon dioxide.
which normally precipitate at lower pH range of 4.7–5.0 than glycinin soy protein, the higher molecular weight soy protein. From the results shown in Figs. 7 and 8, it is clear that the two types of proteins are formed only at 30 bar. This explains why the protein recovery was higher in case of using 30 bar of CO2 for precipitation of protein and at pH of 5.6. 4. Conclusions High pressure carbon dioxide was applied to recover protein from defatted soymeal. This process was found to produce protein in pure form. The advantage of using pressurized carbon dioxide in precipitating food protein is to achieve the precipitation at higher pH with minimum possible pressure, to save energy and to get high protein recovery and more purified protein than that usually obtained using mineral acids, which needs further treatments. CO2, unlike acids, will separate easily from the solution by depressurizing it leaving the solution clean. Highest recovery of soy protein has been achieved at a pressure of 30 bar of CO2 at ambient temperature of 22 ± 1 C, which correspond to a solution pH in the range of 5.4–5.6. The qualitative analysis of RP-HPLC for soy protein precipitate showed two totally separated peaks, which indi-
Aalbersberg, W. Y., Hamer, R. J., Jasperse, P., de Jong, H. H. J., de Kruik, C. G., Walstra, P., et al. (2003). Industrial proteins in perspective. Progress in Biotechnology, 23. Aguilar, M. I. (2003). HPLC of peptides and proteins, methods and protocols. Methods in Molecular Biology, 251, 9–12. Bell, D. J., & Dunnill, P. (1982). The influence of precipitation reactor configuration on the centrifugal recovery of isoelectric soy protein precipitate. Biotechnology and Bioengineering, 24, 2319–2336. Catsimpoolas, N. (1969). Isolaton of glycinin subunit by isoelectric focusing in ureamercaptoethanol. FEHS, 4, 259–261. Demchenco, A. P. (1986). Ultraviolet spectroscopy of proteins. Berlin Heidelberg, New York, London, Tokyo: Springer-Verlag, p. 94. Hammond, E. G., Murphy, P. A., & Johnson, L. A. (2003). Properties and analysis of soybeans. USA: Elsevier Science Ltd., p. 32. Hofland, G. W., van Es, M., van der Wielen, L. A. M., & Witkamp, G. J. (1999). Isoelectric precipitation of casein using high pressure carbon dioxide. American Chemical Society, 38, 4919–4927. Jarzebski, A. B., & Malinowski, J. J. (1995). Potentials and prospects for application of supercritical fluid technology in bioprocessing. Process Biochemistry, 30, 343–352. Johnson, L. A., & Myers, D. J. (1995). Industrial uses for soybeans. In D. R. Erikson (Ed.), A practical handbook of soybean processing and utilization (pp. 380–427). AOCS Press. Liu, K. (1997). Soybeans; chemistry, technology and utilization. New York: International Thomson Publishing, p. 532. Mant, C. T., & Hodges, R. S. (1996). Analysis of peptides by high performance liquid chromatography. Methods in Enzymology, 271, 3–50. Mezaki, M., Mochizuki, M., & Ogawa, K. (2000). Engineering data on mixing. Amsterdam. The Netherlands: Science, p. 15. Olano, A. L., & Calvo, M. M. (1995). Preservation of raw milk with carbon dioxide. Zeitschrift fur Lebensmittel-Untersuchung und-Forschung, 200, 293–296. Salt, D. J., Liliford, R. B., Dunnill, P., & Eur, J. (1982). Factors influencing protein structure during acid precipitation; a study of soy proteins. Journal of Applied Microbiology and Biotechnology, 14, 114–144. Thiering, R., Hofland, G., Foster, N., Witkamp, G. J., & van der Wielen, L. A. M. (2001). Fractionation of soybean proteins with pressurized carbon dioxide as a volatile electrolyte. Biotechnology and Bioengineering, 73, 1. Tomasula, P. M., Grag, J. C., & Boswell, R. T. (1997). A continuous process for casein production using high pressure carbon dioxide. Journal of Food Engineering, 33, 405–419. Van Eldik, R., & Palmer, D. A. (1982). Effects of pressure on the kinetics of the dehydration of carbonic acid and the hydrolysis of carbon dioxide in aqueous solution. Journal of Solution Chemistry, 11, 339–346.