Although the pullulanase of Themzus sp. strain AMD-33 exhib- its bifunctional activity, no amylase activity could be detected in the enzyme produced by E. coli. This lack of amylase activity may be a reflection of differences in gene expression mecha- nisms in E. coli as compared with Thermus sp. strain AMD-33. Such differences exist as illustrated by the fact that the Thermus promoter was not active in E. coli system; in the absence of tac or lac promoters no expression was observed. We intend to perform further studies using a Thermus host-vector system, to clarify whether the pullulanase of Thermus sp. strain AMD-33 has bifunctional amylase and pullulanase activity.

Abbreviations

pCMB, p-chloromercurybenzoate; IAA, monoiodoacetate; SDS, so- dium dodecyl sulfate; EDTA, ethylenediamine tetraacetate; PMSF, phenylmethansulfonylfluoride: DTNB, 5,5’-dithiobis (2-nitrobenzoic acid) and Mer-EtOH, 2-mercaptoethanol.

Acknowledgment

We thank Miss l kuko Uesugi for her technical assistance. We thank W. R. Bellamy of Morinaga Milk Co. for critical reading of our manuscript and valuable discussions.

Bibliography

[ l ] Nakamura, N., N. Sashihara, H. Nagayama, and K. Horikoshi:

[2] Nakamura, N., N. Sashihara, H. Nagayama, and K. Horikoshi:

[3]Sashihara, N., N. Nakamura, H. Nagayama, and K. Horikoshi:

[4] Somogyi, M. J. : J. Biol. Chem. 195 (1952), 195. [5] Lederberg, E. M., and S. M. Cohen: J. Bacteriol. 119 (1974),

[6] Holmes, D. S.. and M. Quigley: Anal. Biochem. 114 (1981), 193.

J. Jpn. SOC. Starch Sci. 34 (1988), 38.

StarchlStarke 41 (1989). 112.

FEMS Microbiol. Lett. 49 (1988), 385.

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[7] Weber, K., and M. Osborn: J. Biol. Chem. 244 (1969). 4406. [a] Hewick, R. M., M. W. Hunkapiller, L. E. Hood, and W. J . Dreyer:

J. Biol. Chem. 256 (1981), 7990. [9] Smith, L. M., J. Z . Sanders, R. J . Kaiser, P. Hughes, C. Dodd, C. R.

Connell, C. Heiner, S. B. H. Kent, and L. E. Hood: Nature 321 (1986), 674.

[lo] Sanger, F., S. Nicklen, and A. R. Coulson: Proc. Natl. Acad. Sci. USA, 74 (1977), 5643.

[ 111 Fukusumi, S., A. Kamizono, S. Horinouchi, and T. Beppu: Eur. J. Biochem. 174 (1988), 15.

[12] Katuragi, N., N. Takizawa, and Y. Murooka: J. Bacteriol. 169 (1987). 2301.

[13] Kuriki, T., J.-H. Park, and T. Imanaka: J. Ferm. Bioeng. 69 (1990). 204.

[14] Amernura, A., R. Chakraborty, M. Fujita, T. Noumi, and M. Futai: J. Biol. Chem. 263 (1988). 9271.

[15] Kuriki, T., and T. Imanaka: J. Gen. Microbiol. 135 (1989.), 1521. [16] Nakajima, R., T. Imanaka, and S. Aiba: J. Bacteriol. 163 (1985),

[17] Nakajima, R., T. Imanaka, and S. Aiba: Appl. Microbiol. Biotech-

[ 181 Ihara, H., T. Sasaki, A. Tsuboi, H. Yamagata, N. Tsukagoshi, and

[I91 Nishizawa, M., and F. Hishinuma: Ann. Rep. Mitsubishi-kasei

[20] Sakai, S., M. Kubota, K. Yamane, T. Nakada, K. Torigoe, 0. Ando,

[21] Kimura, K., S. Kataoka, Y. Ishii, T. Takano, and K. Yamane:

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nol. 23 (1986), 335.

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Institute of Life Sciences 13 (1984), 49.

and T. Sugimoto: J. Jpn. SOC. Starch Sci. 34 (1987), 140.

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Addresses of authors: Dr. Nobuhiro Sashihara *) Author for corre- spondence: Mailing address: Research Institute of Q.P. Corporation, 5-13-1, Sumiyoshi-cho, Fuchu-shi, Tokyo 183, Japan. Nobuyuki Naka- mura, Research Laboratory of Nihon Shokuhin Kako Corporation, 30, Tajima, Fuji-shi, Shizuoka 417, Japan. Koki Horikoshi, Laboratory of Microbiology, The Riken Institute, 2-1, Hirosawa, Wako-shi, Saitama, 351-01, Japan.

(Received: June 1, 1991).

Foam Fractionation of Proteins: Potential for Separations from Dilute Starch Suspensions*)

Ale8 Prokop and Robert D. Tanner, Nashville, TN, (U.S.A.)

Fundamentals of the separation of proteins from foam are reviewed. Thermodynamic, hydrodynamics and kinetic considerations of foam separation from protein mixtures are discussed as a guidance for operating and design parameters. They are useful for suggesting ways to apply this methodology to the starch industry, particularly in the area of protein (zein) removal from starch particles.

1 Introduction Foaming offers an alternative way of separation or fractionating a mixture of components in a solution provided they exhibit ~

*) Presented at “Corn Utilization Conference IV”, June 24-26, 1992, St. Louis, MO (U.S.A).

Schaurnfraktionierung von Proteinen: Moglichkeit zur Abtren- nung aus verdunnten Sttirkesuspensionen. Die Grundlagen der Abtrennung von Proteinen aus Schaum werden besprochen. Ther- modynamische, hydrodynamische und kinetische Betrachtungen der Schaumabtrennung aus Proteinmischungen werden als Richtschnur fur betriebliche und konstruktive Parameter diskutiert. Sie sind niitz- lich als Vorschlag zur Anwendung dieser Methodologie fur die Starkeindustrie, insbesondere im Bereich der Protein(Zein)beseiti- gung von Starkepartikeln.

surface activity. The foaming requires the presence of surface active agents to generate a relatively stable foam which rises to the surface and can be removed, the process being called flotation. In general terms, flotation can convert suspended, colloidal or dissolved substances to floating matter. In our case, proteins are macromolecules present as dissolved substances. By nature, they are also surface-active. In some instances,

150 starchlstarke 45 (1993) Nr. 4, S. 150- 154 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993 0038-9056/93/0404-0150$05.00+.25/0

extraneous surface-active substances can be added to modify surface properties of proteins. By contrast. starch is not usually surface active. By attachment of gas to the suspended or liquid phase, the bulk density of the fluid system may be less than the density of the parent system, the agglomerated suspension or solute aggregate (precipitate) to be floated to the top. The force responsible for the floating of the agglomerate is called buoyancy. Selective flotation or foam fractionation of particles/solutes takes advan- tage of differences in the degree of attachment between species. Those which do not attach to gas bubbles are left behind and not removed from the solution. This article will explore fundamen- tals of foam separation, and basic operating and design parame- ters involved in foam recovery and fractionation of proteins. It will then suggest ways to apply this methodology to the starch industry, particularly in the area of protein removal (e.g., the zein fraction in corn starch) from starch particles.

2 Theory

The controlling step for flotation to proceed is bubble-particle attachment. The success of flotation is due to solid/solute hydrophobicity. bubble-to-particle-size ratio and the degree of turbulency at flotation. For bubble-particle attachment to oc- cur. both thermodynamic and kinetic conditions must be fulfil- led. At equilibrium. the attachment of a particle to a gas bubble is controlled by the hydrophobicity, quantified via a relation between free energy change per unit area of surface (AG) to interfacial tension ( Y G ~ ) , involving the contact angle of the gas (G) bubble-particle 8 (strictly for a flat geometry) in a liquid (L) solution:

It can be shown that the attachment to a bubble is feasible for 8>0. For good flotation to occur, a contact angle over 30 degrees is required. There are simple and quick ways available to measure the contact angle, where a small angle means a low hydrophobicity and high wetting, and a high angle hig hydro- phobicitiy and non-wetting. To facilitate better attachment of proteins to gas bubbles, surface-active agents (collectors) can be used. They diminish the particle-water interaction by rendering the protein surfaces more hydrophobic and facilitate the dis- placement of the wetting water film from the protein surface. Collectors are typically long-chain hydrocarbon molecules con- taining polar groups. The molecule adsorbs onto the protein surface via the charged group with the hydrocarbon chain presented to the aqueous phase. A convenient and practical means of evaluating protein surface activity (or protein-collector-gas bubble system) is via the adsoption isotherm (Figure 1) valid for a twocomponent (one protein) system at equilibrium:

l- I

c, C

Figure 1. Protein adsorption Isithre.

This can be extended to a multi-component system, assuming no interaction between solutes. Another valuable relationship is that of the Gibb’s equation, demonstrating the surface tension-concentration dependence .(Figure 2). The situation is more complex. however, since both types of relationships (isotherm and Gibb’s equation) depend on the pH and ionic strength of the protein solutions. Unfortunately. very few

I

C

Figure 2. Surface tension - concentration Dependence, I-curve with plataeau. 2-curve with a minimum.

experimental data are available for pure protein or protein mixture solutions [l]. The kinetic requirements of bubble-particle attachment in- volve: 0 collision between bubble and particle, 0 thinning of the liquid film (as above) between bubble and

0 rupture of the liquid film, and 0 stable attachment of particle to gas bubble. The bubble-particle collision frequency, dependent mainly on a process hydrodynamics, is closely controlled by the particle and bubble sizes [2] . In addition, electric charges may play a role. For small particles. less than 0.1 mm in size, the collection efficiency, E (log E), has been shown to depend indirectly on the particle sue (in diffusion regime) (Figure 3):

particle,

IogE-lld; (3)

rendering the process quite inefficient. For large particles the

OihRiOfl mbclion

Figure 3. Collection efficiency depends on particle size.

collision efficiency (in the collection regime) depends on the following ratio:

E - (d,/ db)’ (4)

Thus, it might be useful to increase the particle size in order to enhance its attachment to the gas phase. To accomplish this in

starchktarke 45 (1993) Nr. 4, S. 150-154 151

the case of proteins, the addition of electrolytes (polyelectroly- tes, nonionic solvents) may be required. Electrolytes suppress the double electric layer adjacent to the protein molecule and induce protein precipitation, being effectively facilitated in a turbulent field. The same effect can be accomplished by chang- ing the electrostatic charge density via pH change. The adjust- ment of pH to the isoelectric point (by definition the pH where the charge density is minimized or close to zero) is the most useful. The thinning and rupture of the liquid film between the colliding particle and bubble will require a finite time. Only then the stable bubble-particle aggregate will be formed. As bubbles move in a flotation reactor (column) they encounter and collect many particles. During this encounter, the following dynamical- ly unstable situation exists: 0 viscous drag on particles cause an asymmetric bubble-particle

system to partially rotate and oscillate, 0 particles move on the bubble surface and slide down to form a

particle cap, being swept from the bubble front by fluid motion past the bubble (Figure 4, [2]).

Lift force

f

mostly reported at the isoelectric point. The eventual foam callapse is absolutely needed in order to recover a concentrated product stream. The entrained liquid in the film drains down under the force of gravity. Consequently, liquid holdup in the foam section of the column decreases with height, resulting in a concentration gradient of adsorbant (protein) along the column length in this section. This is merely due to the nonequilibrium adsorption, where the protein adsorption is a slow process.

3 Operational Parameters Processing time. Batch flotation process (in a column) is a first order reac- tion:

where k is called flotation rate constant. The value of k is found by plotting recovery vs. flotation time on a semi-logarithmic scale. The residual purity ratio (where c = cR) at a given time is conveniently used:

(7)

and (-k) is obtained from a plot of this ratio vs. time (Figure 5). In the above equation (7), cR is the residual (bulk) protein concentration, Vb is volume of protein solution in the nonfoam-

force

h a Mioh E.r

Figure 4. Particle cap formation.

The rate of rise of gas bubbles in the water (containing protein molecules) is expressed by Stoke’s law, which holds for bubbles with diameters less than 130 pm:

v-kd;

For larger bubble diameters the simple relationships depicted in Figure 3 no longer hold, the rise velocity being dependent in a complex manner on d, [3]. This regime is kinetically more important as it leads to higher capture efficiencies, as noted in Equation (4). The residence time of bubbles in a foam fractionation reactor is a critical factor. The sufficiently long hydraulic residence time will allow a complete saturation and a high removal efficiency to be achieved. Beyond a critical time, further extension of the bubble residence time is not favorable [3]. The bubble stability is another important factor. In foam flotation the solute to be removed from the bulk solution is surface active so that it will adsorb to the surface of rising bubbles and be removed in the foam. The foam bed consists of gas bubbles of different sizes separated by liquid film (lamel- lae). The rupture of these films leads to the coalescence of neighbouring bubbles and to essential collapse of the foam bed. The column hydrodynamics are the major factors controlling the film and foam stability. Of the physical factors, surface viscosity and density at the interface are generally the most important [4]. The foam stability is also under the influence of surface tension and ionic strength, the maximum stability being

I

t Figure 5. Flotation is a first-order reaction.

ing section and the subscripts o and t refer to initial and terminal time of the foaming process [5]. Column performance. The separation ratio is a convenient efficiency measure for the batch process and is defined as the ratio of the product (protein) concentration in the foam (collected above the column liquid and collapsed mechanically) to the residual liquid product concentration:

The performance of continuous foam columns is evaluated via enrichment and recovery ratios. Enrichment is defined as the ratio of the product concentration (in the foam) to the feed concentration:

e = cplcF (9)

and recovery as:

r = (cPP)/(cFF) = e(P/F) (10)

where P and F are the product and feed stream flow rates,

152 starchktarke 45 (1993) Nr. 4, S. 150-154

respectively. Both high enrichment and high recoveries are desirable. Equation (8) can be also used for continuous column operation. Experiments with bovine serum albumin (BSA) [6] have shown that as the protein concentration in the feed (cF) increases protein concentration in the residue (cR) increases and concentration in the foam liquid (cp) decreases, reducing both s and e. Thus foam separation (at least for BSA) is most effective for dilute protein solutions. As expected from Equa- tion 10, an increase in feed flow rate F results in the lowering of r, eventually being insensitive to flow changes. The enrich- ment e exhibits a maximum due to entrained liquid in the foam layer [4]. The above holds for pure protein solutions. The solute feed composition may have an effect on column performance. The isolation of a single protein from a protein mixture should be governed solely by its surface activity. The proteins with higher surface activity will be selectively adsorbed to bubbles slowly rising through the liquid and concentrated in the foam. Some deviations (from equilibrium) have been noted especially at high gas flow rates [5 ] . Medium pH and ionic strength. As pH effects the net charge of protein molecules, it effects the protein enrichment as well. The maximum enrichment is fre- quently observed at the isoelectric pH (PI) (e.g., [l]). This is due to a minimum in surface tension at that pH. In addition, proteins typically exhibit the minimum in solubility (and maximum surface activity) at their PI values. Typical enrichment-pH curves at different protein concentrations are depicted in Figure 6. However, quite opposite data have also been observed [4].

l-----

Figure 6. Enrichment dependence on pH and protein Concentra- tion.

The ionic strength effect provides a basis for explaining these contradictory data (Figure 7, [7]). The measurements of surface tension exhibit a minimum at the isoelectric point of the protein studied, but a different dependency away from PI. Thus, combined pH and electrolyte effects play an important role in determining actual enrichment ratios. These differing effects also demonstrate the need for obtaining more experimental data to help clarify these interactions.

I

Ionic strength

Figure 7. Surface tension vs. ionic strength (pH varies).

The importance of electrolytes should be also stressed. “Struc- ture makers”. which reduce protein solubility in the bulk liquid, do not affect the s ratio, but other electrolytes (“structure breakers”, e.g., sodium sulfate) and alcohols (below 1% by volume) increase the solubility and s ratio as well, primarily through increased foaminess [6]. Water-soluble nonionic emul- sifiers (collectors) also increase enrichment at low concentra- tions. However, at higher concentrations both alcohols and emulsifiers depress the separation in foams [8]. All these chemical additives (electrolytes, alcohols and emulsifiers) effect to a different degree the extent of gas-liquid interfacial area (i.e.. bubble size distribution). As such, all are considered fundamen- tal operating variables, to a large degree controlled by physical principles. Modem approaches. Modem biotechnology provides tools which can dramatically modify physico-chemical properties of foam separation. Two examples are: 0 One, foam fractionation of proteins can be specifically af-

fected via selection of the proper solute treatment before flotation. Wheat germ agglutinin (WGA) is a plant protein (lectin) with specificity for N-acetyl-glucosamine. As such, WGA does not lend itself to flotation by air bubbles. The affinity to air bubbles is dramatically increased by making a complex between WGA and chitosan (polysaccharide rich in N-acetyl-glucosamine). Bubbles specifically bind to chi tosan and lift the WGA-chitosan precipitate to the surface [91.

0 Two, modern genetic engineering provides another specific possibility. Blue-green algae can adjust their buoyancy in lakes via synthesis of gas vesicle protein (GV protein), generating a highly hydrophobic surface. The GV protein gene has been isolated and cloned into other microorganisms to improve their surface activity and flotation properties [lo].

4 Design Parameters Column geometry. With increasing liquid column length, cR diminishes, hence, s, e and r increase ([l] [4] [6]). By lengthening the column, the contact time (residence time) between bubbles and liquid is increased, allowing more time for equilibrium near the interface to be approached and, thus better separation. This slow time for equilibrium is due to relatively low diffusivities of proteins, as they are quite large molecules. As the foam height increases, the liquid holdup at the top of the foam decreases, leading to higher enrichments and lower recoveries [6] [ll]. An increase in the column diameter increases both the enrich- ment and recovery. This is because a larger bubble sue in a larger diameter column (with negligible wall effects) allows for better drainage, leading to sharper separation. Above a critical column diameter, however, purification falls sharply [5]. Gas phase characteristics. Enrichment increase by lowering superficial gas velocities (the process is driven towards equilibrium) and with larger bubble sizes [4] [ll]. At higher gas flow rates more liquid entrainment in the foam occurs and, hence, less enrichment is observed. Large bubble sizes exhibit faster rates of drainage. Bubble size can in part be controlled by the porosity of the aerator. When the pore diameter of this aerator is reduced, cR diminishes and hence s increases [6]. In addition, the bubble size is under interfacial tension control, as discussed above. which in turn influences the bubble residence time. Column operation mode. One should distinguish between the batch column and that of the continuous operation column. The semi-batch single stage is the most common mode of batch operation, where the feed

starchlstiirke 45 (1993) Nr. 4. S. 150-154 153

material is added at one time and the products are drawn off continuously. Multi-stage columns have also evaluated, sho- wing as expected, higher enrichments [6]. Modelling Successful scale-up of foam fractionating requires a quantifica- tion of various operating parameters (see review in [12]). A variety of models for foam fractionation have been developed. Many take into consideration the observations listed above. Very few, however, consider the time-dependent, dynamic operation of the foam flotation column [3]. The nonequilibrium nature of protein adsorption has been largely avoided. A model for the evolution in time of the concentration stratification in foams has been suggested [3].

5 Significance of Foam Fractionation in the Starch Industry

The separation of proteins from starch presents a major chal- lenge in the starch industry. Protein components are always present no matter the source of starch (tubers, cereals). As a result of the milling process, both starch and protein are typically present in distrinct particles. In addition, some pro- teins may be present in the resulting liquid solution. Traditio- nally a hydrocyclone separation has been used for separation of the two polymers, based on size and specific density differences. As proteins exhibit surface activity they can be collected in the foam fraction of a gas is introduced into this complex dispersion. Hydrophobicity of proteins, as compared to starches, can be further adjusted (pH, ionic strength) or through applying certain biotechnology tools as mentioned. Plant proteins are commonly a mixture in terms of molecular weight and species. Foam fractionation offers distinct advantages for their separa- tion and fractionation. It will be especially convenient for handling of dilute wastes generated in food processing indu- stries (e.g., potato wastes). A patent has been issued for separating wheat gluten from starch through foam separation [13]. Most of the wheat starch and some solubles readily drain in the downward flow and are collected at the column bottom. The protein rich layer is collected at the top of column. The adjustment of p H to 5.0 (presumably the dominant o r average protein isoelectric point) facilitated maximal gluten recovery in the foam (Table 1). The product (starch) quality (color) was superior when compared to the unfoamed starch process.

Table 1 . pH Effect on Starch and Protein Yields (% of total product). (US Patent 3,868,355. 1975) 1131.

PH 3.0 3.4 3.8 4.2 5.0 5.8

Starch 65.3 68.8 50.6 52.5 50.9 54.8 Protein 13.7 19.4 26.1 34.3 35.6 31.9

Further protein fractionation is feasible via a n application of an “isoelectric focusing”, sequential p H gradient methodology [7] 1141, perhaps followed by an ultrafiltration concentration step. Such treatment would expand applications of protein fractions in the different areas of the food industry and may create new products.

6 Conclusions

Foam flotation and fractionation offer attractive means of removing proteins or their mixtures from other biological macromolecules or material like starch. The advantages are numerous:

0 high separation efficiency (enrichment up to 10 times, reco- veries up to 90%), particularly from dilute solutions (typical for many streams derived from food processing),

0 specific removal of individual proteins from complex mixtu- res of proteins o r other materials via rational strategies (e.g.. “isoelectric focusing”),

0 low energy requirement and maintenance costs, 0 low cost (no additives other than air on inexpensive gases like

C02, which are not retained by the protein and starch products),

0 retainment of biological activity in many instances, potential for further improvement in specificity.

7 Outlook Faster progress can be achieved if more data of a fundamental nature are available, particularly in terms of the gas-liquid interface (e.g., adsorption isotherms for pure proteins and their mixtures, kinetics of adsorption, competitive adsorption of proteins and collectors). In addition. data on surface tension of proteins as influenced by the complex influence of ionic strength and p H are critically needed. Moreover, the methods of characterization of surface activity and of its modification should be further developed.

Bibliography [l] Schnepf; R. E., and E. L. Gaden: Foam fractionation of proteins:

Concentration of aqueous solutions of bovine serum albumin. J. Biochem. Microbiol. Technol. Eng. 1 (1959), 1-8.

[2] Reay, D. V., and G. A. Ratcliff: Removal of fine particles from water by dispersed air flotation: Effects of bubble sue and particle size on collection efficiency. Canad. J. Chem. Eng. 51 (1973), 178-185.

[3] Kiefer, J. E., and D. J. Wilson: Time-dependent foam flotation stripping column operation. Sep. Sci. Technol. 16 (1981), 147-171.

[4] Brown, L., P. Bhattacharya, and P. C. Wankat: Foam fractionation of globular proteins. Biotechnol. Bioeng. 36 (1990), 947-959.

[5] Sarkar, P., P. Bhattacharya, R. N. Mukherjea, and M . Mukherjea: Isolation and purification of protease from human placenta by foam fractionation. Biotechnol. Bioeng. 29 (1987), 934-940.

[6] Gehle, R. D., and K. Schiigerl: Protein recovery by continuous flotation. Appl. Microbiol. Biotechnol. 20 (1984). 133- 138.

[7] Ostermeier, K. , and B. Dobias: Flotative separation of proteins in pH-gradient. Colloid Surfaces 14 (1985), 199-208.

[S] Ahmed, S. I.: Laws of foam formation and foam fractionation: 11. The influence of different association conditions on surfactants, glycerides, sugar, and salts on the foam fractionation of albumin. Sep. Sci. 10 (1975), 689-700.

[9] Seansrad, C., and B. Mattiasson: Purification of wheat germ aggluti- nin using affinity flocculation with chitosan and a subsequent centrifugation or flotation step. Biotechnol. Bioeng. 34 (1989).

[lo] Institute de Pasteur, Paris: Expression of DNA sequence encoding gas vesicle. WO Pat. 9010071 (1990).

I l l] Ahmad, S. J.: Laws of foam formation and foam fractionation: I. The effect of different operating parameters on the foam fractionation of albumin from a solution containing organic and inorganic materials. Sep. Sci. 10 (1975), 673-688.

[12] Uruizee, F., and G. Narsimhan: Foam fractionation of proteins and enzymes. 11. Performance and modelling. Enzyme Microbiol. Technol. 12 (1990). 315-316.

[13] Rodgers, N. E.: Foam separation of gluten and starch. U.S. Pat. 3868355 (1975).

[14] Potter, F. J. , A . H . G. DeSouza, R. D. Tanner, and D. J . Wilson: Modeling in-situ protein separation by bubble fractionation in: Baker’s yeast fermentation process. Sep. Sci. Technol. 25 (1990),

Address of authors: AleS Prokop and Robert D. Tanner, Research Professor, Chemical Engineering Department, Vanderbilt University, Nashville, TN 37235, (U.S.A.). (Received: July 8, 1992).

387-393.

673 -687.

154 starchlstarke 45 (1993) Nr. 4, S. 150-154