The present invention concerns a single-pass pulsed membrane emulsification process and apparatus.

In particular, the invention concerns a single pass pulsed cross flow membrane emulsification method and an apparatus for performing such method.

Membrane emulsification is a technique that permits to produce emulsion droplets through extrusion of one liquid phase (dispersed phase) into a second liquid phase (continuous phase) using a microporous membrane. During droplet formation, dispersed phase droplets grown at the membrane pores opening are detached in the continuous phase thought a drop-by-drop mechanism. The principal membrane emulsification technologies developed include:

- dynamic membrane emulsification in which:

i. static and fixed membranes are used and the continuous phase is moved (cross-flow or stirred membrane emulsification) ;

ii. moving membranes are used to generate relative motion between the membranes and the continuous phase (rotating or vibrating membrane emulsification) ;

- static membrane/microchannel emulsification in which droplets are detached from the membrane pore when they reach a certain size in absence of additional moving force .

Cross-flow membrane emulsification is a well- studied technique [A. J. Abrahamse, 2002; S.J. Peng, 1998; G. De Luca 2004] in which the continuous phase is circulated in tangential direction along the membrane lumen side. In the stirred membrane emulsification the continuous phase is stirred using a rotator or a paddle stirrer positioned over the membrane [Stillwell M., 2007; Egidi E. , 2008] . The main advantages of membrane emulsification process compared to the conventional mechanical emulsification processes (ultrasound, high- pressure systems, rotor-stator systems) are the low energy consumption per unit of product made [H. Schubert, 2006] with high energy efficiency, quality and functionality of delicate used ingredients and the precise manufacture of emulsions with controlled droplets size and size distribution.

A considerable challenge in membrane emulsification remains in obtaining emulsions having a narrow droplet size distribution (coefficient variation < 5%) at high dispersed phase fluxes (productivity of 50 - 100 L/h) sufficient to make the process suitable for industrial application. This problem has been addressed using micro-engineered membranes etched in silicon [Kobayashi, 2003] . These membranes are thin and have a well-defined uniform pore size, leading to high disperse phase fluxes and a narrow droplet size distribution [Kobayashi, 2003] compared to what is obtained with traditional SPG or polymer membranes. Recent studies [Yuan Q. , 2009 a, 2009 b] discuss the factors that determine the membrane emulsification productivity, which include membrane properties, like pore surface and pore shape. For example, noncircular pores offer significant process benefits in terms of high productivity and size distribution control compared to circular pores. Different phenomena can occur associated with the non circular geometric factors, including interfacial instability determined by pore shape effects. Slotted pores produce uniform droplets at much higher droplet formation rate with the rotation technology (2,5-4 mm/s) than with the crossflow technique (0,45 mm/s) [Yuan Q. , 2009 b] .

The interaction between the disperse phase and the pore is a significant factor in the productivity enhancement. The membrane pore wall having good wettability to the disperse phase allows the oil phase to permeate more quickly in the pores, and hence results in significantly higher productivity. The addition of one miscible alcohol to viscous oil was proven to increase oil flux much more than expected based on its viscosity.

With respect to the oils, the alcohol is water soluble and can increase the wettability of the oil phase on the pore wall with a consequent increase in the oil flux [Yuan Q. , 2009 a]. Results suggest that the modification of the pore wall in order to increase hydrophobic properties will permit to increase the dispersed phase flux while the hydrophilic detachment part retained will avoid the spread of oil phase over the membrane for emulsion quality control. This knowledge can be used to design high-productivity membranes .

Another strategy used to increase membrane emulsification process productivity is using repeated premix membrane emulsification [Park et al . , 2001; Altenbach-Rehm et al., 2002; Suzuki et al., 1998]. In this process, the preliminary emulsified oil-in-water (O/W) or water-in-oil (W/O) emulsion is dispersed into the continuous phase forcing the coarse emulsion through the membrane [Akmal Nazir, 2010] . The drawback of premix emulsification is membrane fouling that may become serious depending on the formulation components [A. Trentin, 2009] , and related to their interaction with the membrane and their ease of removal.

A novel approach to decrease the average droplet size while maintaining high disperse phase fluxes is the use of moving membranes . The use of high membrane shear rates has long been recognized in filtration process (ultra filtration UF, micro filtration MF, nano filtration NF, reverse osmosis RO) [Jaffrin, 2008] as one of the most efficient factors for increasing permeate flux as it reduces fouling phenomena. Dynamic or shear-enhanced filtration consists in creating the shear rate at the membrane by a moving part such as a rotating membrane, or a disk rotating near a fixed circular membrane or by vibrating the membrane either longitudinally or torsionally around a perpendicular axis .

The rotating membrane concept has been applied earlier in dynamic membrane filters, which can be designed as rotating disk membranes [J. Engler, 2000] or rotating cylindrical membranes [C.K. Choi, 1999] . These filters are most applicable to the clarification of very high concentration suspensions and the separation of biological products [A. Brou, 2003] .

Schadler and Windhab [V. Schadler, 2006] have studied continuous production of water-in-oil (W/O) emulsions using a rotating nickel membrane with carbon coating deposited on the porous substrate. The rotating membrane was mounted coaxially inside an outer cylinder.

A new rotating membrane emulsification system in which a stainless steel membrane tube was rotated inside a stationary glass cylinder was developed by Vladisavljevic and Williams [Goran T. Vladisavlj evic, 2006] . The oil phase was introduced inside the membrane tube and permeated through the porous wall moving radially into the continuous phase in the form of individual droplets .

In these preliminary experiments, increasing the membrane rotational speed increased the wall shear stress which resulted in a smaller average droplet diameter being produced. Results suggested the potential for the rotating membrane methods as an emulsification method for manufacturing droplets with a consistent and selectable droplet size. Q. Yuan et al . demonstrated the performance of slotted pores in producing size-controlled uniform droplets using rotating membrane emulsification enhancing the membrane emulsification productivity using a rotating membrane with non circular pores [Q. Yuan, 2009a, 2009b] .

Promoted by successful application of vibration in membrane separation modules [O. Al Akoum, 2002, J. Postlethwaite 2004] , Zhu and Barrow [Zhu J, 2005] reported the first investigation into the efficacy of transversal excitation in membrane emulsification using a micro machined silicon nitride membrane excited by a piezoactuator system. Zhu and Barrow found a decrease in droplet size for frequencies around 10 Hz and cross- flow velocities on the order of centimeters per second.

Kelder et al . carried out a numerical study intruding the influence of inertia in droplet detachment model [Kelder, 2007] . The simulations show that membrane excitation potentially has a strong effect on the average droplet size in membrane emulsification, but that successful exploitation will require careful design of membrane and process. First estimates seem to indicate that systems with lower excitation frequency and larger excitation amplitude may perform better, but this will require experimental verification.

Holdich et al. [Holdich R. , 2010] described the application of vibrating membrane emulsification at low frequency oscillation in order to control the production of droplets larger than 30 \i .

To stimulate a fluid stream to disperse into mono- disperse droplets, a new method and a rationed rotating membrane system using periodical switch-on between the rotating membrane and micro-nozzle were introduced (Y.B. Li, 2011) . The dispersed phase is forced to flow through the nozzle to form a fluid stream which later is divided into rationed parts by a rotating membrane. Under the disturbance introduced by periodically switch-on between the nozzle and membrane, the quantitative volume of the dispersed phase will transform into droplets as it flies out of the membrane. The droplet size decreases with the increase of the rotation speed and with the decrease of the jetting velocity.

An important point in emulsion manufacturing industry is the production of uniform size distribution and small size particles (according to the end use of particles) which guarantee the emulsion stability during the storage and use.

A general rule in membrane emulsification is the use of high shear rate to generate uniform small droplets. High shear rates at the membrane surface are obtained by:

- increasing the tangential continuous phase velocity along the membrane and reducing membrane tube diameter in cross- flow membrane emulsification;

- increasing angular velocity in stirring membrane emulsification;

- increasing the moving membranes velocity in rotating (membrane rotational speed) and vibrating (frequency oscillation membrane) membrane emulsification.

One problem related to the use of high shear stress is the break-up of the droplets previously formed within the pump and fitting in cross-flow system or in the paddle stirred system in stirred membrane emulsification. This is particularly important for example in the production of: i) lager droplets that have the application in food and flavor encapsulation, medical diagnostic particles, controlled delivery systems, etc.; ii) bioactive functionalized particles containing value-added functional products targeted in food, pharmaceutical, cosmetic, personal care sectors or protein with targeted function in biotechnology applications; iii) viscous dispersed materials like oil-in-water or oil-in-water-in-oil emulsions, polymer- based materials.

Static membrane emulsification process in the absence of shear flow at the membrane surface is potentially suitable for the production of less viscous and/or larger droplets with uniform size. In addition, it is expected that the simple experimental set-up and the low energy input required makes the static membrane emulsification process interesting from a technology point of view. However, at AP/P _C > 1,13, polydispersed droplets were generated [M . Kukizaki, 2009] and the membrane controlled droplets formation is possible only at low dispersed phase flux with low productivity.

An alternative method for increasing the shear- stress at the membrane surface while maintaining a low value along the circuit out of the membrane during emulsification process was recently introduced [A. Koris, 2011] . Using turbulence static promoters was possible to obtain very good emulsion quality in terms of droplet size distribution and stability, even operating at high dispersed phase flux values.

In the light of the above, it is evident the need for an emulsification method that guarantees appropriate shear stress at the membrane level while preventing the shear stress effect related to the recirculation of emulsion droplets along the circuit within the pump and fitting in cross- flow membrane emulsification.

In this context it is proposed the solution according to the present invention, which aims to provide a membrane emulsification method using pulsed liquid continuous phase at the lumen level of a microporous membrane. The method allows obtaining highly concentrated uniform liquid droplets without any damaging due to mechanical stress.

The purpose of the present invention is therefore to provide a method which allows to overcome the limits of the solutions according to the prior art and to obtain the previously described technical results.

Further object of the invention is that said method can be implemented with substantially low costs.

Another object of the invention is to provide a method which is substantially simple, safe and reliable.

It is therefore a first specific object of the present invention a membrane emulsification method, wherein a continuous phase is present on a first side of a membrane wall and a dispersed phase is pressed on the other side of the membrane wall, so that the dispersed phase passes through the pores of the membrane, wherein the continuous phase is moved back- and-forward in a direction tangential to the membrane wall, until an emulsion is obtained with the desired concentration of the dispersed phase within the continuous phase.

In particular, according to the present invention, said emulsion is removed from the membrane only after the desired concentration of the dispersed phase within the continuous phase is obtained.

Preferably, according to the invention, surfactants are added in the continuous phase .

In particular, according to the present invention, said back-and-forward movement has an amplitude comprised between 10 and 400 mm and a frequency comprised between 0,1 and 5Hz .

It is a second specific object of the present invention a membrane emulsification apparatus, wherein a membrane module is fed with a continuous phase on a first side of a porous membrane wall and a dispersed phase on the other side of the membrane wall, said dispersed phase being pressed against the membrane wall by pressure means so that the dispersed phase passes through the pores of the membrane wall, further comprising means for moving said continuous phase back- and-forward in a direction tangential to said membrane wall .

Preferably, according to the invention, said membrane module comprises at least one tubular membrane, and more preferably said continuous phase is fed tangentially to a lumen of said at least one tubular membrane .

Moreover, always according to the present invention, said means for moving said continuous phase comprise a pump with reverse flow direction function.

The advantages of the method and apparatus of the present invention are evident, the pulsed membrane emulsification method eliminating the shear stress outside of the membrane related to the recirculation of emulsion droplets along the pump and fitting circuit as it happens in conventional cross-flow membrane emulsification. In fact, according to the present invention, the emulsion is collected within the shaking phase within the lumen (which serves as batch system) and then collected in the reservoir in a single pass without recirculating it. In effect, the process can be considered a cross-flow batch system operating in a semicontinuous mode.

Results obtained showed that it was possible obtaining uniform emulsion droplets also when high concentration of dispersed phase was reached with high productivity comparing the traditional cross-flow membrane operation.

Consequently, the pulsed membrane emulsification method and apparatus according to the present invention are suitable for the production of particles containing bioactive molecule or viscous dispersed materials or large droplets with uniform size.

The invention will be hereinafter described for illustrative but not limitative purposes, with particular reference to some illustrative examples and to figure 1, showing a schematic representation of an apparatus according to the present invention.

In particular, according to the present invention, a novel dynamic membrane emulsification method is proposed utilizing a static and fixed tubular membrane. The system has been tested in the preparation of an oil-in-water emulsion.

Making reference to figure 1A, a fixed volume of continuous phase A is fed tangentially to the membrane lumen 1 and the flow direction 2 is reverted at appropriate frequency in such a way that the volume is kept in the lumen 1. In other words, the continuous phase A is tangentially agitated by inverting the flow direction 2 back-and-forward within the membrane lumen 1, which contains the "batch" volume. The dispersed phase B passes radially through the pores 3 of the porous membrane wall 4 and forms droplets C into the shaking continuous phase A. The continuous phase volume is then removed from the membrane lumen 1 once the desired dispersed phase concentration has been obtained.

The method can be applied in the preparation of simple emulsions such as oil-in-water (O/W) and water- in-oil (W/O) emulsions.

Oil-in-water emulsions can be prepared using as dispersed phase vegetal oil such as (but not limited to) soybean oil, sunflower oil, olive oil and corn oil or other organic compounds such as (but not limited to) squalene or limonene or hydrocarbons such as (but not limited to) isooctane or hexane . Different water soluble surfactants can be dissolved in the water continuous phase such as (but not limited to) Tween 20 ^® or Tween 80 ^® (non-ionic surfactants) or sodium dodecyl sulfate (SDS, ionic surfactant) . Biomolecules such as (but not limited to) proteins ( β-lactoglobuline or bovin serum albumin (BSA) or lipase) can also be used without structural modifications to promote emulsion stability or to give specific functional activities.

The method according to the present invention is particularly suitable for the preparation of formulation containing labile biomolecules because no strong mechanical stress is applied. The present invention allows the generation of new products having properties that could not be achieved using conventional methods. For example (but not limited to), in the production of viscous high concentrated emulsions or polymeric suspensions.

Water-in-oil emulsions can be prepared using different combination of water and oil phases as indicated for the preparation of oil- in-water emulsions. Oil soluble surfactants can be dissolved in the oil continuous phase such as (but not limited to) Span™ 80, Span™ 85, SY Glyster PO-5S.

Multiple emulsions can also be prepared using a primary emulsion as dispersed phase.

The method can also be used to prepare solid particles by polymerization process or by direct chemical precipitation. Polymeric solutions such as (but not limited to) polyvynil acetate (PVA) or chitosan can be used to produce solid particles by emulsion droplets polymerization. Using high-melting point solid lipid material such as (but not limited to) cocoa butter, hard wax it is also possible to produce solid particles by direct precipitation by cooling down method. The temperature of the membrane emulsification process is kept at a higher temperature than the melting point of the oil phase. Emulsion droplets are produced through the temperature-controlled membrane emulsification step while the resultant emulsions are immediately cooled to solidify the oil phase.

The method is particularly suitable for this specific application because the temperature-controlled emulsification step can be carried out by controlling the temperature operative conditions at the membrane module level where the emulsion is obtained while the solidification cooling step will be obtained outside of the membrane module. This is possible because the emulsion is not recirculated along the circuit. When solid lipid particles are produced by conventional cross-flow membrane emulsification, it is necessary to keep the equipment, included dispersed phase and continuous phase circuit, under controlled temperature conditions to prevent the solidification of the dispersed phase at the membrane level without production of particles.

In particular, in the method according to the present invention, the back-and-forward movement is described by two parameters: amplitude and frequency. The appropriate combination between said parameters permits to control the production of uniform emulsion droplets. In particular, according to the present invention, amplitude comprised between 10 and 400 mm and a frequency comprised between 0,1 and 5 Hz were used in the production of 0/W emulsions. A specific range of frequency and amplitude must be chosen for a selected formulation. In general, high frequency and low amplitude permit to generate uniform droplets with size equal to three times the pore diameter.

Preferably, the method was proven with membranes having 3 micron as pore size in a range between 10-400 mm as amplitude and 0,1-5 Hz as frequency in the preparation of oil-in-water emulsions. The method of the present invention may be adapted to produce particles with membranes having both lower and higher pore size, in principle with no limitations but preferably in the range of 0,2 - 20 micron pore size. The range of amplitude and frequency could be adapted on the basis of pore size (especially for the larger size) and physical chemical properties.

With reference to figure 1, a schematic figure of the apparatus used for pulsed membrane emulsification according to the present invention is shown, wherein a membrane module 10 is present. A dispersed phase B, coming from a dispersed phase vessel 11, is fed to the membrane module 10, on a first side of the porous membrane wall 4, having a first and a second side, by a dispersed phase inlet conduit 12. The dispersed phase B is pressed through the membrane wall 4 by a pressurized gas, contained in a gas vessel 13.

The continuous phase A, coming from a continuous phase vessel 14, is fed to the membrane module 10, on the second side of the membrane wall 4, by a continuous phase inlet conduit 15 and "shaked" using a programmable pump 16 with reverse flow direction function.

Pressure gauges 17 and valves 18 are also present in order to control pressure and flow within the membrane module 10.

Once the desired dispersed phase concentration in the continuous phase has been obtained, the valve 19 is open, so the emulsion is removed from the lumen 1 of the membrane module 10 through an emulsion outlet conduit 20 and stored in an emulsion vessel 21. A dispersed phase outlet conduit 22 is also present, and provided with a valve 23. Example 1

An oil-in-water emulsion was prepared using commercial grade soybean oil as dispersed phase in (p = 0,919 g/cm3, η = 69 mPa) and Tween 80® 2% wt as continuous water phase (p = 0,998 g/cm3, η = 1,38 mPa) . An SPG (Shirasu porous glass) tubular membrane (8,7 mm inner diameter x 0,65 mm wall thickness) from SPG Technology Co., Ltd. (Japan) with nominal pore size of 3,lym was used. The effective membrane area was 31,3 cm ² . The membrane was wetted in the continuous phase under vacuum and ultrasonic field before the installation. Oil pressure was ensured by N ₂ gas and it was injected from the shell side of the membrane. The continuous phase was recirculated or pulsed on the lumen side of the membrane by peristaltic pump (Digi- Staltic double-Y Masterflex ^® pump Micropump, model GJ- N23. JF1SAB1) . The applied transmembrane pressure (TMP) was 0,7 bar. The axial flow rate velocity along the circuit was 850 ml/min that correspond to an axial velocity of 0,26 m/s. When pulsed membrane emulsification method was used, a fixed continuous phase volume of 2,8 ml was fed tangentially to the membrane lumen and the flow direction was reverted at 2,53 Hz frequency in such a way that the volume was kept in the lumen. The disperse phase flux was determined by volume as a function of time that passed through the membrane area. The volume was measured upon the oil consumption from a graduated feed tube. The experiments were carried out at room temperature.

Dispersed phase flux, oil %, droplets size and droplets size distribution were evaluated and monitored as a function of time during pulsed membrane emulsification experiments. The initial continuous phase volume used was 30 ml. Around 30% of oil was obtained in approximately 2,5 hours. After that 30% oil was obtained, emulsion was removed and 30 ml of fresh continuous phase was replaced. The process was carried out until 30% oil was obtained in 120 ml of emulsion total volume. The dispersed phase flux maintained a constant value of 1 ± 0,08 1/hm ² for the duration of the experiment . Uniform droplets with span values of 0,8 were obtained. No difference in droplet size and droplets size distribution was observed when the oil % in emulsion increased. The efficiency of pulsed membrane emulsification was proven comparing data with those obtained from experiments carried out in the same condition (PTM/P _C and axial velocity) and with the same phases composition using cross -flow membrane emulsification process. Table 1 reports the results of this comparison. In this case, droplet size and droplet size distribution changed during the time under the effect of continuous phase recirculation along the circuit and increase of the incidence of collision between particles.

In particular, Table 1 shows that when cross-flow membrane emulsification method was used droplets size uniformity was lost when highly concentrated emulsions are produced and span changed from 0,8 to 3,5. The most significant effect on droplets size was observed in the volume-weighted mean particle diameter (D[4,3]) which is more sensitive to the presence of any large particles and D[4,3] changed from 9,8 micron to 16,5 micron.

Table 1 Membrane 0/W% Mean Span Emulsification Particles

Method Diameter

μπι

D[3,2] D[4,3]

Present

5 8,7 9.6 0,8 invention

Cross- flow 5 8,7 9.8 0,8

Present

30 8,7 9.6 0,8 invention

Cross- flow 30 9 16.5 3,4

This emulsification method guarantees appropriate shear stress at the membrane level while preventing the shear stress effect related to the recirculation of emulsion droplets along the circuit within the pump and fitting in cross-flow membrane emulsification. This innovative technique is suitable for the production of bioactive functionalized particles, viscous dispersed materials and large droplets with uniform size .

Pulsed membrane emulsification technology has many advantages compared to the conventional emulsification methods and the other membrane emulsification mode operations used.

a) Pulsed membrane emulsification technology guarantees appropriate shear stress at the membrane level while preventing the shear stress effect related to the recirculation of emulsion droplets along the circuit within the pump and fitting in cross -flow membrane emulsification.

b) The tangentially back-and- forward agitation of the continuous phase within the membrane lumen prevents droplets destabilization phenomena related to the recirculation during cross-flow membrane emulsification.

c) Pulsed membrane emulsification technology permits to obtain uniform emulsion droplets also using trans-membrane pressure value higher than the critical pressure and with high final dispersed phase concentration .

d) Pulsed membrane emulsification technology is suitable for the production of bioactive functionalized particles, viscous dispersed materials and large droplets with uniform size.

e) Pulsed membrane emulsification technology permits to operate for hours in continuous mode without decrease of dispersed phase flux and perfect control in terms of droplets size and size distribution.

f) Pulsed membrane emulsification technology is related to the development of a new method for collecting droplets in a single pass in the continuous phase along the lumen of the microporous membrane. The method allows obtaining highly concentrated uniform liquid droplets without damaging. The great advantage of the single pass pulsed membrane emulsification is that it eliminates the shear stress outside of the membrane related to the recirculation of emulsion droplets along the pump and fitting circuit as it happens in conventional cross- flow membrane emulsification.

g) Pulsed membrane emulsification technology permits to eliminate the shear stress to which the particles are subjected in the circuit outside of the membrane since emulsion is produced in the membrane with a single pass mode . The present invention has been described for illustrative but not limitative purposes, according to preferred embodiments, but it is to be understood that variations and/or modifications can be made by those skilled in the art without departing from the relevant scope of protection, as defined by the enclosed claims.