Patent Application
20240057624
This invention relates to the inhibition of protein agglomeration.
Protein agglomeration is a major industrial process-engineering problem in a variety of industries, e.g. milk and whey-protein-concentrate solutions in the dairy industry, and therapeutic antibodies in the bio-pharmaceuticals industry to give just two examples. The skilled person will appreciate that protein-containing liquids are used and processed in many other industries also (food and beverage production and processing, enzymes used in chemical processes, and biotechnology generally.
Agglomeration of proteins in, for example, milk or whey frequently results in bio-fouling of plate-and-frame heat exchangers and is a major problem in the dairy industry, with the deposition of agglomerated-protein biofilms on metal surfaces. In the medical arena, protein coagulation is also a major problem in blood processing, with thinning and anti-coagulation agents like warfarin taking their toll on patients vis-à-vis challenging side effects. Similar problems arise in other industries where proteins that are susceptible to agglomeration or coagulation are used.
In the context of milk processing, proteins comprise 26% of total solids content of milk with casein and β-lactoglobin (β-Lg) accounting for 76% and 9%, respectively, of total protein content. The most commonly used heat transfer device is a plate heat exchanger (HE). Tubular HEs are also employed, particularly at higher temperatures, and are more prone to fouling the plate HEs due to lower turbulence leading to higher rates of deposition onto the HE surfaces. Milk is heat-treated to pasteurise it, with different heating regimes employed to produce different products. The heating inherent in pasteurisation leads to fouling, resulting in process down-time due to the requirement for more frequent periodic cleaning of process equipment by Clean-In-Place (CIP) methods, in turn reducing productivity and increasing energy costs and operational expenditure.
Agglomeration may be the result of free-energy minimisation via many-body protein aggregation and ‘self-assembly’ towards thermodynamically (meta-)stable protein-agglomerated clusters.
To address the problem of unwanted agglomeration, several methods are currently in use, such as: (i) solvent exchange, (ii) glycerol addition, (iii) changing pH, (iii) changing salt concentration, (iv) adding an osmolyte, addition of metal ions, (v) addition of stabilising polymers, (vi) use of a phosphate buffer, and (vii) coating of surfaces to inhibit deposition of agglomerated proteins. The large economic expense and operationally challenging circumstances of removing any added agents post-processing is a substantial and ongoing problem, in addition to potential modification of protein properties by these additives (e.g. taste, purity standards etc. for milk).
Pulsed (static, time-invariant) electric fields [see references 1-10 at the end of the description] have been applied to protein solutions in an effort to control agglomeration properties, although little consensus exists. This unclear picture presents mixed results, other than a typical tendency for static and pulsed electric fields to lead to enhanced agglomeration [11]. Of course, an important goal in food or dairy process-engineering (and in other fields of science) is to reliably and controllably suppress rather than promote this phenomenon.
WO 2010/106296 discloses treating cows milk and whey protein concentrate by applying a low frequency electromagnetic field of 13.7 kHz, 20 kHz or 28 kHz in order to reduce the protein fouling observed in a heat exchanger.
There is provided a method of method of inhibiting protein agglomeration in a protein-containing liquid, carried out in an industrial processing plant, the method comprising:
It has been found that exposing a protein-containing liquid to a microwave electromagnetic field can induce an anti-agglomerative effect that lasts for a period of time after the exposure. In particular, it has been found that it is possible to induce such a change within a larger industrial process, such that the change persists for sufficient time to allow for a subsequent processing step to take place with reduced agglomeration of protein, and thereby reduce the incidence of fouling, blockages, and so on in the subsequent processing step.
The electric field strength of the microwave field is controlled to avoid significant heating of the liquid, as the goal is not to heat the liquid but rather to induce a decrease in viscosity and a consequential reduced fouling tendency during a later processing step, in particular during a subsequent processing step in which the liquid is heated and where fouling due to agglomeration would be expected to occur to a greater extent in the absence of the microwave treatment.
Without being bound to any theory, it is believed that the field-reversal frequency observed in microwave fields (wherein the electric- and magnetic-field components change direction continuously, back and forth for each sinusoidal field cycle) can be tailored to overlap with fundamental oscillatory translational and rotational modes in proteins. This is an example of bio-mechanical ‘resonance’—which usurps and subverts proteins' propensity to agglomerate by undermining the energy-relaxation or “self-assembly” processes. In effect, the resonant energy transfer may enhance the amplitude of natural “wiggling” and “jiggling” protein motions, discouraging their assembly together in an ordered coagulated or agglomerated arrangement—assisting entropy's ‘guiding hand’.
In addition, these microwave-based protein-disaggregation methods can be used to suppress and disaggregate existing agglomerates.
The method is energy efficient, and highly adaptable to industrial processing for a number of reasons: it provides the ability to retrofit and increase the capacity of existing footprint-constrained plant/heat exchangers; it is readily amenable to scale-up and continuous-mode flow operation; it can be provided using a portable apparatus, providing ease of shipping equipment (e.g., skid-mounted) for process-pipe ‘plug-in’; it is an additive and contact-free method, which is especially important for regulatory approval in the biopharma industry and for consumer acceptability in the food and drink industries; it is naturally reversible, in that the anti-agglomeration effects relax slowly upon field removal and ultimately dissipate; it has no adverse effects in protein function.
Preferably, said microwave electromagnetic field has an average field strength of at least 0.25 kV/m.
Further preferably, said microwave electromagnetic field has an average field strength of at least 0.4 kV/m.
Preferably, said microwave electromagnetic field has an average field strength of no more than 10 kV/m.
More preferably, said microwave electromagnetic field has an average field strength of no more than 5 kV/m.
Further preferably, said microwave electromagnetic field has an average field strength of between 0.5 kV/m and 5 kV/m.
References to average field strengths herein relate to the root mean square (r.m.s.) field intensity during the “On” part of any duty cycle, averaged across the volume within which the liquid is exposed to the field.
Preferably, the liquid is exposed to the microwave electromagnetic field for at least one minute, during which the microwave electromagnetic field provided as a pulsed or continuous field.
Preferably, the microwave electromagnetic field is provided as a pulsed field, with a duty cycle ratio of on time to off time of from 1:800 to 1:1.
Providing a pulsed field reduces the overall energy delivered to the liquid, as one would expect. Surprisingly, however, it has been found that even with off times which are orders of magnitude longer than the on times, the effect of the microwave treatment is maintained with very little reduction in effect. For example, off times which are 200 to 500 times longer than the on times have been found to result in similar reductions in effect on agglomeration (compared to continuous microwave fields) while drastically reducing the amount of heating of the liquid during the microwave processing step for a given duration of treatment, and reducing the energy consumption of the treatment process.
Preferably, the duty cycle ratio has an on time to off time of from 1:600 to 1:100.
In preferred embodiments, the microwave electromagnetic field is provided as a pulsed field with a duty cycle of an on time between 0.1 and 10 microseconds and an off time between 10 and 80 microseconds.
Preferably, the microwave electromagnetic field is provided as a pulsed field with a duty cycle of an on time between 0.1 and 1 microseconds and an off time between 20 and 80 microseconds.
Preferably, the microwave electromagnetic field has a frequency of between 0.1 and 30 GHz.
More preferably, the microwave electromagnetic field has a frequency of between 0.25 and 10 GHz.
In particularly preferred embodiments, the microwave electromagnetic field has a frequency of between 0.5 and 5 GHz.
It is preferred to tailor the frequency to a resonant frequency of the protein-containing liquid, that is a frequency which is observed to exhibit an increased anti-agglomeration tendency.
Preferably, the method further comprises the step of thermostatically controlling the exposure of the liquid to the microwave electromagnetic field, and thereby preventing the liquid from being heated above a predetermined temperature.
The predetermined temperature below which the liquid should remain will depend on the protein in the liquid, since different proteins will be affected differently at different temperatures. However, preferably said predetermined temperature is less than 60° C., more preferably less than 50° C., 40° C. or 30° C.
In certain embodiments, the inlet conduit and the outlet conduit are the same conduit.
In some embodiments, the liquid is exposed to said microwave electromagnetic field in a batched operation.
In other embodiments, the liquid is exposed to said microwave electromagnetic field in a continuous flow mode of operation wherein the liquid flows through said cavity at a volumetric rate that provides a predetermined level or range of exposure to said microwave electromagnetic field.
Preferably, said liquid is an aqueous liquid.
The method has particular application wherein said liquid is milk or a derivative thereof.
In many embodiments of interest, said further industrial processing step comprises heating said liquid to a temperature higher than the maximum temperature reached during the exposure of the liquid to said microwave electromagnetic field.
Preferably, said further industrial processing step comprises passing said liquid through a heat exchanger to perform said heating of said liquid.
As the skilled person will be aware, the fouling problem is frequently most pronounced when a protein-containing liquid is passed through a heat exchanger. By performing the microwave processing step (with minimal heating) and then, within a predetermined time while the effect of agglomeration reduction is still evident, performing the required heat exchanger step, fouling is reduced.
In preferred embodiments, said further industrial processing step heats said liquid to a temperature of greater than 70° C., and the maximum temperature reached during exposure of the liquid to said microwave electromagnetic field is no greater than 60° C., more preferably 50° C., more preferably 40° C., more preferably 30° C.
Preferably, said heating step is performed within a time of 30 minutes after exposure to said microwave electromagnetic field, more preferably 20 minutes, more preferably 10 minutes.
In a preferred embodiment the method further comprises, prior to the step of exposing the liquid within said microwave resonance cavity to a microwave electromagnetic field: performing a molecular dynamic simulation to determine the effect of different microwave frequencies on inter-molecular spacing between molecules of a protein found in said liquid; and determining from said simulation an effective frequency to increase said inter-molecular spacing; and wherein said step of exposing the liquid within said microwave resonance cavity to a microwave electromagnetic field employs a microwave electromagnetic field having a frequency determined to be effective in said simulation.
In this way, the microwave treatment can be tailored towards the resonant frequencies that are found, in the simulation, to have a significant effect on inter-molecular distances, and a consequent reduction in agglomeration for a window of time following the treatment.
Preferably, said step of determining an effective frequency identifies a frequency or range of frequencies for which said inter-molecular spacing is increased beyond a minimum level relative to a zero-field equilibrium spacing for said protein.
More preferably, said identified frequency or range of frequencies maximises said inter-molecular spacing.
There is also provided an assembly of an industrial processing plant comprising:
NEMD simulations were run for four β-lactoglobulin proteins in a large simulation box of water on all-atom basis, using the CHARMM and SPC/E potential models [3], under zero-field conditions and also in 2.45 and 100 GHz microwave fields. The root-mean square (r.m.s.) microwave field intensity range was 0.005 to 0.05 V/A; the former level is around the dielectric-saturation threshold of liquid water and aqueous phases, whilst the latter abuts a linear-to-non-linear field-response transition [1]. For each field condition, we performed two independent simulations with Maxwell-Boltzmann velocity randomisation, so as to average over each for increased statistical certainty (and observe reproducibility).
Shown in
Upon microwave field removal, the spacings reverted back to the zero-field level, with no substantial change in protein geometry and configurations. Naturally, this reversibility upon field removal is a very important desideratum for industrial anti-agglomeration and subsequent post-processing retaining of desired physico-chemical and rheological/taste properties.
It can be seen form
Following on from the simulations of
The proteins simulated in
A larger-system NEMD simulation of eight EG2-hFc proteins, i.e. at double the concentration (akin to 4 and 10%-fat milk for DLS) show that there is a more pronounced disaggregating effect than for the lower-concentration case, which was also observed for milk.
Further simulations of four EG2-hFc proteins at 0.5, 1, 2.45, 10, 20, 50 & 100 GHz confirmed that 1 GHz is the most effective deagglomerative frequency in this case (see Table 2).
NEMD simulations demonstrate the proof of concept and the theoretical basis for the underlying phenomenon. In such simulations a pulse of tens of nanoseconds shows a tangible effect in inter-molecular distances, when very high field strengths are applied for those very short pulses. However, for practical applications, lower field strengths are used for longer periods.
Further simulations were performed on different proteins to identify the frequency at which the maximum anti-agglomeration effect was observed, the results being as follows:
The identified frequencies are not guaranteed to provide the absolute maximum of effect; rather they are the optimum frequencies from a number of candidate frequencies for which simulations were run for each protein. Nevertheless, by determining the frequency providing maximum effect from a range of candidate frequencies, the microwave treatment process can be significantly improved by being tailored to the species of interest in the liquid.
In the above NEMD, the general trend is that larger proteins typically have lower resonant frequencies, owing to their larger translational and rotational inertia. However, other factors, such as backbone stability and hydrogen-bond lifetimes, particular to secondary structure per se, were also seen to have second-order importance.
The viscosity of whole-fat retail milk (approximately 4% fat), was measured at about 1.85 cP at 20° C., unaffected by exposure to e/m fields, using a Malvern Zetasizer Ultra. This apparatus uses Dynamic Light Scattering (DLS) to measure viscosity as described in refs. 13-17. In brief, one extracts the particle (protein) mean-squared displacement from DLS within Stokes-Einstein continuum theory, describing also the auto-correlation function (ACF) of the time-domain light-scattering signal. From frequency-domain (Fourier) analysis, one then extracts from the ACF by fitting to their real-/imaginary-space analogues the micro-rheological Stokes-Einstein properties, including the frequency-dependent (complex) viscosity—and, thence, the viscosity per se.
50 ml virgin milk samples were placed in a small-scale microwave field exposure cell at 2.45 GHz and E=2 kV/m for 10 mins in a steady state. There was no physical contact with the electrodes generating the microwave field. The samples were then placed immediately in the DLS. Within about one minute of DLS probing, the viscosity was measured at 1.27+/−0.19 cP, i.e. a reduction in viscosity of close to one-third.
Measuring every 5 minutes thereafter DLS, it took of the order of 15-20 mins to reach 90-95% of the original viscosity i.e. 1.85 cP.
Given the importance of undesired agglomeration of casein and β-lactoglobulin proteins, which cause biofilm clogging of heat exchangers and which are a primary culprit of problems in the production, respectively, of powdered and liquid milk—this reversible control over, and manipulation of, protein-solution viscosity is very important. Simply put, a one-third reduction in viscosity (suggested by preliminary experiments) would increase flow by a third, and lead to operational expenditure falling by close to that fraction.
The same experiment was repeated using a pulsed microwave field, for 10 minutes, with a duty cycle (on time: off time) of 1:200. The viscosity-reduction performance was again observed, with an efficacy of approximately 70% compared to the reduction in viscosity found in the steady state (reduction to approximately 1.45 cP). Thus, with only 0.5% of the energy input to the system, it was still possible to achieve an impressive reduction in the viscosity of the liquid, with similar duration of effect, i.e. 15-20 minutes), resulting in a very significant energy saving and reduction in the energy absorbed by the liquid.
A natural question arises as to whether this technology is effective for more concentrated dairy-protein solutions (with intermediate, pre-Pasteurisation milk-process fluids at about 40-45% solids). Bearing this in mind, we also carried out two experiments using ‘heavier’ (approximately 10%-solids) milk obtained from gentle heating.
Using the same set-up as Example 1, these experiments showed about 36% and 37% reductions respectively in viscosity by the same EMF-exposure strategy, with reversibility time lasting about 30 minutes for 95% re-establishment of the pre-exposure viscosity. Again when repeated with a 1:200 pulse train the reduction was observed at about 70% of the steady-state case.
Therefore, both the extent of the EMF-based viscosity-reduction effect and reversibility timescale have been found to be increased for more concentrated solutions.
Given that process-fluid residence times in dairy-processing heat exchangers and membranes are usually less than 5-10 minutes, this method will be effective for temporary protein-coagulation and viscosity suppression and control.
It is important to note that there is already a degree of natural (thermodynamically metastable) colloidal suspension in milk, reflecting partially-agglomerated β-lactoglobulin clusters. The fact that partial, time-reversible EMF-mediated disaggregation was observed via the direct proxy measure of viscosity signals prima fade demonstrates that this technology is not only effective in inhibiting protein agglomeration from the outset, but also in reducing this after coagulation or agglomeration has occurred naturally. This opens up designing and implementing industrial, rheology-monitoring strategies in-line for real-time EMF-based process control.
Experimentally, solvated BSA and thrombin (at around 1.4-1.7 wt %) underwent DLS viscosity-determination after 2.45 GHz exposure, in an identical manner to Examples 1 and 2, and there were averaged 23 and 18% reductions in viscosity—still appreciable given the slightly lower concentration than in full-fat dairy milk tested in the previous examples.
Accordingly, it can be seen that the methods disclosed herein are applicable beyond the dairy industry and into all areas where protein-containing liquids are industrially processed, In particular for processing biological liquids and in the biopharma industry.
In the plate heat exchanger 16, the milk passes successively though a number of heat exchanger stages. It first enters a pre-heating and pre-cooling exchanger 22, in which heat is transferred into the milk in section 22a from the milk in section 22b. The pre-heated milk from section 22a enters a clarification unit 24 to remove particles from the milk. At this point the milk could also be centrifuged to separate different fractions and alter the fat content.
The milk then enters the heating stage 26, which is heated by hot water (or steam) circulated from a hot water supply 28. The heated milk passes through a holding coil 30 to ensure sufficient residence time at the required temperature to achieve pasteurisation, before re-entering stage 26 where in section 26b it continues to be heated by the hot water supply 28.
From section 26b the milk passes into section 22b, where it is cooled by transfer of heat to section 22a as previously noted. Then it enters the main cooling stage 32 where, in section 32b, it is cooled by heat transfer with refrigerated water circulated from a cold water supply 34. The cooled and pasteurised milk is then transferred into the pasteurised milk storage 18.
Referring to
Microwave-frequency alternative current is carried in a conductor or waveguide 36 which is sheathed with an insulating sheath 38, and takes the form of an elongated coil 40 within the walls of the pipeline 20 (shown in partial cut-away). The coil's entry and exit from the pipeline are sealed by a pair of respective gaskets 42, 44, to prevent leakage or contamination of the milk.
A microwave frequency electromagnetic field is generated within the pipeline by this coil 40, and the milk passing through the section of pipeline is thereby exposed to a field of a desired field strength for a desired period of time (determined by the flow rate and residence time within the cavity formed by this section of pipeline 20).
The microwave-frequency current in the conductor or waveguide 36 is generated by a microwave RF signal generator and power supply 50 under the control of a computer or programmable logic controller denoted as PC/PLC 52. This controller can determine the frequency, intensity, duty cycle, and other characteristics of the microwave field to which the milk is exposed, and is typically integrated with the overall plant control, e.g. to ensure that these characteristics are tailored to the composition of the milk and the flow rate.
As described previously and as demonstrated by the experimental data herein, this exposure inhibits protein agglomeration in the milk for an extended period of time, which is sufficient for the milk to pass fully through the plate heat exchanger 16 of
It will be appreciated that while the invention has been illustrated in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020202.4 | Dec 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/086517 | 12/17/2021 | WO |
