CELLULOSE

Background of the Invention

Field of the Invention

[0001] The present invention relates to the preparation of stable oil-in-water emulsions from mechanically fibrillated MCC, to be used as an emulsion stabilizer in products of the food industry.

Description of Related Art

[0002] Lignocellulosic biomass is the most abundant natural resource on earth, and it contains components, such as cellulose, hemicelluloses, and lignin, that can be beneficial for different technological applications also in the food sector, specifically as hydrocolloids.

[0003] Several cellulose based products have already been approved for use as food additives in different food related functions, such as stabilization. However, often some form of modification is required to achieve desirable techno-functional properties. For example, one of the simplest modifications that can be done to make cellulose more suitable for food applications is to remove its amorphous parts via hydrolysis, typically carried out using concentrated mineral acid, leaving only the crystalline fraction of cellulose. This yields a product called microcrystalline cellulose (MCC), which is a well- established food ingredient. This degradation also results in the removal of hemicellulose.

[0004] The preparation of MCC from cellulose is typically a chemical process, wherein the fibrous structure is degraded, for example with the help of a mineral acid, whereby hemicellulose is removed from the fiber at the same time. In the past, the preparation of MCC has been such an expensive process that the utilization of MCC has been largely avoided, but lately new production processes have been developed that have made MCC a more convenient raw material. [0005] The cellulose crystals of microscale that are obtained in the MCC production are natural stabilizers because of their amphiphilic properties, the surface of the crystal being hydrophobic, while the free hydroxyl groups on the crystal surface confer hydrophilic characteristics. Because MCC is non-toxic, sustainable, biodegradable, renewable, and has excellent physicochemical properties, it is a good candidate to be considered for interfacial stabilization, such as in water/oil systems, even in food products.

[0006] One type of MCC that has stabilizing properties is colloidal MCC (cMCC).

In food applications, the used MCC is usually colloidal MCC. Manufacturing cMCC takes place via mechanical disintegration, where strong shear forces break up the cellulose aggregates of the MCC. After mechanical disintegration, the microcrystals are coprocessed with an amphiphilic dispersant, e.g. sodium carboxymethyl cellulose (CMC), thus forming colloidal MCC (cMCC). Thus, the production of cMCC always requires the use of a synthetic or chemically modified additive. The dispersant acts by preventing reaggregation, thus further facilitating the formation of a stable dispersion. However, the use of such synthetic dispersants in food applications is always a disadvantage.

[0007] In oil/water systems, MCC functions by thickening the continuous phase between oil droplets and by forming a three-dimensional network around them, thus working as a mechanical barrier to oil droplet coalescence, while the dispersant (e.g. CMC) acts as the dispersing agent and a protective colloid for the MCC.

[0008] WO202 1/053268 describes such a use of undried MCC in preparing a homogeneous gel-like fiber mixture in a 2-step mechanical process by using MCC mixed with water, oil or an oil/water mixture. However, the procedure described in this publication is not suitable for preparing liquid emulsions.

[0009] Alfassi et al., in turn, describes a process, wherein dried MCC is dissolved in sodium hydroxide (7 %wt) and coagulated to achieve a cellulose hydrogel which is further used as an emulsifier. This method harnesses the amphiphilic properties of cellulose through cellulose regeneration, making it possible for it to entrap hydrophobic molecules such as oil. However, the procedure described involves the pretreatment of MCC via cellulose regeneration with sodium hydroxide, which is demanding through use of chemicals, water, and energy. [0010] Thus, there is still a need for further development of more economical and sustainable stabilizing systems, particularly for use in food products of a wider variety, including also drinks.

Summary of the Invention

[0011] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

[0012] According to a first aspect of the present invention, there is provided a method for producing a stable oil-in-water emulsion.

[0013] According to a second aspect of the present invention, there is provided a method and product that utilize micro crystalline cellulose (MCC) as a stabilizer.

[0014] According to a further aspect of the invention, there is provided a low viscosity oil-in-water emulsion containing mechanically fibrillated microcrystalline cellulose (fMCC) to prevent coalescence of oil droplets.

[0015] Stabilization is the key concern when preparing oil-in-water dispersions and emulsions. Conventional cellulose-based stabilizers are typically required in high amounts, resulting in mainly gel-like dispersions. Liquid emulsions require stabilizers that are effective in smaller amounts, or smaller consistencies, or that have been combined with synthetic emulsifiers.

[0016] The inventors studying stabilizing systems based on cellulose crystals have now surprisingly found that stable liquid emulsions can be obtained, without the use of synthetic emulsifiers, by using mechanically treated MCC. In other words, they found that stable dispersions require the mechanical fibrillation of the microcrystalline cellulose, thus obtaining fMCC. The use of synthetic or chemically treated cMCC is, however, avoided. [0017] The used fMCC is produced by using chemically produced microcrystalline cellulose (MCC) as raw material, from which hemicellulose has been substantially removed. The fMCC gel is used to stabilize oil-in-water dispersions to create low viscosity emulsions suitable for low viscosity and low- fat food emulsion applications.

[0018] The stabilization is achieved by a high pressure homogenization of an aqueous gel-like mixture of the fMCC, together with vegetable or animal oil. The stability of the system is based on a physical stabilization mechanism of the fibrillated MCC matrix, where the individual fibrils trap the small oil droplets within the cellulose matrix, forming a mechanical barrier to oil droplet coalescence.

[0019] Several advantages are achieved using the present invention. Among others, the invention makes it possible to use MCC for efficient emulsion stabilization, without using any colloidal additive. The product of the invention is thus entirely non-toxic, and meets the requirements of the food and pharmaceutical industries. The oil is selected from edible vegetable and animal oils, while the MCC is produced in a manner providing microscale particles, excluding nano-scale particles, the health effects of which are still unknown.

[0020] Since the fMCC can be used in lower consistencies, it has now become possible to obtain low-viscosity, or liquid, emulsions using entirely organic stabilizing systems, based on cellulose stabilizers. For example, fMCC emulsions with cellulose to oil ratios of 1 : 10 or below can form emulsions that are physically stable up to 40 days.

[0021] Achieving stable liquid emulsions has, in turn, made it possible to use these emulsion stabilizers in a wider range of products, including in addition to the thick gel-like end-products of the known high-consistency dispersions, also liquid products, such as beverages.

Brief Description of the Drawings

[0022] FIGURE 1 is a drawing illustrating the function of a single-inlet microfluidizer that is suitable for use in the high-pressure homogenization step of the present invention. [0023] FIGURE 2 is a graphical illustration of the change in Turbiscan Stability Index (TSI) values over time for oil-in-water emulsions prepared using different fMCC concentrations (Figs. 2 A and 2B) and different oil concentrations.

[0024] FIGURE 3 shows microscopic images illustrating the change in droplet size over time for oil-in-water emulsions prepared using different fMCC concentrations (Figs. 3A and 3B) and different oil concentrations.

[0025] FIGURE 4 shows the obtained Cryo-SEM images of the samples, Fig. 4A and 4B for the 1.0% fMCC containing 0.5% oil, and Fig. 4C and 4D for the 1.0% MCC containing 5.0% oil.

[0026] FIGURE 5 is a graphical illustration of the oil droplet size of the present emulsions, in Fig. 5A for 0.5% fMCC emulsions, and in Fig. 5B for 1.0% fMCC emulsions.

[0027] FIGURE 6 is a graphical illustration of the change in diameters for the droplets in oil-in-water emulsions prepared using 0.5% fMCC, and different oil concentrations, with Fig 6A showing the results for the surface-weighted diameter D[3,2] and Fig 6B showing the results for the volume-weighted diameter D[4,3],

[0028] FIGURE 7 is a graphical illustration of the change in diameters for the droplets in oil-in-water emulsions prepared using 1.0% fMCC, and different oil concentrations, with Fig 7 A showing the results for the surface-weighted diameter D[3,2] and Fig 7B showing the results for the volume-weighted diameter D[4,3],

Embodiments of the Invention

[0029] Definitions

In the present context, the term “MCC” or “microcrystalline cellulose” comprises particulate cellulose of micro-scale crystals, produced from cellulose by chemical degradation, simultaneously causing hemicellulose removal. It can be either unbleached or bleached, whereby it either contains lignin or not.

The term “cMCC” or “colloidal microcrystalline cellulose” encompasses MCC that has been further subjected to strong shear forces that break up the cellulose aggregates of the MCC, followed by co-processing with an amphiphilic dispersant, e.g. sodium carboxymethyl cellulose (CMC). Due to its manufacturing technique, via mechanical disintegration, cMCC essentially lacks the impurities, such as sulphur, that are present in cellulose materials that are produced by harsher acid treatment, such as CNC.

The term “fMCC” or “mechanically fibrillated microcrystalline cellulose” encompasses MCC that has been subjected to a mechanical disintegration.

The term “homogenization” is obviously intended to mean an even distribution of the particles or droplets in a mixture but is herein used in a slightly more narrow context than what is common, as it is used to describe a procedure taking place in a high-pressure homogenizer, such as a microfluidizer, thus being based on mechanical action combined with high pressure, typically > 100 bar, causing further deterioration of particle structures, and a more even distribution of the particles in the product mixture or emulsion. Further, based on the known meaning of the term “microfluidization” the high-pressure homogenization is intended to describe a procedure, where fine droplets are created in order to form emulsions.

[0030] The present invention relates to a method for producing a stable oil-in-water emulsion that is stabilized by microcrystalline cellulose (MCC). In this method, a mechanically fibrillated microcrystalline cellulose (fMCC) is provided as an aqueous gel, at a pH of 4-7, and mixed with a vegetable or animal oil into a cellulose-to-oil ratio of 1 : 1- 1 :20, and finally the mixture is homogenized at high pressure.

[0031] The mixing step is typically carried out using a common mixing device, without added pressure, e.g. using a stirrer, a blender, a mixer or a low-intensity homogenizer. [0032] The mixing step typically requires <15 min, preferably 5-10 min, and the homogenization <10 min, preferably about 5 min.

[0033] The cellulose-to-oil ratio is preferably 1 :1-1 :10. A suitable cellulose-to-oil ratio can be achieved e.g. by providing the aqueous gel at a cellulose concentration of 0.5- 1 ,0w-%, and then mixing the gel with 0.5-10w-% of the oil.

[0034] The pH of the fMCC aqueous gel typically does not require separate adjustment. Instead, the desired pH is achieved already during the preparation of the raw material, i.e. the preparation of the microcrystalline cellulose, which is followed by a washing step, and no neutralizing chemicals remain in the raw materials at the mixing step of the present invention. Thus, no neutralizing chemicals, such as alkalis, are used when mixing the fMCC gel with the oil, and the obtained mixture consists of said combination of fMCC aqueous gel and oil. A preferred pH of the fMCC aqueous gel is 7.

[0035] The temperature is typically maintained at, or close to, room temperature, during the entire method.

[0036] If cellulose fiber products are not bleached, they contain lignin and hemicellulose in addition to cellulose. When bleaching, lignin is removed, leaving only fiber containing hemicellulose and cellulose. Since the MCC used in the present invention has been prepared via steps that result in removal of the hemicellulose, it is thus either unbleached MCC containing both cellulose and lignin, or bleached MCC containing cellulose without lignin.

[0037] The used microcrystalline cellulose is specifically selected to be the mechanically fibrillated microcrystalline cellulose (fMCC), since this fMCC is formed of micro-scale crystals, by excluding nano-scale particles, and without requiring any colloidal additive. Thus, the fMCC is non-toxic. The individual fibrils of the fMCC facilitates the desired stabilization effect, as the fibrils form a mechanical barrier to oil droplet coalescence.

[0038] The oil used to prepare the oil-in-water mixtures is vegetable or animal oil. Preferably, the oil is selected from liquid oils or oils that become liquid when the temperature rises to temperatures of >20°C, more preferably from edible oils such as linseed oil, mustard oil, almond oil, soybean oil, hemp oil, palm oil, peanut oil, castor oil, coconut oil or com oil, typically from edible vegetable oils such as rapeseed oil, canola oil, sunflower oil, olive oil, or cocoa butter.

[0039] In an embodiment of the invention, a pre-homogenization step is carried out after mixing the aqueous gel with the oil, to convert the oil-in-water mixture into a coarse emulsion before the high-pressure homogenizing step is carried out. Typically, such a pre- homogenization step is carried out using a dispersing homogenizer, preferably a rotor- driven homogenizer.

[0040] The following high-pressure homogenization of the oil-in-water mixture of the fMCC is typically carried out at a pressure of >100bar, preferably >300bar.

[0041] One suitable alternative for the high-pressure homogenization is to feed the mixture through a microfluidizer, which creates small-droplet emulsions from oil- water mixtures by utilizing a sufficiently high pressure, combined with a suitable number of passes. Also the concentration of the agents in the mixture being fed through the microfluidizer will have an impact on the obtained droplet size.

[0042] In such a microfluidizer, it is preferred to use a pressure of 500 to 1200 bar, more preferably 700 to 900 bar, and most suitably about 800 bar. The mixture can be processed continuously or in batches, with 4 - 7 passes, preferably 5 passes.

[0043] For a microfluidizing step, it is preferred to use a single-inlet microfluidizer (see Fig. 1), where the oil-in-water mixture is prepared before the micro fluidizing step, and the mixture is fed through a single inlet into the microfluidizer, preferably as a prehomogenized coarse mixture. In such a single-inlet microfluidizer, the microfluidizing effect (i.e. the formation of small droplets) is achieved by colliding the coarse droplets of the mixture fed into the microfluidizer at high pressure with the surfaces in the microfluidizer, thus causing the formation of smaller droplets. Due to the presence of fMCC, a stabilizing network is formed on the surfaces of the oil droplets, avoiding coagulation of the formed small droplets and providing the stable emulsion of the invention. [0044] As stated above, the present invention allows the use of lower MCC consistencies as compared to the consistencies of known MCC stabilizer systems, while the use of synthetic additives can be avoided. Thus, the consistency of the oil-in-water mixture of the organically produced fMCC, when feeding it through the high-pressure homogenizing step is 0.5-3.0% by weight, preferably 0.5- 1.5% by weight, with respect to the cellulose fiber.

[0045] The particle size for fMCC emulsions is 20-50 pm, preferably 30-40 pm, for cellulose-oil aggregates, and 0.5-5 pm, preferably 1-2 pm for individual oil-droplets.

[0046] The present invention also relates to the homogeneous low- viscosity product that has been prepared from MCC using the above-described method, as well as to the use of the homogeneous oil-in-water fiber emulsion thus produced in fat- and oil-based foods. Particularly, these emulsions are suitable for use in mayonnaises, chocolates, mustards, ketchups, yoghurts, in milk products, salad dressings or yoghurts, or in juices or sports drinks.

[0047] It is to be understood that the embodiments of the invention disclosed are not limited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0048] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term such as, for example, about or substantially, the exact numerical value is also disclosed.

[0049] As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. In addition, various embodiments and examples of the present invention may be referred to herein along with alternatives for the various components thereof It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another but are to be considered as separate and autonomous representations of the present invention.

[0050] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In this description, numerous specific details are provided to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details.

[0051] While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

[0052] The following non-limiting examples are intended merely to illustrate the advantages obtained with the embodiments of the present invention.

EXAMPLES

Example 1 - Emulsion preparation

[0053] Emulsions were prepared by diluting fMCC in MilliQ water to 0.1%, 0.5% and 1.0% concentration of cellulose, and by adding 0.5-10.0% rapeseed oil. The mixture was then coarsely homogenized using Ultra-Turrax (UT) at 22000 rpm for 2 min. The coarse emulsion was then immediately homogenized with a micro fluidizer HOY (Microfluidics, Westwood, MA, USA) at 80 MPa for 5 passes in batches to produce a fine emulsion. [0054] The stability of the prepared emulsions was observed, and the results shown in the below Table 1.

Table 1. Visual observations on fMCC emulsions

[0055] Thus, all 0.1% emulsions separated immediately after micro fluidizing, while the emulsions containing higher cellulose concentrations formed visually stable and homogeneous emulsions. However, 0.5% fMCC emulsions with the highest used oil concentrations showed small droplets forming on the surface of the emulsion, indicating that not all oil was fully emulsified by the homogenization process. Other samples did not show any sign of leakage or oil droplets on the surface, confirming that the critical surfactant-to-oil ratio (SOR) for fMCC is 0.1.

[0056] Control samples were prepared using a similar procedure with 0.5% and 1.0% cellulose, without any oil. Each sample was prepared in triplicates. The samples were stored in room temperature.

Example 2 - Analyzing the physical stability of the prepared emulsions

[0057] After emulsion production, it was observed that no oil layer or other phase separation was forming during storage, except a serum layer formed at the bottom of the samples. To study the stability of the fMCC emulsions, Turbiscan measurements were carried out, as well as particle size measurements and microscopic imaging.

Turbiscan measurements:

[0058] Emulsions, as well as micro fluidized suspensions of 0.5% and 1.0% cellulose, with the 0.5-10.0% concentrations of rapeseed oil used also in Example 1, were monitored by Turbiscan Lab Expert analyser (Formulaction, Toulouse, France) during a storage time of 40 days. Approximately 20 mF of sample was poured into transparent glass vials and kept in room temperature undisturbed. For measurement, each sample was scanned vertically from bottom to top by a near-infrared light (X = 880 nm) source. Detectors measured synchronously the intensity of transmitted and backscattered light at 180 and 45, respectively. The Turbiscan software (v. 1.2) was used to calculate the Turbiscan stability index (TSI), which is the sum of all changes in the measuring cell, which are calculated from the change in backscattering values and sample height. The results are shown in Fig. 2A and 2B, with Fig. 2A showing the results for the 0.5% fMCC emulsions.

[0059] Increased TSI is known to indicate increased instability, and for these samples the obtained results show that stability had been achieved for both the 0.5% (Fig. 2A) and for the 1.0% (Fig. 2B) emulsions, which were both stable throughout the followup period (40 days). The 0.5% fMCC emulsions with <5.0% oil are relatively stable (TSI < 2 indicates “very stable”), while emulsions containing >5.0% oil destabilize after 7 days. As can be seen when comparing Fig. 2A to Fig. 2B, 1.0% fMCC emulsions are generally even more stable than 0.5% fMCC emulsions, due to the higher viscosity.

Microscopy:

[0060] To get a better understanding of droplet diameters and morphology, optical microscopy was carried out for the emulsions. The used optical microscope (AxioVision, Carl Zeiss, Microimaging GmbH, Jena, Germany) was connected with AxioCam MRm digital camera (Carl Zeiss, Microimaging GmbH, Jena, Germany), and was used to optically analyse the emulsion morphology. Prior to analysis the emulsions were diluted to 1 :10 ratio with MilliQ water. One mF of diluted emulsion was placed in an Eppendorf tube. Samples were vortexed for 15 s and then analysed by placing one drop of emulsion on a glass slide, covering it with a glass cover slip. Samples were analysed with lOOx objective lens, under brightfield light. AxioVision v.4.7.1.0 (Carl Zeiss, Inc., Germany) application software was used to acquire images.

[0061] Fig. 3 shows the Brightfield microscopy images, Fig. 3A for the 0.5% fMCC and Fig. 3B for the 1.0% fMCC. As observed from the images, the emulsion contains spherical oil droplets as well as larger cellulose particles. It can also be observed that the oil droplets are growing as oil concentration is increased.

[0062] To understand how the entrapment of oil droplets happens and how the cellulose network develops around the droplets, cryogenic-scanning electron microscopy (Cryo-SEM) was also performed on selected emulsions, i.e. on 1.0% fMCC emulsions with oil concentrations of 0.5% and 5.0%.

[0063] The emulsions were cryopreserved with a high-pressure freezer (Leica EM HPM100, Leica Microsystems GmbH, Wetzlar Germany) using a 2 x 100 pm planchette. The two planchettes were placed in a holder under liquid nitrogen and moved to a vitreous cryo-transfer shuttle (Leica EM VCT100, Leica Microsystems GmbH, Wetzlar, Germany). Emulsions were cracked and sublimated for 1 min at -90°C, after which they were sputter- coated with 6 nm thick carbon/platinum coating (Leica EM MED020, Leica Microsystems GmbH, Wetzlar, Germany). After coating, the emulsions were examined with a SEM (LEI Quanta 3D, Thermo Fisher Scientific, Massachusetts, USA) at an accelerating voltage of 2 kV.

[0064] Fig. 4 shows the obtained Cryo-SEM images of the samples, Fig. 4A and 4B for the 1.0% fMCC containing 0.5% oil, and Fig. 4C and 4D for the 1.0% MCC containing 5.0% oil.

[0065] The cryo-SEM images show the morphology of the formed cellulose network, also emphasizing the fact that fMCC produces a network on the oil droplets, instead of the conventional film obtained using cMCC. In images with lower amount of oil (Fig. 4A and 4B), the entangled crowded cellulose system can be observed, while the images with higher amount of oil (Fig. 4C and 4D) show how the cellulose fibrils cover a single oil droplet. The Cryo-SEM images confirm the hypothesis that the stabilization mechanism of fMCC is based on physical entrapment of the oil droplets by cellulose fibrils.

Particle size measurements:

[0066] The particle size distributions, as well as the surface- and volume- weighted diameter values D[3,2] and D[4,3], were analyzed for the 0.5% and 1.0% fMCC emulsions, with 0.5-10.0% rapeseed oil over a period of 4 weeks. [0067] Mastersizer 3000 static light scattering equipment with Hydro EV dispersion accessory (Malvern Instruments Ltd, Malvern, UK) was used to study the particle size distribution of the oil droplets in emulsions, after preparation and during storage. In addition to particle size distribution, surface-weighted diameter D[3,2] and volume- weighted diameter D[4,3] were measured. Mastersizer v.3.62 application software (Malvern Instruments Ltd.) was used to operate the equipment. Prior to analysis, each emulsion was carefully mixed by turning the sample bottle upside down 10 times. To avoid multiple scattering effects, the emulsions were diluted in MilliQ water directly into the dispersion accessory. The refractive indices of water and rapeseed oil were 1.33 and 1.47, respectively. Each sample was measured in triplicates.

[0068] The droplet size results are shown in Eig. 5A (for 0.5% fMCC emulsions) and Fig. 5B (for 1.0% fMCC emulsions). The diameter values are, in turn, shown in Figs 6 and 7, with Fig. 6 showing the results for the 0.5% fMCC and Fig. 7 showing the results for the 1.0% fMCC, while Figs. 6A and 7A show the results for the surface-weighted diameters D[3,2] and Figs. 6B and 7B show the results for the volume-weighted diameters D[4,3],

[0069] As expected, the results indicate that the particle size distribution increased slightly over time for all samples. Both emulsions, with different fMCC concentrations, show increase in their D[3,2] values and their D[4,3] values over time.

Industrial Applicability

[0070] The present method can be used to prepare cellulose-based emulsion stabilizing systems, e.g. for use in maintaining physical stability in low-viscosity oil-in- water emulsions. Due to the use of non-toxic materials, the product obtained in said method can be used even in edible products.

[0071] In particular, the present material is useful in preparing non-toxic, edible emulsion stabilizing systems for use in food products, such as mayonnaises, chocolates, mustards, ketchups, yoghurts, milk products, salad dressings, juices or sports drinks. Citation List Patent Literature

WO202 1/053268

Non-Patent Literature

Alfassi, G., et al., Journal of Chemical Technology and Biotechnology (2018), vol. 94, No. l, p. 178-184