WO2015042683A1

WO2015042683A1 - Biopolymer compositions comprising a plurality of treated single-celled microorganisms

Info

Publication number: WO2015042683A1
Application number: PCT/CA2013/000823
Authority: WO
Inventors: Firoozmand HASSAN; Derick ROUSSEAU
Original assignee: Hassan Firoozmand; Rousseau Derick
Priority date: 2013-09-26
Filing date: 2013-09-26
Publication date: 2015-04-02

Abstract

The present invention relates to a plurality of single-celled microorganisms comprising at least one substantially inactivated single-celled microorganism wherein the at least one substantially inactivated single-celled microorganism further comprises at least one altered surface property, the preparation of these microorganisms and their use in biopolymer compositions.

Description

TITLE OF THE INVENTION

Biopolymer Compositions Comprising a Plurality of Treated Single-Celled Microorganisms

FIELD OF THE INVENTION

This invention relates to biopolymer compositions modified by a plurality of treated single- celled microorganisms wherein at least one single-celled microorganism has been substantially inactivated and comprises at least one surface-altered property.

BACKGROUND OF THE INVENTION

A phase-separated solution of thermodynamic incompatible biopolymers is known as a water-in-water emulsion (Tolstoguzov, V. B. (1998). Functional properties of protein- polysaccharide mixtures. In S. E. Hill, D. A. Ledward, & J. R. Mitchell (Eds.), Functional properties of food macromolecules (2^nd ed) (pp. 252-277). Gaithersburg, MD: Aspen Publishers). The stability of such a system can be addressed by conventional emulsion theories (Foster, T. J., Underdown, J., Brown, C. R., Ferdinando, D. P., & Norton, I. T. (1997). Emulsion behavior of non-gelled biopolymer mixtures. In E. Dickinson & B. Bergenstahl (Eds.), Food colloids: Proteins, lipids and polysaccharides (pp. 346-355). Cambridge, UK: Royal Society of Chemistry, Woodhead Publishing Limited). The distinct characteristic of biopolymers emulsion is the extremely low value of its interfacial tension in comparison with that of conventional oil + water emulsions. In biopolymer (water-in-water) emulsions, the interfacial tension between the aqueous phases rich in the two different polymers is remarkably low (Wolf, B., Scirocco, R., Frith, W. J., & Norton, I. T. (2000). Shear-induced anisotropic microstructure in phase-separated biopolymer mixtures. Food Hydrocolloids, 14, 217-225; Scholten, E., Tuinier, R., Tromp, R. H., & Lekkerkerker, H. N.^' W. (2002). Interfacial tension of a decomposed biopolymer mixture. Langmuir, 18, 2234- 2238). Therefore, using molecules or particles as interfacial material to produce a water-in- water emulsion raises the question of whether such a system can be emulsified or not (Norton, I. T., Norton, A. B, Spyropoulos, F., Reverend, B. J. D. L., & Cox, P. (201 1). Rheological control and understanding necessary to formulate healthy everyday foods. In I. T. Norton, F. Spyropoulos, & P. Cox (Eds.), Practical food rheology (pp. 219-253). Chichester, UK: Wiley-Blackwell; Simon, K. A., Sejwal, P., Gerecht, R. B., & Luk, Y.-Y. (2007). Water-in-water emulsions stabilized by non-amphiphilic interactions: Polymer- dispersed lyotropic liquid crystals. Langmuir, 23, 1453-1458; Balakrishnan, G., Nicolai, T., Benyahia, L., & Durand, D. (2012). Particles trapped at the droplet interface in water-in- water emulsions. Langmuir, 28, 5921-5926). However, it has been shown that in a phase- separated water-in-water emulsion, adsorption of colloidal particles at the water-water interface clearly increases the stability of the emulsion (Poortinga, A. T. (2008). Microcapsules from self-assembled colloidal particles using aqueous phase-separated polymer solutions. Langmuir, 24, 1644-1647).

In a phase-separating system, the incompatible components try to minimize their contact to decrease an unfavorable interaction. When a two-phase system of fluids is near its critical point (to phase separate), introducing a third component into the system can minimise the contact between the two original phases, and if one of the original components completely wets the newly introduced third component, the phase separation condition is altered (Cahn, J. W. (1977). Critical-point wetting. Journal of Chemical Physics, 66, 3667-3672). As an example, Tanaka and co-workers (Tanaka, H., Lovinger, A. J., & Davis, D. D. (1994). Pattern evolution caused by dynamic coupling between wetting and phase-separation in binary-liquid mixture containing glass particles. Physical Review Letters, 72, 2581-2584) showed that the addition of glass particles as a third phase to a binary mixture of immiscible liquids affects the phase separation condition and the resulting morphology of the mixture. Tanaka et al. (1994) described this phenomenon as the spatial and shape-pinning effect of particles, which originates from the interplay between the dynamic wetting of the particles and the phase separation in the mixture.

If the added particles have greater attraction toward one of the phases, this phase preferentially wets and includes most of the particles within itself. A layer of wetting fluid surrounds the particles (Araki, T., & Tanaka, H. (2008). Dynamic depletion attraction between colloids suspended in a phase-separating binary liquid mixture. Journal of Physics- Condensed Matter, 20, 072101) and the attractive phase absorbs more particles as the phase separation proceeds (Tanaka, H., Lovinger, A. J., & Davis, D. D. (1994). Pattern evolution caused by dynamic coupling between wetting and phase-separation in binary-liquid mixture containing glass particles. Physical Review Letters, 72, 2581-2584; Karim, A., Douglas, J. F., Nisato, G., Liu, D. W., & Amis, E. J. (1999). Transient target patterns in phase separating filled polymer blends. Macromolecules, 32, 5917-5924; Lee, B. P., Douglas, J. F., & Glotzer, S. C. (1999). Filler-induced composition waves in phase-separating polymer blends. Physical Review E, 60, 5812-5822; Tanaka, H. (2001). Interplay between wetting and phase separation in binary fluid mixtures: Roles of hydrodynamics. Journal of Physics-Condensed Matter, 13, 4637-4674; Chung, H. J., Taubert, A., Deshmukh, R. D., & Composto, R. J. (2004). Mobile nanoparticles and their effect on phase separation dynamics in thin-film polymer blends. Europhysics Letters, 68, 219-225). The mobility of the particles in an immiscible two-phase liquid system induces a complex dynamic between phase separation and wetting, which can result in various morphologies ( Karim, A., Douglas, J. F., Nisato, G., Liu, D. W., & Amis, E. J. (1999). Transient target patterns in phase separating filled polymer blends. Macromolecules, 32, 5917-5924; Lee, B. P., Douglas, J. F., & Glotzer, S. C. (1999). Filler- induced composition waves in phase-separating polymer blends. Physical Review E, 60, 5812-5822; Tanaka, H. (2001). Interplay between wetting and phase separation in binary fluid mixtures: Roles of hydrodynamics. Journal of Physics-Condensed Matter, 13, 4637- 4674; Chung, H. J., Taubert, A., Deshmukh, R. D., & Composto, R. J. (2004). Mobile nanoparticles and their effect on phase separation dynamics in thin-film polymer blends. Europhysics Letters, 68, 219-225). As the phase separation proceeds, the liquid phase(s) promotes rearrangements of the particles within, but particles can sometimes restrain the phase separation by hindering the coalescence and coarsening of particle-filled domains (Van't Zand, D. D., Schofield, A. B., Thijssen, J. H. J, & Clegg, P. S. (201 1). Hindered coarsening of a phase-separating microemulsion due to dispersed colloidal particles. Langmuir, 27, 13436-13443).

In a phase-separated biopolymer solution, the assembly of colloidal particles at the liquid— liquid interface and at the consequent viscoelastic particle-laden interface has the potential to control and enable the development of a new type of texture for food manufacturing (Foster, T., & Wolf, B. (201 1). Hydrocolloid gums-Their role and interactions in foods. In I. T. Norton, F. Spyropoulos, & P. Cox (Eds.), Practical food rheology: An interpretive approach (pp. 61-84). Chichester, UK: Wiley-Blackwell). However, readily available food-grade colloidal particles are scarce. Production techniques for food-grade particles are restricted by legislative regulations and are limited to small/lab-scale applications. Thus, there has existed a long-felt need for a cost-effective and safe food-grade colloidal particle. One of the applications of biopolymer mixture is encapsulation (Benichou, A.; Aserin, A. & Garti, N. (2002). Protein-polysaccharide interactions for stabilization of food emulsions. Journal of Dispersion Science and Technology, 23, 93-123; Tolstoguzov, V. B. (2002). Thermodynamic aspects of biopolymer functionality in biological systems, foods, and beverages. Critical Reviews in Biotechnology, 22, 89-174; Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17, 25-39; Tolstoguzov, V. B. (2003). Some thermodynamic considerations in food formulation. Food Hydrocolloids, 17, 1 -23; McClements, D. J. (2006). Non-covalent interactions between proteins and polysaccharides. Biotechnology Advances, 24, 621-625; McClements, D. J.; Decker, E. A.; Park, Y. & Weiss, J. (2009). Structural design principles for delivery of bioactive components in nutraceuticals and functional foods. Critical Reviews in Food Science and Nutrition, 49, 577-606); a process in which an amount of substance is trapped within a shield formed by biopolymer(s). The formed shield isolates the substance from its surrounding (Zuidam, N. J. & Shimoni, E. (2010). Overview of microencapsulates for use in food products or processes and methods to make them. In: Zuidam, N. J. & Nedovic, V. (Eds.) Encapsulation technologies for active food ingredients and food processing. New York, USA, Springer, (pp. 3-29)) and acts as a protective barrier, preserves the entrapped substance against undesirable physiochemical changes. Encapsulation may be triggered spontaneously by thermodynamic incompatibility between biopolymers solutions and liquid-liquid phase separation. As the phase separation progresses, the solubility of each biopolymer is reduced and each biopolymer concentrates into a separate domain. These newly-formed domains can encompass and trap their embedded materials within and, therefore, effectively encapsulate their contained substances (Weiss, J.; Takhistov, P. & Mcclements, D. J. (2006). Functional materials in food nanotechnology. Journal of Food Science, 71, R107-R1 16; Tolstuguzov, V.B. & Rivier, V. (1997). Encapsulated Particles in Protein from a Polysaccharide-Containing Dispersion. European Patent Application, EP 0 797 925 B l . Nestle SA, Vevey, Switzerland). Moreover, deposition of the cells (particles) on the protein-polysaccharide interface can provide an extra protective shield around the formed domains and it can enhance the encapsulation process further. Accumulation of probiotic bacteria (e.g., Lactobacillus paracasei) on dextran- methylcellulose interface, (polysaccharide-polysaccharide interface) as a means of new encapsulation material has been reported earlier (Poortinga, A. T. (2008). Microcapsules from self-assembled colloidal particles using aqueous phase-separated polymer solutions. Langmuir, 24, 1644-1647).

Single cells performed as active filler particles when added into the gel. Incorporation of active particles in the gel matrix causes an increase in gel modulus (Richardson, R. K.; Robinson, G.; Ross-Murphy, S. B. & Todd, S. (1981). Mechanical spectroscopy of filled gelatin gels. Polymer Bulletin, 4, 541-546; Ring, S. & Stainsby, G. (1982). Filler reinforcement of gels. Progress in Food and Nutrition Science, 6, 323-329; Van Vliet, T. (1988). Rheological properties of filled gels - influence of filler matrix interaction. Colloid and Polymer Science, 266, 518-524). Based on the nonspecific interactions of single cells with the gel matrix and the nature of each group of added cells, we obtained different rheological responses with time/temperature treatments (T_h = 40 °C and ¾ = 30 sec and 10 min). In general, it has been reported that single cells of microalgae can act as active and inactive filler particles based on the interaction of microalgae and biopolymers (Batista, A. P.; Nunes, M. C; Fradinho, P.; Gouveia, L.; Sousa, I.; Raymundo, A. & Franco, J. M. (2012). Novel foods with microalgal ingredients - Effect of gel setting conditions on the linear viscoelasticity of Spirulina and Haematococcus gels. Journal of Food Engineering, 110, 182- 189; Batista, A. P.; Nunes, M. C; Raymundo, A.; Gouveia, L.; Sousa, I.; Cordobes, F.; Guerrero, A. & Franco, J. M. (201 1). Microalgae biomass interaction in biopolymer gelled systems. Food Hydrocolloids, 25, 817-825). Incorporation of an active filler reinforces mechanical strength of the gel (Yost, R. A. & insella, J. E. (1992). Microstructure of whey- protein isolate gels containing emulsified butterfat droplets. Journal of Food Science, 57, 892-897) and inactive fillers show little influence on gel rheology (Langley, K. R. & Green, M. L. (1989). Compression strength and fracture properties of model particulate food composites in relation to their microstructure and particle-matrix interaction. Journal of Texture Studies, 20, 191-207). SUMMARY OF THE INVENTION

Acronyms:

G' = shear storage modulus

G" = shear loss modulus G* = complex shear modulus

γ = strain

Th = heating temperature (°C)

¾ = holding time

As used herein, the term "biopolymer" refers to a polymer that is produced by a living organism.

As used herein, the term "altered surface property" refers to at least one single-celled microorganism with at least one altered cell surface property.

As used herein, the term "treated" refers to the altering, preferably substantially inactivating, of at least one characteristic of a single-celled microorganism using means such as, but not limited to, chemical, thermal, biochemical and radiation. The treatment comprises contacting the single-celled microorganism with at least one substance that results in substantial inactivation and preferably alteration of the surface of the microorganism, and/or an alcohol, or a combination thereof. In one aspect, the at least one substance is selected from a basic compound or a salt, or the like. The basic compound is preferably an alkali metal hydroxide, most preferably sodium hydroxide, or the like. The salt is preferably a sodium or potassium salt, most preferably sodium chloride. In another aspect, the at least one substance is an enzyme preferably a protease, or the like. In another aspect, the at least one substance is an acid preferably hydrochloric acid, or the like. The alcohol is preferably selected from the group consisting of methanol, ethanol, n-propanol and isopropanol, or the like, most preferably ethanol.

As used herein, the term "substantially inactivated" refers to a single-celled microorganism that is no longer viable.

As used herein, the term "soft material" refers to a polymer, a biopolymer or a mixture of polymers or biopolymers thereof

As used herein, the term "biopolymer solution" refers to a dispersion, suspension or solution of at least one biopolymer or mixture of biopolymers. In one aspect of the invention, there is provided a plurality of single-celled microorganisms comprising at least one substantially inactivated single-celled microorganism wherein said microorganism further comprises at least one altered surface property.

In another aspect of the invention, there is provided a process for treating at least one single- celled microorganism, preferably a plurality of single-celled microorganisms, resulting in at least one substantially inactivated single cell microorganism with at least one altered surface property, said process comprising:

a) Substantially inactivating said at least one single-celled microorganism, and b) Substantially altering said surface.

In another aspect of the invention, there is provided a use of a plurality of single-celled microorganisms comprising at least one substantially inactivated single cell microorganism with at least one altered surface property, in altering at least one property of a soft material. In one embodiment the soft material may be a biopolymer. In another embodiment, the biopolymer may be a protein or polysaccharide, such as gelatin, starch, or starch-derived material. In another aspect of the invention there is provided a soft material comprising a plurality of substantially inactivated single-celled microorganisms with at least one altered surface property. Preferably said microorganisms are present in said soft material between about 0.1% and about 30% by weight, preferably between about 0.5% and about 10% by weight and most preferably between about 1% and about 5% by weight.

In one embodiment, the single-celled microorganism is selected from the group consisting of fungi, algae and bacteria. The fungi are most preferably a yeast such as Saccharomyces cerevisiae. The algae are most preferably chlorella or spirulina. The bacteria are most preferably selected from lactic acid bacteria, preferably Lactobacillus bulgaricus and/or Streptococcus thermophilus.

In another embodiment, the at least one altered surface property of the single-celled microorganisms comprises denaturation of proteins on the cell wall, preferably selected from denaturing mannoprotein in a yeast cell wall {Saccharomyces cerevisiae) denaturing peptidoglycan in a spirulina cell wall, denaturing glucosamine in a chlorella cell wall and denaturing peptidoglycan in a Lactobacillus bulgaricus or Streptococcus thermophilus cell wall. In another embodiment, in the process for treating a plurality of single-celled microorganisms the substantially inactivating step and the substantially altering step may occur simultaneously or sequentially, preferably simultaneously. In another embodiment, the substantially inactivating step involves the use of at least one of a substance, heat, microwave, ultraviolet light or irradiation or a combination thereof. In yet another embodiment the substantially inactivating and/or substantially altering steps are carried out by contacting the single-celled microorganism with at least one substance selected from an enzyme, a basic substance, an acidic substance or an alcohol or a combination thereof, preferably at a predetermined temperature. Preferably, the single-celled microorganisms are substantially inactivated and substantially altered by this process. In another embodiment, the treated single-celled microorganisms are subjected to a washing step.

In another aspect of the invention there is provided a process for modifying a biopolymer solution using a plurality of single-celled microorganisms comprising the steps:

1) Providing at least one biopolymer or a mixture of biopolymers;

2) Optionally providing at least one sweetener;

3) Providing a plurality of treated single-celled microorganisms comprising at

least one substantially inactivated single cell microorganism with at least one altered surface property, and

4) Contacting said at least one biopolymer with said plurality of single-celled

microorganisms and optionally with said at least one sweetener.

In another embodiment, the biopolymer is in the form of a biopolymer solution which contains about 0.1 % to about 30% by weight of biopolymer, preferably about 1 % to about 20%) by weight of biopolymer and most preferably about 3% to about 15% by weight of biopolymer. Preferably the biopolymer is a protein, polysaccharide or a combination thereof, most preferably the biopolymer is gelatin, starch or a starch-based material. In other embodiments, the sweetener is present in an amount of about 0% to about 60% by weight, preferably about 50% by weight. In one embodiment, the sweetener may be crystalline, noncrystalline or glassy in form, in another embodiment, the sweetener may be in a solid or a liquid form. Preferably the sweetener is selected from saccharides, said saccharides are selected from monosaccharides, disaccharides or polysaccharides, derivatives and/or modified forms thereof. Preferably in another embodiment, said sweeteners are selected from combinations thereof. Most preferably the sweetener is sucrose or glucose syrup or combinations thereof.

In another embodiment, the biopolymer solution may further comprise at least one flavouring agent. In yet another embodiment, the biopolymer solution may further comprise at least one colouring agent. In another embodiment, the biopolymer solution may further comprise at least one acidulant. In another embodiment, the biopolymer solution may further comprise at least one salt. In another embodiment, the biopolymer solution may further comprise at least one antioxidant. In another embodiment, the biopolymer solution may further comprise at least one edible oil. In another embodiment, the biopolymer solution may further comprise at least one emulsifier. In another embodiment, the biopolymer solution may further comprise at least one glazing agent. In another embodiment, the biopolymer solution may further comprise at least one vitamin. In another embodiment, the biopolymer solution may further comprise at least one lipophilic agent. In another embodiment, the biopolymer solution may further comprise at least one mineral. Further and other ingredients known in the food industry to a person of ordinary skill in the art may be added to the biopolymer solution without compromising the present invention.

In another embodiment, substantially inactivating a plurality of single-celled microorganisms results in and alteration of the rheological property of the biopolymer solution. In yet another aspect, there is provided the use of said treated plurality of single-celled microorganisms of the present invention as a food-grade colloidal particle.

Other uses for said invention include but are not limited to, uses as filler particles, a texture modifier, a rheology modifier, a protein gel binder, a structure modifier, for example as precursors for developing microstructures, and as interfacial particles for encapsulation of small molecules, including but not limited to pharmaceuticals, nutraceuticals, agrochemicals, health supplements, personal health care products and cosmetics. In a preferred embodiment of the present invention there is provided a a plurality of treated single-celled microorganisms comprising at least one substantially inactivated single-celled microorganism wherein said microorganism further comprises at least one altered surface property. The single-celled microorganisms are most preferably selected from yeast, algae and bacteria. Most preferably the algae are chlorella and spirulina, the bacteria are selected from lactic acid bacteria, for example, Lactobacillus bulgaricus or Streptococcus thermophilus, and the yeast are selected from Saccharomyces cerevisiae

Preparation of the substantially inactivated single-celled microorganism preferably involves contacting the microorganism with at least one substance resulting in substantial inactivation and preferably alteration of the surface of the single-celled microorganism, selected from a basic compound, an acidic compound, a salt, and an alcohol at a predetermined temperature. The basic substance is preferably an alkali metal hydroxide, most preferably sodium hydroxide. The acidic compound is preferably hydrochloric acid, or the like. The salt is preferably a sodium or potassium salt, most preferably sodium chloride. In one aspect, the predetermined temperature at which the inactivation process may be carried out is preferably between about 70°C and about 1 10°C, more preferably between about 80°C and 100°C and most preferably about 95 °C. The duration of the inactivation process may be about 20 minutes with optional agitation every about 5 to about 7 minutes.

Preferably, when the substance is a basic compound, an acidic compound or a salt, the substance substantially inactivates and substantially alters the surface of the single-celled microorganism. In another aspect, when the substance is an alcohol selected from the group consisting of methanol, ethanol, n-propanol and isopropanol, most preferably ethanol, the predetermined temperature at which alteration of the surface is preferably between about 15°C and about 60°C, more preferably between about 20°C and 50°C and most preferably between about 25°C and 45°C. The duration of the alteration process may be between about 12 hours and six days, most preferably between about 16 hours and five days.

In another aspect, when the substance is selected from an enzyme, the treatment comprises: 1) optionally substantially inactivating the single-celled microorganism by means discussed herein; 2) contacting the optionally substantially inactivated single-celled microorganism with at least one enzyme for substantial alteration of the surface of the single-celled microorganism at a predetermined time and temperature from about 35°C to about 65°C resulting in substantial alteration; 3) substantially deactivating, preferably fully deactivating, the at least one enzyme, preferably at a deactivation temperature, more preferably at a deactivation temperature that is dependent on the selected enzyme.

The treated single-celled microorganisms are preferably washed, preferably with distilled water, to remove base, acid, salt, alcohol and/or enzyme from the treated single-celled microorganisms.

The biopolymer to be modified is preferably a polypeptide, a protein or polysaccharide, most preferably gelatin, starch or a starch-derived material. The formation of a solution of the biopolymer is carried out by dissolving the biopolymer in water at an elevated temperature with mixing. The preferred temperature is from about 65°C to about 100°C, preferably from about 80°C to about 100°C and most preferably about 90°C. Mixing may be accomplished by any means known to a person of ordinary skill in the art. In one embodiment, the biopolymer solution further comprises at least one sweetener, preferably a saccharide and most preferably sugar or a glucose syrup or a combination thereof. Further and other aspects of the present invention will become apparent to the person of skill in the art.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a confocal laser scanning microscopy (CLSM) of gel made of 6wt% gelatin and 6wt % maltodextrin subjected to T_h = 40 °C, t_h = 30sec.

Figure 2 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin subjected to T_h = 40 °C, t_h = lO min. Figure 3 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% Lactobacillus subjected to T_h = 40 °C, t_h = 30 sec. Figure 4 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% Lactobacillus subjected to T_h = 40 °C, ¾ = 10 min.

Figure 5 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% chlorella subjected to T_h = 40 °C, t_h = 30 sec.

Figure 6 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% chlorella subjected to T_h = 40 °C, t_h = 10 min. Figure 7 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% yeast subjected to T_h = 40 °C, t_h = 30 sec.

Figure 8 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% yeast subjected to T_h = 40 °C, ¾ = 10 min.

Figure 9 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% spirulina subjected to T_h = 40 °C, t_h = 30 sec.

Figure 10 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% spirulina subjected to Th = 40 °C, t_h = 10 min.

Figure 1 1 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing lwt% NaCl subjected to T_h = 40 °C, t_h = 30sec. Figure 12 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% chlorella and lwt% NaCl subjected to T_h = 40 °C, t_h = 30 sec.

Figure 13 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% chlorella and lwt% NaCl subjected to T_h = 40 °C, t_h = 30 sec.

Figure 14 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% spirulina and lwt% NaCl subjected to T_h = 40 °C, t_h = 30 sec. Figure 15 is a CLSM of gel made of 6wt% gelatin and 6wt% maltodextrin containing 2wt% spirulina and lwt% NaCl subjected to T_h = 40 °C, ¾ = 30 sec.

Figure 16 is a time-dependent change in storage modulus (G^of gel made with 6wt% gelatin and 6wt% maltodextrin subjected to T_h = 40 °C and ¾ = 30 sec: without added cell■; with 2 wt% Lactobacillus ·; with 2wt% chlorella,♦; with 2wt% yeast, A; with 2wt% spirulina, *. Loss modulus (G") is adjacent to X axis

Figure 17 is a time-dependent change in G' of gel made with 6wt% gelatin and 6wt% maltodextrin subjected to T_h = 40 °C and t_h = 10 min: without added cell■; with 2 wt% Lactobacillus ·; with 2wt% chlorella,♦; with 2wt% yeast, A; with 2wt% spirulina, *. G" is adjacent to X axis

Figure 18 is a time-dependent change in G' of gel made with 6wt% gelatin and 6wt% maltodextrin subjected to T_h = 40 °C and ¾ = 30 sec: without salt, T; with lwt% salt, *; with lwt% salt and 2wt% spirulina# ; with lwt% salt and 2wt% chlorella,♦.

Figure 19 is a strain-dependence complex modulus (G*) of gel made with 6wt% gelatin and 6wt% maltodextrin subjected to T = 40 °C and ¾ = 30 sec after 1 hour ageing at 25 °C: without added cell,□; with 2wt% Lactobacillus, O; with 2wt% chlorella, ; with 2wt% yeast, Δ; with 2wt% spirulina,

Figure 20 is a G' of gel made with 6wt% gelatin and 6wt% maltodextrin subjected to T_h = 40 °C for t_h = 30 sec (T) and T_h = 40 °C for t_h = 10 min (□) after 1 hour ageing at 25 °C: without added cell (control); 6wt% gelatin and 6wt% maltodextrin with 2wt% Lactobacillus (cont+lact); 6wt% gelatin and 6wt% maltodextrin and 2wt% chlorella (cont+chlo); 6wt% gelatin and 6wt% maltodextrin and 2wt% yeast (cont+yeas); 6wt% gelatin and 6wt% maltodextrin and 2wt% spirulina (cont+spir). Figure 21 is a I % | loss of G' for 1 -hour aged gels at 25 °C, 100 x[G' of (T_h = 40 °C for t_h = 10 min) - G' of (T_h = 40 °C for t_h = 30 sec)]/G' of (T_h = 40 °C for t_h = 30 sec), 6wt% gelatin and 6wt% maltodextrin without added cell (control); 6wt% gelatin and 6wt% maltodextrin with 2 wt% Lactobacillus (cont+lact); 6wt% gelatin and 6wt% maltodextrin and 2wt% chlorella (cont+chlo); 6wt% gelatin and 6wt% maltodextrin and 2wt% yeast (cont+yeas); 6wt% gelatin and 6wt% maltodextrin and 2wt% spirulina (cont+spir).

Figure 22 is a CLSM of a sample made of 3wt% gelatin and 10wt% maltodextrin containing 0.1 wt% spirulina and lwt% substantially inactivated yeast (by 1 M solution of NaOH) subjected to T_h = 40 °C, t_h = 30 sec.

Figure 23 is a CLSM (higher magnification) of a sample made of 3wt% gelatin and 10wt% maltodextrin containing 0.1 wt% spirulina and lwt% substantially inactivated yeast (by 1 M solution of NaOH) subjected to T_h = 40 °C, t_h = 30 sec.

Figure 24 is a CLSM of sample made of 3wt% gelatin and 10wt% maltodextrin containing 1 wt% olive oil and lwt% substantially inactivated yeast (by 1 M solution of NaOH) subjected to T_h = 40 °C, t_h = 30 sec.

Figure 25 is a CLSM (higher magnification) of sample made of 3wt% gelatin and 10wt% maltodextrin containing 1 wt% olive oil and lwt% substantially inactivated yeast (by 1 M solution of NaOH) subjected to T_h = 40 °C, t_h = 30 sec. Figure 26 is a time-dependent change in G' of gel made with 6wt% gelatin-only gels subjected to T_h = 40 °C and t_h = 30 sec: without added cell■; with 2wt% chlorella,♦; with 2 wt% Lactobacillus ·; with 2wt% yeast (treated with NaOH), A; with 2wt% spirulina, *. G" is adjacent to X axis. Figure 27 is a time-dependent change in G' of gel made with 5wt% gelatin and 3 wt% inulin gels subjected to Th = 40 °C and t_h = 30 sec: without added cell■; with 2 wt% Streptococcus thermophilus ·. G" is adjacent to X axis.

Figure 28 is a time-dependent change in G' of gel made with 6wt% gelatin and 6 wt% starch and 20wt% sugar and 30 wt% glucose syrup subjected to T_h = 40 °C and t_h = 30 sec: without added cell ■; with 2 wt% yeast substantially inactivated/modified with enzymatically treatment ·. G" is adjacent to X axis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

With reference to Figure 1, confocal laser scanning microscopy (CLSM) shows a gelatin- maltodextrin biopolymer sample (without cells) subjected to T_h = 40 °C for ¾ = 30 sec. Its microstructure resembles a typical water-in-water emulsion, where the gelatin-rich phase 10 forms a continuous phase containing the droplet-like maltodextrin-rich phase 12. The configuration of the microstructure clearly indicates that nucleation and growth served as the phase separation pathway.

With reference to Figure 2, CLSM shows a gelatin-maltodextrin biopolymer sample (without cells) held at a longer holding time of t_h = 10 min at T_h = 40 °C. This holding time intensified the extent of phase separation and coarsened the sample microstructure. Maltodextrin-rich droplets 20 merged to form larger droplets in the microstructure. Secondary phase separation also occurred in the system, with gelatin droplets appearing inside the larger maltodextrin- rich droplets.

With reference to Figure 3, CLSM shows a gelatin-maltodextrin biopolymer with substantially deactivated Lactobacillus (2wt%) subjected to T = 40 °C for t_h = 30 sec. Cells 30 reside on the interface and associate with the gelatin phase 32, and the gelatin forms continuous structures seemingly out of equilibrium.

With reference to Figure 4, CLSM shows a gelatin-maltodextrin biopolymer with substantially deactivated Lactobacillus (2wt%) held for a longer holding time of ¾ = 10 min at T_h = 40 °C. The phase separation proceeds further when the sample is kept at 40 °C for 10 minutes, with the microstructure reaching an apparent degree of equilibrium. The coarsened, cell-coated gelatin droplets 40 in the sample merged into a continuous gelatin phase, and some of them formed larger droplets with extended features. Maltodextrin-rich regions 42 appeared as void regions depleted of gelatin and cells. Gelatin-rich domains manifested a high accumulation of cells in the form of clusters, but some dark (maltodextrin-rich) patches also existed in cell clusters within these gelatin-rich domains.

With reference to Figure 5, CLSM shows a gelatin-maltodextrin biopolymer with substantially inactivated chlorella (2wt%) subjected to T_h = 40 °C for t_h = 30 sec. In this sample, cells were associated with the gelatin phase 50 and built up a continuous phase. Maltodextrin forms included phases in the shape of individual droplets 52 (dark circles). This microstructure indicates that phase separation proceeded through nucleation and growth. Interestingly, cells were not associated with the interface, and maltodextrin regions at the interface resemble void regions with blunt edges.

With reference to Figure 6, CLSM shows a gelatin-maltodextrin biopolymer with substantially inactivated chlorella held at a longer holding time of t_h = 10 min at T_h = 40 °C. Over this holding time, the sample coarsened and phase separation proceeded further. Scattered maltodextrin-rich regions 60, in the form of crescents and incomplete circles, appeared in the microstructure. These features partially resembled open-cell foam, spongelike microstructures, exhibiting inter-connected gelatin-rich stretched domains 62 filled with small maltodextrin-rich (excluded phase) droplets. These stretched domains intersect in the nodes of the built scaffolding, which holds the larger blobs of maltodextrin-rich phase inside the cells.

With reference to Figure 7, CLSM shows a gelatin-maltodextrin biopolymer with substantially inactivated yeast (2wt%) subjected to T_h = 40 °C for t_h = 30 sec. The microstructure displays bicontinuous features, with cells associated mostly with the gelatin phase 70. The lack of any round droplets in the microstructure suggests that the pathway of phase separation was spinodal decomposition without secondary phase separation. However, the presence of cells in the system profoundly affects the microstructure: the interface boundaries are not smooth and do not possess a curvature similar to conventional spinodal- type microstructures. On the contrary, the interface boundaries are rather linear and jagged. The alignment of the cells on the interface influenced this interface morphology.

With reference to Figure 8, CLSM shows a gelatin-maltodextrin biopolymer with substantially inactivated yeast (2wt%) held at a longer holding time of th = 10 min at T_h = 40 °C. Here, the domains exhibit more expansion due to additional coarsening caused by this longer thermal treatment. The bicontinuous structure and interfacial features of the phase boundaries of the microstructure were preserved; i.e., they remained more or less linear and jagged. The domains 80 had irregular spacing distances, and some edges of the gelatin-rich domains 82 appeared narrow and starched. These are the regions where gelatin defused inside the domains, and cells on the interface formed ductile-type features. With reference to Figure 9, CLSM shows a gelatin-maltodextrin biopolymer with substantially inactivated spirulina (2wt%) subjected to T_h = 40 °C for ¾ = 30 sec. The microstructure of gelatin-maltodextrin gels containing spirulina 90 is fairly similar to that of gelatin-maltodextrin gels containing chlorella. However, the number and size of maltodextrin-rich domains 92 in the sample containing spirulina appeared lower than those of the sample containing chlorella.

With reference to Figure 10, CLSM shows a gelatin-maltodextrin biopolymer with substantially inactivated spirulina held at a longer holding time of ¾ = 10 min at T_h = 40 °C. The microstructure of gelatin-maltodextrin gels containing spirulina held for this length of time resembled their counterpart sample containing chlorella, except that the size and number of maltodextrin-rich domains - in the form of partial crescents and incomplete circles - were smaller than those in the sample containing chlorella. However, the size and number of maltodextrin-rich droplet-like features 100 (included phase) within the gelatin-rich domains 102 appear higher than those in the sample containing chlorella.

With reference to Figure 1 1, CLSM shows a gelatin-maltodextrin biopolymer sample (without cells) containing lwt% NaCl that was subjected to T_h = 40 °C for t_h = 30 sec. The addition of lwt% salt to gelatin-maltodextrin gels under this thermal treatment significantly enhanced the extent of phase separation. The sample microstructure was much more coarsened than that of samples without salt subjected to similar thermal treatment (Figure 1 above), including that of the sample with a longer holding time of ¾ = 10 min (Figure 2 above). Some maltodextrin-rich droplets 1 10 appeared larger than those in samples without salt (Figures land 2 above), and the presence of larger gelatin droplets within the maltodextrin-rich droplets 1 10 indicated that secondary phase separation had also progressed further.

With reference to Figures 12 and 13 (higher magnification), CLSM shows a gelatin- maltodextrin biopolymer with substantially inactivated chlorella (2wt%) with lwt% NaCl subjected to T_h = 40 °C for t_h = 30 sec. The addition of salt to the gelatin-maltodextrin gel containing chlorella caused drastic changes in the system microstructure, such as phase inversion and the adsorption of cells on the gelatin-maltodextrin interface. In the system containing salt, cells associated with the gelatin phase 120 were similar to those in the system without salt. However, with the addition of salt to the system, the continuous gelatin phase transformed into a discontinuous phase, forming individual gelatin droplets 120 scattered in a maltodextrin continuous phase 122. A very distinctive feature of the microstructure is the association of the cells with interfacial regions. As we can clearly see, cells 130, 132 are adsorbed at the interface. The microstructure well-represents a water-in-water emulsion, consisting of a continuous maltodextrin-rich phase 134 and flocculated droplets of a gelatin- rich phase 136. With reference to Figure 14 and 15 (higher magnification), CLSM shows a gelatin- maltodextrin biopolymer with substantially inactivated spirulina (2wt%) with lwt% NaCl subjected to T_h = 40 °C for ¾ = 30 sec. The addition of salt to a gelatin-maltodextrin gel containing spirulina caused immense changes in system microstructure. The phase separation pathway form nucleation and growth changed to spinodal decomposition, and consequently the microstructure transformed from droplets and a continuous matrix to a semi-bicontinuous microstructure. Predominantly, the gelatin-rich phase 140 constructs the continuous phase, and the maltodextrin-rich phase 142 forms non-spherical blobs as an included phase. In contrast to the sample without salt (Figure 9), cells normally associated with the gelatin-rich phase were adsorbed at the gelatin-maltodextrin interface 144.

The modified biopolymers of the present invention can be subjected to rheological measurements, preferably small-deformation oscillatory measurements to demonstrate the improved characteristics of the biopolymers modified by the plurality of inactivated single- celled microorganism. Measurement of storage modulus (G') and loss modulus (G") of phase-separated biopolymer gels may be carried out at about 25°C.

With reference to Figure 16, time-dependent G' and G" of phase-separated gels at 25 °C, illustrating samples containing single cell microorganisms as well as a control sample without added cells. Prior to taking measurements, all samples were quenched from « 90 °C to 40 °C and subjected to T_h = 40 °C for t_h = 30 sec before being quenched to 25 °C. After 1 min equilibrium at 25 °C, the measurements commenced and continued for 1 hour. The gelatin plus maltodextrin system showed the lowest G' after 1 hour (926 ± 13.4 Pa). In contrast, systems containing added single cells (2wt%) showed higher G' than that of the sample without added cells. With Lactobacillus, G' increased 4.7 % to 970 ± 26 Pa. With chlorella, G' was raised by 54.4 % to 1430 ± 17.32 Pa. The addition of yeast caused a 97.2% increase in G' to 1826.6 ± 35.1 Pa. Finally, the incorporation of spirulina caused a pronounced 131.4 % increase in G', to 2143.3 ± 37.8 Pa. In the second set of experiments, we changed the holding time at 40 °C from 30 sec to 10 min (T_h = 40 °C for t_h = 10 min), followed by a similar set of rheological measurements.

With reference to Figure 17, time-dependent G' and G" of phase-separated gels at 25 °C, consisting of samples containing single cell microorganisms and one control sample without added cells (each held at 40 °C for 10 min). As in the previous experiments, all samples containing single cell organisms (2wt%) showed higher G' than that of samples without added cells. Of the gels with added cells, the gelatin plus maltodextrin system showed the lowest G' after 1 hour (648.6 ± 12 A Pa). With Lactobacillus, G' increased 42.34 ± 0.1 % to 923.3 ± 30 Pa. With chlorella, G' increased 104% to 1323 ± 40.41 Pa. The addition of yeast caused a 155.3%) increase in G' to 1656.6 ± 41.6 Pa, and the incorporation of spirulina caused a 215 % increase in G' to 2043.3 ± 70.9 Pa.

With reference to Figure 18, time-dependent G' and G" of phase-separated gels at 25 °C, illustrating three samples - one containing 2wt% chlorella, one with 2wt% spirulina, and a control sample without added cells - in the presence of lwt% salt (NaCl). As in the first set of experiments (Figure 16), prior to taking measurements all samples were quenched from « 90 °C to 40 °C and held at this temperature for 30 sec (T_h = 40 °C for t_h = 30 sec), then cooled down to 25 °C. After 1 min equilibrium, the measurements commenced and continued for 1 hour. The addition of as little as lwt% of salt to the gelatin plus maltodextrin system caused a significant reduction of G' in comparison to the system without salt: a 38% loss, reducing the G' from 926 ± 13.4 Pa to 574 ± 12.12 Pa. In this salt-containing system, the addition of 2wt% spirulina improved the G' by 17.4%, to 674 ± 20 Pa. Similarly, the addition of 2wt% chlorella increased the G' by 79.44%, raising the initial value (926 ± 13.4 Pa) to 1030 ± 10 Pa. With reference to Figure 20, nonspecific interactions of single cells with the gel matrix and the nature of each group of added cells were observed by the different rheological responses with time/temperature treatments (Th = 40 °C and ¾ = 30 sec and 10 min). With reference to Figures 22 and 23 (higher magnification) CLSM micrographs show phase- separated samples encapsulating 0.1 wt% spirulina 150, 152 within a discontinuous 3 wt% gelatin phase 154 scattered in a 10 wt% maltodextrin continuous phase 156. Gelatin domains are encapsulated with substantially inactivated 1 wt% yeast (by 1 M solution of NaOH).

With reference to Figures 24 and 25 (higher magnification) CLSM micrographs show phase- separated samples encapsulating 1 wt% olive oil 160, 162 within discontinuous 3 wt% gelatin phase 164 scattered in a 10 wt%. maltodextrin continuous phase 166. Gelatin domains are encapsulated with substantially inactivated 1 wt% yeast 168, 169 (by 1 M solution of NaOH).

These micrographs demonstrate partitioning of the particles (spirulina) and oil droplets in the dispersed gelatin phase due to liquid-liquid phase separation. Since this encapsulation method does not require organic solvents or other chemicals, sensitive substances like cells and vulnerable lipophilic agents can be preserved within the phase-separated domains with the extra protection of the absorbed particles (yeast cells) at the liquid-liquid interface.

With reference to Figure 26, time-dependent G' and G" of gelatin-only gels, (single phase, containing one biopolymer) at 25 °C, illustrate samples containing single cell microorganisms as well as a control sample without added cells. Prior to taking measurements, all samples were quenched from « 90 °C to 40 °C and subjected to T_h = 40 °C for t_h = 30 sec before being quenched to 25 °C. After 1 min equilibrium at 25 °C, the measurements commenced and continued for 1 hour. The gelatin without added cells system showed the lowest G' after 1 hour (447 ± 8 Pa). In contrast, systems containing added single cells (2wt%) showed higher G' than that of the sample without added cells. With chlorella, G' increased 58.2 % to 707.3 ± 20 Pa. With Lactobacillus, G' was raised by 64.7 % to 736.3 ± 22.8 Pa. The addition of yeast caused a 1 1 1.5% increase in G' to 945.6± 17 Pa. Finally, the incorporation of spirulina caused a pronounced 155.7 % increase in G', to 1 143.3 ± 55 Pa.

With reference to Figure 27, time-dependent G' and G" of gelatin-inulin gels (single phase, containing two biopolymers) at 25 °C, illustrate samples containing single cell microorganisms as well as a control sample without added cells. Prior to taking measurements, all samples were quenched from « 90 °C to 40 °C and subjected to T_h = 40 °C for ¾ = 30 sec before being quenched to 25 °C. After 1 min equilibrium at 25 °C, the measurements commenced and continued for 1 hour. The G' of gelatin-inulin sample without added cells reached to 261.3 ± 1 1.54 Pa after 1 hour. In contrast, systems containing added single cells (2wt%) showed higher G' than that of the sample without added cells. With Streptococcus thermophilus, G' increased 69.5 % to 443 ± 7 Pa.

With reference to Figure 28, time-dependent G' and G" of gelatin 6 wt% and starch 6 wt% and sugar 20 wt% and glucose syrup 30 wt% gels at 25 °C, illustrate samples containing single cell microorganisms as well as a control sample without added cells. Prior to taking measurements, all samples were quenched from « 90 °C to 40 °C and subjected to T_h = 40 °C for t_h = 30 sec before being quenched to 25 °C. After 1 min equilibrium at 25 °C, the measurements commenced and continued for 1 hour. The G' of gelatin/starch/sugar/glucose syrup sample without added cells reached to 3573.3 ± 87.3 Pa after 1 hour. In contrast, systems containing 2 wt% single cells (substantially inactivated/modified yeasts with enzymatic treatment) showed higher G than that of the sample without added cells. With added 2 wt% enzymatically treated yeast, G' increased 30.5 % to 4666.6 ± 104. Pa.

Strain-sweep tests at a constant frequency may be carried out on the modified biopolymer solutions to assess viscoelastic properties of the modified biopolymer. With reference Figure 19, strain sweep tests at constant frequency (1 Hz) and temperature (25 °C) on samples after the completion of small oscillatory deformation measurements (samples subjected to T_h = 40 °C for ¾ = 30 sec). In this test, the changes in complex shear modulus (G*) as a function of shear strain amplitude in the oscillatory mode were monitored. At the lower limit of strain γ, 0.01 (1%), the initial G*of each sample matched its G' valve. Results showed that all samples had a long linear viscoelastic region. Samples containing 2wt% Lactobacillus showed strain- softening behaviour (decreasing the G* under strain); samples containing 2wt% spirulina and 2wt% yeast displayed similar behaviour, but to a lower extent with spirulina and even more moderately in the sample containing yeast. For all samples, increasing the strain to approximately 1-2.5 (100-250%) fractured the sample on the macroscopic scale, causing a more than 5% decrease in the initial value of G*. Before reaching the fracture point, all samples showed strain hardening behaviour, becoming resistant to flow under greater deformation. Without being bound by theory phase separation dictates the microstructure morphologies in a thermodynamic-incompatible biopolymer solution. A phase-separated sample with no added treated single-celled microorganisms, such yeast, chlorella, spirulina or Lactobacillus, represented a typical water-in-water emulsion, with spherical domains of one phase being surrounded by the other continuous phase in the matrix. CLSM observations revealed that the microstructure of gelatin-maltodextrin gel was altered in the presence of a plurality of treated single-celled microorganisms. All added cells resided in the gelatin-rich phase - presumably, the adsorption of protein at the particle surface lowered the interfacial energy contact of the inactivated single-celled microorganisms, which in turn favoured the particles residing in the protein-rich phase.

In one embodiment of the present invention, improvement of the rheological properties of the binary (gelatin-maltodextrin) gels was achieved, but also to minimize the adverse effect of phase separation on G'. With reference to Figure 21 , the | % I difference of G' for 1-hour aged gels at 25 °C (subjected to T_h = 40 °C, t_h = 30 sec and T_h = 40 °C, t_h = 10 min). Phase separation reduced the G' substantially: 29.94% from its original value in the gel sample without adding treated single-celled microorganisms. This G' loss significantly improved to 9.3% with the addition of 2wt% yeast and further to 7.4% with the addition of 2wt% chlorella. The G' loss reached a minimum of 4.8% with 2wt% Lactobacillus and 4.6% with 2 wt% spirulina. These results clearly indicate that the incorporation of a plurality of treated single-celled microorganisms into a phase-separated gelatin-maltodextrin gel could stabilize the rheological property of the gels against the adverse effect of phase separation.

The presence of a plurality of treated single-celled microorganisms in the gelatin- maltodextrin gel had a pronounced effect on the fracture behavior of gels under increased strain. In general, large deformation of gels is related to fracture failure and rupture propagation in the gel matrix. In microscopic level cracks, the initiation and its formation determine the macroscopic fractures. Gelatin-maltodextrin gel is considered a filled gel and, depending on the continuous and included phase, it could be classified as a gelatin gel filled with a maltodextrin included phase or vice versa.

The addition of a plurality of treated single-celled microorganisms to a phase-separated gelatin-maltodextrin gel significantly alters the developing microstructures under phase separation conditions. By using the single-celled microorganisms as colloidal particles, it is possible to create a group of microstructures that never existed before. The presence of a plurality of treated single-celled microorganism enhances the rheological properties of the phase-separated gel. It can also enhance the G' of the gel and, more importantly, it reduces the negative impact of phase separation on G'. Most likely, as the biopolymer-cell forms stiffer chains, the resulting gel becomes more resistance to rheological changes induced by phase separation. Treated single-celled microorganisms can be used as colloidal particles in thermodynamic-incompatible biopolymer solutions, or thermodynamic-compatible biopolymer solutions or in biopolymer solutions as filler particles. Different groups of treated single-celled microorganisms create different types of microstructures and impart different rheological properties to the mixture. These treated cell-incorporated biopolymers emulsions ("cibe") could be considered as an addition to food gels, providing diverse microstructures and enhanced rheological properties. Non-limiting examples are provided as follows.

Examples

Material

Food-grade gelatin from acid-treated porcine skin (Type A, pi 7.0 - 9.0, Bloom 300 ± 25, moisture content < 12.0 wt %, ash content < 1.0 wt %) was supplied by Sigma-Aldrich (Oakville, ON, Canada). Eliane™ MD 2, maltodextrin obtained by enzymatic conversion of potato starch moisture content <8 wt %, ash content < 1 wt %) was kindly supplied by National Starch (Bridgewater, NJ, USA). Yeast (product #51475) (Sacchromyces cerevisiae) was supplied by Sigma-Aldrich (Oakville, ON, Canada). Spirulina and Chlorella were supplied by PureBulk Inc. (Roseburg, OR, USA). Lactobacillus bulgaricus, LB-12, in a form of frozen pellet (DVS) was kindly offered by Chr. Hansen (Milwaukee, WI, USA). FITC (fluorescein-5-isothiocyanate, 90% HLPC grade) was provided by Sigma-Aldrich (Oakville, ON, Canada). Anhydrous ethyl alcohol was supplied by Commercial Alcohols Inc. (Brampton, ON, Canada). Sodium hydroxide (NaOH) and salt (NaCl) was supplied by Fisher Scientific (Ottawa, ON, Canada). Distilled water was used throughout. Streptococcus thermophilus, ST-M5 in the form of freeze-dried pellets were supplied from Chr. Hansen (Milwaukee, WI, USA). Protease enzyme, Protin NY 100, was kindly supplied by Amano Enzyme INC (Nagoya, Japan). Hydrochloric acid A.C.S. 36.5-38% was supplied by Fisher Scientific (Ottawa, ON, Canada). Sucrose (sugar) was provided by Sigma-Aldrich (Oakville, ON, Canada). Perfectamyl™ Gel-EMP, Oxidised potato starch obtained by oxidation of native potato starch with sodium hypochlorite moisture content <12 wt %, ash content < 2 wt %) was kindly supplied by National Starch (Bridgewater, NJ, USA). Inulin, Fiberrific™ was supplied by Pure-le Natural (Barrie, ON, Canada). Glucose syrup, DE 40-46 and olive oil were purchased from local supermarket.

Example 1 - Cell preparations

Single-celled microorganisms were treated with NaOH, HC1, saturated salt solution, enzyme or alcohol solutions. After treatment the single-celled microorganisms were washed repeatedly with distilled water until soluble substances substantially extracted from the cells and centrifugation of cell/distilled water dispersion resulted in a substantially clear and transparent supernatant (water) and homogenous and uniform sediment (cells). After washing pH was adjusted to 7 ±0.2 for cells treated with NaOH or HC1. Moisture (water content) of all prepared cells was adjusted to 89-95 wt% by measuring the solid contents of the dispersion of each prepared cell by means of water evaporations at 50 °C using a thermostat controlled oven. The electric charge of the cells and zeta potential (ζ) were measured with a 90 Plus™ particle size analyzer provided by Brookhaven Instruments Corporation (Holtsville, USA). Example 2 - Yeast preparation

Yeast cells were suspended in a 1 M solution of NaOH at a concentration of 10 wt% in a sealed screw cap glass bottle. The yeast-containing bottle was placed in water bath at 95 °C for 20 min and it was shaken regularly every 5-6 min for 15-20 sec. Then, the glass bottle was removed from the water bath and left at room temperature to sediment the undissolved fraction of yeast cells from the solution. The supernatant was discarded and each 15 ml of sediment was transferred to a 50 ml centrifuge tube and washed 7-9 times with 30 ml of water by repeated re-suspension/centrifugation cycles (5 min, 3000 rpm). After each stage of centrifugation, the slimy portion at the top of the sediment and the solid grain-like material at the bottom of the sediment in the centrifuge tubes were discarded. At the final stage of centrifugation, a clear transparent supernatant (water) and a homogeneous and uniform sediment were obtained. The pH of the final solution was adjusted to 7 ± 0.2, zeta potential ζ = -16 ± 1.06 mV Example 3 - Spirulina preparation

Spirulina cells were prepared similarly to the yeast cells, but after thermal treatment, the whole suspension was transferred to a centrifuge tube and longer cycles (15 min, 3000 rpm) were applied (zeta potential ζ = -14 ± 1.88 mV).

Example 4 - Chlorella preparation

Chlorella cells were suspended in a saturated salt solution with a concentration of 10 wt% in a sealed screw cap glass bottle. The Chlorella-containing bottle was placed in a water bath at 95 °C for 20 min and it was shaken regularly every 5-6 min for 15-20 sec. Then, the glass bottle was removed from the water bath and left at room temperature to sediment the chlorella cells from the solution. The supernatant was discarded and the sediment was mixed with an equal volume of 50% (v/v) ethanol solution. The mixture was kept in a sealed glass bottle for 5 days at 45 °C in a thermostat-controlled oven. During the inculcation, the alcohol fraction of the mixture phase-separated from the solution, 3 times the alcohol-rich supernatant was replaced with fresh ethanol, and the sample-containing bottle was shaken manually. After incubation, the alcohol fraction of the mixture was discarded, and each 15 ml of sediment was transferred to a 50 ml centrifuge tube and washed 5 times with 30 ml of water by repeated re-suspension/centrifugation cycles (5 min, 3000 rpm) until a clear transparent supernatant was obtained at the last stage of centrifugation (zeta potential ζ = - 55.79 ± 1.08 mV).

Example 5 - Lactobacillus bulgaricus preparation

Lactobacillus cells in the form of frozen pallets were thawed and transferred to a 50 ml centrifuge tube and kept in a water bath at 95 °C for 20 min followed by centrifugation for 15 min at 3000 rpm. Then, supernatant and solid sediments were separated from cell dispersions. Each 15-20 ml of cell dispersion was transferred to a 50 ml centrifuge tube and washed 4-5 times with 30 ml water by repeated re-suspension/centrifugation cycles (15 min, 3000 rpm). After each stage of centrifugation, the solid, cake-like sediment at the bottom of the sediment in the centrifuge tubes was discarded. Next, the cell suspensions were mixed with an equal volume of 50% (v/v) ethanol solution in a sealed glass bottle using a magnetic stirrer overnight (16 hours) at room temperature. Then, once again, cell dispersions were transferred to a 50 ml centrifuge tube and were washed 5-7 times with 30 ml of water by repeated re- suspension/centrifugation cycles (15 min, 3000 rpm). After each stage of centrifugation, the solid cake-like sediment at the bottom was discarded until a clear transparent supernatant and a monotonous sediment were obtained (zeta potential ζ = +5.96 ± 3.07 mV).

Example 6 - Preparation of solutions

The gelatin and maltodextrin solutions were prepared in separate, sealed, screw-cap glass vials (20 ml) by placing them in a 90 °C water bath for 10 min for maltodextrin and for 3 min for the gelatin solution, with vortexing at 10-15 sec intervals at 3000 rpm (Fisher Scientific, Nepean, ON, Canada). The cell suspension was added to the gelatin-containing vial and vortexed as above. For the final sample preparation, solutions of twice the required final biopolymer concentrations were prepared and mixed with equal weights of the biopolymer solutions. The final blends were mixed at « 90 °C and again placed in a water bath at « 90 °C for 1-2 min with repeated vortexing. This was followed by ultrasonication for « 10 s at « 90 °C to remove any air bubbles (Eumax® UD50SH-2L ultrasonic bath, Kwun Wah Int. Ltd, Hong Kong). For the confocal laser scanning microscopy of the control sample (without cell), the gelatin solution also contained ~ 0.001 wt % FITC.

Example 7 - Thermal treatment and small-deformation rheometry

A Physica MCR 301 rheometer (Anton Paar GmbH, Graz, Austria), equipped with a Peltier plate temperature control unit (P-PTD 200), was used to perform small-deformation oscillatory rheometery. All measurements were carried out with parallel plate geometry (PP 25/TG) with a diameter of 25mm. The time-dependent storage (G') and loss moduli (G") were measured at 25 °C at a constant frequency of 1 Hz and a target strain of 0.2% for up to 70 min. To avoid sample drying, the measuring geometry was covered with a solvent trap containing a moist strip of tissue paper.

Preceding each measurement, the temperature of the Peltier plate was set at 40 °C and the mixed hot biopolymer solution (at 90 °C) was poured directly onto the hot plate. The geometry was then lowered onto the sample to an operating gap width (1 mm) and the sample was carefully trimmed and held at 40 °C temperature of holding (T_h) for 2 different holding times (¾) of 30 seconds and 10 minutes to initiate and increase the extent of phase separation. After thermal treatments at 40 °C, the temperature of the rheometer cell was decreased from 40 °C to 25 °C at 16 °C min^"1, and the sample was kept for 1 minute at 25 °C to ensure the equilibrium. At this point, measurements were commenced and lasted for 1 hr. Using this standard thermal treatment (i.e., T_h = 40 °C for t_h = 30 sec), a group of phase-separated samples with a relatively low extent of phase separation were produced. And with another thermal treatment (T_h = 40 °C for t_h = 10 min), another group of samples with a higher extent of phase separation was produced. All results herein reported are based on more than triplicate runs.

Example 8 - Confocal laser scanning microscopy (CLSM)

Confocal microscopy was performed using an upright Zeiss LSM 510 (Carl Zeiss, Toronto, ON, Canada). The CLSM was operated in fluorescent mode with an Ar laser source (488 nm). The emission spectra were collected with 1 channel set at 505 nm. No fluorescent labeling was used for cell-containing samples, since autofluorescence properties of the cell were sufficient for CLSM observations. To subject the CLSM samples to a thermal treatment similar to the rheology experiments, a flat metal washer with 2 cover slips was used as a sample holder, and the sample was sandwiched within the metallic washer between the 2 cover slips. This holder was placed on the Peltier plate temperature control unit of the rheometer, equilibrated, and then samples were processed as per the thermal conditioning used for the rheological measurements. After a 10 min hold at 25 °C, the sample holder was transferred to the CLSM stage for characterization. 10 χ and 20 χ objective lenses were used. Images were recorded at 25 °C at a resolution of 1024 χ 1024 pixels. Image optimization was performed using the LSM 510's built-in image analysis software. Images shown herein are representative of the microstructure seen for a given composition.

Example 9 - Streptococcus thermophilus preparation

Streptococcus thermophilus cells in the form of freeze-dried pallets were soaked and dispersed by distilled water and transferred to a 50 ml centrifuge tube. Centrifugation for 15 min at 3000 rpm and re-suspension were repeated 4-5 times until the cake-like sediments (remnants of the cell culture) were separated from the cells. Next, hydrochloric acid (32-38% solution) was added to the cell suspensions (around 50 wt% cells) to obtain 1 M acid cell dispersion. The acid-cell solution was transferred to a sealed screw cap glass bottle and placed in a water bath at 95 °C for 20 min and it was shaken regularly every 5-6 min for 15- 20 sec.

Then the cell suspension was transferred to a 50 ml centrifuge tube and washed 6-7 times with water by repeated re-suspension/centrifugation cycles (15 min, 3000 rpm). After each stage of centrifugation, any remaining cake-like materials at the bottom of the sediment in the centrifuge tubes were discarded. At the final stage of centrifugation, a clear transparent supernatant (water) and homogeneous and uniform sediment (cell) were obtained. The pH of the final solution was adjusted to 7 ± 0.2, zeta potential ζ = -5.39 ± 0.17 mV Example 10 - Yeast preparation with enzymatic treatment

Yeast cells were suspended in a 2 wt% enzyme (Protin NY 100) solution with a concentration of 10 wt% in a sealed screw cap glass bottle. The yeast-containing bottle was placed in a thermostat-controlled oven at 45 °C for 24 hours. Then, the glass bottle was removed from the thermostat-controlled oven and it was placed in a water bath at 95 °C for 50 minutes. Then, the glass bottle was removed from the water bath and left at room temperature to sediment the yeast cells from the solution, the supernatant was discarded and each 15 ml of sediment was transferred to a 50 ml centrifuge tube and washed 7-8 times with 30 ml of water by repeated re-suspension/centrifugation cycles (5 min, 3000 rpm). After each stage of centrifugation, the slimy portion at the top of the sediment and the solid grain-like material at the bottom of the sediment in the centrifuge tubes were discarded until a clear transparent supernatant was obtained at the last stage of centrifugation (zeta potential ζ = - 14.37± 0.14 mV).

Although the above examples are on a laboratory scale, a person of ordinary skill in the art reading the above examples will be able to carry out the present invention on an industrial scale.

As many changes can be made to the preferred embodiment of the invention without departing from the scope thereof, it is intended that all matter contained herein be considered illustrative of the invention and not in a limiting sense.

Claims

I . A plurality of single-celled microorganisms comprising at least one substantially inactivated single-celled microorganism wherein said at least one substantially inactivated single-celled microorganism further comprises at least one altered surface property.

2. The microorganisms of claim 1 wherein the single-celled microorganisms are selected from the group consisting of fungi, algae and bacteria.

3. The microorganisms of claim 1 wherein the at least one altered surface property comprises a denatured surface protein.

4. The microorganisms of claim 2 wherein the bacteria is selected from lactic acid bacteria.

5. The microorganism of claim 4 wherein the lactic acid bacteria are selected from Lactobacillus bulgaricus or Streptococcus thermophilus.

6. The microorganisms of claim 2 wherein the fungi is selected from a yeast.

7. The microorganism of claim 6 wherein the yeast is Saccharomyces cerevisiae.

8. The microorganisms of claim 2 wherein the algae is selected from a single-celled group.

9. The microorganism of claim 8 wherein the single-celled group is selected from chlorella or spirulina.

10. A process for treating at least one single-celled microorganism, preferably a plurality of single-celled microorganisms, resulting in at least one substantially inactivated single cell microorganism with at least one altered surface property, said process comprising: a) Substantially inactivating said at least one single-celled microorganism, and b) Substantially altering said surface.

I I . The process of claim 10 wherein step a) comprises inactivating said at least one single-celled microorganism with at least one of a substance, heat, microwave, ultraviolet or irradiation

12. The process of claim 10 wherein step b) comprises treating said at least one single- celled microorganism with at least one of an enzyme, a basic substance, an acidic substance, a salt or an alcohol and combinations thereof.

13. The process of claim 12 wherein the basic substance is sodium hydroxide.

14. The process of claim 12 wherein the alcohol is selected from the group consisting of methanol, ethanol, n-propanol and isopropanol and combinations thereof.

15. The process of claim 12 wherein the acid is hydrochloric acid.

16. The process of claim 12 wherein the salt is sodium chloride.

17. The process of claim 12 wherein the enzyme is a protease.

18. The use of a plurality of single-celled microorganisms comprising at least one substantially inactivated single cell microorganism with at least one altered surface property, in altering at least one property of a soft material.

19. The use of claim 18 wherein the soft material is a biopolymer or a mixture of biopolymers.

20. The use of claim 18 wherein the single-celled microorganism is selected from the group consisting of fungi, algae and bacteria.

21. The use of claim 20 wherein the bacteria is Lactobacillus bulgaricus or Streptococcus thermophilus.

22. The use of claim 20 wherein the algae is chlorella or spirulina.

23. The use of claim 20 wherein the fungi is a yeast Saccharomyces cerevisiae.

24. A soft material comprising a plurality of inactivated single-celled microorganisms with at least one altered surface property wherein said microorganisms are present between about 0.1% and about 30%.

25. The soft material of claim 24 wherein the soft material is a polymer, a biopolymer or mixture of polymers or biopolymers thereof.

26. A process for preparing a modified biopolymer solution using a plurality of single- celled microorganisms comprising the steps:

1) Providing a biopolymer or a mixture of biopolymers

2) Optionally providing at least one sweetener; 3) Providing a plurality of treated single-celled microorganisms comprising at

4) Contacting said biopolymer or mixture of biopolymers with said plurality of

single-celled microorganisms and optionally with said at least one sweetener.

27. The process of claim 26 wherein the biopolymer is selected from the group consisting of gelatin, starch and combinations and-derivatives thereof.

28. The process of claim 26 wherein the sweetener is selected from a monosaccharide, disaccharide or polysaccharide, derivatives and/or modified forms thereof.

29. The process of claim 26 wherein the sweetener is selected from sucrose or glucose syrup or a combination thereof.

30. The use of a plurality of single-celled microorganisms comprising at least one substantially inactivated single cell microorganism with at least one altered surface property as a food-grade colloidal particle.

31. The use of a plurality of single-celled microorganisms comprising at least one substantially inactivated single-celled microorganism with at least one altered surface property in the preparation of a modified biopolymer solution.

32. The use of claim 31 wherein the biopolymer solution further comprises at least one ingredient selected from the group consisting of a flavouring agent, a colouring agent an acidulant, a salt, an antioxidant, an edible oil, an emulsifier and a glazing agent.