METHODS OF MAKING SAME

Technical Field

[0001] Some embodiments of the present invention pertain to microcapsules containing probiotics, probiotics and prebiotics, and/or synbiotics. Some embodiments of the present invention pertain to methods of making microcapsules containing probiotics, probiotics and prebiotics, and/or synbiotics.

Background

[0002] Probiotics are 'live microorganisms' or 'live microbial feed supplements' that offer health benefits to their host, and include primarily bacteria from the Lactobacillus and

Bifidobacterium genera (Fuller, 1989; Araya et al., 2002; Famworth, 2007). Probiotics have been reported to offer enhanced health, if administered at therapeutic doses (approx. 10 live cells per gram of intestinal contents). Beneficial effects on the host are thought to include: promoting digestion and mineral absorption; maintaining intestinal microbial balance; improving immune system response and resistance to pathogens; preventing intestinal tract infection; reducing the risk of cardiovascular disease, cancer, obesity and type-2 diabetes; regulating lipid levels and serum cholesterol; ameliorating lactose intolerance; as well as treating certain symptoms, such as inflammation, autoimmune responses, or allergies (Gibson & Roberfroid, 1995; Collins & Gibson, 1999; Shortt, 1999; Hooper et al., 2001; Pridmore et al., 2004; Sartor, 2004; Bielecka, 2007; Rastall, 2007; Sarkar, 2007).

[0003] Bifidobacteria are non-motile, non-spore forming and strictly anaerobic Gram-positive bacteria that grow at pH values between 4.5 and 8.5 (Scardovi, 1986; Rokka and Rantamaki, 2010). They are indigenous to the human intestine (Fuller, 1991) and play an important beneficial role in inhibiting proliferation of potentially harmful microorganisms in the gastrointestinal tract (Hoover, 1993; Gibson & Roberfroid, 1995; Yaeshima, 1996; Holzapfel, Haberer, Snel, Schillinger, & Huis in't Veld, 1998; Alander et al., 1999). Exemplary

Bifidobacteria species include Bifidobacteria adolescentis, Bifidobactertium animalis,

Bifidobacterium bifidum, Bifidobacterium breve or Bifidobacterium longum. [0004] Lactobacilli are large, non-spore forming, Gram-positive rods, that have anaerobic or microaerophilic respiration. Gram-positive bacteria have a thick peptidoglycan layer as part of their cell wall whereas Gram-negative bacteria have a thin layer. Anaerobic bacteria will not grow in the presence of oxygen and therefore an atmosphere devoid of oxygen must be present in order for their growth to occur. Microaerophilic organisms are organisms that require a lower concentration of oxygen than that found in air. Lactobacillus bacteria are microaerophilic and are commonly used as a starter culture in yogurt production and are the most commonly used probiotics in foods. Exemplary Lactobacillus species used as probiotics include L. delbreuckii subspecies bulgaricus, L. acidophilus, L. casei, L. germentum, L. plantarum, L. brevis, L.

cellobious, L. lactis and L. reuteri.

[0005] Prebiotics are compounds that support probiotic growth. Prebiotics act to increase the number and activity of beneficial bacteria which already colonize the colon. Exemplary prebiotics include non-digestible carbohydrates such as: resistant starch (starch which is not hydrolysed in the small intestine), non-starch polysaccharides (hemicellulose, pectins, gums), and oligosaccharides (galactooligosaccharides, fructoligosaccharides, maltoligosaccharides), lactulose, inulin, and the like.

[0006] Synbiotics are nutritional supplements containing both probiotics and prebiotics. In some cases the combination of prebiotics and probiotics produces synergistic health benefits, hence the term "synbiotics" is used.

[0007] Various factors such as antibacterial drugs, stress, gastrointestinal disorders, and aging can contribute to a decline in the number of naturally-occurring beneficial bacteria (including Bifidobacteria and Lactobacillus) in the gut, leading to chronic gastrointestinal diseases with symptoms such as abdominal cramps, diarrhea, fever, vomiting, etc. (Mitsuoka, 1982; Gothefors, 1989; Hoover, 1993). Therefore, oral administration of probiotics (including Bifidobacteria and Lactobacillus) is supposed to have potential beneficial effects to relieve or even cure such diseases. However, probiotic strains are sensitive to the harsh environmental conditions within foods, during processing and during transit through the gastrointestinal tract (Charteris, Kelly, Morelli, & Collins, 1998; Clark & Martin, 1994; Truelstrup Hansen, Allan- Wojtas, Jin, & Paulson, 2002). Thus, the widespread use of probiotics in foods and as nutraceuticals is hindered by the harsh food environment, and the inability to deliver high numbers of viable cells through the acidic barrier of the stomach and into the lower gastrointestinal (GI) system where they proliferate and exert their beneficial effects. Similar issues arise for use as a feed/pet food supplement, where probiotics are expected to withstand feed processing (i.e., pelletization) and long-term storage so as to remain viable during transit through the animal's GI system.

[0008] To circumvent these problems, encapsulation technology may be used to deliver therapeutic levels of both prebiotics and probiotics to the GI system, providing maximum health benefits of these agents. Encapsulation technology involves encasing the sensitive core ingredients within a biopolymer shell, which can release its contents at controlled rates once triggered by an external sensor (e.g., temperature, pH, enzymes, etc.). Although a wide variety of biopolymers have been employed in the literature, alginate-based capsules seem to dominate. Alginate is a linear heteropolysaccharide comprised of D-mannuronic and L-guluronic acids; the latter being highly- sensitive to divalent calcium ions resulting in the formation of strong egg box-like junction zones. However, past alginate-probiotic capsules have been shown to be ineffective at adequately protecting probiotic bacteria subjected to simulated gastric juice.

[0009] Other efforts have been made to enhance the survival of probiotics under these conditions using microencapsulation prepared from biopolymers originating from seaweed (carrageenan, alginate), plants (starch and gum Arabic), bacteria (gellan, xanthan), and animal proteins (milk, gelatin) (Guerin, Vuillemard, & Subirade, 2003; Rao, Shiwnarain, & Maharaj, 1989; Ravula & Shah, 1998; Sun & Griffiths, 2000; Truelstrup Hansen et al., 2002; Annan, Borza, & Truelstrup Hansen, 2008; Rokka & Rantamaki, 2010).

[0010] In some cases, small capsules (<100 μιη) can be produced using an emulsification technique, allowing capsules to be incorporated into foods without affecting their sensory attributes (Cui, Goh, Kim, Choi, & Lee, 2000; Lee & Heo, 2000; Truelstrup Hansen et al., 2002; Chandramouli, Kailasapathy, Peiris, & Jones, 2004). [0011] Plant proteins are becoming increasingly important to the food industry as a replacement for animal-derived proteins (e.g., gelatin and whey) for use as food and encapsulating ingredients. Plant proteins represent an attractive alternative because of their low cost, renewability and functionality and as replacements for animal proteins based on consumer choices (e.g., vegans) and religious practices. They also are advantageous when used in combination with polysaccharides as wall materials, as they can be designed to have pH- or enzymatic triggers for controlled release purposes. The application of plant proteins as encapsulating agents for probiotic delivery is currently very limited; however, this practice could be developed for use in non-dairy markets and products. Klemmer, Korber, Low, and Nickerson (2011) recently determined that pea protein isolate-alginate mixed capsules prepared by extrusion offered significant protection to B. adolescentis within simulated gastric juice (SGJ) over 2 h/37°C, and showed prolonged release within simulated intestinal fluids (SIF). However, capsule sizes were too large (~2 mm) for use as food supplements.

[0012] There is a need for improved plant protein-based microcapsules having a smaller particle size acceptable for incorporation into food products that does not adversely affect the sensory attributes of the food product (i.e. less than about 100 μιη diameter). Microcapsules having a smaller particle size have a greater surface area to volume ratio, leading to increased exposure to surface area and decreasing the survival of probiotics. Polysaccharide-based microcapsules can be made, but provide poor survival rates for probiotics.

[0013] The foregoing examples of the related art and limitations related thereto are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.

Summary

[0014] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements. [0015] In one embodiment, a microcapsule for protecting a probiotic and/or delivering a probiotic to an intestine of a subject is provided. The microcapsule comprises a biopolymer, a plant protein and a probiotic, and the microcapsule is prepared by crosslinking the biopolymer in an emulsion of the biopolymer, the plant protein, the probiotic, and an oil to encapsulate the probiotic.

[0016] In another embodiment, a method of preparing a microcapsule is provided. The method includes combining a plant protein, a biopolymer and a probiotic to form an aqueous phase, adding an oil to the aqueous phase to form an emulsion, adding a crosslinker to crosslink the biopolymer, and separating the microcapsules from the oil phase of the emulsion.

[0017] In some embodiments, the plant protein is chickpea protein isolate, pea protein isolate, and/or soy protein isolate. In some embodiments, the biopolymer is alginate, iota-carrageenan, and/or deacylated gellan gum. In some embodiments, the probiotic is from the Lactobacillus or Bifidobacterium genera. In some embodiments, the probiotic is encapsulated together with a prebiotic.

[0018] In some embodiments, the microcapsules have a diameter of between about 20 and about 100 micrometers. In some embodiments, the microcapsules are incorporated into a food product. In some embodiments, the food product is yogurt, fruit juice, a cereal product, or a dried food product. In some embodiments, the microcapsule provides delivery of the encapsulated probiotic to the intestine of a mammalian subject.

[0019] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions.

Brief Description of the Drawings

[0020] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. [0021] Figure 1 shows the survival of B. adolescentis entrapped in chickpea protein isolate (CPI, 10% w/w) capsules cross linked with genipin (0.2% w/v), or in the presence of alginate or κ- carrageenan (0.2% w/v) as a function of time during an acid challenge (pH 2.0/25°C). Data represent the mean + one standard deviation (n = 2).

[0022] Figure 2 shows the survival of B. adolescentis entrapped in chickpea protein (10% w/w) - alginate (0.1% w/v) capsules as a function of time within synthetic gastric juice (pH 2.5/37°C). Data represent the mean + one standard deviation (n = 2).

[0023] Figure 3 shows the survival of free B. adolescentis and those released from chickpea protein (10% w/w) - alginate (0.1% w/v) capsules as a function of time within simulated intestinal fluid (pH 6.5/37°C). Data represent the mean + one standard deviation (n = 2).

[0024] Figure 4 shows the survival of B. adolescentis entrapped in alginate microcapsules incorporating different plant-based proteins (pea, soy, faba, and lentil) as a function of time within synthetic gastric juice.

[0025] Figure 5 shows the release of B. adolescentis entrapped in alginate microcapsules incorporating different plant-based proteins (pea, soy, faba, and lentil) as a function of time within simulated intestinal fluid.

[0026] Figure 6 shows the survival of B. adolescentis entrapped in pea protein (10% w/w) with iota-carrageenan or deacylated gellan gum (0.1% w/v) microcapsules as a function of time within synthetic gastric juice (pH 2.5/37 °C). Data represent the mean + one standard deviation (n = 2).

[0027] Figure 7 shows survival of B. adolescentis released from pea protein (10% w/w) with iota-carrageenan or deacylated gellan gum (0.1% w/v) microcapsules as a function of time within simulated intestinal fluid (pH 6.5/37 °C). Data represent the mean + one standard deviation (n = 2). [0028] Figure 8A shows an SEM (scanning electron microscopy) image of a microcapsule prepared from chickpea protein isolate and alginate at 625x magnification. Figure 8B shows an SEM image of a microcapsule prepared from chickpea protein isolate and alginate at 2500x magnification.

[0029] Figure 9A shows an SEM image of a microcapsule prepared from pea protein isolate and alginate at 625x magnification. Figure 9B shows an SEM image of a microcapsule prepared from pea protein isolate and alginate at 2500x magnification.

[0030] Figure 10A shows an SEM image of a microcapsule prepared from soy protein isolate and alginate at 625x magnification. Figure 10B shows an SEM image of a microcapsule prepared from soy protein isolate and alginate at 2500x magnification.

[0031] Figure 11 A shows an SEM image of a microcapsule prepared from faba bean protein isolate and alginate at 625x magnification. Figure 11B shows an SEM image of a microcapsule prepared from faba bean protein isolate and alginate at 2500x magnification.

[0032] Figure 12A shows an SEM image of a microcapsule prepared from lentil protein isolate and alginate at 625x magnification. Figure 11B shows an SEM image of a microcapsule prepared from lentil protein isolate and alginate at 2500x magnification.

Description

[0033] Throughout the following description specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0034] Some embodiments of the present invention pertain to micron- sized plant protein-based capsules for carrying probiotic bacteria, and/or mixtures of prebiotics and probiotic bacteria, and/or probiotic-prebiotic synbiotics. In some embodiments, the capsules can protect the probiotic bacteria against conditions of the stomach (e.g. gastric pH) and facilitate targeted delivery of the probiotic bacteria to the intestines of a mammalian subject. In some

embodiments, the capsules are suitable for use as a food ingredient, being sufficiently small in size to avoid adverse effects on a tongue of a subject. In some embodiments, the capsules are used as nutritional supplements or ingredients in human food products and/or animal feed.

[0035] As used herein, the terms "about" and "approximately" mean a value that is within plus or minus 10% of the stated value.

[0036] The terms "comprises" and "comprising" are used in an open-ended sense to mean "including, but not limited to".

[0037] "Gastric conditions" are conditions similar to the conditions that would be experienced by a microcapsule in the stomach of a mammal. In some embodiments, the gastric conditions are similar to the conditions that would be experienced by a microcapsule in the stomach of a human. In some embodiments, "gastric conditions" that would be experienced in the stomach of a mammal means a pH of about 1.5-4.1 and a temperature of between about 36.5°C and 39.5°C. In some embodiments, gastric conditions comprise approximately 0.5% HC1 with large quantities of KCl and NaCl (Kararli, 1995). In some embodiments, gastric conditions that would be experienced in the stomach of a human means a pH of about 1.5 to 3.5 and a temperature of between about 36.5°C and about 37.5°C. In some embodiments "gastric conditions" includes the presence of other compounds such as enzymes such as lysozyme and pepsin.

[0038] "Intestinal conditions" are conditions similar to those that would be experienced by a microcapsule in the small intestine of a mammal. In some embodiments, the intestinal conditions are similar to those that would be experienced by a microcapsule in the small intestine of a human. In some embodiments, "intestinal conditions" that would be experienced in the small intestine of a mammal means a pH in the range of approximately 5.0-8.0 and a temperature of about 36.5°C to 39.5°C. In some embodiments, intestinal conditions that would be experienced in the small intestine of a human means a pH in the range of 5.0 to 7.0 and a temperature of about 36.5°C to 37.5°C. In some embodiments, "intestinal conditions" includes the presence of other compounds such as bile and enzymes such as pancreatin (a mixture of amylase, lipases and proteases).

[0039] Some embodiments of the present invention pertain to microcapsules made from a biopolymer and a plant-based protein. In some embodiments, the microcapsules are used to contain a probiotic, a probiotic and a prebiotic, or a synbiotic. In some embodiments, the microcapsules can be used to deliver a probiotic to an intestine of a mammalian subject by protecting the probiotic from the conditions prevailing in the stomach of the subject and releasing the probiotic under the conditions experienced in the intestine of the subject. In some embodiments, the microcapsules can be used to deliver a probiotic and a prebiotic, or a synbiotic, to an intestine of a mammalian subject by protecting the probiotic and prebiotic, or synbiotic, from the conditions prevailing in the stomach of the subject and releasing the probiotic under the conditions experienced in the intestine of the subject.

[0040] Examples of potentially suitable biopolymers for use in some embodiments of the present invention include alginate, iota-carrageenan, or gellan gum. In some embodiments, the biopolymer is alginate. In some embodiments, the biopolymer is iota-carrageenan. In some embodiments, the biopolymer is deacylated gellan gum.

[0041] In some embodiments, the biopolymer is present in the initial aqueous phase prior to emulsion at a concentration of between about 0.05 and 0.50% w/v or any value therebetween, e.g. 0.07, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 or 0.45% w/v in the initial aqueous phase prior to emulsion. In some embodiments, a higher concentration of biopolymer is used where there is a higher concentration of protein present.

[0042] In some embodiments, the plant-based proteins are chick pea protein, pea protein and/or soy protein.

[0043] In some embodiments, the plant-based protein is present at a concentration of between about 5 and 10% w/w in the initial aqueous phase prior to emulsion, or any value therebetween, e.g. about 6, 7, 8 or 9% w/w in the initial aqueous phase prior to emulsion. In some embodiments, the plant-based protein is present at a concentration of 10% w/w in the initial aqueous phase prior to emulsion. Higher concentrations of plant-based protein could potentially be used if desired, but sufficiently high concentrations of plant-based protein could lead to solubility and/or viscosity issues.

[0044] In some embodiments, the biopolymer is crosslinked to provide the microcapsules. In embodiments in which the biopolymer is alginate, iota-carrageenan, or gellan gum, suitable crosslinkers include calcium (e.g. supplied as CaCl ₂ ), or magnesium (e.g. supplied as MgCl ₂ ).

[0045] In some embodiments, an excess of crosslinker is added to the water-in-oil emulsion to cross-link the biopolymer. In some embodiments the crosslinker is present in a concentration in the range of about 0.1 to 0.5 M in the water-in-oil emulsion or any value therebetween, e.g. 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, or 0.45 M in the water-in-oil emulsion.

[0046] Some embodiments provide micron-sized plant protein-based microcapsules capable of carrying probiotic bacteria, mixtures of probiotic and prebiotics, or synbiotics through conditions of gastric pH to provide targeted delivery of the contents of the capsules within the intestines of a mammalian subject. In some embodiments, the capsules are less than approximately 100 micrometers in diameter, less than approximately 20 micrometers in diameter, or any value therebetween, e.g. less than approximately 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35 or 30 micrometers in diameter. In some embodiments, the capsules are between about 20 and about 100 micrometers in diameter. In some embodiments, the capsules are useful as feed and food supplements or ingredients.

[0047] In some embodiments, the encapsulated probiotic is from the Lactobacillus or

Bifidobacterium genera. In some embodiments, the probiotic is Bifidobacteria adolescentis, Bifidobactertium animalis, Bifidobacterium bifidum, Bifidobacterium breve or Bifidobacterium longum. In some embodiments, the probiotic is Lactobacillus delbreuckii subspecies bulgaricus, L. acidophilus, L. casei, L. germentum, L. plantarum, L. brevis, L. cellobious, L. lactis or L. reuteri. In some embodiments, the encapsulated probiotic is a combination of one or more of the preceding species. In some embodiments, the probiotic is Bifidobacteria adolescentis.

[0048] In some embodiments, the encapsulated prebiotic is a carbohydrate. In some

embodiments, the encapsulated prebiotic is a non-digestible carbohydrate such as: resistant starch (starch which is not hydrolysed in the small intestine); a non-starch polysaccharide such as hemicellulose, pectins, or gums; an oligosaccharide such as a galactooligosaccharide, fructoligosaccharide, maltoligosaccharide; lactulose; inulin; or the like. In some embodiments, the encapsulated prebiotic is a fructooligo saccharide, lactulose, transgalactooligosaccharide, or inulin.

[0049] In some embodiments, the encapsulated probiotic is combined with a suitable prebiotic to provide a synbiotic.

[0050] In some embodiments, the microcapsules protect the encapsulated probiotic from conditions experienced during the processing and storage of a feed product such as animal feed. In some embodiments, the microcapsules protect the encapsulated probiotic from conditions experienced during pelletization of the feed product. In some embodiments, the microcapsules protect the encapsulated probiotic to retain viability of the probiotic during long term storage of the feed product, for example for a period of time greater than two weeks to six months or more, or any period of time there between, e.g. four weeks, six weeks, eight weeks, ten weeks, three months, four months, five months, or the like.

[0051] In some embodiments, the microcapsules protect the encapsulated probiotic from conditions experienced during preparation and/or storage of a food product, for example, low pH, extrusion, drying or the like. Examples of food products in which the microcapsules according to embodiments of the present invention might be used include dairy products (e.g. yogurt having a pH in the range of about 3.5 to 4.0), cereals, dried fruit products, and/or fruit juices having moderately low pH.

[0052] In some embodiments, the capsules are prepared using an emulsion technique that involves dispersing a mixture of the plant-based protein, the biopolymer and the probiotic as droplets within a continuous oil medium to create an emulsion. In some embodiments, the emulsion is formed in a vegetable oil. In some embodiments, the emulsion is formed in canola oil.

[0053] After a set period of time, the emulsion is broken using a solution of cross-linker (to cross-link the biopolymer), followed by a hardening step. Microcapsules are then hardened and harvested.

[0054] In some embodiments, the microcapsules are dried after being harvested. Any suitable drying method that is compatible with the encapsulated probiotic can be used to dry the microcapsules. In some embodiments, drying of the microcapsules is done by freeze-drying.

[0055] In some embodiments, the microcapsules provide protection against substantial loss of viability of the probiotic contained in the microcapsules for at least two hours under simulated gastric conditions. As used in the present specification, "substantial loss of viability" means any loss of viability greater than about 2 log CFU/g. In some embodiments, the microcapsules provide protection against a loss of viability greater than about 1 log CFU/g. In some embodiments, the microcapsules provide protection against a loss of viability greater than about 3 log CFU/g.

[0056] In some embodiments, the microcapsules provide release of the probiotic contained in the microcapsules under simulated intestinal conditions. In some embodiments, the probiotics contained in the microcapsules may provide a positive influence on the gastrointestinal system of a mammalian subject, even in cases where the probiotics are not delivered live to the small intestine of the subject.

[0057] Based on the experimental results described herein, it can be soundly predicted that some embodiments of the microcapsules can be used to provide targeted delivery of the encapsulated probiotic or combination of probiotic and prebiotic, or synbiotic, to the intestines of a

mammalian subject by protecting the encapsulated material against a substantial loss of viability due to gastric conditions during transit through the stomach of a mammalian subject, and then releasing the encapsulated material upon exposure to intestinal conditions in the intestine of the mammalian subject.

[0058] In some embodiments, the microcapsules are used to deliver a probiotic, probiotic and prebiotic, or synbiotic to the intestine of a mammalian subject. The microcapsules are stable at gastric pH to protect the probiotic from degradation in the stomach of the mammalian subject, as can be observed by the extended survival of prebiotics under simulated gastric conditions. The microcapsules release the probiotic, probiotic and prebiotic, or synbiotic in the intestine of the mammalian subject under intestinal conditions.

[0059] In some embodiments, the microcapsules are prepared by forming a water-in-oil emulsion in a solution containing the plant-based protein, the biopolymer, and the material to be encapsulated. The mixture is mixed to achieve homogeneity and is then added to oil. In some embodiments, the oil is a vegetable oil such as canola oil. The resulting mixture is stirred. A crosslinking agent is added. In some embodiments, the crosslinking agent is calcium chloride or magnesium chloride. The aqueous phase containing the microcapsules is separated from the oil phase. In some embodiments, the microcapsules are rinsed with a suitable detergent to maintain good capsule dispersability after the microcapsules have been separated from the oil phase. In some embodiments, the microcapsules are dried in any suitable manner, for example, by freeze- drying.

[0060] Some embodiments of the present invention are further described with reference to the following examples, which are intended to be illustrative and not limiting in nature.

Example 1.0 - Materials and Methods

Example 1.1 - Materials

[0061] Alginic acid sodium salt from brown algae, κ-carrageenan, L-cysteine hydrochloride monohydrate, proteose peptone, bile salt, pepsin from porcine gastric mucosa, lysozyme from egg white, pancreatin from porcine pancreas, and Tween™ 80 (polysorbate 80) were all supplied from Sigma-Aldrich (Mississauga, ON, Canada); De man, Rogosa, Sharpe (Lactobacilli MRS). Broth, calcium chloride (CaCl ₂ ), sodium bicarbonate (NaHC0 ₃ ) and sodium chloride (NaCl) were purchased from EMD Chemicals Inc. LTD (Darmstadt, Germany); alkaline peptone and granulated Difco agar were obtained from Becton Dickinson & Company (Sparks, MD, USA); genepin was purchased from Challenge Bioproducts Co., LTD (Taiwan, China); hydrochloric acid (HC1) was purchased from Ricca Chemical Company (Arlington, TX, USA); potassium chloride (KC1) was purchased from VWR International LLC. (Radnor, PA, USA); sodium hydroxide (NaOH) was purchased from Fisher Scientific (Fair Lawn, NJ, USA); water (MQW) was purified by Milli-Q water purification system purchased from Millipore Corp. (Billerica, MA, USA).

Example 1.2 - Preparation of a chickpea protein isolate

[0062] Chickpea seeds, kindly donated by the Crop Development Centre (Saskatoon, SK), were initially ground using a bowl grinder (Cuisinart Mini-Prep Plus) followed by a fine grind (IKA Al l basic. IKA Works Inc., Wilmington, NC) to give flour. The flour was then defatted with hexane (L'Hocine, Boye, & Arcand, 2006) and then concentrated utilizing an isoelectric precipitation procedure (Can Karaca et al., 201 la). In brief, the defatted flour was dispersed in MQW at a 1 to 10 (w/v) protein/MQW ratio, followed by pH adjustment to 9.0 with 1.0 M NaOH so as to facilitate protein solubility. The resulting solution was stirred at 1000 rpm

(Ikamag Ret-G, IKA Labortechnik, Germany) for 1 h, and then centrifuged at 5000 x g for 20 min at 4°C (Sorvall RC6+; Thermo Fisher Scientific, Waltham, MA). The supernatant was collected for later use, and the process was repeated with a 1 to 5 (w/v) pellet/MQW ratio.

Supernatants from both extractions were pooled and adjusted to pH 4.6 with 1.0 M HC1 so as to facilitate protein precipitation. The precipitate was collected by centrifugation (5000 x g, 20 min, 4°C), washed with 25 mL of MQW, frozen (-30°C), and then freeze-dried (Labconco FreeZone, Kansas City, MO) to yield a free flowing powder. Protein isolates were stored at 4°C in sealed tubes until use.

[0063] The crude ash, lipid, moisture and protein (%N x 6.25) contents for the resulting isolate was determined according to the Association of Official Analytical Chemists (AOAC, 2003) methods: 923.03, 920.85, 925.10, and 920.87, respectively. The carbohydrate content was determined on the basis of percent differential from 100%. Chemical analysis of the isolate indicated protein, moisture, lipid, ash and carbohydrate levels were 85.76%, 2.39%, 0.83%; 4.41% and 6.89%, respectively.

Example 1.3 - Bacteria and Culture Conditions

[0064] Bifidobacterium adolescentis (ATCC 15703) was stored at -70°C in a 1/1 (v/v) suspension of 15% (w/v) glycerol and 5.22% (w/v) MRS broth. Cultures were streaked onto RCM agar supplemented with 0.05% (v/v) L-cysteine HC1 (RCM-cys) plates at 37°C under anaerobic conditions (80% N ₂ , 10% C0 ₂ and 10% H ₂ ) in an anaerobic chamber (Forma

Scientific Inc., Marietta, GA, USA). A preliminary growth study was conducted in RCM-cys broth to determine the stationary phase of growth according to Klemmer et al. (2011). Briefly, RCM-cys broth was autoclaved at 121°C for 15 minutes and left to equilibrate overnight within an anaerobic chamber. Two isolated colonies of B. adolescentis were used to inoculate 5 mL of sterile RCM-cys broth which was then incubated at 37°C for 24 hours under anaerobic conditions. A ΙΟΟ-μί aliquot of the starter culture was then transferred to 100 mL of RCM-cys broth followed by incubation under anaerobic conditions for 40 hours at 37°C. The optical density (600 nm) of the suspensions was measured over time using a Sequoia-Turner Model 340 spectrophotometer (Pegasus Scientific, Rockville, MD, USA). Determination of cell

concentrations at time 15, 20, 24 and 40 hours was performed, providing confirmation that the stationary phase of growth initiated at 20 hours with cell numbers of 9 log CFU mL ^"1 .

[0065] A total of 10.0 mL of 5.22% (w/v) MRS broth supplemented with 0.05% (w/v) L- cysteine-HCl and 1.5% (w/v) agar was inoculated with two isolated colonies of B. adolescentis for 20 hours at 37 °C under anaerobic conditions. After centrifugation for 5 minutes at 1000 x g, the resulting pellet was re-suspended in 1.00 mL of sterile 0.1% (w/v) alkaline peptone water (APW). Probiotics were then applied to encapsulation study and the final concentration (log CFU mL ^"1 ) of probiotics grown under anaerobic conditions at 37°C for 48 hours was determined through the spread plate count method conducted in duplicate.

Example 1.4 - Encapsulation of B. adolescentis

[0066] Microcapsules were prepared with the chickpea protein isolates using emulsion-based technology which employed a two-phase system to make a water-in-oil emulsion (Truelstrup Hansen et al, 2002; Butler, Ng, & Pudney, 2003; Krasaekoopt, Bhandari, & Deeth, 2003; Winder et al., 2003). Briefly, 9.0 niL of a chickpea protein (1.18 g protein power containing 1.00 g protein) solution (10% (w/w) with respect to the final volume of the aqueous phase, 10.0 mL) was prepared in MQW, adjusted to pH 7.0 with 0.5 M NaOH, and stirred at room temperature overnight (16 hours). Genipin, alginate or κ-carrageenan powder was added to the solution to reach a final concentration of 0.2% (w/v). Genipin and alginate powder could be added directly to the solution; however, in the case of κ-carrageenan, the powder was dissolved first at 60°C in advance and then cooled to room temperature before use. One mL of bacterial suspension was subsequently added to the protein-crosslinker mixture to bring the final volume of the aqueous wall material solution to 10.0 mL.

[0067] The mixture was stirred (750 rpm) for 5 minutes to achieve homogeneity and then added into 100.00 g of canola oil, and the resulting mixture was stirred with an overhead Real Torque Digital Stirrer (Caframo, Wiarton, ON, Canada) at 1000 rpm. In the case of genipin, the emulsion was stirred for 6 hours so as to afford time for crosslinking, followed by the addition of 100.00 g of MQW to break the emulsion in order to harvest the microcapsules. In contrast, in the presence of alginate or κ-carrageenan, emulsions were stirred for 0.5 hours, followed by the addition of 100.00 g of 0.1 M CaCl ₂ and 0.3 M KC1, respectively, to facilitate microcapsule formation via ionic crosslinking. The resulting suspension was then centrifuged for 5 minutes at 1000 x g to fully separate the oil phase from the capsule-aqueous phase. The aqueous phase was rinsed with 10.0 mL of a 1.0% Tween™ 80 (polysorbate 80) solution so as to maintain good capsule dispersability. The aqueous capsule phase was then transferred to a 50 mL separatory funnel and allowed to settle for 5 minutes prior to the final capsule harvest to ensure full removal of the oil phase. The concentration of viable encapsulated organisms (CFU mL ^"1 ) was then determined by plate counting on MRS-cys agar plates.

[0068] Microcapsules were prepared using pea, soy, faba bean and lentil protein isolate in the same manner as described above, with the alternate protein being substituted for chickpea protein isolate. Microcapsules were prepared using pea protein-iota carrageenan and pea protein-deacyl gellan gum mixtures in the same manner as described above, with the alternate biopolymer being substituted for alginate. Example 1.5 - Capsule Size

[0069] Capsule size (geometric mean diameter + standard deviation) was determined by light scattering using a Mastersizer 2000 equipped with a Hydro 2000S wet sample cell (Malvern Instruments, Westborough, MA, USA). Measurement conditions used in the Mastersizer included % obscurity (10 - 20%), a pump speed of 850 rpm, a sample absorbance default of 0.1 and refractive index values of 1.45 and 1.33 for the sample (protein) and dispersant (MQW), respectively. Experiments were performed using each capsule formulation for duplicate batches, with size analysis completed in duplicate.

Example 1.6 - Survival of Free and Entrapped B. adolescentis in pH 2.0 Solution

[0070] The viability of B. adolescentis released from chickpea protein- alginate microcapsules, as compared to free cells, was assessed in duplicate after microencapsulated bacteria were treated in acid challenge conditions at pH 2.0 and at room temperature (25°C). Briefly, 90.0 mL of MQW was adjusted to pH 2.0 with 2.0 M HC1 and 0.5 M NaOH. Chickpea capsules were produced as previously described, washed with 10.0 mL 0.1% of Tween™ 80 (polysorbate 80), and harvested by a centrifuge at 5000 x g for 10 minutes at 4°C, followed by a collection of the pellet. After harvesting capsules, 10.0 mL of the capsule solution was immediately added to the acid challenge solution under stirring (750 rpm). At 0, 5, 10, 20, 30, 60, 90 and 120 minutes after addition, a 100 μΐ _^ aliquot was sampled and serially diluted in 900 μΐ _^ sterile 0.1% APW to neutralize pH. The first dilution of each sample was homogenized (Omni International Inc., GA, USA) at 13,000 rpm for 30 seconds to break up the capsule wall. The number of viable surviving bacteria (as log CFU mL ^"1 ) was determined by plate counting after 48 hours anaerobic incubation at 37°C for each time tested. Replicate plates were counted at each time interval during the survival study, and then repeated in duplicate.

Example 1.7 - Survival of Free and Entrapped B. adolescentis in Synthetic Gastric Juice (SGJ)

[0071] SGJ was prepared with a total of 8.3 g of proteose peptone, 3.5 g of glucose, 2.05 g of NaCl, 0.6 g of KH ₂ P0 ₄ , 0.11 g of CaCl ₂ , 0.37 g of KC1, 0.05 g of bile, 0.1 g of lysozyme, and 13.3 mg of pepsin in 1 L of MQW with adjustment to pH 2.5 by 1 M HC1. SGJ was heated to 37°C for 30 min and sterile filtered before use. The ability of free and encapsulated B.

adolescentis to survive in SGJ [0.08 M HC1 and 0.2% NaCl (w/v)] was tested (Cotter, Gahan, & Hill, 2001). Chickpea protein-alginate capsules were produced and harvested as previously described. The free cell and the entrapped probiotics in chickpea- alginate capsules were re- suspended with 2.0 mL of 0.1% APW, and then incubated in 48.0 mL of SGJ with stirring (750 rpm) for 2 hours under anaerobic conditions at 37°C. Sampling was carried out at 10, 30, 60, and 120 minutes. A 100 μΐ _^ aliquot was sampled and serially diluted in 900 μΐ _^ sterile 0.1% APW to relieve SGJ stress. The first dilution of each sample was homogenized (Omni International Inc., GA, USA) at 13,000 rpm for 30 seconds to break up the capsule wall. The number of viable surviving bacteria (log CFU mL ^"1 ) was determined by plate counting after 48 hours anaerobic incubation at 37°C for each time tested. Replicate plates were counted at each time interval during the survival study, and then repeated in duplicate.

Example 1.8 - Release of B. adolescentis in Simulated Intestinal Fluid (SIF)

[0072] The ability of encapsulated B. adolescentis to be released from the chickpea protein- alginate capsules in SIF [1.25% (w/v) NaHC0 ₃ , 0.6% (w/v) oxgall dehydrated fresh bile, and 0.09% (w/v) pancreatin] was tested using a modified method from Laird et al. (2007). The free cell and entrapped probiotics in chickpea- alginate capsules were re-suspended with 2 mL of 0.1% APW, and then added into 48.0 mL SIF, pH 6.5 (adjusted with 0.1 M NaOH), with stirring (750 rpm) for 3 hours under anaerobic conditions at 37°C. Sampling was carried out at 10, 30, 60, 120, and 180 minutes. The diluted samples were directly incubated without mechanical homogenization to break the capsules. The number of viable surviving bacteria (log CFU mL ^"1 ) was determined by plate counting after 48 hours anaerobic incubation at 37°C for each time tested. Replicate plates were counted at each time interval during the release study, and repeated in duplicate.

Example 1.9 - Statistical Analysis

[0073] All experiments in this study were conducted in duplicate for each treatment. A two- tailed paired Students T-Test was used to test for statistical differences (p<0.05) between mean log CFU mL ^"1 values: 1) before and after encapsulation; 2) after exposure to pH 2.0 for 2 hours; and 3) after exposure to synthetic gastric juice for 2 hours. A similar test was used to determine differences between mean capsule sizes. All statistical analyses were performed using Microsoft Office Excel 2007 (Microsoft Canada Co., Mississauga, ON, Canada). Example 1.10 - SEM (Scanning Electron Microscopy) Analysis

[0074] Freshly-made microcapsules were analyzed using SEM to investigate differences in morphology. SEM images were obtained by conventional methods using microcapsules prepared from different plant proteins (chickpea, pea, soy and faba bean) in conjunction with alginate polysaccharide as the biopolymer.

Example 2.0 - Results and Discussion

Example 2.1 - Encapsulation and Survival of B. adolescentis Within Chickpea Protein-Based Capsules During an Acid Challenge

[0075] Bifidobacterium adolescentis was entrapped within a 10.00% (w/w) chickpea protein capsule crosslinked with (0.20% w/v) genipin or in the presence of (0.20% w/v) alginate or κ- carrageenan. Overall, the entrapment process did not significantly suppress the growth of B. adolescentis regardless of the capsule formulation (p>0.05). Prior to encapsulation, the number of viable B. adolescentis was 8.6 + 0.1 log CFU mL ^"1 , whereas afterwards cell counts ranged between 8.5 + 0.1 and 8.7 + 0.1 log CFU mL ^"1 for the protein capsules with alginate or κ- carrageenan, respectively. In contrast, for capsules prepared with genipin, viable cell counts were reduced to 7.8 + 0.1 log CFU mL ^"1 . Figure 1 shows viable log CFU mL ^"1 over a 2 hour acid challenge (pH 2.0/25°C) for encapsulated and free cells, and free cells with genipin. Viable cell counts after 2 hours were significantly reduced in all capsule formulations relative to time zero (p<0.01), as well as amongst themselves (p<0.01). Chickpea protein- alginate capsules offered the greatest protection to B. adolescentis (4.6 + 0.1 log CFU mL ^"1 ), followed by chickpea protein capsules prepared with K-carrageenan (3.5 + 0.3 log CFU mL ^"1 ) and genipin (1.8 + 0.1 log CFU mL ^"1 ) (Figure 1). In contrast, the majority of free cells died within the first 30-90 minutes of exposure to pH 2.0/25°C following a similar trend as the encapsulated CPI-alginate/κ- carrageenan (Figure 1). Viable cells within the CPI-genipin capsule showed the majority of losses within the first 30-40 minutes, below that of free cells alone. A similar trend was observed for the control of free cells in the presence of genipin (Figure 1). Without being bound by theory, it is postulated that the presence of genipin somehow induced cell death (Sung, Huang, Chang, Huang, & Hsu, 1999; Tsai, Huang, Sung, & Liang, 2000). [0076] The geometric mean capsule diameter for the different chickpea protein isolate formulations were all significantly different (p<0.01). Capsules size was largest for chickpea protein isolate capsules prepared in the presence of K-carrageenan (838.5 + 31.3 μιη), followed by genipin (749.5 + 2.3 μιη) and then alginate (21.9 + 1.2 μιη). Based on these findings, chickpea protein-alginate capsules appeared to offer better survival at pH 2.0/25°C than the other formulations and is of a more appropriate size (<100 μιη) size. However at these sizes, the high surface area to volume ratio can potentially lead to reduced survival under simulated gastric conditions (Sultana et al., 2000).

[0077] In the present study, without being bound by theory, differences in survival during an acid challenge and with capsule diameter were thought to reflect differences in the capsule wall, a consequence of biopolymer type and crosslinking. It is proposed that the wall within the chickpea protein-alginate capsules was more ordered than the other two designs due to the ionic crosslinking of a-L-guluronic acids of alginate with calcium (Rokka & Rantamaki, 2010) to form a more compact matrix with smaller pores, as evidenced by a much smaller capsule size.

Alginate's sensitivity to calcium ions is reported in the literature. In contrast, pores formed within chickpea protein-K-carrageenan capsules were presumed to be larger, due to the formation of a weaker polysaccharide network. Unlike ionic crosslinking which is almost instantaneous, genipin acts to form both inter- and intra-molecular covalent crosslinks between primary amines over time (Muzzarelli, 2009; Butler, Ng, & Pudney, 2003). As a result, the capsule was less compact and thought to have larger pores than the capsules prepared with alginate. Based on the acid challenge and size data, further experiments were conducted in the presence of alginate only.

Example 2.2 - Chickpea Protein- Alginate Capsules: The Effect of Alginate Concentration on the Survival of B. adolescentis During an Acid Challenge

[0078] In the present study, the effect of alginate concentration (0.05, 0.10 and 0.20%, w/v) within a 10.00% (w/w) chickpea protein capsule was investigated for their ability to protect B. adolescentis during a similar acid challenge experiment (pH 2.0/25°C). Overall, the entrapment process resulted in a slight decline in viable cells from 8.5 + 0.1 log CFU mL ^"1 (pre- encapsulation) with decreasing alginate concentration, where cell numbers were found to be 8.6 + 0.1, 8.4 + 0.3 and 8.1 + 0.2 log CFU mL ^"1 for capsules in the presence of 0.20%, 0.10% and 0.05% (w/v) alginate, respectively. Table 1 shows changes to viable cell counts for the different formulations and loss reductions over a 2 hour period at pH 2.0/25°C. After 2 hours, all capsule formulations were found to be significantly lower than time zero (p<0.01) and also different amongst themselves (p<0.01). The greatest protection was found for capsules prepared with 0.10% (w/v) alginate (0.6 + 0.3 log CFU mL ^"1 reduction), followed by 0.05% (w/v) (1.5 + 0.2 log CFU mL ^"1 reduction) and then 0.20% (w/v) (2.8 + 0.4 log CFU mL ^"1 reduction) (Table 1).

Table 1. Survival of B. adolescentis entrapped in chickpea protein (10% w/w) capsules in the presence of varying alginate concentrations (0.05-0.2% w/v), and entrapped within a 0.1% (w/v) alginate capsule (absence of chickpea protein) as a function of time during an acid challenge (pH

2.0/25°C). Data represent the mean + one standard deviation (n = 2).

Viable cells (log CFU mL ¹ )

Capsule design Log reduction

Time (0 h) Time (2 h)

CPI-0.05% (w/v) alginate 7.2 : b 0.2 5.7 d b O. l 1.5 : b 0.2

CPI-0.10% (w/v) alginate 7.1 : b 0.3 6.4 d 0.2 0.6 : b 0.3

CPI-0.20% (w/v) alginate 7.5 : b 0.4 4.6 d b O. l 2.8 : b 0.4

0.10% (w/v) alginate 7.5 : b 0.9 3.8 d b O. l 3.7 : b 0.7

[0079] Without being bound by theory, it is proposed that an optimal alginate to chickpea protein concentration ratio exists, resulting in differences in survival. At lower alginate hickpea ratios alginate-calcium linkages form larger pores. At higher alginate: chickpea ratios alginate chains undergo greater self-association to form thicker fibers and ultimately larger pores than at the optimal ratio.

[0080] Alginate capsules (0.10% w/v) without chickpea protein were also prepared and subjected to the same acid challenge, to show a 3.7 + 0.7 log CFU mL ^"1 cell number reduction over the 2 hour period (Table 1), which is significantly greater than when chickpea protein was present (p<0.05). [0081] Without being bound by theory, it is proposed that an alginate-Ca network developed to maintain capsule integrity, with the globular chickpea proteins packed within interstitial spaces acting to fill pores within the capsule. The formation of a 'cage-like network' within the chickpea protein-alginate capsule likely follows a similar mechanism as described by Sheu and Marchall (1993) for an alginate alone capsule crosslinked by Ca ²⁺ through an external gelation process. Alginate is a linear polysaccharide comprised of L-guluronic and D-mannuronic acid residues as homo- or co-block polymeric regions. As CaCl ₂ is added to the Na ⁺ -alginate- probiotic emulsion, gelation occurs immediately at the periphery of the matrix forming a solid Ca ²⁺ -alginate bead, followed by the movement of a gelling front as Ca ²⁺ ions diffuse into the interior of the capsule until all of the Na ⁺ ions have been replaced with Ca ²⁺ ions (Gilson, Thomas, & Hawkes, 1990; Quong & Neufeld, 1998, Quong, Neufeld, Skjak-Brak, & Poncelet, 1998). Calcium reacts strongly with the homogeneous block regions of L-guluronic residues to form a characteristic 'egg-box-like' junction zone. As the Ca ²⁺ ions migrate inwards, alginate chains also are drawn towards the ion front creating pore-like structures capable of housing live cells (Quong, Neufeld, Skjak-Braek, & Poncelet, 1998). Without being bound by theory, the excessive gelation at the capsule periphery is proposed to lead to greater compaction of the capsule structure, to reduce cell leakage and to increase protection to the entrapped cells.

Example 2.3 - Survival of Free and Entrapped B. adolescentis Within Synthetic Gastric Juice

[0082] The survival of free and entrapped B. adolescentis within a chickpea protein-alginate (0.1% w/v) capsule under synthetic gastric juice conditions (pH 2.5/37°C) was investigated over a 2 hour incubation period, as shown in Figure 2. Unlike the acid challenge above, SGJ contains various enzymes (e.g., pepsin and lysozyme) that could potentially lead to degradation of the chickpea protein during the assay, thereby releasing the bacteria. Over the 2 hour period, free and entrapped cell numbers were significantly reduced relative to initial values (p<0.01), as reflected by a 6.3 + 0.3 and 1.1 + 0.3 log CFU mL ^"1 reduction, respectively. In the case of free cells, viable cell numbers were reduced from 8.7 + 0.2 (0 h) to 2.4 + 0.2 log CFU mL ^"1 (2 hours); whereas, viability of entrapped cells dropped slightly from 8.7 + 0.2 to 7.8 + 0.2 log CFU mL ^"1 within the first 10 min and then became stable at approximately 7.5 + 0.2 log CFU mL ^"1 for the remainder of the 2 hour period (Figure 2). Example 2.4 - Release of Entrapped B. adolescentis in Simulated Intestinal Fluid

[0083] The release of B. adolescentis from chickpea protein-alginate (0.1% w/v) capsules within SIF was investigated at pH 6.5 37°C under anaerobic conditions over 3 hours. Almost all of the entrapped B. adolescentis cells (-7.8 + 0.6 log CFU mL ^-1 ) were released to give cell counts of -7.1 ± 0.1 log CFU mL ^"1 at T = 0 and -7.6 + 0.2 log CFU mL ^"1 after 10 min, followed by no further release (Figure 3). In contrast, free cells exposed to SIF became reduced in numbers from -7.0 + 0.1 log CFU mL ^"1 at time zero to 6.0 + 0.2 log CFU mL ^"1 after 30 min, hypothetically due to the presence of bile salt within the SIF which could result in reduced survival of the probiotics (Sahadeva et al, 2011). After 30 minutes, no further decline in cell viability occurred (Figure 3). Without being bound by theory, the higher viable cell count for cells upon release from the capsules relative to free cells may be attributed to protective effects of biopolymers free in solution (not in encapsulating form). The release of probiotics in SIF might be due to

destabilation of the alginate matrix by high concentration of Na ⁺ (Smidsrod, & Skjakbraek, 1990) or degradation of entrapped protein by proteases present in SIF (Klemmer et al., 2011).

[0084] The present examples demonstrate the potential for entrapping probiotics within a chickpea protein-based capsule, either crosslinked by genipin or in the presence of alginate or κ- carrageenan (plus salts). In this example, a chickpea protein capsule in the presence of 0.10% (w/v) alginate offered the best protection under the tested conditions to B. adolescentis within synthetic gastric juice. Capsules produced using this design were <100 μιη in size, and as such, there would be no perceived adverse effects on the sensory attributes of this ingredient into foods by consumers. Within simulated intestinal fluid, a burst-release of B. adolescentis was observed, likely due to high surface area:volume ratio of the capsules, with almost all cells being released within the first 5 minutes. These examples establish a sound prediction that chickpea protein- alginate microcapsule designs could serve as a suitable probiotic carrier intended for food applications to provide targeted delivery of a probiotic to the intestines of a mammalian subject, including a human subject.

Example 3.0 - Other Plant-Based Proteins

[0085] Experiments were conducted under similar conditions to assess microcapsules containing crosslinked alginate (at 0.1% w/v) and pea, soy, faba bean or lentil protein. [0086] The capsule sizes of the microcapsules so obtained were 18.380 μιη (pea), 21.001 μιη (soy), 21.046 μιη (faba bean), and 869.840 μιη (lentil). It was found through SEM images discussed below that chickpea-alginate and pea-alginate microcapsules formed spherical structures, whereas soy-alginate, faba bean-alginate and lentil-alginate microcapsules resembled a mass of biopolymer with no specific shape. Without being bound by theory, it is believed that the chickpea-alginate and pea-alginate microcapsules were stronger and maintained their shape and integrity better than alginate microcapsules formed with the other plant proteins.

[0087] Figure 4 shows the protection of B. adolescentis from simulated gastric conditions by the alginate microcapsules with pea, soy, faba bean or lentil protein. Figure 5 shows the release of B. adolescentis from the microcapsules under simulated intestinal conditions.

[0088] These results demonstrate that microcapsules made from a combination of alginate and pea protein or alginate and soy protein provide strong protection to an encapsulated probiotic under simulated gastric conditions, while allowing release of the encapsulated probiotic under simulated intestinal fluid. Thus, it can be soundly predicted that microcapsules made from a combination of alginate and pea protein or alginate and soy protein will be able to provide targeted delivery of a probiotic through the stomach of a mammalian subject to its intestine.

Example 4.0 - Other Biopolymers

[0089] Microcapsules were prepared using pea protein-iota carrageenan (P-I) and pea protein- deacyl gellan gum (P-D) mixtures to determine if either polysaccharide could be used instead of alginate. Entrapped B. adolescentis within synthetic gastric juice underwent a 2 log reduction in viable CFU/mL over the 2 h period (Figure 6). However, release studies within simulated intestinal fluid over a 3 h duration indicated a similar release profile as seen for other proteins (Figure 7). Once released, probiotics remained in a viable state. The data in Figures 6 and 7 can be compared with the data for survival of free cells shown in Figures 2 and 4, and Figures 3 and 5, respectively. These results demonstrate that microcapsules prepared using iota-carrageenan or deacyl gellan gum as the biopolymer can provide protection to probiotics under simulated gastric conditions while allowing release under simulated intestinal conditions. It can therefore be soundly predicted that iota-carrageenan and deacyl gellan gum can be used as the biopolymer in some embodiments of the present invention to provide targeted delivery of probiotics, probiotics and prebiotics, and/or synbiotics to the intestine of a mammalian subject, including a human subject.

Example 5.0 - SEM (Scanning Electron Microscopy)

[0090] SEM images were obtained using microcapsules prepared from different plant proteins (chickpea, pea, soy, faba bean or lentil protein) in conjunction with alginate polysaccharide. SEM images indicated that the microcapsules formed using chickpea or pea protein were more distinct and spherical (Figures 8A/8B and 9A/9B). In contrast, microcapsules formed using soy, faba bean or lentil protein isolate formed using alginate appeared more as a aggregated mass rather than a distinct capsule (Figures 10A/10B, 11 A/1 IB and 12A/12B), and was less-effective at protecting the probiotic bacteria during acid challenge experiments.

[0091] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are consistent with the broadest interpretation of the specification as a whole.

References

The following references are related to the subject matter disclosed herein, and each such reference is hereby incorporated by reference herein in its entirety:

Alander, M., De Smet, I., Nollet, L., Verstraete, W., von Wright, A., & Mattila-Sandholm, T. (1999). The effect of probiotic strains on the microbiota of the simulator of the human intestinal microbial ecosystem. International Journal of Food Microbiology, 46, 71-79.

Annan, N. T., Borza, A. D., & Truelstrup Hansen L. (2008). Encapsulation in alginate-coated gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis 15703T during exposure to simulated gastro-intestinal conditions. Food Research International, 41, 184-193.

AOAC. (2003). Methods 920.87. In: Official Method of Analysis, 17th Edn (Washington, DC: Association of Official Analytical Chemists).

Araya, M., Morelli, L., Reid, G., Sanders, M. E., Stanton, C, Pineiro, M., & Ben Embarek, P.

(2002). Guidelines for the evaluation of probiotics in food - Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food, London, ON, Canada.

Bielecka, M. (2007) Probiotics in food. In: Sikorshi, Z. E. (Eds), Chemical and Functional Properties of Food Components (pp. 413-426). New York: CRC Press.

Butler, M. F., Ng, Y. F., & Pudney, P. D. A. (2003). Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups and genipin. Journal of Polymer Science Part A: Polymer Chemistry, 41, 3941-3953.

Can Karaca, A., Low, N. H., & Nickerson, M. T. (2011a). Emulsifying properties of chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction. Food Research International, 44, 2742-2750.

Can Karaca, A., Nickerson, M. T., & Low, N. H. (2011b). Lentil and chickpea protein-stabilized emulsions: optimization of emulsion formulation. Journal of Agricultural and Food

Chemistry, 59(24), 13203-13211.

Chandramouli, V., Kailasapathy, K., Peiris, P., & Jones, M. (2004). An improved method of microencapsulation and its evaluation to protect Lactobacillus spp. in simulated gastric conditions. Journal of Microbiological Methods, 56(1), 27-35.

Charteris, W. P., Kelly, P. M., Morelli, L., & Collins, J. K. (1998). Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic

Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. Journal of Applied Microbiology, 84, 759-768. Clark, P. A., & Martin, J. H. (1994). Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: III- Tolerance to simulated bile concentrations of human small intestines. Cultured Dairy Products Journal, 29, 18-21.

Collins, M. D., & Gibson, G. R. (1999). Probiotics, prebiotics, and synbiotics: approaches for modulating the microbial ecology of the gut. American Journal of Clinical Nutrition, 69, 1052S-1057S.

Cotter, P. D., Gahan, C. G. M., & Hill, C. (2001). A glutamate decarboxylase system protects Listeria monocytogenes in gastric fluid. Molecular Microbiology, 40, 465-475.

Cui, J., Goh, J., Kim, P., Choi, S., & Lee, B. (2000). Survival and stability of bifidobacteria loaded in alginate poly-l-lysine microparticles. International Journal of Pharmaceutics, 210(1-2), 51-59.

Farnworth, R. (2007). Probiotics and prebiotics. In: Wildman R. E. C. (Eds), Handbook of

Nutraceuticals and Functional Foods (pp. 335-352). New York: CRC Press.

Fuller, R. (1989). Probiotics in man and animals. Journal of Applied Bacteriology, 66, 365-378.

Fuller, R. (1991). Probiotics in human medicine. Gut, 32, 439-442.

Gibson, G. R., & Roberfroid, M. B. (1995). Dietary modulation of the human colonic microbiota - introducing the concept of prebiotics. Journal of Nutrition, 125, 1401-1412.

Gilson, C. D., Thomas, A., & Hawkes, F. R. (1990). Gelling mechanism of alginate beads with and without immobilized yeast. Process Biochemistry International, 25, 104-108.

Gothefors, L. (1989). Effects of diet on intestinal flora. Acta paediatrica Scandinavica

Supplement, 351, 118-121.

Gruber, P., Longer, M.A. and Robinson, J.R. 1987. Some biological issues in oral, controlled drug delivery. Adv. Drug Delivery Rev., 1: 1-18.

Guerin, D., Vuillemard, J. C, & Subirade, M. (2003). Protection of bifidobacteria encapsulated in polysaccharide-protein gel beads against gastric juice and bile. Journal of Food Protection, 66, 2076-2084.

Holzapfel, W. H., Haberer, P., Snel, J., SchiUinger, U., & Huis in't Veld, J. H. (1998). Overview of gut flora and probiotics. International Journal of Food Microbiology , 41, 85-101.

Hooper, L. V., Wong, M. H., Thelin, A., Hansson, L., Falk, P. G., & Gordon, J. I. (2001).

Molecular analysis of commensal host-microbial relationships in the intestine. Science, 291, 881-884.

Hoover, D. G. (1993). Bifidobacteria: activity and potential benefits. Food Technology 47, 120- 124.

Kararli, T.T. 1995. Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharmaceutics & Drug Disposition. 16: 351-380.

Klemmer, K. J, Korber, D. R., Low, N. H, & Nickerson, M. T. (2011). Pea protein-based capsules for probiotic and prebiotic delivery. International Journal of Food Science and Technology, 46, 2248-2256.

Kotikalapudi, B. L., Low, N. H., Nickerson, M. T., & Korber, D. R. (2010). In vitro

characterization of probiotic survial, adherence and antimicrobial resistence: candidate selection for encapsulation in a pre proein isolate-alginate delivery system. International Journal of Probiotics and Prebiotic s, 5, 1-12.

Krasaekoopt, W., Bhandari, B., & Deeth, H. (2003). Evaluation of encapsulation techniques of probiotics for yogurt. International Dairy Journal, 13, 3-13.

Laird, B. D., Van de Wiele, T. R., Corriveau, M. C, Jamieson, H. E., Parsons, M. B., Verstraete, W., & Siciliano, S. D. (2007). Gastrointestinal microbes increase arsenic bioaccessibility of ingested mine tailings using the simulator of the human intestinal microbial ecosystem.

Environmental Science and Technology, 41, 5542-5547.

Lee, K., & Heo, T. (2000). Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution. Applied and Environmental

Microbiology, 66(2), 869-873.

L'Hocine, L., Boye, J. I., & Arcand, Y. (2006). Composition and functional properties of soy protein isolates prepared using alternative defatting and extraction procedures. Journal of Food Science: C - Food Chemistry and Toxicology, 71, C137-C145.

Mitsuoka, T. (1982). Recent trends in research on intestinal flora. Bifidobacteria Microflora, 1, 3-24.

Muzzarelli, R. A. A. (2009). Genipin-crosslinked chitosan hydrogels as biomedical and

pharmaceutical aids. Carbohydrate Polymers, 77, 1-9.

Pridmore, R. D., Berger, B., Desiere, F., Vilanova, D., Barretto, C, Pittet, A. C, Zwahlen, M. C, Rouvet, M., Altermann, E., Barrangou, R., Mollet, B., Mercenier, A., Klaenhammer, T., Arigoni, F., & Schell, M. A. (2004). The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proceedings of the National Academy of Sciences of the United States of America, 104, 2512-2517.

Quong, D., & Neufeld, R. J. (1998). DNA protection from extracapsular nucleases, within chitosan- or poly-L-lysine-coated alginate beads. Biotechnology and Bioengineering, 60, 124- 134.

Quong, D., Neufeld, R. J., Skjak-Braek, G., & Poncelet, D. (1998). External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation. Biotechnology and Bioengineering, 57, 438-446. Rao, A. V., Shiwnarain, H., & Maharaj, I. (1989). Survival of microencapsulated

Bifidobacterium pseudolongum in simulated gastric and intestinal juices. Canadian Institute of Food Science and Technology Journal, 22, 345-349.

Rastall, B. (2007) Prebiotics. In: Sikorski (Eds), Chemical and Functional Properties of Food Components(pp. 391-411). New York: CRC Press.

Ravula, R. R., & Shah, N. P. (1998). Viability of probiotic bacteria in fermented frozen dairy desserts. Food Australia, 50, 136-139.

Reid, A., VuiUemard, J. C, Britten, M., Arcand, Y., Famworth, E., & Champagne, C. P. (2005). Microentrapment of probiotic bacteria in a Ca2+-induced whey protein gel and effects on their viability in a dynamic gastro-intestinal model. Journal of Microencapsulation, 22, 603- 619.

Rokka, S., & Rantamaki, P. (2010). Protecting probiotic bacteria by microencapsulation:

challenges for industrial applications. European Food Research and Technology, 231, 1-12.

Sahadeva, R. P. K., Leong, S. F., Chua, K. H., Tan, C. H., Chan, H. Y., Tong, E. V., Wong, S. Y. W., & Chan, H. K. (2011). Survival of commercial probiotic strains to pH and bile.

International Food Research Journal, 18(4), 1515-1522.

Sarkar, S. (2007). Potential of prebiotics as functional foods-a review. Nutrition and Food

Science, 37, 168-177.

Sartor, R. B. (2004). Therapeutic manipulation of the enteric microflora in inflammatory bowel disease: antibiotics, probiotics, and prebiotics. Gastroenterology, 126(6), 1620-1633.

Scardovi, V. (1986). Genus Bifidobacterium. In: Mair, N.S. (Eds), Bergey's Manual of

Systematic Bacteriology, Vol. 2. Williams and Wilkins (pp. 1418-1434). New York: Bergey's Manual Trust.

Smith, H.W. 1965. Observations of the flora of the alimentary tract of animals and factors affecting its composition. J. Pathol. Bacterid., 89: 95-122.

Sung, H. W., Huang, D. M., Chang, W. H., Huang, R. N., & Hsu, J. C. (1999). Evaluation of gelatin hydrogel cross-linked with various cross-linking agents as bioadhesives: In vitro study. Journal of Biomedical Materials Research, 46, 520-530.

Sheu, T. Y. & Marshall, R. T. (1993). Microentrapment of lactobacilli in calcium alginate gels. Journal of Food Science, 58, 557-561.

Shortt, C. (1999). The probiotic century: historical and current perspectives. Trends in Food Science and Technology, 10, 411-417.

Smidsrod, O., & Skjakbraek, G. (1990). Alginate as immobilization matrix for cells. Trends in Biotechnology, 8, 71-78.

Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., & Kailasapathy, K. (2000). Encapsulation of probioitc bacteria with alginate- starch and evaluation of survival in simulated gastrointestinal conditions and in yogurt. International Journal of Food

Microbiology, 62, 47-55.

Sun, W., & Griffiths, M. W. (2000). Survival of bifidobacteria in yogurt and simulated gastric juice following immobilization in gellan-xanthan beads. International Journal of Food Microbiology, 61, 17-25.

Truelstrup Hansen, L., Allan- Wojtas, P. M., Jin, Y. L., & Paulson, A. T. (2002). Survival of Ca- alginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions. Food Microbiology, 19, 35-45.

Tsai, C. C, Huang, R. N., Sung, H. C, & Liang, H. C. (2000). In vitro evaluation of the genotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. Journal of Biomedical Materials Research, 52, 58-65.

Winder, R. S., Wheeler, J. J., Conder, N., Otvos, I. S., Nevil, R., & Duan, L. (2003).

Microencapsulation: a strategy for formulation of inoculum. Biocontrol Science and

Technology, 13, 155-169.

Wood, K. (2010). Synbiot Production and Microencapsulation. M.Sc. Thesis. Saskatoon:

University of Saskatchewan.

Yaeshima, T. (1996). Benefits of bifidobacteria to human health. Bulletin of the International Dairy Federation, 313, 36-42.