WO2013188626A2

WO2013188626A2 - Probiotic-containing particles having improved probiotic stability when in aqueous formulations

Info

Publication number: WO2013188626A2
Application number: PCT/US2013/045577
Authority: WO
Inventors: Liang Hong; Krishna Madduri; Kelsey PRESSLER; Christopher J. Tucker; Caroline Woelfle-Gupta
Original assignee: Dow Global Technologies Llc
Priority date: 2012-06-15
Filing date: 2013-06-13
Publication date: 2013-12-19
Also published as: WO2013188626A3

Abstract

Probiotic-containing compositions are provided which comprise a probiotic, an oxygen carrying compound, and a bio-compatible binder. The probiotic-containing compositions have stable probiotic viability and are suitable for incorporation in aqueous personal care formulations, such as cosmetic and non-cosmetic lotions, soaps, creams, shampoos, body washes, and the like.

Description

PROBIOTIC-CONTAINING PARTICLES HAVING IMPROVED

PROBIOTIC STABILITY WHEN IN AQUEOUS FORMULATIONS

Field of the Invention

The present invention relates to probiotic-containing compositions having stable probiotic viability when incorporated in aqueous personal care formulations. The probiotic- containing compositions comprise a biocompatible polymer, a probiotic and an oxygen carrying compound. Background

Probiotics are live microorganisms, such as bacteria or yeast, which when topically applied or orally administered, in adequate amounts to a subject, confer a health benefit to the subject. It is well-known to include probiotics in food products, personal care products, and beverage products, among others, as means for delivering such health benefits. Regardless of the type of probiotic or benefit to be conferred, an effective quantity of the probiotic must be alive ("viable") in the product at the time of application or administration for the benefit to be conferred.

Various conditions occurring during manufacture, transport, storage, application and administration of probiotic-containing products affect the viability of the probiotic. "Live" or "viable" probiotics are those members, or "units," which are capable of metabolizing nutrients and reproduction, i.e., colony forming units (cfu). The probiotic viability should also be "stable," i.e., remain viable over a meaningful period of time, such as days, weeks, or even years, to allow for manufacture, transport and storage. Stable probiotic viability may be measured as a decrease in the quantity of colony forming units (cfu). For example, a log reduction in cfu is a ten-fold decrease in the quantity of cfu, which represents a ten-fold decrease in the viability of the overall probiotic population.

Even when manufacturing, transport and storage processes have been carefully designed and performed to avoid exposing the probiotic to typical adverse conditions such as heat, light, shearing forces, etc., the conditions which exist during application or administration, or even as a consequence of the type of product in which the probiotic has been formulated or the environment in which the probiotic is being applied or administered, may reduce viability of the probiotics over time. To date, efforts to maintain and improve the stability of probiotic viability have concentrated on freeze drying the probiotic of interest and then keeping it dry, usually by using a hydrophobic coating or other encapsulating technique. For example, U.S. Patent Application Publication No. 2008/0260893 discloses a foodstuff which includes probiotics coated or encapsulated in polysaccharide, fat, starch, protein or sugar to protect the probiotics from inactivation by processing and storage conditions. Considerable research has been done on strategies for dry shelf stability, as well as enteric coatings to protect the probiotics as they pass through a subject's stomach. Nonetheless, viability of probiotics appears to decrease over time, even in dry environments. Furthermore, in environments where the moisture is about 10% or greater water, activity drops precipitously (see, U.S. Patent Application Publication No. 2005/0266069, paragraph [0025], asserting a 4 log reduction in 14 days at 37°C for one variety of encapsulated probiotic in a 10% water environment). This would seem to recommend against incorporating probiotics in aqueous personal care formulations, or least to expect relatively short shelf lives due to the presence of water.

International Patent Application Publication No. WO2010-013182 describes the cosmetic use of probiotics, such as Lactobacillus or Bifidobacterium species, for treating scalp disorders including dandruff. At the time of application, the probiotics may be viable (live) or inactivated (dead) and are delivered in a composition which may include hydrophilic gelling agents (e.g., carboxylic polymers, poly aery lamides, hydroxyalkylcelloloses and natural gums such as guar gum, xantham gum), or lipophilic gelling agents (e.g., modified clays, metal salts of fatty acids, ethylcellulose or polyethylene).

U.S. Patent Application Publication No. 2003/0194423 describes a ready-to-use food composition for providing supplemental nutrition which may include probiotics, as well as one or more humectants (e.g., a sugar, a polyhydroxyl alcohol), one or more fatty acids (e.g., lauric acid, strearic acid, oleic acid) and vegetable oils (coconut oil, cotton seed oil, sunflower oil) for controlling the probiotic population. The food composition may be in the form of an aqueous solution, liquid concentrate, or a colloidal suspension, and may have the physical characteristics of a gel, a sol-gel, a gravy or a syrup.

U.S. Patent Application Publication No. 2006/0067984 discloses micro-pellets capable of controlled release of physiologically active substances, such as probiotics, amino acids, enzymes, etc., although no actual examples including probiotics are provided. The micro-pellets have diameters between 0.5 and 5 millimeters and are multilayered. More particularly, the micro-pellets described in US 2006/0067984 have a core comprising the active substance and a matrix carrier which may include binders such as rubber, cellulose, starch, waxes, and fats. The micro-pellets also have an external coating which comprises a hydrophobic film (e.g., made from lauric acid, stearic acid) as the initial layer, and an impact and temperature resistant second layer (e.g., made from vegetable, paraffin and synthetic waxes).

U.S. Patent Application Publication No. 2009/0041911 describes a concentrated, clear, liquid beverage which is shelf stable and may include nutritional additives such as probiotics, antioxidants, amino acids, fiber, collagen, and glucosamine, among others. The concentrated beverage may also include a thickening agent comprising collagen (gelatin), gellan gum, carbohydrate gel-forming polymers (e.g., pectin, starches, sugars), carrageenan, carbo bean, gum, alginates, xanthan and carboxymethyl cellulose. However, there is no detailed discussion or examples of such concentrated liquid beverages actually containing probiotics, so any difficulties or issues relating to inclusion of probiotics would not have been encountered by these applicants.

International Patent Application Publication No. WO 2000/40252 discloses that polymer formulations containing perfluorinated compounds for biological applications can be used to improve cell metabolism and survival as a result of the oxygen carrying capacity of the perfluorinated compounds. Generally, liquid perfluorinated compounds have a high solubility for oxygen, carbon dioxide and other non-polar gases. Furthermore, the lack of chemical bonds between oxygen and PFC (oxygen molecules simply occupy cavities between the molecules of the liquid PFCs) allows the easy release of oxygen to adjacent media or substrates (aqueous solution, skin or hair, etc.). This easy release of oxygen from perfluorinated compounds recommends against their inclusion in probiotic-containing compositions since too much oxygen could be delivered to dry, dormant encapsulated probiotics by the perfluorinated compounds and cause oxygen toxicity, thereby reducing viability of the probiotics.

Thus, while probiotics are known and used to confer various health benefits when incorporated in aqueous formulations, their stability suffers in such environments due, at least in part, to the presence of water. Thus, there remains a need for improving the viability stability of probiotics incorporated in aqueous formulations, such as topically applied aqueous personal care formulations. Improvements in the stability of probiotic viability in such formulations would extend their useful shelf life and also permit use of smaller quantities of probiotics therein. It would also be useful if probiotic-containing compositions also contained other beneficial components which did not interfere with the probiotic activity or viability. The present invention addresses this need by providing probiotic-containing compositions which also include oxygen carrying compounds and have stable probiotic viability, even when incorporated into aqueous formulations. Summary of the Invention

The present invention provides a probiotic-containing composition having stable viability of the probiotic when incorporated into aqueous personal care formulations. The composition comprises: A) a binder; B) a probiotic; and C) an oxygen-carrying compound.

The binder is one or more bio-compatible polymers which may be selected from the group consisting of: methylcellulose, ethylcellulose, alginate, gellan gum, and combinations thereof.

In some embodiments of the present invention, the probiotic in the composition experiences a reduction in viable population of less than 3.5 log cfu.

In some embodiments of the present invention, the probiotic-containing composition comprises microspheres which comprise components A), B) and C). In some embodiments, the microspheres have an average particle size less than 500 microns.

In some embodiments, the probiotic-containing composition also comprises an effective amount of bacteriostatic agent, such as lauric acid.

Detailed Description

Hereinafter, each recited range includes all combinations and subcombinations of ranges, as well as specific numerals contained therein.

The probiotic-containing compositions of the present invention also contain an oxygen-carrying compound and have stable probiotic viability when formulated in aqueous products. These probiotic-containing compositions are, therefore, useful for formulating various aqueous personal care products such as, but not limited to, skin care lotions, soaps, detergents, hair care products, hand sanitizers, body washes, as well as other topically applied cosmetic and non-cosmetic products. More particularly, the probiotic-containing compositions of the present invention comprise a binder, a probiotic and an oxygen carrying compound.

Examples of suitable probiotic micro-organisms include yeasts such as Saccharomyces, Debaromyces, Candidaw Pichia and Torulopsis, molds such as Aspergillus, Rhizopus, Mucor, Torulopsis, and Penicillium and bacteria such as the genera Bifidobacterium, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Streptococcus, Enterococcus, Lactococcus, Kocuriaw, Staphylococcus, Peptostrepococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, Oenococcus and Lactobacillus. Non-limiting examples of Lactobacillus include L. acidophilus, L. alimentarius, L. amylovorus, L. crispatus, L. brevis, L. case4 L. curvatus, L. cellobiosus, L. delbrueckii ss. bulgaricus, L farciminis, L. fermentum, L. gasseri, L. helveticus, L. lactis, L. plantarum, L. johnsonii, L. reuteri, L. rhamnosus, L. sakei, and L. salivarius.

Suitable probiotics may be obtained from Jarrow Formulas, for example,

Lactobacillus plantarum 299v (active ingredient of Ideal Bowel Support™ 299v; 10 billion live organisms) or Custom Probiotics, for example L. plantarum, L. rhamnosus, or L. acidophilus.

"Stable" probiotic viability, in the context of the present invention, means that the probiotic retains sufficient viability to confer its health benefit over a reasonable period of time. Such amounts are known to those skilled in the art for each strain of probiotic, based on the intended use of the probiotic. For example, in some embodiments of the composition of the present invention, "stable" viability of the probiotic is accomplished when the probiotic experiences a reduction in viable population of no more than 3.5 log cfu, , or no more than 2.5 log cfu, or even no more than 1.5 log cfu. In other embodiments, stable viability of the probiotic may be accomplished when the probiotic experiences a reduction in viable population of such as no more than 1.3 log cfu, or no more than 1.0 log cfu, or no more than 0.9 log cfu, or even no more than 0.8 log cfu.

The binder should comprise one or more biocompatible polymers. A biocompatible polymer may be synthetic or natural and is capable of replacing part of a living system or functioning in intimate contact with living tissue without interfering with the health and operation of the living tissue. Generally, biocompatible materials interface with biological systems to evaluate, treat, augment or replace bodily tissues, organs, or functions.

Various synthetic, natural, and modified polymers are suitable for use as the biocompatible polymer in the probiotic -containing composition of the present invention, including, without limitation, keratin; casein; albumin; collagen; gellan gum; glutelin; glucagon; gluten; zein; gelatins and derivatives thereof; polymers derived from chitin or from chitosan; polysaccharide polymers such as cellulose-based polymers, for example, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, carboxymethylcellulose and quaternized cellulose derivatives; starches and derivatives thereof; acrylic polymers or copolymers such as polyacrylates; polymethacrylates and copolymers thereof; polyvinyl alcohols; as well as polymers of natural origin, which are optionally derivitized, such as gum arabic, guar gum, xanthan derivatives or karaya gum, alginates, carrageenans, ulvanes and other algal colloids, glycoaminoglycans, hyaluronic acid and its derivatives, shellac, sandarac gum, dammar resins, elemi gums and copal resins, deoxyribonucleic acid, mucopolysaccharides such as hyaluronic acid, chondroitin sulphate, caprolactams, pullulan, pectin, mannan and galactomannans, and glucomannans, and derivatives thereof. Optionally, two or more such biocompatible polymers may be blended for use as the binder in accordance with the present invention. Non-limiting examples of preferred binders include ethylcellulose and alginate.

In some embodiments of the present invention, the biocompatible polymer is crosslinked to form gel beads comprising the probiotic and oxygen carrying compound. Such gel beads may, for example, be formed by contacting the polymer with an ionic or nonionic crosslinking agent.

Typical gel-forming cations capable of crosslinking such polymers include, but are not limited to, alkaline earth metals, transition metals, and poly ammonium ions. Exemplary cations include magnesium (Mg²⁺), calcium (Ca²⁺), strontium (Sr²⁺), barium (Ba²⁺), aluminum (Al³⁺), boron (B³⁺), lead (Pb⁴⁺ or Pb²⁺), copper (Cu⁺ or Cu²⁺), cadmium (Cd²⁺), zinc (Zn²⁺), nickel (Ni²⁺), manganese (Mn⁴⁺ or Mn²⁺), iron (Fe²⁺ or Fe³⁺), dichromate (Cr₂0₇ ² ), ⁺H₃N(-CH₂)_n -CH((CH₂-)_m-NH₃ ⁺)((CH₂)_p-NH₃ ⁺-) and ⁺H₃N(-CH₂)_n-NH₃ ⁺ or where n is an integer ranging from 1 to 8, and m and p are integers ranging from 0 to 8 ions, or a combination thereof. Specific crosslinking ions include salts of alkaline earth elements along with aluminum; for example, magnesium (Mg²⁺), calcium (Ca²⁺), strontium (Sr²⁺), barium (Ba²⁺), aluminum (Al³⁺), or a combination thereof. A more specific crosslinking cation is calcium (Ca²⁺).

Suitable anions may be derived from polybasic organic or inorganic acids for use as crosslinking agents. Appropriate crosslinking anions include, but are not limited to, phosphate, sulfate, citrate, borate, succinate, maleate, adipate, and oxalate ions. Non-ionic crosslinking agents include, aldehydes, epoxides, and melamine. Mixtures of various crosslinking agents may be used, if desired.

Furthermore, in some embodiments of the present invention, the probiotic-containing compounds may be in the form of microspheres. The microspheres may be formed in any manner, known now or in the future, such as by fluid bed agglomeration, gel bead formation, spray drying, or foam granulation. For example, to form gel beads, generally, the probiotic and the oxygen carrying substance may be blended together and then added to a solution containing one or more binders, after which gel-forming agents are added to form microspheres. In some embodiments, conditions may be controlled to produce microspheres having an average a particle size less than 500 microns. Typically, the probiotic is lyophilized and then either coated, dispersed, or otherwise entrained in the microsphere. In some embodiments, the microspheres may have a multilayer composition.

Fluid bed coating methods provide another way of producing particulate compounds containing both probiotics and perfluorodecalin, in accordance with the present invention. Various different types of fluidized bed coating processes are well-known to persons of ordinary skill in the relevant art and are suitable for use in the present invention, including, without limitation, top-spray coating, tangential- spray coating, and bottom spray-coating methods. For instance, the "Wurster" process is a bottom spray type of process very commonly used in the pharmaceutical industry to coat particles since it has the advantage of consistently providing particles having very homogenous coatings. More particularly, the Wurster process is characterized by the location of a spray nozzle at the bottom of a fluidized bed of solid particles suspended in a fluidized bed coater chamber using an air or nitrogen stream. The nozzle sprays an atomized flow of coating solution, or suspension on the particles, which creates a uniform coating around the particles. In accordance with the present invention, more particularly, a bed of probiotics, in freeze-dried powder form, would be fluidized in a fluidized bed coater chamber. A solvent based solution of perfluorodecalin (perfluorodecalin is miscible with some hydrocarbons (e.g., hexane in some cases)) would then be injected into the chamber and onto the powder, followed by injection of a solvent based solution of a biocompatible polymer (binder) suitable for personal care applications. The solvent based solutions would form one or more coatings on the freeze-dried probiotics particles, thereby producing microspheres having a particle size between 50 and ΙΟΟΟμιη.

Water activity is a measure of the intensity with which water associates with various non-aqueous constituents included in an aqueous system (i.e., mixture or solution). Generally, water activity measures the energy status of the water in a system. Water activity is defined as [the partial pressure of water vapor at the surface of the product at a given temperature] divided by [the saturation pressure of the partial pressure of water vapor above pure water at the same temperature]. Thus, for example, the water activity of pure distilled water is 1.0. In the context of foods and beverages, water activity of >0.3 to <0.5 is considered semi-dry, >0.5 to <0.7 is considered semi-moist, and >0.7 or above is considered moist. Of course, aqueous formulations intended for use as personal care products may have water activities almost anywhere along the same categories.

In some embodiments of the present invention, probiotic -containing compositions in accordance with the present invention are incorporated in aqueous personal care formulations, i.e., having water in an amount of greater than 20% water, such as greater than 40% water, or greater than 60% water, or even greater than 80% water.

Aqueous formulations (i.e., those comprising water in an amount of 10 wt % or greater based on the total weight of the formulation) are relatively low oxygen environments. As already mentioned, it is known and expected for probiotics to experience a significant loss of viability when incorporated in aqueous formulations, even if the probiotics are first dried and protected by coating or encapsulation. Furthermore, incorporation of an oxygen carrying compound with coated or encapsulated probiotics might be expected to result in an excessive supply of oxygen in contact with the probiotics, resulting in oxygen toxicity and lower or unstable viability of the probiotics. Without wishing to be bound by theory, it has been surprisingly discovered that the low oxygen aqueous media of aqueous formulations appears to prevent significant transport of oxygen to the probiotics by the oxygen carrying compound and, thereby, prevents oxygen toxicity to the probiotics. Thus, incorporation of a composition comprising an oxygen carrying compound along with the probiotics and the binder in aqueous personal care formulations to be topically applied to a subject should have probiotic stability as well as providing the benefits of oxygen delivery to the subject's skin, nails and hair.

Oxygen carrying compounds suitable for use in the probiotic-containing compositions of the present invention include perfluorocarbons (PFCs), cyclohexanes, and derivatives or combinations thereof. PFCs suitable for use as oxygen carrying compounds in the probiotic- containing compositions of the present invention may be, for example, without limitation, selected from the group consisting of: perfluorodecalin, perfluorotributylamine, perfluorooctyl bromide, bis-perfluorbutyl-ethene and perfluoro-n-octane, dimethylcyclohexane. In addition, cyclohexanes suitable for use as the oxygen carrying compound according to the present invention may comprisedimethylcyclohexane, among others.

The probiotic-containing compositions of the present invention may include other ingredients along with the binder, probiotic and oxygen carrying compound, such as other active ingredients that are not incompatible with the probiotic, such as pigments, colorants, or fragrances, or even additional agents to control the stability of the probiotic, such as an effective amount of a bacteriostatic agent, for example, nisin, decanoic acid, lauric acid, chitosan, sodium benzoate, fumaric acid, potassium sorbate, PABA, propionic acid, TRICLOSAN, and linoleic acid. Where the bacteriostatic agent is lauric acid, it may be present in a wt % range (relative to the total weight of the composition) from about 0.3 to about 15, such as from about 0.3 to about 12, or from about 0.4 to about 11, or even from about 0.5 to about 10.

Without intending to limit the way in which the probiotic-containing compounds are formed, one method for producing gel bead microspheres is to use perfluorodecalin as the oxygen carrying substance, probiotics in dry powder form, and alginate as the binder in the form of a solution of sodium alginate. First, perfluorodecalin and the probiotic are blended together and then the resulting blend is dispersed into a solution of sodium alginate. The resulting formulation is then pipetted into a calcium chloride solution in order to crosslink the sodium alginate and create gel beads. Typical particle sizes are in the 1-2 millimeter range.

EXAMPLES

The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. All percentages are by weight unless otherwise specified.

EXAMPLE 1

Table 1 lists the composition of perfluorodecalin gel beads that were made by first dispersing probiotics into perfluorodecalin, then dispersing the resulting mixture into a 2% sodium alginate solution, and pipetting the resulting formulation into a 1M calcium chloride solution. Once formed, the gel beads were transferred immediately into DI water. Table 2 lists the viability obtained for unencapsulated L. plantarum (Lp299v from Jarrow Formulas), and L. plantarum encapsulated in perfluorodecalin-containing gel beads, where both were maintained in water for 3 weeks. No significant loss in viability was observed for the L. plantarum encapsulated with the perfluorodecalin/sodium alginate formulation when compared to the naked strain. It is hypothesized that the loss of viability will be lower for encapsulated probiotics incorporated into cosmetic products such as creams where the water activity should be lower than in water.

Table 1. Composition of perfluordecalin/probiotics gel beads

Table 2. Viability results for naked L.plantarum and encapsulated L.plantarum, after 3 weeks in water

EXAMPLE 2

Nine different types of gel beads were produced by the method stated in Example 1 and their compositions are listed below in Table 3A, in grams, and in Table 3B, as weight percents. Each type of gel bead contained perfluorodecalin in the same quantity, and one of three different probiotics, L. plantarum, L. salivarius and L. acidophilus, in the same quantity. Furthermore, sodium alginate (binder) was used in each of the types of gel beads, in three different amounts. Gel Beads 4-6 also contained METHOCEL polymer (binder) and Gel Beads 7-9 contained gellan gum polymer (binder).

Table 3A: Composition of Gel Beads, in Grams

Each of Gel Beads 1-9 was tested initially for number of viable probiotic units, which was recorded as cfu/gram of gel beads, and then again after 3 weeks immersion in water. The resulting viable cell counts, at time zero and three weeks, as well as the log reduction in cfu, are provided in Table 4. Table 4: Bacterial cell count per one gram of gel beads (cfu/g of gel beads)

[Key: #.##E+NN equals #.## x (T ]

Table 5: Bacterial cell count per one gram of freeze-dried bacterial powder (cfu/g beads) assuming 40% of water present in the beads

Claims

Claims:

1. A probiotic-containing composition having stable viability of the probiotic when incorporated into aqueous personal care formulations and which comprises:

A) a binder;

B) a probiotic; and

C) an oxygen-carrying compound.

2. The composition of Claim 1, wherein said binder is one or more bio-compatible polymers selected from the group consisting of: methylcellulose, ethylcellulose, alginate, gellan gum, and combinations thereof.

3. The composition of Claim 1, wherein the probiotic experiences a reduction in viable population of less than 3.5 log cfu.

4. The composition of Claim 1, wherein said probiotic-containing composition comprises microspheres which comprise components A), B) and C).

5. The composition of Claim 4, wherein said microspheres have an average particle size less than 500 microns

6. The composition of Claim 5, wherein each of said microspheres has a multilayer architecture.

7. The composition of Claim 1, further comprising an effective amount of bacteriostatic agent.

8. The composition of Claim 8, wherein the bacteriostatic agent is lauric acid.