WO2017063909A1

WO2017063909A1 - A process for preparing metabolites by reaction of a prebiotic component with a probiotic component

Info

Publication number: WO2017063909A1
Application number: PCT/EP2016/073608
Authority: WO
Inventors: Debjani GHOSH; Mruthyunjaya Swamy MATHAPATHI; Nivedita Jagdish PATIL; Smitha Ashok Upadhyaya
Original assignee: Unilever N.V.; Unilever Plc; Conopco, Inc., D/B/A Unilever
Priority date: 2015-10-13
Filing date: 2016-10-04
Publication date: 2017-04-20

Abstract

Disclosed is a process for preparing metabolites by the reaction of a prebiotic component with a probiotic component, comprising the steps of: (i) contacting a prebiotic component which is oat fibres, having less than 10 cfu/g bacteria in it, with a culture medium, in the presence of a nutrient; (ii) contacting the mix of step (i) with at least one probiotic component which is a lactic acid bacteria; (iii) incubating the mix of step (ii) at 30 °C to 40 °C under partially anaerobic conditions; (iv) separating, by any means, said culture of step (iii) into supernatant and pellet; and, filtering said supernatant of step (iv) and optionally freeze-drying it to obtain said metabolites. The metabolites are useful for variety of applications in skin care.

Description

A PROCESS FOR PREPARING METABOLITES BY REACTION OF A PREBIOTIC COMPONENT WITH A PROBIOTIC COMPONENT

Field of the invention

The present invention relates to a process for preparing metabolites by reaction of a prebiotic component with a probiotic component. The metabolites are useful for enhancing the innate immunity of cells and attenuate skin photoaging and inflammation. Background of the invention

The epithelial barrier of skin, oral cavity or the gut of animals, in particular human beings acts as a first line of defence against all external factors such as heat, light, humidity, germs, pathogens and pollution. The skin thereby acts like a natural barrier against all adverse conditions and factors.

This barrier function and innate immunity of the epithelial barrier of skin, oral cavity or the gut of animals is primarily responsible for protecting us against such adverse conditions and factors. The epidermal skin barrier is a barrier which acts like the natural protective layer of the body. The epidermal skin barrier forms an effective two-way protection barrier from the environment by preventing invasion of unwanted chemicals from outside and by preventing unregulated loss of water and nutrients from within the cells. The human skin consists of two major layers. There is a thicker layer at the bottom called dermis and a thinner layer at the top which is epidermis. Dermis provides strength, elasticity and the thickness to the skin. With aging, the thickness of the dermal layer reduces and this is believed to be partially responsible for formation of wrinkles in aged skin. Keratinocytes form majority of the cells in the epidermis. Within the epidermis, the keratinocytes reside in four distinct stages of differentiation.

Epidermal differentiation is important for providing a protective barrier against environmental factors and for prevention or reduction of trans-epidermal water loss. Formation of a cornified envelope is the final stage of keratinocyte differentiation. Ultraviolet rays (UV) from the sun have damaging effects on the skin like photoaging, oxidative stress and inflammation. UV rays induce the various tissue degrading enzymes in the skin eg. Matrix metalloproteinases (MMPs) which bring about the higher degradation of matrix proteins (eg. Collagen) which give strength and resilience to the skin. This degradation process leads to photoaging where there is aggravated appearance of skin aging effects (such as wrinkles and skin sagging).

Small chain fatty acids and derivatives thereof, e.g., acetates, propionate and butyrates are closely associated with certain important functions of skin, which include, defence against bacteria, barrier function, and innate immunity, induction of antimicrobial peptides, tissue repair, and moisturisation. Although these short-chain compounds molecules are products of metabolic pathways associated with number of beneficial functions in human beings, these are also metabolites of the interaction of certain good bacteria on various substrates.

There are cosmetic compositions that contain live non-pathogenic bacteria or lysates. A user needs to apply the compositions to the skin where the bacteria are allowed to multiply and/or interact with the skin and deposit some beneficial by-products directly on to the skin. WO2009/53564 A1 (L'Oreal) discloses use of lysate of Bifidobacterium species in a cosmetic composition to reinforce the barrier function. A lysate is a material which is obtained after destruction or dissolution of biological cells via cell lysis, thus causing the release of the intracellular biological constituents naturally contained within the microorganism. A lysate can be used in various forms such as solution or powder.

W013122932 A1 (P&G) discloses topical use of a prebiotic substance to improve the health of the skin microbiome, thereby potentially improving the condition and/or appearance of the skin. The publication also discloses topical cosmetic compositions that include the prebiotic. The topical cosmetic compositions may include a

dermatologically acceptable carrier and an effective amount of prebiotic, and may be used in conjunction with one or more oral or topical prebiotics, probiotics and/or probiotic lysates. US2005/196480 A1 (Sullivan Michael) discloses that extracts of Lactobacillus can stimulate the production of beta-defensins in skin cells, generally in a dose-dependent manner. Further, it is disclosed that several different forms of Lactobacillus extract, including extracts containing both water-soluble and water insoluble materials are able to elicit beta-defensin production in skin cell cultures. The exemplified extracts are based on beef or bean-based broth.

EP2742942 A1 (Unilever, 2014) relates to new use of niacinamide for triggering the generation of AMPs (antimicrobial peptides) on skin. This has application in improving the immunity of skin, scalp and oral cavity against attack by microorganisms. This publication does not disclose effects of lactic acid bacteria or any fibres on human skin.

Kedia G et.al in "Enzymatic digestion and in vitro fermentation of oat fractions by human lactobacillus strains", in the journal Enzyme And Microbial Technology, have studied the fermentation of soluble, insoluble and non-digestible fractions of oat fractions separated by debranning, whole oat flour and bran by human Lactobacilus strains to test its in vitro prebiotic potential. The assessment is based on kinetic parameters of this cultures, obtained by numerical adjustment of the results to the logistic equation. The article compares growth of Lactobacillus strains in the soluble, insoluble and indigestible components of oat fractions and flour.

On the other hand, the same authors have studied the interaction of bran-rich fractions of oats and whole oat flour for their prebiotic properties using a mixed culture of human fecal bacteria. The study suggest that oat bran fraction obtained by debranning is digested by the gut ecosystem and increases the population of beneficial bacteria in the indigenous gut microbiota. The authors conclude by disclosing that lactobacilli and bifidobacteria are able to grow in a fecal mixed culture. Neither of the journal articles discloses the use of metabolites procured from the reaction of a lactic acid bacteria and oat fibres.

Niyonsaba F et.al., in their article titled "Induction of a microbicidal protein psoriasin (S100A7), and its stimulatory effects on normal human keratin ocytes", published in the Journal of Dermatological Science, vol. 52, no. 3, 1 December 2008, discloses the role of psoriacin on keratinocytes.

Although such compositions are considered safe for human beings, there could be problems in factories where such bacteria need to be handled at mass scales.

Probiotics are live microorganisms that are beneficial to humans. Such microflora include yeast and certain types of bacteria. It is believed that these microorganisms help the body's natural gut microflora to grow and/or increase their metabolic activity, which in turn has beneficial effect on the body's physiological, biochemical and immune systems. Long-term exposure to certain bacteria may induce autoimmune response. Therefore, it is advisable to prevent, or at least reduce such exposure. A further drawback of compositions containing live microbes is that the microbes may or may not survive long enough in the compositions. On the other hand, even if they survive, there is always a possibility that the organisms may not remain functional.

Prebiotics are non-microbial substances that can exert beneficial effect on the human body by helping the body's natural gut microflora to grow and/or by increasing their metabolic activity. They generally are used in combination with probiotic

microorganisms. Examples of prebiotic substances include plant-derived edible fibres and their constituents, which can both be soluble (i.e. able to dissolve in water, e.g. certain types of β D-glucans) and insoluble (e.g. cellulose) which can be found in the walls of plant cells). Several food compositions contain probiotics and prebiotics. WO 91 17672 A1 (Nestle) discloses a food product obtained by fermenting oat bran using microorganisms. This food product is made by fermenting cereal bran (from barley, wheat, rice and millet) in an aqueous mixture using live Lactobacilli microorganisms. These microorganisms include species such as Lactobacillus or other lactic-acid bacteria, propionic-acid bacteria, and other similar bacteria. Particular microorganisms include Lactobacillus

GG, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus sp., Lactobacillus thermophilus, Lactobacillus casei and Streptococcus sp.

US2010098805 A (Velle RW) discloses a process for preparing a probiotic oat-based fluid food product comprising a first fermentation step, involving the fermentation of oat material; mechanically treating one or more unfermented oat-derived substances in the presence of water to form an aqueous suspension containing 1 ,3-1 ,4 β D-glucan in dissolved and/or suspended form. The average molecular weight is at least 1 ,500,000 Da. The next step involves combining the products from the first fermentation step with the oat-derived substances and the water, before, during or after the formation of the aqueous suspension; and a second fermentation step, involving the fermentation of the aqueous suspension in the presence of Lactobacteria and/or Bifidobacteria. US2012052152 A1 (Tate and Lyle Ingredients Americas LLC) discloses a synbiotic product composition comprising a blend of a prebiotic carbohydrate and probiotic spore-forming bacilli. Examples of prebiotic carbohydrates useful in synbiotic products include arabinoxylan, arabinoxylan oligosaccharides, xylose, soluble fiber dextrin, soluble corn fiber, and polydextrose. Also disclosed are human foodstuffs and animal feed comprising such synbiotic products and methods of increasing the titer of spore- forming bacteria in the intestinal tracts of mammals by administration of symbiotic products.

A research paper published in Beneficial Microbes (2014), Volume 5, Number 2, pp. 99-107 ISSN: 1876-2883 discloses the role of pre- and probiotics on skin health by modulating cutaneous microbiota. Topical applications of probiotic bacteria have a direct effect at the site of application by enhancing the skin's natural defence barriers.

Probiotics as well as resident bacteria can produce antimicrobial peptides that benefit cutaneous immune responses and eliminate pathogens.

We have now determined that metabolites formed as a result of interaction of specific probiotics with specific prebiotics under specified conditions can stimulate the release of antibacterial peptides from keratinocytes in a culture.

The metabolites reduce the levels of matrix metalloproteinases and inflammatory cytokines induced by ultravioletrays, which bring about skin matrix degradation leading to skin-aging and inflammation respectively. This method is safer and is a highly efficacious alternative to known methods to stimulate the release of AMPs to improve the health and immunity of epithelial cells of mammals. The metabolites are free of bacteria. Summary of the invention

Disclosed in accordance with a first aspect is a process for preparing metabolites by reaction of a prebiotic component with a probiotic component, comprising the steps of: (i) contacting at least one prebiotic component which is oat fibres having less than 10 cfu/g bacteria in it, with a culture medium in the presence of extract of yeast;

(ii) contacting the mix of step (i) with at least one probiotic component which is a lactic acid bacteria;

(iii) incubating the mix of step (ii) at 30 °C to 40 °C under partially anaerobic

conditions;

(iv) separating, by any means, said culture of step (iii) into supernatant and pellet; and,

filtering said supernatant of step (iv) and optionally freeze-drying it to obtain said metabolites.

Detailed description of the invention

The process of the invention

The process of the invention comprises the following steps: A first step involves contacting oat fibres having less than 10 cfu/g bacteria in it with a culture medium, in the presence of a nutrient medium. Presence of bacteria in the fibres may cause undesired reactions, therefore it is necessary to have less than 10 cfu/g bacteria. In order to ensure that the number of colony forming units of bacteria is less than 10 cfu/g, it is preferred that the oat fibres are pre-washed with dilute solution of a strong acid, preferably hydrochloric acid, more preferably 0.1 N hydrochloric acid. This is followed by treatment with a dilute solution of a strong base, preferably sodium hydroxide, until pH of the medium is neutral. The fibres are then washed with deionized water to remove traces of alkali, if any.

It is preferred that the culture medium is saline solution having of pH 6 to 8. Hank's Balanced Salt Solution (HBSS) is a preferred medium but any other suitable culture medium may be used instead. The nutrient serves as a source of proteins and sugars for the growth of bacterial (probiotic) colonies so that metabolites are produced in appreciable quantity. A preferred nutrient is yeast extract but any other suitable nutrient may be used. For example, tryptone, peptone or soy extract may be used.

The second step involves contacting the mix of step (i) with at least one probiotic component which is a lactic acid bacteria. It is preferred that the lactic acid bacteria is selected from Lactobacillus or Bifidobacterium species. It is preferred that the amount of the probiotic component, i.e., the lactic acid bacteria, is such that there are 10⁶ cfu/ml to 10¹⁰ cfu/ml.

When the probiotic component is a Lactobacillus species, it preferably is one or more of the following organisms:

Lactobacillus acidophilus (NCFB 1748); Lactobacillus amylovorus, Lactobacillus casei (Shirota), Lactobacillus rhamnosus (strain GG), Lactobacillus brevis, Lactobacillus crispatus, Lactobacillus delbrueckii (subsp bulgaricus, lactis), Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus johnsonii (CNCM 1-1225), Lactobacillus paracasei, Lactobacillus plantarum,

Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus alimentarius, Lactobacillus curvatus, Lactobacillus casei, Lactobacillus sake and Lactococcus lactis.

When the probiotic component is a Bifidobacterium, it preferably is one or more of the following organisms: Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium infantis or Bifidobacterium pseudocatenulatum. A Lactobacillus and a Bifidobacterium may be used in combination.

A third step involves incubating the mix of step (ii) at 30 °C to 40 °C under partially anaerobic conditions. It is preferred that this step is carried out in an atmosphere of carbon dioxide (CO2), more preferably under 5% CO2. It is preferred that thereafter, the bacteria are allowed to grow for at least 24 hours after incubation.

The fourth step involves separating, by any means, the culture of step (iii) into a supernatant and a pellet. The pellet (residue) may be discarded. The final step involves filtering said supernatant of step (iv) and optionally freeze-drying it to obtain the metabolites. The supernatant may be used as it is, i.e., in aqueous form, or may be freeze-dried to a powder. It is preferred that the supernatant is filtered by passing through a filter of 0.2 μηη, so as to remove particulate material, in particular, dead bacteria.

The product obtainable by the process of the invention:

In accordance with a second aspect is disclosed metabolites obtainable by a process of the first aspect. The exact composition of the metabolites is unknown but it is believed to contain proteins, sugars and short chain fatty acids/esters in a largely aqueous medium which forms bulk of the supernatent.

It is further, and particularly preferred that the metabolites are substantially free of the prebiotic component and the probiotic component. This is to avoid chances of

contamination or adverse reactions on the skin of consumers. In accordance with another aspect of the invention is disclosed metabolites obtainable by the process of the first aspect for use to stimulate the release of AMPs to improve health and immunity of epithelial cells of mammals. Also disclosed is metabolites obtainable by the process of the first aspect for use to enhance the release of psoriasin from epithelial cells of mammals.

Release of psoriasin is an indication of enhancement of inherent or innate immunity of the cells. More psoriasin implies more enhancement.

In yet another aspect is disclosed metabolites obtainable by a process of the first aspect for use to reduce the levels of matrix-metalloproteinases (Pro-MMP-1 ) under UV stress for improving skin anti-ageing effects in fibroblasts of mammals In yet further aspect is disclosed metabolites obtainable by a process of the first aspect for use to reduce the inflammatory cytokine IL-6 under UV stress for improving skin antiinflammatory effect in keratinocytes of mammals.

In other words, it brings about reduction in the release of Pro-MMP-1 from dermal fibroblasts of mammals under UV stress. MMP-1 brings about degradation of important skin matrix protein- Collagen. Reduced release of Pro-MMP-1 is an indication of anti- aging effects of the skin cells. Less Pro-MMP-1 indicates lesser skin matrix degradation and better anti-aging effects. Reduced release of IL-6 is an indication of anti-inflammatory effects of the skin cells. Less IL-6 indicates lesser skin inflammation.

Compositions and uses Metabolites obtainable by the process according to the invention can be used in various compositions. Thus in accordance with another aspect is disclosed a composition comprising metabolites obtainable by a process of the first aspect. The compositions may be food composition or a personal care composition. This is better and safer option to live, or inactive or dead bacteria or lysates thereof.

The cosmetic compositions may, for example be useful for skin health, scalp health or oral care. The food compositions may be useful for innate immunity, gut health, general well- being. Food compositions may also be beverage or refreshment compositions. Examples include ice cream, jams, jellies, squashes, fruit juices, fruit juice concentrates, coffee, tea, ice tea, yoghurt, frozen desserts, confectionary and bakery products. Non-limiting benefits include improvement in skin moisturisation, reduced occurrence of infection, faster wound healing, improvement in intestinal health, reduced occurrence of gut infections.

In accordance with a further aspect is disclosed a composition comprising metabolites obtainable by a process in accordance with the first aspect for use to stimulate the release of AMPs for improving health and immunity of epithelial cells of mammals, such as the skin. In particular, it is the immunity of oral tissues.

In accordance with a further aspect is disclosed a composition comprising metabolites obtainable by a process of the first aspect for use to enhance the release of psoriasin from epithelial cells of mammals, such as the skin. In particular, it is the release from oral tissues.

The metabolites may be used for therapeutic or non-therapeutic applications.

In a further aspect is disclosed use of metabolites obtainable by a process of the first aspect in a therapeutic method to stimulate the release of AMPs for improving health and immunity of epithelial tissues of mammals, such as the skin. In particular, it is health and immunity of oral tissues.

In one more aspect is disclosed use of metabolites obtainable by a process of the first aspect in a non-therapeutic method to stimulate the release of AMPs for improving health and immunity of epithelial tissues of mammals, such as the skin. In particular, it is the immunity of oral tissues.

In a yet further aspect is disclosed use of metabolites obtainable by a process of the first aspect in a therapeutic method for enhancing the release of Psoriasin from epithelial tissues of mammals, such as the skin. In particular, it is the release from oral tissues.

In yet another aspect is disclosed use of metabolites obtainable by a process of the first aspect in a non-therapeutic method for enhancing the release of Psoriasin from epithelial tissues of mammals, such as the skin. In particular, it is the release from oral tissues.

In yet another aspect is disclosed use of metabolites obtainable by a process of the first aspect in a therapeutic method for reducing the levels of matrix-metalloproteinases (Pro- MMP-1 ) under UV stress for improving skin anti-ageing effects in fibroblasts of mammals

In yet another aspect is disclosed use of metabolites obtainable by a process of the first aspect in a non-therapeutic method for reducing the levels of matrix-metalloproteinases (Pro-MMP-1 ) under UV stress for improving skin anti-ageing effects in fibroblasts of mammals

In yet further aspect is disclosed the use of metabolites obtainable by a process of the first aspect in a non-therapeutic method for reducing the inflammatory cytokine IL-6 under UV stress for improving skin anti-inflammatory effect in keratinocytes of mammals.

In yet further aspect is disclosed the use of metabolites obtainable by a process of the first aspect in a therapeutic method for reducing the inflammatory cytokine IL-6 under UV stress for improving skin anti-inflammatory effect in keratinocytes of mammals.

In yet another aspect is disclosed use of metabolites obtainable by a process of the first aspect in the manufacture of a composition for reducing the levels of matrix- metalloproteinases (Pro-MMP-1 ) under UV stress for improving skin anti-ageing effects in fibroblasts of mammals. In yet another aspect is disclosed use of metabolites obtainable by a process of the first aspect in the manufacture of a composition for reducing the inflammatory cytokine IL-6 under UV stress for improving skin anti-inflammatory effect in keratinocytes of mammals. The invention will now be explained further with the help of the following non-limiting examples.

Examples Example 1 : Preparation of 100 g (aqueous) of metabolites using Bifidobacterium lactis

One gram of commercial fibres (refer Table 1 for details) were weighed out accurately and were transferred to a 500 ml flask. Hydrochloric acid (16.5 ml, 0.1 M) was added to the flask and the fibres were allowed to remain in contact with the acid for 90 minutes. Thereafter, 2.3 ml of 0.25 M NaOH was added to the fibres to adjust the pH of the medium to neutral. The fibres were washed with water. Enough amount of Hanks Balanced Salt solution (HBSS) was the added to the flask to make up the total volume of the flask to 100 ml.

Contents of the flask were then centrifuged at 4000 rpm for 10 minutes and the supernatant was discarded. The pellet (residue) was washed once again with HBSS and the tube was centrifuged again. To the pellet of the second centrifuge, 80 ml of HBSS and 20 ml of nutrient in the form of Bacto Yeast extract (2%) was added. The culture medium was then examined for signs/evidence of bacterial growth.

Ten ml of Bifidobacterium lactis Bb12 with optical density (OD) of 0.1 (adjusted in HBSS) was then added until a point where the final OD was 0.01. Some tubes were maintained as controls as well (no fiber/no bacteria). The tubes were incubated for 24 hours at 37 °C in an atmosphere of 5% carbon-di-oxide.

A 96-well plate was set up to observe and record the kinetics of bacterial growth with HBSS or MRS. The culture was removed from the medium and centrifuged at 4500 rpm for 10 minutes. The supernatant was filtered through 0.2 μηη filter and aliquots were prepared. These were stored at -20 °C.

Stimulation of psoriacin in keratinocytes usinq the supernatant:

Human keratinocytes (Isolated from new born skin tissue; NHEK) were cultured in cell culture plates with 500 ul of Keratinocyte Growth Medium (KGM). The plates were incubated at 37 ^°C in an atmosphere of 5% CO2 for 48 hours. After 48 hours, the cells were treated with the culture supernatant obtained from a symbiotic combination of the prebiotic and the probiotic (1 :10 diluted in KGM) or short chain fatty acids in KGM and incubated in a carbon-di-oxide incubator at 37 ^°C for further 72 hours.

After 72 hours of incubation, the cell culture supernatant was collected and aliquoted and stored at -70 ^°C until used for ELISA.

The amount of AMP (psoriasin) secreted in response to the bacterial culture supernatant was quantified by ELISA.

Example-2: Preparation of 100 q (aqueous solution) of metabolites usinq Lactobacillus rhamnosus GG

In summary, the procedure for Example 1 was repeated using L.rhamnosus instead of B.lactis. The data pertaining to Examples 1 and 2 is summarised in Table 1.

Table 1

Organism Sample ng/ml of

Psoriasin

No Bacteria Basal media containing 1.66

salts and yeast extract

but no fibre

L.rhamnosus Basal media 1.08

Maize fibres 1.86

Sugar beet fibres 0.92

Pea fibres 1.75

Oat fibres 3.45 B.lactis Basal media 1.27

Maize fibres 2.27

Sugar beet fibres 2.33

Pea fibres 1.80

Oat fibres 3.59

Release of Psoriasin indicates of enhancement of inherent or innate immunity of the cells. More Psoriasin implies more enhancement. The data indicates that oat fibres led to release of significantly more amount of proriasin. In contrast, the other fibres failed to bring about appreciable release.

Example-3: Modulation of Pro-MMP-1 in dermal fibroblasts using the supernatant:

Human adult dermal fibroblasts (HDFa) were cultured in cell culture plates with 300 ul of Fibroblast Growth Medium (FGM). The plates were incubated at 37 ^°C in an atmosphere of 5% CO2. After 24 hours incubation, UVA exposure was done to half the cells. First, media was removed and cells were washed with 1xPBS. Thin layer of 1 X PBS was added to each well and cells were exposed to UVA light (108mJ/cm2) for three consecutive days which corresponds to 325mJ/cm2. Control wells were not exposed to UVA (sham). After every exposure fresh media was added. After last 3^rd exposure, media with the culture supernatant obtained from a symbiotic combination of the prebiotic and the probiotic (1 :10 diluted in FGM) was added and incubated in a carbon-di-oxide incubator at 37 ^°C for further 24 hours. After 24 hours of incubation, the cell culture supernatant was collected and aliquoted and stored at -70 ^°C until used for ELISA.

The amount of Pro-MMP-1 secreted in response to the bacterial culture supernatant at basal levels and under UV stress was quantified by ELISA. The Pro-MMP-1 results are summarised in Table 2.

Table 2

Organism Sample ng/ml of ng/ml of Pro-

Pro-MMP-1 MMP-1

Basal Under UV stress No Bacteria Basal media containing 3.27 5.34 salts and yeast extract

but no fibre

L.rhamnosus Basal media 3.34 5.01

Oat fibres 2.63 5.59

B.lactis Basal media 2.88 4.23

Oat fibres 2.55 3.89

Release of more amount of Pro-MMP-1 under UV stress indicates greater degradation of skin dermal matrix. Higher Pro-MMP-1 leads to signs of skin-aging. The data indicates that oat fibers caused significant reduction in Pro-MMP-1 release under UV stress.

Example 4: Modulation of IL-6 in epidermal keratinocytes using the supernatant:

Human epidermal keratinocytes (NHEK) were cultured in cell culture plates with 500 ul of Keratinocyte Growth Medium (KGM). The plates were incubated at 37 ^°C in an atmosphere of 5% CO2 for 24 hours. After 24 hours, the cells were treated with the culture supernatant obtained from a symbiotic combination of the prebiotic and the probiotic (1 :10 diluted in KGM) and incubated in a carbon-di-oxide incubator at 37 ^°C for further 24 hours. After 24 hours incubation, UVA exposure was done to half the cells. First, media was removed and cells were washed with 1xPBS. Thin layer of 1 X PBS was added to each well and cells were exposed to UVA light (22.5mJ/cm2). After exposure, media with the culture supernatant obtained from a symbiotic combination of the prebiotic and the probiotic (1 :10 diluted in FGM) was added again and incubated for 6 hours. The cell culture supernatant was collected and aliquoted and stored at -70 ^°C until used for ELISA.

The amount of IL-6 secreted in response to the bacterial culture supernatant at basal levels and under UV stress was quantified by ELISA. The Pro-MMP-1 results are summarised in Table 3.

Table 3 Organism Sample pg/ml of IL-6 pg/ml of IL-6

Basal Under UV stress

No Bacteria Basal media containing 72 131

salts and yeast extract

but no fibre

L.rhamnosus Basal media 57 59

Oat fibres 64 70

B.lactis Basal media 54 67

Oat fibres 70 69

Release of more of IL-6 by keratinocytes under UV stress indicates of higher inflammation in skin epidermis. More of IL-6 cytokine leads to skin inflammation. The data indicates that oat fibers caused significant reduction in IL-6 release under UV stress.

Claims

1. A process for preparing metabolites by the reaction of a prebiotic component with a probiotic component, comprising the steps of:

(i) contacting a prebiotic component which is oat fibres, having less than 10 cfu/g bacteria in it, with a culture medium, in the presence of a nutrient;

(iii) incubating the mix of step (ii) at 30 °C to 40 °C under partially anaerobic conditions;

2. Metabolites obtainable by a process as claimed in claim 1.

3. Metabolites as claimed in claim 2 wherein said metabolites are substantially free of said prebiotic component and said probiotic component.

4. Metabolites as claimed in claim 2 or 3 for use to stimulate the release of AMPs to improve health and immunity of epithelial cells of mammals.

5. Metabolites as claimed in any one of the preceding claims 2 to 4 for use to enhance the release of psoriasin from epithelial cells of mammals.

6. Use of metabolites obtainable by a process as claimed in claim 1 in a therapeutic method to stimulate the release of AMPs for improving health and immunity of epithelial cells of mammals.

7. Use of metabolites obtainable by a process as claimed in claim 1 in a non- therapeutic method to stimulate the release of AMPs for improving health and immunity of epithelial cells of mammals.

8. Use of metabolites obtainable by a process as claimed in claim 1 in a therapeutic method for enhancing the release of psoriasin from epithelial cells of mammals.

9. Use of metabolites obtainable by a process as claimed in claim 1 in a non- therapeutic method for enhancing the release of psoriasin from epithelial cells of mammals.

10. A composition comprising metabolites obtainable by a process as claimed in claim 1.

11. A composition comprising metabolites obtainable by a process as claim 1 for use to stimulate the release of AMPs for improving health and immunity of epithelial cells of mammals.

12. A composition comprising metabolites obtainable by a process as claimed in claim 1 for use to enhance the release of psoriasin from epithelial cells of mammals.

13. Metabolites as claimed in claim 2 or 3 for use to reduce matrix metalloproteinases and inflammatory cytokines induced by UV rays in skin cells of mammals.

14. A composition comprising metabolites obtainable by a process as claim 1 for use to reduce the matrix metalloproteinase pro-MMP-1 under UV stress for improving skin anti-aging effects in fibroblasts of mammals.

15. A composition comprising metabolites obtainable by a process as claim 1 for use to reduce the inflammatory cytokine IL-6 under UV stress for improving skin antiinflammatory effect in keratinocytes of mammals.