Givaudan UK’s Heather Carolan, Stephen Watkins & David Bradshaw explore the cosmetic opportunities for prebiotics
In cosmetics, we are always looking for the next big claim, a headlining ingredient that can keep our marketing colleagues happy and satisfy the consumer’s need for new and exciting products. Conversely, we are being driven more and more to exclude certain categories of ingredients from our formulations. Rightly or wrongly we are seeing more products that are paraben and preservative-free.
This presents chemists with some practical difficulties. While it is understandable that the consumer wants the mildest products possible for their skin, they must also be safe and effective. One way to find solutions to these needs is to look for inspiration from related industries, such as foods and dietary supplements.
Consumers have long regarded foodstuffs as safe and appealing for the skin, and have often been subject to prolonged advertising extolling the virtues of particular foodstuffs, so a message about their cosmetic benefits is often easy to communicate.
A recent case in point is the growth of probiotic and prebiotic products. This segment has grown from the original probiotic yogurts and dairy drinks, to encompass many foods and supplements. But what do we mean by these terms?
PREBIOTIC DEFINITION
The arena of functional foods has seen the launch of many probiotic products, where a food contains live cultures of potentially beneficial bacteria. Many of these species are naturally found in the human gut. The bacteria are beneficial to maintaining a healthy digestive system, reducing unpleasant symptoms such as stomach cramps and aiding nutrient uptake. Bacterial populations may be depleted, for example by taking antibiotics, and their decrease may allow pathogenic species to thrive, to the detriment of our health.
Probiotic bacterial cultures (taken as pills, or incorporated into drinks or yogurts) are intended to add back the good gut bacteria, helping the body’s naturally occurring gut flora to re-establish a healthy level.
In contrast to a probiotic, the term prebiotic was coined by Prof Gibson & Dr Roberfroid in 1995. Prebiotics are defined as substances able to “selectively stimulate the growth and/or activity of beneficial bacteria”.
The rationale is to provide a substrate that preferentially favours the growth of friendly organisms over some of the less friendly bacteria, reducing the chance of harmful species proliferating.
Prebiotics are generally short chain carbohydrates, found naturally in some fruits and vegetables. One of the most effective is inulin, a fructan or polymer of fructose, the natural sugar found in many fruits and vegetables. Inulin is used by these plants as a means of storing energy and is typically found in the roots or rhizomes. Most plants that synthesize and store inulin do not store other materials such as starch.
Inulin is found naturally in a number of plants such as wild yam, chicory, oats, leek and garlic, and has been widely studied. Commercially, it is usually extracted from chicory (endive) by using hot water before being dried, and it is used as a supplement in a variety of foodstuffs as diverse as breakfast cereals, breads, drinks, petfood and infant formula, to support a prebiotic claim.
PREBIOTICS IN SKIN CARE
Skin is our natural defensive barrier to the outside world; it is constantly in contact with microorganisms naturally present in the environment. Our skin becomes colonised from birth with representatives of various microbial species, forming a complex microbial ecosystem, with mixtures of long-term resident populations of bacteria and fungal species (mainly staphylococci, coryneforms, including Corynebacterium, Propionibacteria and Brevibacterium) and a high frequency of short-term, transient organisms.
In addition, the skin microflora is heavily influenced by environmental conditions, so changes to the host (such as antibiotic use, changes in water level, pH, lipids etc associated with washing) or environment may lead to depletion of the natural skin microflora or the colonization of the skin by different, more resistant, species.
To take an extreme example, MRSA is a well known problem for hospital patients with weakened immunity, where the bacterium can colonise open wounds and is then resistant to treatment with common antibiotics. MRSA kills those with compromised immunity by continuing to grow, eventually spreading to vital organs, causing sepsis and toxic shock.
Full surveys, utilising the latest gene sequencing techniques for accurate identification, are now enhancing our knowledge of the species routinely found on skin, and how the populations vary over time and subject. There is a difficulty in comparing the early studies, where populations were assessed by laboratory culture, with the later work, so the evidence currently remains somewhat contradictory about the variability and stability of populations across people and time.
For example in this survey, the authors found 91 different species on the forearm skin of the six people studied; six genera were present on all six subjects (Propionibacteria, Corynebacterium, Staphylococcus, Streptococcus, Acinetobacter and Finegoldia) and accounted for 63% of the organisms analysed. However, 62 genera were found on only one person each, leading the authors to conclude that the skin microflora was highly diversified, with a low level of interpersonal consensus.
In addition, differences were seen between a subject’s arms, with only 50-77% of genera found on that individual being found on both arms. When the subjects were re-tested eight months later, there was a significant difference in the species found at the two time points.
These differences will be mainly due to environmental factors – such as temperature, humidity and light exposure – and host factors – including gender, genotype, immune status, sweat production, medication, especially antibiotic use. Cosmetic use, particularly antimicrobial formulations may all affect microbial population size and community structure. Many of these factors are continually changing so the microbial community is in a constant state of flux.
Additionally, different areas of the body harbour different species: aerobic bacteria multiply better in warm moist areas; specific structures such as the hair follicle or sebaceous glands may also have associated microflora, and tend to be the areas where most organisms are found; there are comparatively very few microbes found on the dry, exposed skin, and not all squames are colonised. Since bacteria are quite small, skin could potentially harbour a considerable number of colony-forming units [CFU] on a given square centimetre of skin, but in practice the above factors, and especially food supply, limit their numbers.
For example, a 1980 survey found 1 x 106 CFU/cm2 on the scalp, 5 x 105 CFU/cm2 in the axilla, 4 x 104 CFU/cm2 on the abdomen, and 1 x 104 CFU/cm2 on the forearm. Another found counts of up to 107 aerobic bacteria per cm2 in moist areas such as the axilla, compared to 102 or fewer in dry areas such as the forearm or trunk.
The microbes gain nutrition from lipids and protein (keratin), and the different species are always in competition for the resources available. They must also adhere to skin; cell turnover, washing etc will physically remove loosely attached organisms and this can upset the balance. With such a finely balanced ecosystem, only slight shifts in any one of these parameters could lead to conditions that favour one species, or result in the eradication of another, leading to the possibility of overgrowth of pathogenic species and potential problems for the host.
PROBLEMS WITH CERTAIN SPECIES
Several species found on the skin are directly implicated in skin or scalp conditions, such as Propionibacterium acnes (acne) and Malassezia spp. (dandruff). Many organisms are also known to be harmful to the body, such as Staphylococcus aureus, which can cause skin infections, and Escherichia coli, a normal gut coloniser. In addition, some organisms present on the skin provide no physical harm to the host but are associated with problems such as body odour (Corynebacterium spp., Brevibacterium spp.).
Many papers have shown the presence of Propionibacterium acnes bacteria on the skin of those suffering from acne vulgaris. However, the same organisms are also found on the skin of persons without acne, and changes in the microflora during antibiotic treatment (measured by cultivation studies) do not correlate with acne remission. The antibiotics must be having a beneficial effect, and several hypotheses have been proposed, but whether it is a weakness of the test methodology or an example of a more complicated aetiology is not yet known.
A similar problem is associated with dandruff; at various times of life, such as puberty, sebum production increases on the scalp. Malassezia (a yeast species previously known as Pityrosporum ovale) exists on the skin, breaking down triglycerides produced by the sebaceous glands and using the saturated fatty acids produced for nutrition. Greater sebum availability can allow increases in a relatively low population, leading to proliferation. As the population grows, the saturated fats are increasingly depleted and unsaturates left on the skin lead to scalp itching and flaking.
For many years, the Malassezia species were implicated in dandruff aetiology, but the mechanism was unknown as, like acne, antifungal agents are effective treatments, but the size and presence of Malassezia populations are not always correlated with dandruff severity. It now appears that individuals with dandruff have a greater sensitivity to these free fatty acids than unaffected individuals, possibly due to increased penetration into the stratum corneum.
CAN SKIN BE TOO CLEAN
Consumers often consider that a reduced bacterial population would be a benefit, but commensal organisms may help to inhibit the growth of more harmful species. For example, microbial lipases generate free fatty acids from secreted triglycerides, which may be antimicrobial and help to limit the types of microorganisms that can exist on skin.
Other protective effects of the normal skin populations include the production of bacteriocins (species-specific proteinaceous antimicrobials given off by bacteria to inhibit the growth of other bacterial strains); production of metabolites toxic to other species; depletion of essential nutrients; and their physical presence limits the growth of other populations - the different bacterial populations are competing for location, adherence, nutrition and other resources necessary for growth and multiplication.
Indirectly, they can also reinforce the host’s immune system by triggering antibody and cytokine production. This low level of circulating antibodies will cross-react with certain related pathogenic species, preventing infection or invasion.
The presence of some of these species on the skin can therefore actually be beneficial to skin health. In addition, repeated washing can have a negative effect on the skin barrier, by stripping lipids etc. A US government Centre for Disease Control review found that “no reductions beyond an equilibrium level are attained even with use of antiseptic preparations, which substantially reduce counts of hand flora. The numbers of organisms spread from the hands of nurses who washed frequently with an antimicrobial soap actually increased after a period of time; this increase is associated with declining skin health. Nurses with damaged hands were twice as likely to be colonized with S. hominis, S. aureus, gram-negative bacteria, enterococci and Candida spp. and had a greater number of species colonizing the hands”. They concluded that “the goal should be to identify skin hygiene practices that provide adequate protection from transmission of infecting agents while minimizing the risk for changing the ecology and health of the skin and increasing resistance in the skin flora”.
PREBIOTICS IN PERSONAL CARE
Many modern handwash products include antimicrobial agents to effectively kill bacteria, but even washing with plain soap mechanically removes bacteria adhering to the surface corneocytes. In addition, as discussed above, the skin microflora is heavily influenced by environmental conditions, so changes to the host (such as antibiotic use, changes in water level, pH, lipids etc associated with washing) or environment may lead to depletion of the natural skin microflora, or the colonisation of the skin by different, more resistant, species. This may lead to a change in the resident species, even allowing the opportunistic colonization of aggressive, pathogenic species. These are often associated with unpleasant conditions, and their by-products, such as acids and enzymes, can cause such symptoms as redness and itching. In addition, the regular use of antimicrobial products by those with healthy skin and no medical need may be associated with the emergence of resistant strains of bacteria.
While it would probably not be advantageous to use probiotics to add new bacterial populations to the skin (as each person’s microflora is unique), the addition of prebiotic agents could theoretically selectively nourish the commensal organisms already present on the skin.
Once an imbalance occurs on the skin, due to product use or one or more of the other factors identified above, it is possible to wait for the normal recolonisation that will occur gradually over time and hope that in the meantime more aggressive species do not predominate. This should be acceptable in normal healthy individuals, but the process can be accelerated, by preferentially supporting the growth of protective or commensal bacteria through the use of a prebiotic agent.The use of prebiotics offers an opportunity to reduce the harmful bacteria without the side effects and negative perceptions now widely associated with antibacterial agents.
In the past few years, Henkel has made a number of studies on prebiotic materials from plant extracts, using a number of advanced techniques, such as 16s rDNA sequencing and fluorescence in situ hybridisation (FISH) to overcome the problems associated with classical culture methods; a number of these species are hard to grow in vitro, particularly in coculture situations. In addition, the researchers identified several plant extracts that reduced populations of P. acnes on volunteers’ skin, while maintaining the populations of other species, notably Staph. epidermidis, supporting a claim that their product regime helped to rebalance the skin population. This was commercialised as the Aok First Beauty range for teenage, problem skin.
Yoghurt has now been accepted as a beneficial ingredient for cosmetic products, helping to moisturise and firm the skin. Powdered Yogurt can easily be combined with inulin to provide a single ingredient to take advantage of the technical properties of both the individual ingredients, while offering additional marketing concepts and support; due to the activity of food suppliers, consumers are well aware of the health benefits of yogurt and, while they may not fully understand the terms prebiotic and probiotic, they have absorbed the message that these are good for health.
While both yogurt powder and inulin have separately been proven to have a prebiotic effect in vitro, it was important to prove that the blend also had a measurable effect. To this end, commensal and pathogenic species were co-cultured so that they were competing for nutrients etc.
study 1: Activity of Yogurt/ inulin blend on the relative growth of microorganisms
A 10% yogurt/ inulin blend was mixed with deionised water and autoclaved to produce a sterilised test solution. Standard cultures of Staphylococcus aureus and Staphylococcus epidermidis were cultured separately overnight.
A standard tryptone soya broth (TSB) culture medium was prepared and split into two portions. To one portion of the broth, 10% by weight of the sterilised yogurt/inulin blend solution was added (labelled TSB+YB). To the second portion of broth, 10% by weight of sterile water was added (labelled TSB) and 100 microlitres of the S. aureus culture and 100 microlitres of the S. epidermidis culture were added to each types of broth: TSB and TSB+YB, making co-culture broth solutions in both TSB or TSB +YB. All the co-culture broth solutions were incubated overnight.
Serial dilutions of each co-culture broth were made and plated out onto agar. After 24 hours of incubation, the number of colonies for each bacterial species was counted and the ratio of ‘pathogenic’ bacteria (S. aureus) to ‘skin friendly’ bacteria (S. epidermidis) calculated.
Results: The plate counts of each organism from the co-cultured broth solutions showed a favourable alteration in the balance of the bacterial populations, with a reduction in some numbers of the pathogenic species of bacteria (Staphylococcus aureus) when cultured with the yogurt/inulin blend versus the broth without the yogurt/inulin blend. This method was replicated with new batches of all media.
study 2: Activity of Yogurt/ inulin blend on the Growth Ratio of Microorganisms
This was a repeat of the first but using a different pathogenic species – Escherichia coli – normally found in the gut and known to cause a variety of infections. The protocol used was identical to the first test.
Results: Results showed a favourable alteration in the balance of the bacterial populations, with a clear reduction in numbers of the pathogenic species of bacteria (Escherichia coli) when cultured with yogurt/inulin blend versus the broth without the yogurt/inulin blend.
The yogurt/inulin blend provides an interesting ingredient, not only for those who are concerned about skin hygiene, but for anyone looking for a more natural way to protect the skin and promote a natural balance, allowing the body’s natural defence systems to work effectively. When communicating the concept to consumers, it is not necessary and probably not helpful to mention skin bacteria, but better to refer to the above terms to convey how the ingredient works with the body’s processes.
It is ideal for use in baby care, handwashes and dishwashing products, without the aggressiveness associated with some antibacterial formulations, making it ideal for formulations for sensitive skin, especially those prone to conditions such as acne.
Contact
Heather Carolan, Givaudan UK
e-mail heather.carolan@givaudan.com
First presented at the SCS Symposium 2008