Use of Probiotics for Dermal Applications

Abstract

The concept of probiotic bacteria is considerably evolving. Clinical and experimental researches extensively document that
beyond probiotic capacity to influence positively the intestinal functions, they can exert their benefits at the skin level
thanks to their peculiar properties. Indeed, scientific and evidence-based reports strengthen the assumption that certain
probiotics can contribute to modulate cutaneous microflora, lipid barrier, and skin immune system, leading to the preservation
of the skin homeostasis. In this chapter, the most relevant evidences available from scientific literature as well as registered
patents have been summarized in relation to actual or potential topical applications of probiotics in the field of dermatology.
Altogether the evidences reported in this review afford the possibility of designing new strategies based on a topical approach
for the prevention and treatment of cutaneous disorders.

Full text

Use of Probiotics for Dermal Applications
Benedetta Cinque, Cristina La Torre, Esterina Melchiorre, Giuseppe
Marchesani, Giovanni Zoccali, Paola Palumbo, Luisa Di Marzio,
Alessandra Masci, Luciana Mosca, Paola Mastromarino,
Maurizio Giuliani, and Maria Grazia Cifone
Contents
1 Introduction ..................................... ............................................ 222
2 Probiotics and Skin Microflora .. . .......................................................... 223
2.1 Production of Acids by Probiotics .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 223
2.2 Antimicrobial Substances .. . ......................................................... 223
2.3 Beta Defensins .. . ..................................................................... 225
2.4 Probiotics and Wound Protection .. . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . 225
3 Probiotics and Disturbed Skin Barrier .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 227
3.1 Cutaneous pH .. . ...................................................................... 227
3.2 Ceramides .. ........................................................................... 228
3.3 Hyaluronic Acid .. . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . 230
3.4 Collagen ............................................................................... 231
B. Cinque • C. La Torre • E. Melchiorre • G. Marchesani • G. Zoccali • P. Palumbo •
M. Giuliani • M.G. Cifone (*)
Department of Health’s Sciences, University of L’Aquila, 67100 Coppito, L’Aquila, Italy
e-mail: benedetta.cinque@univaq.it;crix_latorre@libero.it;esterina.melchiorre@univaq.it;
giuseppe.marchesani@univaq.it;zoccali.giovanni@virgilio.it;paola.palumbo@univaq.it;
maurizio.giuliani@univaq.it;cifone@univaq.it
L. Di Marzio
Department of Drug Sciences, University of Chieti-Pescara Gabriele D’Annunzio, Via dei Vestini,
66013 Chieti, Italy
e-mail: l.dimarzio@unich.it
A. Masci • L. Mosca
Department of Biochemical Sciences, Sapienza University, p.le Aldo Moro, 5, 00185 Rome, Italy
e-mail: alexmasci@katamail.com;luciana.mosca@uniroma1.it
P. Mastromarino
Department of Public Health and Infectious Diseases, Section of Microbiology, Sapienza
University, p.le Aldo Moro, 5, 00185 Rome, Italy
e-mail: paola.mastromarino@uniroma1.it
M.-T. Liong (ed.), Probiotics, Microbiology Monographs 21,
DOI 10.1007/978-3-642-20838-6_9, #Springer-Verlag Berlin Heidelberg 2011
221

4 Probiotics and Skin Inflammatory/Immune System .. . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . .. 231
4.1 Barrier Function and Skin Reactivity .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 231
4.2 Environmental Stress .. . . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. 232
4.3 Protection Against NO .. ............................................................. 234
4.4 Anti-inflammatory Potential .......................................................... 235
5 Conclusions .. . . . . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. 237
References ....................................... ................................................ 237
Abstract The concept of probiotic bacteria is considerably evolving. Clinical and
experimental researches extensively document that beyond probiotic capacity to
influence positively the intestinal functions, they can exert their benefits at the skin
level thanks to their peculiar properties. Indeed, scientific and evidence-based
reports strengthen the assumption that certain probiotics can contribute to modulate
cutaneous microflora, lipid barrier, and skin immune system, leading to the preser-
vation of the skin homeostasis. In this chapter, the most relevant evidences avail-
able from scientific literature as well as registered patents have been summarized in
relation to actual or potential topical applications of probiotics in the field of
dermatology. Altogether the evidences reported in this review afford the possibility
of designing new strategies based on a topical approach for the prevention and
treatment of cutaneous disorders.
1 Introduction
Most often in foods and oral formulations, the probiotics are mainly used as a means
for restoring microbial balance, particularly in the gastrointestinal tract (Williams
2010). This approach appears particularly significant since the intestinal microbiota is
involved in physiological balance and in the intestinal development and maturation of
the host immune system (Oelschlaeger 2010). Thus, in the last years an increasing
interest has been focused on the possible use of ingested probiotics for treating
inflammatory and allergic conditions being specific strains able to modulate the
immune system at the local and systemic levels (Caramia et al. 2008). Arck et al.
(2010) have recently hypothesized a new, unifying model i.e., gut–brain–skin axis,
suggesting that modulation of the microbiome by deployment of probiotics can exert
profound beneficial effects on skin homeostasis, skin inflammation, hair growth, and
peripheral tissue responses to stress. On the other hand, new insights could now
fundamentally change the impact of probiotics on dermatology. Indeed, an emerging
approach to help preventing and treating skin conditions, including the external signs
of aging, acne, rosacea, yeast and bacterial infections, psoriasis, and dermatitis, is
represented by topical probiotics, as shown by the growing marketplace for topical
probiotic formulations available for skin care and antiaging benefits. In this chapter,
the key evidences available from scientific literature as well as registered patents will
be summarized in relation to actual or potential topical applications of probiotics in
the field of dermatology.
222 B. Cinque et al.

2 Probiotics and Skin Microflora
The skin is able to act as a physical barrier exerting several functions such as fluid
homeostasis, thermoregulation, immune responses, neurosensory functions, meta-
bolic functions, and primary protection against infection. The skin microflora plays
a significant role in competitive exclusion of pathogens that are aggressive and
provoke infection in the skin and in the processing of skin proteins, free fatty acids
(FFAs), and sebum. Interestingly the resident microbiota may be regarded as
“beneficial” to the normal, healthy host, but may become dangerous to the host
with disturbed skin integrity. Microorganisms may have a role even in atopic
dermatitis (AD), eczema, rosacea, psoriasis, and acne. Even if there are only very
few studies pursuing on probiotic approach for microflora-related skin disorders, it
is intriguing to suppose that topical probiotic application can be beneficial either for
preventing or for treating altered microflora-associated skin diseases (Krutmann
2009; Simmering and Breves 2009).
2.1 Production of Acids by Probiotics
Preservation of the resident microflora is thought to be an effective way to achieve
maintenance of healthy normal skin functions; however, the number of microbial
species colonizing human skin is limited depending on the hard physical and
biochemical factors. Probiotic microorganisms use different mechanisms, such as
by lowering pH, to preserve skin health and to inhibit the growth of pathogens. The
acidic skin environment is indeed very important as it discourages bacterial coloni-
zation and provides a moisture barrier through absorption or moisture by
aminoacids, salts, and other substances in the acid mantle (Lambers et al. 2006;
Mauro 2006). An interesting property of probiotics is the fermentative metabolism
that involves the production of acid molecules (i.e., lactic acid), thus acidifying the
surrounding environment (Krutmann 2009).
2.2 Antimicrobial Substances
The potential topical use of probiotic strains capable of producing potent antimi-
crobial toxins (i.e., bacteriocins, bacteriocin-like substances, organic acids, and
H
2
O
2
) has received increasing attention to successfully prevent pathogen adhesion
and outcompete undesired species (Oh et al. 2006; Gillor et al. 2008). Topical
compositions containing probiotic bacteria, spores, and extracellular products
and uses thereof represented the basis of the invention of Farmer (2005) suitable
for topical application to the skin which can be utilized to inhibit the growth of
bacteria, yeasts, fungi, viruses, and combinations thereof. The invention also
Use of Probiotics for Dermal Applications 223

disclosed methods of treatment and therapeutic systems for inhibiting the growth of
pathogens and combinations thereof, by topical application of therapeutic
compositions which were comprised, in part, of isolated Bacillus species, spores,
or an extracellular product of Bacillus coagulans comprising a supernatant or
filtrate of a culture of Bacillus coagulans strain. A method and composition were
also provided for application of probiotic microorganisms to a surface to prevent or
inhibit contamination by pathogenic microorganisms (Spigelman and Ross 2008).
The probiotic microorganisms may be bacteria, yeast, or mold. Suitable probiotics
should be selected according to one or more particular properties, being the
preferred properties of their competitive exclusion of pathogenic organisms from
the surface to which they are applied, adherence to human tissue, sensitivity to
antibiotics, antimicrobial activity, acid tolerance, and a high oxygen tolerance. In
particular, the method consists of different application modalities (i.e., lotions,
spraying, wipe paper) of one or more probiotic microorganisms to a wide variety
of surfaces, such as human skin and hospital equipment and fixtures, in an amount
effective to, at least partly, prevent their contamination, colonization, growth, and
cross-contamination by the pathogenic bacteria. The method relies on the probiotic
ability to form isolated colonies producing a protective layer that can inhibit and
exclude pathogenic bacteria generally unable to grow on top of other bacteria. The
probiotic application is recommended for a sufficient time depending upon such
factors as the therapeutically effective amount, the type, the mode of probiotic
application, or the degree of contamination of the biological or nonbiological
surface. Accordingly, the method propones the use of a single or a plurality of
different probiotic microorganisms, applying multiple bacteria serially, in layers to
fight several or single resistant types of pathogenic organisms.
In regards to the potential use of bacteriocin-producing strains as probiotic
and bioprotective agents, a number of bacteriocins produced by various lactic
acid bacteria species including Lactobacillus, Lactococcus, Pediococcus, Propioni-
bacterium, Leuconostoc, and Carnobacterium have been reported (Klaenhammer
1993; Oh et al. 2006). Of interest, Oh et al. (2006) reported the efficacy of the
bacteriocin from Lactococcus sp. HY 449 in controlling skin-inflammation and
acnes by clinical skin irritation test. This study demonstrated that this bacteriocin
was able to inhibit the growth of skin inflammatory bacteria such as Staphylococcus
epidermidis Staphylococcus aureus, Streptococcus pyogenes, and Propioni-
bacterium acnes. The experimental data highlighted that the inhibitory effect
of the bacteriocin employed in this study was due to bacteriolytic action on the
cell wall and cell membranes of P. acnes. Thanks to its antimicrobic properties,
Lactococcus bacteriocin could be used in cosmetic application for a variety of
purposes.
The invention of Teodorescu (1999) disclosed the use of eubiotic product,
consisting of a mixture of three Lactobacillus acidophilus strains, LD-11, LR-13,
and LV-17, associated in equal parts, for the maintenance and treatment of tegu-
ment. In comparison with the common strains, LD-11, LR-13, and LV-17 can also
ferment raffinose, trehalose, and dextrin, respectively. The most important aspect of
this finding lies on an optimum association of three L. acidophilus anallergical
224 B. Cinque et al.

strains for realizing a natural eubiotic product, capable of maintaining the skin pH
at physiological values, to destroy the pathogenic microflora and to be resistant in
cosmetic composition.
2.3 Beta Defensins
Sullivan et al. (2009) claimed that extracts of Lactobacillus could stimulate the
production of beta-defensins in skin cells, which may be useful in the reduction or
prevention of growth of microbial populations on the skin, in a dose-dependent
manner. Effective amounts of Lactobacillus extracts are applied to an open cut or
wound on the skin that may have been in contact with dirt or undesirable microbes;
or on a chronic basis, applied to clean skin to maintain a healthy level of skin flora.
According to the authors, the extracts could also be useful in the treatment of acne.
Indeed, topical compositions containing L. plantarum extract are shown to reduce
the incidence of both inflamed and noninflamed acne lesions when used regularly
over a period of 2 months. The extracts had further been proposed as a preservative
in cosmetic of pharmaceutical products, in particular the L. plantarum, which
possesses a broad spectrum of activity against both Gram-positive and Gram-
negative bacteria. Acne vulgaris is multifactorial condition and is characterized
by hypercolonization with Propionibacterium acnes, inflammation, and immune
responses. The synbiotic ability of probiotic bacteria and Konjac glucomannan
hydrolysates to inhibit the growth of Propionibacterium acnes in an in vivo study
has been recently reported suggesting that the development of a new alternative
involving probiotic therapy for reducing acne episodes in vivo could be encourag-
ing (Al-Ghazzewi and Tester 2009).
2.4 Probiotics and Wound Protection
Chronic wounds are, by definition, the ones that remain in a chronic inflammatory
state and therefore fail to follow the normal patterns of the healing process; factors
that may impede healing must be identified and, if possible, corrected, for healing to
occur. Chronic wounds, and burns in particular, are rarely, if ever, sterile. The burn
wound surface is sterile immediately following injury, although it is repopulated
within 48 h with Gram-positive organisms from hair follicles, skin appendages,
and environment; successively, more virulent Gram-negative organisms replace
the Gram-positive organisms after 5–7 days. Burns produce disruption of the
mechanical integrity of the skin and generalized immune suppression that allows
microorganisms to multiply freely. Currently, the common pathogens isolated
from burn are Staphylococcus aureus,Pseudomonas aeruginosa,Streptococcus
pyogenes, and various coliforms. Other streptococci, anaerobic organisms, and
fungi can also cause infections. Bacterial infections are generally treated by
Use of Probiotics for Dermal Applications 225

administration of antibiotics; however, this is not always efficacious. Contributing
to the lack of successful antibiotic treatment is the ability of colonizing bacteria to
establish themselves and proliferate in a biofilm, a characteristic architecture of
microcolonies embedded in a self-made matrix of biopolymers that offers structural
stability and protection. In this composite state, the bacteria resist the action of
a variety of antimicrobial measures, and moreover they are extremely resistant
to antibiotics, antiseptics, and to the host immune response, so new therapeutic
modalities may be required. An effective approach to prevent or contrast infection
could be bacteriotherapy, i.e., the use of probiotics to displace pathogenic
organisms. Valde
`z et al. (2005) suggested that L. plantarum and/or its products
are potential therapeutic agents in the local treatment of P. aeruginosa burn
infections. The authors showed that the in vitro treatment with L. plantarum was
able to inhibit the production of the P. aeruginosa quorum-sensing signal
molecules, acyl-homoserine-lactones, and two virulence factors controlled by
these signal molecules elastase and biofilm. On the other hand, the subcutaneous
injection of L. plantarum on a burned-mouse model with P. aeruginosa burn
infection, induced inhibition of P. aeruginosa colonization as observed in skin,
liver, and spleen samples taken after 5, 10, and 15 days upon infection, thus leading
to a significant improvement in tissue repair. On the basis of these encouraging
findings, a preliminary study (Peral et al. 2009a) was carried out to determinate the
effect of topical L. plantarum treatment on infected and non-infected second-degree
burn patients and on infected third-degree burn patients. As a result, in non-infected
third-degree burns, the ability of L. plantarum to prevent infection, to promote
granulation tissue, and to heal wounds was comparable to the silver sulphadiazine
cream one; in infected second-degree burns, L. plantarum treatment was as effec-
tive as the silver sulphadiazine one with reference to decrease in the bacterial
load, promotion of the appearance of granulation tissue, and wound healing. In
infected third-degree burns, treatment with L. plantarum would show great efficacy.
A further study of the same group showed the efficacy of L. plantarum
bacteriotherapy on the chronic infected leg ulcers of diabetic and nondiabetic
patients (Peral et al. 2009b).
The antimicrobial efficacy of nitric oxide (NO) is well known. Of note, a recent
report (Jones et al. 2010) showed that the NO-producing probiotic patch device
containing lyophilized alginate-immobilized L. fermentum, glucose, and nitrite
salts can produce sufficient levels of gaseous NO over a therapeutically relevant
duration, to kill common bacterial and fungal pathogens existed in the wounds of
humans.
A recent invention of Hansen and Jespersen (2010) was directed to a wound or
tissue dressing comprising bacteria having the property of producing lactic acid
by fermentation of the sugars, to use in healing wounds or in accelerating the
wound healing. Particularly, preferred species of lactic acid bacteria including
L. sporogenes, L. acidophilus, L. plantarum, L. casei, L. brevis, L. delbruckii, and
L. lactis are present in the dressing, capable of (1) lowering the pH in an open
wound environment, (2) securing an intraspecies competitive exclusion thus
preventing growth of undesirable bacterial species, (3) exerting an
226 B. Cinque et al.

immunomodulatory effect by inducing “wound healing-promoting substances”
(i.e., cytokines, growth factors), and (4) producing certain bacteriocins such as
toxins that can sustain a wound-healing process.
Actual management of chronic wound and burn patients is enormously expen-
sive. Moreover, traditional treatments and caring are often ineffective and fail
to eradicate bacteria, especially in biofilm form. Bacteriotherapy with specific
probiotic strains could represent a new therapeutic modality thanks to their efficacy,
innocuousness, easy access to application, and low costs, even if this issue deserves
further investigation for possible use in topical wound treatments.
3 Probiotics and Disturbed Skin Barrier
Cutaneous pH plays an important role in the barrier function; in particular, human
normal skin pH is acidic and varies from an acidic pH of 3.0 to an almost neutral
pH of 6.5. Acidic skin surface has been attributed to microbial factors, esocrine
gland presence, generation of urocanic acid by histidase-catalyzed deimination
of histidin, secretory phospholipase A
2
-mediated generation of FFAs from
phospholipids, and a nonenergy-dependent sodium–proton exchanger (Feingold
2007; Cinque et al. 2010). Cutaneous pH can control bacterial populations on
skin surface affecting resident microbiota and can regulate epidermal permeability
barrier homeostasis and stratum corneum (SC) integrity (Feingold 2007; Cinque
et al. 2010).
3.1 Cutaneous pH
The two key cutaneous lipid-processing enzymes, b-glucocerebrosidase and acidic
sphingomyelinase (aSMase), which generate a family of ceramides from glucosyl-
ceramide and sphingomyelin (SM), respectively, exhibit low pH optima (Feingold
2007; Cinque et al. 2010). An acidic pH directly impacts lipid–lipid interactions
in the SC extracellular lamellar bilayers confirming the link between SC pH
and barrier homeostasis and SC integrity/cohesion. Elevations of SC pH are
accompanied by perturbed cutaneous permeability barrier homeostasis, and by an
increase in serine protease activity that mediate degradation of corneodesmosome,
resulting in abnormality of SC integrity/cohesion. In conclusion, an acute increase
in SC pH reduces the activity of certain key lipid-processing enzymes in the SC,
resulting in abnormal lipid processing and the formation of defective lamellar
membranes. Moreover, an elevation in SC pH is associated with several cutaneous
disorders, such as acute eczema, atopic dermatitis, and seborrheic dermatitis.
In these diseases the increased pH could adversely affect cutaneous functions
exacerbating these conditions further with more severe clinical manifestations
(Feingold 2007; Cinque et al. 2010). Thus, in order to restore the altered cutaneous
Use of Probiotics for Dermal Applications 227

functions because of increased pH, alternative strategies could be pursued in order
to modulate SC pH. As stated above, an interesting approach could envisage the
use of probiotic as lactic acid bacteria with fermentative metabolism that are able to
produce lactic acid and obtain energy from the fermentation of lactose, glucose, and
other sugars to lactate via homofermentative metabolism (Teodorescu 1999;
Farmer 2005; Chiba 2007)
Of interest, Yadav et al. (2007) reported that the lipolysis of milk fat by probiotic
lactobacilli increases the production of FFAs and produces conjugated linoleic acid
by using internal linoleic acid. This acid-producing mechanism inhibits growth
of other organisms and favors the growth of lactobacilli that thrive in low pH
environments conferring a further health benefit to the host.
3.2 Ceramides
The SC lipids are secreted from keratinocytes in lamellar bodies containing lipid
precursors (i.e., glucosylceramides, phospholipids, and cholesterol sulfate) and
enzymes (b-glucocerebrosidase, acidic SMase, secretory phospholipase A
2
, and
steroid sulfatase) that generate ceramides, FFAs, and cholesterol. Within the epi-
dermal membrane structure, the ceramides are the dominant lipid class by weight
(~50%) and play an essential role in maintaining and structuring the lipid barrier of
the skin. Therefore, a decrease of ceramide in SC will cause water loss and barrier
dysfunction in the epidermis, including a loss of protection against antigens,
including bacterial, and can result in a skin abnormality such as AD (Feingold
2007; Mizutani et al. 2009; Cinque et al. 2010). The rate-limiting enzyme for the
synthesis of ceramides is serine palmitoyl-CoA transferase since its inhibition leads
to delayed barrier repair. Ceramides result also from hydrolysis of cerebrosides and
SM through b-glucocerebrosidase and SMase, respectively. The SC ceramide
levels are thus regulated by the balance among these ceramide-generating enzymes
and the degradative-enzyme ceramidase. Both SM and SMase are present in the
epidermis and are originally contained in lamellar bodies; structure contains
a mixture of ceramides, cholesterol, and FFA. They play a role in the formation
of the lipid component of the skin barrier and in the maintenance of the SC stability.
To effectively maintain the permeability barrier homeostasis, all three main lipidic
components of the stratum corneum, cholesterol, FFA, and ceramides must be
present. The absence or decrease of even one of these components in perturbed
skin can delay barrier recovery (Feingold 2007; Mizutani et al. 2009; Cinque et al.
2010). A previous study of our group (Di Marzio et al. 1999) reported that high
levels of neutral SMase was detected in sonicated Streptococcus salivarium ssp.
thermophilus which was able to induce generation of relevant ceramide levels in
keratinocytes in vitro. Both hydroxyceramide and nonhydroxyceramide levels
strongly and gradually increased in the presence of sonicated S. thermophilus in a
time-dependent manner due to SM hydrolysis in keratinocytes which in turn could
be correlated with the high levels of a neutral SMase activity of S. thermophilus.
228 B. Cinque et al.

These results were confirmed by in vivo studies in healthy and young volunteers.
A relevant increase in SC ceramide levels was indeed observed in all the analyzed
subjects after topical application of experimental cream containing lysed
S. thermophilus (Di Marzio et al. 1999). The presence of high levels of neutral
SMase activity in the volunteers was responsible for the observed increase of SC
ceramide levels, thus leading to an improvement in barrier function and mainte-
nance of SC flexibility. The use of SMase obtained from selected strains of lactic
acid bacteria to increase the levels of skin ceramides, and dermatological and
cosmetic compositions suitable for topical application containing same, represented
the basis of the invention of Cavaliere and De Simone (2001). According to
this patent the bacterial SMase could also be used as a cutaneous permeation or
absorption enhancer, either alone or in admixture with other enhancers, for prepar-
ing pharmaceuticals or cosmetic compositions suitable for transdermal administra-
tion. Thus, a method claimed to prevent or treat all conditions associated with
abnormal ceramide levels (deficiencies), including skin aging, atopic eczema,
dermatosis or dermatitis, atopic dermatitis, psoriasis, ichtyosis, Fabry’s disease,
Gaucher’s disease, Tay-Sachs disease, or Sjogren-Larsson’s syndrome comprising
topical applying on the affected skin with a pharmaceutical composition which
contain neutral SMase obtained from sonicated lactic acid bacteria. To test the
possibility that the use of the S. thermophilus-containing cream could improve SM
dysmetabolism in AD patients, our group previously conducted a study to deter-
mine the effect of sonicated S. thermophilus on the level of ceramides in vivo in the
skin of AD patients (Di Marzio et al. 2003). A 2-week application of the cream
containing a sonicated S. thermophilus in the forearm skin of the patients led to
a significant and relevant increase of skin ceramide amounts, which could result
from the SM hydrolysis through the bacterial SMase. Of note, the topical treatment
consequently led to a reduction of the AD-associated signs and symptoms such as
erythema, scaling, and pruritus in all patients.
Some reports in the last decade have described the changes in total SC with
increasing age, in particular the reduced corneum lipid levels (Cinque et al. 2010).
Indeed, all major lipid species in the stratum corneum of aged human and mice skin
decreased by approximately 30%. In addition, the neutralization of the normally
acidic SC has deleterious effect in permeability barrier homeostasis and SC
integrity/cohesion. Considering the role of ceramides in SC, our group investigated
the short-term topical application of a probiotic formulation on healthy skin of old
Caucasian women (Di Marzio et al. 2008). Increase in skin ceramide levels
in aged subjects following a short-term topical application of bacterial SMase
from S. thermophilus has been reported. Consequently, the skin hydration and
ceramide levels as markers of functional skin were determined. The results of this
study showed hydration effects increase in the skin of subjects treated with
S. thermophilus-containing cream when compared with controls. The hydration
skin increase could be attributed to the enhanced SC ceramide levels probably
due to the SMase presence in S. thermophilus. These findings suggested that
topical application of a sonicated S. thermophilus preparation may contribute
Use of Probiotics for Dermal Applications 229

to the improvement of lipid barrier and a more effective resistance against aging-
associated skin xerosis.
The invention of Gueniche et al. (2006a,b) related to the use of an effective
amount of at least one microorganism belonging to the species L. paracasei or
casei, a fraction thereof or a metabolite thereof, in combination with an effective
amount of at least one microorganism belonging to the species Bifidobacterium
lactis, a fraction thereof or a metabolite thereof, for producing a dermatological
composition intended for treating and/or preventing reactive, irritable and/or intol-
erant, acquired dry skin and/or constitutional dry skin.
The cosmetic and/or dermatologic use of hesperidin (a flavonoid) in combina-
tion with probiotic microorganism for preventing a reduction in and/or for
reinforcing the barrier function of the skin associated with aging and/or photoaging
was also proposed in a recent patent of Gueniche and Castiel (2009). The authors
also presented an invention related to the cosmetic use of an effective amount of
at least one microorganism, especially a probiotic microorganism, or a fraction
thereof, as an agent for preventing the appearance and/or for treating the manifes-
tation of sensations of discomfort and/or cutaneous signs associated with a surface
skin treatment or an invasive treatment for esthetic purposes (Castiel and Gueniche
2009).
Epidermal keratinocyte differentiation is another essential event that coordinates
epidermal turnover and construction of skin barrier function. In this context,
Baba et al. (2006) reported that L. helveticus-fermented milk was able to promote
differentiation of cultured normal human epidermal keratinocytes by enhancing
production of the differentiation-related element profilaggrin, a precursor of a
natural moisturizing factor that controls normal epidermal hydration and flexibility.
3.3 Hyaluronic Acid
Fermentation technology using lactic acid bacteria was also proposed to produce
many other beauty factors, including hyaluronic acid (HA) and collagen, that
provide beneficial activities in maintaining skin health and preventing skin aging
(Chong et al. 2005; Chiba 2007). In the skin, HA represents a predominant volumi-
nous molecule of extracellular matrix (ECM). It is synthesized by keratinocytes and
fibroblasts and performs important biological role in the skin by having specific
rheological characteristics and good water-holding properties. HA has several
applications in medicine and cosmetics, including skin moisturizers. The ability
of Bifidobacterium-fermented soy milk extract (BE) to significantly enhance HA
synthesis in vitro and in vivo has also been performed (Miyazaki et al. 2003,2004).
The authors established that topical application of BE was able to ameliorate the
elasticity and viscoelasticity of mouse skin, to increase HA level and thus preventing
the age-dependent loss of cutaneous HA. The topical application of a gel formula
containing BE on human skin significantly enhanced skin elasticity suggesting
BE as a new cosmetic ingredient to improve skin elasticity through augment of
230 B. Cinque et al.

HA production. Recently, Izawa et al. (2010) described the optimized fermentation
conditions in a skimmed milk-based medium which had significantly (about 20-fold)
increased HA yield from S. thermophilus YIT 2084. Because of the safety of this
bacterium, the fermentation technique would have an impact on the application of the
bacterial HA.
3.4 Collagen
Collagen is a major constituent of the human skin and accounts for a high propor-
tion of the skin’s elasticity and physical properties. It is well documented that
exposure to sunlight damages the skin structures. In response to this damage, the
skin repairs itself through the rapid production of collagen and other associated
dermal components such as polysaccharides. A recent invention of Lieurey and
Watkins (2009) reported that the use of a fermented milk product comprising
nonhydrolysed and casein-free whey proteins improved skin firmness when topi-
cally applied to skin. The milk fermented with classic lactic acid bacteria
(S. thermophilus and L. bulgaricus) could improve the structuring of skin’s collagen
without promoting collagen synthesis. The inventors suggested the topical use of
the fermented milk on damaged skin to improve the collagen natural generation as
part of the repair process, thus preventing a skin condition termed elastosis.
4 Probiotics and Skin Inflammatory/Immune System
Skin consists of stratified epithelium with various cell types, including
keratinocytes that have been specialized to act as the outpost of the innate defense
system and, in a lower proportion, dendritic cells, melanocytes, and Langerhans
cells. Each of these cell types contributes to skin protection. Moreover, the under-
lying dermal compartment harbors leukocytes, mastocytes, and macrophages that
are key actors of cell defense. Probiotics actions on the skin can be mediated by
modulation of host’s immune response including innate as well as adaptive.
4.1 Barrier Function and Skin Reactivity
Some probiotic strains display potent immune-modulatory properties at the skin
level. Recently, the ability of L. paracasei CNCM-I 2116 (ST11) to modulate
reactive skin-associated inflammatory mechanisms has been evaluated (Gueniche
et al. 2010a). The authors showed that ST11 was able to abrogate vasodilation,
edema, mast cell degranulation, and TNF-alpha release which induced by substance
P, compared to control. Moreover, using ex vivo skin organ culture, the authors
showed that ST11-conditioned medium induced a significantly faster barrier
Use of Probiotics for Dermal Applications 231

function recovery after sodium lauryl sulphate disruption, compared to control.
These results support a beneficial role of ST11 on key biological processes
associated with barrier function and skin reactivity. Gueniche et al. (2010b)
performed an in vitro and a clinical trial using B. longum sp. extract proved that
these nonreplicating bacteria forms applied to the skin were able to improve
sensitive skin in various parameters associated with inflammation such as decrease
in vasodilation, edema, mast cell degranulation, and TNF-alpha release. These
findings suggest that new approaches, based on a bacteria lysate, could be devel-
oped for the treatment and/or prevention of symptoms related to reactive skin. A
recent invention of Gueniche (2010) disclosed methods directed to the cosmetic use
of an effective amount of at least one probiotic microorganism especially from the
genus Lactobacillus and/or Bifidobacterium, or a fraction thereof and/or a metabo-
lite thereof, as an active agent for limiting, preventing or treating skin irritation,
and/or irritative skin disorders.
The innate immune system uses pattern recognition receptors such as Toll-like
receptors (TLRs) to recognize microorganisms or their products on the cell
membranes (Guan and Mariuzza 2007). Keratinocytes were found to express
TLR2, which is specifically involved in the recognition of peptidoglycan (PGN),
a mesh-like layer outside the plasma membrane of bacteria forming the cell wall
that protects bacteria from environmental stress. It has been recently demonstrated
that TLR2 can differentially recognize PGN from Gram-positive and Gram-negative
bacteria (Asong et al. 2009). DAP-containing muropeptides were bound with high
affinity to TLR2, whereas only a restricted number of Gram-positive lysine-
containing muropeptides, derived from PGN remodeled by bacterial autolysins,
were recognized. The difference in recognition of the two classes of muropeptides
is proposed to be a strategy by the host to differentially respond to Gram-negative
and Gram-positive bacteria, which produce vastly different quantities of PGN.
Lactobacilli were demonstrated to stimulate innate immune response via TLR2
and nucleotide-binding oligomerization domain-2 thus modulating dendritic cell
function (Zeuthen et al. 2008). They were also demonstrated to induce the produc-
tion of interleukin-12 and other regulatory factors by macrophages (Sun et al. 2005;
Shida et al. 2006; Bleau et al. 2007). Most notably, L. casei strain Shirota, both as
intact cells or as cell wall-derived polysaccharide–peptidoglycan complex (PSPG),
inhibited IL-6 production in lipopolysaccharide (LPS)-stimulated lamina propria
mononuclear cells isolated from murine models of inflammatory bowel disease
and induced an improvement of disease conditions in mice (Matsumoto et al. 2009).
In the light of these findings, it is reasonable to hypothesize a possible role of
Lactobacillus surface molecules in modulating inflammatory response in skin.
4.2 Environmental Stress
Skin is the largest human organ and is directly exposed to environmental stress which
may cause oxidative stress, especially UV irradiation. Indeed, UVB (290–320 nm)
and UVA (320–400 nm) light can both induce the generation of reactive oxygen
232 B. Cinque et al.

and nitrogen species (ROS/RNS) in human skin (Xu and Fisher 2005). When the
generation of ROS/RNS exceeds the skin antioxidant defenses, the consequence is
epidermal oxidative stress. Excess production of ROS/RNS may affect the cell
function leading to apoptotic or necrotic cell death. Oxidative stress is thought to
play a central role in initiating and driving the signaling events that lead to cellular
response following skin UV irradiation. Increased ROS/RNS production induced
by UV light alters gene and protein structure and function, leading to skin damage
through the activation of multiple cytokine and growth factor cell surface receptors
(Rittie
´and Fisher 2002). Many of the molecular alterations observed following
UV irradiation of skin also occur during aging, a condition which is known to be
associated with oxidative stress. Indeed, it is well known that endogenous
antioxidants are decreased in skin and blood during UV exposure and in senescence
(Rittie
´and Fisher 2002). Hence, a treatment aimed at counteracting oxidative stress
may be helpful in the prevention of damages caused by UVB and UVA light or skin
aging. Furthermore, epidemiological studies clearly indicate UV light exposure
as the major cause of skin cancer (Armstrong and Kricker 2001); hence it is
recommended to adopt preventive measures by sunscreens via the topical route,
in addition to antioxidants via the systemic route. A number of reports indicate that
food supplementation with antioxidant molecules (such as vitamins C and E,
carotenoids, flavonoid, polyphenols, thiol compounds, and selenium) is able to
counteract skin damage by UVA and UVB (Greul et al. 2002).
Many evidences indicate that probiotics may be helpful as antioxidant agents,
both in vitro and in vivo. A number of probiotic strains were demonstrated to
possess antioxidative action in vitro. Lin and Yen (1999) and Lin and Chang (2000)
demonstrated that various Lactobacillus and Bifidobacterium strains were able to
exert antioxidant action in vitro. Both intact cells and cell-free extracts were able to
inhibit ascorbate autoxidation, to exert metal-chelating ability, to scavenge super-
oxide anion and other ROS, and to inhibit lipid peroxidation. Probiotics’ ability to
act as antioxidant can be attributed to the presence of antioxidant enzymes such as
superoxide dismutase (Shen et al. 2010) to the release of antioxidant compounds
such as glutathione (Peran et al. 2006) and to the production of extracellular
polysaccharide (EPS) biomolecules that probiotic bacteria release into the sur-
roundings to protect themselves under starvation conditions and also from extreme
pH and temperature conditions (Kodali and Sen 2008). Ingestion of probiotics may
also exert systemic protection from oxidative stress. Lactic acid bacteria extracts or
fermented milks were found to decrease human low-density lipoprotein oxidation
and to prolong the resistance of the lipoprotein fraction to oxidation (Terahara et al.
2001; Kullisaar et al. 2003; Gueniche et al. 2006b,2008; Peguet-Navarro et al.
2008; Bouilly-Gauthier et al. 2010). Thus, probiotics may represent a useful
therapeutic tool for the prevention of epidermal oxidative stress either via the
topical route or via ingestion. On the basis of these premises, our group has recently
performed a number of experiments in order to study the antioxidant activity
of a specific strain of lactic acid bacteria, the S. thermophilus S244 (provided by
VSL Pharmaceuticals, Inc., Gaithersburg, MD, USA) (manuscript in preparation).
Extracts of this bacterium were found to be good free radical scavengers, as
Use of Probiotics for Dermal Applications 233

compared to Trolox used as reference antioxidant compound. The oxygen radical
absorbance capacity of the bacterial lysate, measured as the area under the curve,
revealed that the bacterial extract efficiently inhibited the free radical-dependent
loss of phycoerythrin-E fluorescence in a dose-dependent fashion (not shown).
Encouraged by these results we assessed the photoprotective activity of the bacte-
rial lysate. When human HaCaT keratinocytes were irradiated with UVB light,
a 50% reduction in cells viability was observed. However, when the cells were
treated with UV in the presence of the bacterial extract, a dose-dependent protection
of cell viability was observed (not shown). In the light of our data on the antioxidant
activity of bacterial extracts, we propose that the photoprotective effect may be at
least in part due to the good free radical-scavenging properties of the extract.
4.3 Protection Against NO
The NO pathway has been shown in several cell types that reside in the skin,
including keratinocytes, melanocytes, Langerhans cells, fibroblasts, and endothelial
cells (Bruch-Gerharz et al. 1998). Convincing evidence suggests that NO synthesis
in these cells can be modulated by calcium-mobilizing agonists as well as diverse
inflammatory and immune stimuli, and thereby contributes to the pathogenesis of
several human skin diseases. Characterization of these intrinsic and extrinsic
regulatory stimuli of NO synthesis has afforded substantial insights into the role
of NO in inflammatory, hyperproliferative, and autoimmune skin diseases, as well
as skin cancer, and may ultimately form the basis for future therapeutic interven-
tion. NO is synthesized from arginine and oxygen by various nitric oxide synthase
(NOS) enzymes. NOS is a group of enzymes responsible for the synthesis of NO
from the terminal nitrogen atom of L-arginine in the presence of oxygen and some
cofactors. The presence of arginine deiminase in L. brevis, previously reported by
our group (Di Marzio et al. 2001), was further characterized through activity and
expression studies. L. brevis arginine deiminase, being able to metabolize arginine
to citrulline and ammonia, allows to inhibit NO generation by competing with NOS
for the same substrate, arginine. Considering the role of NO in inflammatory
conditions our group had also analyzed the ability of L. brevis to inhibit NOS
activity as well other inflammatory parameters including IFN-gand PGE
2
release
and MMP expression in murine macrophages activated by LPS (Della Riccia et al.
2007). The results suggested that the presence of L. brevis extracts in cell culture
strongly inhibited inducible NOS activity, IFN-g/PGE
2
production, and MMP
activity in LPS-activated macrophages. These effects could be attributed to
L. brevis’ ability to prevent inducible NOS activity responsible for NO, a key
inflammatory molecule. The invention of De Simone (2003) disclosed the use of
bacteria endowed with arginine deiminase to induce apoptosis and/or reduce an
inflammatory reaction, and pharmaceutical compositions containing such bacteria,
including creams and ointments. The inventor also included a strain of L. brevis
referred to as CD2 highly endowed with arginine deiminase.
234 B. Cinque et al.

4.4 Anti-inflammatory Potential
In the context of the anti-inflammatory potential of probiotics, the in vitro and
in vivo effects of supernatants from L. acidophilus (ATCC strains 4356 and 43121)
on tissue repair and angiogenesis were investigated by Halper et al. (2003). The
authors suggested that Lactobacillus supernatant promoted proinflammatory pro-
cesses including chemoattraction of polymorphonuclear cells, macrophages, and
angiogenesis in addition to previously described stimulation of production of TNFa
and other cytokines including interleukins and interferons.
Trautmann et al. (2001) demonstrated that in atopic dermatitis and allergic
contact dermatitis, skin-activated T cells stimulated Fas-induced keratinocyte apo-
ptosis. In particular, diseased skin-infiltrating T cells produce IFN-gthat increasing
Fas receptor number on keratinocyte membrane renders them susceptible to apo-
ptosis by Fas ligand expressed on or released by T cell surface. The knowledge of
these mechanisms provided a useful molecular model to focus on innovative
therapeutic applications. In order to examine the role of probiotics on inflammatory
skin disease, our group investigated the effect of a selected extract from B. infantis
on human keratinocyte cell line (HaCaT) abnormal apoptosis induced by activated
T-lymphocyte (Cinque et al. 2006). In particular, the probiotic effect on inflamma-
tory skin disease was investigated in the experimental model of AD as proposed by
Trautmann (2001). In this in vitro model of atopic AD, the ability of the probiotic
extract to protect HaCaT from apoptosis induced by soluble factors (IFN-gand
CD95 ligand) released by human T-lymphocytes in vitro, activated with anti-CD3/
CD28 mAbs or phytohemoagglutinin, has been evaluated. The obtained results
highlighted the bacterial extracts’ ability to totally prevent T-lymphocyte-induced
HaCaT cell apoptosis in vitro. The mechanism underlying this inhibitory effect has
been suggested to depend on the ability of the bacterial extracts to significantly
reduce anti-CD3/CD28 mAbs and mitogen-induced T-cell proliferation, IFN-g
generation, and CD95 ligand release. These results may represent an experimental
basis for a potential therapeutic approach mainly targeting the skin disorders-
associated immune abnormalities.
Cutaneous immune responses must be tightly controlled to prevent unnecessary
inflammation in response to innocuous antigens, while maintaining the ability to
combat skin-tropic pathogens. Regulatory T cells (Treg) play an important protec-
tive role against autoimmune response to self-antigens to maintain self-tolerance,
functioning by suppressing the activation, cytokine production, and proliferation of
other T cells. Tregs are CD4- and CD25-positive cells but the most specific marker
for these cells is FoxP3 (forkhead box P3), which is localized intracellularly.
Dysregulation in Treg cell frequency or functions may lead to the development of
autoimmune disease. Treg modulation is considered to be a promising therapeutical
approach to treat some selected disorders, such as allergies, and to prevent allograft
rejection (Clark 2010). In normal human skin these cells represent between 5 and
10% of the T cells resident and proliferating in inflamed skin serving as a brake
for cutaneous inflammation. Indeed their number increases in the skin lesions of
Use of Probiotics for Dermal Applications 235

contact dermatitis and in DTH reactions (Teraki and Shiohara 2003; Vukmanovic-
Stejic et al. 2008). Of note, in a recent study, de Roock et al. (2010) examined the
ability of specific probiotic strains to induce Foxp3-positive Tregs from human
peripheral blood mononuclear cells (PBMC) in vitro.The authors highlighted
a different capacity of tested probiotic strains to stimulate Treg population
demonstrating that L. acidophilus proved to be the most potent Treg inducing
bacterium. Moreover L. acidophilus-induced Foxp3 cells were also able to diminish
effector T cell proliferation. All examined probiotic strains were tested for the
ability to induce cytokine secretion, and in particular the results showed that all
bacteria induced a comparable IFN-grelease while IL-17 and IL-13 were produced
in low levels, and IL-4 did not detect. Induction of regulatory T cells is an attractive
target in the therapy of disorders where an abnormal immune response is present
including autoimmune diseases, asthma, and allergy. For this purpose there is
a need to increase understanding about specific role of each single bacterium in
modulating the Treg function in distinct compartments, both in the intestine and
locally in inflamed tissue. Volz and Biedermann (2009) sustained the topical
application of probiotics for prophylaxis and therapy of overwhelming cutaneous
Fig. 1 Comprehensive model that summarizes the main actions carried out by probiotics in
different skin conditions associated with altered microflora, abnormal oxidative stress, disturbed
skin barrier, and/or inflammatory/immune skin reactions. It is important to note that, for simplic-
ity, the generic term “probiotics” is indicated but it must be implied that the claimed effect should
be attributed to specific species or specific strains of probiotics as indicated in the text
236 B. Cinque et al.

proinflammatory immune reactions, considering it very promising also on the basis
of the ability of specific probiotic strains to trigger the production of tolerogenic
IL-10 by activating anti-inflammatory Treg.
5 Conclusions
Topical probiotic formulations are becoming increasingly available for healthy skin
care, prevention and treatment of skin diseases, and antiaging benefits, thus
representing an emerging area for skin health. The potential benefits of skin
probiotics could strongly depend on how each species or strain is selected as the
specific mechanisms underlying a specific effect on the healthy or disturbed skin. It
appears, therefore, particularly important to stress that it is not possible to general-
ize the effects that each of them, any association or combination thereof or extracts
thereof, has on the skin. A comprehensive model that summarizes the main actions
carried out by probiotics in different skin conditions associated with altered micro-
flora, abnormal oxidative stress, disturbed skin barrier, and/or inflammatory/
immune skin reactions is shown in Fig. 1.
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