Acetate
Sulfate
1 × 10
4
4 × 10	4	Aging	Wrinkles Skin firming	Skin conditioning agent Bioactive: skin moisturizing, anti-aging	Promotion of collagen synthesis and connexins levels.[48][49][113,114]
[	4	]	FucoPol	Enterobacter A47	Anionic, branched Fuc, Gal, Glc, GlcA Succinate, pyruvate, acetate	2–6 × 106	Hydrocolloid, emulsifying, flocculating and film-forming agent Bioactive: antioxidant, wound healing, photoprotection	[50][
Levan
Pseudoalteromonas sp. CNCM I-4150	Anionic Glc, Gal, GlcA, GlcNAc, GalA, Man	8 × 105 *	51	Aging Wrinkles	][52][53][54][55][115,	Improvement of skin moisturizing due to the water retention capacity.	[85116,117,118,119,120]
]	[	GalactoPol	Pseudomonas sp.	Aerobacter sp., Bacillus sp., Halomonas sp., Pseudomonas sp., Streptococcus sp., Zymomonas sp.	Neutral, linear or branched Fru	2 × 106	Water-soluble, strongly adhesive, film former, viscosity enhancer Skin conditioning agent Bioactive: anti-inflammatory, cell proliferative	[2][18][58][59][60][61][62][63][2,33,123,124,125,126,127,128]

It appears that non-marine bacteria produce a diversity of EPS owing functional and/or bioactive properties making them suitable candidates as cosmetic ingredients. Their anionic nature, related to the presence of uronic acids and substituting groups as well as their high-molecular weights seem to determine their valuable functional properties. Some EPS, such as xanthan or HA have been extensively studied since their early discovery and are now widely used in the cosmetic industry.

With ocean representing 70.8% of the Earth surface and the existence of a large number of niches characterized by specific conditions, this wide ecosystem still remains underexplored and represents a source of new biodiversity and chemical diversity. Many industry fields exhibit great interest to these potentially new compounds, including bacterial EPS. In particular, marine bacteria constitute a rich source of innovative EPS, that are explored for their skincare effects in cosmetic products. The main described bacteria are Gammaproteobacteria belonging to the genera Alteromonas, Pseudoalteromonas and Vibrio ^{[3][64][65][66][67][68][69][70]}[3,59,93,129,130,131,132,133]. Alteromonas and Pseudoalteromonas species usually produce highly branched anionic EPS composed of both neutral sugars and uronic acids substituted with sulfate, pyruvate and/or lactate groups. In contrast, Vibrio species synthesize linear anionic EPS containing mainly uronic acids and hexosamines, and can also be substituted with acetyl and/or lactate groups as well as amino acids ^[71][72

69
]
V. alginolyticus
CNCM I-4151
Anionic	GalA, GlcNAc 2 amino acids (alanine and serine)	Anionic, linear Gal, Man, Glc, Rha	2 × 105 *	Aging Inflammation Acne	Reduction of inflammation reduced; improvement of quality of the superficial layers of the epidermis; degradation of the extracellular matrix reduced.	[77][136]
V. alginolyticus CNCM I-5035	Anionic Gal, GlcNAc, GulNAcA	5 × 105	Barrier function Acne	Improvement of physical and chemical barriers function by increasing the keratinocyte differentiation and epidermal renewal. Increasement of immune defense against pathogens involved in acne.	[86][65]
Vibrio sp. CNCM I-4239	Anionic GlcNAc, GlcA, GalNAc	1 × 105–1 × 106 *	Hydration Inflammation	Promotion of the healing process; inhibition of neuronal exocytosis (inflammation; acne; wrinkle reduction).	[87][71]
Vibrio sp. CNCM I-4277	Anionic GlcA, GlcNAc, Glc, Fuc Sulfate	1 × 106	Aging Wrinkles	Increase of hyaluronic acid synthesis.	[88][70]
V. diabolicus CNCM I-1629	GlcA, GlcNAc, GalNAc	1 × 106	Skin regeneration	Collagen structuring and extracellular matrix establishment by dermal fibroblasts	[65][89][90][93,143,144]

Abyssine^TM PF produced by the deep-sea hydrothermal vent bacterium A. macleodii subsp. fijiensis biovar deepsane (HYD657 CNCM I-1285) was firstly developed by Lanatech and is now commercialized by Lucas-Meyer ^[91][27]. Abyssine^TM PF is an anionic heteropolysaccharide of high-molecular weight (1 × 10⁶ g/mol) composed of neutral sugars (galactose, glucose, mannose, fucose), uronic acids (glucuronic acid, galacturonic acid), and substituted with three anionic groups (sulfate, lactate, pyruvate). The EPS is known for its soothing properties and it also reduces irritation and provides photoprotection from UVB irradiations ^[79][138]. Halomonas sp. produces an EPS mainly composed of neutral sugars, however, uronic acids and substituting sulfate groups may also be present at low levels (e.g., H. eurihalina LMG P-28571) [4]. Similar osidic composition was also reported for EPS produced by Pseudoalteromonas sp. CNCM I-4150 ^[85][69]. Other EPS are substituted with various anionic organic and inorganic groups and some of them are also substituted with amino acids. For example, C. marina CNCM I-4353, which produces a sulfated EPS composed of neutral sugar, uronic acid and hexosamine residues, is additionally decorated with amino acids (threonine and serine) ^[75][64]. The native high-molecular weight (1 × 10⁶ g/mol) EPS exhibits anti-inflammatory properties, whereas its low molecular weight derivatives (2 × 10⁵ g/mol) possess anti-aging properties and improve skin appearance, and barrier functions.

EPS synthesized by Vibrio sp. are linear anionic polysaccharides devoid of sulfate mainly composed of uronic acids and hexosamines that may also be substituted with amino acids. For instance, a high molecular weight (1 × 10⁶ g/mol) EPS, diabolican, produced by V. diabolicus CNCM I-1629 displays original structural features close to those of hyaluronic acid as it is composed of glucuronic acid and N-Acetylglucosamine as well as N-Acetylgalactosamine ^[65][93]. The EPS produced by V. alginolyticus CNCM I-5034 is composed of glucuronic acid, N-Acetylglucosamine, galactose and galacturonic acid and it is substituted with alanine and lactate groups ^[73][94]. Epidermist 4.0^TM produced by V. alginolyticus CNCM I-5035 is a linear EPS composed of galactose, N-Acetylglucosamine and N-Acetylguluronic acid, with 30% of acetyl groups ^[86][65].

To modulate the EPS bioactivities, depolymerization can be performed to reduce the molecular weight of the native polymer. Several techniques are used to decrease the molecular weight of the polymer, such as chemical depolymerization using free radicals or acids ^[92][93][145,146], as well as mechanical ^[94][147] or ultrasonic degradations ^[95][148]. Free-radical depolymerization with hydrogen peroxide and copper, used as a metal catalyst, is reproducible and can be controlled through pH regulation and adjustment of hydrogen peroxide concentration, leading to low molecular weight derivatives between 20,000 and 100,000 g/mol ^[93][96][146,149]. Two patented derivatives were obtained using radical depolymerization of the native EPS from Pseudoalteromonas sp. CNCM I-4150 and Vibrio sp. CNCM I-4239 strains ^[85][87][69,71]. Even if depolymerization using acids or free radicals is suitable to decrease the molecular weight of polysaccharides, this method is not specific and may lead to the loss of substituents or sugar residues. Enzymatic hydrolysis remains more specific and more sustainable for polysaccharide depolymerization. However, it is highly challenging due to specific EPS structures, which implies the use of appropriate enzymes. Indeed, it was shown that enzymatic depolymerization of infernan (GY785 EPS) produced by the deep-sea hydrothermal vent bacterium A. infernus was not effective although various commercially available enzymes were tested. Only intracellular protein extract of this bacterium, containing a polysaccharide lyase, was able to depolymerize the EPS that it produces ^[97][98][16,150]. Another patented depolymerization process which was successfully applied to prepare low molecular weight derivatives is based on supercritical fluid-accelerated hydrothermolysis ^[99][151]. It allows partial depolymerization without altering monosaccharide pattern, decorating amino acids and sulfate groups. This technique using carbon dioxide heated to 200 °C and high pressures up to 250 bars was applied to depolymerize native EPS from C. marina CNCM I-4353 ^[75][76][64,135] and V. alginolyticus CNCM I-4151 ^[77][136].

EPS-producing marine bacteria were isolated from different environments, including atypical ones presenting extreme conditions such as the Antarctic marine environment, sea ice, marine snow, microbial mats, hypersaline environments, shallow and deep-sea hydrothermal vent environments ^{[67][72][100]}[58,60,130]. Some of the marine bacteria producing the EPS presented in Section 6.2 were isolated from extreme environments, such as A. macleodii subsp. fijiensis biovar deepsane (HYD657 CNCM I-1285) ^[66][129] and V. diabolicus CNCM I-1629 ^[65][93], both isolated from the polychaeta annelid Alvinella pompejana living close to hydrothermal vents located on the East Pacific Rise ^[101][160].

In addition, C. marina strains, psychrophilic bacteria evolving in cold water, were isolated from coastal sea samples ^[102][161] and mussels ^[103][162]. C. marina KMM 296 could even grow in the wide range of temperatures from 4 to 42 °C ^[103][104][162,163]. H. eurihalina is a moderately halophilic bacterium which can spread in diverse saline environments, such as solar salterns, intertidal estuaries, hydrothermal vents, hypersaline lakes and open ocean ^[104][105][163,164]. H. eurihalina MS1 isolated from saline soil in Alicante (Spain) was shown to be an EPS producing strain ^[106][107][165,166]. Another Halomonas sp., H. anticariensis strains FP35 and FP36 isolated from saline soils (Spain) also produced EPS ^[106][107][165,166]. Pseudoalteromonas strains are only found in marine environments, they possess environmental adaptation capacities as they can survive in extreme habitats, such as hydrothermal vents and polar areas ^[67][69][130,132]. Moreover, V. alginolyticus is a halophilic bacterium growing in the ocean or estuary environment ^[108][167]. These marine bacteria can grow in harsh conditions, and EPS production is important for their survival. Although isolated from extreme environments, these strains’ physiological requirements and tolerances are compatible with classical conditions of production, i.e., mesophilic temperature and neutral pH. These production conditions can be kept upon scaling up of an industrial process.

Commercial production of marine bacteria relies on the ability of the strain to grow in classical conditions of production, as high or low temperatures induce high energy demand and require specifically designed bioreactors, and salt concentration needs to be decreased to limit equipment corrosion. Bacteria growing at extreme values of pH require the use of acids and alkalis that induce corrosion and are risky to handle at industrial scale. Moreover, the use of piezophiles cannot be considered as it requires specific and costly equipment associated with high risks due to high pressure. However, among marine extremophiles, only thermophilic and psychrophilic bacterial strains have been reported to produce EPS. None of them are currently commercialized or have been considered as actives for cosmetics.

EPS production yield in marine thermophiles is lower than mesophilic marine strains ^[109][168]. Requirement of high temperature also imposes shake flasks as bioreactors are not developed for such high temperature. Fermentation for psychrophiles is longer than mesophiles. All of these specificities may hinder development of EPS from extremophiles unless more efforts are conducted for optimizing fermentation process conditions including further developments for scale-up production of EPS in bioreactor.

EPS are produced in response to biotic and abiotic stresses, and their secretion is one of the mechanisms used to tolerate harsh conditions including cold stress and ice crystal damage for psychrophiles, high temperature for thermophiles eventually exposed to large temperature gradients, high salts for halophiles, and acidic or toxic metal-containing environments for acidophiles or metal resistant microorganisms ^{[110][111][112]}[184,185,186]. Some microorganisms also have the ability to thrive in environments with multiple extreme conditions, such as deep-sea hydrothermal vents with intermittent extreme physical and chemical gradients between vent fluids and surrounding seawater, or Kopara that are Polynesian microbial mats found in pools of Polynesian atolls and subjected to variations in salinity, dessication and sun exposure. This protective role of EPS relies on the formation of a mucous slime around bacterial cells and is claimed in the bioactivities of final ingredient, emphasizing their biomimetic action on skin, including protection against low-temperature for psychrophiles ^[113][114][179,187], chelation of trace metals and binding of heavy metals ^[115][116][177,188] although activity features of EPS from extremophiles were not studied for cosmetics. However, extremophilic bacteria represents a new biodiversity source of EPS, strengthening their chemical diversity.

Marine-derived EPS presented in Section 6.2 exhibit interesting bioactivities for cosmetic applications. They are further detailed in this section. Examples of assays used to demonstrate these bioactivities are also presented, they can be applied to various active candidates depending on molecular weight, solubility and targeted bioactivities.

Due to regulatory requirements and safety assessments, EPS bioactivities need to be demonstrated on skin models for their commercial development. The EPS presented in Table 1 possess interesting biological activities, which were assessed using in vitro and ex vivo techniques. Examples of targeted bioactivity assays among selected EPS are shown in Table 34. These EPS possess anti-aging ^[84][62], anti-inflammatory ^[77][136] and anti-acne ^[77][136] properties as well as moisturizing ^[88][70] and slimming effects ^[84][62]. They promote vascularization ^[75][64] and improve skin barrier function ^[77][87][71,136]. The first step in assessing the EPS bioactivities is to determine the cytotoxicity of the compound on the selected cell culture or skin model, to verify that the tested compound can be applied at a suitable concentration without adverse effects. Cytotoxicity data are not systematically given in patents. However, cell viability is often assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or AlamarBlue assays.

Strain	Claims	Cell Culture Model	Activity and Analysis	Refs.
C. marina CNCM I-4353	Vascularization	Co-culture of human dermal fibroblasts (NHDF) and human umbilical vein endothelial cells (HUVECs) infected with a lentivirus that express green fluorescent protein	Quantification of angiogenesis (fluorescence levels expressed by HUVECs)	[75][64]
H. anticariensis LMG P-27891	Anti-aging	Human dermal fibroblasts (2D)	Type I collagen synthesis (ELISA assay)	[84][62]
H. anticariensis LMG P-27891	Slimming	Human subcutaneous pre-adipocytes in a complete differentiation medium (2D)	Reduction of the lipid accumulation “adipogenesis” (fluorescence assay)	[84][62]
V. alginolyticus CNCM I-4151	Anti-inflammation	Skin explants inflamed by lipopolysaccharides addition	Interleukin production quantification (IL-8 levels of expression)	[77][136]
V. alginolyticus CNCM I-4151	Anti-inflammation Anti-acne	Inflamed reconstructed human skin (3D)	Inflammation level studied by metalloproteinase expression (MMP3 mRNAs levels of expression)	[77][136]
V. alginolyticus CNCM I-4151	Barrier function	Reconstructed aged human skin (3D)	Late Cornified Envelop Proteins (LCEs) proteins of the stratum corneum (gene expression of LCE3)	[77][136]
Vibrio sp. CNCM I-4277	Moisturizing	Human dermal fibroblasts (2D)	Hyaluronic acid synthesis (ELISA assay)	[88][70]
Vibrio sp. CNCM I-4239	Barrier function	Human keratinocytes (2D)	Healing test (microscopic observations of cells compared before and after treatment on the scrap region)	[87][71]
Vibrio sp. CNCM I-4239	Cytotoxicity	Human dermal fibroblasts (2D)	Proliferation assay to measure cell viability (fluorescence assay)	[87][71]
V. diabolicus CNCM I-1629	Promotion of fibroblast proliferation	Dermal equivalent matrices with human dermal fibroblasts (3D)	Proliferation and migration of fibroblasts and production of an extracellular matrix	[89][90][143,144]

Typically, two-dimensional (2D) and three-dimensional (3D) cell culture models are used to further assess the potential activities of the EPS. For instance, the EPS from H. anticariensis LMG P-27891 was applied in 2D cell culture of dermal fibroblasts to assess its effect on type I collagen synthesis ^[84][62]. Similar 2D model was used to demonstrate moisturizing properties of the EPS from Vibrio sp. CNCM I-4277, which was shown to increase HA synthesis ^[88][70]. 2D cell culture of keratinocytes was selected to assess the barrier function of the skin upon treatment with EPS from Vibrio sp. CNCM I-4239 ^[87][71]. A particular example of 2D cell culture of subcutaneous pre-adipocytes was used to assess the potential slimming effect, i.e., the decrease of lipid accumulation, in the presence of EPS produced by H. anticariensis LMG P-27891 ^[84][62]. 2D cell co-cultures composed of dermal fibroblasts and vein endothelial cells were used to study more complex processes, such as the formation of blood vessels (angiogenesis), to trigger the vascularization stimulation effect of EPS from C. marina CNCM I-4353 ^[75][64].

More complex 3D cell culture models were also used to demonstrate EPS properties, such as reconstructed human skin. Specific reconstructed human skin with induced inflammation or aging was used to assess the potential anti-inflammatory activities of the EPS produced by V. alginolyticus CNCM I-4151. Inflammation was studied by measuring the expression of metalloproteinase 3 (MMP3), the level of which increases in the case of acne lesions. EPS from V. alginolyticus CNCM I-4151 on this model led to decreased MMP3 levels. Moreover, the effect of this EPS on reconstructed aged human skin improved barrier function by increasing the expression of proteins of the stratum corneum ^[77][136].

Skin explant, an ex vivo skin model, was also used to demonstrate EPS bioactivities. For example, skin explants exhibiting induced inflammation by the addition of LPS were used to study the anti-inflammatory activity of the EPS from V. alginolyticus CNCM I-4151. An inflammatory cytokine known to stimulate sebum production in the skin, Interleukin-8 (IL-8) was quantified, its production level decreased after treatment with the studied EPS ^[77][136]. Furthermore, a high molecular weight EPS produced by the deep-sea hydrothermal vent bacterium V. diabolicus CNCM I-1629 was shown in a dermal equivalent model (composed of collagen I and EPS and containing living human dermal fibroblasts) to promote both collagen structuring and fibroblast colonization (migration and proliferation) with an extracellular matrix synthesis by the cells ^[89][90][143,144]. As an active ingredient, Epidermist 4.0^TM improves skin barrier functions and increases skin defense against pathogens involved in acne ^[86][65]. These examples emphasize the interesting bioactivities of some marine bacterial EPS and demonstrate their value as cosmetic active ingredients.

It was shown through the cited examples that bacterial EPS present a wide diversity not only in terms of structural features but also of molecular weights, which further determine their functional properties relevant for cosmetic products. Anionic high molecular weight xanthan and gellan gums are mainly used as functional ingredients providing textural properties to cosmetic formulations, while HA constitutes a multifunctional ingredient as it possesses both textural and bioactive properties. Marine bacterial EPS are mainly used for their various biological activities, which make these polymers good candidates as active ingredients. However, structure-function relationships of EPS for cosmetic applications are not easy to identify due to important diversity of their compositions and structures (if known), their molecular weights and molecular conformations. Nevertheless, some hypotheses can be proposed. Due to their anionic nature resulting from the presence of negative charges of uronic acids and sulfate, acetate or pyruvate groups, EPS can efficiently bind positively charged components (e.g., proteins, growth factors) through ionic interactions. Such a role is largely known for non-sulfated (hyaluronic acid) and sulfated (heparan, heparan sulfate, chondroitin sulfate) GAG of mammalian tissues. Indeed, through interactions with multiple proteins, GAG regulate cellular processes (adhesion, migration, proliferation, differentiation) and are thus involved in physiological processes ^[117][189]. These interactions have been shown important for skin repair or regeneration activities, where the presence of EPS was shown to stimulate the synthesis of the extracellular matrix rich in HA and collagen by the cells.

Polysaccharides in cosmetic formulations are also used to maintain skin structural integrity and health due to their moisturizing, soothing, and anti-wrinkle activities, as well as whitening action and UV protection. Since polysaccharides are highly hydrophilic polymers, their moisturizing properties result from their high-water binding capacity due to the hydrogen bond formation between their multiple polar groups and water molecules, which confers moisturizing activity and further prevents from the loss of water from the skin surface ^[118][21]. The presence of different functional groups in the EPS structure (hydroxyl, sulfate, carboxyl, carbonyl, secondary amine) also provides metal chelating activity, an important property for protective effect against pollution ^[119][120][190,191]. Skin whitening is an important cosmetic market in Asia and is mainly due to inhibition of tyrosinase activity involved in melanin biosynthesis. This property has been discussed in relation of mannose presence in polysaccharide extracts ^[121][192]. Polysaccharides endowed with anti-oxidant, in particular through reactive oxygen species (ROS) scavenging ^[118][21], or anti-inflammatory activities are valuable in anti-aging application, and UV protection of skin. Besides its whitening action, mannose was also shown to inhibit inflammatory damage in skin ^[121][192]. However, the underlying structural basis of action mechanisms is still limited.

Biological activities of bacterial EPS on skin also depend on the molecular weight of polymers, i.e., the length of the polysaccharide chain, and its molecular conformation. Similarly to other polysaccharides from plant and animal origin, bacterial EPS are highly flexible macromolecules adopting helical conformations in solution ^{[122][123][124]}[14,193,194]. With increasing length, polysaccharide chains display tendency to high entanglement reinforced by hydrophilic and ionic interactions. Therefore, low molecular weight polymers can easier cross the skin barrier and activate various biological pathways (e.g., anti-aging), compared to high molecular weight native polymers, acting at the epidermis level through moisturizing, barrier function and anti-wrinkle smoothing effects. Regarding human skin, hyaluronic acid penetration was shown to depend on the molecular weight, as HA of high molecular weight (1000 to 1400 kDa) remained at the surface of the stratum corneum, and HA of low molecular weight (20 to 300 kDa) was able to crosse the stratum corneum ^[125][195].