EFFECT OF GALACTO-OLIGOSACCHARIDES ON STAPHYLOCOCCUS EPIDERMIDIS GROWTH AND EXAMINATION OF THEIR DIFFUSION FROM COSMETIC FORMULATIONS

According to the current definition, a prebiotic is described as “a substrate that is selectively utilized by host microorganisms conferring a health benefit”. Originally, the concept of prebiotics was defined for the gut microbiota. However, it can be also applied to modulate the composition of other microbiological populations, for example, skin microbiota. In recent years, scientific interest in the composition and function of the dermal microbiota has been growing, as it has been established that certain skin diseases are associated with dysbiosis of the skin microbiota. Consequently, for the cosmetics industry, it is of utmost importance to develop products that have a prebiotic effect and thus can provide a balanced composition of the skin microbiota. In this paper, an assessment of the galacto-oligosaccharides (GOS) prebiotic activity was performed by following the effect of the addition of these oligosaccharides on the growth of Staphylococcus epidermidis, a member of the permanent skin microbiota. Simultaneously, changes in nutrient medium composition during the fermentation were monitored using high-performance liquid chromatography. In the end, bearing in mind the future implementation of GOS in cosmetic formulations, the possibility of controlled release was determined using the Franz cell diffusion study. The obtained results showed that GOS has a stimulating effect on the growth of S. epidermidis since it has been noticed that the number of vegetative cells increases with increasing concentration of GOS up to the concentration of 5 % (w/v). Based on the change in the composition of soluble sugars in nutrient media enriched with oligosaccharides, it was confirmed that the bacteria S. epidermidis can utilize GOS. Finally, the effective diffusion coefficients of GOS, that have been incorporated into two different cosmetic formulations for previously defined optimal concentration (5 % w/v), were determined. Namely, the best results were obtained for formulation with Heliogel as a gelling agent - the effective diffusion coefficients of GOS trisaccharides (GOS3) and GOS tetrasaccharides (GOS4) were 1.904·10-6 cm2/s and 4.696·10-9 cm2/s, respectively. On the other hand, the effective diffusion coefficient of GOS3 in hydrogel Aristoflex AVC was 5.148·10-8 cm2/s, while the diffusion coefficient of GOS4 could not be determined, due to the low diffused concentrations. This indicates that GOS3 diffuse significantly faster than GOS4 and the rate and mechanism of transport of these molecules are highly dependent upon the type and characteristics of the formulation

Selective Immobilization of Fructosyltransferase onto Glutaraldehyde Modified Support and Its Application in the Production of Fructo-oligosaccharides

In recent decades, the scientific community has recognized the growing importance of prebiotics, and therefore, numerous studies are focused on their economical production due to their low presence in natural resources. It has been confirmed that prebiotics are a source of energy for probiotics in the gastrointestinal tract (GIT) and enable their proliferation, consequently leading to the normal functioning of the intestinal microbiota. Also, products of their fermentation are short-chain fatty acids (SCFA), which play a key role in maintaining and improving the health not only of the GIT but also of the whole organism. Among several confirmed prebiotics, fructo-oligosaccharides (FOS) are considered interesting candidates for use in a wide range of products in the food industry. They are characterized as low-calorie and non-cariogenic substances that represent an adequate sugar substitute and can be considered suitable for use in products intended for diabetics. The subject of this research will be the production of FOS by transforming sucrose using a fructosyltransferase (FTase) present in commercial preparation Pectinex® Ultra SP-L, with special emphasis on the development of adequate FTase immobilization method that would enable selective isolation of the enzyme responsible for the synthesis of FOS from the complex enzymatic mixture. This would lead to considerable enzyme purification and allow its direct incorporation into different sucrosebased products, without the fear that action of the other hydrolytic enzymes may adversely affect the products functional characteristics. Accordingly, the possibility of selective immobilization of the enzyme using support with primary amino groups, Purolite® A109, which was previously activated and modified using glutaraldehyde (GA), was investigated. In the initial phase of the research, the effects of individual immobilization parameters such as pH, enzyme concentration and immobilization time were investigated to optimize the process using support chemically activated with 15% and 0.5% GA to form dimers and monomers, respectively. It was determined that highly active immobilized preparations (371.8 IU/g of support - dimer and 213.8 IU/g of support – monomer) were achieved under acidic conditions (pH 4) provided that an enzyme concentration was 50 mg/g of support after 7 h and 3 h, respectively. Bearing in mind the obtained results of the expressed activity, it is noticeable that the formation of dimers showed higher reactivity compared to the form of monomers. Also, in the case of support modification using 15% GA, the value of the ratio of FTase and pectinase (as dominant enzyme mixture component) activity immobilization yields was 16.45, indicating the high feasibility of selective immobilization of FTase on modified polystyrene resin. After obtaining immobilized preparations of satisfactory features, they were tested in a reaction of FOS synthesis under determined optimal conditions. The maximum FOS yields of approximately 50% of total carbohydrates in the reaction mixture were recorded after 21 h. Finally, it can be concluded that the examined immobilization method yielded highly active, stable and more importantly refined enzyme preparation that can be further utilized on a larger scale for development of continual processes for FOS synthesis, as well as for modification of different sucrose-based mediums

Transdermal diffusion of enzymatically synthesized oil-derived phloridzin esters from oil in water emulsion

Phloridzin is flavonoid known for its antioxidant and UV-protective effects on the skin. However, it has limited solubility in both hydrophilic and lipophilic media, therefore it should be derivatized prior to incorporation in final products. Hereby, we applied lipase-catalyzed acylation to produce different phloridzin ester mixtures using coconut and linseed oil as acyl donors and examined their transdermal delivery. The new derivatives were incorporated in oil in water emulsion to examine their diffusion through artificial skin-mimicking Strat-M® membrane using Franz diffusion cell. The transport of esters was successfully modelled by modified Kosrmeyer-Pepas equation. Lag phase, lasting 90-170 min, was detected for all esters. For coconut oil-derived esters process was entirely controlled by diffusion, while for linseed oil-based esters polymer relaxation was observed, as well. Effective diffusion coefficients of medium-chain esters obtained using coconut oil were one order of magnitude higher compared to long-chain ones. Zeta potential of both formulations was constant during six month storage period, indicating their high stability. Obtained results underscore suitability of natural triglycerides application in the production of phloridzin esters for topical products.European Biotechnology Congress 2023, Ljubljana, Slovenia, October 4-6, 202

Valorization of sunflower meal toward the production of emerging prebiotics

Recently, there is an increasing interest in utilizing biomass and waste potential for development of new products and materials. The example of this is sunflower meal, by-product of sunflower seed oil industry, that is currently used as a cheap component of animal feed owing to a poor protein content. The major drawback for their application is high content of complex carbohydrates (15.27% hemicellulose, 12.54% cellulose and 5.88% lignin). Therefore, extraction and enzymatic transformation of carbohydrates (primarily xylan) represent an attractive valorization approach. Since xylan extraction from the plant cell wall is highly restricted by lignin presence, different alkaline extraction methods have been proposed. This work is focused on xylan extraction using ethanol extraction and subsequent delignification approach. The ethanol extraction step yielded fraction rich in simple sugars, polyphenols (predominantly chlorogenic acid) and colorants that proved to have great prebiotic potential for application in skin products enabling proliferation of beneficial skin commensal and suppressing the pathogen one. Introduction of additional delignification step enabled high xylan yields (91.35%). This fraction was subjected to the enzymatic treatment using commercial xylanase (ROHALASE® SEP-VISCO) to obtain xylo-oligosaccharides (XOS), compounds that comprise of 2–10 xylose residues linked via β-1,4-glycosidic bonds with high prebiotic activity and excellent application properties (high temperature and pH stability, good sweetening power and low caloric value) for food industry. As result of the sunflower meal xylan treatment under optimum reaction conditions (60°C, pH6 and enzyme concentration of 0.5% per substrate), XOS with a polymerization degree of 2–6 were produced (with predominance of XOS2), without xylose generation. The prebiotic activity of the obtained XOS fraction was confirmed by fermentation with beneficial microbial cultures and pathogenic gut microbiota representative. Therefore, it was shown that significant portion of meal can be converted to added-value functional ingredients, while remaining product retains higher protein content

In situ transformation of sucrose in maple syrup in order to produce fructo-oligosaccharide enriched product

It is well known that maple syrup, a product obtained by processing maple tree sap, is widely used in many households due to its sweetness and beneficial ingredients. It is most often used as a topping for pancakes, waffles, donuts, and other desserts, but also as a sweetener for numerous beverages such as coffee, tea, lemonade, and many others. This natural syrup, in which, in addition to numerous minerals, antioxidants, carbohydrates and other components, sucrose predominates, presents an excellent substrate for the synthesis of bioactive molecules, i.e. fructo-oligosaccharides (FOS). These indigestible oligosaccharides which belong to a group of established prebiotics are very suitable from the standpoint of human health because they enable the normal functioning of the gastrointestinal tract, have a positive effect on the immune system as well as many other benefits for the human organism. With the approach which include the conversion of sucrose into FOS, it is possible to obtain a product with improved characteristics, i.e. higher functional and lower caloric values. Accordingly, in this study, a detailed optimization of the enzymatic synthesis of FOS was performed using maple syrup as a source of sucrose and commercial enzyme mixture Pectinex® Ultra SP-L as a source of fructosyltransferase. Namely, by individual varying of enzymatic synthesis key factors such as temperature (30-80 °C), enzyme concentration (1, 3 and 5%) and reaction time (0-25 h), optimal conditions were selected. It was determined that by performing the synthesis reaction at a temperature of 60 °C with an enzyme concentration of 3% for 12h, a remarkable sucrose hydrolysis degree of 82% and a FOS yield of approximately 55% were achieved. In this case, the estimated caloric value of the obtained product is about 2.2 kcal/mL, which is around 1.6-fold lower compared to the initial value of 3.6 kcal/mL. The product obtained in this manner could represent a low-calorie sweetener with high fiber content and could potentially be included in the diet of diabetics and obese people, as well as all those who aim to maintain better overall healt

NANOFILTRATION AS A TOOL FOR HIGH-YIELD PURIFICATION OF DIETARY OLIGOSACCHARIDES

Fructo-oligosaccharides (FOS) are linear bioactive molecules consisting of one terminal glucose and other fructose units and belong to the group of prebiotics with scientifically proven benefits for human health. Physiological active compounds like trisaccharides (FOS3), tetrasaccharides (FOS4) and pentasaccharides (FOS5) can be produced from sucrose using enzyme-fructosyltransferase. The reaction mixture obtained in this way contains, in addition to the desired prebiotics, glucose, fructose and an unreacted amount of sucrose. Direct incorporation in products of food and pharmaceutical origin is limited due to the presence of unwanted carbohydrates (mono- and disaccharide), and to increase the functional value of the mixture, the attention was focused on their removal. In this regard, membrane separation techniques present a very suitable solution for obtaining a product with a higher FOS proportion. This study aimed to determine the optimal conditions for purification process of produced FOS mixture using nanofiltration membrane modules. Therefore, the influence of membranes with different MWCO (300-500Da and 600-800Da), temperature (20-45°C) and carbohydrate concentration in feed solution (1-5%) at a constant flow rate of 22 mL/min was investigated. From the initial mixture containing 60.2% FOS, 29.7% monosaccharides and 10.1% sucrose, after purification process under determined optimal conditions (membrane 300-500Da, temperature 35°C and carbohydrate concentration 3%), the product with 88.8% FOS, 10.0% sucrose and 1.2% monosaccharides in total carbohydrates was obtained. Purification yield was greater than 95% and the rejection coefficients for glucose, fructose, sucrose, FOS3, FOS4 and FOS5 were 0.17, 0.20, 0.95, 0.99, 1.00 and 1.00, respectively. Based on the calculated purification factor, it can be concluded that the initial mixture is refined 1.47 times. This result suggests a significant increase in the purity of the final mixture, which can be used as a sugar substitute or addition to numerous products that would be suitable for all consumers, including those suffering from diabetes

IN VITRO RELEASE AND (TRANS)DERMAL DELIVERY STUDY OF BLACKCURRANT ANTHOCYANINS INCORPORATED IN COSMETIC FORMULATION

Blackcurrant is anthocyanin-rich berry with proven antioxidant and photoprotective activity and emerging prebiotic potential, widely applied in cosmetic products. Although beneficial effects of its extract are well known, release from skin care products and (trans)dermal permeation were not previously investigated. In this study, enzyme-assisted extraction of polyphenols from lyophilized blackcurrant was performed and obtained extract was incorporated into an Aristoflex® AVC based hydrogel. Furthermore, Franz diffusion cell experiments were conducted in order to examine the release of these molecules from the prepared formulation using cellulose acetate membrane and transdermal and dermal delivery using human skin mimicking membrane (Strat® M). Obtained results revealed that all four dominant anthocyanins readily permeated from hydrogel since 17.5%, 32.8%, 33.8% and 39.2% of delphinidin 3-glucoside, delphinidin 3- rutinoside, cyanidin 3-glucoside and cyanidin 3-rutinoside, respectively, were detected in receptor fluid after 24 h. Experimental values were successfully fitted with the Peppas and Sahlin diffusion model. On the other hand, after 72 h long experiment with transdermal skin diffusion model (Strat® M membrane), no detectable amounts of anthocyanins were present in receptor fluid and only 0.5% of the initial quantity from donor compartment was extracted from the membrane itself, indicating weak dermal delivery. Present study revealed that hydrogel is suitable carrier system for the topical delivery of bioactive anthocyanins from blackcurrant, while dermal and transdermal delivery of these molecules is very limited. This implies applicability of blackcurrant extract for treatments targeting skin surface (i.e. antioxidant, prebiotic, photoprotective)

Enzyme-assisted extraction of various bioactive components from blackcurrant (Ribes nigrum)

According to experts, berries are a major source of phytochemicals, disease-fighting components, which can help to boost the immune system and lower the risk of many agingrelated conditions. The simplest way to isolate these compounds is through solvent extraction. However, this method is not entirely successful since, in addition to free phytochemicals, there are also significant amounts of bound components that are trapped in the cell wall. For these reasons, the addition of enzymes such as cellulases and pectinases provides the hydrolysis of the cell wall, facilitate the release of molecules and increase the extraction yield of phytochemical. Therefore, in this work, enzyme-assisted extraction of lyophilized blackcurrant (LCR), a fruit that is increasingly used for pharmaceutical and cosmetic purposes, was examined. Extraction was performed in acetate buffer pH 4.5, at 50 ̊ C using four enzymes: Pectinex® Ultra SP-L, Viscozyme® L, Cellic® CTec3, Rohapect® MC. In the first step of the experiment, the extraction was optimized by different-time varying, from 10 to 120 min, and after choosing the optimal time, the influence of the solid-liquid ratio (1:4, 1:10, 1:40) on the extraction efficiency was examined. The progress of extraction was monitored spectrophotometrically, through antioxidant properties, the total content of polyphenols, flavonoids, and phenolic acids from all blackcurrant extracts and compared to the control (extract without enzyme). Regarding the total content of polyphenols, it was revealed that 60 min was the optimal extraction time. On the other hand, it was found that for a solid-liquid ratio of 1:4, all results were about 1.5 times higher than the control values. In terms of enzymes, Viscozyme® and Pectinex® Ultra SP-L enabled the recovery of the highest total phenolic content (~28 mg GAE/g LCR) and phenolic acids (~3 mg CA/g LCR), while, on the other hand, Rohapect® MC enhanced the extraction of antioxidants (FRAP 380 µmol Fe2+/g LCR), as well as total flavonoids (2.85 mg QE/g LCR). Obtained results open new possibilities for blackcurrant extracts treated with different enzymes as value-added ingredients for the cosmetic and food industr

Evaluation of Herbal Extracts for Their Potential Application as Skin Prebiotics

One of the fundamental requirements for overall human well-being is a stable and balanced microbiome. Aside from the microorganisms that reside within the body, a large number of microorganisms, especially bacteria, swarming the human skin are in homeostasis with the host and represent a skin microbiota. Even though the immune system of the skin is capable of distinguishing between commensal and potentially harmful transient bacteria, the cutaneous microbial balance can be disrupted under certain circumstances. In that case, reduction in the skin microbiota diversity, as well as changes in metabolic activity, result in dermal infections and inflammation. Probiotics and prebiotics have the potential to play a significant role in the treatment of these skin disorders. The most common resident bacteria found on the skin, Staphylococcus epidermidis, can act as a potential skin probiotic, contributing to the protection of healthy skin from pathogen colonization, such as Staphylococcus aureus, which is related to atopic dermatitis exacerbation. However, as it is difficult to meet regulations in cosmetic products, another therapy approach could be topical prebiotic supplementation of the skin microbiota. In recent research, polyphenols are attracting scientists' interest as biomolecules with possible prebiotic effects on the skin microbiota. This research aimed to determine how herbal extracts rich in different polyphenolic compounds (lemon balm, St. John's wort, coltsfoot, pine needle and yarrow) affected the growth of S. epidermidis and S. aureus. The first part of the study involved screening of plants to determine if they could be regarded as probable candidates to be skin prebiotics. The effect of each plant on bacterial growth was examined by supplementing the nutrient medium with their extracts and comparing it with control samples (without extract). The results obtained after 24 h of incubation showed that all tested extracts influenced the growth of the examined bacteria to some extent. Since lemon balm and St. John's wort extracts displayed bactericidal activity against S. epidermidis, whereas coltsfoot inhibited both bacteria equally, they were not explored further. On the other hand, pine needle and yarrow extract led to an increase in S. epidermidis/S. aureus ratio, making them prospective candidates to be used as skin prebiotics. Byexamining the prebiotic effect of two extracts at different concentrations, it was revealed that, in the case of yarrow, 0.1% of extract dry matter in the fermentation medium was optimal, while for the pine needle extract, a concentration of 0.05% was preferred, since it selectively stimulated S. epidermidis growth and inhibited S. aureus proliferation. Additionally, total polyphenols and flavonoids content of two extracts were determined, revealing different concentrations and polyphenol profiles. Since yarrow and pine extracts affected the growth of skin bacteria in a dose-dependent manner, by carefully selecting the quantities of these extracts, and thus polyphenols content, it is possible to achieve desirable alterations of skin microbiota composition, which may be suitable for the treatment of atopic dermatitis

DISCOVERING POTENTIAL OF POLYPHENOL COMPOUNDS FROM BLUEBERRY, CRANBERRY AND CHOKEBERRY EXTRACTS AS SKIN PREBIOTICS

Berries are known to be one of the richest sources of polyphenols which can offer various health benefits. Dietary supplementation with berries has a positive influence on the gut microbiota, which directly affects overall health, including skin health. However, topical application of berry polyphenols has been used mainly for its antioxidant activity to prevent premature aging and improve the skin's appearance. Therefore, this study examined the content of different polyphenol classes of cranberry (Vaccinium macrocarpa), chokeberry (Aronia melanocarpa) and blueberry (Vaccinium angustifolium) extracts, investigating their antioxidant properties and potential impact on skin as topical prebiotics. The prebiotic capacity of these extracts in applied concentrations range of 0.015-0.05 mg GAE/mL, was determined against two cutaneous bacteria - beneficial Staphylococcus epidermidis and opportunistic pathogen Staphylococcus aureus, since the disrupted balance between them may contribute worsening of atopic dermatitis. The obtained results showed that the total polyphenol content was highest in chokeberry extract (9.88 mg GAE/g DM), followed by cranberry extract (8.78 mg GAE/g DM), and the lowest in blueberry extract (6.48 mg GAE/g DM). Chokeberry extract was also richest in flavonoids, flavonols, anthocyanins and phenolic acids. Notably, cranberry extract had the highest concentration of tannins, almost three times higher compared to blueberry extract. According to DPPH and ABTS methods, the antioxidant activity was significantly high in chokeberry extract, while FRAP method revealed that cranberry extract is the most potent antioxidant. Regarding prebiotic capacity, positive values (0.10-0.48) were observed at all concentrations of cranberry extract, with a trend indicating a decrease in prebiotic capacity as polyphenol concentration increases. Prebiotic capacities of blueberry and chokeberry extracts had either negative values or values equal to zero, indicating that these extracts do not positively impact the microorganisms’ ratio. Obtained findings suggest cranberry extract potential for enhancing both antioxidant defense of skin and rebalancing cutaneous microbiota