Characterization of Exopolysaccharides Isolated from Donkey Milk and Its Biological Safety for Skincare Applications

Abstract

Kefiran is a heteropolysaccharide that is considered a postbiotic and is obtained by kefir grains fermented in cow’s milk, while little is known about the donkey milk (DM) variety. Postbiotics are recognised as having important human health benefits that are very similar to probiotics but without the negative effects associated with their ingestion. Donkey is a monogastric animal, as are humans, and when used as an alternative food for infants who suffer from cow milk protein allergies, DM could therefore display more biocompatibility. In this study, the DM kefiran was extracted by ultrasound from kefir grains cultured in donkey milk and fully characterized for its structural and physicochemical properties by Fourier-transform infrared spectroscopy (FT-IR), High-Performance Liquid Chromatography- Refractive Index (HPLC-RI), Scanning electron microscope (SEM), Differential Scanning Calorimeters (DSC) and rheological analyses. In addition, tests were conducted on keratinocytes cell lines and human red blood cells to assess the nontoxicity and haemolysis degree of the polymer. The extraction yield of the DM kefiran was 6.5 ± 0.15%. The FT-IR analysis confirmed the structure of the polysaccharide by showing that the stretching of the C-O-C and C-O bonds in the ring, which formed two bands at 1157 and 1071 cm⁻¹, respectively, and the anomeric band at 896 cm⁻¹ indicates the β configuration and vibrational modes of glucose and galactose. Results were confirmed by HPLC-RI analysis indicating that the ratio glucose/galactose was 1:0.87. Furthermore, the SEM analysis showed a porous and homogeneous structure. The rheological analysis confirmed the pseudoplastic nature of the polymer, while the DSC analysis highlighted excellent thermal resistance (324 °C). Finally, DM kefiran was revealed to have biologically acceptable toxicity, showing a haemolytic activity of less than 2% when using fresh human red blood cells and showing no cytotoxicity on human keratinocytes. Therefore, kefiran obtained by DM shows an excellent biocompatibility, establishing it as a promising polymer for bioengineering human tissue for regenerative applications.

1. Introduction

Kefiran is a microbial polysaccharide obtained by kefir grains, which are usually used as a culture starter for milk fermentation [1]. This heteropolysaccharide, consisting mainly of glucose and galactose, is completely soluble in water. Theoretically, kefiran, the polymer obtained, has the same ratio of glucose to galactose as lactose; the disaccharide presents in the fermentation substrate. Nevertheless, variations in the glucose to galactose ratio have been reported, ranging from 1:0.4 to 1:1.88, as well as variations in its molecular weight [2]. Factors that can influence these parameters as well as the chemical physical and rheological characteristics are fermentation conditions, extraction and purification methods [3]. Today, this polymer is recognised as postbiotic and has GRAS (generally recognised as safe) status, and its production is carried out by lactic acid bacteria, mainly Lactobacillus kefiranofaciens [4]. Furthermore, kefiran has been reported to possess several beneficial impacts on human health [5]. The main recognised functions of kefiran are antimicrobial and antioxidant activity [6], as well as the ability to form edible films with good mechanical and barrier properties for use in food packaging [7] and the ability to improve the rheological features of fermented milks [8] due to its physicochemical properties in increasing viscosity and viscoelasticity [9]. Furthermore, with the development of the field of application of polysaccharides, cosmetic activities [10] such as moisturising, whitening and anti-ageing [11] have paid increasing attention to polysaccharides [12], as they can be easily obtained from plants [13] and are also synthetic products of bacteria [14]. In addition, recent studies have shown that the monosaccharide composition and infrastructure bonds can directly influence the biological activity of polysaccharides [10]. Yao et al. showed that exopolysaccharides containing high proportions of glucose, galactose and arabinose exhibit strong antioxidant activity [15].

Kefiran is usually isolated from kefir grains cultured in cow milk, and, to the best of our knowledge, little evidence exists in the literature about the donkey milk (DM) variation. Donkey is a monogastric animal, as are humans, and DM is used as an alternative food for infants who suffer from cow milk protein allergies [16]. Recently, Cirrincione et al. [17] evaluated the bioactive peptide-profile form of fermented DM, which was assessed using the targeted fermentation procedure. The proteins identified were β-casein, with 31 peptides, and κ-casein, with 10 peptides [17]. In recent years, our research group started studying symbiotic fermentation in order to obtain knowledge about biocompound shelf-life, fortified food [1,18,19] and the extraction methodology of kefiran from kefir grains [20]. In the present work, kefiran was obtained from milk kefir grains after the fermentation of DM, and then biocompatibility testing was performed due to the important skin-healing properties of exopolysaccharides, including kefiran, which is used in cosmetic industries. The obtained kefiran was fully characterized for its structural, physicochemical and biological properties through a complete set of technologies such as Fourier-transform infrared spectroscopy (FT-IR), High Performance Liquid Chromatography equipped with refractive index (HPLC-RI), differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and rheology. Then, the cytocompatibility of kefiran was also assessed by quantifying its haemolytic activity and its impact on the metabolic activity of exposed HaCat cells.

2. Materials and Methods

2.1. Extraction of Exolysaccharides

Kefir grains were purchased by Kefiralia (Arrasate, Gipuzkoa, Spain). Kefir grains were activated in cow milk for 7 days, then subsequently fermented in DM for one day. The grains in the DM, 10 g per 100 mL, were separated by filtration through a sieve and used for extraction. Briefly, kefir grains were immersed in water and sonicated for 10 min using ultrasonic (24 kHz as frequency and 100 W as power) (Ultrasonic Processor Hielscher Model UP400S, Hielscher Ultrasonic, Teltow, Brandburg, Germany). The sample was centrifuged at 10,000 rpm for 15 min. The supernatant was added to cooled absolute EtOH at −20 °C (1:1 V/V) and stored at −20 °C overnight. The precipitated exopolysaccharide was separated by centrifugation (10,000 rpm, t = 20 min, T = 4 °C). This treatment was carried out two times to obtain a sediment. The polysaccharide samples were finally freeze-dried (Lyophilizer Telstar freeze-dryer, mod. Cryodos, Telstar, Terrassa, Barcelona, Spain) [20].

2.2. Fourier Transform Infrared

The obtained polymer was characterised by an FT-IR (Bruker ALPHA FT-IR spectrometer, Bruker, Billerica, MA, USA) equipped with an A241/D reflection module (Bruker, Billerica, MA, USA). The pulverized sample was prepared by mixing it with KBr and compressing the resulting powder with a press at a force of 6 tons [21]. The spectra were recorded in the wavelength range of 400–4000 cm⁻¹.

2.3. Monosaccharide Composition

To assess the monosaccharide profile, a hydrolysis of polymer (10 mg) was carried out at 70 °C for 72 h, with 0.2 M trifluoroacetic acid (TFA). Then, the sample was filtered through a microfilter 0.45 µm (Chromafil Xtra PVDF, Macherey-Nagel, Duren, Germany), and 20 µL of the clear diluted sample was isocratically separated by an HPLC (HPLC LC-NetII/ADC PU-2089 plus) equipped with refractive index RI-4030, autosampler As-2050 plus, oven co-2060 Plus (Jasco Technologies, Santa Clara, CA, USA) on a Rezex ROA-Organic Acid column H+ (8%) 300 × 7.8 mm i.d., 8 µm, (Phenomenex, Aschaffenburg, Germany) by a flow of 0.4 mL/min with 5 mM H₂SO₄ at 75 °C [22]. Glucose and galactose were quantified by 5-point calibration with external standards in a range of 0.01–1 mg/mL.

2.4. Differential Scanning Calorimetry

The thermal properties of the sample (10 mg) were ascertained using differential scanning calorimetry (SETARAM 131 evo, LabWrench, Midland, ON, Canada). Analyses were performed from −20 °C to 500 °C at a temperature scan rate of 20 °C/min under nitrogen flow [23].

2.5. Rheological Measurement

The rheological measurement of kefiran was performed at a fixed concentration of 3% in water using a strain-controlled rheometer RFS III (Rheometrics, TA Instruments, New Castle, DE, USA) and a cone and plate geometry with a 50 mm diameter. The cone angle was 0.04 rad, and the gap was set at 0.048 mm. This experiment determined the shear viscosity as a function of the shear rate, from 0.1 to 100 s⁻¹ at 25 °C [24].

2.6. Scanning Electron Microscope Measurement

The morphological features of exopolysaccharide (EPS) were characterised using a Field Emission SEM FEI Quanta 200 (ThermoFisher Scientific, Hillsboro, OR, USA) at 15 KV. First, a significant part of the sample was attached to a typical 12 mm diameter support of the SEM Analyser by a biadhesive. The particulate remnants on the sample and support were blown off with air prior to the sputtering/metallisation process. Secondly, sputter coating was carried out with a conductive material, specifically a 5-nm layer of carbon, using a QUORUM Q150T-ES Carbon Coater (ThermoFisher Scientific, Hillsboro, OR, USA). Morphological images were acquired from the scattered electron signal, and the crystal characteristics were observed by backscattered electrons (BSE signal).

2.7. Molecular Weight Evaluetion

The viscosities of dilute solutions (at concentrations of 0.02, 0.04, 0.06, 0.08, 0.10 and 0.12% w/V) were determined by using a strain-controlled RFS III (Rheometrics, TA Instruments, New Castle, DE, USA) equipped with double-gap concentric cylinder. The temperature was controlled by a water circulator apparatus (±0.1 °C). To prevent errors due to evaporation, the measuring geometries were surrounded by a solvent trap containing water. The intrinsic viscosities [h] were determined by measuring relative viscosities (hr = h/hs, where h is the viscosity of the solution, and hs is that of the solvent). The flow curves show Newtonian behaviour. Therefore, the intrinsic viscosities (h) were computed by plotting the relative and reduced viscosity versus concentration using the following equations, in which the intercept of the relative and the reduced curves with the y axis was the intrinsic viscosity [25].

2.8. Haemolysis Assay

About 5 mL of residual human red blood cells (RBC) (extracted with sodium citrate 3.2%) was added to 45 mL of PBS and immediately centrifuged at 1700 rpm for 5 min, and the supernatant was then rejected. This process was repeated until the PBS solution was clear. Then, 50 µL RBC was deposited in each well of a 96-well plate, with 50 µL of different amounts of the kefiran sample. Kefiran from cow milk was also tested. PBS and Triton X-100 10% (Sigma-Aldrich, St. Louis, MO, USA) were used as the negative and positive controls, respectively. During 1 h, the 96-well plate was incubated at 37 °C and then centrifuged at 1700× g for 5 min. Finally, 50 µL of each well was transferred into a 96-well-plate, and the absorbance at 405 nm was measured (Synergy H1 by BioTeck, Winooski, VT, USA). The experiments were performed in triplicate using DM kefiran polymer (50, 100 and 200 µg/mL). For each sample, the data were normalized with the controls, as described in Sæbø et al., 2023 [26], using the following equation where the test compounds (OD sample) were normalized relative to the positive (Triton X-100; OD pos) and negative (PBS; OD neg) control samples to give the haemolysis (%):

Haemolysis rate (%) = [(OD sample − OD neg)/(OD pos − OD neg)] × 100

2.9. Cytotoxicity Assay

MTT, which was chemically 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was performed to assess the cell viability [27]. The cells were seeded at the density of 1000 cells/well in 96-well plates and allowed to attach overnight. Media containing DM kefiran at concentrations of 50, 100 and 200 μg/mL were added and incubated for one day at 37 °C. The cells were treated with 25 µL of MTT reagent (2 mg/mL) and incubated in the dark for 6 h. The medium was discarded, the cells washed with PBS and 200 µL of DMSO was added to dissolve the coloured formazan crystals. The plates were agitated on a shaker for 5 min under basic conditions, and the absorbance was measured at 545 nm using a multimode microplate reader (Synergy H1 by BioTeck, Winooski, VT, USA). The results obtained were normalized to the percent of the control (wells without DM kefiran) and plotted against the DM kefiran concentration. The cell viability was calculated using the following equation:

Viability (%) = (Sample OD/Control OD) × 100

2.10. Statistical Analysis

Unless specified, all data were expressed as mean ± standard deviation (SD). The ordinary two-way ANOVA test followed by the Sidak Multiple Comparison Test for the biocompatibility assessment. GraphPad software (version 9.1.0) was used for the statistical analyses (GraphPad Software, San Diego, CA, USA). Differences were considered statistically significant at p < 0.05.

3. Results and Discussion

3.1. Yield of Polysaccharides from Kefir Grains

The kefir grains (10% w/V) were cultured in whole DM, reaching a growth rate of about 11.2% (w/w). Extraction was performed on 10 g of the grains, and the yield obtained was 0.65 g ± 0.015 (6.5%). Currently, comparison of the extractive yield is possible only with the exopolysaccharide isolated from the grains after the fermentation of cow milk. The most recent work on kefiran was developed by Liao et al. [28] in 2023. Their results, in terms of yield (11.0 ± 0.15%), were higher with respect to the kefiran obtained from DM.

3.2. Polymer Structural Analysis by FTIR Spectroscopy

In order to further characterise the kefiran isolated by the DM kefir grains and to identify the fundamental groups present in its structure, an infrared radiation (IR) analysis was performed (Figure 1). The stretching of the O-H group in the constituent sugar residues formed an intense peak at 3435 cm⁻¹. The relevant peak at 2917 cm⁻¹ can be associated with the symmetric and asymmetric vibrations of the C-H in the sugar ring. The peak around 1634 cm⁻¹ was formed from the bending vibration of water molecules trapped in the polysaccharide matrix. In the 1200–1000 cm⁻¹ range, the stretching of the C-O-C and C-O bonds in the ring formed two bands at 1090 and 1025 cm⁻¹, respectively, which confirmed the structure of the polysaccharide [29]. According to Guo et al. [30], the peak between 1000 and 1200 cm⁻¹ indicates the presence of the pyranose ring, confirming the presence of monosaccharides in the structure. The peaks at 600–800 cm⁻¹ can be attributed to the absorbance of low-molecular-weight carbohydrates, polyols and monosaccharides [31].

The presence of bands at 895 and 872 cm⁻¹ indicate the presence of the anomeric region, α and β-configuration, respectively, indicating the glycosidic bond between the monomeric units [32,33].

3.3. Identification of Monosaccharide

Monosaccharides form the main skeleton of polysaccharides. According to HPLC data (Figure 2), monosaccharides separated from the kefiran solution of 0.1 mg/mL were represented by glucose and galactose in a molar ratio of 1:0.87, as determined from the peak areas and by comparison with calibrated solutions of glucose and galactose. Comparable results were obtained by Pop et al. [34] on kefiran after fermentation in cow milk. Their results showed a kefiran polymer consisting of glucose and galactose in a 0.94:1.1 ratio. On the other hand, Padro et al. [35] highlighted the presence of glucose, galactose and mannose. Evaluating the composition in monosaccharides could be especially useful in order to be able to study the properties the polymer could exhibit. Jiang et al. [36] have studied how the presence of certain monosaccharides, such as galactose, in polysaccharides can cause an increase in antioxidant activity.

3.4. Thermal Capacity of Donkey Milk Kefiran

DSC was used to evaluate the thermophysical and kinetic properties with the temperature of DM kefiran (Figure 3). The sample presents three classic peaks usually observed in kefiran [32]. In particular, the kefiran of DM had three peaks at 112.4 ± 9.5, 280.6 ± 0.7 and 324.2 ± 3.19 °C. All peaks were endothermic. The transition around 100 °C could be related to its melting point, which was explained by the hydrophilic nature of DM kefiran’s functional groups. This peak also reveals the presence of the water bond: the stronger the bond, the more the peak shifts toward higher temperatures [32].

3.5. Rheological Properties of Donkey Milk Kefiran

Figure 4 shows data of the shear viscosity measured as a function of the shear rate. This plot shows data of the viscosity measured as a function of the shear rate. It was shown that the viscosity declined as the shear rate augmented in kefiran at 25 °C (2.01 ± 0.1, 1.20 ± 0.1, 0.67 ± 0.1 and 0.23 ± 0.001 Pa·s for 0.1, 1, 10 and 100 s⁻¹, respectively). This tendency could be explained by the fact that polymers have pseudoplastic behaviour, as observed by Muksing et al. [37]. An asymmetric molecular chain, in fact, can become entangled and orient itself randomly. Under stress, the molecular chains become oriented, and the number of tangles is reduced, resulting in a decrease in viscosity. At higher shear rates, an almost Newtonian behaviour can be observed, that is, the deformation rate was directly proportional to the shear rate.

3.6. Morphological Propierties of Donkey Milk Kefiran

The SEM analysis of the kefiran cryogels, obtained after fermentation in cow milk, showed a homogeneous morphology with a porous and spongy structure [38]. This sponge-like structure explains the polymer’s high water-holding capacity.

Donkey kefiran showed the same morphology, as seen in Figure 5, with a porous and filamentous surface with high water-holding capacity, so it could have applications in the food industry.

3.7. Molecular Weight Assessment

The method used for the determination of the average molecular weight of polymer involves dissolution in solvent and determination of the intrinsic viscosity. The MW is then obtained by applying the Mark–Houwink–Sakurada (MHS) equation as reported in the reference [39], using values for a and K of 0.72 and 3.8 × 10⁻⁴, respectively. The calculated intrinsic viscosity was around 20 dl/g; consequently, the Mw was around 3.5 × 10⁶ Da.

Molecular weight has an important role in characterising polymers, In particular, studying the units that constitute a polymer can determine its future use. The monosaccharide composition, for example, influences the molecular weight, but especially the thermal stability of the polymer. Ozen et al. [40] have studied the thermal resistance of beta-glucans, which consist only of glucose, and the DSC analysis of this polymer showed a thermal stability of no more than 150 °C. Conversely, Fernandez et al. [41] have studied the thermal stability of agarose, a polymer consisting mainly of a galactose unit, and the thermal resistance in the latter case does not exceed 90 °C. In our case, however, a high molecular weight, higher than 3.5 × 10⁶ Da, and the combination of glucose and galactose units resulted in a markedly improved thermal stability of over 300 °C. La Torre et al. [20], in 2022, demonstrated how the fermentation substrate can change the thermal capabilities of the polymer as well as the extraction conditions. In fact, using classic extraction methods such as simple temperature (90 °C for 1 h) can decrease thermal stability. In fact, the same polymer obtained from fermenting cow and goat milks showed a temperature degradation of less than 280 °C, while the polymer obtained from fermenting buffalo milk and using ultrasonic extraction led to a greater yield, but principally to a greater thermal stability (≅323 °C).

3.8. Biological Properties of Donkey Milk Kefiran

The biologically acceptable toxicity of materials is a crucial aspect in many applications, including tissue engineering and drug delivery systems [42]. Thus, extensive in vitro analyses according to ISO-10993-4 are required prior to clinical applications [43]. Haemolysis assays are commonly employed to evaluate the biologically acceptable toxicity of materials by assessing their impacts on red blood cells. When a haemolysis index of a substance is less than 5%, it is considered safe [44]. This classification system aligns with the American Society for Testing and Materials (ASTM)’s stringent criteria for ensuring the safety and compatibility of materials with blood components; an adequate material should not generate more than 2% of the haemolysis effect.

Table 1. Haemolysis rate (%) of DM and cow (reference sample) kefiran at three different concentrations (50, 100 and 200 µg/mL) in contact with blood cells.

Samples Kefiran Polymer	Haemolysis Rate (%)
	Donkey	Cow (Reference Sample)	p-Value
50 µg/mL	1.48 ± 0.04	1.76 ± 0.09	** p < 0.01
100 µg/mL	1.71 ± 0.14	1.94 ± 0.02	* p < 0.05
200 µg/mL	1.87 ± 0.06	2.04 ± 0.08	ns

Data are mean ± Standard deviation (SD). ** p < 0.01, * p< 0.05; ns not significant.

shows that the results demonstrated that kefiran after fermentation in donkey milk did not present a haemolytic effect, and its haemolysis rate was slightly lower than kefiran in cow milk. In this context, the research has demonstrated that kefiran could display biocompatible properties, as indicated by its lack of haemolytic activity [45].

3.9. Cytotoxicity Assessment of Donkey Milk Kefiran

Biologically acceptable toxicity is an essential feature for skincare applications, and DM kefiran exhibited a non-cytotoxic nature (100 ± 10, 105.7 ± 12.1, 100.3 ± 10.3 and 124.9 ± 9.8 for control, 50, 100 and 200 µg/mL, respectively), as shown in Figure 6.

The keratinocytes used as the cell model constitute the main cellular component in the epidermis [46]. For this motive, the HaCat cell line was chosen. Herein, we have demonstrated that the DM kefiran at different concentrations remains biocompatible. Moreover, because the cytotoxicity assay results pointed out that the DM kefiran did not display any toxic effect on cell availability, it can be considered safe for biomaterial application that directly acts on skin lesions [47]. Many polysaccharides show excellent anti-inflammatory activities, slowing down the inflammation process and, on the other hand, accelerating the wound repair process [48]. Several research studies have shown that appropriate modification promotes the analysis of the structure-activity relationship of polysaccharides. Furthermore, the addition of sulphuric or phosphate groups ensures greater exposure of the active sites and thus the improved ability to deliver hydrogen ions and, consequently, improved radical scavenging activity [49].

4. Conclusions

The ultrasound extraction method resulted in a good polymer extraction yield in just 24 h of milk kefir fermentation, using an unusual milk such as donkey milk. Characterisations demonstrated the excellent thermal and rheological capacities of the polymer, which could guarantee its use in the food industry. The polymer obtained here showed a lower toxicity than that obtained after fermentation in cow milk, the most commonly used milk. These characteristics make it a valuable biopolymer for a variety of biomedical and biotechnological applications.