Exopolysaccharides from camel milk-derived Limosilactobacillus reuteri C66: Structural characterization, bioactive and rheological properties for food applications

Abstract

Microbial exopolysaccharides (EPS) are valued as safe, functional bio-ingredients in food. Produced by microorganisms like lactic acid bacteria (LAB), they enhance the rheological and sensory properties of food products. In this study, Limosilactobacillus reuteri C66 (EPS-C66), was utilized to produce EPS. EPS-C66 was isolated, purified, and characterized for its rheological properties and biofunctionalities. Molecular weight was 3.7 × 10⁵ Da. Three monosaccharides (arabinose, mannose, and glucose) were identified. At 10 mg/mL, EPS-C66 exhibited significant scavenging activity against DPPH and ABTS radicals, achieving 67.9 % and 31.3 %, respectively. Additionally, it reduced the viability of Caco-2 and MCF-7 cancer cells by 93.7 % and 61.4 %, respectively. EPS-C66 also demonstrated antibacterial effects against S. aureus, S. typhimurium, L. monocytogenes, and E. coli, with ranges of 5.9, 6.1, 6.0, and 5.7 log CFU/mL, respectively. The current study highlights that EPS-C66 can enhance the health benefits and rheological properties of foods, contributing to the development of novel functional foods.

Highlights

• •Characterized EPS-C66 from Limosilactobacillus reuteri C66 isolated from camel milk.
• •EPS-C66 exhibits antioxidant, antidiabetic, anticancer, and antibacterial activities.
• •Rheological properties of EPS-C66 influenced by ionic strength and pH.
• •EPS-C66 enhances food texture and stability, especially in dairy products.
• •Potential application of EPS-C66 as a functional ingredient in the food industry.

1. Introduction

Microbial exopolysaccharides (EPS) are multifaceted molecules composed of repeated monosaccharide subunits and are produced outside the microbial cell. EPS are generally synthesized by a range of microorganisms including LAB and non-LAB, such as Lactobacillus and Bifidobacterium species (Nabot et al., 2022). They are especially well-suited for biotechnological applications in the food sector because they are generally recognized as safe (Saadat et al., 2019). Microbial EPSs differ primarily depending on the species and strains. Additional factors that could influence their characteristics include the composition of the medium, the culture environment, the structure of monosaccharides, charge, linkages between units, and the presence of repetitive side chains and substitutions.

EPS can be categorized into two primary types: homopolysaccharides, which are composed of a single type of sugar molecule, and heteropolysaccharides, which consist of several different sugar molecules (Zhang et al., 2024). EPS production mechanisms vary with microorganisms. L. bulgaricus and S. thermophilus utilize Wzx/Wzy-dependent pathways for homo-exopolysaccharide production while employing the extracellular synthetic pathway for heteropolysaccharide production (Zhou et al., 2021). These polysaccharides can alter the flavor, texture, and appearance of fermented foods. The viscoelastic properties of EPS, such as energy retention and release (especially storage (G') and loss (G") moduli), significantly impact their performance (Ayyash, Abu-Jdayil, Olaimat, et al., 2020).

The rheological properties of exopolysaccharides (EPS) vary due to differences in their molecular weight, the types of bonds between sugar molecules, the sugar composition, and the functional groups attached to them (Zhou et al., 2019). In the food industry, they are valuable for their ability to form gels, mix ingredients, and act as emulsifiers, stabilizers and thickeners.

EPS in dairy products improve thickness, stability, and texture by acting as thickening agents, enhancing water retention, and reducing syneresis. They also help retain water and fat, contributing to a creamier texture and higher yield without altering the taste (Xu et al., 2019b). Concerning the pharmaceutical sector, EPS has been utilized as an antioxidant, antibacterial, and antitumor agent (Ayyash, Abu-Jdayil, Itsaranuwat, et al., 2020; Jeong et al., 2017; Wang et al., 2015). Discovering natural compounds that can combat tumors, heart diseases, and diabetes is crucial to minimizing the negative impacts of numerous chemotherapy medications and preventing the body from developing resistance to specific drugs (Ayyash, Abu-Jdayil, Olaimat, et al., 2020). Dairy LABs contain a variety of bioactive compounds that could exert such protective effects. Consuming fermented probiotic dairy products may help prevent breast cancer, bladder cancer, stomach cancer, and colorectal cancer (Kober et al., 2024).

Although microbial exopolysaccharides (EPS) are gaining attention for their functional benefits in the food industry, research on the bioactive and rheological properties of EPS from L. reuteri, especially those derived from camel milk, remains limited. In contrast, EPS from other lactic acid bacteria, such as L. casei, have shown promising bio-functionalities, including antihypertensive effects through ACE inhibition (Sørensen et al., 2022), the EPS from L. reuteri remains understudied in this context. Few studies have explored the strain-dependent differences in EPS production and composition among L. reuteri strains (İspirli et al., 2019; Miao et al., 2015; Zhao et al., 2022), leaving a gap in understanding the specific functional properties of EPS produced by L. reuteri C66. Moreover, no comprehensive evaluation of both the rheological and bioactive properties, such as antioxidant, antibacterial, and anticancer activities, has been conducted for this strain.

Camel milk-derived Limosilactobacillus reuteri C66 exopolysaccharides (EPS) offer superior bio-functional and rheological properties compared to EPS from other sources, attributed to camel milk's unique biochemical composition, including high levels of bioactive peptides, lactoferrin, and essential minerals. These EPS exhibit potent α-amylase and α-glucosidase inhibition, highlighting their potential for managing diabetes by controlling postprandial glucose levels. Additionally, they possess strong antioxidant, antimicrobial, and cytotoxic activities, aligning with applications in pharmaceutical and functional food sectors. Compared to EPS from other lactic acid bacteria (LAB) such as L. casei, camel milk-derived EPS exhibit enhanced stability under extreme conditions and hypoallergenic properties, making them suitable for a broader consumer base (Bakry et al., 2021; Wu et al., 2023; Xu et al., 2019a; Zhang et al., 2023). Despite these advantages, strain-specific variations and comprehensive industrial evaluations remain underexplored, necessitating further research to fully harness their potential. A deeper evaluation of their performance in real-world food and pharmaceutical applications is needed, especially their interaction with other ingredients. Therefore, the present study was undertaken to characterize the EPS-C66 produced by a potential probiotic strain L. reuteri C66 isolated previously from camel milk and to assess its biological activities, including antioxidant capacity, amylase and glucosidase inhibition, ACE-inhibition, cytotoxicity, and antipathogenic activity. Furthermore, the study examined the rheological properties of EPS-C66 as influenced by different salts (NaCl and CaCl₂) and pH values (4.0 and 6.0).

2. Materials and methods

2.1. Bacteria and propagation

Limosilactobacillus reuteri C66 was isolated by Abushelaibi et al. (2017) and stored in a solution with 50 % glycerol at −80 °C. It was then grown in MRS broth (de Mann Rogosa Sharpe; LAB-M Limited, Lancashire, UK) at 37 °C for 20 h. After incubation, it was kept at 4 °C for short-term storage (less than a week). Two successive cultivations (37 °C/20 h in MRS broth) of L. reuteri C66 were performed before EPS extraction each time.

2.2. Isolation and purification of EPS-C66

The extraction of EPS from L. reuteri C66 fermented broth was achieved in three steps following the detailed procedure of Ayyash, Abu-Jdayil, Itsaranuwat, et al. (2020). Firstly, L. reuteri C66 was cultured in an MRS broth containing 20 g/L sucrose. Cell removal was achieved using centrifugation and filtration after incubating the inoculated M-17 broth at 25.0 ± 0.1 °C for 48 h and centrifuging at 4000 ×g for 20 min at 4 °C to obtain a cell-free supernatant (CFS). In the second step, a precipitating agent (chilled absolute ethanol) was added to precipitate the EPS from the CFS, followed by refrigeration at 4 °C for 24 h. The crude extract was then collected by centrifugation at 8000 ×g for 30 min at 4 °C. In the third step, the crude extract was mixed with trichloroacetic acid to achieve a final concentration of 8 %. The partially purified EPS was lyophilized at −80 °C, with some stored at −20 °C in a sealed container for later analysis. A sample was scanned for traces of nucleic acids and proteins using a UV–Vis spectrometer at 260 and 280 nm (Epoch2, Bio-Tek, VT, US) to detect DNA and protein traces, respectively. No absorbance was detected, indicating the absence of nucleic acids or proteins.

2.3. EPS characterization

2.3.1. Determination of average molecular mass and monosaccharide composition

EPS-C66 solution was filtered through 0.22 μm syringe filters before injection into the SIL-20 AC autosampler of the Shimadzu HPLC system (Kyoto 604–8511, Japan), equipped with a refractive index detector (RID-20 A) (Bamigbade et al., 2024). The EPS-C66 Mw was calculated using calibration curves derived from pullulan standards with molecular weights of 800, 400, 200, 110, 50, 22, 10, 6, 1.3, and 0.342 kDa. The monosaccharide composition of the EPS-C66 was assessed using the method reported by Bamigbade et al. (2024). In brief, the EPS-C66 was initially treated with 2 M trifluoroacetic acid for hydrolysis and then reacted with 1-phenyl-3-methyl-5-pyrazolone (PMP) for derivatization. The resulting derivatized MPS was then passed through a 0.45 μm membrane filter and introduced into the Shodex C18 column (250 × 4.6 mm, 5 μm) of the Shimadzu HPLC system. This system is fitted with an SPD-M20A photodiode array detector set at PDA-245 nm and runs on a gradient program with a variable mobile phase (A: 0.1 M ammonium acetate, B: acetonitrile), a flow rate of 1.5 mL/min, and a column temperature of 30 °C.

2.3.2. Fourier transform-infrared spectroscopy (FT-IR)

The infrared analysis for functional groups in the EPS of L. reuteri C66 was conducted using an attenuated total reflectance FT-IR spectrophotometer in the spectrum range of 400–4000 cm–1 at room temperature. A diamond/ZnSe crystal plate was used, and the EPS powder was positioned on the plate.

2.3.3. Nuclear magnetic resonance (NMR) analysis

NMR analysis was performed on an Avance III Bruker spectrometer operating at 600 MHz with a cryoprobe, following the procedure of Bubb (2003). One-dimensional spectra (¹H and ¹³C) and two-dimensional spectra were used to determine total correlation spectroscopy and heteronuclear multiple bond correlation. The CASPER program (www.casper.organ.su.se/casper) was used to predict the EPS structure. The procedure and analysis were conducted as described previously (El-Deeb et al., 2018).

2.3.4. Thermal properties

A differential scanning calorimeter was used to assess how EPS reacts to different temperatures. A small amount (25 mg) of EPS was heated in a special dish from 20 °C to 350 °C. A thermogram was recorded following the previously explained protocol (Sasikumar et al., 2017).

2.3.5. Scanning electron microscopy (SEM)

Surface morphology of the EPS was analyzed using a scanning electron microscope (Akishima, Tokyo, Japan) operated at a high voltage (20 kV) to obtain high-resolution images. A small amount (5 mg) of EPS was air-dried and coated with gold using a Cressington 108 Auto Sputter Coater (Ted Pella Inc., Redding, CA, USA).

2.3.6. Zeta potential and particle size analysis

Zeta potential was measured to understand the surface charge and stability of the EPS. The analysis was performed using a Nano Plus Zeta potential and particle size analyzer (Particulate System, GA, USA) as per the previously described protocol (Sasikumar et al., 2017).

2.4. Evaluation of EPS bioactivities

2.4.1. Antioxidant capacity

The EPS was tested for its antioxidant capacity using in vitro 2,2-azino-bis (3-ethylbenzo-thiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assays (Ayyash, Abu-Jdayil, Olaimat, et al., 2020). The scavenging activity was evaluated at two different concentrations of EPS (5 and 10 mg/mL). The DPPH and ABTS scavenging activities were calculated using Eq. 1 to determine the percentages.

$$\text{Scavenging Rate}\left(\%\right)=\left[1-\frac{{\mathrm{A}}_{\text{sample}-}{\mathrm{A}}_0}{{\mathrm{A}}_{\text{control}-}{\mathrm{A}}_0}\right]\times 100$$ (1)

where A₀ is the blank absorbance (negative control). A_control is the absorbance of the radical of Vitamin C as a positive control.

2.4.2. Cytotoxicity activity

The cytotoxicity activity of EPS was evaluated using Caco-2 and MCF-7 cells, derived from colon and breast cancers, respectively, at 5 and 10 mg/mL concentrations. The protocol utilized was thoroughly explained previously (Ayyash, Abu-Jdayil, Olaimat, et al., 2020). Cytotoxicity percentage was calculated using Eq. 2.

$$\text{Cytotoxicity}\left(\%\right)=\left[1-\frac{{\mathrm{R}}_{\text{sample}-}{\mathrm{R}}_0}{{\mathrm{R}}_{\text{control}-}{\mathrm{R}}_0}\right]\times 100$$ (2)

where R_sample is the absorbance ratio of OD570/OD605 of the EPS, R_control is the absorbance of the control sample (without EPS) and R_o is the average background, considered as a negative control with no cells.

2.4.3. Antimicrobial activity

The antimicrobial activity of EPS produced by L. reuteri C66 was assayed against four foodborne pathogenic bacteria: E. coli O157, Salmonella Typhimurium 02–8423, Listeria monocytogenes, and Staphylococcus aureus ATCC 25923. The response of bacteria to higher EPS concentrations was examined, as mentioned previously (Jeong et al., 2017). A 100 μL solution of EPS (5 mg/mL) was combined with 250 μL of activated pathogen in brain heart infusion (BHI, LAB-M), then incubated at 37 °C for 18 h. The pathogen was then counted on BHI agar (LAB-M) and incubated aerobically at 37 °C for an additional 24 h.

$$\text{Inhibition}\left(\%\right)=\left(1-\frac{{\mathrm{Abs}}_{\text{sample}-}{\mathrm{Abs}}_{\text{blank}}}{{\mathrm{Abs}}_{\text{control}}}\right)\times 100$$ (3)

where Abs_sample: OD of inoculated BHI with EPS, Abs_blank: OD of BHI with EPS but no bacteria, and Abs_control of inoculated BHI without EPS.

2.4.4. Antidiabetic activity

The in vitro antidiabetic activity was assessed by determining the ability of the produced EPS to inhibit α-amylase and α-glucosidase. According to Ayyash, Abu-Jdayil, Olaimat, et al. (2020), the inhibition of the two enzymes was assessed using different concentrations of the new EPS. The inhibition percentage was calculated using Eq. 3. Where Abs_sample: OD of essay with EPS, Abs_blank: OD of essay without, and Abs_control of essay without EPS (enzyme and substrate).

2.5. Rheological properties

After EPS-C66 samples were prepared, rheological tests were conducted using a Discovery Hybrid Rheometer (TA Instruments, New Castle, DE, US). Aqueous solutions were prepared by adding 0.1 M CaCl₂ or 0.1 M NaCl to a 5 mg/mL EPS-C66 aqueous solution. The solutions were adjusted to two pH values (pH 4.0 and pH 6.0). The rheological characteristics of the water-based solution were assessed using cone and plate geometries (with a 1° cone angle, 50 mm gap, and 50 mm diameter). The plate was kept at a constant temperature of 25.0 ± 0.1 °C.

2.5.1. Apparent viscosity

The flow characteristics of the prepared EPS-C66 solutions were evaluated for their response to varying shear rates (ranging from 10 s⁻¹ to 1000 s⁻¹) in both upward and downward directions. The assessments were performed at a temperature of 25.0 ± 0.1 °C. The difference between the upward and downward curves was utilized to gauge the extent of thixotropy. The flow patterns of the innovative EPS were characterized using the power law model (Eq. 4).

$$\mathrm{\tau }=\mathrm{mY}\dot{}\mathrm{ƞ}\dots$$ (4)

where τ is the shear stress, m is the consistency coefficient, Y ˙ is the shear rate, and ƞ is the flow behavior index.

2.5.2. Temperature-dependent behavior

The apparent viscosity of the new EPS-C66 was evaluated by ramping up the temperature to 80 °C at a speed of 3 °C/min while maintaining a constant shear rate of 20 s⁻¹. The flow activation energy of EPS-C66 was computed using the Arrhenius equation (Eq. 5).

$$\mathrm{n}=n_{0}e\frac{E_a}{\mathit{RT}}$$ (5)

where $\mathrm{n}$ is the apparent viscosity, $n_0$is the Arrhenius constant, $E_a$ is the flow activation energy, R is the gas constant, and T is the temperature in Kelvin.

2.5.3. Amplitude and frequency sweep tests

To assess the linear viscoelastic region of EPS-C66, a test measuring amplitude strain was conducted. This test utilized a consistent frequency of 1 Hz and explored strain levels ranging from 0.1 % to 20 %. Subsequently, a frequency sweep test was performed within a frequency spectrum of 0.1–20 Hz at a steady strain under 2 %, which falls within the linear viscoelastic range. This was to analyze the viscoelastic characteristics of EPS, specifically the storage modulus (G′), loss modulus (G′′), and the damping factor (tan δ). Both the amplitude and frequency sweep tests were executed at a stable temperature of approximately 25.0 ± 0.1 °C.

2.6. Statistical analysis

All measurements were taken in triplicate during the experiments. A one-way ANOVA was applied to identify any significant differences between the data sets. The results were presented as mean values with their corresponding standard deviations (mean ± SD), and a significance level of P < 0.05 was used to determine statistical relevance. Fisher's test was conducted to compare the mean values at this significance level.

3. Results and discussion

3.1. Molecular weight and monosaccharide composition of EPS-C66

To ascertain the biological and functional characteristics of EPS, it is crucial to investigate their chemical structure and content. The average molecular weight of EPS-C66 was determined to be 3.7 × 10⁵ Da (Fig. 1A). This result is relatively comparable to the molecular weight of EPS produced by L. reuteri SK24.003 (2.5 × 10⁷ Da) (Miao et al., 2015) and L. reuteri DSM 17938 (6.5 × 10⁵ Da) (Kšonžeková et al., 2016). The larger molecular weight significantly contributes to the rheological properties and viscosity of food products (Zhou et al., 2019). As illustrated in Fig. 1B and C, EPS-C66 is composed of three monosaccharides: mannose, glucose, and galactose in a molar ratio of 1.0:9.5:0.5. This variation in monosaccharides shows that EPS-C66 is a hetero-EPS. The monosaccharide composition significantly influences the functionalities of the EPS (Zhou et al., 2019). Our results differ from the monosaccharide composition of EPS produced by L. reuteri DSM 17938 (Kšonžeková et al., 2016) and L. reuteri SK24.003 (Miao et al., 2015) who reported only one monosaccharide type (glucose) as a composition. This indicates that the current EPS-C66 possesses different functionalities compared to those previously reported.

Fig. 1.

Molecular weight (A), monosaccharides standard (B), and EPC-C66 monosaccharide composition.

3.2. FT-IR and NMR analysis

FT-IR functional group analysis is shown in Fig. 2. The presence of the α- and β-configurations of monosaccharides was indicated by peaks at 529.9 cm⁻¹ and 814.6 cm⁻¹. The FT-IR spectra revealed various peak positions: the stretching vibration of CH formed a peak at 2926.1 cm⁻¹, while the broad stretching vibration at 3299.2 cm⁻¹ indicated hydroxyl groups. Additionally, the pyranose ring vibration demonstrated a distinct peak at 1213.8 cm⁻¹, and the presence of monosaccharides generated a peak at 917.1 cm⁻¹. The current FITR spectrum is comparable to the FTIR spectrum of EPS produced by L. reuteri E81 (İspirli et al., 2019). The difference is mainly due to the monosaccharide composition between the EPS-C66 and EPS-E81.

Fig. 2.

FTIR spectrum of EPS-C66.

Concerning NMR analysis, the ¹H, ¹³C, ¹H—¹H TOCSY, ¹³C—¹H HQSC, and ¹³C—¹H HMBC NMR spectra are shown in Fig. 3A, B, C, D, and E, respectively. The ¹H peaks between 3 and 4 ppm were assigned to the protons of C2–C6 (Ye et al., 2018). The anomeric region appeared at chemical shifts in the range of 4.5–5.5 ppm. The occurrence of peaks at 172.7, 176.5, and 177.1 ppm in ¹³C NMR spectra (Fig. 3B) and the observation of a strong HSQC correlation linking a carbon at 76.8 ppm to a proton at 5.37 ppm, accompanied by a weaker one at 72.6 and 4.62 ppm (Fig. 3C), could be considered the signature of the presence of uronic acid functions in EPS-3, in either α- and β-anomeric configuration (Ma et al., 2018). The ¹H—¹³C NMR HQSC spectrum (Fig. 3C) displayed seven major anomeric correlation signals, which may be ascribed to α/β links of d-glucopyranose, D-mannopyranose, and d-galactopyranose (Zhou et al., 2019). The current NMR spectra differ from those reported by Kšonžeková et al. (2016) for EPS produced by L. reuteri DSM 17938. This suggests that the EPS structure is strain-dependent. The EPS structures produced by the current strain and L. reuteri DSM 17938 are different. The CASPER computed assignments (Lundborg & Widmalm, 2011) of the experimental anomeric CH correlations obtained from ¹H—¹³C NMR HQSC are presented in Table 1. Based on the results from CASPER, the suggested backbone structure of EPS-3 was →4)α-d-Man(1 → 4)α-d-Glc(1 → 4)β-d-Gal(1 → 6)β-d-Man(1 → 3)β-d-Glc(1 → 6)α-d-Glc(1 → 2)β-d-Glc(1 → 6)α-d-Gal(1 → .

Fig. 3.

1D and 2D NMR analysis of EPS-C66. A) Region from 1D ¹H spectrum. B) Region from 1D ¹³C spectrum. C) Region from 2D ¹H TOCSY. D) Region from 2D ¹³C—¹H HSQC spectrum. E) Overlay of 2D ¹³C—¹H HSQC (black contours) and 2D ¹³C—¹H HMBC (red-blue contours). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1.

Chemical shifts assignments of NMR spectra.

Residues	Chemical shifts (ppm)
	1	2	3	4	5	6,6’
→6)α-d-Gali(1→	102.67	69.44	69.44	69.44	71.64	69.44
	5.29	3.83	3.83	3.83	4.79	3.65,3.83
→2)β-d-Glcii(1→	104.86	79.75	75.92	n.d.	77.47	61.91
	5.04	3.49	3.76	n.d.	3.63	3.73,3.93
→6)α-d-Glciii(1→	100.91	72.23	73.06	69.44	71.64	68.78
	5.11	3.47	3.83	3.65	4.72	3.94,4.52
→3)β-d-Glciv(1→	104.65	72.81	82.45	69.44	76.52	61.87
	4.99	3.55	4.03	3.65	3.32	3.74,3.92
→6)β-d-Manv(1→	102.13	71.64	72.81	66.50	76.54	68.22
	5.18	4.69	3.55	4.09	3.56	4.00,5.39
→4)β-d-Galvi(1→	105.57	72.30	73.00	81.31	76.74	61.15
	4.96	3.70	3.91	4.11	4.08	4.98,5.01
→4)α-d-Glcvii(1→	102.90	72.81	75.53	79.54	72.63	61.49
	5.14	3.55	4.31	3.95	4.89	3.78,3.81
→4)α-d-Manviii(1→	103.25	71.64	72.73	77.96	73.06	63.74
	5.22	4.21	4.07	4.10	3.83	3.75,4.99

3.3. SEM, thermal properties, zeta potential, and particle size

3.3.1. SEM and thermal properties

Scanning electron microscopy (SEM) has become recognized as an effective technique for examining the surface morphology of polysaccharides (Zhao et al., 2021). SEM images at 800× (Fig. 4A) and 1800× (Fig. 4B) magnifications of EPS-C66 revealed a layer-like, flake-like, and compact structure. The layer-like structure provides firm structural qualities that may be caused by the presence of α- and β-linkages (Zhou et al., 2019). Additionally, the SEM scan showed that EPS-C66 had a porous structure. The porous network potentially facilitates the retention of excess moisture, which is an advantageous feature for food production, such as cheesemaking (Bamigbade et al., 2023) and fermented milk.

Fig. 4.

SEM images at magnifications 800× (A) and 1800× (B) and DSC thermogram (C) of EPS-C66.

The thermodynamic curves are indicated in Fig. 4C. The transition temperature (T_g) of EPS-C66 was found to be 50.03 °C. A prominent peak indicating the melting temperature was found to be at 161.08 °C. At the T_m, the enthalpy energy was 204.00 J/g. EPS-C66's current Tm is higher than EPS generated by L. reuteri SK24.003 (T_m 147.7 °C) (Miao et al., 2015) and L. plantarum BR2 (Tm 97.3 °C) (Sasikumar et al., 2017). However, T_m of EPS-C66 is lower than EPS produced by L. reuteri E81, which has a T_m of 290 °C (İspirli et al., 2019). The higher T_m values indicated that EPS-C66 was stable at higher temperatures, which is advantageous for thermal processing in the food and dairy manufacturing industries.

3.3.2. Zeta potential and particle size

The particle size analyzer determined that the average size and zeta potential of EPS-C66 were 1510.4 nm and − 314.5 mV, respectively. The size and charge of EPS-C66 are comparable to those reported for EPS produced by Pediococcus pentosaceus M41 (Ayyash, Abu-Jdayil, Olaimat, et al., 2020). Negative charge is typically associated with notable bioactive properties of EPS (Xu et al., 2019b). The negative charge of EPS-C66 may be attributed to hydroxyl and carboxyl groups present in the EPS structure.

3.4. Bioactive properties

3.4.1. Antioxidant capacity

At a concentration of 5 mg/mL, EPS-C66 demonstrated radical scavenging activities of 42.7 % and 27.1 % in the DPPH and ABTS assays, respectively. When the concentration was increased to 10 mg/mL, the radical scavenging activity significantly (P < 0.05) increased to 67.9 % in the DPPH assay and 31.3 % in the ABTS assay (Table 2). These results are consistent with those obtained in previous studies (Amiri et al., 2019; Ayyash, Abu-Jdayil, Olaimat, et al., 2020). The concentration and structure of the produced EPS may contribute to their antioxidant capacity. Certain structural features, such as the presence of functional groups or molecular weight, can influence their ability to scavenge free radicals and protect against oxidative stress (Zhou et al., 2019).

Table 2.

Bioactivities of EPS-C66: antioxidant capacity (5 mg and 10 mg), antidiabetic (100 μg and 200 μg), anticancer activities (5 mg and 10 mg) and antipathogenic activity (5 mg/mL). Values are mean ± standard error of triplicates.

Attributes	EPS-C66 concentration
Antioxidant capacity	5 mg/mL	10 mg/mL
DPPH (%)	42.7 ± 2.73aB	67.9 ± 7.69aA
ABTS (%)	27.1 ± 1.28bB	31.3 ± 1.28aA
Antidiabetic activities	100 μg /mL	200 μg /mL
Amylase inhibition (%)	87.1 ± 1.51aA	88.4 ± 1.85aA
Glucosidase inhibition (%)	87.2 ± 0.53aA	88.2 ± 0.22aA
Anticancer activities	5 mg/mL	10 mg/mL
Caco-2	88.9 ± 14.22aA	93.7 ± 8.32aA
MCF-7	32.0 ± 0.61bB	61.4 ± 11.32bA
Antipathogenic activities	Log CFU/mL	Reduction
Staphylococcus aureus	5.9 ± 0.49	3.13
Salmonella Typhimurium	6.1 ± 0.17	2.89
Listeria monocytogenes	6.0 ± 0.24	2.98
E. coli O157:H7	5.7 ± 0.45	3.27

3.4.2. Antidiabetic activity

Inhibition of α-amylase is an indirect approach to controlling diabetes by reducing sugar intake from carbohydrate/starch hydrolysis. EPS-C66 showed potent inhibitory activities against α-amylase (87.1 %) and α-glucosidase (87.2 %) at 100 μg/mL, and 88.4 % and 88.2 %, respectively, at 200 μg/mL (Table 2). The values of antidiabetic activity obtained in the present study are comparable to those reported in another study (Ayyash, Abu-Jdayil, Itsaranuwat, et al., 2020), except for the α-amylase value (88.4 %) at 200 μg/mL. Comparatively, the inhibitory rate of α-amylase was found to be 90.6 % for L. acidophilus (Raj et al., 2024), which was higher than that in the present study. However, the inhibitory rate of α-glucosidase was 68.7 % for L. acidophilus (Raj et al., 2024), which were lower than that in the present study (Raj et al., 2024). Additionally, it was observed that the α-glucosidase inhibition rate of LAB strains ranged from 19.8 % to 72.3 % (Jeong et al., 2021). The α-glucosidase inhibition rates of L. gasseri MG4524, L. rhamnosus MG4502, and L. reuteri MG5149 were found to be 63.8 %, 63.4 %, and 60.3 %, respectively (Jeong et al., 2021), which were lower than our findings. The glycosidic linkages in EPS might have the ability to inhibit the activity of both α-amylase and α-glucosidase enzymes (Robyt, 2005). L. rhamnosus GG (LGG), one of the most well-studied probiotic LAB, significantly decreased blood glucose levels and improved hyperglycemia in neonatal streptozotocin-induced diabetic rats, thus proving its antidiabetic potential (Jeong et al., 2021).

The inhibitory impact of EPS on α-amylase and α-glucosidase may stem from competitive and non-competitive interactions between EPS and the enzymes' active or allosteric sites. EPS can also function as a physical barrier or engage directly with enzymes, hindering substrate access. Additionally, the functional groups in EPS, such as hydroxyl and carboxyl groups, might establish hydrogen bonds or ionic interactions with essential residues in the enzyme active sites, which further boosts their inhibitory potential (Zhang et al., 2023). These findings suggest that EPS-C66 could directly influence glucose metabolism. Moreover, it might impact the composition and activity of the gut microbiota. A balanced gut microbiota has been associated with improved metabolic health, including better glucose regulation and insulin sensitivity, which are key factors in managing diabetes.

3.4.3. Antibacterial activity

The antibacterial activity of EPS-C66 was assessed against four prevalent foodborne pathogens. Table 2 demonstrates the remarkable antibacterial activity of EPS-C66, with pathogen population reductions ranging from 2.89 log to 3.27 log CFU/mL. EPS-C66 can achieve an approximate decrease of 3.0 logs, which is advantageous considering the high initial bacterial load (9 logs). This indicates that EPS-C66 can effectively inhibit or even kill pathogens. There was no significant difference in log reduction among the four pathogens (Table 2). Our results are similar to those obtained previously (Ayyash, Abu-Jdayil, Olaimat, et al., 2020), except for S. aureus and S. typhimurium. The formation of an antibiofilm by EPS has been observed in some studies (Liu et al., 2017; Wang et al., 2015), but the effectiveness of the drug for this purpose remains uncertain (Wang et al., 2015). Several factors could be responsible for the remarkable antibacterial activity of EPS-C66, such as: (i) EPS-C66 might interfere with biofilm formation or destabilize existing biofilms, making bacteria more susceptible to immune clearance or antibiotic treatment. (ii) The observed antibacterial action of generated EPS-C66 may be partially explained by the disruption of bacterial cell membranes, which allows for the loss of cellular contents, as well as the suppression of vital bacterial enzymes involved in activities like DNA replication and protein synthesis (Jeong et al., 2017; Xu et al., 2019b).

3.4.4. Anticancer activity

The colon (Caco-2) and breast (MCF-7) cancer cell lines were significantly inhibited by EPS-C66, as shown in Table 2. EPS-C66 demonstrated an antitumor inhibition rate of 88.9 % and 32.0 % at 5 mg/mL, against Caco-2 and MCF-7, respectively. EPS-C66 showed a significant increase (P < 0.05) in its ability to inhibit tumors at 10 mg/mL to 93.7 % and 61.4 % for Caco-2 and MCF-7 cells, respectively. The level of inhibition of Caco-2 cells in this study is similar to that observed for EPS produced by R. mucilaginosa CICC (Ma et al., 2018). The antitumor properties of EPS-C66 have been found to be particularly effective against colon cancer cells (Caco-2) and breast cancer (MCF-7) cells (Table 2). The anticancer activities of L. acidophilus LA-EPS-20079 against both Caco-2 and MCF-7 cells reached 80.65 % and 71.86 %, respectively, at 10 mg/mL in cellular proliferation (BRDU cellular incorporation), being significantly different from the non-treated cells (El-Deeb et al., 2018). The specific mechanism(s) behind EPS's anticancer effects is not entirely clear. Several potential reasons exist for the anticancer effects of EPS-C66, including the promotion of apoptosis in cancer cells and competition with growth factors like tumor necrosis factor at cell receptors. Additionally, EPS may stimulate autophagy by influencing the Beclin-I protein. It might also inhibit cancer cell receptors, thereby preventing growth promoters from accessing the cells (Kim et al., 2010).

3.5. Rheological behaviours

3.5.1. Apparent viscosity and flow curves of EPS-C66

The EPS-C66 solutions showed a decrease in apparent viscosity (η) as the shear rate increased from 0 to 1000 s⁻¹ (Fig. 5A). The apparent viscosity of EPS-C66 solutions decreased when NaCl or CaCl₂ was added. This implies that the behavior of EPS-C66 is non-Newtonian (shear-thinning). The electrolyte's screening effect on intermolecular hydrogen bonding could be responsible for the decrease in EPS-C66 viscosity and may also result in a decrease in the strength of the three-dimensional network (Abid et al., 2021). This result is consistent with EPS generated by Mesorhizobium loti (de Oliveira et al., 2018). Furthermore, by curling polymer coils, the intramolecular electrostatic repulsions between the negatively charged groups in EPS-C15 molecules can reduce this viscosity (Abid et al., 2021).

Fig. 5.

Apparent viscosity (A) and flow curves fitted with power law mode (B) of EPS-C66 in CaCl₂ pH 6.0 (◇), CaCl₂ pH 4.0 (☐), NaCl pH 6.0 (○), NaCl pH 4.0 (△), H₂O pH 6.0 (+), H₂O pH 4.0 (✕).

When shear rates were below 100 s⁻¹, the impact of pH was more noticeable than that of salt types. Specifically, EPS solutions with a pH of 6.0 displayed a higher viscosity than those with an adjusted pH of 4.0 (Fig. 5A). This suggests that the type of salt did not affect the apparent viscosity, which is explained by the fact that the ionization degree of carboxyl groups in polysaccharide molecules increased as the pH value increased (Bamigbade et al., 2023). However, in a different investigation, the presence of CaCl₂ was found to increase the viscosity of EPS generated by Sporidiobolus pararoseus JD-2 (Han et al., 2016). These variations across studies could be attributed to differences in the molecular weight (Mw) and monosaccharide composition of EPS produced by microbes of different genera, species, and strains. Overall, EPS-C66 demonstrated stability when exposed to monovalent ions (Na⁺) (Fig. 5A). The Power law model fits the flow curves well (R² > 0.99) (Fig. 5B; Table S1).

The rheological properties of EPS-C66 solutions, which display shear-thinning behavior, are highly relevant to food systems, particularly in areas such as food texture, yogurt viscosity, and creaminess. The reduction in apparent viscosity as shear rate increases and the presence of electrolytes like NaCl and CaCl2 indicate that the viscosity modulation by EPS-C66 is shaped by its molecular interactions, encompassing electrostatic repulsions and the disruption of hydrogen bonds. The pH-dependent behavior of viscosity, with notable increases at pH 6.0, implies that EPS-C66 could improve the creaminess and mouthfeel of products like yogurt, where consistent viscosity and texture are essential.

3.5.2. Amplitude and frequency sweeps

The amplitude sweep curves for G' and G“ of EPS-C66, displayed in Fig. S1, show a general decrease in modulus with increasing strain across different pH levels and ionic conditions. Both moduli G' and G" are highest in CaCl₂ at pH 4.0 and lowest in H₂O at pH 4.0, indicating that ionic strength and pH significantly influence the mechanical properties of EPS-C66 (Fig. S1).

The frequency sweep curves for G' and G“ of EPS-C66 indicate that both the storage modulus (G') and loss modulus (G") increase with frequency across all conditions (Fig. S2). The highest values for both moduli are observed in CaCl₂ at pH 4.0, suggesting that the ionic environment and lower pH enhance the viscoelastic properties of EPS-C66. Conversely, the lowest values are seen in H₂O at pH 4.0, highlighting the significant role of ionic strength in modulating the mechanical behavior. These results demonstrate that the viscoelastic properties of EPS-C66 are highly dependent on the ionic composition and pH of the surrounding medium, with calcium ions providing the most substantial reinforcement effect (Fig. S2). Tan delta (δ) of EPS-C66 shows a decrease with increasing frequency across all conditions (Fig. S3). Higher tan delta values, indicating more liquid-like behavior, are seen in H₂O at both pH levels. Lower values, indicating more solid-like behavior, are observed in CaCl₂, particularly at pH 4.0, highlighting the strong influence of calcium ions and acidic conditions in enhancing the elastic nature of EPS-C66. The higher storage modulus (G') and loss modulus (G") observed with CaCl2 at pH 4.0 indicate that the presence of calcium ions improves the rigidity and stability of the exopolysaccharide, making it more suitable for applications in food systems where texture and stability are key. Additionally, the reduced tan delta values in CaCl2 at pH 4.0, which suggest a more solid-like consistency, mean that EPS-C66 is particularly effective for enhancing the texture of food items like yogurt and dressings by boosting creaminess, stability, and viscosity. These characteristics can aid in stabilizing emulsions, enhancing mouthfeel, and ensuring consistency, especially in products that demand a smooth and uniform texture, showcasing EPS-C66's potential as a natural stabilizer and texturizer in the food industry.

3.5.3. Temperature-dependent behavior

The apparent viscosity as a function of temperature is displayed in Fig. 6, illustrating how all EPS-C66 solutions behaved differently depending on temperature. With rising temperatures, viscosities of all EPS-C66 solutions decreased (Fig. 6). Significant activation was seen in the EPS-C66 solution containing CaCl₂ at pH 6.0 (Table S1). This is consistent with EPS-C66's apparent viscosity. The temperature-dependent behavior was significantly influenced by the pH value (6.0) and salt type (Ca²⁺). This could be explained by the formation of intermolecular cross-linkage bridges by Ca²⁺ at pH 6.0. We hypothesize that the intermolecular linkage bridges formed by Ca²⁺ were not supported by pH 4.0. The temperature-dependent behavior of EPS-C66 in the presence of monovalent Na⁺ was demonstrated to be stable in Fig. 6. The differences in apparent viscosities between the EPS-C66 solutions decreased until 80 °C after 55 °C. This result implies that high-heat processes are unsuitable for the current EPS-C66. This outcome is consistent with the temperature-dependent behaviours of EPS-JD2 (Han et al., 2016).

Fig. 6.

Temperature-dependent behavior of EPS-C66 in CaCl₂ pH 6.0 (◇), CaCl₂ pH 4.0 (☐), NaCl pH 6.0 (○), NaCl pH 4.0 (△), H₂O pH 6.0 (+), H₂O pH 4.0 (✕).

4. Conclusion

EPS are valuable biopolymers with a wide range of potential applications in pharmaceuticals, food ingredients, adhesives, textiles, and cosmetics. Due to its significant bioactive properties and relatively high molecular weight, EPS-C66 is a promising candidate as a functional ingredient that can be used in various food formulations. All EPS-C66 samples exhibited shear-thinning characteristics. The rheological properties were influenced by pH level and salt content. EPS-C66 produced by L. reuteri C66 has the potential to enhance the appearance, rheological properties, texture, and mouthfeel of fermented dairy products such as yogurt. Consequently, EPS-C66 is considered an attractive ingredient in the food industry, potentially serving as a viscosifier, stabilizer, emulsifying agent, or gelling agent.

CRediT authorship contribution statement

A.K.M. Humayun Kober: Writing – original draft, Formal analysis. Mohamed Abdin: Writing – original draft, Formal analysis. Athira Subhash: Writing – review & editing, Investigation. Shao-Quan Liu: Writing – review & editing, Validation. Enes Dertli: Writing – review & editing, Validation. Basim Abu-Jdayil: Writing – review & editing, Investigation. Pau-Loke Show: Writing – review & editing, Validation. Mutamed Ayyash: Writing – review & editing, Visualization, Validation, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.