Partial Purification, Characterization, and Application of Exopolysaccharides Produced by Lactobacillus plantarum NS1905E in Yogurt

Exopolysaccharides (EPS) of lactic acid bacteria (LAB) has gained special interests in the dairy industry due to their health-promoting properties and contribution to the rheology of fermentation milk products. In the present study, an EPS designated as EPS-NS1905E was partially purifed and characterized secreted from L. plantarum NS1905E. Te EPS-NS1905E had an average molecular weight of 2.658 × 10 5 Da and was composed of glucose, arabinose, rhamnose, mannose, and galactose in an approximate molar ratio of 58.83:17.4: 6.31 :5.86: 2.74. Te FTIR spectroscopy showed the prevalence of carboxyl and hydroxyl groups. Te EPS-NS1905E had a thermal stability and exhibited higher viscosity in the NaCl solution and low acidity conditions (pH 2–4). Furthermore, the EPS-NS1905E possessed strong scavenging abilities against DPPH radicals (96%). Te addition of EPS-NS1905E in skim milk signifcantly shortened the fermentation time of yogurt, and the water-holding capacity (WHC) and viscosity of yogurt were improved during storage. Te results indicated that the EPS-NS1905E has great potential for use as a food additive in the food industry.


Introduction
Microbial exopolysaccharides (EPS) are produced by a wide variety of bacteria in the natural ecological environments, and they played a critical role in the prevention of desiccation and protection against environmental stresses and adherence to surfaces [1][2][3].Several natural EPS produced by bacteria have been characterized and used as biofocculants, bioabsorbents, and drug delivery agents in the food, cosmetic, and pharmaceutical industries [4].In the last decades, the EPS produced by lactic acid bacteria (LAB) have received increasing attention due to their food-grade status [5,6].EPS-producing LAB has been widely employed in yogurt and cheese for improving the rheology, texture, and mouth feel [7,8].In addition, there has been an increasing interest in characterizing and exploiting the EPS produced by LAB for their potential biological activities including antioxidant, antitumor, immune stimulatory, and cholesterol lowering [9][10][11].
L. plantarum is one of the most studied species of LAB and frequently isolated from many food products.Te diferences in characteristic and functional properties of EPS produced by L. plantarum strains were observed.Due to the biological activity and health benefts, more EPS produced by novel L. plantarum strains attracted a great deal of interest.Liu et al. [12] reported that EPS produced by L. plantarum HY exhibited considerable antioxidant and α-amylase inhibitory activities.EPS produced by L. plantarum C70 exhibited potential anticancer, antidiabetic, and antioxidant bioactivities [10].EPS derived from L. plantarum WLPL09 inhibited the proliferation of HepG-2 and HCT-8 cells [13].
Raw milk is a complex matrix that facilitates the development of microorganisms.Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, and Pediococcus, often presented in raw milk, are associated with the manufacturing of dairy products.In the previous study, we have evaluated the EPS-producing abilities of LAB strains through the EPS content determination, and the isolated strain L. plantarum NS1905E (which was isolated from fresh milk in Nanshan Pasture, Nanshan Animal Husbandry Co., Ltd.) exhibited an excellent EPS-producing ability [14].In the present study, we aim to characterize the partial purifed EPS produced by L. plantarum NS1905E, and its in vitro antioxidant activity and application in yogurt were evaluated.

Isolation of EPS-NS1905E
. L. plantarum NS1905E was cultured in MRS broth at 37 °C overnight.After incubation, culture medium was centrifuged (9000 × g, 20 min, 4 °C) to remove cells.Tree volumes of cold ethanol were added to the supernatant at 4 °C for 48 h for extracting the EPS.Te precipitate was collected by centrifugation (9000 × g, 15 min at 4 °C) and dissolved in distilled water (ca. 1 g/L).Te protein was precipitated by adding Sevag agent (chloroform : n-butylalcohol = 4 : 1, v/v) and removed by centrifugation (9000 × g, 15 min at 4 °C).Furthermore, the solution was dialyzed against distilled water for 72 h.Te polysaccharide aqueous solution after dialysis was lyophilized for advanced study.

Basic Components Analysis of EPS-NS1905E.
Total sugar content of EPS was determined according to the method described by Dubois et al. [15] with glucose as standard.Te total protein content was assessed by the Bradford method [16].

Molecular Weight (Mw) and Monosaccharide Composition Analysis of EPS-NS1905E.
Te Mw of partially purifed EPS-NS1905E was determined according to the method described by Zhao et al. [17] via gel permeation chromatography-multiangle laser light scattering (GPC-MALLS).

Fourier-Transform Infrared (FTIR) Spectroscopy of EPS-NS1905E.
Te analysis of functional groups presented in EPS-NS1905E was conducted by Fourier-transform infrared spectroscopy.Te spectra from 400 to 4000 cm −1 were recorded by a FTIR-8400S spectrophotometer (Shimadzu, Japan).

Rheological Characterization of EPS-NS1905E.
Te aqueous solutions of EPS-NS1905E were prepared by dissolved lyophilized EPS in 2 M Ca 2+ , Mg 2+ , Na + , and K + solutions and distilled water at a concentration of 1% (w/w), respectively.Te aqueous solution of EPS-NS1905E was adjusted to diferent pH values (2, 4, 7, 10, and 12) by 1 N HCl and NaOH solutions.To evaluate the efect of temperature on the viscosity of the EPS-NS1905E, the aqueous solution of EPS-NS1905E was exposed to diferent temperatures (−20, 25, and 90 °C), respectively.Te rheological properties of EPS-NS1905E aqueous solution were measured by a HAAKE RS6000 rheometer (Termo Fisher Scientifc Inc., MA, USA) with a cone-plate geometry (35 mm diameter) at 25 °C.

Biological Activity of EPS-NS1905E.
Te DPPH radical scavenging activity of EPS-NS1905E was detected according to the method described by Wang et al. [9].DPPH scavenging activity was expressed as a percentage and calculated by the following equation:

DPPH scavenging activity (%) �
Absorbance of blank − absorbance of sample Absorbance of blank × 100. ( Te antitumor activity of EPS-NS1905E was evaluated according to the protocol described by Ayyash et al. [10].Te inhibition efect of EPS-NS1905E on Caco-2 was calculated via the following formula: where M 1 and M 2 are mass of precipitate (g) and yogurt sample (g), respectively.

Rheological Properties' Analysis.
Te change in viscosity of yogurt samples during storage was measured by HAAKE RS6000 rheometer (Termo Fisher Scientifc Inc., MA, USA) at 25 °C.In brief, the fxture model is P35TiL and the shear rate range is 0.01-150 s −1 .
2.12.Statistical Analysis.Each measurement was conducted in triplicate.Te signifcant diferences of results were evaluated by one-way ANOVA of the Tukey method (SPSS 16.0).

Basic Components' Analysis of EPS-NS1905E.
Carbohydrates and proteins in the purifed EPS not only afect the formation of EPS three-position gel network but also the physical properties and chemical activity of EPS [18,19].In the present study, the composition of the partially purifed EPS was determined.Te total sugar content in EPS-NS1905E was estimated to be 84.44 ± 0.01%, and a low total protein content (1.35 ± 0.03%) was observed (data not shown).Similar results were reported in previous studies [20].

Structure Analysis of EPS-NS1905E.
Te FTIR spectrum of EPS-NS1905E was distinguished in the region between 400 cm −1 and 4000 cm −1 (Figure 2).Te absorption at 3391.80 cm −1 was assigned to the O-H stretching vibration of the hydroxyl group [26].Te peak at 2941.53 cm −1 could be attributed to C-H groups [27].Te absorption at 1649.59 cm −1 may correspond to the vibration of C�O bonds [28,29].Te region below 1500 cm −1 corresponding to the fngerprint region was known as the polysaccharide [18].Te absorption peak at 1449.92 cm −1 was attributable to the stretching vibration of the C�O bond.Te peaks between 1403.30 and 1242.74cm −1 may correspond to C-O stretch groups, C-H bond, and S�O stretching vibrations [28].Te bands observed within 900-1150 cm −1 may correspond to the vibration of the C-O-C bond [30].Te peaks at 1000-1200 cm −1 were assigned to the pyranose ring [9], which should be further confrmed by the NMR analysis.
Te EPS-NS1905E in diferent salt solutions exhibited less viscosity than in water (Figure 3(a)).Te EPS-NS1905E in the NaCl solution has shown more viscosity than in other salt solutions over the lower shear rate range.Tis result may be related to the binding strength of salt ions on negative EPS sites.Te salt resistance of EPS is a beneft of its application in the dairy industry [31].A higher viscosity of the EPS-NS1905E solution at acidic pH (4) was observed (Figure 3(b)), which would be benefcial for application of this EPS to improve the texture of the yogurt.Similarity, Wang et al. [23] observed that the EPS from L. plantarum YW11 exhibited higher viscosity at acidic pH (4 and 6).Te rheological characteristics of EPS-NS1905E at diferent temperatures is shown in Figure 3(c); a stable rheological property of EPS-NS1905E was observed with the increasing temperature from −20 to 90 °C.Wang et al. [23] and Journal of Food Biochemistry 3 4 Journal of Food Biochemistry Kanmani et al. [32] stated that the EPS exhibited decreased viscosity with increasing temperatures.Te thermal properties of EPS-NS1905E make it suitable as a potential additive used in food processing.Tese diferences in rheological properties of EPS under diferent treatment conditions may be caused by changes in Mw, type of glycosidic bond, monosaccharide composition, functional groups, and substitutions [10,33].

Biological Activity of EPS.
Te scavenging ability of EPS-NS1905E on DPPH free radicals was measured, and the result is shown in Figure 4(a).A strong scavenging activity of EPS-NS1905E towards DPPH free radicals was observed.At a concentration of 1% (m/m), EPS-NS1905E showed 96% of DPPH radical scavenging activity.Wang et al. [23] reported that a lower DPPH elimination activity (30%) of EPS is produced by L. plantarum YW32.Te antioxidant activity of EPS may depend on its molecular weight, monosaccharide composition, and the purifcation methods used [34].
Te antitumor activities of EPS-NS1905E against Caco-2 cells were investigated, and the results showed that the exhibited inhibition activities were observed in a concentration-dependent manner (Figure 4(b)).Te inhibition ratio of EPS-NS1905E (1%, m/m) against Caco-2 cells was 21%.Wang et al. [9] reported that the inhibition ratios of r-EPS1 and r-EPS2 (600 μg/mL) produced by L. plantarum 70810 against Caco-2 were 25.94% and 35.04%, respectively.Te diferences in the antitumor activities of these EPS might result from molecular weight, monosaccharide composition, and specifc structure [35,36].

Efects of EPS-NS1905E on Storage of Yogurt.
Te changes of pH and acidity of EPS-NS1905E fortifed and control yogurt samples were investigated.As shown in Figure 5, as predicted, the titratable acidity was negatively correlated with pH values.Te skim milk fortifed with 0.1% (m/m) EPS-NS1905E showed the lowest pH reduction and shorter fermentation time during fermentation (data not shown).

Journal of Food Biochemistry
Te EPS-NS1905E fortifed yogurt exhibited lightly higher acidity than the non-EPS yogurt result from the fact that the added EPS acts as an additional carbon source in the lactic acid fermentation [37].Te EPS-NS1905E fortifed and control yogurt samples showed a signifcant decrease in pH during the storage and was in accordance with the results reported by Prasanna et al. [38].

Efects of EPS-NS1905E on Yogurt Water-Holding
Capacity.Te water-holding capacity (WHC) of yogurts fortifed with EPS-NS1905E during storage was studied, and results are shown in Figure 6.During the frst 7 days of storage, the WHC of yogurt decreased signifcantly.Te WHC of yogurts fortifed with EPS-NS1905E were increased in a concentration-dependent manner, and the yogurt with 0.1% EPS-NS1905E showed a signifcant (p ≤ 0.05) increase.Tere was a consensus that EPS could improve WHC of the yogurt by interacting with biological macromolecule of milk, such as proteins and micelles [37,39].However, no signifcant diference (p > 0.05) of WHC was found between EPS-NS1905E fortifed and control yogurt samples along with the storage.

Efects of EPS-NS1905E on Yogurt Viscosity during
Storage.Te viscosity of all yogurt samples during storage is shown in Figure 7.At the beginning of storage (day 1), the yogurt with 0.1% EPS-NS1905E has shown more viscosity  EPS can improve the structure of yogurt by binding with the serum phase of yogurt [37].

Conclusions
In this study, the EPS produced by L. plantarum NS1905E had shown to consist of glucose, arabinose, rhamnose, mannose, and galactose in an approximate molar ratio of 58.83 : 17.4 : 6.31 : 5.86 : 2.74 and had a molecular weight of 2.658 × 10 5 Da.Te EPS was thermally stable and had a higher viscosity in acidic pH.Furthermore, the EPS had good DPPH scavenging antioxidant activity.Te addition of EPS in skim milk shortened the fermentation time and improved the WHC and viscosity of yogurt during storage.Terefore, the EPS produced by L. plantarum NS1905E could be exploited as a food additive for application in functional foods.

Figure 6 :
Figure 6: Changes in water-holding capacity of yogurt with EPS-NS1905E during cold storage.
Journal of Food Biochemistry yogurt cups.Te incubation was carried out at 42 °C until the pH reached 4.6.Te fermentation was stopped in a cold room and stored at 4 °C for 21 days.
°C in waterbath, samples were inoculated with 3% of yogurt starter (Danisco YO-MIX 883) and distributed in 2