Extracellular polymeric substances (EPS) are high-molecular-weight biomolecules secreted by microorganisms into their environment [1]. Lactic acid bacteria (LAB) are known to be the common group of EPS producers [2]. EPS are mainly composed of exopolysaccharides and proteins but include other macromolecules, such as nucleic acids and lipids [3]. The production of EPS can be stimulated by various environmental stresses as a form of cellular protective response and bioflm formation enhancement [4, 5]. Promoting EPS production benefts microorganisms in terms of tolerant improvement to environmental stresses [1] because this can simultaneously form a protective layer for the cells [2]. Bacteria are encased in these polymers to form complex bioflm structures, therefore they can better resist in hostile environments [6]. In recent years, probiotics have been widely used due to their health benefits [7]. In the food and pharmaceutical industries, freeze-drying is the preferred method to increase the stability of sensitive and valuable products such as probiotics in dry-solid states [8]. During the drying process, there is a possibility of significantly losing the probiotic viability due to changes in the physical properties of lipid membranes or in the structure of sensitive cellular proteins [9]. To improve the viability of probiotics during freeze-drying, different approaches have been investigated. Many studies have focused on microencapsulating cells with protective agents to increase cell viability [10–12]. However, the encapsulation of cells with protective agents may have certain limitations such as uneven distribution of the microencapsulation agent to all cells [13, 14] and the breaking instability of the microencapsulation under negative environmental influences [15]. The addition of other external ingredients may also increase the risk of contamination during encapsulation. In addition, in pharmaceutical applications that require high-purity probiotics, the addition of protective agents, such as whey protein isolate and maltodextrins, sometimes leads to undesirable effects for consumers that could give problem to subject that are allergic to these ingredients [16–18]. Therefore, encapsulation by stimulating EPS production in probiotics to form a protective layer firmly attached to the cell wall is a promising alternative. To determine the survival rate, freeze-dried probiotics are currently counted colonies after re-suspending the lyophilized powder in saline or phosphate-buffered saline solution. The survival rate is calculated by dividing the viable counts of cell suspension after freeze-drying by the viable ones before freeze-drying [10, 19]. Thus, to obtain the survival rate, the density of cells must be determined twice before and after drying. This work may be laborious and therefore raises the necessity to construct a correlation equation between the amount of EPS synthesized and the survival rate after freeze-drying, by which can calculate the survival rate based on the determination of EPS content before freeze-drying. In this study, three probiotic strains, namely L. plantarum, L. acidophilus,and B. bifidumafter being exposed to different environmental challenges, such as temperature, pH, and increased carbon dioxide  (CO 2 ) concentration, would be determined in terms of the EPS content and freeze-dried survival of cells. Simultaneously, the study also analyzed the correlation between the amount of synthesized EPS and the freeze-dried survival rate of the strains. 

Materials and Methods 

Materials

L. plantarumVAL6, L. acidophilusVAR1, and B. bifdum VAG1 were obtained from the Department of Biotechnology, An Giang University, Vietnam National University Ho Chi Minh City, Vietnam. Man, Rogosa and Sharpe (MRS) medium used for bacterial culture was purchased from Qingdao Hope Bio-Technology Co., Ltd. (Qingdao, China).

Bioreactor Operating Conditions for Environmental Adaptation 

The cultures were carried out in a 5-L bioreactor (BIOSTAT, Sartorius Stedim, Germany). Briefy, 5 L of MRS medium was inoculated with 100 mL of overnight bacterial culture (OD 595= 1.5). The pH was maintained to 6.8 by titration with 10 M NaOH, the temperature was kept at 37 °C, and the agitation rate was set at 250 rpm. After 24 h of cultures, environmental challenges were then performed independently as shown in Table 1. 

EPS Extraction and Quantifcation 

EPS were extracted from cell suspensions collected following the method described by Salazar et al. [20] and Nguyen et al. [21]. Accordingly, total EPS were harvested after 24 h by mixing 500 mL of supernatants with an equal amount of 2 M NaOH and gently stirring overnight at room temperature. Supernatants were then recovered by centrifugation at 8200 × gfor 15 min and EPS were precipitated from the supernatants by adding double the volume of 96% (vol/vol) of cold ethanol. The precipitation was carried out at 4 °C for 24 h. After the second centrifugation step at 8200 × gand at 4 °C for 20 min, EPS were dried at 55 °C until constant weight. Freeze‑Drying and Survival Rate Determination Freeze-drying operating conditions and viability determination as described by [22]. Samples were frozen at − 70 °C for 1 h and later freeze-dried (0.6 MPa, − 50 °C) for 24 h in a vacuum freeze-dryer (Heto-FD4 freeze-dryer, Heto-Holten, Denmark). The number of viable cells (CFU) before and after freeze-drying was determined by the serial dilution method Statistical Analysis All experiments in this study were performed with three biological replicates. The signifcance of the disparity was evaluated with one-way ANOVA. Duncan’s multiple range tests were applied to the individual variables to compare means and to assess if there was a signifcant diference. Results Efect of Environmental Stresses on the EPS Synthesis and Viability of Probiotics The increased EPS synthesis under environmental challenges could improve the survival of probiotics is proven in our study. Accordingly, we determined the EPS yield of three bacterial strains included L. plantarum, L. acidophilus, and B. bifdumin diferent conditions of temperature, pH, and increased   CO2 concentration and ultimately analyzed the freeze-dried survival of acclimatized cells. As a result, the tested environmental challenges signifcantly enhanced EPS production, resulting in improved cell viability after freeze-drying 

Conclusion

Environmental challenges stimulated EPS synthesis in the probiotics to form microencapsulation, resulting in the enhancement of their viability after freeze-drying. The EPS content and freeze-dried survival rate of the probiotics were proportional. This experimental correlation is expressed by a mathematical equation y = ax 2 + bx + c, in which y-value is the survival rate of the probiotics and x-value is the EPS content. This could be a novel method for determining the freeze-dried viability of probiotic strains based on the measured EPS content