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Effects of 2′-Fucosyllactose-Based Encapsulation on Probiotic Properties in Streptococcus thermophilus

Effects of 2′-Fucosyllactose-Based Encapsulation on Probiotic Properties in Streptococcus... applied sciences Article Effects of 2 -Fucosyllactose-Based Encapsulation on Probiotic Properties in Streptococcus thermophilus Shadi Pakroo, Gloria Ghion, Armin Tarrah * , Alessio Giacomini and Viviana Corich Department of Agronomy Food Natural Resources Animal and Environment (DAFNAE), University of Padova, Viale dell’Università 16, 35020 Legnaro, PD, Italy; shadi.pakroo@phd.unipd.it (S.P.); gloria.ghion@studenti.unipd.it (G.G.); alessio.giacomini@unipd.it (A.G.); viviana.corich@unipd.it (V.C.) * Correspondence: author: armin.tarrah@unipd.it Abstract: Streptococcus thermophilus is widely used in dairy fermentation as a starter culture for yogurt and cheese production. Many strains are endowed with potential probiotic properties; however, since they might not survive in adequate amounts after transit through the human gastrointestinal tract, it is advisable to improve cell survivability during this passage. The present study evaluates the use of 2 -fucosyllactose, a prebiotic molecule from human milk, compared with other known molecules, such as gelatin and inulin, to form alginate-based microcapsules to fulfill these requirements. Such microcapsules, obtained by the extrusion technique, were evaluated in terms of encapsulation efficiency, storage stability, gastrointestinal condition resistance, and cell release kinetics. Results reveal that microcapsules made using 2 -fucosyllactose and those with inulin resulted in the most efficient structure to protect S. thermophilus strain TH982 under simulated gastrointestinal conditions (less than 0.45 log CFU/g decrease for both agents). In addition, a prompt and abundant release of encapsulated cells was detected after only 30 min from microcapsules made with sodium alginate plus 2 -fucosyllactose in simulated gastrointestinal fluid (more than 90% of the cells). These encouraging Citation: Pakroo, S.; Ghion, G.; results represent the first report on the effects of 2 -fucosyllactose used as a co-encapsulating agent. Tarrah, A.; Giacomini, A.; Corich, V. Effects of 2 -Fucosyllactose-Based Encapsulation on Probiotic Properties Keywords: microencapsulation; probiotic; prebiotic; Streptococcus thermophilus TH982; 2 -fucosyllactose in Streptococcus thermophilus. Appl. Sci. 2021, 11, 5761. https://doi.org/ 10.3390/app11135761 1. Introduction Academic Editors: Athina S. Tzora The genus Streptococcus includes more than 70 species, but to date, only S. thermophilus and Antonio Valero possesses the GRAS (generally recognized as safe) status due to its long history of safe application in food production [1]. For this reason, this is the only species of the genus Received: 29 April 2021 used in the food industry and represents the second most important species of industrial Accepted: 18 June 2021 lactic acid bacteria (LAB) after Lactococcus lactis and one of the primary starters of yo- Published: 22 June 2021 gurt [2,3]. Furthermore, many S. thermophilus strains have several technological properties, such as sugar metabolism, galactose utilization capability, proteolytic activity, and urease Publisher’s Note: MDPI stays neutral activity [4]. with regard to jurisdictional claims in Some strains have also shown potential as a probiotic, having revealed various health published maps and institutional affil- benefits [5,6]. S. thermophilus is a bacterium of non-human origin and, although known iations. to be sensitive to the acidic gastric conditions, it was able to survive the gastrointestinal tract and moderately capable of adhesion to intestinal epithelial cells [7], thus making it a transient probiotic [7,8]. Nowadays, probiotics are one of the driving forces in the design of functional foods, particularly dairy products; hence it is advisable to improve Copyright: © 2021 by the authors. cell survivability during the gastrointestinal passage. The inclusion of probiotics into Licensee MDPI, Basel, Switzerland. microcapsules seems to be a promising solution and also a means to control the release of This article is an open access article cells into the target tissues [9]. distributed under the terms and Several techniques are available for encapsulation, which differ on the nature of their conditions of the Creative Commons core (content) and intended use [10]. Encapsulation techniques applied to the food in- Attribution (CC BY) license (https:// dustry include spray-drying, fluid bed coating, spray-chilling or spray cooling, extrusion, creativecommons.org/licenses/by/ emulsification, coacervation, co-extrusion, inclusion complexation, liposome entrapment, 4.0/). Appl. Sci. 2021, 11, 5761. https://doi.org/10.3390/app11135761 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 5761 2 of 11 centrifugal extrusion, encapsulation by a rapid expansion of supercritical fluid (RESS), freeze- or vacuum drying, and nanotechnological approaches [11]. The use of extrusion technology has many advantages for the encapsulation of probiotic cells. It is a simple and inexpensive method using mild operations, and the procedure does not involve any harmful solvent. Moreover, this technique appears to promote probiotic cell viability [12–15]. Combining alginate with some prebiotic molecules can also enhance cell protection in food systems [10]. Prebiotics form three-dimensional microcrystal networks that interact together, forming small aggregates that contribute to the better protection of the cells [16]. The most frequently used prebiotics in co-encapsulation are oligosaccharides, inulin, and resistant starches [16]. 2’-fucosyllactose (2 -FL), is a neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose and is the most abundant oligosaccharide in human milk (HMO), accounting for about 30% of all of HMOs, i.e., 2–3 g/L [17]. This prebiotic molecule has been reported to display numerous health advantages in humans [18–20]. Another attractive characteristic of 2 -FL is its simplicity, which facilitates its de novo synthesis using microbial, enzymatic, or chemical methods [21]. In this study, we aimed at evaluating the protective effect of 2’-FL on S. thermophilus TH982 cells, a potential probiotic with the ability to produce high levels of folate [22] compared to some other common co-encapsulating materials in alginate beads. 2. Materials and Methods 2.1. Bacteria and Growth Conditions S. thermophilus TH982 [23] is part of the collection of the Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Italy. S. ther- mophilus TH982 was stored at 80 C in M17 broth (Sigma-Aldrich, St. Louis, MI, USA) containing glycerol (25% v/v) and was activated before each experiment by culturing it in M17 (Sigma-Aldrich) broth at 37 C for 24 h. 2.2. Encapsulation Procedure The components used for encapsulation of S. thermophilus TH982 were sodium alginate (S, Sigma-Aldrich), gelatin (G, from bovine skin, Sigma-Aldrich), inulin (I, from chicory, Sigma-Aldrich) and 2 -fucosyllactose (F, Sigma-Aldrich). Fifty milliliters of reactivated cells in M17 broth were centrifuged (Hettich, Westphalia, Germany) at 5500 rpm for 5 min. The bacterial pellets were washed twice with sterile saline solution (0.85%) (Sigma-Aldrich) and added to the encapsulation matrix. The encapsulating matrix was prepared using the extrusion technique through the combination of sodium alginate with either inulin, gelatin, or 2 -fucosyllactose, each one at a concentration of 2% (w/v). Sodium alginate alone was also used as a control. The different matrices were mixed and added to the S. thermophilus TH982 pellet suspension. The mixture was then injected using a sterile syringe with a 450 m needle into sterile 0.3 M CaCl (Sigma-Aldrich) hardener solution. The distance from the bottom of the nozzle and the surface of the CaCl solution was kept at 10 cm (Figure 1). The microcapsules were left in the hardening solution (CaCl ) for 30 min at room temperature. Finally, capsules were transferred in 0.1% (w/v) sterile peptone water solution and stored at 4 C until further use. The morphology of the microcapsules was examined using an optical stereomicroscope (Olympus, Tokyo, Japan). Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 11 Appl. Sci. 2021, 11, 5761 3 of 11 Figure 1. Scheme of microencapsulation procedure of S. thermophilus TH982 cells using the extrusion technique. (A) The Figure 1. Scheme of microencapsulation procedure of S. thermophilus TH982 cells using the extru- sodium alginate alone as control. (B) Sodium alginate with other agents. sion technique. (A) The sodium alginate alone as control. (B) Sodium alginate with other agents. 2.3. Encapsulation Efficiency 2.3. Encapsulation Efficiency One milliliter o One f eac milliliter h type ofof ca each psule type solution w of capsule as added to solution 9 m wasLadded of saline to sol 9 mL ution of saline solution (0.85%), serially (0.85%), diluted, serially and pl diluted, ated on M and 17 plated agar m on edium M17 t agar o det medium ermine th toe v determine iable cell the viable cell concentration in capsule solutions. One gram of each type of capsule was homogenized in concentration in capsule solutions. One gram of each type of capsule was homogenized 9 mL of 10% (w/v) sterilized sodium citrate by vortexing for 1 min, serially diluted, and in 9 mL of 10% (w/v) sterilized sodium citrate by vortexing for 1 min, serially diluted, and plated on M17 to get the number of entrapped cells per g of capsule. The enumeration plated on M17 to get the number of entrapped cells per g of capsule. The enumeration of of viable cells from both the solution and the capsule was done using the micro drop viable cells from both the solution and the capsule was done using the micro drop tech- technique by placing 20 L aliquots on the surface of M17 agar plates and incubating at nique by placing 20 µL aliquots on the surface of M17 agar plates and incubating at 37 °C 37 C for 48 h. The encapsulation efficiency (EE%) was determined using the equation [24]: for 48 h. The encapsulation efficiency (EE%) was determined using the equation [24]: EE% = (N1/N0) × 100 EE% = (N /N )  100 1 0 where N1 is the number of viable entrapped cells (log CFU/g) released after the encap- where N is the number of viable entrapped cells (log CFU/g) released after the encapsula- sulation procedure and N0 is the number of viable cells (log CFU/mL) added to the mix- tion procedure and N is the number of viable cells (log CFU/mL) added to the mixture ture before encapsulation. before encapsulation. 2.4. Storage Stability in Skimmed Milk 2.4. Storage Stability in Skimmed Milk One gram of microcapsules was suspended in 9 mL of 10% (w/v) reconstituted One gram of microcapsules was suspended in 9 mL of 10% (w/v) reconstituted skimmed milk, stored under refrigerated conditions (4 °C) and sampled at different time skimmed milk, stored under refrigerated conditions (4 C) and sampled at different time intervals, namely after 0, 7, 14 and 21 days to evaluate the survival of encapsulated S. intervals, namely after 0, 7, 14 and 21 days to evaluate the survival of encapsulated thermophilus TH982 [25]. The survival of free (non-encapsulated) bacterial cells was de- S. thermophilus TH982 [25]. The survival of free (non-encapsulated) bacterial cells was termined by adding 9 mL of 10% (w/v) reconstituted skimmed milk (Sigma-Aldrich) to 1 determined by adding 9 mL of 10% (w/v) reconstituted skimmed milk (Sigma-Aldrich) mL of free cells as well. At each time interval, 1 g aliquot of capsules was aseptically to 1 mL of free cells as well. At each time interval, 1 g aliquot of capsules was aseptically centrifuged at 5500 rpm for 5 min. The supernatant was discarded and the cells were al- centrifuged at 5500 rpm for 5 min. The supernatant was discarded and the cells were lowed to release in 9 mL of 10% (w/v) sodium citrate by vortexing for 1 min, serially di- allowed to release in 9 mL of 10% (w/v) sodium citrate by vortexing for 1 min, serially luted, and plated on M1 diluted, and 7 agplated ar, followed on M17 by incub agar, followed ation at 37 by °C incubation for 48 h. The s at 37 urv Civ for al w 48 as h. The survival determined as the was number determined of ceas lls the recov number ered duri of cells ng dirff ecover erent stora ed during ge time i differ nterva ent storage ls with time intervals respect to the number o with respect f init to iathe l ent number rapped cel of initial ls. entrapped cells. 2.5. Survivability under Simulated Gastrointestinal Conditions 2.5. Survivability under Simulated Gastrointestinal Conditions The simulated gastrointestinal conditions were obtained using basic fluid, gastric The simulated gastrointestinal conditions were obtained using basic fluid, gastric fluid, and intestinal fluid. The basic fluid was prepared by dissolving (per liter): 1.12 g fluid, and intestinal fluid. The basic fluid was prepared by dissolving (per liter): 1.12 g potassium chloride (Sigma-Aldrich), 2.0 g sodium chloride (Sigma-Aldrich), 0.11 g calcium potassium chloride (Sigma-Aldrich), 2.0 g sodium chloride (Sigma-Aldrich), 0.11 g cal- chloride (Sigma-Aldrich), and 0.4 g potassium dihydrogen phosphate (Sigma-Aldrich) in cium chloride (Sigma-Aldrich), and 0.4 g potassium dihydrogen phosphate (Sig- saline solution (0.85%). The basic fluid was sterilized by autoclaving at 121 C for 15 min. ma-Aldrich) in saline solution (0.85%). The basic fluid was sterilized by autoclaving at 121 Appl. Sci. 2021, 11, 5761 4 of 11 The simulated gastric fluid (SGJ) and simulated intestinal fluid (SIJ) were prepared based on a modified method published by Singh et al. [25]. The gastric fluid consisted of 0.01 g/L of swine pepsin (Sigma-Aldrich) and 0.01 g/L of swine mucin (Sigma-Aldrich) added directly to the sterile basic juice. The pH was adjusted to 2.5 with 1 N HCl, and the liquid was filter-sterilized using a 0.22 m membrane filter (Sigma-Aldrich). The intestinal fluid contained (per liter of basic fluid): 0.01 g pancreatin (Sigma- Aldrich), 0.08 g Ox-bile extract (Sigma-Aldrich), and 0.01 g lysozyme (Sigma-Aldrich). The pH was adjusted to 7.5 with 1 N NaOH, and the juice was filter sterilized. Both gastric and intestinal fluids were prepared fresh on the day of the experiment. Overnight bacterial cultures were obtained after subculturing in M17 broth, and capsules were prepared as described in the encapsulation procedure. One gram of each type of capsule was added to 9 mL of SGJ and incubated at 37 C for 1 h to evaluate the survivability of S. thermophilus TH982 under gastric conditions. For the survival of S. thermophilus TH982 under gastrointestinal conditions, 9 mL of SIJ were added following gastric fluid incubation, and the capsules and free cells with simulated gastrointestinal fluid were left at 37 C for a further 2 h. The cell viability was evaluated for free and encapsulated S. thermophilus after gastric and gastrointestinal incubations by plating on M17 agar and using the micro drop technique, followed by incubation at 37 C for 48 h. 2.6. Release Kinetics The release kinetics from capsules were determined using phosphate solution without enzymes. The release of encapsulated S. thermophilus TH982 was evaluated according to Shi et al. [26] with some modifications. A phosphate solution without the addition of intestinal enzymes (6.8 g/L of K HPO , 50 mM) was prepared, the pH was adjusted 2 4 to 7.5 with 1 N HCl and subsequently sterilized by autoclaving at 121 C for 15 min. Microcapsules (1 g) from different matrices were added to 9 mL of the abovementioned phosphate solution and incubated at 37 C with agitation (100 rpm). At predetermined time intervals (0, 30, 60, 90, 120, 150, and 180 min), 100 L of solution were taken from each capsule type, serially diluted, and plated using the micro drop technique on M17 agar to detect the number of released cells. The number of released cells was determined after 48 h of incubation at 37 C and expressed as log CFU/mL of K HPO solution. 2 4 2.7. Statistical Analysis All the experiments were performed in triplicate. Data were analyzed using a one- way analysis of variance (ANOVA). Tukey’s test was used as a post-hoc analysis by the GraphPad Prism software (version 7, GraphPad Software, Inc., San Diego, CA, USA). Results were considered significantly different for p values lower than 0.05. 3. Results and Discussion 3.1. Morphology and Encapsulation Efficiency All capsules appeared globular and irregular in shape with a rough surface and a drop- like shape. This result is probably due to the high surface tension of the used hardening solution (CaCl ) that determines an imperfect sphere formation [27]. However, no surface cavities or fractures were visible. The average size of capsules was 3.5 0.52 (mm) without any significant difference among the different microcapsules types. The capsules with S + G showed a more dense structure, as confirmed by the viscosity of G solution, due to electrostatic interactions between amino groups of G and carboxyl groups of S, which make cells more resistant to enzymatic and acidic hydrolysis [28]. Encapsulation efficiencies (EE%) of the four microcapsule types are reported in Table 1. EE% is a parameter that describes both the survival of viable cells and the efficacy of entrapment of the encapsulation procedure [29]. The yield of S. thermophilus TH982 viable cells co-encapsulated using S + F (96.13%) was significantly higher (p < 0.01) than that of cells encapsulated with S alone (91.07%), used as control, and was similar (not statistically Appl. Sci. 2021, 11, 5761 5 of 11 different) to S + I (98.61%). The high EE% obtained with S + F and S + I matrices might be due to the decrease in the porosity of the gel-beads and, consequently, to a reduction in the leakage of entrapped S. thermophilus TH982 cells [30]. Besides, incorporating prebiotics as material for encapsulation may better protect probiotics in food systems and inside the gastrointestinal tract due to mutual cells/prebiotic interactions [31]. By contrast, the number of viable cells encapsulated in S alone and S + G showed no significant difference (EE% 91.07 and 90.50, respectively). The main reason why EE% is normally lower than 100% is linked to cell damage due to detrimental conditions caused by the encapsulation process itself, such as shear stress and the use of concentrated solutions. Furthermore, during the time required for the hardening of capsules, a physical loss of cells can occur in significant numbers [29]. It should also be noted that a dissolution process is required to determine the number of viable cells in the microcapsules, and therefore an incomplete disintegration can underestimate the calculated EE% value. In particular, for the solubilization of S-based capsules, sodium citrate (10% w/v) was used, which is a chelating agent [30] that can remove calcium from the “egg-box” structure, leading to the destabilization of alginate coating and to the effective release of cells [32]. Table 1. Encapsulation efficiency (EE%) of S. thermophilus TH982 using different encapsulating matrices. Cell in the Encapsulation Solution Cells after Encapsulation Encapsulation Efficiency Capsule Composition (log CFU/mL) (log CFU/g) (EE%) a a a Sodium alginate 9.46  0.04 8.61  0.19 91.07 a b b Sodium alginate + Inulin 9.17  0.21 9.04  0.12 98.61 a a a Sodium alginate + Gelatin 9.39  0.09 8.50  0.04 90.50 b b Sodium alginate + Fucosyllactose 9.34  0.03 8.97  0.07 96.13 Values are the mean  standard deviation (SD) of triplicate experiments (n = 3). Superscript letters in each column indicate statistically significant (p < 0.05) differences among different formulations. 3.2. Storage Stability in Skimmed Milk In order to test the viability of S. thermophilus cells inside capsules during prolonged storage, we maintained them in skimmed milk at refrigeration conditions (4 C) for 21 days (Figure 2). At the end of the period, cell viability showed 0.46, 1.62, 1.79, and 1.97 log decrease for S. thermophilus TH982 cells encapsulated with S + G, S + F, S, and S + I, respectively. Capsules containing G as a co-encapsulating agent showed a better perfor- mance after 21 days with respect to other co-encapsulating agents. However, data show that free cells had the highest viability level throughout the storage period since there was no significant difference between initial (8.90  0.08 log CFU/mL; day0) and final (8.94  0.05 log CFU/mL; day21) values. Interestingly, S, S + I, and S + F capsules revealed a similar, not significantly different, viability reduction (p < 0.05). Unlike some studies that indicate an increase in surviv- ability during refrigerated storage conditions, which could be linked to the difference in species/strain used. S. thermophilus is frequently used in the production of different dairy products, and it is highly adapted to the dairy environment. It was reported that encapsulation improved the stability of L. plantarum during storage [33], and encapsulation of bacteria in alginate was found to improve survivability when compared to unprotected cell counts stored in skimmed milk for 24 h [14]. Since FAO and the International Dairy Fed- eration (IDF) recommend that the minimum content of probiotic cells should be 10 CFU/g of product at the time of consumption [34], the four encapsulating agents used in this study were efficient in maintaining the required viability of S. thermophilus TH982 after refriger- ated storage in skimmed milk. The high numbers suggested by IDF have been proposed to compensate for the possible numerical reduction of probiotic organisms during passage through the human stomach and intestine. The cells must remain viable throughout the projected shelf-life period of a product so that when consumed, the product contains a sufficient number of viable cells. Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 11 must remain viable throughout the projected shelf-life period of a product so that when consumed, the product contains a sufficient number of viable cells. The viability of probiotic bacteria in food products is affected by many intrinsic and extrinsic aspects such as dissolved oxygen and oxygen permeation through the package, post acidification in fermented products (lactic and acetic acids), pH, storage and incu- bation temperature, duration of fermentation, production of hydrogen peroxide due to bacterial metabolism, and processing conditions [35,36]. Moreover, the specific strains used, the interaction between species, availability of nutrients, and growth promoters and inhibitors can affect viable cells’ survivability [14]. The great survivability of free S. thermophilus TH982 cells in skimmed milk is prob- ably due to the fact that S. thermophilus is a species highly adapted to grow on lactose as its energy and carbon source, that is internalized by lactose permease (LacS) and hydro- lyzed to glucose and galactose by β-galactosidase (LacZ) [37]. It is likely that entrapment of S. thermophilus TH982 cells into capsules of different matrices probably led to a re- duced diffusion of lactose inside the capsule layer or membrane, limiting the possibility Appl. Sci. 2021, 11, 5761 6 of 11 of its use by the entrapped cells [35]. Figure 2. Figure 2.Su Survivability rvivability of en of encapsulated capsulated an andd free free S. S thermophilus . thermophilus TH982 TH982 cellscells during 21 days of during 21 days of storage storage in skimmed milk at 4 °C. in skimmed milk at 4 C. The viability of probiotic bacteria in food products is affected by many intrinsic and 3.3. Survivability under Simulated Gastrointestinal Conditions extrinsic aspects such as dissolved oxygen and oxygen permeation through the package, The survival of probiotics under gastrointestinal conditions represents a significant post acidification in fermented products (lactic and acetic acids), pH, storage and incubation issue for their effectiveness, and microencapsulation could represent a valid method to temperature, duration of fermentation, production of hydrogen peroxide due to bacterial provide significant protection [15]. The survival of free and encapsulated S. thermophilus metabolism, and processing conditions [35,36]. Moreover, the specific strains used, the TH982 cells after 1, 2, and 3 h of incubation in simulated gastrointestinal conditions are interaction between species, availability of nutrients, and growth promoters and inhibitors presented in Figure 3. After 1 h of gastric fluid incubation, the highest reduction of viable can affect viable cells’ survivability [14]. cells was found, as expected, for free S. thermophilus TH982, as the initial number of 10.40 The great survivability of free S. thermophilus TH982 cells in skimmed milk is probably ± 0.10 log CFU/mL was diminished by 0.95 log CFU/mL. A notable decrease in viability due to the fact that S. thermophilus is a species highly adapted to grow on lactose as its after 3 h of gastrointestinal conditions was confirmed for free S. thermophilus TH982 cells, energy and carbon source, that is internalized by lactose permease (LacS) and hydrolyzed reduced by approximately 1.60-log CFU/mL, dropping to 8.79 ± 0.02 log CFU/mL. By to glucose and galactose by -galactosidase (LacZ) [37]. It is likely that entrapment of contrast, for cells encapsulated with S alone, only 1.05 log CFU/g reduction of viable cells S. thermophilus TH982 cells into capsules of different matrices probably led to a reduced was observed, implying that microencapsulation provided protection compared to the diffusion of lactose inside the capsule layer or membrane, limiting the possibility of its use free cells. This protection offered by sodium alginate might be related to the establish- by the entrapped cells [35]. ment of a hydrogel barrier through an external layer of sodium alginate that has retarded 3.3. Survivability under Simulated Gastrointestinal Conditions The survival of probiotics under gastrointestinal conditions represents a significant issue for their effectiveness, and microencapsulation could represent a valid method to provide significant protection [15]. The survival of free and encapsulated S. thermophilus TH982 cells after 1, 2, and 3 h of incubation in simulated gastrointestinal conditions are presented in Figure 3. After 1 h of gastric fluid incubation, the highest reduction of viable cells was found, as expected, for free S. thermophilus TH982, as the initial number of 10.40  0.10 log CFU/mL was diminished by 0.95 log CFU/mL. A notable decrease in viability after 3 h of gastrointestinal conditions was confirmed for free S. thermophilus TH982 cells, reduced by approximately 1.60-log CFU/mL, dropping to 8.79  0.02 log CFU/mL. By contrast, for cells encapsulated with S alone, only 1.05 log CFU/g reduction of viable cells was observed, implying that microencapsulation provided protection compared to the free cells. This protection offered by sodium alginate might be related to the establishment of a hydrogel barrier through an external layer of sodium alginate that has retarded the permeation of simulated fluids into the capsules and thus the interaction with the probiotic cells [38]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 11 the permeation of simulated fluids into the capsules and thus the interaction with the Appl. Sci. 2021, 11, 5761 7 of 11 probiotic cells [38]. Figure 3. Survivability of encapsulated and free S. thermophilus TH982 cells during exposure to Figure 3. Survivability of encapsulated and free S. thermophilus TH982 cells during exposure to simsimulated ulated gast gastr rointestinal ointestinal conditions conditions. . DiffDif erent le ferentters t letters indicate indicate significant differences w significant differencesithin the within the same incubation step (p < 0.05) among different encapsulating matrix types. same incubation step (p < 0.05) among different encapsulating matrix types. S + G capsules showed low survivability during gastric environment; indeed, the S + G capsules showed low survivability during gastric environment; indeed, the reduction of viable cells was higher (0.52 log CFU/g decrease) than encapsulated cells with reduction of viable cells was higher (0.52 log CFU/g decrease) than encapsulated cells S + I (no reduction detected) after 1 h of gastric condition (pH 2.5). This fact might be due with S + I (no reduction detected) after 1 h of gastric condition (pH 2.5). This fact might be to a variation of pH, which changes the gelatin charge of amino and carboxyl groups so due to a variation of pH, which changes the gelatin charge of amino and carboxyl groups that the modification of cross-links and structure of the chain could influence the swelling so that the modification of cross-links and structure of the chain could influence the behavior of gelatin capsules [39]. swelling behavior of gelatin capsules [39]. On the other side, an interesting outcome was given by the two types of prebiotics used On the other side, an interesting outcome was given by the two types of prebiotics as a co-encapsulating agent with sodium alginate. After 3 h of exposure to gastrointestinal used as a co-encapsulating agent with sodium alginate. After 3 h of exposure to gastro- conditions, S + I and S + F revealed the highest cell survivability without any significant intestinal conditions, S + I and S + F revealed the highest cell survivability without any difference, leading to a low cell reduction (less than 0.45 log CFU/g decrease for both significant difference, leading to a low cell reduction (less than 0.45 log CFU/g decrease agents). Prebiotics, such as 2 -FL and inulin, which could provide good protection and even for both agents). Prebiotics, such as 2′-FL and inulin, which could provide good protec- promote cell proliferation, appeared to contribute to the growth of S. thermophilus TH982. In tion and even promote cell proliferation, appeared to contribute to the growth of S. addition, oligosaccharides are difficult to decompose by enzymes in digestive fluid but can thermophilus TH982. In addition, oligosaccharides are difficult to decompose by enzymes be metabolized by beneficial bacteria in the colon [30]. It has been further demonstrated that in digestive fluid but can be metabolized by beneficial bacteria in the colon [30]. It has oligosaccharides contained in human milk have an extraordinary resistance to hydrolysis been further demonstrated that oligosaccharides contained in human milk have an ex- by digestive enzymes of the small intestine [40]. The combination of calcium alginate with traordinary resistance to hydrolysis by digestive enzymes of the small intestine [40]. The prebiotics such as inulin improves the viability of probiotics and facilitates the formation combination of calcium alginate with prebiotics such as inulin improves the viability of of an integrated structure of capsules. Researchers found better protection of Lactobacillus probiotics and facilitates the formation of an integrated structure of capsules. Researchers casei and Bifidobacterium bifidum cells in coated capsules with prebiotics such as inulin after found better protection of Lactobacillus casei and Bifidobacterium bifidum cells in coated the gastrointestinal transit. The addition of prebiotics could be synergistic in gelling, and capsules with prebiotics such as inulin after the gastrointestinal transit. The addition of as a result, may help to maintain and improve the degree of protection of the bacterial prebiotics could be synergistic in gelling, and as a result, may help to maintain and im- cells [41]. prove the degree of protection of the bacterial cells [41]. Lastly, the free cell suffered more from the gastric conditions than that during the 3-h Lastly, the free cell suffered more from the gastric conditions than that during the gastrointestinal transition. In contrast, encapsulated bacteria evidenced greater reductions 3-h gastrointestinal transition. In contrast, encapsulated bacteria evidenced greater re- during gastrointestinal transit than under gastric conditions alone. This behavior may ductions during gastrointestinal transit than under gastric conditions alone. This behav- be due to the fact that alginate is stable in low-pH solutions: the ionotropic alginate gel ior may be due to the fact that alginate is stable in low-pH solutions: the ionotropic algi- 2+ formed by Ca cross-linking of carboxylate groups is insoluble at low pH, but exposure to neutral pH or higher solubilize the alginate, causing swelling [42]. Appl. Sci. 2021, 11, 5761 8 of 11 3.4. Release Kinetics The release of encapsulated S. thermophilus TH982 after incubation in simulated in- testinal fluid was evaluated after 30, 60, 90, 120, 150, and 180 min, as reported in Figure 4. After 30 min of agitation at 100 rpm at 37 C in 50 mM K HPO a great release of cells 2 4, encapsulated with S + F was detected. Considering the initial number of cells entrapped in this type of capsules (9.73  0.04 log CFU/g of capsules), the number of cells released in the simulated intestinal fluid after 30 min was 8.83  0.04 log CFU/mL of K HPO , 2 4 representing a very high value compared with the other types of encapsulating matrices after 180 min, which resulted about 2-log lower. This result confirms what was visible during the experiment: after 30 min, there was an evident solubilization of S + F capsules, a characteristic not displayed by the others (S, S + I, and S + G). Moreover, the number Appl. Sci. 2021, 11, x FOR PEER REVIEW of released cells suggests that once liberated, S. thermophilus TH982 can withstand 9 of well 11 simulated intestinal conditions, since after 180 min the number of cells released from S + F capsules was 9.72  0.05 log CFU/mL, which means 99.89% of the initial entrapped cells. Figure 4. Release kinetics of entrapped S. thermophilus TH982 cells from different capsule formulations Figure 4. Release kinetics of entrapped S. thermophilus TH982 cells from different capsule formula- during incubation in a simulated intestinal environment. tions during incubation in a simulated intestinal environment. It is essential to ensure that the capsules give protection through the simulated gas- A possible mechanism that could be involved in the fast release of S. thermophilus trointestinal passage and ensure that the encapsulation matrix allows a release of viable and TH982 cells encapsulated with sodium alginate and 2′-fucosyllactose could be that this metabolically active cells in the intestine [30,43]. The release of the cells from microcapsules small soluble milk glycan used as a co-encapsulating agent in this experiment is formed in the colon is essential for the growth and possible colonization of probiotics [30,44]. In the by fucose linked to the two positions of β-Gal residues of lactose; hence no electrostatic absence of this property, the entrapped organisms and the capsules will be washed out from repulsions may occur with the negatively charged carboxyl groups of sodium alginate the body without exerting a significant beneficial effect [45]. Many studies investigated the structure. Therefore, the complete release of the cells is probably due to the vigorous in- release of encapsulated probiotics in simulated intestinal fluid; however, to the best of our 2+ teraction of the prebiotic structure with the divalent cations (Ca ) of the sodium alginate knowledge, there is no similar outcome from any encapsulating agent reported in the liter- network, resulting in disintegration of the “egg-box” structure in K2HPO4. This process is ature to date [25,30,43]. Sabikhi et al. [45] reported the release of encapsulated L. acidophilus well known and recognized to explain the inulin capsules releasing behavior in simu- in alginate-starch microspheres using a simulated colonic fluid with the same formulation lated intestinal fluid, a prebiotic commonly used as encapsulating material [33]. (50mM KH PO , pH 7.4  0.2 during 2.5 h). They found that the cell count after 150 min 2 4 of incubation was 7.45 log CFU/mL, suggesting that all the microencapsulated cells were 4. Conclusions released at that time (initial number of 7.47 log CFU/g of capsule). Although 150 min is This study represents the first report among the available literature on the use of about the time needed for intestinal transit of microcapsules, they did not find a significant 2′-fucosyllactose, a trisaccharide from human milk, used as a prebiotic agent in micro- release after 30 min [45]. In another study, a fully released L. plantarum encapsulated encapsulation of S. thermophilus cells. The alginate-based microcapsules showed inter- with sodium alginate and sodium alginate with inulin in a 60-min exposure to simulated esting features, including the capability to protect bacterial cells from harsh simulated intestinal fluid was reported. From the results, it was concluded that the release mechanism gastrointestinal conditions. Compared to other molecules used in microencapsulation was probably due to the replacing of calcium ions in the encapsulation matrices. Also, together with alginate, such as gelatin and inulin, 2′-fucosyllactose evidenced an ex- tremely quick (30 min) and abundant release of bacterial cells from the capsules inside a simulated intestinal fluid. These results encourage further studies aimed at testing these properties under in vivo conditions. Author Contributions: S.P.: investigation, data curation, and writing–original draft. G.G.: investi- gation and writing–original draft. A.T.: conceptualization, data curation, and writing–original draft. A.G.: funding acquisition, writing–review, and editing. V.C.: funding acquisition, writing– review, and editing. All authors have read and agreed to the published version of the manuscript. Funding: This work has been funded in part by the Ministero dell’Università e della Ricerca Scien- tifica (MIUR, Dotazione Ordinaria Ricerca, DOR). Conflicts of Interest: The authors declare that there are no conflicts of interest. Appl. Sci. 2021, 11, 5761 9 of 11 capsules with inulin showed a faster release rate during the first 20 min, which could have been induced by the addition of inulin in sodium alginate affecting the binding of calcium ion [33]. A possible mechanism that could be involved in the fast release of S. thermophilus TH982 cells encapsulated with sodium alginate and 2 -fucosyllactose could be that this small soluble milk glycan used as a co-encapsulating agent in this experiment is formed by fucose linked to the two positions of -Gal residues of lactose; hence no electrostatic repulsions may occur with the negatively charged carboxyl groups of sodium alginate structure. Therefore, the complete release of the cells is probably due to the vigorous 2+ interaction of the prebiotic structure with the divalent cations (Ca ) of the sodium alginate network, resulting in disintegration of the “egg-box” structure in K HPO . This process is 2 4 well known and recognized to explain the inulin capsules releasing behavior in simulated intestinal fluid, a prebiotic commonly used as encapsulating material [33]. 4. Conclusions This study represents the first report among the available literature on the use of 2 -fucosyllactose, a trisaccharide from human milk, used as a prebiotic agent in microen- capsulation of S. thermophilus cells. The alginate-based microcapsules showed interesting features, including the capability to protect bacterial cells from harsh simulated gastroin- testinal conditions. Compared to other molecules used in microencapsulation together with alginate, such as gelatin and inulin, 2 -fucosyllactose evidenced an extremely quick (30 min) and abundant release of bacterial cells from the capsules inside a simulated intesti- nal fluid. These results encourage further studies aimed at testing these properties under in vivo conditions. Author Contributions: S.P.: investigation, data curation, and writing–original draft. G.G.: investiga- tion and writing–original draft. A.T.: conceptualization, data curation, and writing–original draft. 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Effects of 2′-Fucosyllactose-Based Encapsulation on Probiotic Properties in Streptococcus thermophilus

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applied sciences Article Effects of 2 -Fucosyllactose-Based Encapsulation on Probiotic Properties in Streptococcus thermophilus Shadi Pakroo, Gloria Ghion, Armin Tarrah * , Alessio Giacomini and Viviana Corich Department of Agronomy Food Natural Resources Animal and Environment (DAFNAE), University of Padova, Viale dell’Università 16, 35020 Legnaro, PD, Italy; shadi.pakroo@phd.unipd.it (S.P.); gloria.ghion@studenti.unipd.it (G.G.); alessio.giacomini@unipd.it (A.G.); viviana.corich@unipd.it (V.C.) * Correspondence: author: armin.tarrah@unipd.it Abstract: Streptococcus thermophilus is widely used in dairy fermentation as a starter culture for yogurt and cheese production. Many strains are endowed with potential probiotic properties; however, since they might not survive in adequate amounts after transit through the human gastrointestinal tract, it is advisable to improve cell survivability during this passage. The present study evaluates the use of 2 -fucosyllactose, a prebiotic molecule from human milk, compared with other known molecules, such as gelatin and inulin, to form alginate-based microcapsules to fulfill these requirements. Such microcapsules, obtained by the extrusion technique, were evaluated in terms of encapsulation efficiency, storage stability, gastrointestinal condition resistance, and cell release kinetics. Results reveal that microcapsules made using 2 -fucosyllactose and those with inulin resulted in the most efficient structure to protect S. thermophilus strain TH982 under simulated gastrointestinal conditions (less than 0.45 log CFU/g decrease for both agents). In addition, a prompt and abundant release of encapsulated cells was detected after only 30 min from microcapsules made with sodium alginate plus 2 -fucosyllactose in simulated gastrointestinal fluid (more than 90% of the cells). These encouraging Citation: Pakroo, S.; Ghion, G.; results represent the first report on the effects of 2 -fucosyllactose used as a co-encapsulating agent. Tarrah, A.; Giacomini, A.; Corich, V. Effects of 2 -Fucosyllactose-Based Encapsulation on Probiotic Properties Keywords: microencapsulation; probiotic; prebiotic; Streptococcus thermophilus TH982; 2 -fucosyllactose in Streptococcus thermophilus. Appl. Sci. 2021, 11, 5761. https://doi.org/ 10.3390/app11135761 1. Introduction Academic Editors: Athina S. Tzora The genus Streptococcus includes more than 70 species, but to date, only S. thermophilus and Antonio Valero possesses the GRAS (generally recognized as safe) status due to its long history of safe application in food production [1]. For this reason, this is the only species of the genus Received: 29 April 2021 used in the food industry and represents the second most important species of industrial Accepted: 18 June 2021 lactic acid bacteria (LAB) after Lactococcus lactis and one of the primary starters of yo- Published: 22 June 2021 gurt [2,3]. Furthermore, many S. thermophilus strains have several technological properties, such as sugar metabolism, galactose utilization capability, proteolytic activity, and urease Publisher’s Note: MDPI stays neutral activity [4]. with regard to jurisdictional claims in Some strains have also shown potential as a probiotic, having revealed various health published maps and institutional affil- benefits [5,6]. S. thermophilus is a bacterium of non-human origin and, although known iations. to be sensitive to the acidic gastric conditions, it was able to survive the gastrointestinal tract and moderately capable of adhesion to intestinal epithelial cells [7], thus making it a transient probiotic [7,8]. Nowadays, probiotics are one of the driving forces in the design of functional foods, particularly dairy products; hence it is advisable to improve Copyright: © 2021 by the authors. cell survivability during the gastrointestinal passage. The inclusion of probiotics into Licensee MDPI, Basel, Switzerland. microcapsules seems to be a promising solution and also a means to control the release of This article is an open access article cells into the target tissues [9]. distributed under the terms and Several techniques are available for encapsulation, which differ on the nature of their conditions of the Creative Commons core (content) and intended use [10]. Encapsulation techniques applied to the food in- Attribution (CC BY) license (https:// dustry include spray-drying, fluid bed coating, spray-chilling or spray cooling, extrusion, creativecommons.org/licenses/by/ emulsification, coacervation, co-extrusion, inclusion complexation, liposome entrapment, 4.0/). Appl. Sci. 2021, 11, 5761. https://doi.org/10.3390/app11135761 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 5761 2 of 11 centrifugal extrusion, encapsulation by a rapid expansion of supercritical fluid (RESS), freeze- or vacuum drying, and nanotechnological approaches [11]. The use of extrusion technology has many advantages for the encapsulation of probiotic cells. It is a simple and inexpensive method using mild operations, and the procedure does not involve any harmful solvent. Moreover, this technique appears to promote probiotic cell viability [12–15]. Combining alginate with some prebiotic molecules can also enhance cell protection in food systems [10]. Prebiotics form three-dimensional microcrystal networks that interact together, forming small aggregates that contribute to the better protection of the cells [16]. The most frequently used prebiotics in co-encapsulation are oligosaccharides, inulin, and resistant starches [16]. 2’-fucosyllactose (2 -FL), is a neutral trisaccharide composed of L-fucose, D-galactose, and D-glucose and is the most abundant oligosaccharide in human milk (HMO), accounting for about 30% of all of HMOs, i.e., 2–3 g/L [17]. This prebiotic molecule has been reported to display numerous health advantages in humans [18–20]. Another attractive characteristic of 2 -FL is its simplicity, which facilitates its de novo synthesis using microbial, enzymatic, or chemical methods [21]. In this study, we aimed at evaluating the protective effect of 2’-FL on S. thermophilus TH982 cells, a potential probiotic with the ability to produce high levels of folate [22] compared to some other common co-encapsulating materials in alginate beads. 2. Materials and Methods 2.1. Bacteria and Growth Conditions S. thermophilus TH982 [23] is part of the collection of the Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Italy. S. ther- mophilus TH982 was stored at 80 C in M17 broth (Sigma-Aldrich, St. Louis, MI, USA) containing glycerol (25% v/v) and was activated before each experiment by culturing it in M17 (Sigma-Aldrich) broth at 37 C for 24 h. 2.2. Encapsulation Procedure The components used for encapsulation of S. thermophilus TH982 were sodium alginate (S, Sigma-Aldrich), gelatin (G, from bovine skin, Sigma-Aldrich), inulin (I, from chicory, Sigma-Aldrich) and 2 -fucosyllactose (F, Sigma-Aldrich). Fifty milliliters of reactivated cells in M17 broth were centrifuged (Hettich, Westphalia, Germany) at 5500 rpm for 5 min. The bacterial pellets were washed twice with sterile saline solution (0.85%) (Sigma-Aldrich) and added to the encapsulation matrix. The encapsulating matrix was prepared using the extrusion technique through the combination of sodium alginate with either inulin, gelatin, or 2 -fucosyllactose, each one at a concentration of 2% (w/v). Sodium alginate alone was also used as a control. The different matrices were mixed and added to the S. thermophilus TH982 pellet suspension. The mixture was then injected using a sterile syringe with a 450 m needle into sterile 0.3 M CaCl (Sigma-Aldrich) hardener solution. The distance from the bottom of the nozzle and the surface of the CaCl solution was kept at 10 cm (Figure 1). The microcapsules were left in the hardening solution (CaCl ) for 30 min at room temperature. Finally, capsules were transferred in 0.1% (w/v) sterile peptone water solution and stored at 4 C until further use. The morphology of the microcapsules was examined using an optical stereomicroscope (Olympus, Tokyo, Japan). Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 11 Appl. Sci. 2021, 11, 5761 3 of 11 Figure 1. Scheme of microencapsulation procedure of S. thermophilus TH982 cells using the extrusion technique. (A) The Figure 1. Scheme of microencapsulation procedure of S. thermophilus TH982 cells using the extru- sodium alginate alone as control. (B) Sodium alginate with other agents. sion technique. (A) The sodium alginate alone as control. (B) Sodium alginate with other agents. 2.3. Encapsulation Efficiency 2.3. Encapsulation Efficiency One milliliter o One f eac milliliter h type ofof ca each psule type solution w of capsule as added to solution 9 m wasLadded of saline to sol 9 mL ution of saline solution (0.85%), serially (0.85%), diluted, serially and pl diluted, ated on M and 17 plated agar m on edium M17 t agar o det medium ermine th toe v determine iable cell the viable cell concentration in capsule solutions. One gram of each type of capsule was homogenized in concentration in capsule solutions. One gram of each type of capsule was homogenized 9 mL of 10% (w/v) sterilized sodium citrate by vortexing for 1 min, serially diluted, and in 9 mL of 10% (w/v) sterilized sodium citrate by vortexing for 1 min, serially diluted, and plated on M17 to get the number of entrapped cells per g of capsule. The enumeration plated on M17 to get the number of entrapped cells per g of capsule. The enumeration of of viable cells from both the solution and the capsule was done using the micro drop viable cells from both the solution and the capsule was done using the micro drop tech- technique by placing 20 L aliquots on the surface of M17 agar plates and incubating at nique by placing 20 µL aliquots on the surface of M17 agar plates and incubating at 37 °C 37 C for 48 h. The encapsulation efficiency (EE%) was determined using the equation [24]: for 48 h. The encapsulation efficiency (EE%) was determined using the equation [24]: EE% = (N1/N0) × 100 EE% = (N /N )  100 1 0 where N1 is the number of viable entrapped cells (log CFU/g) released after the encap- where N is the number of viable entrapped cells (log CFU/g) released after the encapsula- sulation procedure and N0 is the number of viable cells (log CFU/mL) added to the mix- tion procedure and N is the number of viable cells (log CFU/mL) added to the mixture ture before encapsulation. before encapsulation. 2.4. Storage Stability in Skimmed Milk 2.4. Storage Stability in Skimmed Milk One gram of microcapsules was suspended in 9 mL of 10% (w/v) reconstituted One gram of microcapsules was suspended in 9 mL of 10% (w/v) reconstituted skimmed milk, stored under refrigerated conditions (4 °C) and sampled at different time skimmed milk, stored under refrigerated conditions (4 C) and sampled at different time intervals, namely after 0, 7, 14 and 21 days to evaluate the survival of encapsulated S. intervals, namely after 0, 7, 14 and 21 days to evaluate the survival of encapsulated thermophilus TH982 [25]. The survival of free (non-encapsulated) bacterial cells was de- S. thermophilus TH982 [25]. The survival of free (non-encapsulated) bacterial cells was termined by adding 9 mL of 10% (w/v) reconstituted skimmed milk (Sigma-Aldrich) to 1 determined by adding 9 mL of 10% (w/v) reconstituted skimmed milk (Sigma-Aldrich) mL of free cells as well. At each time interval, 1 g aliquot of capsules was aseptically to 1 mL of free cells as well. At each time interval, 1 g aliquot of capsules was aseptically centrifuged at 5500 rpm for 5 min. The supernatant was discarded and the cells were al- centrifuged at 5500 rpm for 5 min. The supernatant was discarded and the cells were lowed to release in 9 mL of 10% (w/v) sodium citrate by vortexing for 1 min, serially di- allowed to release in 9 mL of 10% (w/v) sodium citrate by vortexing for 1 min, serially luted, and plated on M1 diluted, and 7 agplated ar, followed on M17 by incub agar, followed ation at 37 by °C incubation for 48 h. The s at 37 urv Civ for al w 48 as h. The survival determined as the was number determined of ceas lls the recov number ered duri of cells ng dirff ecover erent stora ed during ge time i differ nterva ent storage ls with time intervals respect to the number o with respect f init to iathe l ent number rapped cel of initial ls. entrapped cells. 2.5. Survivability under Simulated Gastrointestinal Conditions 2.5. Survivability under Simulated Gastrointestinal Conditions The simulated gastrointestinal conditions were obtained using basic fluid, gastric The simulated gastrointestinal conditions were obtained using basic fluid, gastric fluid, and intestinal fluid. The basic fluid was prepared by dissolving (per liter): 1.12 g fluid, and intestinal fluid. The basic fluid was prepared by dissolving (per liter): 1.12 g potassium chloride (Sigma-Aldrich), 2.0 g sodium chloride (Sigma-Aldrich), 0.11 g calcium potassium chloride (Sigma-Aldrich), 2.0 g sodium chloride (Sigma-Aldrich), 0.11 g cal- chloride (Sigma-Aldrich), and 0.4 g potassium dihydrogen phosphate (Sigma-Aldrich) in cium chloride (Sigma-Aldrich), and 0.4 g potassium dihydrogen phosphate (Sig- saline solution (0.85%). The basic fluid was sterilized by autoclaving at 121 C for 15 min. ma-Aldrich) in saline solution (0.85%). The basic fluid was sterilized by autoclaving at 121 Appl. Sci. 2021, 11, 5761 4 of 11 The simulated gastric fluid (SGJ) and simulated intestinal fluid (SIJ) were prepared based on a modified method published by Singh et al. [25]. The gastric fluid consisted of 0.01 g/L of swine pepsin (Sigma-Aldrich) and 0.01 g/L of swine mucin (Sigma-Aldrich) added directly to the sterile basic juice. The pH was adjusted to 2.5 with 1 N HCl, and the liquid was filter-sterilized using a 0.22 m membrane filter (Sigma-Aldrich). The intestinal fluid contained (per liter of basic fluid): 0.01 g pancreatin (Sigma- Aldrich), 0.08 g Ox-bile extract (Sigma-Aldrich), and 0.01 g lysozyme (Sigma-Aldrich). The pH was adjusted to 7.5 with 1 N NaOH, and the juice was filter sterilized. Both gastric and intestinal fluids were prepared fresh on the day of the experiment. Overnight bacterial cultures were obtained after subculturing in M17 broth, and capsules were prepared as described in the encapsulation procedure. One gram of each type of capsule was added to 9 mL of SGJ and incubated at 37 C for 1 h to evaluate the survivability of S. thermophilus TH982 under gastric conditions. For the survival of S. thermophilus TH982 under gastrointestinal conditions, 9 mL of SIJ were added following gastric fluid incubation, and the capsules and free cells with simulated gastrointestinal fluid were left at 37 C for a further 2 h. The cell viability was evaluated for free and encapsulated S. thermophilus after gastric and gastrointestinal incubations by plating on M17 agar and using the micro drop technique, followed by incubation at 37 C for 48 h. 2.6. Release Kinetics The release kinetics from capsules were determined using phosphate solution without enzymes. The release of encapsulated S. thermophilus TH982 was evaluated according to Shi et al. [26] with some modifications. A phosphate solution without the addition of intestinal enzymes (6.8 g/L of K HPO , 50 mM) was prepared, the pH was adjusted 2 4 to 7.5 with 1 N HCl and subsequently sterilized by autoclaving at 121 C for 15 min. Microcapsules (1 g) from different matrices were added to 9 mL of the abovementioned phosphate solution and incubated at 37 C with agitation (100 rpm). At predetermined time intervals (0, 30, 60, 90, 120, 150, and 180 min), 100 L of solution were taken from each capsule type, serially diluted, and plated using the micro drop technique on M17 agar to detect the number of released cells. The number of released cells was determined after 48 h of incubation at 37 C and expressed as log CFU/mL of K HPO solution. 2 4 2.7. Statistical Analysis All the experiments were performed in triplicate. Data were analyzed using a one- way analysis of variance (ANOVA). Tukey’s test was used as a post-hoc analysis by the GraphPad Prism software (version 7, GraphPad Software, Inc., San Diego, CA, USA). Results were considered significantly different for p values lower than 0.05. 3. Results and Discussion 3.1. Morphology and Encapsulation Efficiency All capsules appeared globular and irregular in shape with a rough surface and a drop- like shape. This result is probably due to the high surface tension of the used hardening solution (CaCl ) that determines an imperfect sphere formation [27]. However, no surface cavities or fractures were visible. The average size of capsules was 3.5 0.52 (mm) without any significant difference among the different microcapsules types. The capsules with S + G showed a more dense structure, as confirmed by the viscosity of G solution, due to electrostatic interactions between amino groups of G and carboxyl groups of S, which make cells more resistant to enzymatic and acidic hydrolysis [28]. Encapsulation efficiencies (EE%) of the four microcapsule types are reported in Table 1. EE% is a parameter that describes both the survival of viable cells and the efficacy of entrapment of the encapsulation procedure [29]. The yield of S. thermophilus TH982 viable cells co-encapsulated using S + F (96.13%) was significantly higher (p < 0.01) than that of cells encapsulated with S alone (91.07%), used as control, and was similar (not statistically Appl. Sci. 2021, 11, 5761 5 of 11 different) to S + I (98.61%). The high EE% obtained with S + F and S + I matrices might be due to the decrease in the porosity of the gel-beads and, consequently, to a reduction in the leakage of entrapped S. thermophilus TH982 cells [30]. Besides, incorporating prebiotics as material for encapsulation may better protect probiotics in food systems and inside the gastrointestinal tract due to mutual cells/prebiotic interactions [31]. By contrast, the number of viable cells encapsulated in S alone and S + G showed no significant difference (EE% 91.07 and 90.50, respectively). The main reason why EE% is normally lower than 100% is linked to cell damage due to detrimental conditions caused by the encapsulation process itself, such as shear stress and the use of concentrated solutions. Furthermore, during the time required for the hardening of capsules, a physical loss of cells can occur in significant numbers [29]. It should also be noted that a dissolution process is required to determine the number of viable cells in the microcapsules, and therefore an incomplete disintegration can underestimate the calculated EE% value. In particular, for the solubilization of S-based capsules, sodium citrate (10% w/v) was used, which is a chelating agent [30] that can remove calcium from the “egg-box” structure, leading to the destabilization of alginate coating and to the effective release of cells [32]. Table 1. Encapsulation efficiency (EE%) of S. thermophilus TH982 using different encapsulating matrices. Cell in the Encapsulation Solution Cells after Encapsulation Encapsulation Efficiency Capsule Composition (log CFU/mL) (log CFU/g) (EE%) a a a Sodium alginate 9.46  0.04 8.61  0.19 91.07 a b b Sodium alginate + Inulin 9.17  0.21 9.04  0.12 98.61 a a a Sodium alginate + Gelatin 9.39  0.09 8.50  0.04 90.50 b b Sodium alginate + Fucosyllactose 9.34  0.03 8.97  0.07 96.13 Values are the mean  standard deviation (SD) of triplicate experiments (n = 3). Superscript letters in each column indicate statistically significant (p < 0.05) differences among different formulations. 3.2. Storage Stability in Skimmed Milk In order to test the viability of S. thermophilus cells inside capsules during prolonged storage, we maintained them in skimmed milk at refrigeration conditions (4 C) for 21 days (Figure 2). At the end of the period, cell viability showed 0.46, 1.62, 1.79, and 1.97 log decrease for S. thermophilus TH982 cells encapsulated with S + G, S + F, S, and S + I, respectively. Capsules containing G as a co-encapsulating agent showed a better perfor- mance after 21 days with respect to other co-encapsulating agents. However, data show that free cells had the highest viability level throughout the storage period since there was no significant difference between initial (8.90  0.08 log CFU/mL; day0) and final (8.94  0.05 log CFU/mL; day21) values. Interestingly, S, S + I, and S + F capsules revealed a similar, not significantly different, viability reduction (p < 0.05). Unlike some studies that indicate an increase in surviv- ability during refrigerated storage conditions, which could be linked to the difference in species/strain used. S. thermophilus is frequently used in the production of different dairy products, and it is highly adapted to the dairy environment. It was reported that encapsulation improved the stability of L. plantarum during storage [33], and encapsulation of bacteria in alginate was found to improve survivability when compared to unprotected cell counts stored in skimmed milk for 24 h [14]. Since FAO and the International Dairy Fed- eration (IDF) recommend that the minimum content of probiotic cells should be 10 CFU/g of product at the time of consumption [34], the four encapsulating agents used in this study were efficient in maintaining the required viability of S. thermophilus TH982 after refriger- ated storage in skimmed milk. The high numbers suggested by IDF have been proposed to compensate for the possible numerical reduction of probiotic organisms during passage through the human stomach and intestine. The cells must remain viable throughout the projected shelf-life period of a product so that when consumed, the product contains a sufficient number of viable cells. Appl. Sci. 2021, 11, x FOR PEER REVIEW 6 of 11 must remain viable throughout the projected shelf-life period of a product so that when consumed, the product contains a sufficient number of viable cells. The viability of probiotic bacteria in food products is affected by many intrinsic and extrinsic aspects such as dissolved oxygen and oxygen permeation through the package, post acidification in fermented products (lactic and acetic acids), pH, storage and incu- bation temperature, duration of fermentation, production of hydrogen peroxide due to bacterial metabolism, and processing conditions [35,36]. Moreover, the specific strains used, the interaction between species, availability of nutrients, and growth promoters and inhibitors can affect viable cells’ survivability [14]. The great survivability of free S. thermophilus TH982 cells in skimmed milk is prob- ably due to the fact that S. thermophilus is a species highly adapted to grow on lactose as its energy and carbon source, that is internalized by lactose permease (LacS) and hydro- lyzed to glucose and galactose by β-galactosidase (LacZ) [37]. It is likely that entrapment of S. thermophilus TH982 cells into capsules of different matrices probably led to a re- duced diffusion of lactose inside the capsule layer or membrane, limiting the possibility Appl. Sci. 2021, 11, 5761 6 of 11 of its use by the entrapped cells [35]. Figure 2. Figure 2.Su Survivability rvivability of en of encapsulated capsulated an andd free free S. S thermophilus . thermophilus TH982 TH982 cellscells during 21 days of during 21 days of storage storage in skimmed milk at 4 °C. in skimmed milk at 4 C. The viability of probiotic bacteria in food products is affected by many intrinsic and 3.3. Survivability under Simulated Gastrointestinal Conditions extrinsic aspects such as dissolved oxygen and oxygen permeation through the package, The survival of probiotics under gastrointestinal conditions represents a significant post acidification in fermented products (lactic and acetic acids), pH, storage and incubation issue for their effectiveness, and microencapsulation could represent a valid method to temperature, duration of fermentation, production of hydrogen peroxide due to bacterial provide significant protection [15]. The survival of free and encapsulated S. thermophilus metabolism, and processing conditions [35,36]. Moreover, the specific strains used, the TH982 cells after 1, 2, and 3 h of incubation in simulated gastrointestinal conditions are interaction between species, availability of nutrients, and growth promoters and inhibitors presented in Figure 3. After 1 h of gastric fluid incubation, the highest reduction of viable can affect viable cells’ survivability [14]. cells was found, as expected, for free S. thermophilus TH982, as the initial number of 10.40 The great survivability of free S. thermophilus TH982 cells in skimmed milk is probably ± 0.10 log CFU/mL was diminished by 0.95 log CFU/mL. A notable decrease in viability due to the fact that S. thermophilus is a species highly adapted to grow on lactose as its after 3 h of gastrointestinal conditions was confirmed for free S. thermophilus TH982 cells, energy and carbon source, that is internalized by lactose permease (LacS) and hydrolyzed reduced by approximately 1.60-log CFU/mL, dropping to 8.79 ± 0.02 log CFU/mL. By to glucose and galactose by -galactosidase (LacZ) [37]. It is likely that entrapment of contrast, for cells encapsulated with S alone, only 1.05 log CFU/g reduction of viable cells S. thermophilus TH982 cells into capsules of different matrices probably led to a reduced was observed, implying that microencapsulation provided protection compared to the diffusion of lactose inside the capsule layer or membrane, limiting the possibility of its use free cells. This protection offered by sodium alginate might be related to the establish- by the entrapped cells [35]. ment of a hydrogel barrier through an external layer of sodium alginate that has retarded 3.3. Survivability under Simulated Gastrointestinal Conditions The survival of probiotics under gastrointestinal conditions represents a significant issue for their effectiveness, and microencapsulation could represent a valid method to provide significant protection [15]. The survival of free and encapsulated S. thermophilus TH982 cells after 1, 2, and 3 h of incubation in simulated gastrointestinal conditions are presented in Figure 3. After 1 h of gastric fluid incubation, the highest reduction of viable cells was found, as expected, for free S. thermophilus TH982, as the initial number of 10.40  0.10 log CFU/mL was diminished by 0.95 log CFU/mL. A notable decrease in viability after 3 h of gastrointestinal conditions was confirmed for free S. thermophilus TH982 cells, reduced by approximately 1.60-log CFU/mL, dropping to 8.79  0.02 log CFU/mL. By contrast, for cells encapsulated with S alone, only 1.05 log CFU/g reduction of viable cells was observed, implying that microencapsulation provided protection compared to the free cells. This protection offered by sodium alginate might be related to the establishment of a hydrogel barrier through an external layer of sodium alginate that has retarded the permeation of simulated fluids into the capsules and thus the interaction with the probiotic cells [38]. Appl. Sci. 2021, 11, x FOR PEER REVIEW 7 of 11 the permeation of simulated fluids into the capsules and thus the interaction with the Appl. Sci. 2021, 11, 5761 7 of 11 probiotic cells [38]. Figure 3. Survivability of encapsulated and free S. thermophilus TH982 cells during exposure to Figure 3. Survivability of encapsulated and free S. thermophilus TH982 cells during exposure to simsimulated ulated gast gastr rointestinal ointestinal conditions conditions. . DiffDif erent le ferentters t letters indicate indicate significant differences w significant differencesithin the within the same incubation step (p < 0.05) among different encapsulating matrix types. same incubation step (p < 0.05) among different encapsulating matrix types. S + G capsules showed low survivability during gastric environment; indeed, the S + G capsules showed low survivability during gastric environment; indeed, the reduction of viable cells was higher (0.52 log CFU/g decrease) than encapsulated cells with reduction of viable cells was higher (0.52 log CFU/g decrease) than encapsulated cells S + I (no reduction detected) after 1 h of gastric condition (pH 2.5). This fact might be due with S + I (no reduction detected) after 1 h of gastric condition (pH 2.5). This fact might be to a variation of pH, which changes the gelatin charge of amino and carboxyl groups so due to a variation of pH, which changes the gelatin charge of amino and carboxyl groups that the modification of cross-links and structure of the chain could influence the swelling so that the modification of cross-links and structure of the chain could influence the behavior of gelatin capsules [39]. swelling behavior of gelatin capsules [39]. On the other side, an interesting outcome was given by the two types of prebiotics used On the other side, an interesting outcome was given by the two types of prebiotics as a co-encapsulating agent with sodium alginate. After 3 h of exposure to gastrointestinal used as a co-encapsulating agent with sodium alginate. After 3 h of exposure to gastro- conditions, S + I and S + F revealed the highest cell survivability without any significant intestinal conditions, S + I and S + F revealed the highest cell survivability without any difference, leading to a low cell reduction (less than 0.45 log CFU/g decrease for both significant difference, leading to a low cell reduction (less than 0.45 log CFU/g decrease agents). Prebiotics, such as 2 -FL and inulin, which could provide good protection and even for both agents). Prebiotics, such as 2′-FL and inulin, which could provide good protec- promote cell proliferation, appeared to contribute to the growth of S. thermophilus TH982. In tion and even promote cell proliferation, appeared to contribute to the growth of S. addition, oligosaccharides are difficult to decompose by enzymes in digestive fluid but can thermophilus TH982. In addition, oligosaccharides are difficult to decompose by enzymes be metabolized by beneficial bacteria in the colon [30]. It has been further demonstrated that in digestive fluid but can be metabolized by beneficial bacteria in the colon [30]. It has oligosaccharides contained in human milk have an extraordinary resistance to hydrolysis been further demonstrated that oligosaccharides contained in human milk have an ex- by digestive enzymes of the small intestine [40]. The combination of calcium alginate with traordinary resistance to hydrolysis by digestive enzymes of the small intestine [40]. The prebiotics such as inulin improves the viability of probiotics and facilitates the formation combination of calcium alginate with prebiotics such as inulin improves the viability of of an integrated structure of capsules. Researchers found better protection of Lactobacillus probiotics and facilitates the formation of an integrated structure of capsules. Researchers casei and Bifidobacterium bifidum cells in coated capsules with prebiotics such as inulin after found better protection of Lactobacillus casei and Bifidobacterium bifidum cells in coated the gastrointestinal transit. The addition of prebiotics could be synergistic in gelling, and capsules with prebiotics such as inulin after the gastrointestinal transit. The addition of as a result, may help to maintain and improve the degree of protection of the bacterial prebiotics could be synergistic in gelling, and as a result, may help to maintain and im- cells [41]. prove the degree of protection of the bacterial cells [41]. Lastly, the free cell suffered more from the gastric conditions than that during the 3-h Lastly, the free cell suffered more from the gastric conditions than that during the gastrointestinal transition. In contrast, encapsulated bacteria evidenced greater reductions 3-h gastrointestinal transition. In contrast, encapsulated bacteria evidenced greater re- during gastrointestinal transit than under gastric conditions alone. This behavior may ductions during gastrointestinal transit than under gastric conditions alone. This behav- be due to the fact that alginate is stable in low-pH solutions: the ionotropic alginate gel ior may be due to the fact that alginate is stable in low-pH solutions: the ionotropic algi- 2+ formed by Ca cross-linking of carboxylate groups is insoluble at low pH, but exposure to neutral pH or higher solubilize the alginate, causing swelling [42]. Appl. Sci. 2021, 11, 5761 8 of 11 3.4. Release Kinetics The release of encapsulated S. thermophilus TH982 after incubation in simulated in- testinal fluid was evaluated after 30, 60, 90, 120, 150, and 180 min, as reported in Figure 4. After 30 min of agitation at 100 rpm at 37 C in 50 mM K HPO a great release of cells 2 4, encapsulated with S + F was detected. Considering the initial number of cells entrapped in this type of capsules (9.73  0.04 log CFU/g of capsules), the number of cells released in the simulated intestinal fluid after 30 min was 8.83  0.04 log CFU/mL of K HPO , 2 4 representing a very high value compared with the other types of encapsulating matrices after 180 min, which resulted about 2-log lower. This result confirms what was visible during the experiment: after 30 min, there was an evident solubilization of S + F capsules, a characteristic not displayed by the others (S, S + I, and S + G). Moreover, the number Appl. Sci. 2021, 11, x FOR PEER REVIEW of released cells suggests that once liberated, S. thermophilus TH982 can withstand 9 of well 11 simulated intestinal conditions, since after 180 min the number of cells released from S + F capsules was 9.72  0.05 log CFU/mL, which means 99.89% of the initial entrapped cells. Figure 4. Release kinetics of entrapped S. thermophilus TH982 cells from different capsule formulations Figure 4. Release kinetics of entrapped S. thermophilus TH982 cells from different capsule formula- during incubation in a simulated intestinal environment. tions during incubation in a simulated intestinal environment. It is essential to ensure that the capsules give protection through the simulated gas- A possible mechanism that could be involved in the fast release of S. thermophilus trointestinal passage and ensure that the encapsulation matrix allows a release of viable and TH982 cells encapsulated with sodium alginate and 2′-fucosyllactose could be that this metabolically active cells in the intestine [30,43]. The release of the cells from microcapsules small soluble milk glycan used as a co-encapsulating agent in this experiment is formed in the colon is essential for the growth and possible colonization of probiotics [30,44]. In the by fucose linked to the two positions of β-Gal residues of lactose; hence no electrostatic absence of this property, the entrapped organisms and the capsules will be washed out from repulsions may occur with the negatively charged carboxyl groups of sodium alginate the body without exerting a significant beneficial effect [45]. Many studies investigated the structure. Therefore, the complete release of the cells is probably due to the vigorous in- release of encapsulated probiotics in simulated intestinal fluid; however, to the best of our 2+ teraction of the prebiotic structure with the divalent cations (Ca ) of the sodium alginate knowledge, there is no similar outcome from any encapsulating agent reported in the liter- network, resulting in disintegration of the “egg-box” structure in K2HPO4. This process is ature to date [25,30,43]. Sabikhi et al. [45] reported the release of encapsulated L. acidophilus well known and recognized to explain the inulin capsules releasing behavior in simu- in alginate-starch microspheres using a simulated colonic fluid with the same formulation lated intestinal fluid, a prebiotic commonly used as encapsulating material [33]. (50mM KH PO , pH 7.4  0.2 during 2.5 h). They found that the cell count after 150 min 2 4 of incubation was 7.45 log CFU/mL, suggesting that all the microencapsulated cells were 4. Conclusions released at that time (initial number of 7.47 log CFU/g of capsule). Although 150 min is This study represents the first report among the available literature on the use of about the time needed for intestinal transit of microcapsules, they did not find a significant 2′-fucosyllactose, a trisaccharide from human milk, used as a prebiotic agent in micro- release after 30 min [45]. In another study, a fully released L. plantarum encapsulated encapsulation of S. thermophilus cells. The alginate-based microcapsules showed inter- with sodium alginate and sodium alginate with inulin in a 60-min exposure to simulated esting features, including the capability to protect bacterial cells from harsh simulated intestinal fluid was reported. From the results, it was concluded that the release mechanism gastrointestinal conditions. Compared to other molecules used in microencapsulation was probably due to the replacing of calcium ions in the encapsulation matrices. Also, together with alginate, such as gelatin and inulin, 2′-fucosyllactose evidenced an ex- tremely quick (30 min) and abundant release of bacterial cells from the capsules inside a simulated intestinal fluid. These results encourage further studies aimed at testing these properties under in vivo conditions. Author Contributions: S.P.: investigation, data curation, and writing–original draft. G.G.: investi- gation and writing–original draft. A.T.: conceptualization, data curation, and writing–original draft. A.G.: funding acquisition, writing–review, and editing. V.C.: funding acquisition, writing– review, and editing. All authors have read and agreed to the published version of the manuscript. Funding: This work has been funded in part by the Ministero dell’Università e della Ricerca Scien- tifica (MIUR, Dotazione Ordinaria Ricerca, DOR). Conflicts of Interest: The authors declare that there are no conflicts of interest. Appl. Sci. 2021, 11, 5761 9 of 11 capsules with inulin showed a faster release rate during the first 20 min, which could have been induced by the addition of inulin in sodium alginate affecting the binding of calcium ion [33]. A possible mechanism that could be involved in the fast release of S. thermophilus TH982 cells encapsulated with sodium alginate and 2 -fucosyllactose could be that this small soluble milk glycan used as a co-encapsulating agent in this experiment is formed by fucose linked to the two positions of -Gal residues of lactose; hence no electrostatic repulsions may occur with the negatively charged carboxyl groups of sodium alginate structure. Therefore, the complete release of the cells is probably due to the vigorous 2+ interaction of the prebiotic structure with the divalent cations (Ca ) of the sodium alginate network, resulting in disintegration of the “egg-box” structure in K HPO . This process is 2 4 well known and recognized to explain the inulin capsules releasing behavior in simulated intestinal fluid, a prebiotic commonly used as encapsulating material [33]. 4. Conclusions This study represents the first report among the available literature on the use of 2 -fucosyllactose, a trisaccharide from human milk, used as a prebiotic agent in microen- capsulation of S. thermophilus cells. The alginate-based microcapsules showed interesting features, including the capability to protect bacterial cells from harsh simulated gastroin- testinal conditions. Compared to other molecules used in microencapsulation together with alginate, such as gelatin and inulin, 2 -fucosyllactose evidenced an extremely quick (30 min) and abundant release of bacterial cells from the capsules inside a simulated intesti- nal fluid. These results encourage further studies aimed at testing these properties under in vivo conditions. Author Contributions: S.P.: investigation, data curation, and writing–original draft. G.G.: investiga- tion and writing–original draft. A.T.: conceptualization, data curation, and writing–original draft. A.G.: funding acquisition, writing–review, and editing. V.C.: funding acquisition, writing–review, and editing. All authors have read and agreed to the published version of the manuscript. Funding: This work has been funded in part by the Ministero dell’Università e della Ricerca Scien- tifica (MIUR, Dotazione Ordinaria Ricerca, DOR). Conflicts of Interest: The authors declare that there are no conflicts of interest. References 1. Tarrah, A.; Noal, V.; Giaretta, S.; Treu, L.; Duarte, V.D.S.; Corich, V.; Giacomini, A. Effect of different initial pH on the growth of Streptococcus macedonicus and Streptococcus thermophilus strains. Int. Dairy J. 2018, 86, 65–68. [CrossRef] 2. Tarrah, A.; Noal, V.; Treu, L.; Giaretta, S.; Duarte, V.D.S.; Corich, V.; Giacomini, A. Short communication: Comparison of growth kinetics at different temperatures of Streptococcus macedonicus and Streptococcus thermophilus strains of dairy origin. J. Dairy Sci. 2018, 101, 7812–7816. [CrossRef] [PubMed] 3. 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Journal

Applied SciencesMultidisciplinary Digital Publishing Institute

Published: Jun 22, 2021

Keywords: microencapsulation; probiotic; prebiotic; Streptococcus thermophilus TH982; 2′-fucosyllactose

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