Hydrogel Derived from Glucomannan-Chitosan to Improve the Survival of Lactobacillus acidophilus FNCC 0051 in Simulated Gastrointestinal Fluid

The probiotic encapsulating hydrogel derived from porang (Amorphophallus oncophyllus) glucomannan and chitosan was investigated with regard to its encapsulation efficiency, physical properties, prebiotic activity, and survival under simulated gastrointestinal conditions. The hydrogel's encapsulation efficiency was improved by varying the number of the Lactobacillus acidophilus FNCC 0051, which also served to increase the diameter (2-3 mm), polydispersity index (1.23–1.65), positive zeta potential, whiteness, and brightness of the hydrogel. Moreover, the hydrogel's prebiotic activity score was higher than that of inulin after 24 h of incubation, reflecting its role as a cell encapsulant, particularly when it comes to maintaining cells during exposure to simulated gastrointestinal fluid. The cell viability increased from 86% to 100% when immersed in intestinal juice, which is comparable to the increase achieved using alginate and konjac glucomannan hydrogels. Future animal studies are required to determine the cell viability in actual gastrointestinal conditions and assess the health effects of the hydrogel.


Introduction
Glucomannan is a functional polysaccharide that can be extracted from Amorphophallus tubers. While the glucomannan obtained from Amorphophallus konjac has a number of popular and commercial uses, several research groups are currently investigating the potential of glucomannan derived from other sources. Amorphophallus oncophyllus, which is commonly known as porang, is a local glucomannan source in Indonesia [1,2]. It has several characteristics that difer from those of konjac, including mannose/glucose molar ratio, degree of polymerization, and degree of acetylation, leading it to exhibit diferent solubility, viscosity, water-holding capacity, and gelation properties [1,2]. Terefore, the applications of porang may also difer depending on the function.
A hydrogel is a kind of technological glucomannan product that leverages its gelation properties. Hydrogels are formed through interactions between glucomannan and other polymers that lead to the formation of a threedimensional polymeric network [3]. Tis characteristic results in hydrogels exhibit potential as encapsulants. A previous study used a hydrogel created by crosslinking konjac, glucomannan, and chitosan, which was found to have many advantages, including natural formation without the need for a crosslinker, self-assembly, tolerance to different pH levels, and demonstrable ability to encapsulate drugs, proteins, and enzymes [4,5]. A similar study involving hydrogels formed by means of the interaction between porang glucomannan and chitosan investigated the production of the primary carboxymethyl glucomannan material, the compatibility of the substitution degree of the carboxymethyl glucomannan involved in the hydrogel formation, the efect of the polymer concentration on the glucomannan properties, and the application in relation to probiotic encapsulation [6][7][8]. Te key innovation of the study was the use of porang, which has characteristics that difer from those of other glucomannan sources, such as the solubility, viscosity, water-holding capacity, degree of polymerization, degree of acetylation, purity, and X-ray diffraction (XRD) pattern [1,2]. Te other diferences include the type of modifcation used (carboxymethylation) and the use of the hydrogel as a probiotic encapsulant. By contrast, prior studies made use of the oxidation method [5] and encapsulated drugs, proteins, and enzymes [4,5]. Te use of carboxymethyl konjac glucomannan-chitosan as a probiotic encapsulant was recently studied, but it was combined with a calcium-alginate hydrogel bead system [9]. Tey were also found to be used as a secondary emulsion to carry curcumin [10].
However, given that living cells have diferent characteristics to inanimate compounds, the role of this new hydrogel in encapsulating probiotics needs to be further studied. Indeed, the new capsules should ensure the survival of the probiotics during food processing and storage, in addition to ensuring sufcient delivery when consumed (>10 6 -10 7 colony-forming units [CFU]/mL). Furthermore, the capsules need to allow the probiotics to reach the lower gastrointestinal tract if they are to have a benefcial efect on humans. Tus, the survival of the capsules during gastrointestinal digestion and their ability to increase probiotic growth in the colon are important.
We previously studied the properties of the hydrogel produced in the diferent glucomannan concentration and evaluated its probiotic encapsulation efciency, also its role in protecting cells during pasteurization and cold storage [8]. Encapsulation efciency could not only be improved by varying the concentration of added polymer but also added core [11]. Te impact of probiotic cells number as the core on the encapsulation efciency and the properties of the hydrogel in this work remain unexplored. Te present study sought to improve the probiotic encapsulation efciency by varying the number of cells and to evaluate the hydrogel's physical properties. It also examined the ability of the hydrogel to maintain probiotics during simulated gastrointestinal exposure and its potency as a prebiotic.

Materials.
Te primary material used in this study was glucomannan derived from porang tubers (A. oncophyllus), which was obtained from the Faculty of Agricultural Technology, Universitas Gadjah Mada (Yogyakarta, Indonesia). Te carboxymethylation of the glucomannan was performed using sodium chloroacetate, as previously described [7]. Te utilized chitosan, which had a degree of deacetylation of 85%-89%, meaning that it met established food quality criteria, was obtained from PT Biotech Surindo (Cirebon, West Java, Indonesia).

Preparation of the Lactobacillus acidophilus FNCC 0051
Cells. Te L. acidophilus FNCC 0051 cells used in this study were obtained from the Food and Nutrition Culture Collection (FNCC) of the Laboratory of Food Microbiology, Center for Food and Nutrition Studies, Universitas Gadjah Mada. Te cells, which were stored in a skim milk-glycerol suspension, were rejuvenated in de Man, Rogosa, and Sharpe (MRS) broth at 37°C overnight and then grown twice. Subsequently, the cell biomass was harvested by means of centrifugation at 2400 g for 9 min at 4°C and then rinsed with saline solution.

Production of the Hydrogel and Determination of its
Encapsulation Efciency. Te hydrogel was created by mixing porang glucomannan with chitosan using the complex coacervation method [8]. Te encapsulation of the probiotics in the hydrogel was performed using three different cell numbers, namely 8 log CFU/mL, 9 log CFU/mL, and 10 log CFU/mL. Te cells were mixed with glucomannan prior to the start of the coacervation process. Te hydrogel's encapsulation efciency was determined by releasing the cells trapped within it using a bufer solution at pH 8 and 37°C for 24 h [7]. Te released cells were then grown in MRS agar to allow for the enumeration of the total viable cells. To calculate the encapsulation efciency, the total viable cell number was divided by the number of initial cells added to the hydrogel mixture [12].

Determination of the Hydrogel's Properties
2.4.1. Particle Size, Polydispersity Index, and Zeta Potential. Te particle size was estimated based on the hydrogel's diameter and simultaneously measured on the basis of the polydispersity index using a particle size analyzer (SZ-100 series; Horiba, Kyoto, Japan). Te hydrogel's zeta potential was measured using a Nano ZS Zetasizer (v.6.20; Malvern Instruments Ltd., Malvern, UK).

Color.
Te hydrogel was freeze-dried and ground prior to the color measurement. Te redness (a * ), yellowness (b * ), and lightness (L * ) values were determined using a CR200 chroma meter (Minolta, Osaka, Japan). Te whiteness index was calculated as previously described [13].

Crystallinity Percentage.
Te XRD of the hydrogel was determined using a LabX XRD-6000 difractometer (Shimadzu, Kyoto, Japan) equipped with a Cu Kα target at 40 kV and 30 mA, which had a scanning rate of 4°/min. Te pattern was collected in the 2θ range between 3.02°and 90°. Te crystallinity percentage (%) was calculated by dividing the area under the peaks by the total area under the curve [14].

Determination of the Prebiotic Activity
Score. Te prebiotic activity score was calculated by subtracting the ratio of probiotic cell growth with prebiotics and glucose from the ratio of enteric cell growth with prebiotics and glucose, as previously described [15]. Te probiotic used was L. acidophilus FNCC 0051, whereas the enteric cells used were Escherichia coli FNCC 0091. Te test was performed by adding 1% (volume/volume [v/v]) probiotic cells into MRS broth containing 2% (weight/volume [w/v]) glucose or prebiotic and adding 1% (v/v) enteric cells into M9 broth containing 2% (w/v) glucose or prebiotic. Te cells were incubated at 37°C for 0 h, 24 h, and 48 h and then enumerated by means of the plate count method using MRS and nutrient agar. Each test was performed three times.

Determination of L. acidophilus FNCC 0051 Survival during Exposure to Simulated Gastrointestinal Conditions.
Te utilized simulated gastric and intestinal juices were prepared according to the method described by Xu et al. [16]. More specifcally, the gastric juice was prepared by mixing 7 mL of pepsin in hydrochloric acid, 2 g of sodium chloride, and 1 M of sodium hydroxide. Te intestinal juice was prepared by mixing 1% pancreatic powder, 6.8 g of potassium dihydrogen phosphate, and 77 mL of 0.2 N sodium hydroxide. Next, 1 g of either free or encapsulated cells (in hydrogel derived from porang glucomannan-chitosan, konjac glucomannan-chitosan, and calcium alginate) was mixed with 9 mL of simulated gastrointestinal juices and incubated at 37°C for 120 min. Te samples were withdrawn at intervals of 0 min, 30 min, 60 min, and 120 min to refect gastric juice digestion and 0 min, 60 min, 90 min, and 120 min to refect intestinal juice digestion [17]. Te hydrogel was then rinsed twice with acetate bufer. Te cells were enumerated using the pour plate technique on MRS agar after 48 h of incubation. Te number of viable cells following exposure was divided by the initial number of cells in order to determine the cell survival rate during exposure to simulated gastrointestinal conditions [12]. Te hydrogel's appearance during exposure to simulated gastrointestinal conditions was observed using an optical BX51 microscope (Olympus Corp., Tokyo, Japan) and an OptiLab prodigital camera (PT Miconos, Indonesia).

Encapsulation Efciencies of Hydrogels with Diferent
Numbers of Cells. Te encapsulation efciencies of hydrogels with diferent numbers of initial cells are shown in Table 1. Te data revealed that the encapsulation efciencies of the hydrogels ranged between 44.37% and 85.03%. Te highest encapsulation efciency was achieved when 10 log CFU/mL of cells was added to the mixture, which exceeded the Food and Agricultural Organization of the United Nations (FAO) criteria for probiotic products (>6-7 log CFU/mL; [18]). Previous studies using diferent encapsulants obtained different encapsulation efciencies. For instance, the encapsulation of L. acidophilus in hydrogel formed from sodium alginate and soy protein isolates achieved an encapsulation efciency of 95%-98%, whereas the encapsulation of Lactobacillus rhamnosus and Lactobacillus plantarum in an emulsion achieved an encapsulation efciency of 97%-99% [12,19]. Te diferences in the achieved encapsulation effciencies might refect the diferent encapsulant types and encapsulation methods used [12]. We previously showed that the same ratio of glucomannan and chitosan afected the encapsulation efciency due to the chemical bonding of both polymers as well as due to the diference in electrostatic values between the core and the polymer infuencing the degree of cell entrapment [8].

Properties of the Hydrogels with Diferent Cell Numbers.
Te appearance of the hydrogels generated from glucomannan and chitosan containing L. acidophilus is shown in Figure 1. Te polymer solution was clear before the encapsulation process, although it became turbid after the encapsulation process. Tis provided evidence of the formation of particles that infuenced the turbidity of the solution. After the drying process, the hydrogels exhibited a shape similar to that of white cotton. Te particle sizes and color values of the hydrogels will be explained.
Te sizes of the hydrogels encapsulating L. acidophilus were found to be in the range of 0.7 μm to 9 μm, with most having a diameter of 2 μm to 3 μm (Table 2). Tose hydrogels determined to be <100 μm in diameter were classifed as microgels. Te cell concentration signifcantly infuenced the hydrogels' particle size (p < 0.05). In fact, the more cells encapsulated within a given hydrogel, the greater its diameter. Te particle size was also correlated with the encapsulation efciency (Table 1), as more cores could be trapped within larger hydrogel particles. Te other factors found to infuence the particle sizes were the concentration and viscosity of the solution [8,12].
Te polydispersity indexes of the hydrogel-encapsulated cells were all >1 (Table 2), indicating the broad distribution of particles of various sizes. Overall, the index began to change when the initial cell number was 10 log CFU/mL. Moreover, the greater the initial cell number, the higher the polydispersity index. Tis result contrasts with the result of a previous study that found the glucomannan concentration to not infuence the polydispersity index [8].
Te hydrogels' zeta potentials became more electropositive as the cell number increased from 8 to 9 log CFU/ mL but then decreased as the cell number reached 10 log CFU/mL (Table 2). An increase in the number of cells should result in a reduction in the hydrogel's charge due to the positive charge of empty hydrogels and the negative charge of cells [8], including L. acidophilus [20]. Te observed pattern might stem from the zeta potential being measured on the hydrogel's surface, meaning that it could have been afected by the pH of the surrounding environment [21].
Te L * , b * , and whiteness values of the hydrogels increased after the addition of cells, whereas the a * value decreased (Table 3). Te utilized instrument determined these values based on the refection by the cells of a direct light beam from a chroma meter. Terefore, the more cells encapsulated within the hydrogel, the greater the refection. Bacteria may also generate distinct shades of colors such as red. Based on the fndings of a prior study, Lactobacillus pluvialis could refect an orange color from the pigment of canthaxanthin [22]. Tis fnding is in agreement with the present result, especially in terms of the increase in the b * value following the addition of L. acidophilus.
Te XRD spectra represent the interaction between the difraction intensity and the angle (Figure 2). Moreover, a crystalline state is indicated by the sharp difraction peak, whereas an amorphous and solid state is indicated by the declivous peak [2]. Te X-ray difractogram patterns of all the hydrogels showed a very broad band at 2θ between 5°and 90°. In addition, all the hydrogels exhibited nearly identical highest peaks at around 2θ 7.06°-10.46°, 7.62°-11.00°, 7.48°-10.94°, and 7.16°-11.20°for those hydrogels without cells and with cells at numbers of 8 log CFU/mL, 9 log CFU/ mL, and 10 log CFU/mL, respectively. Tese results difer from those concerning porang glucomannan, which exhibited its highest peaks at around 19°-20°and 35° [2]. However, there was a small peak in all the samples at around 2θ 10.5°, indicating the presence of chitosan [23]. Tis observation suggests that the mixture of glucomannan hydrogel and cells strengthened the associated chemical interaction, which is consistent with previous Fouriertransform infrared spectroscopy (FTIR) fndings [8]. It also suggests that some chitosan did not interact with glucomannan. A prior study reported that Schif's crosslinking between glucomannan aldehyde groups and chitosan amino groups could suppress the chitosan's crystalline state, which is usually strengthened by the hydrogen bond    between the amino and hydroxyl groups [23]. We also found evidence of low crystallinity, with values of 26%, 25%, 17%, and 21% being determined for the hydrogels without cells and with cells at numbers of 8 log CFU/mL, 9 log CFU/mL, and 10 log CFU/mL, respectively. Te addition of L. acidophilus appeared to have no efect on the difraction peak, indicating that the entrapment of microbes within the hydrogel did not afect the interaction between glucomannan and chitosan.

Prebiotic Activity of the Hydrogels. Te L. acidophilus
and E. coli cell density increased during 0 h, 24 h, and 48 h of incubation in the presence of carbohydrates, glucose, inulin, and hydrogel (Table 4). Both bacteria showed no signifcant increase in almost all the carbohydrates, except for L. acidophilus with inulin and E. coli with glucose. Tese data suggest that only inulin is able to specifcally stimulate the growth of good bacteria and suppress the growth of enteric bacteria, which is consistent with its widespread use as a commercial prebiotic. Te prebiotic potential of the hydrogel was compared with that of inulin on the basis of the prebiotic activity scores (Figure 3). Te prebiotic activity score of the hydrogel was higher than that of inulin after 24 h of incubation, although it was reduced after 48 h, suggesting that the hydrogel was the preferred energy source for the cells. Tis result is consistent with the XRD fndings, which confrmed the hydrogel to have an amorphous state and no long-range order, making it easier to digest. Moreover, the amount of carbohydrates will decrease with time. By contrast, the known prebiotic inulin [24] required a longer time to be available for the bacteria due to its long polymeric carbon chains-that is, chains of around 2-60 molecules [25].

Cell Survival during Exposure to Simulated
Gastrointestinal Conditions 3.4.1. Cell Survival during Exposure to Gastric Juice. Te L. acidophilus showed good viability during exposure to gastric juice at pH 2, whether in its free form or when encapsulated in hydrogel ( Figure 4). Generally, the growth of lactic acid bacteria is optimum at pH 6-7 (close to neutral pH). Some metabolic reactions change when the pH is <5 or <4.4. Indeed, some minerals will be lost at pH ≤2, while prolonged storage at a low pH will increase the risk of cell death [26]. Our results in this regard are consistent with those of previous studies [3,12]. Furthermore, studies are required to determine the efect of solid or solid-enriched macronutrient foods with a longer transit time [27]. In addition, a shorter exposure time within the stomach enables cells to maintain homeostasis between the internal and external pH, which potentially infuenced the good viability found in this study. Te present study also found that porang glucomannanchitosan hydrogel might exhibit a similar ability to protect cells from the gastric environment as both konjac glucomannan-chitosan hydrogel and calcium-alginate hydrogel (p > 0.05). Tis fnding accords with the ability of alginate to protect L. plantarum [17] and Lactobacillus rhamnosus from this harsh environment over the course of 3 h of exposure [28].
Te hydrogel was stable in the simulated gastric juice throughout 120 min of exposure ( Figure 5), which is consistent with the result of a previous swelling ratio study [8] that determined the hydrogel to not deswell at a pH <5. Deswelling causes the hydrogel to become smaller, which was previously thought to result in the release of cells from the hydrogel. However, the cells are still trapped in the hydrogel ( Figure 5), which perhaps refects the stronger electrostatic interaction between the glucomannan carbonyl   group and chitosan amine group in an acid environment [8].
Te cells remain in the hydrogel because this interaction maintains the core. Tus, deswelling could not be maximized, leading to only a small number of cells being released from the hydrogel. It is possible that some empty hydrogels will shrink to the extent that they are no longer visible after 60 min of exposure. Tese results are consistent with those of other studies using hydrogels made from oxidized glucomannan and chitosan to trap diclofenac drugs, which found <1% of cells to be released during exposure to simulated gastric fuid at pH 1.2 [5]. Tis shows that the hydrogel cores were not released when the hydrogel was exposed to low pH conditions.

Cell Survival during Exposure to Intestinal Juice.
Te viability of the free cells decreased signifcantly during exposure to intestinal juice for 60 min ( Figure 6; p < 0.05). Yet, the viability of the cells encapsulated in the hydrogel was maintained over 120 min of exposure, indicating that the encapsulation increased the viability of the L. acidophilus. A decrease in the number of free cells may refect cell death, which can be caused by factors other than the pH of the medium. Priya et al. [20] reported that while bacteria showed good growth at pH 6.8, the presence of pancreatin (comprising amylase, trypsin, lipase, ribonuclease, and protease) damaged the encapsulation wall, thereby resulting in cell death. Figure 6 indicates that the porang glucomannan hydrogel exhibited the same level of good protective efect as the konjac-chitosan glucomannan and calcium-alginate hydrogels. In this study, the alginate-based hydrogel was used for the purpose of comparison because it is widely used as an encapsulant due to its low price, good biocompatibility, and nontoxicity. A prior study found that the probiotic encapsulation of alginate increased the viability of the trapped cells when compared with the free cells during exposure to a simulated gastrointestinal condition [3]. Terefore, the porang-chitosan glucomannan hydrogel shows potential as a bacterial encapsulant.
Te hydrogel's microscopic appearance was used to confrm the cell viability data. Here, the porang glucomannan-chitosan hydrogel remained stable for up to 2 h in the intestinal fuid. However, it was found to be larger after 60 min of exposure than after 0 min (Figure 7), potentially refecting its swelling behavior at pH 6.8. We have previously shown that porang glucomannan-chitosan hydrogel begins to swell at pH >5 [8]. Te swelling of the hydrogel was evident until it reached 90 min of exposure. Moreover, many small hydrogels and cells were visible in the solution after 120 min of exposure. Te swelling weakened the interaction of the hydrogels, leading to some parts being dissolved, which resulted in both smaller hydrogels and the release of cells from the hydrogels. Tis result is consistent with that of another study that found konjac glucomannancarboxymethyl chitosan hydrogel with a bovine serum albumin core to show greater core release at pH 7.4 than at pH 5 due to the swelling enlarging its pores [4]. Tis core release also occurred when a chitosan-oxidized glucomannan hydrogel was exposed to simulated intestinal fuid for 2-8 h [5].

Conclusions
Te encapsulation of L. acidophilus in hydrogel made from glucomannan and chitosan was improved by varying the number of cells added. In fact, higher numbers were found   to be associated with greater encapsulation efciency, diameter (2-3 mm), polydispersity index (1.23-1.65), positive zeta potential, whiteness, and brightness. In addition, the hydrogel exhibited potential as a prebiotic, particularly after 24 h of incubation. Moreover, the hydrogel protected the encapsulated cells, maintaining them during exposure to simulated gastrointestinal fuid. Furthermore, the cell viability increased from 86% to 100% when the hydrogel was exposed to intestinal juice, which was comparable to the performance of the alginate and konjac glucomannan hydrogels. Furthermore, animal studies are required to determine the cell viability in actual gastrointestinal conditions and assess the health efects of the hydrogel.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.