Mechanism of co-encapsulating highland barley β-glucan to improve the gastrointestinal bioavailability of probiotics

Abstract

Probiotics have shown considerable health benefits, especially concerning gastrointestinal health; however, environmental stress during production and transit through the gastrointestinal tract adversely impact their viability. This study aimed to explore the protective effects of co-encapsulating highland barley β-glucan (HBBG) with probiotics on cell bioavailability during gastrointestinal digestion. The incorporation of HBBG (0.5% w/w) significantly improved probiotic viability and enhanced the microcapsule morphology during spray drying. Both in vivo and in vitro gastrointestinal digestion assays demonstrated superior probiotic survival rates (>8.5 Log CFU per g) within HBBG-containing microcapsule samples. Furthermore, the production of short-chain fatty acids, particularly acetic acid, reached 9.22 mM and the growth of beneficial gut microbiota (e.g., Lactobacillus and Bifidobacterium) was enhanced significantly. The findings highlighted the prominent prebiotic potential of HBBG and its synergistic role in improving probiotic stability and functionality during gastrointestinal digestion.

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1 Introduction

According to FAO (2006), probiotics are live microorganisms that boost the health of the host when given in sufficient amounts, such as treating intestinal inflammation, improving immunity, preventing obesity, etc.^1–6 To exert the abovementioned health effects, a minimum number of 10⁶–10⁷ CFU g⁻¹ viable bacteria in the product when consumed should be guaranteed.^7,8 However, probiotics are generally susceptible to environmental stressors, such as low pH, high osmotic pressure, water activity, etc., resulting in the loss of viability during production, storage, and passage through the gastrointestinal tract.^5,9,10 Microencapsulation technology is thus devised to overcome such limitations, and it entraps probiotics within a protective matrix to form an effective physical barrier that safeguards the probiotics from detrimental conditions, preserving sufficient viable cells to deliver their anticipated beneficial health effects.^11–14

Recent studies have demonstrated that the co-encapsulation of prebiotics with probiotics serves as an innovative method to enhance the oral delivery of viable probiotics, facilitating their targeted release in the host intestine.¹⁵ Prebiotics are non-digestible carbohydrates, such as oligosaccharides and polysaccharides, and selectively promote the growth and activity of specific probiotics. This enhancement contributes to host health by intervening in inflammatory bowel diseases, diabetes, constipation, various types of cancers, and immune diseases.¹⁶ After co-encapsulating prebiotics with probiotics, synergetic interactions between these two active substances occur, wherein prebiotics serve as substrates for probiotics and provide nutritional raw materials, improve their viability in the colon, and accordingly, enhance their biological effects in the large intestine.¹⁷ For example, Zheng et al. co-encapsulated Lactiplantibacillus plantarum M5 and 3% Goji Berry extract in alginate beads and found that co-microcapsule Goji Berry extract and probiotics can greatly increase the survival of probiotics (remaining at 7.17 Log CFU per g) during gastrointestinal digestion.¹⁸ Vázquez-Maldonado et al. co-encapsulated Bacillus clausii and resveratrol in an inulin and lactose matrix and also found that the probiotic activity increased by 0.64 Log CFU per g in inulin microcapsules compared to that without prebiotics during gastrointestinal digestion.¹⁹ Currently, prebiotics that are suitable for co-encapsulation with probiotics are primarily restricted to inulin, fructooligosaccharides, galactooligosaccharides (GOS), isomaltooligosaccharides, and xylooligosaccharides, which cannot meet the increasing requirements of precisely controlled release and targeted therapy of probiotics.^20–22 Consequently, the search for innovative prebiotics to augment probiotic resilience against gastrointestinal digestion and to attain effective targeted release has emerged as a primary objective in the field of probiotic microencapsulation.

β-Glucan is a natural polysaccharide made up of D-glucose units connected by β-glycosidic bonds, predominantly located in yeast, fungi, and cereals.^23,24 It exhibits various biological effects, such as anti-inflammatory properties, modulation of the gut microbiota, and immunomodulatory functions.^25–27 Bai et al. employed an in vitro fermentation model to investigate the prebiotic potential of oat β-glucan and found that the gut microbiota could effectively utilize β-glucan for short-chain fatty acid (SCFA) production, concurrently with a significant increase in Lactobacillus abundance and a notable decrease in Escherichia–Shigella levels.²⁸ Furthermore, Aoe et al. used animal models and demonstrated that the ingestion of low-molecular-weight β-glucans can enhance the abundance of Bifidobacterium and Bacteroides while simultaneously augmenting the production of SCFAs, notably acetate and butyrate.²⁹ However, due to the poor solubility of β-glucan, it is difficult to directly utilize it as a wall material for probiotic encapsulation. Therefore, microencapsulation technology must be employed to achieve co-encapsulation of prebiotics with probiotics.³⁰ Few studies have reported that the incorporation of β-glucan into probiotic microcapsules could effectively improve the gastric digestion properties of probiotic bacteria.³¹ For example, Loyeau et al. added β-glucan with whey proteins (WPI) to encapsulate Bifidobacterium lactis subsp. lactis INL1 and found that the addition of β-glucan significantly enhanced the survival rates of probiotics during simulated gastrointestinal digestion.³¹ Analogously, Yuan et al. utilized WPI and β-glucan to encapsulate Lactiplantibacillus plantarum 550 and found a significant improvement in the ability of L. plantarum 550 to withstand the gastrointestinal digestion environment.³² Nevertheless, current studies remain primarily focused on investigating probiotic survival rates in the gastrointestinal tract, while the mechanistic basis and practical in vivo activity alterations remain largely unexplored, highlighting the requirement for further comprehensive investigations.

Highland barley is widely planted in the alpine agricultural areas of northwest and southwest China, and its main active ingredient is glucan that possesses contents ranging from 3.66% to 8.62%, which is much higher than those in wheat and oats.³³ The highland barley β-glucan (HBBG) molecule is a linear homopolymer composed of β-(1 → 3) and (1 → 4)-D-glycosidic linkages, characterized by the presence of two or three consecutive β-(1 → 4) linkages that are interrupted by a single β-(1 → 3) linkage.³⁴ In our previous research, high-purity β-glucan was extracted from highland barley (HBBG) utilizing the hot water extraction method.³² Thus, the aim of this study was to, firstly, co-encapsulate HBBG with probiotics in a soybean protein isolate (SPI)–pectin (PEC) matrix and evaluate its impact on probiotic microcapsule morphology and viability during spray drying; secondly, explore the protective effects of co-encapsulated HBBG on the cell bioavailability during in vivo and in vitro gastrointestinal digestion; lastly, investigate the effect of co-microencapsulation of prebiotics and probiotics on the generation of SCFAs and growth of beneficial gut microbiota.

2 Materials and methods

2.1 Materials

Sichuan Gaofuji Biological Technology Co., Ltd (Chengdu, China) supplied L. plantarum 550, which was originally obtained from pickle samples sourced in Sichuang. Bile salts were obtained from Macklin (Shanghai, China). The SPI, PEC, pepsin (3000 U mg⁻¹), trypsin (250 U mg⁻¹), MRS broth and MRS agar were sourced from Solarbio (Beijing, China). HBBG (M_w = 5.64 × 10⁵ Da, β-glucan content = 79.05 ± 4.18%) was obtained from our previous study.³² Potassium dihydrogen phosphate (KH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), sodium chloride (NaCl), and other reagents were from Aladdin (Shanghai, China).

2.2 Preparation of the probiotic suspension

MRS broth was utilized to inoculate the appropriate quantity of L. plantarum 550 powder, and the culture was performed twice. Following the addition of 1% activated bacterial solution to MRS broth, the mixture was incubated at 37 °C under a constant relative humidity of 80% for 20 h. Subsequently, the bacterial suspension was prepared for future use by washing twice with sterile normal saline.

2.3 Microencapsulation of probiotic cells

Probiotic microcapsules were prepared based on the condensation reaction between SPI and PEC, along with the gelation of HBBG. Briefly, in a 100 mL liquid system, 2 g of PEC and 10 g of SPI were hydrated overnight to formulate the microcapsule shell solution. The HBBG solution with mass fractions of 0%, 0.1%, 0.5% and 1.0% were prepared. Subsequently, a 5% probiotic suspension (10¹⁰ CFU mL⁻¹) was added to the HBBG solution to formulate the microcapsule core layer solution. Finally, the shell layer solution of the microcapsule and the core layer solution were combined in a 1 : 1 (v/v) ratio and stirred, and the pH of the system was adjusted to 4 with 5% citric acid solution to facilitate the condensation of PEC and SPI. Consequently, probiotics microcapsules of SLP, S-L-0.1%H-P, S-L-0.5%H-P, and S-L-1.0%H-P with varying formulations were obtained.

2.4 Viability of L. plantarum 550 after spray drying

The final solutions were spray-dried utilizing a laboratory-scale spray dryer (B-290, BUCHI Ltd, Switzerland). According to Yuan et al., the inlet temperature is regulated at 135 ± 5 °C, whilst the outlet temperature is maintained at 70 ± 5 °C.³² Samples of dried powder were collected from the base of the cyclone and stored at 4 °C in tightly sealed, sterile containers. With minor adjustments, entrapped bacteria were liberated from the microcapsule according to Zhou et al.³⁵ Briefly, 0.1 g of microcapsules were subjected to high-speed shearing at 4000 rpm for 10 minutes after re-suspension in 19.9 mL of phosphate-buffered saline (PBS) (0.2 M, pH 7.2). The samples were subsequently diluted to 10^–4via the serial dilution method, and L. plantarum 550 was counted using the pure plate method with MRS agar and incubated for 48 hours at 37 °C. Using the provided equation, the survival rate following spray drying was determined:

SR = (N/N₀) × 100% (1)

SR: the survival rate, expressed in percentage, N: the number of cells released from the microparticles after spray drying (Log CFU per g), and N₀: the number of free cells before spray drying (Log CFU per g).

2.5 Morphology and particle size of probiotic microcapsules

Scanning electron microscopy (SEM) (SU8010, Hitachi, Japan) was employed to observe and analyze the morphology of probiotic microcapsules. An appropriate amount of probiotic-containing spritzed microcapsule was applied on the conductive silica gel, followed by gold plating using a vacuum coating apparatus. The morphologies of the gold-plated samples were analyzed at magnifications of 500×, 2k×, and 5k× utilizing a cold field emission SEM with an electron beam acceleration voltage of 5 kV. The particle sizes of SLP and SLHP were determined via dynamic light scattering utilizing a NanoBrook 90 Plus Zeta instrument (Brookhaven Instrument Corp., New York, USA).

2.6 Fourier transform infrared spectroscopy (FT-IR) analysis

Using Fourier transform infrared spectroscopy (Nicolet 5700, Thermo Fisher Scientific, MA, US), the chemical structure of probiotic microcapsules was examined. After being thoroughly crushed and compressed into KBr pellets, the spray-dried samples were combined with KBr at a mass ratio of 1 : 100. To obtain the spectral signals with a wavenumber range of 400–4000 cm⁻¹ with an average of 32 scans at a resolution of 1 cm⁻¹, KBr discs holding the sample were loaded.

2.7 In vitro simulation of gastrointestinal digestion

The method outlined by Liu et al. was adjusted marginally for in vitro digestion experiments.⁸ The pH was adjusted to 2.0, and simulated gastric fluid (SGF) was prepared using 0.32% (w/w) pepsin and 0.2% (w/w) NaCl. A PBS solution (0.2 M, pH = 7.2) was utilized to formulate simulated intestinal fluid (SIF), comprising 0.1% (w/w) pancreatin and 0.08% (w/w) bile salts. After being preheated for 10 minutes at 37 °C, the digestive fluid was filtered through a 0.22 μm membrane to ensure sterilisation. After that, sterilisation tubes containing 9.9 mL of freshly prepared and filter-sterilized SGF were filled with either 0.1 g of microparticles or 0.1 mL of free cells. Following vortexing, the samples were incubated for two hours at 37 °C. After performing a gradient dilution with PBS and plating an MRS plate for bacterial enumeration, a 0.1 mL aliquot was extracted from each sample at specified time intervals (0.5, 1, 1.5, and 2 hours). After stomach digestion, the remaining SGF was transferred to SIF, and cell counts were determined at intervals of 3, 4, 5, and 6 hours, as previously described.

2.8 In vivo gastrointestinal digestion using the mouse model

2.8.1 Animal experiment design. Male C57BL/6J mice were randomly assigned to the negative control (NC) group, free cell group (FC), SLP group, and SLHP group (HBBG solution with a mass fraction of 0.5%) following a week of adaptive feeding. Mice in the free cell group were given 0.2 mL of unembedded bacterial suspension (5 × 10⁹ CFU kg⁻¹ d⁻¹) every day, SLP and S-L-0.5%H-P groups were given the same amount of embedded probiotic suspension (5 × 10⁹ CFU kg⁻¹ d⁻¹) every day, and the NC group was gavaged with the same amount of normal saline every day. All groups of mice were gavaged continuously for 14 days. During gavage, feces were collected for viable counts. The mice were euthanized at the end of the fourteenth day of gavage, and samples of their gastrointestinal contents were taken and stored at −80 °C. All animal experiments were approved by the Animal Care and Use Committee of Nanchang University (Approval number: SYXK (Gan) 2021-0004).

2.8.2 Bacterial viability assay. Following the modified method of Liu et al. mouse feces were collected to investigate the survival of probiotic microcapsules after traversing the gastrointestinal tract of mice.³⁶ 100 mg of mouse feces was dissolved in 900 μL of sterile saline and then homogenized, and the supernatant was collected after centrifugation for viable bacteria enumeration as described above.

2.8.3 Determination of SCFAs. A gas chromatography method was used to determine the SCFAs, according to a prior study.^37–39 Briefly, fecal material (100 mg) was diluted in 0.9 mL of saline to obtain a homogenate, and 0.5 mL diethyl ether was added to it. After centrifugation at 12000 rpm for 5 minutes, a 0.22 μm membrane was used to filter the organic phase. Gas chromatography (Agilent Technology 7890A, USA) was used to synthesize and determine concentrations of various SCFAs in organic fractions.

2.8.4 Intestinal microbiota analysis by 16S rRNA. Four cecal feces samples per group were selected randomly for microbiota 16S rRNA analysis. Microbial genomic DNA was extracted from the samples in accordance with the instructions of the manufacturer, utilizing a TIANamp DNA stool kit (TIANGEN, Beijing, China). Agarose gel electrophoresis confirmed the successful isolation of DNA, and the V3–V4 region of the bacterial 16S rRNA genes was amplified via PCR using the universal primer pairs. PCR amplicons were purified with Vazyme VAHTSTM DNA Clean Beads (Vazyme, Nanjing, China) and quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA), and pair-end sequencing was performed using the Illumina Nova Seq platform. The pretreated samples were submitted to Shanghai Personal Biotechnology Co., Ltd for testing. The operational taxonomic unit (OTU) clustering analysis was subsequently conducted using the valid data, encompassing OTU sequences, analysis, species annotation, and both alpha and beta diversity metrics.

2.9 Statistical analysis

Every measurement was performed three times. Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by the post hoc Tukey's multiple comparison test, which was presented as mean ± standard deviation. When *p < 0.05, differences were deemed significant.

3 Results and discussion

3.1 Physicochemical characteristics of probiotic microcapsules after spray drying

As shown in Table 1, the viability of microencapsulated probiotics with various formulations varied considerably during spray drying. Free probiotic cells lost all viability after spray drying, while the survival rate of probiotics encapsulated in the S-L-P matrix significantly increased to 38.03 ± 1.70%, which may be due to the physical barrier effect of microencapsulation that slowed down the heat stress of probiotics during spray drying. The survival rate of the S-L-0.1%H-P sample was similar to that in S-L-P, however, when the addition of the HBBG concentration increased to 0.5%, the survival rate of probiotics markedly increased to 57.23 ± 2.39%, which can be due to the excellent film-forming properties of HBBG that formed a dense glass state shell and ultimately enhanced the resistance of probiotics to heat shock during spray drying. Polysaccharides with pronounced film-forming properties can immobilize water molecules to generate a three-dimensional gel network, which restricts the permeation of intracellular components and enhances cell membrane stability, thereby shielding probiotics from spray-drying-induced adverse stresses.⁴⁰ Furthermore, according to the water substitution hypothesis,⁴¹ the hydroxyl moieties of HBBG may competitively displace water molecules and form intermolecular interactions with the polar head groups of phospholipids in the bacterial cell membrane, thus preserving the membrane structural integrity of probiotics.^42–44 A similar phenomenon was found by Arepally and Goswami, who encapsulated Lactobacillus acidophilus NCDC 016 in whey isolate-gum arabic-maltodextrin matrix microcapsules and found that maltodextrin was able to partially replace water molecules to interact with the cell membrane and alleviate the damage of probiotic bacteria.⁴² Nonetheless, when the concentration of HBBG was elevated to 1%, the survival rate of probiotic bacteria diminished to 36.45 ± 2.57%, which may be primarily due to the substantial increase in the viscosity of the feed solution that impaired the interaction between the wall materials and resulted in a reduced embedding effect. Furthermore, the elevated viscosity of the feed solution adversely affected spray-drying efficiency, as it promoted particle adhesion to the inner surface of the cyclone separator chamber and prolonged the exposure to high temperature conditions, inducing cellular damage and viability losses of encapsulated probiotics.^32,45 The moisture contents of probiotic microcapsules produced by different recipes during spray drying are presented in Table 1. The moisture content of S-L-P microcapsules was determined to be 7.78 ± 0.14%. After incorporation of HBBG, the moisture content of microcapsules decreased as the concentration of HBBG increased, which can be ascribed to the presence of water-insoluble β-(1 → 4) glycosidic bonds within the HBBG molecular chains, leading to stronger hydrophobicity of microcapsules.^46,47 Besides, the elevated viscosity of HBBG solution also prolonged the duration of exposure to high temperature conditions during spray drying, inducing further moisture losses of probiotic microcapsules.⁴⁸

Table 1 The survival rates and moisture contents of probiotic microcapsules after spray drying

Samples	Survival rates (%)	Moisture contents (%)
Values are expressed as the mean ± standard deviation (n = 3). Columns with different superscripts (a–c) are significantly different (p < 0.05).
F-C	0.00 ± 0.00^c	—
S-L-P	38.03 ± 1.70^b	7.78 ± 0.14^a
S-L-0.1%H-P	37.17 ± 1.75^b	7.53 ± 0.04^a
S-L-0.5%H-P	57.23 ± 2.39^a	7.18 ± 0.02^b
S-L-1.0%H-P	36.45 ± 2.57^b	6.74 ± 0.05^c

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3.2 Morphological characteristics of probiotic microcapsules

The SEM images of different probiotic microcapsules are presented in Fig. 1a. For the SLP sample, the microcapsules tended to shrink and fracture, with some irregular-shaped particles and pores existing on the surface. When HBBG was added, the morphology of microcapsules became smoother with a low degree of wrinkles and correspondingly, the number of tiny pieces gradually reduced. According to Yuan et al., polysaccharides had a positive effect on improving the morphology of protein-based microcapsules and eventually provided better protection for probiotics.³² With the elevation of the HBBG concentration, the degree of surface wrinkles of microcapsules decreased, accompanied by improved sphericity, which could be due to the gelation and film-forming properties of HBBG that mitigate microcapsule folding and shrinkage resulting from dehydration during spray drying. Similarly, Mao et al. incorporated Bifidobacterium longum into the SPI–carrageenan complex coacervates and observed that increasing carrageenan concentration not only enhanced the structural compactness of the microcapsules but also promoted deeper embedding of bacterial cells within the coacervate matrix.⁴⁹ Following the incorporation of 1% HBBG into the SPI-PEC matrix, the morphological characteristics of the microcapsules were largely consistent, displaying particle agglomeration, which can be attributed to the increased viscosity that facilitates interparticle adhesion.⁵⁰ In addition, through FT-IR analysis, after the addition of HBBG, the O–H stretching vibration peak of SLP shifted from 3302 cm⁻¹ to 3403 cm⁻¹, indicating that the addition of HBBG led to the formation of hydrogen bonds in the agglomerate, especially for the hydroxyl groups (Fig. 1b).⁵¹ Liu et al. found that the structural integrity of microcapsules exerted a significant influence on probiotic protection efficacy, as microcapsules exhibiting uniform morphology and intact architecture demonstrated superior barrier properties, effectively shielding probiotics from environmental stressors.³⁶ Yan et al. also illustrated that microcapsules with a regular shape and compact structure prevented the penetration of digestive enzymes and gastric acid, thus improving the survival rate of embedded probiotics during digestion.⁵²

Fig. 1 Morphology and structural characteristics of probiotic microcapsules: (a) scanning electron microscopy (SEM), (b) Fourier transform infrared (FTIR) spectroscopy, and (c) particle size distribution.

The particle size distributions of different probiotic microcapsules are shown in Fig. 1c. All the samples exhibited a unimodal distribution, indicating a homogeneous particle distribution with sizes ranging from 140 to 160 μm. Besides, with the addition of HBBG, the particle size distribution peaks generally exhibited a pronounced leftward shift, signifying the reduction in particle sizes, which can be attributed to the increased density of intermolecular cross-linking of molecular chains that resulted in a more compact structure (Fig. 1a).⁵³

3.3 In vitro gastrointestinal digestion of probiotic microcapsules

The survival rates of microencapsulated probiotics under in vitro gastrointestinal conditions are presented in Fig. 2a. After one hour of simulated gastric digestion, no viable bacteria were detected in the FC group, which can be attributed to the high H⁺ concentration that induced denaturation of cell membrane proteins, breakage of the cell structure, and increase in cell permeability, facilitating the penetration of digestive juices into the interior of cells that eventually caused the death of probiotics.¹³ High H⁺ levels would result in a decrease in cytoplasmic pH within probiotics, inhibiting glycolytic enzyme activity and subsequently affecting the function of the F1F2-ATPase proton pump, which impedes normal cellular metabolism and ultimately leads to the death of cells.⁵ For the SLP sample, after 6 h of gastrointestinal digestion, the survival number increased to 7 Log CFU per g (still above the WHO recommended value of 6 Log CFU per g).⁵⁴ This significant improvement in viability can be attributed to two factors: on the one hand, the enhanced structural density and physical barrier effects of microcapsules, and on the other hand, SPI possesses the pH buffering capacity under H⁺ conditions that prevents the penetration of digestive fluids into the cell cytoplasm and alleviates the denaturation of biological macromolecules such as proteins and nucleic acids.^55–57 Besides, for the SLP sample, during the entire digestion stage, the loss of probiotic viability mainly occurred in the gastric digestion stage (decreased from 100 ± 3.01% to 13.47 ± 0.95% within 2 h). As the gastric pH (2.0) was lower than the pK_a (4.6) of pectin, the ionization of carboxyl groups in pectin was constrained, which diminished the ionic interaction between SPI and PEC molecules and reduced the physical barrier effect of microcapsules to probiotics.^32,58,59 Under SIF conditions, the pH (7.2) was well above the pK_a (4.6) of pectin and the isoelectric point of SPI, thus both biopolymers exhibited net negative charges and resulted in the tendency of mutual repulsion.⁵⁸ As SIF digestion continued, the SLP microcapsules tended to dissolve and expose the probiotics to bile salts, which possessed antibacterial properties and served as detergents and DNA-damaging agents that disrupted the cell membranes of probiotics, leading to a further decrease in probiotic survival from 13.47 ± 0.95% to 1.13 ± 0.19%.⁶⁰

Fig. 2 The survival rates of free and encapsulated cells during in vitro gastrointestinal digestion.

After incorporation with HBBG, the survival rates of probiotics increased significantly, meanwhile, as the concentration of HBBG increased from 0.1% to 0.5%, the viability of cells after digestion increased from 4.34 ± 0.17% to 40 ± 3.3%. This continuous improvement in probiotic viabilities can be attributed to two factors: on the one hand, the resistance of HBBG to digestive enzymatic hydrolysis, which well preserved the rigid conformation of microcapsules, effectively inhibiting the penetration of digestive juices and providing substantial protection,^53,61 on the other hand, as potential prebiotics, HBBG can be degraded and metabolized by cells to generate energy and maintain the viability of probiotics during gastrointestinal digestion.⁶² Atia et al. co-encapsulated 5% inulin with probiotics in sodium alginate microbeads and found that this prebiotic not only improved the survival of Lactobacillus salivarius during gastrointestinal digestion but also facilitated the adhesion and colonization of probiotics on the intestinal mucosa.²⁰ A similar phenomenon was also reported by Krasaekoopt and Watcharapoka, who found that the gastrointestinal survival rate of microencapsulated probiotics containing GOS was 1000 times higher than that of probiotic microcapsules without GOS.²¹ However, when the addition of HBBG was increased to 1.0%, the survival rate of probiotics decreased significantly, which was mainly attributed to the high concentration of HBBG that led to a sharp increase in the viscosity of the solution and prolonged the exposure time to high temperature conditions accompanied by increased cellular damage (Table 1) and ultimately decreased the resistance of probiotics to gastric acid digestion. In addition, the excessive presence of high-molecular-weight HBBG chains created a large excluded volume, physically interfering with the electrostatic cross-linking between SPI and pectin.⁶³ This interference prevented the formation of a dense and continuous encapsulation network, resulting in a more porous shell structure that failed to effectively shield the probiotics from lethal thermal exposure during spray drying.

3.4 In vivo gastrointestinal digestion of probiotic microcapsules

The cell viabilities in mouse feces post-gavage at different time intervals are shown in Fig. 3. The free cells passed through the whole gastrointestinal tract within 2 h, and with the increase in digestion time, the detected viable cells decreased gradually (Fig. 3b). After encapsulation in SLP microcapsules, a significant increase was observed at all collection times, which correlated well with the result of in vitro gastrointestinal digestion (Fig. 2), confirming the effective protective role of the SLP microcapsule matrix. Besides, the peak concentration occurred 2 h later than in the FC sample, which could be attributed to the additional degradation and release of probiotics from SLP microcapsules (Fig. 3c). After incorporation of HBBG, the concentration of viable cells further increased as compared with that in SLP samples, which could be attributed to the prebiotic effects of HBBG that provided extra energy for probiotics to resist the harsh gastrointestinal conditions (Fig. 3d).

Fig. 3 The viabilities of free and encapsulated cells during in vivo gastrointestinal digestion: (a) experimental scheme of sample collection, (b–d) cell viabilities in feces post-gavage (0–10 h) of FC, SLP and S-L-0.5%H-P samples respectively, and (e) cell viabilities in feces post-gavage (0–10 d) of free and encapsulated cells.

Fig. 3e illustrates the survival rates of free and encapsulated probiotics as they moved through the gastrointestinal tract of mice at different gavage days. As expected, the free cell group showed the lowest survival among all gavage times, however, there were still more than 5.5 Log CFU per g viable probiotics detected in the feces, which can be attributed to the comparatively moderate digestive conditions, shorter residence of probiotics, and the presence of food residues in the mouse stomach that induced higher survival rates of free probiotics compared to that in in vitro digestion. After encapsulating probiotics in SLP microcapsules, the cell survival at different gavage times increased above 7.5 Log CFU per g, which can be primarily due to the physical barrier effect of microencapsulation and the effective pH buffering capacity of SPI that relieved the erosion from gastrointestinal contents in vivo. With the incorporation of HBBG, further increases in cell viability were found compared with that in SLP samples and the highest survival of 8.5 Log CFU per g was observed on the tenth day of gavage, which can be due to the prebiotic effects of HBBG that can be metabolized by probiotics and selectively promote the proliferation of cells.⁶⁴ Generally, the in vivo viabilities of microencapsulated cells showed a similar tendency as compared to that in in vitro digestion, which well confirmed the effectiveness of the wall material used in this study that possesses the ability to protect cells from harsh gastrointestinal conditions.

3.5 SCFA analysis

The degradation of carbohydrates is normally accompanied by the production of organic acids, such as acetic acid, propionic acid, and butyric acid, which are necessary for the health of the host. Acetic acid can be absorbed by the intestinal epithelium and provides energy to the heart, brain, and peripheral tissues.⁶⁵ Propionic acid exhibits anti-inflammatory and antibacterial activities, reduces liver cholesterol synthesis, enhances fat metabolism and provides protection against intestinal pathogens in humans.⁶⁶ Butyric acid plays a critical role as an energy substrate for colonocytes and is essential in regulating cellular differentiation and homeostasis.⁶⁷ Following uptake by free cells, a significant increase in butyric acid production was observed, whereas acetic acid and propionic acid levels remained unchanged. As a result, the total acid amount in the free cell sample increased significantly compared to that in the NC group, which can be ascribed to the fermentation of ingested probiotics that generated organic acids and reduced intestinal pH. Huang et al. investigated the effect of Lactobacillus plantarum PS128 on the structure of intestinal flora and found that supplementation with L. plantarum PS218 significantly increased acetic acid production as well as the abundance of Bifidobacterium and Akkermansia.⁶⁸ For SLP samples, in contrast to the marked increase in survival rates during gastrointestinal digestion (Fig. 2), no significant variation of organic acid generation was found between the SLP samples and the FC group. For the HBBG sample, the amounts of different organic acids were significantly higher than those in the NC sample, and among these, the concentration of propionic acid was significantly higher than that in FC and SLP samples. HBBG can facilitate the proliferation of propionate-producing bacteria during degradation and metabolism, hence enhancing the synthesis of propionic acid.⁶⁹ Simultaneously, the glucose produced by the degradation of HBBG is prone to produce propionic acid and butyric acid through the 6-phosphofructose–butyric acid and 6-phosphofructose–succinic acid–propionic acid pathways.⁶⁹ Carlson et al. also observed that after 12 hours of fermentation by human fecal microbiota, the propionate levels in the β-glucan group were considerably elevated compared to those in the xylooligosaccharide and inulin groups.⁷⁰ Inspiringly, after adding HBBG into SLP microcapsules, further significant increases in the amount of all organic acids were observed, accompanied by the remarkable rise in the amount of total acid generation, which can be attributed to the fact that after the disintegration of microcapsules in the colon, HBBG and encapsulated probiotics were released simultaneously, and due to the significant prebiotic effect of HBBG, the released probiotics could degrade and metabolize HBBG readily and spontaneously, which ultimately led to the high-efficiency generation of SCFAs (Fig. 4).

Fig. 4 SCFA production variations in HBBG and probiotic microcapsule samples: (a) experimental scheme of the SCFA detection process, (b) acetic acid, (c) propionic acid, (d) butyric acid, (e) total acid, and the (f) mechanism of SCFA production.

3.6 Gut microbiota analysis

To investigate the effects of probiotics (FC), HBBG, and their compound (SLHP) on the diversity and abundance of the gut microbiota in mice, 16S rRNA gene amplicon sequencing was performed on fecal samples from mice. The α-diversity of the gut microbiota was not significantly different among these five groups, as manifested by the Chao1 index or Shannon index (Fig. 5a and b). Subsequently, ANOSIM and NMDS analyses (Fig. 5c) were used to evaluate β-diversity and it was found that there was a significant (R = 0.3212, p = 0.001) difference in microbial communities among different groups. The SLHP group was located distant from the NC group, while the position of the FC group was closer to the NC group than to the SLHP group (Fig. 5c).

Fig. 5 Analysis of diversity and abundance of the gut microbiota in mice: (a and b) alpha diversity by the Chao1 index and Shannon index respectively, (c) NMDS analysis of beta-diversity based on the Bray–Curtis distance (Stress = 0.054). Significant differences in community structure were confirmed by ANOSIM (R = 0.3212, P < 0.01), (d) relative abundance of the gut microbiota at the phylum level, (e) heatmap of the gut microbiota at the genus level, (f–k) LDA scores between NC, FC, HBBG, SLP, and SLHP groups, and (l) intestinal flora function predication.

Comparative analysis of gut microbiota composition among each group was then conducted (Fig. 5d). At the phylum level, Bacteroidetes was the first major category in all samples, followed by Firmicutes and Proteobacteria. Interestingly, oral supplementation with FC, SLP, HBBG, and SLHP in normal mice all effectively suppressed the abundance of pathogenic Proteobacteria, accounting for 2.54%, 2.38%, 2.65% and 2.5%, respectively, demonstrating their beneficial roles in sustaining intestinal microbial homeostasis.⁷¹ At the genus level, each group presented a similar profile of gut microbiota composition, which was dominated by Lactobacillus, Oscillospira, and Odoribacter (Fig. 5e). These genera were recognized as beneficial bacteria with potential health-promoting effects, among which Lactobacillus, playing a key role in maintaining intestinal barrier integrity and modulating inflammation, was more abundant in the SLHP group.⁷²

The differentially abundant taxa within these groups were further revealed by linear discriminant analysis (LDA, LDA Score ≥3) and effect size (LEfSe, p < 0.05). Compared to the NC group, the FC group effectively inhibited the relative abundance of f_Helicobacteraceae (known as harmful bacteria, p < 0.05) and increased the relative abundance of beneficial bacteria, such as g_Bifidobacterium (known to alleviate intestinal inflammation, p < 0.05) and s_Parabacteroides_distasonis (known to be negatively correlated with obesity and metabolic dysfunctions, p < 0.01) (Fig. 5f and g).^73–75 In our results, for the HBBG group, the abundances of g_Lactobacillus, g_Bifidobacterium, and s_Parabacteroides_distasonis were significantly enriched, and all of these taxa were associated with SCFA metabolism, as SCFAs can be produced from the microbial fermentation of indigestible carbohydrates and were the key mediators of the beneficial effects elicited by the gut microbiota (Fig. 5h).⁷⁶ Among the bacteria that differed significantly between the NC and HBBG groups, g_Lactobacillus occupied the largest mean proportion (∼7%) (Fig. 5i). Parabacteroides_distasonis can metabolize polysaccharides to generate SCFAs, thereby reducing colonic pH and inhibiting pathogen growth.^24,77 Research conducted by Zhang et al. also indicated a rise in beneficial bacteria, including Lactobacillus and Parabacteroides in IBD mice following the administration of barley β-glucan.⁷⁸ The SLHP group further increased the relative abundance of s_Bifidobacterium_pseudolongum (can produce acetate and suppress hepatocellular carcinoma) and g_Lactobacillus, whereas the relative abundance of IBD-related s_Ruminocossus_gnavus was remarkably decreased (p < 0.01) (Fig. 5j and k).^78,79 These results indicated that HBBG possessed the ability to selectively promote the proliferation of beneficial bacteria and decrease the abundance of certain harmful bacteria, and co-microencapsulation of HBBG with probiotics can further increase the abundance of beneficial bacteria and improve the structure of intestinal flora.

To determine the functional variation of microbiota, we used Tax4Fun2 to predict the functions of KEGG pathways from 16S rRNA sequencing data. The results showed that the KEGG (level 2) pathways in the SLHP group were similar to those in the FC, SLP, and HBBG groups; however, they were higher than those in the NC group (Fig. 5l). Notably, the SLHP group exhibited several enhanced functions, such as aging, cancers, cardiovascular disease, etc. The intestinal microbiome was a proven immune system modulator, which played an important role in the development of cancers and may affect the effectiveness of anti-cancer therapy.⁸⁰ Herein, the enhanced functions related to cancer may be regulated by the superior improvement effect of SLHP intervention on the composition of the gut microbiota in mice, i.e., g_Bifidobacterium and g_Lactobacillus. The richness of Bifidobacterium spp. can improve the anti-tumor specific immunity by activating antigen-presenting cells and cytotoxic T cells within the tumor, and Lactobacillus intestinalis can facilitate tumor-derived CCL5 to recruit dendritic cells and suppress colorectal tumorigenesis.^81,82

4 Conclusion

This study demonstrated that HBBG can serve as an efficacious prebiotic substrate and a protective matrix for probiotic encapsulation. Co-encapsulation utilizing HBBG significantly improved probiotic viability under both spray-drying conditions and simulated gastrointestinal stress. Moreover, the HBBG-probiotic symbiotic system possessed the ability to concomitantly enhance the production of SCFAs, while exhibiting a pronounced bifidogenic effect, selectively enriching beneficial gut microbiota (Lactobacillus, Bifidobacterium, etc.). The developed co-encapsulation system presented considerable potential for probiotic applications in functional foods. Further optimization of this HBBG-based probiotic delivery platform may yield viable commercial strategies for next-generation nutraceuticals and bioactive food formulations.

Author contributions

Wei Liao: methodology, investigation, formal analysis, and writing – original draft. Ronghai Hu: formal analysis, data curation, software, and writing – review & editing. Huan Liu: conceptualization, methodology, writing – review & editing, project administration, and funding acquisition. Jielun Hu: formal analysis, writing – review & editing, project administration, and funding acquisition.

Conflicts of interest

There are no conflicts to declare.

Abbreviations

FT-IR	Fourier transform infrared
GOS	Galactooligosaccharides
HBBG	Highland barley β-glucan
OTU	Operational taxonomic unit
PBS	Phosphate-buffered saline
PEC	Pectin
SCFAs	Short-chain fatty acids
SEM	Scanning electron microscopy
SGF	Simulated gastric fluid
SIF	Simulated intestinal fluid
SPI	Soybean protein isolate
WPI	Whey proteins

Data availability

All data presented in this study are available on request from the corresponding authors.

Acknowledgements

This work was supported by the National Natural Science Fund for Excellent Young Scholars (32222065), the National Natural Science Foundation of China (32360581), the National Key Research and Development Program of China (2023YFF1103403 and 2024YFF1106002), the Key Research and Development Project of Jiangxi Province (20224BBF62002, 20244AFH82002, 20223BBF61023, and 20252ABF010002), the Natural Science Fund for Distinguished Young Scholars of Jiangxi Province (20252BAC220068), the Natural Science Foundation of Chongqing (CSTB2023NSCQ-MSX0976). Cultivation Program of Jiangxi Medicine Academy of Nutrition and Health Management.

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