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. 2023 May 22;97:106450. doi: 10.1016/j.ultsonch.2023.106450

Ultrasound-assisted multilayer Pickering emulsion fabricated by WPI-EGCG covalent conjugates for encapsulating probiotics in colon-targeted release

Xian He a, Wanshui Yang a,⁎, Xinsheng Qin a,b,⁎
PMCID: PMC10238641  PMID: 37224638

Graphical abstract

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Keywords: Ultrasound, Multilayer Pickering emulsion, Pasteurization and GIT passage viability, Probiotics, Colon-targeted release

Highlights

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    Ultrasonic homogenization on the W1/O/W2 double emulsions were studied.

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    Double emulsion particles were coated with chitosan, alginate, and CaCl2.

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    Encapsulated probiotic was more sensitive to heating treatment than GIT digestion.

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    Double emulsion droplets with 3 layers were available for probiotic encapsulation.

Abstract

This study demonstrated the influences of ultrasound-assisted multilayer Pickering double emulsion capsules on the pasteurization and gastrointestinal digestive viability of probiotic (L. plantarum) strain liquid. Firstly, the role of ultrasonic homogenization on the morphology of W1/O/W2 double emulsions were studied. The double emulsion formed by ultrasonic intensity at 285 W had a single and narrow distribution with smallest droplet size. The double emulsion particles were then coated with chitosan(Chi), alginate (Alg), and CaCl2(Ca). The multilayer emulsion after pasteurization and gastrointestinal digestion both had the highest viability at 5 coating layers, but its particle size (108.65 μm) exceeded the limit of human oral sensory (80 μm). It could be noted that the deposition of 3–4 layers of coating had similar activity after pasteurization/GIT digestion. And droplets with 3 layers of coating were the minimum and most available formulation for encapsulated probiotics (L. plantarum). Hence, the results suggest that the use of ultrasound-assisted multilayer emulsions encapsulated with probiotics in granular food and pharmaceutical applications is a promising strategy.

1. Introduction

Maladjustment of the microbiota in human colon can result in serious and deadly diseases commonly known as inflammatory bowel disease (IBD) (including CD and UC) [1]. The beneficial roles of probiotics are (1) enhancement of the gut epithelial screen, (2) inhibition of combination or growth of pathogens by competing the binding points or secreting antibacterial materials, and (3) regulation of the host immune reaction [2], [3]. The metabolism of probiotics is beneficial to the growth of host gastrointestinal microorganisms, which is one of the ways to maintain the homeostasis of intestinal flora [4]. Based on the World Health Organization (WHO) and International Dairy Federation (IDF) for Probiotics, the human body should consume at least 109 colony-forming units (CFU) of probiotics in food daily for health [5]. Probiotics such as Lactobacillus, Bifidobacterium and certain yeasts have been applied as food supplements since 1980 s in dairy drinks and fermented foods. A major problem in the oral delivery procedure of probiotics is that the viability of the bacteria decreases under rigorous conditions in human gastrointestinal tract, for example, low pH and pepsin in gastric fluid, and pancreatin and bile salt in small intestine fluid. Building capsules to deliver probiotics that preserve them through the gastrointestinal tract and ultimately release them in the colon is becoming increasingly popular. Currently, the microencapsulation techniques for probiotics package have been widely developed by means of emulsification, extrusion, spray drying, and freeze drying [6]. More recently, Qin et al. [7] have prepared double network emulsion gels using a new one-step continuous cold gelation strategy to investigate the effects of whey protein isolate (WPI) / (−) -epigallocatechin-3-gallate (EGCG) covalent conjugates/gellan gum double network emulsion gels on the digestive viability of probiotics. The results provided a promising strategy for promoting the application of double network emulsion gels coated probiotics.

W1/O/W2 double emulsions have been widely used for the packaging of hydrophilic active ingredient as a deliver carrier [8], [9]. The emulsification technique is a gentle method to encapsulate probiotics in the internal water (W1) phase. W1/O/W2 double emulsions are generally prepared in two steps. A W1/O emulsion is formed firstly using polyglycerol polyricinoleate (PGPR) as surfactant, and then emulsified with external water (W2) phase using high-speed homogenization or high-pressure homogenization [10], [11]. In our previous research, WPI-EGCG covalent conjugate nanoparticles (814 nm, contact angle = 53.0°) formed by free-radical induction reaction could be used to stabilize emulsion droplets in the external aqueous phase via the Pickering mechanism [12], [13]. In addition, the high-speed homogenization or high-pressure homogenization of the second step emulsification with excessive external force input may cause the demulsification of the W1/O emulsion. In this research, we plan to use ultrasonic homogenization as the external water phase emulsification technique for double emulsion formation. However, several studies have displayed that these double emulsions are vulnerable to damage in gastrointestinal tract. Multiple layers of positively and negatively charged biopolymers on emulsion droplets has been used to envelops biochemical substances, such as bioactive substances, enzymes, and antigens, to stabilize their properties [14].

Different from traditional Pickering emulsions with one-fold interfacial construction, a multilayer emulsion was formed by additional layers covering emulsion droplets, and fabricated via layer by layer self-assembly with inversely charged biopolymers based on electrostatic attraction [15]. Cationic and anionic biopolymers can be consecutively deposited around emulsion particle templates to regulate their interfacial characters, for example, size, charge, penetrability, and rheology [16]. Multilayer emulsions with thicker interface layer generally have a good stability resist emulsion droplets flocculation and coalescence. The multilayer emulsions formed by layer by layer self-assembly on double emulsion templates as designed carrier with efficient encapsulation and control release to be developed [17]. For example, β-carotene bilayer emulsions could be coated by chitosan-ferulic acid conjugates and propylene glycol alginate using the layer by layer technique, which improved the emulsifying stability against different surrounding conditions [18]. Depositing alginate on the interface of bilayer emulsions (the first layer is whey protein isolate, and the second one is gum arabic) was verified to facilitate its stability, storage ability, and control release of packaged hydrophobic bioactive compounds [19]. Proteins and polysaccharides are generally applied as the ingredient for layer by layer deposition because of their nontoxic and biodegradable characters. Recent studies have shown that quinoa protein Pickering emulsion (QPE) can reduce ice crystal size in fish protein gels and slow protein oxidation after F-T cycle [20], [21]. Chi and Alg are commonly used as layer by layer materials due to their opposite charge. In theory, the survival rate of probiotics could be improved by layer by layer deposition on the surface of the emulsion droplets. However, it is favorable to remain the particle size less than 80 μm for the fake of human tongue does not “feel” the particles. Thus, it is urgent to solve the following problems: (i) what is the most available and minimum number of layers needed to stabilize the particles, (ii) how response capacity is the layer by layer deposition particles to environmental conditions, and (iii) whether these modified capsules can effectively release probiotics to specific parts of the colon. Hence, there is necessary to exploit a new type of multilayer emulsion capsules formed by layer by layer self-assembly on double emulsion templates able to deliver probiotics by controlling the number of layers.

In this study, Chi and Alg layer by layer self-assembly microcapsule with WPI-EGCG covalent conjugates stabilized ultrasound-assisted Pickering double emulsion templates were used to encapsulate probiotic strain liquid (L. plantarum). Firstly, the double emulsion was stabilized by ultrasonic homogenization after preparation of the conjugated compounds. The role of ultrasonic intensity on the droplet size and morphology of double emulsion was measured. Secondly, the particle properties such as ζ-potentials, particle sizes, morphology, FTIR, and stability of multilayer emulsions by controlling the number of layers were characterized. Finally, the viability of L. plantarum encapsulated in a multilayer emulsion deposited layer by layer with the assistance of ultrasound was evaluated after pasteurization and gastrointestinal digestion.

2. Materials and methods

2.1. Materials

(-)-Epigallocatechin-3-gallate (EGCG, concentration > 90%), whey protein isolate (WPI, concentration > 90%), ascorbic acid (Vc), H2O2, Man Rogosa Sharpe (MRS) broth medium, and bile salt (Yuanye Biological Technology Co., Ltd., China.). Porcine pepsin (≥250 U/mg), porcine pancreatin (8 × USP specifications) were acquired from Sigma-Aldrich, Inc., China. MCT, polyglycerol polyricinoleate (PGPR), fructooligosaccharides (FOS), chitosan (Chi), alginate (Alg), and CaCl2 (Ca) (Aladdin CO., LTD. Shanghai, China). The strains of L. plantarum were obtained from Prof. Jiguo Yang’s research group (School of Food Science and Engineering of SCUT). All reagents were of analytical grade.

2.2. Preparation of WPI-EGCG covalent conjugate nanoparticles

Under redox initiator matrix, WPI-EGCG covalent conjugates had been synthesized by free-radical induced reaction [22]. The WPI dispersions (1%, w/v) were formed by immediately dissolved WPI powders in distilled water at 25 °C with thorough stirring. Then oxidizing reagent (H2O2, 1%, v/v) and reducing reagent (Vc, 0.25%, w/v) were added to the compound at 25 °C and left to stand for 2 h without stirring. After adding EGCG (0.1%, w/v),the pH of the compound should be adjusted to 9. After incubation for 24 h, excess unreacted EGCG was removed with dialysis (MWCO: 7000 Da, Yuanye Biological Technology Co., Ltd., China) for 48 h at 25 °C. Finally, all compounds should be freeze-dried prior to use.

2.3. Preparation of W1/O/W2 double emulsions by ultrasound homogenization

W1/O/W2 double emulsions were formed using ultrasonic homogenization with two-step emulsion method [23]. (i) Formation of W1/O emulsions: FOS (as prebiotic agent, 2%, w/v) directly dissolved in distilled water served as W1 phase. The PGPR (as lipophilic surfactant, 3%, w/v) immediately dispersed in MCT acted as oil phase. Then, the W1 phase with oil phase (oil fraction = 0.6) was mixed under agitation at 240 rpm for 20 min. (ii) Formation of W1/O/W2 emulsions: the WPI-EGCG covalent conjugates were dissolved in distilled water with stirring as hydrophilic emulsifier (W2 phase, particle concentrations = 3%, w/v). The formative W1/O emulsions were further mixed with the W2 phase in a ratio of 1:1 (v/v) with a SCIENTZ-II D ultrasonic cell disruptor (Noise Isolating Tamber, Ningbo Scientz Biotechnology CO., LTD, rated intensity = 950 W) for 10 min (5 s on, 2 s off) at different ultrasonic intensity (95 W, 190 W, 285 W, 380 W, 475 W). During ultrasonic homogenization, the samples were put into beaker loaded with a cooling device below 30 °C. The W1/O/W2 double emulsions were kept at 25 °C for 7 days storage.

2.4. Measurement of W1/O/W2 double emulsions

To identify the effects of different ultrasonic intensities on the properties of W1/O/W2 double emulsion, the size distribution of droplets and optical microscopy were explored. Volume-mean diameter (D43) values and the size distribution of droplets were determined using a Malvern Mastersizer analyzer 2000 (Malvern Instruments, U.K.) [24]. The droplet was dispersed in distilled water at 1.095 of absorption index and 2500 rpm of pump speed. The microstructure of the double emulsions was measured by light microscope BH2 (Olympus Co. Ltd., Japan) with a photographic camera [25]. The emulsion droplet was moved to a glass slide and covered with a cover glass. The images were measured and observed under magnifying glasses of 200, and 500-times.

2.5. Cultivation and enumeration of L. Plantarum

Before encapsulation, L. plantarum strains were incubated in MRS broth at 37 °C for 18 h (with two continuous incubations), centrifuged for 10 min (4 °C, 3000 rpm). To quantify the number of viable bacteria, the L. plantarum strains were increasingly diluted to a suitable concentration. Next, under anaerobic environment, the strain solution was plated on MRS broth medium for 18 h at 37 °C. The usable number of colony-forming units (CFU) was calculated by plate counting between 30 and 300 [26].

2.6. Layer by layer self-assembly of W1/O/W2 double emulsions encapsulated with L. Plantarum

Firstly, based on the preparation described above, Alg solution (1%, w/v) was additional dissolved in W2 phase. The L. plantarum strain liquid was encapsulated in inner W1 phase of W1/O/W2 double emulsions by two-step emulsion method. To enhance the stability, the layer by layer deposition of biopolymers were used to further coat the double emulsions. The first layer of emulsion was coated by dispersed the double emulsion in Chi solution (0.1%, w/v) or Ca solution (100 mM) and mixed under agitation at 100 rpm for 10 min. To coat more layers, repeat this operation with commutative solutions of Alg and Chi/Ca. The multilayer emulsions after different layer coatings were labeled as Emulsion (Alg)-Ca, Emulsion (Alg)-Chi, Emulsion (Alg)-Chi/Alg, Emulsion (Alg)-Chi/Alg/Ca, Emulsion (Alg)-Chi/Alg/Chi, Emulsion (Alg)-(Chi/Alg)2, Emulsion (Alg)-(Chi/Alg)2Ca. The W1/O/W2 double emulsions with layer by layer deposition were stored for 15 days at 4 °C [27].

2.7. Characterization of multilayer emulsion particles

2.7.1. Determination of ζ-potential

ζ-potentials of multilayer emulsions with different layer coatings were measured by a SZ-100Z nanoparticle Analyzer (HORIBA, Japan) [25]. Extracted 1 mL liquid diluted with distilled water (10 mg/mL, w/v) and put it into the test electrophoresis pool (25 °C).

2.7.2. Particle size measurement

The particle size (volume-mean diameter, D43) of multilayer emulsions with different layer coatings, were measured by light scattering using a Malvern Mastersizer analyzer 2000 (Malvern Instruments, U.K.). The emulsion particles were dispersed in distilled water at 1.095 of absorption index and 2500 rpm of pump speed [28].

2.7.3. Optical microscopy

According to this method, the microstructure of multilayer emulsions with different layer coatings was measured by light microscope BH2 (Olympus Co. Ltd., Japan) with a photographic camera [24]. An emulsion droplet was moved to a glass slide and covered with a cover glass. The images were measured and observed under a magnifying glass of 500-times.

2.7.4. FTIR spectrometry

The molecular interactions in the particles can be detected by FITR spectra [29]. The final spectra were measured using a VERTEX 70 FTIR spectrometer (Bruker Co., Germany) connected to triglycine deuterated sulfate. The test slice was pressed with sample and KBr. The measurement was employed in the wavenumber scale from 4000 to 400 cm−1 with 64 scans at a resolution rate of 4 cm−1. The spectrum data was analyzed by OMNIC software.

2.8. Viability of encapsulated L. Plantarum in multilayer emulsion after pasteurization

The cultured free probiotics and probiotics encapsulated in multilayer emulsion were heated in a water bath to explore the number of viable probiotics after thermal process. The samples were all heated at 63 °C for 30 min to simulate the thermal pasteurization procedure. The usable number of probiotics was counted by the plate counting on MRS broth medium between 30 and 300 [30].

2.9. Viability of encapsulated L. Plantarum in multilayer emulsion after digestion in vitro

The free probiotics cultured in vitro and probiotics encapsulated in multilayer emulsion were digested in a simulated gastrointestinal tract [31]. Firstly, the simulated gastric fluid (SGF) containing 85 mM NaCl and 3 g/L pepsin were mixed evenly with the samples, and then adjusted by HCl to pH 2.0 at 37 °C for 2 h [32]. After the stomach digestion, the simulated intestinal fluid (SIF) including 10 mM CaCl2,85 mM NaCl, 1 g/L bile salt and 1 g/L pancreatin were added before the pH regulated to 7.0. Similarly, the mixture was digested with SIF at 37 °C for 2 h. Finally, the above liquid was added to pH 6.8 PBS buffer, which was used as simulated colonic fluid (SCF), and incubated at 37 °C for 2 h with sufficient stirring. The usable number of probiotics in the eventual mixture was counted by the plate counting on MRS broth agar plates between 30 and 300. Simultaneously, living cell growth was measured by recording and observing the optical density (OD600) of the samples after digestion in three stages of the stomach, small intestine, and colon simulated in vitro. Apart from digestive enzymes, all ingredients and reagents used in the above experiments were sterilized.

2.10. Statistical analysis

ANOVA using SPSS software was used to calculate the statistical significance between the means (p less than 0.05). All the results were expressed as the average value of the three experiments, and the error bar shows the standard deviation.

3. Results and discussion

3.1. Effect of ultrasonic intensity on the formation of W1/O/W2 double emulsions

In this study, we explored the ultrasonic homogenization as the external water phase emulsification technique for W1/O/W2 double emulsion formation. We maintained the system temperature below 30 °C using a circular cooling bath device. According to Fig. 1, the effect of ultrasonic intensity on the droplet size and distribution of W1/O/W2 double emulsions was measured. The droplet size of the double emulsions decreased from 116.96 to 30.12 μm with increasing ultrasonic intensity from 95 to 285 W. The improvement of cavitation phenomenon of the ultrasonic homogenization might be responsible for the reduction of droplet size [33]. Nevertheless, after larger ultrasonic intensity from 285 to 475 W, the D43 values of the double emulsions increased from 30.12 to 741.72 μm. These results might be explained by the micro-steaming, shear force, and turbulent forces of the greater ultrasonic treatment, which could destroy the primary W1/O emulsion [25]. Furthermore, the droplet size of the double emulsions formed at 95 and 190 W expressed a multimodal distribution. These emulsions formed with lower ultrasonic treatment had a peak of W1/O droplet around 1–20 μm, revealing that not all W1/O droplets formed W1/O/W2 double emulsion. The droplet size distribution of the double emulsions formed at 285 W had a single and narrow distribution without W1/O droplets. This indicated that the W1/O/W2 double emulsion was completely emulsified with optimal ultrasonic homogenization of 285 W. Some large size oil droplets were found in W1/O/W2 double emulsion formed at 380 and 475 W, indicating that greater ultrasonic treatment dissociated the W1/O and W1/O/W2 emulsion droplets. However, it is obtained that partial double emulsion formed with high-speed homogenization or high-pressure homogenization containing two peaks of slight W1/O droplets and vast W1/O/W2 droplets. Therefore, proper ultrasonic homogenization could form a W1/O/W2 double emulsion with uniform emulsion droplets.

Fig. 1.

Fig. 1

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Visual observations, typical optical micrographs, and droplet sizes of W1/O/W2 double emulsions as a function of the ultrasonic intensity (95 W, 190 W, 285 W, 380 W, 475 W).

To further explore the structure of the W1/O/W2 double emulsion, microstructure tests were definitely visualized by optical microscopy. As can be seen from Fig. 1, only a small number of W1/O droplets were observed in W1/O/W2 droplets, indicating that the constructed emulsions were favorably formed. Consistent with the result described above, the size of W1/O/W2 droplets in the observed microstructure also varied with the change of ultrasonic intensity [34]. With the increasing ultrasonic intensity from 95 to 285 W, the W1/O/W2 emulsion droplet size gradually reduced. Moreover, a greater ultrasonic treatment at 380 and 475 W could lead to the formation of W1/O/W2 droplets with larger sizes. The microstructure of the double emulsion formed by ultrasonic treatment at 475 W was filled with a large number of oil droplets, which indicated that the strong ultrasonic treatment broken the formed emulsion droplets.

For the emulsion stability, the W1/O/W2 double emulsions treated with different ultrasonic-assisted techniques had different physical stability. After 7 days, the double emulsion formed by ultrasonic treatment at 285 W maintained the whole emulsified state without layered. The stability of W1/O/W2 double emulsions were mainly determined by the droplet size of emulsion formation through a Pickering mechanism [35]. Proper ultrasonic homogenization treatment could provide to a significant anti-flocculation compact structure on the surface of the emulsion droplets.

3.2. Characterization of multilayer emulsion particles

3.2.1. Determination of ζ-potential

The ζ-potential values of multilayer emulsion particles after layer by layer deposition are shown in Fig. 2. The Emulsion (Alg)-Ca particles showed a negative value (-35.5 mV). The Emulsion (Alg) with Chi solution deposition changed the ζ-potential to a positive value (+31.55 mV). Emulsion (Alg)-Ca, Emulsion (Alg)-Chi/Alg, Emulsion (Alg)-Chi/Alg/Ca, Emulsion (Alg)-(Chi/Alg)2, and Emulsion (Alg)-(Chi/Alg)2Ca particles were negatively charged, which could be due to the high negative charge of the Alg solution deposition [36]. Nevertheless, the Emulsion (Alg)-Chi and Emulsion (Alg)-Chi/Alg/Chi particles displayed positively charged when they were coated with Chi solution. These results showed that electrostatic interaction was the prime acting force for the formation of multilayer emulsion particles after layer by layer coating. The inversion of the ζ-potential values was visualized after each deposition step, demonstrating the successive coating of oppositely charged biopolymers (Alg and Chi) [37]. A similar phenomenon has also been studied for β-carotene bilayer emulsions coated by chitosan-ferulic acid conjugates / propylene glycol alginate and bilayer emulsions deposited with alginate.

Fig. 2.

Fig. 2

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Change in ζ-potentials of multilayer emulsion particles after layer by layer deposition.

3.2.2. Particle size measurement

Fig. 3 represents macroscopic observations and typical optical micrographs of multilayer emulsion particles after layer by layer deposition. The W1/O/W2 double emulsion with ultrasonic treatment at 285 W was more uniform distribution but little larger in droplet size (30.12 μm) compared to that generated in previous research with high-speed homogenization way (21.57 μm). To decrease the potential harm of excessive external force input to probiotic strain, we used ultrasonic homogenization that showed a low shear stress. To improve the stability of double emulsion particles as well as to increase the capacity of encapsulated probiotic strain, we coated several layers of biopolymers on the double emulsion template. As many researches have confirmed, the biopolymer multilayers formed by layer by layer deposition were quite stable because of the interaction of oppositely charged biopolymers [38]. As can be seen from Fig. 3, the sizes of multilayer emulsion particles after layer by layer deposition suggested that the coating of 4 layers led to an improve in the particle size from 30.5 to 108.65 μm. These results were compatible with another study, which represented that the incorporation of biopolymers largely increased the size of double emulsion particles. It is supposed that the Chi interacted with Alg in the external layer of the emulsion particles, thus leading them to accumulate more compactly. Interestingly, it is favorable to remain the particle size less than 80 μm for the fake of human tongue does not “feel” the particles [30]. The size of the double emulsion particles could be regulated precisely in a scope from ten to hundred micrometers by adjusting the number of layers. According to Fig. 3, only the particle size of Emulsion (Alg)-(Chi/Alg)2Ca in the experimental samples was>80 μm. The double emulsion particles with up to 4 coating layers were well under human perceptual limit indicated that they are properly applied as food materials.

Fig. 3.

Fig. 3

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Visual observations, typical optical micrographs, and particle sizes of multilayer emulsion particles after layer by layer deposition.

3.2.3. Optical microscopy

To further investigate the structure of multilayer emulsion particles, the microstructure measurement by optical microscopy confirmed that Chi was evenly deposited on the Emulsion (Alg) particles. Compared to the microstructure of double emulsion template, the coating of additional biopolymers layers changed the packing of particle surfaces observably. In accordance with droplet size determination, the size of multilayer emulsion particles in the obtained microstructure also increased with the increasing deposition layer. After 15 days storage, it could be observed that double emulsion template with different deposition layer had different physical stability. The multilayer emulsion formed by Ca deposition kept the whole emulsified state without layered. These results might be explained by the Ca formed a hard coating on the outer layer of the multilayer emulsion particles against flocculation. To remove the unreacted biopolymers solution, we collected the multilayer emulsion particles by filtration. In conclusion, from the aspect of particle size and stability, the Emulsion (Alg)-Chi/Alg/Ca with 3 coating layers is the most appropriate formula.

3.2.4. FTIR spectrometry

FTIR was applied to explore the character of functional groups and interactions occurring inside the particles. FTIR spectra of Alg, Chi, and multilayer emulsions (Emulsion (Alg)-Ca, Emulsion (Alg)-Chi, and Emulsion (Alg)-Chi/Alg) are illustrated in Fig. 4. For Alg, the peak at 3469 cm−1 was attributed to the stretching vibration of the O-H groups, and the peak at 1030 cm−1 was ascribed to vibrations of the C-O-C groups [18]. Carboxylate salt groups generated both symmetric and asymmetric stretching absorption band, which resulted in the peaks at 1417 and 1621 cm−1, respectively. In the spectra of Chi, the absorption peak at 3426 cm−1 attributed to the N-H stretching vibration. NH3+ groups produced both C = O stretching (amide I) and –CH-OH in cyclic alcohols and C-O stretch (secondary O-H group), which led to the absorption bands at 1655 and 1077 cm−1, respectively [30]. The FTIR spectra of Emulsion (Alg)-Ca were similar to those of Alg, but the absorption peaks were shifted slightly, in accordance with Alg molecular structures. As for Emulsion (Alg)-Chi, when Chi was combined to Emulsion (Alg), the absorption band related to the amide II bands moved from 1621 to 1633 cm−1, which indicated that there were electrostatic interactions between Alg and Chi within the particles [36]. The absorption peak of N-H groups (amide A) shifted from 3469 to 3432 cm−1, which indicated that there was hydrogen bonding between these two biopolymers. Electrostatic interactions and hydrogen bonding both played an important effect in the Emulsion (Alg)-Chi formation. Moreover, the absorption peaks near 1030 and 1417 cm−1 disappeared, which possibly attributed to the ionic interactions between COO– and NH3+ groups of Emulsion (Alg)-Chi. After the second Alg deposition, the absorption peaks of Emulsion (Alg)-Chi/Alg were rarely moved from that of Emulsion (Alg)-Ca, which confirmed that the Alg were successfully deposited on the surface of Emulsion (Alg)-Chi particles.

Fig. 4.

Fig. 4

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Full FTIR spectra of Alg, Chi, and multilayer emulsions (Emulsion (Alg)-Ca, Emulsion (Alg)-Chi, and Emulsion (Alg)-Chi/Alg).

3.3. Viability of encapsulated L. Plantarum in multilayer emulsion after pasteurization

Thermal pasteurization procedure (63 °C for 30 min) is currently used in food industry to insure food health and security. Thus, the total number of living cells in the multilayer emulsion (L. plantarum) was recorded and compared before and after pasteurization by MRS plate counting, which was shown in Fig. 5A. Usually, pasteurization procedure generated prominent reduction in total living cell count of probiotics. However, compared with L. plantarum without multilayer emulsion encapsulation, the layer by layer self-assembly multilayer emulsion encapsulation significantly increased the number of viable cells, and the number of viable bacteria increased with the increase of the number of layers. This could be attributed to more deposition layers improved inhibition of heating treatment, which was beneficial to enhance the resistance of L. plantarum to the pasteurization procedure. The highest viable cell count (5.97 × 107 CFU/mL) of the L. plantarum displayed in the multilayer emulsion with 5 coating layers (Emulsion (Alg)-(Chi/Alg)2Ca). However, combined with the particle size determination, the particle size of the multilayer emulsion particles must be less than 80 μm to avoid tongue touch. It could be noted that the deposition of 3–4 coating layers affected the viable cell count slightly (Emulsion (Alg)-Chi/Alg/Ca, Emulsion (Alg)-Chi/Alg/Chi, Emulsion (Alg)-(Chi/Alg)2), and the results showed that the minimum-necessary formulation to maintain the survival of L. plantarum during pasteurization was 3 coating layers (emulsion (Alg)-Chi/Alg/Ca) [31].

Fig. 5.

Fig. 5

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(A) Total viable cell count of probiotic strain liquid (L. plantarum) encapsulated by multilayer emulsions after layer by layer deposition before and after pasteurization / GIT digestion. (B) OD600 values of probiotic strain liquid (L. plantarum) encapsulated by multilayer emulsions after layer by layer deposition during each step of the GIT digestion.

3.4. Viability of encapsulated L. Plantarum after digestion in vitro in multilayer emulsion

Furthermore, we explored the encapsulation ability of multilayer emulsion with different number of layers in the gastrointestinal passage. Fig. 5A presents the number of total viable cell count after the exposure to the simulated gastrointestinal tract. In the case of multilayer emulsion encapsulated L. plantarum, the particles were destroyed with violent oscillation and applied to MRS plate counting. As showed in Fig. 5A, the viable cell count of free probiotics after GIT digestion was extremely low, suggesting that L. plantarum almost died after digestion. Once probiotics were encapsulated in the Emulsion (Alg)-Ca, an obvious increase in the viable cell count by 20 orders of times after GIT digestion was obtained (2.62 × 107 CFU/mL). However, this viability of L. plantarum still maintains low, implying that only 31.6% of the probiotics were survived. The encapsulation ability of L. plantarum was significantly improved by continuous deposition of biopolymer layers on multilayer emulsions. This may be due to increased layers of sediment and increased resistance to digestive enzymes [39]. Multilayer emulsion with 3 coating layers (Emulsion (Alg)-Chi/Alg/Ca) observed the maintenance of 75% viable probiotics after GIT digestion (6.0 × 107 CFU/mL). The received results have explicitly indicated that the deposition of three coating layers onto double emulsion particles was sufficient to remain the viable cell count of probiotics during the GIT digestion, which took about 6 h. It could be observed from the Fig. 5A that the viable cell count of probiotics in all multilayer emulsions after pasteurization procedure was lower than that after GIT digestion. Thus, the L. plantarum was more sensitive to heating treatment than gastrointestinal passage [40].

On the other hand, in order to illustrate the ability of L. plantarum to survive in each process of the GIT digestion, the OD600 values were recorded, as shown in Fig. 5B. The free probiotics and multilayer emulsions after layer by layer deposition encapsulated probiotics were digested in three stages of the gastrointestinal tract for 2 h each [41]. At first, after gastrointestinal digestion, the OD600 values of free probiotics dropped sharply, indicating that about 50% of free probiotics died during the course of the stimulating environment of SGF. For multilayer emulsions encapsulated probiotics, there was only a small loss of OD600 value due to the resistance of the deposit layers after 2 h in the stomach. After 2 h of digestion by the small intestine, the OD600 values of the probiotics, free or coated with multiple emulsions, were further reduced. The encapsulation of L. plantarum into double emulsion and further deposited with biopolymer multilayers resulted in a slightly decrease in OD600 values, and multilayer emulsion with 3 coating layers attained the suitable maintenance of L. plantarum viability. The OD600 values of the multilayer emulsions remained no loss basically after digestion in the colon. In general, the multilayer emulsion with 5 coating layers (Emulsion (Alg)-(Chi/Alg)2Ca) had the highest OD600 values after GIT digestion, but its particle size exceeded 80 μm. It could be concluded that the deposition of 3–4 coating layers had similar OD600 values, suggesting that Emulsion (Alg)-Chi/Alg/Ca was the minimum and most available formulation for the L. plantarum encapsulation under GIT digestion [42].

4. Conclusions

In summary, microcapsules of multilayer emulsions deposited layer by layer under ultrasonic assistance can be used to improve the viability of L. plantarum strain solution under pasteurization and GIT digestion (Fig. 6). Firstly, ultrasonic homogenization was used to fabricate the W1/O/W2 double emulsion. The effects of ultrasonic intensity on the droplet size and morphology of constructed emulsions were studied. The double emulsion formed by ultrasonic treatment at 285 W had a single and narrow distribution with smallest droplet size. The double emulsion particles were then coated with Alg, Chi, and Ca. The formation of multilayer emulsions was quantitatively proved by ζ-potentials, morphology, and FTIR measurements. Because of most probiotics cannot survive under pasteurization and gastrointestinal passage, L. plantarum strain liquid was encapsulated into the double emulsion with coating layers added. After pasteurization / GIT digestion, the viability of coated L. plantarum increased with the number of coating layers. This could be ascribed to more deposition layers enhanced resistance of heating treatment and digestive enzymes. However, the L. plantarum encapsulated in multilayer emulsions was more sensitive to heating treatment than gastrointestinal passage. The multilayer emulsion after pasteurization and GIT digestion both had the highest viability at 5 coating layers (Emulsion (Alg)-(Chi/Alg)2Ca), but its particle size (108.65 μm) exceeded the limit of human oral sensory (80 μm). It could be noted that the deposition of 3–4 coating layers had similar viability after pasteurization / GIT digestion, implying that the minimum and most available formulation for L. plantarum to be encapsulated was emulsion (Alg)-Chi/Alg/Ca (46.34 μm). Hence, multilayer emulsions encapsulation of probiotics strain solutions using ultrasound-assisted technology is a promising strategy for oral application in granular food products (solid beverages, pressed confectionery or juice drinks) and pharmaceuticals.

Fig. 6.

Fig. 6

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The process flowchart of layer by layer assembled multilayer capsule with WPI-EGCG covalent conjugates stabilized Pickering double emulsion templates for L. plantarum encapsulation.

CRediT authorship contribution statement

Xian He: Methodology, Investigation, Data curation, Software, Writing – original draft. Wanshui Yang: Validation, Writing – review & editing, Visualization, Supervision. Xinsheng Qin: Conceptualization, Methodology, Investigation, Data curation, Software, Writing – original draft, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

Acknowledgments

Financial support from the University Natural Science Research Project of Anhui Province (KJ2021A0223) and Peak Discipline Construction Project in School of Public Health of Anhui Medical University in 2022-2023 (Research and development of key technologies in the production of pre-packaged food for calcium homeostatic colloid delivery system for nutritional support therapy of colorectal cancer).

We thank Zhigang Luo from School of Food Science and Engineering, South China University of Technology for Project administration, Supervision and Funding acquisition (National Natural Science Foundation of China, 21878106 and 21576098).

Contributor Information

Wanshui Yang, Email: wanshuiyang@gmail.com.

Xinsheng Qin, Email: qinxinsheng@ahmu.edu.cn.

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