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. 2022 Jul 19;31(11):1463–1472. doi: 10.1007/s10068-022-01134-8

Sodium alginate-based wall materials microencapsulated Lactobacillus plantarum CICC 20022: characteristics and survivability study

Lihua Zhang 1,2,3, Peixin Tang 1, Shunfeng Li 4, Xia Wang 1, Wei Zong 1,2,3,✉
PMCID: PMC9433622  PMID: 36060564

Abstract

In this study, microcapsules of Lactobacillus plantarum CICC 20022 (L. plantarum CICC 20022) were prepared by extrusion technique using sodium alginate (SA), sodium alginate-sodium caseinate (SA-SC) and sodium alginate-whey protein isolate (SA-WPI) as wall materials, respectively. Results showed that the best encapsulation yield of L. plantarum CICC 20022 was SA-WPI. Morphology and texture analysis showed that the microcapsules prepared by the SA-WPI system presented a more compact internal structure and higher resistance to external pressure. Fourier-transform infrared spectroscopy and low field nuclear magnetic resonance analysis demonstrated that the hydrogen bonding ability and network structure of the SA were improved by the addition of WPI. The survivability of L. plantarum CICC 20022 entrapped with the SA-WPI system was improved during freeze-drying and simulated gastrointestinal digestion. Therefore, the SA-WPI system can potentially be used as the vector of L. plantarum CICC 20022 in food applications.

Graphical abstract

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Keywords: Lactobacillus plantarum CICC 20022, Sodium alginate, Whey protein isolate, Microcapsule, Simulated gastrointestinal digestion

Introduction

As a kind of probiotic (Saxelin, 2008), Lactobacillus plantarum (L. plantarum) has been widely used in fermented vegetable juice and fruit juice (Chung et al., 2020), kimchi (Khan et al., 2016), and various dairy products (Song et al., 2016) due to the special flavor and health benefits for the host after fermentation. However, the use of L. plantarum in food carriers was limited by its sensitivity to various factors during storage, processing, and gastrointestinal digestion as reported by many studies (Rather et al., 2017; Wang et al., 2020a; 2020b). Microcapsules can provide a physical barrier to resist severe environmental conditions such as freeze-drying and gastrointestinal digestion, which has been considered a promising technology for introducing live probiotics into food. For the microencapsulation of probiotics, selecting appropriate wall materials was very important. The wall materials of microcapsules should have good stability, film-forming ability, and security for probiotics. A variety of food-grade polymers (Liu et al., 2019; Massounga et al., 2019), such as carbohydrates (Gutierrez-Zamorano et al., 2019) and protein (Savedboworn et al., 2020) were commonly used for microencapsulation.

Sodium alginate (SA) is the most widely used polymer for encapsulation owing to its excellent attributes such as being cheap, non-toxic, and biocompatible, which is exchanged with Ca2+ or other divalent cations to form calcium alginate gel with mechanical strength and elasticity (Zhang et al., 2016). However, the SA gel has high porosity, lower mechanical strength, and lower diffusivity, which may lead to a lower encapsulation potency and a quicker unleash of the encapsulate and could not ensure that large-scale commercial applications and the embedded active substance withstand severe environmental conditions such as gastrointestinal digestion. SA was often compounded with other high molecular polymers to enhance the resistance of probiotic bacteria to the external adverse environment (Su et al., 2021). Studies had shown that adding proper amounts of cellulose nanofibers and lecithin to the outer layer of the SA gel, could enhance the strength of the SA gel and the survival rate of the embedded materials (Huq et al., 2017). Vaziri et al. (2018) studied the use of sodium tripolyphosphate as the ionic double cross-linking agent to embed L. plantarum PTCC 1058 in SA-chitosan composite microspheres, which effectively improved the vitality of bacteria. SA-WPI has been applied because the wall material of microcapsules protected probiotics and improved the quality of microcapsules.

Whey protein isolate (WPI) is a protein obtained by further extraction from the concentrated whey protein, with a protein content of more than 90%. When the protein is heated, disulfide bonds can be formed between β-lactoglobulin, then cross-linked into a dense network structure, which has good film-forming properties. It was a good encapsulation wall material for a series of biologically active substances because of forming a non-covalent bond "calcium bridge" with salt ions such as Ca2+ to enhance the cross-linking between protein molecules and then form a gel structure (Heidebach et al., 2009). Sodium caseinate (SC) is the sodium salt of casein which is the main protein in milk. It contains more hydrophilic and hydrophobic groups, which have excellent surface properties and can effectively improve the stability and bioavailability of the embedded nutrients (Bai et al., 2019). The above-mentioned showed potential within the encapsulation of probiotic bacteria because of the property to act with alternative polymers for the formation of complexes. The study proved that Lactobacillus casei (L. casei) cells were effectively entrapped into caseinate and gellan gum mixture, and the viability of encapsulated cells increased by 2 times than control after 120 min in simulated gastric fluid. Another research about sodium caseinate gelled (SCG) with transglutaminase enzyme (TE) for Lactobacillus paracasei (L. paracasei) and Bifidobacterium lactis (B. lactis) encapsulation found that the survival of entrapped probiotics was increased significantly by 2 log CFU/g than control after 90 min in simulated gastric fluid at pH 2.5 (Nag et al., 2011). Han et al. (2020) prepared Ca-alginate-whey protein macromolecule isolate (Ca-Alg-WPI) microcapsules for Lactobacillus eubacterium paracasei (L. paracasei) and Lactobacillus eubacterium bulgaricus (L. bulgaricus), which showed the Ca-Alg-WPI wall system keeps probiotics from the thermal condition. To our knowledge, there were few studies on SA-WPI, SA-SC, and SA as wall materials of microcapsules to improve the survival of L. plantarum CICC 20022. Previous studies had shown that L. plantarum fermentation could significantly improve the nutritional quality of carrot slices with jujube juice (Zhang et al., 2020). However, due to the poor tolerance of L. plantarum CICC 20022, acid production by fermentation in food had a negative impact on the growth of L. plantarum CICC 20022 (Zhang et al., 2022). Therefore, this study aimed to compare the morphology of microcapsules formed by three different wall materials and their effects on the survival rate of L. plantarum CICC 20022, so as to provide a reference for improving the processing performance of SA microcapsules and the tolerance of L. plantarum.

In this study, WPI and SC were added in SA and were used as wall materials to prepare the L. plantarum CICC 20022 microcapsules by the extrusion method, respectively. In simulating gastrointestinal juice and freeze-dried environment, the encapsulation yield, surface morphology, colloidal structure of microcapsules, and survival rate of the resulting microcapsules were evaluated.

Materials and methods

Materials

Freeze-dried Lactobacillus plantarum CICC 20022 (L. plantarum CICC 20022) commercialized as probiotic strains were supplied by the China Center of Industrial Culture Collection (Beijing, China). Firstly, the freeze-dried culture of L. plantarum CICC 20022 was cultured at 37 °C in MRS broth (sterilized at 121 °C, 15 min) for 48 h. After propagation, L. plantarum CICC 20022 cells were obtained at 6000×g, 4 °C, 10 min by centrifugation (HC-3618R, Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd, China), and subsequently washed twice with saline solution (sterilized at 121 °C, 15 min), then the L. plantarum CICC 20022 cells were dissolved in sterile saline solution. A series of dilutions were followed and prepared for the same diluent. Populations of L. plantarum CICC 20022 were enumerated by pouring plate technique, using 1 mL of per dilution in MRS agar, and then cultured at 37 °C for 48 h. The count of viable cells was 9.62 ± 0.02 log CFU/mL using serial dilutions in MRS agar.

MRS broth was purchased from Beijing Aobox Biotechnology Co. Ltd (Beijing, China). Sodium Alginate and Sodium Caseinate were obtained from Linxia Human Biological Products Co. Ltd (Gansu, China). WPI was purchased from Zhejiang Yinuo Biological Technology Co. Ltd (Zhejiang, China).

Microencapsulation of L. plantarum CICC 20022

Microencapsulation was produced by the extrusion technique as reported by Yan et al. (2014) with some modifications. L. plantarum CICC 20022 was encapsulated by three different wall materials: SA, SA-SC, and SA-WPI. Briefly, SA powder (20 g/L) was dissolved in sterile water while heating with stirring for 2 h at 70 °C and cooled overnight. 2 mL of bacterial cell suspension (9.62 ± 0.02 log CFU/mL) were added to 5 mL of SA solution, and mixed further at 150×g for 5 min before freeze-drying operations. After 20 g of SC powder was added to a beaker containing 1 L of sterile distilled water, it was stirred on a magnetic stirrer to dissolve SC. 2.5 mL of SA solution was mixed with 2.5 mL of SC solution and homogenized at 150×g for 5 min, and the next 2 mL of bacterial cell suspension (9.62 ± 0.02 log CFU/mL) was mixed with SA-SC mixture at 150×g for 5 min before freeze-drying operations.

WPI powder (20 g/L) was mixed with sterile water gently using a magnetic stirrer (78-2, Changzhou Guohua Electric Co., Ltd, China), and then kept at 90 °C for 30 min and cooled to room temperature. WPI solution (2.5 mL) was blended with SA solution (2.5 mL) and homogenized at 150×g for 5 min and then the SA-WPI mixture was mixed with 2 mL of bacterial cell suspension (9.62 ± 0.02 log CFU/mL), mixed further at 150×g for 5 min before freeze-drying operations.

Three sample mixtures were extruded dropwise into 2% calcium chloride solution (w/v) (sterilized at 121 °C, 15 min) using a disposable syringe (0.5 × 10 cm, Henan Shuguang Huizhikang Biotechnology Co., Ltd, China). After the alginate microcapsules were formed, they were quietly matured in the calcium chloride-containing sterile saline solution for 30 min to complete gelation and then washed. Following, the capsules were dried in a vacuum freeze drier (XY-FD-18, Shanghai Xinyu Instrument Co., Ltd, China) for 14 h.

Encapsulation yield

The viability of L. plantarum CICC 20022 was measured by the standard plate counting method. Bacterial fluid was 10 times gradients diluted in a sterile saline solution. 100 μL of appropriate bacterial solution were poured into MRS agar plates, which were then cultured at 37 ± 1 °C for 48 h to count. According to the methodology described by Darjani et al. (2016) with some modifications. 1 g microcapsule was homogenized with 9 mL of 0.06 mol/L sodium citrate solution and 0.2 mol/L sodium bicarbonate, then adjusted the pH between 7.5 and 7.8 with 0.1 mol/L NaOH and HCl, and then vortex for 10 min to dissolve the microcapsule and release the lactic acid bacteria completely. The encapsulation yield of bacterial cells was calculated by the following formula:

Encapsulationefficiency%=lgNlgN0×100

where N is the number of entrapped bacterial cells loaded inside the bead, CFU/g, and N0 is the amount of free bacterial cells in the solution during the microencapsulation process, CFU/g.

Texture profile analysis (TPA)

TPA of dry microcapsules was evaluated by TA-XT Plus physical property analyzer (Stable Micro Systems, Britain) with a probe of TP-6.5 g load cell at a test speed of 1.0 mm/s, pre-test speed of 1.0 mm/s, post-test speed 1.0 mm/s was used to reach a 50% compression and repeated 5 times for each sample.

Low field nuclear magnetic resonance (LF-NMR) measurement

The moisture distribution of microencapsulated L. plantarum CICC 20022 was performed by NM120 LF-NMR (Shanghai Newman Electronic Technology Co., Ltd, China). 2 g of microcapsules were put in a 25 mL of NMR tube, and set the T2 relaxation time scan with the CPMG sequence, the echo time was 400 μs, the number of echoes was 8000, the waiting time was 8 s, each measurement was scanned 8 times, and the temperature was 25 °C.

Fourier-transform infrared spectroscopy (FT-IR) investigation

FT-IR spectroscopy (Vertex 70, Thermo Fisher, USA) was often used to analyze the freeze-dried microspheres’ group structure. 25 mg of crushed microcapsules were mixed with 500 g of potassium bromide and pressed to pastilles after being homogenized in a mortar with a pestle. Then it was placed in the infrared spectrum channel for testing. Infrared spectra were collected in the wavelength range 4000–400/cm, the resolution was 4/cm, and the repeat scan was 32 times.

Morphological characterization of the microcapsules

The shape and surface texture of microspheres were observed by JSM-7001F field emission scanning electron microscope (SEM) (JSM-7001F, Electronics Corporation, Japan). Freeze-dried microcapsules were mounted on a metal stub, using double-sided conductive tape, gold-coated under vacuum, and examined at 3 kV.

Survival assays of microencapsulated L. plantarum CICC 20022 under simulated gastrointestinal conditions

The tolerance of microencapsulated L. plantarum CICC 20022 under simulated gastric conditions was referred to the method described by Zou et al. (2011) with modifications. Initially, the simulated gastric fluid (SGF) was prepared using 0.3 g/L pepsin solution (2 g/L salines). The pH was adjusted to 2.0 with 0.1 mol/L HCl solution to simulate the variable acidity in the stomach. Then, the sample (1 g) was homogenized in 9 mL SGF and incubated at 37 °C for 2 h with the agitation of approximately 150×g. To study the changes in the viability of encapsulated cell viability under different digestion times, intervals of 0, 5, 30, 60, and 120 min were predetermined. Subsequently, the microspheres were sampled and dissolved in the 9 mL of sterile buffer (the mixture of 0.2 mol/L sodium bicarbonate and 0.06 mol/L sodium citrate), then shaken gently for 10 min until completely dissolved. The L. plantarum was counted by the drop plate method on MRS agar.

The simulated digestive tract was continuous, which included the esophagus-stomach pathway, the duodenum step, and the ileum step. The viability measurement of microencapsulated L. plantarum CICC 20022 in simulated digestive juice (SDJ) was evaluated following the method of Madureira et al. (2011) with some modifications. Firstly, 5 g/L pepsin was dissolved in MRS broth for esophagus-stomach simulation, adjusted the pH to 5.5, 4.6, 3.8, 2.8, 2.0 by 0.1 mol/L HCl, respectively. The duodenal simulant solution used 3 g/L bile salt and 10 g/L trypsin to be dissolved in MRS liquid broth medium, adjusted to pH 5.0 by 1 mol/L HCl. Finally, the ileum step was stimulated by an increase of the duodenum simulation fluid pH to 6.5 using 0.1 mol/L NaHCO3. 1 g of microcapsule samples were weighed and then added into the above simulation solution in sequence. SDJ consists of successively incubating for 10 min in pH 5.5, 4.6, and 3.8 solutions, respectively, then vibrated orderly in the solutions with pH 2.8 and 2.0 for 20 min, next shaken in a solution with pH 5.0 for 90 min, in the end at pH 6.5 for 150 min of incubation. The SDJ was changed each time, centrifuged at 8000×g for 5 min (4 °C) to precipitate the microcapsules, and then aspirated the supernatant with a pipette. Every time the SDJ was replaced, mixed well by vortex for 30 s. Then, the dilution method in MRS agar was performed to determine the viability of L. plantarum CICC 20022.

Freeze drying of microencapsulated L. plantarum CICC 20022

1 g of beads was pre-cooled at − 26 °C for 8 h and dried with an XY-FD-18 freeze dryer for 14 h. Then, dried microcapsules were mixed with 9 mL of sterile saline solution, and vortexed for 10 min until completely dissolved. The enumeration of L. plantarum CICC 20022 was performed by drop plate method on MRS agar.

Statistical analysis

Each analysis was made in triplicate and the results were reported as mean and standard deviation. Analysis of variance (ANOVA) and Duncan's multiple comparison tests were used to compare all results. When the confidence interval was less than 5% (P < 0.05), the difference between the means was considered significant. The analysis was carried out by SPSS software (IBM; Armonk, New York, USA). The figures were executed by origin Pro 8.5.

Results and discussion

Encapsulation efficiency of microcapsules

The encapsulation efficiency of the three kinds of L. plantarum CICC 20022 microcapsules was shown in Fig. 1. It was suggested the encapsulation efficiency of the SA-WPI microcapsule was 70.40 ± 2.8%, significantly higher than that of SA-SC (46.08 ± 2.5%) and SA (42.72 ± 0.48%), respectively. The encapsulation efficiency of WPI microcapsule was similar to that of L. plantarum NCDC201 (72.48 ± 2.31%) with a double alginate layer (Rather et al., 2017), while lower than WPI microcapsules containing L.bulgaricus and L.paracasei (89.08 ± 1.79%) and L. casei ATCC 393 (85.69 ± 4.82%) encapsulated with pea protein isolate-alginate (Xiao et al., 2020). This is related to the interactions between bacterial cells and wall materials. The surface of bacterial cells is mainly composed of glycoproteins, which can establish interaction with wall materials. The mixture of protein and carbohydrate has a supplementary embedding effect because they are combined through electrostatic attraction, hydrogen bond, and van der Waals forces (Mishra et al., 2001), which can improve the compactness of the wall materials and the delivery efficiency of microcapsules. Burgain et al. (2014) pointed out that the cell surface of L. rhamnosus GG has a stronger adhesive property with whey protein than micellar casein. On the other hand, the number of active cells of the SA-WPI microcapsule (9.47 log CFU/g) was significantly higher than the SA-SC microcapsule (9.28 log CFU/g) and SA microcapsule (9.25 log CFU/g), respectively. Rajam et al. (2012) observed that the cell viability of L. plantarum CICC 20022 encapsulated with a denatured WPI-alginate wall system was 9.48 log CFU/mL after freeze-drying, which was higher than that of undenatured WPI treatment (9.15 log CFU/mL). It indicates that heat-denatured whey protein has better-encapsulating properties in cells due to the unfolded structure of heat-induced denatured whey proteins exhibiting stronger film than native whey protein (Cai et al., 2018).

Fig. 1.

Fig. 1

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Effect of different microcapsule wall materials on encapsulation efficiency. Values are means of triplicate determination ± standard deviation. Different capital letters indicate a significant difference (P < 0.05) in encapsulation efficiency of L. plantarum CICC 20022 capsulated by three wall materials. Different lowercase letters displayed a significant difference (P < 0.05) in survivable cells among different microcapsule wall materials. sodium alginate, sodium alginate-sodium caseinate, and sodium alginate-whey protein isolate were abbreviated to SA, SA-SC, and SA-WPI

Texture profile analysis of microcapsules

It can be seen from Table 1, the texture properties of dry microcapsules encapsulated with SA and SA-SC were inferior to that of those encapsulated with SA-WPI, especially the hardness and gumminess. SA-WPI dry microcapsule showed significantly higher hardness and gumminess than that of SA microcapsule, which was related to the modification of closely folded protein molecules of whey protein from their natural form to unfolded state when the solution was heated to above 90 °C, thus protein–protein interactions, disulfide cross-linking and hydrogen bond formation. These interactions make the denatured whey protein stiffer, stronger, and more stretchable. This result was on the basis of the studies of Wang et al. (2020a, 2020b) and Zhou et al. (2014) On the other hand, SA-WPI and SA-SC microcapsules showed significantly higher springiness and cohesiveness compared to the SA encapsulated samples, respectively, which indicated that the addition of WPI or SC is more conductive to stabilizing the gel network structure of sodium alginate micelles (Tyuftin et al., 2020).

Table 1.

Qualitative and structural characteristics of microcapsules

Type of encapsulating material Hardness/N Springiness/mm Cohesiveness Gumminess/N
SA 82.39 ± 1.06b 0.59 ± 0.06b 0.54 ± 0.0b 40.40 ± 1.28b
SA-SC 84.16 ± 1.65ab 0.73 ± 0.04a 0.57 ± 0.0a 42.32 ± 1.33ab
SA-WPI 87.19 ± 2.49a 0.76 ± 0.04a 0.58 ± 0.0a 45.45 ± 2.04a
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Values are means of triplicate determination ± standard deviation. Different lowercase letters expressed remarkably differences in qualitative and structural characteristics of microcapsules among different coated wall materials. Sodium alginate, sodium alginate-sodium caseinate, and sodium alginate-whey protein isolate were abbreviated to SA, SA-SC, and SA-WPI

The moisture distribution and FT-IR characterization of beads

According to the difference in the degree of freedom of water, the binding state of water can be roughly divided into three categories: bound water (T21: 1–10 ms); immobilized water (T22: 10–100 ms), and free water (T23: 100–1000 ms) (Li et al., 2020). The moisture status proportion of microcapsules encapsulated with SA, SA-SC, and SA-WPI was shown in Table 2. It showed that the area percentage of bound water A21 of SA-SC beads and SA-WPI beads significantly increased as compared to that of SA, and the difference between SA-SC and SA-WPI was not significant. This phenomenon was attributed to the formation of polysaccharide-protein structure, which leads to limited mobility of water due to more water molecules being incorporated into the gel microstructure (Li et al., 2021). The A22 of the SA-WPI microcapsule was significantly reduced, and the corresponding A23 was significantly increased (P < 0.05). It also can be seen from Fig. 2(A), that the T22 and T23 of SA beads shift to a higher relaxation time after SC and WPI were added, which may be the hydrophobic effect of the film was fortified due to the combination of sodium alginate and polar groups in the protein. This caused the immobilized water in the beads to be released and converted into free water (Ullah et al., 2019).

Table 2.

The moisture distribution of microcapsules changed

Type of encapsulating material Percentage of bound water peak area A21/% Percentage of immobilized water peak area A22/% Percentage of free water peak area A23/%
SA 1.39 ± 0.46b 84.38 ± 1.47a 14.22 ± 1.60b
SA-SC 2.17 ± 0.31a 82.98 ± 0.41a 14.85 ± 0.48b
SA-WPI 2.18 ± 0.50a 77.32 ± 1.65b 20.90 ± 1.50a
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Different lowercase letters expressed remarkably differences in the moisture distribution of microcapsules changed among different coated wall materials

Fig. 2.

Fig. 2

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The moisture distribution (A), and FT-IR spectroscopy (B) of microparticles were influenced by different coated wall materials

The FT-IR of microcapsules encapsulated with SA, SA-SC, and SA-WPI were demonstrated in Fig. 2(B). The spectrum of SA beads presented three typical regions: the first at 3100–3520/cm reflected the changes in O–H and N–H stretching vibrations related to the degree of hydrogen bond association in the system; the second at 1630/cm corresponded to the stretching vibrations of C=C in alkene; the third at 1000–1100/cm is the fingerprint area of polysaccharides, which is mainly related to the coupling of C–O or the stretching of C–C and the bending mode of C–O–H (Flamminii et al., 2020). The positions of these characteristic peaks of microcapsules encapsulated with SA-SC and SA-WPI did not shift significantly compared to SA bead, indicating that new chemical bonds were not generated in the process of ion cross-linking to form colloidal particles, which was in accordance with studies of Zhou et al. (2014) Further observation, the intensity of three representative peaks of SA-SC and SA-WPI were aggrandized referred to SA, especially at 3100–3520/cm, probably the C=O and N–H in the protein are easily combined with O–H in polysaccharides to form intermolecular hydrogen bonds, which can reduce the free hydrogen bound to water in the capsule wall, thereby improve the water-resistance and mechanical properties of SA bead (Wang et al., 2014). However, there was also observed a weak stretching vibration of protein characteristic absorption peak at about 1550/cm in SA-WPI and SA-SC microcapsules other than in SA microcapsules. The transmittances of SA-WPI and SA-SC microcapsules were lower than that of SA microcapsules. And the shoulders at 2000–3000/cm in SA-WPI and SA-SC microcapsules were much sharper than that in SA microcapsules. This phenomenon may be related to the addition of WPI and SC. This result was consistent with the result of LF-NMR [Fig. 2(A)], which further proved that SA-WPI can better maintain the network structure of the sodium alginate gel by strengthening the intermolecular hydrogen bond interaction.

Morphological characterization by SEM

The surface morphology of the dried microcaps L. Plantarum CICC 20022 was shown in Fig. 3(A–D) at the magnifications of 10,000×g. It showed that microcapsules were a typical rod-like form with a flat surface and a size of about 1.0 × 0.5 μm [Fig. 3(A)]. The surface of the SA microcapsule [Fig. 3(B)] was rough and had pore-like gaps, and the L. plantarum CICC 20022 encapsulated with SA was mainly distributed on the surface of the colloidal particles. When protein was incorporated into the SA system, the morphology of the microcapsules changed significantly, the entrapped cells were invisible and it indicated that the SA-SC, SA-WPI wall matrix completely encapsulated the bacterial cell. Moreover, the surfaces of the microcapsules encapsulated with SA-WPI [Fig. 3(D)] were more smooth and tighter than SA-SC [Fig. 3(C)] and SA [Fig. 3(B)], respectively, indicating that a large amount of L. plantarum CICC 20022 was encapsulated in the inside of beads. In addition, the surface of SA-WPI beads [Fig. 3(D)] was glossier and more complete, showing a denser surface morphology. Xiao et al. (2020) pointed out that the freeze-drying process along with the loss of water and formation of ice crystals caused much damage (exposed probiotics, surface dents, blow-holes) to the probiotic microcapsules if the cryoprotectants were absent. Therefore, The SA-WPI microcapsules were more compact due to the whey protein-Ca2+ crosslinked alginate to prevent the deterioration or loss of core material and promote the forming of a smooth surface without any cracks (Dehkordi et al., 2020).

Fig. 3.

Fig. 3

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Scanning electron microscopy (SEM) images of the microcapsule encapsulated by different wall materials (A): Lactobacillus plantarum CICC 20022, (B): SA, (C): SA-SC, (D): SA-WPI). The SEM images were magnified 10,000 times

Survival of encapsulated L. plantarum CICC 20022 in simulated digestion and freeze-drying conditions

The viability of encapsulated L. plantarum CICC 20022 decreased continuously in the SGF containing low pH with 0.3% pepsin during 120 min digestion [Fig. 4(A)]. The cell viable number of SA microcapsule was decreased from 9.57 log CFU/g to 8.17 log CFU/g after 120 min, which was a decrease of 1.40 orders of magnitude, thus, the ability of SA wall material to protect L. plantarum CICC 20022 from the acid environment was limited. Meanwhile, the SA-WPI microcapsule has higher cell viability and remained at 8.31 log CFU/g after 120 min. This result was consistent with the research of Rajam et al. (2012). The cell viability of SA-SC and SA-WPI microcapsules was markedly higher than that of SA microcapsules (P < 0.05) for the first 1 h, which declared that the combination of SA and WPI attenuated the damage of cells from acid more effectively. The previous literature also showed that adding the WPI in wall material systems has the effect of reducing the negative effect of the acidic environment of L. bulgarius and L.paracasei (Huq et al., 2017).

Fig. 4.

Fig. 4

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Effects of simulated gastric fluid (A), simulated digestive juice (B) and vacuum freeze-drying (C) on the activity of Lactobacillus plantarum CICC 20022 encapsulated by different wall materials

The survival of the encapsulated cells in SDJ was depicted in Fig. 4(B). The survival of L. plantarum CICC 20022 encapsulated with SA-SC and SA-WPI showed more than 9.0 log CFU/g while they were exposed to SDJ during the whole process of 310 min, which is significantly higher than that of SA microcapsule. The reason was that the bile salts were limited diffusion in SA-WPI and SA-SC microcapsules and avoided damage to probiotics caused by bile salts (Huq et al., 2017). It was found that the SA encapsulated cells were reduced by more than 0.61 log CFU/g after 310 min in SDJ, while SA-WPI and SA-SC encapsulated cells decreased by about 0.33 and 0.52 log CFU/g. This change in wall materials stability may be attributed to the stronger and insoluble film-forming characteristics of SA-WPI (Table 1), which limits the release of L. plantarum CICC 20022 in SDJ. The considerable sustained release of encapsulated L. plantarum CICC 20022 containing denatured WPI and SA under simulated gastrointestinal conditions was also recorded by Rajam et al. (2012). Therefore, SA-WPI microcapsules have the best protective effect on L. plantarum CICC 20022 in the SDJ.

Survival cell numbers of freeze-dried microencapsulated L. plantarum CICC 20022 were given in Fig. 4(C). It was found that the viability of SA microcapsules dropped to 7.94 log CFU/g, while SA-SC and SA-WPI respectively increased by 0.25 log CFU/g and 0.52 log CFU/g, respectively. The high survival of SA-SC and SA-WPI microcapsules could be explained by possible protecting cells from osmotic shock, which is caused by delayed water replenishment caused by the microenvironment of microcapsules (Tonnis et al., 2015). Furthermore, the mixture of SA and WPI was beneficial to improve the mechanical strength and formed the physical barrier between the L. plantarum CICC 20022 and the external environment. Meanwhile, a viscous layer formed around L. plantarum CICC 20022 bacteria by using SA-WPI as an encapsulant, which can inhibit the formation of ice crystals, thereby enhancing the vitality of L. plantarum CICC 20022 in freeze-drying by delaying the damage of the severe environment to the L. plantarum CICC 20022 during processing and preservation (Song et al., 2014).

In the study, L. plantarum CICC 20022 was encapsulated with SA, SA-SC, and SA-WPI by extrusion technique, respectively. The yield of SA-WPI microcapsules was 70.40 ± 2.8%, significantly higher than that of SA-SC microcapsules (46.08 ± 2.5%) and SA microcapsules (42.72 ± 0.48%). Morphology of SA-WPI microcapsules and SA-SC microcapsules showed smooth surfaces while SA had thin surfaces. Texture, FT-IR, and LF-NMR analysis demonstrated that the hydrogen bonding ability and network structure of the SA were improved by the addition of WPI. The survival of L. plantarum CICC 20022 encapsulated with SA-WPI was improved during freeze-drying and simulated gastrointestinal digestion, remaining more than 8.2 log CFU/g. Therefore, it can be concluded that the SA-WPI system could be available as wall materials and be used as the vector of L. plantarum CICC 20022 in food applications for providing higher survival after freeze-drying and simulated gastrointestinal digestion.

Acknowledgements

This study was funded by the Study on Zhengzhou Science and Technology Bureau Plan (2020CXZX0086).

Author contributions

LZ: Writing, Reviewing and Editing. PT: Conceptualization, Methodology, Software, Data curation, Writing, Original draft preparation. SL: Supervision, Software, Validation. XW: Conceptualization, Software, Data curation. WZ: Visualization, Investigation.

Declarations

Conflict of interest

We declare no competing financial interest, and our manuscript "Sodium alginate-based wall materials microencapsulated Lactobacillus plantarumCICC 20022: Characteristics and survivability study"is not under review in any other journals.

Footnotes

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