Thallus-exopolysaccharide co-embedded envelope state microcapsule and preparation method thereof

Abstract

The invention discloses a thallus-exopolysaccharide co-embedded envelope state microcapsule and a preparation method thereof, and belongs to the technical field of microcapsule preparation. The preparation method comprises the following steps: (1) dissolving sodium alginate, pectin and whey protein isolate, stirring for 3-4 hours, and sterilizing to obtain a composite wall material solution; and (2) inoculating the activated bacterial liquid into a composite wall material solution, then adding the enveloping matrix exopolysaccharide, uniformly stirring and mixing, then adding a calcium chloride solution to form gel, then removing redundant gel, and cleaning to prepare the thallus-exopolysaccharide co-embedded enveloping state microcapsule. The co-embedded microcapsule prepared by the invention not only has good storage stability, high temperature resistance, osmotic pressure stress resistance and the like, but also can effectively promote the proliferation of thalli.

Description

Technical Field

[0001] The present invention belongs to the technical field of microcapsule preparation, and in particular relates to a membrane-state microcapsule co-encapsulated with bacteria and extracellular polysaccharide and a preparation method thereof. Background Art

[0002] In the field of biotechnology, microencapsulation technology, which emerged alongside the development of immobilized cell technology, involves encapsulating biological materials such as cells using microcapsules. Microencapsulation technology refers to the use of natural or synthetic polymers to encapsulate tiny core materials—solid, liquid, or even gaseous—into semipermeable or sealed microcapsules with diameters ranging from 1 to 5000 μm (typically 5 to 400 μm). Microencapsulation can protect probiotics from bacteriophages, improve their survival rate during freeze-drying, and enhance storage stability. Encapsulating probiotics within a microencapsulation wall material can also reduce environmental damage to the cells and enable the reuse of multiple batches of probiotic cells. However, currently manufactured microencapsulated products often suffer from issues such as insufficient stability and poor bacterial protection. Summary of the Invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a bacterial-extracellular polysaccharide co-encapsulated membrane-state microcapsule and a preparation method, which can not only effectively improve the viability of the bacterial body and maintain stability, but also give the microcapsule more functional activity.

[0004] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: The present invention aims to provide a method for preparing bacterial-exopolysaccharide co-encapsulated membrane-state microcapsules, which comprises the following steps: (1) Dissolve sodium alginate, pectin, and whey protein isolate, stir for 3-4 hours, and then sterilize to prepare a composite material solution; (2) The activated bacterial solution is inoculated into the composite wall material solution, and then the membrane matrix extracellular polysaccharide is added. After stirring and mixing, calcium chloride solution is added to form a gel. The excess gel is then removed and washed to obtain a membrane-state microcapsule co-encapsulated with bacteria and extracellular polysaccharide.

[0005] Furthermore, in step (1), the mass ratio of sodium alginate, pectin and whey protein isolate is 1.5~3:1.5~3:2~4.

[0006] Furthermore, in step (2), the exopolysaccharide of the membrane matrix is ​​derived from Lactobacillus plantarum LR-1, and its inoculation amount is 1-1.5%.

[0007] Furthermore, the preparation process of the membrane matrix extracellular polysaccharide is as follows: S1. Strain culture The activated Lactobacillus plantarum LR-1 was inoculated into the liquid culture medium and dispersed in a 6-well polystyrene microtiter plate (Costar 3524, Corning) at a volume of 10 mL per well, and cultured statically at 37 °C for 48 h. S2. Extraction and separation of extracellular polysaccharides from the membrane matrix After centrifugation, the supernatant was collected and neutral protease was added to react for 2-3 h. The supernatant was then collected by centrifugation, and anhydrous ethanol was added to the supernatant, and the polysaccharide was precipitated at 4°C. The precipitate was collected by centrifugation, redissolved with deionized water, and separated by dialysis to obtain the membrane matrix extracellular polysaccharide.

[0008] Furthermore, the liquid culture medium in S1 is skim milk or mMRS liquid culture medium, and the cultured strain is a model strain or a planktonic strain.

[0009] Furthermore, the isolated extracellular polysaccharide of the membrane matrix is ​​TB EPS0 (which is cultured in skim milk). L. paraplantarum LR-1 capsule matrix derived polysaccharide) or mB EPS0 (mMRS cultured L. paraplantarum LR-1 capsule matrix derived polysaccharide).

[0010] Furthermore, in step (2), the final concentration of the extracellular polysaccharide of the membrane matrix in the composite wall material solution is 200-800 μg / mL.

[0011] Furthermore, the concentration of the calcium chloride solution added in step (2) is 700-750 mmol / L.

[0012] Another object of the present invention is to provide a membrane-encapsulated microcapsule containing bacteria and extracellular polysaccharide, which is prepared by the above method.

[0013] Beneficial effects of the present invention: Microencapsulation co-encapsulation technology allows for the co-encapsulation of active substances and bacterial cells. Therefore, selecting the appropriate substance to serve as both a functional core material and a protective wall material for microencapsulation of live bacteria achieves two goals with one stone, offering promising application prospects. The co-encapsulated microcapsules produced by this invention not only exhibit excellent storage stability, high temperature resistance, and resistance to osmotic stress, but also effectively promote bacterial proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The UV spectrum analysis diagrams of different exocellular polysaccharides; Figure 2 Fourier transform infrared spectra of different exopolysaccharides; Figure 3 This is an electron microscope image of the co-embedded microcapsules prepared in the present invention; Figure 4 This is an infrared spectrum analysis diagram of the co-embedded microcapsules prepared in the present invention; Figure 5 This is an X-ray diffraction analysis diagram of the co-embedded microcapsules prepared in the present invention; Figure 6 This is a live bacteria detection diagram of the co-embedded microcapsules prepared by the present invention; Figure 7 This is a graph showing the heat resistance test results of the co-embedded microcapsules prepared in the present invention; Figure 8 This is a graph showing the osmotic stress resistance test results of the co-embedded microcapsules prepared in the present invention; Figure 9 This is a graph showing the freeze-drying stress resistance test results of the co-embedded microcapsules prepared in the present invention; Figure 10 This is a diagram showing the results of a simulated gastrointestinal stress test of the co-embedded microcapsules prepared in the present invention; Figure 11 This is a graph showing the storage stability test results of the co-embedded microcapsules prepared in the present invention; Figure 12 This is a graph showing the release characteristics test results of the co-embedded microcapsules prepared in the present invention. DETAILED DESCRIPTION

[0015] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0016] Example 1 Preparation of membrane matrix and supernatant exopolysaccharide 1. Exopolysaccharide of membrane matrix S1. Cultivation of Encapsulated Strain The activated plantarum-like Lactobacillus LR-1 was inoculated into skim milk and mMRS liquid culture medium at a 1% inoculum volume, respectively, and dispersed into a 6-well polystyrene microtiter plate (Costar 3524, Corning) at a volume of 10 mL per well. The plate was cultured at 37°C for 48 h to obtain the encapsulated strain.

[0017] S2. Extraction and separation of extracellular polysaccharides from the membrane matrix The encapsulated strain was centrifuged at 5000 r / min for 10 minutes, and the supernatant was collected. Neutral protease (final concentration 2 g / L) was then added to the supernatant, and the reaction was shaken for 2 hours to degrade protein impurities. After the reaction was complete, the mixture was centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. Three volumes of pre-chilled anhydrous ethanol were added, and the mixture was allowed to stand at 4°C for 12 hours to allow the polysaccharide to precipitate. After precipitation, the mixture was centrifuged again at 8000 r / min for 15 minutes, the supernatant removed, and the precipitate reconstituted with an appropriate amount of deionized water. The reconstituted solution was transferred to a dialysis bag with a molecular weight cutoff of 8000-12000 Da and dialyzed at 4°C for 24 hours, with the deionized water replaced every 4 hours. The dialyzed solution was pre-frozen at -80 °C for 24 h, then freeze-dried in a vacuum freeze dryer (-40 °C, 40 MPa) for 24 h, and then eluted and separated by cellulose DEAE-52 ion exchange chromatography column to obtain the extracellular polysaccharides TB EPS0 and mB EPS0 of the membrane-bound LR-1 derived from skim milk and mMRS medium.

[0018] 2. Exopolysaccharides in the planktonic supernatant S1. Planktonic strain culture The activated LR-1 was inoculated into skim milk and mMRS liquid culture medium at a 1% inoculum volume, respectively, and cultured at 37°C and 150 rpm for 48 h to obtain a planktonic strain.

[0019] S2. Extraction and separation of extracellular polysaccharides from the supernatant The planktonic strain was centrifuged at 5000 r / min for 10 minutes, and the supernatant was collected. Neutral protease (final concentration 2 g / L) was then added to the supernatant, and the reaction was shaken for 2 hours to degrade protein impurities. After the reaction was complete, the mixture was centrifuged at 8000 r / min for 15 minutes, and the supernatant was collected. Three volumes of pre-chilled anhydrous ethanol were added, and the mixture was allowed to stand at 4°C for 12 hours to allow the polysaccharide to precipitate. After precipitation, the mixture was centrifuged again at 8000 r / min for 15 minutes, the supernatant removed, and the precipitate reconstituted with an appropriate amount of deionized water. The reconstituted solution was transferred to a dialysis bag with a molecular weight cutoff of 8000-12000 Da and dialyzed at 4°C for 24 hours, with the deionized water replaced every 4 hours. The dialyzed solution was pre-frozen at -80°C for 24 h, then freeze-dried in a vacuum freeze dryer (-40°C, 40 MPa) for 24 h, and then eluted and separated by cellulose DEAE-52 ion exchange chromatography column to obtain planktonic LR-1 exopolysaccharides TP EPS0 and mP EPS0 derived from skim milk and mMRS medium.

[0020] The specific elution method of the above-mentioned extracellular polysaccharide is as follows: When the NaCl concentration of the elution solution was 0 mol / L, more extracellular polysaccharide components were eluted, and EPS was not eluted at other salt concentrations. Therefore, the EPS eluted from the skim milk membrane state and the planktonic state LR-1 were TB EPS0 (skim milk cultured) and TB EPS1 (skim milk cultured). L. paraplantarum LR-1 capsule matrix derived polysaccharide) and TP EPS0 (skim milk cultured polysaccharide) L. paraplantarum LR-1 supernatant-derived polysaccharide).

[0021] When the NaCl concentration of the elution solution is 0 mol / L, the recovery rate of the eluted extracellular polysaccharide components is relatively high. The polysaccharide content eluted by the 0.1 mol / L salt solution is small and difficult to collect. However, the polysaccharide is not eluted at other salt solution concentrations. Therefore, the EPS of the mMRS membrane state and the planktonic LR-1 eluted by deionized water are mB EPS0 (mMRS cultured) and mB EPS1 (mMRS cultured). L. paraplantarum LR-1 capsule matrix derived polysaccharide) and mP EPS0 (mMRS cultured L. paraplantarum LR-1 supernatant-derived polysaccharide).

[0022] Example 2 A bacterial cell-exopolysaccharide co-encapsulated membrane-state microcapsule, the preparation method of which is as follows: (1) Weigh 3 g of sodium alginate, 3 g of pectin, and 4 g of whey protein isolate and dissolve them in 200 mL of distilled water. Stir with a stirring bar for 3 h to fully dissolve them. Sterilize at 105°C for 3 min to prepare a composite material solution. (2) The activated LR-1 bacterial solution was inoculated into the composite wall material at a ratio of 1%, and then the extracellular polysaccharide TB EPS0 obtained by skim milk culture was added according to the volume of the composite wall material solution at concentrations of 200 μg / mL, 500 μg / mL, and 800 μg / mL. The mixture was fully shaken and mixed on a vortexer. The mixture was allowed to stand for a period of time until the bubbles dissipated. A 1 mL medical sterile syringe was used to drop the suspension into a 750 mmol / L CaCl2 solution. The suspension was allowed to gel for 20 min, and the excess CaCl2 solution was removed. The microcapsules were washed three times with sterile water. The microcapsules were then cultured in a polystyrene six-well cell culture plate containing MRS culture medium, with 5 mL of culture medium and 1 g of microcapsules per well. The microcapsules were incubated in a 37°C incubator for 48 h until the final concentration reached 10 10 CFU / g, and washed three times with sterile water to obtain endogenous microcapsules with composite wall materials co-encapsulating TB EPS0 and SPW-LR-1-TB EPS0.

[0023] Example 3 A bacterial cell-exopolysaccharide co-encapsulated membrane-state microcapsule, the preparation method of which is as follows: (1) Weigh 3 g of sodium alginate, 3 g of pectin, and 4 g of whey protein isolate and dissolve them in 200 mL of distilled water. Stir with a stirring bar for 3 h to fully dissolve them. Sterilize at 105°C for 3 min to prepare a composite material solution. (2) The activated LR-1 bacterial solution was inoculated into the composite wall material at a ratio of 1%, and then the extracellular polysaccharide mB EPS0 obtained by mMRS culture was added according to the volume of the composite wall material solution at concentrations of 200 μg / mL, 500 μg / mL, and 800 μg / mL. The mixture was fully shaken and mixed on a vortexer. The mixture was allowed to stand for a period of time until the bubbles dissipated. A 1 mL medical sterile syringe was used to drop the suspension into a 750 mmol / L CaCl2 solution. The suspension was allowed to gel for 20 min, and the excess CaCl2 solution was removed. The microcapsules were washed three times with sterile water. The microcapsules were then cultured in a polystyrene six-well cell culture plate containing MRS culture medium, with 5 mL of culture medium and 1 g of microcapsules per well. The microcapsules were incubated in a 37°C incubator for 48 h until the final concentration reached 10 10 CFU / g, and washed three times with sterile water to obtain endogenous microcapsules with composite wall materials co-encapsulating TP EPS0 and SPW-LR-1-mB EPS0.

[0024] Comparative Example 1 The difference from Example 2 is that the planktonic supernatant extracellular polysaccharide TP EPS0 was used, and the rest of the process was the same as Example 2.

[0025] Comparative Example 2 The difference from Example 2 is that the planktonic supernatant exopolysaccharide mP EPS0 was used, and the rest of the process was the same as Example 2.

[0026] Test example 1. Detection of extracellular polysaccharides (1) Ultraviolet spectrum analysis The purified 4 groups of extracellular polysaccharides were dissolved in distilled water to prepare 1 mg / mL polysaccharide solution, and the UV full-band scanning was performed at 200-400 nm. The results are shown in Figure 1 .

[0027] like Figure 1As shown, none of the four EPS samples showed significant characteristic absorption peaks at wavelengths of 260 nm and 280 nm. This spectral feature confirms that the content of protein and nucleic acid substances in the extracted polysaccharide fraction is extremely low. Specifically, the absorbance baseline at 260 nm is stable, consistent with the characteristics of purified polysaccharides reported in the literature, indicating that there is no nucleic acid contamination in the sample. Although a weak signal response was observed at 280 nm, the absorption intensity was significantly lower than the detection threshold of typical proteins, suggesting that only trace amounts of protein impurities may be present.

[0028] (2) Fourier transform infrared spectroscopy Fourier Transform Infrared Spectroscopy (FT-IR) was used to characterize the structures of the four main components of the purified EPS. The specific steps are as follows: First, the EPS sample, which had been dried to a constant mass, was accurately weighed and mixed with potassium bromide (KBr) at a mass ratio of 1:40. The mixture was then ground thoroughly in an agate mortar for 5 minutes until uniformly mixed. The mixed powder was then pressed into transparent sheets using a hydraulic tablet press. Finally, the prepared sample was placed in the sample chamber of an infrared spectrometer and analyzed at 4000-400 cm -1 The full band scan is performed within the wave number range to obtain the infrared absorption spectrum of the sample. The results are shown in Figure 2 .

[0029] like Figure 2 As shown, the four EPS at 3400 cm –1 There are strong absorption peaks at 2930 cm −1 The absorption peaks near 1640 cm are caused by CH stretching vibration. Both absorption peaks are characteristic peaks of polysaccharides. −1 The absorption peak near 1410 cm is the characteristic peak of bound water, indicating that there is a strong interaction between polysaccharide and water molecules. -1 The absorption peak near 1148 cm comes from the deformation vibration of CH. -1 and 1030 cm -1 Peaks indicating the presence of pyranose in the four polysaccharides. 846 and 927 cm -1 The absorption peaks at 1730 cm indicate the presence of α and β glycosidic bonds, respectively. -1 There is no absorption peak at , indicating that the four EPS do not contain uronic acid and are neutral polysaccharides.

[0030] 2. Microcapsule morphology and structure detection (1) Electron microscopy Using a scanning electron microscope (Gemini 300, ZEISS), the fully dried samples (microcapsules prepared in Examples 2-5) were fixed to a conductive copper sample stage, gold-coated using a vacuum sputtering coater, and then placed in the SEM sample chamber. A series of microscopic images were acquired at different magnifications to analyze the multi-scale surface morphology of the samples. The results are shown in Figure 2. Figure 3 , among them, (A) SPW; (B) SPW-LR-1; (C) SPW-LR-1-TB EPS0200 (μg / mL); (D) SPW-LR-1-TBEPS0500 (μg / mL); (E) SPW-LR-1-TB EPS0800 (μg / mL); (F) SPW-LR-1-TP EPS0200 (μg / mL); (G) SPW-LR-1-TP EPS0500 (μg / mL); (H) SPW-LR-1-TP EPS0800 (μg / mL); (I) SPW-LR-1-mBEPS0200 (μg / mL); (J) SPW-LR-1-mB EPS0500(μg / mL); (K) SPW-LR-1-mB EPS0800(μg / mL); (L) SPW-LR-1-mP EPS0200 (μg / mL); (M) SPW-LR-1-mP EPS0500 (μg / mL); (N) SPW-LR-1-mBEPS0800 (μg / mL).

[0031] like Figure 3 As shown in the figure, compared with the control SPW, the probiotic microcapsules containing LR-1 were more rounded after freeze-drying, indicating that the combination of LR-1 loading and the wall material stabilized the microcapsule structure. Compared with microcapsules without EPS, microcapsules co-encapsulated with EPS had a sporadic particle structure and a more compact surface. Furthermore, as the co-encapsulated EPS concentration increased, the microcapsule surface became more rounded and compact.

[0032] (2) Infrared spectroscopy analysis 1 mg of each freeze-dried microcapsule sample from Examples 2 to 5 was placed in an agate mortar, thoroughly mixed and ground with 200 mg of KBr, pressed into transparent sheets, and placed on a sample holder. The resulting samples were analyzed using a Fourier transform infrared spectrometer (Spectrum Two FT-IR, PerkinElmer) with a resolution of 4 cm and a spectral range of 4000–500 cm -1 , Origin software was used to analyze the data and draw the map. The results are shown in Figure 4, In the figure, (A) SPW-LR-1-TB EPS0; (B) SPW-LR-1-TP EPS0; (C) SPW-LR-1-mB EPS0; (D) SPW-LR-1-mP EPS0.

[0033] like Figure 4 As shown, the microcapsules prepared in each embodiment are approximately 3400 cm -1 The broad peak of the blank microcapsule indicates the OH stretching vibration, which is the characteristic peak of OH. -1 The bands at 3418-3435 cm-1 are attributed to the stretching of OH- in sodium alginate, and the hydroxyl stretching peak of the co-embedded EPS microcapsules shifts to a lower wavenumber, shifting to 3418-3435 cm-1. -1 This is due to the strong hydrogen bonds in the polysaccharide ion complex or the hydrogen bonds formed between EPS and the wall material SPW. Substances rich in hydroxyl groups tend to form a large number of hydrogen bonds, making them more prone to vibration, thus producing a wider infrared spectrum band. The two absorption peaks are at 2900 cm -1 and 2800 cm -1 , mainly originating from CH stretching vibrations, which are characteristic of the polymer matrix. 1600 cm -1 and 1400 cm -1 The absorption peaks are related to the symmetrical vibration of carboxylate ions on alginate and the mixture.

[0034] After adding EPS, microcapsules were formed at 1630 cm -1 -1642 cm -1 The longitudinal peak at 1000 cm-1 is deeper, but its peak is smaller than that of the blank microcapsules, indicating that EPS may be successfully combined with sodium alginate. -1 The weak absorption peak near the pyran ring is mainly the CO vibration absorption peak of the main chain. Protein has a peak at 1600-1690 cm -1 and 1480-1575 cm -1 The characteristic spectra at 1645 cm-1 correspond to amide I (CO stretching) and amide II (NH bond), respectively. The amide I peaks of SPW-LP and SPW-LP-EPS appear at 1645 cm-1, respectively. -1 The positions of the amide II peaks of the SPW-LP and SPW-LP-EPS groups shifted. These changes may be attributed to the electrostatic and hydrophobic interactions between SPW-LP and EPS.

[0035] (3) X-ray diffraction analysis The XRD patterns of the microcapsule samples prepared in Examples 2 to 5 were analyzed using an X-ray diffractometer. Figure 5 .

[0036] The parameters were set as: Cu-Ka ray as the excitation source, scanning range 5°-90°(20), 40 kV maintenance tube voltage, 40 mA tube current, and scanning speed set to 2 (°) / min.

[0037] like Figure 5 As shown, a characteristic peak at 2θ = 33° was observed in all LR-1-encapsulated microcapsules, indicating successful encapsulation of LR-1. Furthermore, all microcapsules exhibited a semicrystalline structure. The addition of EPS did not alter the structural properties of the microcapsules. The WPI-GA complex coacervates formed a semicrystalline structure with a slightly sharper diffraction peak. The semicrystalline structure plays an important role in the properties and performance of microcapsules. Semicrystalline materials typically possess high mechanical strength and toughness, making the microcapsules more durable during handling and application. Furthermore, the semicrystalline structure improves the thermal stability of the microcapsules, enabling them to maintain good performance even at higher temperatures. Due to the distinct properties of the crystalline and amorphous regions within the semicrystalline structure, the microcapsules can achieve better controlled release, enabling the release of drugs or active ingredients at a specific rate. After successful co-encapsulation of EPS with the microcapsules, no characteristic peaks were observed in the co-encapsulated microcapsules, indicating that EPS likely interacts with the wall material and exists in an amorphous form.

[0038] 3. Viable bacteria count detection SPW-LR-1, SPW-LR-1-TB EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), SPW-LR-1-TP EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), SPW-LR-1-mB EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), and SPW-LR-1-mP EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL) microcapsules prepared according to the above method were cultured in a constant temperature incubator at 37°C for 48 h. The counting method was as follows: 1.0 g of microcapsules was dissolved in 10 mL of 0.1 mol / L sodium citrate to fully release the bacteria. After incubation at 37°C for 48 h, the viable bacteria were counted by plate count method. The results are shown in Table 2. Figure 6 .

[0039] like Figure 6As shown in the figure, with the increase of EPS concentration, the number of viable bacteria in the co-encapsulated EPS group gradually increased compared with that in SPW-LR-1. When the EPS concentration of each group reached 800 μg / mL, the number of viable bacteria in each group of microcapsules co-encapsulated with EPS in situ coating reached the maximum value. Among them, the viable bacterial counts of SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mBEPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) groups were 10.24 ± 0.02 lg CFU / g, 10.20 ± 0.04 lg CFU / g, 10.38 ± 0.01 lg CFU / g, and 10.26 ± 0.01 lg CFU / g, respectively, which increased by 0.15 lg CFU / g, 0.13 lg CFU / g, 0.31 lg CFU / g, and 0.19 lg CFU / g compared with SPW-LR-1, respectively.

[0040] It can be seen that the microcapsule groups SPW-LR-1-mB EPS0800 (μg / mL) and SPW-LR-1-mP EPS0800 (μg / mL) co-embedded with mB EPS0 and mP EPS0 have higher viable bacterial counts than the microcapsule groups SPW-LR-1-TB EPS0800 (μg / mL) and SPW-LR-1-TP EPS0800 (μg / mL) co-embedded with TB EPS0 and TP EPS0. This shows that the mMRS-derived EPS has a stronger effect on the proliferation of strains than the skim milk-derived EPS. At the same time, under the conditions of mMRS culture medium, the viable bacterial count of the group SPW-LR-1-mB EPS0800 (μg / mL) co-embedded with mMRS-derived membrane matrix EPS was higher than that of the group SPW-LR-1-mP EPS0800 (μg / mL) co-embedded with mMRS-derived supernatant EPS. EPS0800 (μg / mL), which shows that under the same culture medium conditions, the proliferation effect of the membrane matrix exopolysaccharide is stronger than that of the planktonic supernatant exopolysaccharide.

[0041] 4. Tolerance testing Biofilm-LR-1, SPW-LR-1, SPW-LR-1-TB EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), SPW-LR-1-TP EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), SPW-LR-1-mB EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), SPW-LR-1-mP EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL) were subjected to environmental stress, such as heat stress, osmotic stress, freeze-drying, and simulated gastrointestinal stress in vitro. Before the experiment, the number of viable bacteria in each group was uniformly adjusted to 10 10 CFU / g.

[0042] (1) Heat stress resistance Each group was treated at 63°C for 30 min, and the viable bacteria were counted using the plate count method, and the survival rate was calculated. The results are shown in Figure 7 .

[0043] like Figure 7 As shown in the figure, the viable bacterial count of Biofilm-LR-1 after treatment at 63 ℃ was 23.33 ± 3.01%. The figure shows that with the increase of EPS concentration, the survival rate of the co-encapsulated EPS group gradually increased. When the EPS concentration of each group reached 800 μg / mL, the viable bacterial rate of the co-encapsulated EPS in situ film-state microcapsules reached the maximum. The viable bacterial rates of the SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) groups were 44.66 ± 0.57%, 43.80 ± 0.52%, 46.70 ± 1.15%, and 43.63 ± 0.98%, respectively, which increased by 7.66%, 6.8%, 9.7%, and 6.63% compared with SPW-LR-1, respectively. Among them, the survival rates of the co-encapsulated mMRS-derived mB EPS0 and mP EPS0 groups SPW-LR-1-mB EPS0800 (μg / mL) and SPW-LR-1-mP EPS0800 (μg / mL) were higher than those of the co-encapsulated skim milk-derived TB EPS0 and TPEPS0 groups SPW-LR-1-TB EPS0800 (μg / mL) and SPW-LR-1-TP EPS0800 (μg / mL). This shows that EPS from different sources have different protective properties under heat-resistant conditions, and SPW-LR-1-mB EPS0800 (μg / mL) has the highest viable bacterial survival rate under heat-resistant stress conditions. (2) Resistance to osmotic stress The 14 treatment groups were placed in 10% and 20% NaCl solutions for 2.0 h, and the viable bacteria were counted using the plate count method and the survival rate was calculated. The results are shown in Figure 8 , in the figure, (A) 10% osmotic stress; (B) 20% osmotic stress.

[0044] Depend on Figure 8 It can be seen that under the condition of 10% NaCl osmotic pressure, the viable bacterial rate of Biofilm-LR-1 was 39.26 ± 0.97%, and the viable bacterial survival rate increased by 13.74% after microencapsulation. With the increase of EPS concentration, the number of viable bacteria in the co-encapsulated EPS groups gradually increased. When the EPS concentration of each group reached 800 μg / mL, the survival rate of the co-encapsulated EPS in situ membrane-state microcapsules reached the maximum. The viable bacteria rates of the SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) groups were 56.93 ± 2.30%, 54.84 ± 1.58%, 58.63 ± 1.31%, and 53.64 ± 2.32%, respectively, which increased by 3.93%, 1.84%, 5.63%, and 0.64% compared with those of the SPW-LR-1 groups, respectively. EPS0800 (μg / mL) had the highest survival rate. Under the same culture medium conditions, the coating matrix EPS had a higher protective property, which may be due to the strong negative charge (lower Zeta potential) of the coating matrix EPS and the surface charge of the probiotics (usually weakly negative) forming a gradient electrostatic interlocking, optimizing the internal spatial distribution of the microcapsules.

[0045] (3) Resistance to freeze-drying stress The 14 treatment groups were freeze-dried for 36 hours. The freeze-dried microcapsules were placed in epoxies containing sodium citrate (0.1 mol / L) and placed at 37°C for 2 hours to allow the wall material to disintegrate and release cells. The plate count method was used to count viable bacteria and calculate the survival rate. Biofilm-LR-1 was used as a control to analyze its freeze-drying tolerance. The results are shown in Figure 9 .

[0046] like Figure 9As shown in the figure, the live bacterial rate of Biofilm-LR-1 was 53.80 ± 056%, and the live bacterial rate was significantly increased to 75.63 ± 3.71% after microencapsulation. With the increase of EPS concentration, the number of viable bacteria in the co-embedded EPS in situ coated microcapsules gradually increased. When the EPS concentration of each group reached 800 μg / mL, the survival rate of the co-embedded EPS in situ coated microcapsules reached the maximum. The viable bacterial rates of the SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) groups were 87.43 ± 0.81%, 80.93 ± 0.70%, 92.10 ± 1.78%, and 85.23 ± 1.00%, respectively, which increased by 11.8%, 5.3%, 16.47%, and 9.6% compared with the SPW-LR-1 groups, respectively. This shows that the co-embedded mMRS-derived coating matrix EPS (mB Compared with the co-encapsulated skim milk-derived capsule matrix EPS (TB EPS0) and skim milk-derived supernatant EPS (TP EPS0) microcapsule groups SPW-LR-1-TB EPS0800 (μg / mL) and SPW-LR-1-TP EPS0800 (μg / mL), the comparison between the two showed that under the same culture medium conditions, the protective effect of capsule matrix exopolysaccharide was stronger than that of supernatant exopolysaccharide under freeze-drying environmental stress.

[0047] (4) In vitro simulation of gastrointestinal stress An in vitro digestion model was used to evaluate the stability of the samples in the gastrointestinal environment. Simulated digestive fluid was prepared: artificial gastric fluid (SGF) contained 3.2 g / L pepsin and 2.0 g / L sodium chloride (pH = 1.2), and artificial intestinal fluid (SIF) consisted of 10.0 g / L trypsin and 6.8 g / L potassium dihydrogen phosphate (pH = 7.5 ± 0.1). The 14 groups were subjected to gastric tolerance test (37 ℃, 2h) and intestinal tolerance assessment (37 ℃, 2h). After digestion treatment, the survival rate was determined by plate counting method, and the stability differences of different treatment groups under simulated gastrointestinal conditions were analyzed. The results are shown in Figure 10 In the figure, (A) simulated gastric fluid stress for 2 h; (B) simulated intestinal fluid stress for 2 h.

[0048] like Figure 10As shown in the results, the survival rate of Biofilm-LR-1 was 18.43 ± 2.10% after 2 h of treatment in simulated gastric fluid, and reached 71.95 ± 1.28% after microencapsulation, a significant increase. The number of viable bacteria in the EPS-encapsulated microcapsules gradually increased with increasing EPS concentrations. The survival rate reached its maximum when the EPS concentration in each group reached 800 μg / mL.

[0049] The viable bacterial rates of the EPS in situ coated microcapsules SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) were 78.12 ± 2.31%, 79.56 ± 0.40, 86.80 ± 0.66%, and 80.94 ± 3.17%, respectively. Compared with the SPW-LR-1 group, all groups increased to varying degrees, with SPW-LR-1-mB EPS0800 showing the highest increase of 14.85%. After 2 h of treatment in simulated intestinal fluid, the survival rate of Biofilm-LR-1 was 13.12 ± 2.52%, while the survival rate of SPW-LR-1 after microencapsulation could reach 74.24 ± 1.59%. With the increase of EPS concentration, the number of viable bacteria in the co-encapsulated EPS in situ coated microcapsules gradually increased. When the EPS concentration in each group reached 800 μg / mL, the survival rate of the co-encapsulated EPS in situ coated microcapsules reached its maximum. The viable bacterial rates of the SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) groups were 84.23 ± 0.59%, 83.68 ± 0.42%, 87.49 ± 0.92%, and 85.03 ± 1.43%, respectively. This trend was consistent with the gastric fluid tolerance test mentioned above.

[0050] Compared with unencapsulated Biofilm-LR-1 and SPW-LR-1, co-encapsulation of EPS significantly increased gastrointestinal resistance, with mMRS medium-derived exopolysaccharides exhibiting a stronger protective effect. Furthermore, under mMRS medium conditions, the group co-encapsulating mMRS-derived membrane matrix EPS (mB EPS0) SPW-LR-1-mB EPS0800 (μg / mL) exhibited better tolerance than the group co-encapsulating mMRS-derived planktonic supernatant EPS (mP EPS0) SPW-LR-1-mP EPS0800 (μg / mL). This suggests that membrane matrix exopolysaccharides exhibit a superior protective effect compared to planktonic supernatant exopolysaccharides.

[0051] 5. Storage stability of microcapsules The freeze-dried microcapsules of Biofilm-LR-1, SPW-LR-1, SPW-LR-1-TB EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), SPW-LR-1-TP EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), SPW-LR-1-mBEPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL), and SPW-LR-1-mP EPS0 (200 μg / mL, 500 μg / mL, 800 μg / mL) were stored at −20°C, 4°C, and 25°C for 28 days. Samples were taken every seven days, and the viable bacteria were counted by the plate count method to measure the shelf life. The results are shown in Table 2. Figure 11 , in the figure, (A) -4 ℃; (B) -20 ℃; (C) 25 ℃.

[0052] like Figure 11As shown in the data, the storage stability was significantly improved after microencapsulation under the three temperature storage conditions, and the survival rate of the co-embedded EPS in situ membrane-coated microcapsule group was higher than that of the SPW-LR-1 group, and the storage stability also increased with the increase of the co-embedded EPS concentration. After storage at -20℃ for 4 weeks, the survival rate of Biofilm-LR-1 was 38.46 ± 2.42%, and the survival rate after microencapsulation could reach 65.16 ± 0.58%, with a significant increase in the survival rate. When the EPS concentration of each group reached 800 μg / mL, the survival rate of the co-encapsulated EPS in situ film-state microcapsules reached the maximum. The viable bacterial rates of SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) groups were 65.06 ± 1.06%, 66.36 ± 3.15%, 73.23 ± 1.12%, and 71.56 ± 4.80%, respectively. The experimental results showed that SPW-LR-1-mB EPS0800 (μg / mL) > SPW-LR-1-mP EPS0800 (μg / mL) > SPW-LR-1-TB EPS0800 (μg / mL) > SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL) increased the survival rate by 8.07% compared with SPW-LR-1.

[0053] The results of storage experiments at -4 ℃ and 25 ℃ showed that, compared with the groups of Biofilm-LR-1 and SPW-LR-1, as well as the EPS in situ microcapsules co-embedded with them, the order of SPW-LR-1-mB EPS0800 (μg / mL) > SPW-LR-1-mP EPS0800 (μg / mL) > SPW-LR-1-TB EPS0800 (μg / mL) > SPW-LR-1-TP EPS0800 (μg / mL) > SPW-LR-1 > Biofilm-LR-1 was observed. The survival rate of viable bacteria at different temperatures was ranked as follows: -20 ℃ > 4 ℃ > 25 ℃. Low temperature storage was more conducive to ensuring the stability of the microcapsules and the activity of the strains. Therefore, -20 ℃ was chosen to store the microcapsules.

[0054] 6. Microcapsule release characteristics The freeze-dried microcapsules were immersed in simulated gastric fluid for 4 h, and their transparency was measured every hour at 600 nm using a UV spectrophotometer to study the dissolution rate of the microcapsules in simulated gastric fluid. Subsequently, the freeze-dried microcapsules were placed in simulated intestinal fluid for 2 h, and the transparency was measured every 30 minutes at 600 nm using a UV spectrophotometer to evaluate the release rate of the microcapsules in simulated intestinal fluid. The results are shown in Figure 12 , in the figure, (A) gastric juice solubility; (B) intestinal release.

[0055] like Figure 12 As shown in the figure, after 4 h of treatment in simulated gastric fluid, the microcapsules in each group showed varying degrees of turbidity, and the transmittance showed a downward trend. The turbidity was caused by incomplete embedding of part of the microcapsules and partial dissolution of the wall material on the surface of the microcapsules. The transmittance of SPW-LR-1 decreased the most by 19.63 ± 1.36%. The in situ coated microcapsules co-encapsulated with different EPS showed a trend of decreasing dissolution rate with increasing co-encapsulated EPS concentration. The SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) groups decreased by 14.50 ± 1.32%, 14.63 ± 0.50%, 4.00 ± 1.21%, and 14.86 ± 0.21%, respectively, demonstrating that microcapsules prepared with these four EPSs provide a strong physical barrier for the encapsulated LR-1. This is primarily due to the EPS' ability to enhance the mechanical stress on the surface of the microcapsule wall, making it more compact and providing better protection for the strain.

[0056] As can be seen, the microcapsules prepared by the present invention can significantly improve the tolerance of probiotic LR-1 in gastric juice. The probiotics reach the intestine through gastric juice. Further evaluation of the intestinal release rate of the microcapsules shows that only when the probiotics are present in sufficient numbers can they colonize the intestine. Good enteric solubility means that the microcapsules can resist the attack of gastric acid and digestive enzymes, and only dissolve slightly or in the acidic environment of gastric juice, achieving targeted release and successfully releasing the probiotics into the intestinal fluid.

[0057] like Figure 12 As shown in the figure, after being treated in simulated intestinal fluid for 2.0 h, the microcapsules of each group were basically completely cleaved, indicating that all the LR-1 embedded in the microcapsules were effectively released and had good enteric solubility. This is because the carboxyl groups in the simulated intestinal fluid will react with the protons in the microcapsule structure, weakening the hydrogen bonding between the wall material molecules, thereby causing the microspheres to cleave and release the embedded substances.

[0058] The release rates of SPW-LR-1 and SPW-LR-1 in situ encapsulated microcapsules were 81.74 ± 1.52%, respectively. The release rates of the in situ encapsulated microcapsules with different EPS concentrations increased with increasing EPS concentrations. The release rates of SPW-LR-1-TB EPS0800 (μg / mL), SPW-LR-1-TP EPS0800 (μg / mL), SPW-LR-1-mB EPS0800 (μg / mL), and SPW-LR-1-mP EPS0800 (μg / mL) were 85 ± 1.00%, 84.20 ± 0.36%, 87.17 ± 0.25%, and 85.32 ± 1.06%, respectively. SPW-LR-1-mB EPS0800 (μg / mL) exhibited the best release efficiency, likely due to a slow water absorption phase and smaller, more compact microcapsule structures.

[0059] 7. Microcapsule particle size and water content detection (1) Particle size From each group of freeze-dried microcapsules, samples were randomly selected (20 cases in each experimental group), and the particle size of the selected microcapsule samples was measured with a vernier caliper, and the average value was finally taken.

[0060] (2) Water content Place a clean glass weighing bottle in a 105°C drying oven for 1.0 h, cool it for 0.5 h, and maintain constant weight with a weight difference of no more than 2.0 mg between two consecutive weighings. Place an appropriate amount of microcapsule sample (denoted as M1) into the weighing bottle, which has reached constant weight, and continue drying for another 4.0 h. After drying, cool it for half an hour and then weigh it. To ensure the accuracy of the results, repeat the drying and weighing process until the weight difference between two consecutive weighings is no more than 2.0 mg, maintaining constant weight (denoted as M2). The final result should be the average of the three experiments. Particle size and moisture content test results are shown in Table 1.

[0061]

[0062] Wherein, M1 is the mass of the microcapsules after freeze-drying, g; M2 is the mass of the microcapsules after drying to constant weight, g.

[0063] Table 1 Particle size and water content

[0064] Moisture content affects the stability of microcapsules. High moisture content can make them unstable during long-term storage, leading to hydrolysis or degradation. Low moisture content improves the long-term stability of microcapsules, but may affect the solubility of the active ingredients. Studies have shown that the moisture content of dried probiotic microcapsules for long-term storage should not exceed 10%. As shown in Table 1, all microcapsules produced by the present invention have a moisture content below 10%, which is beneficial for long-term storage of the microcapsules.

[0065] In addition to moisture content, microcapsule particle size also affects the stress resistance and storage stability of the encapsulated probiotics, making it an important indicator for evaluating the potential applications of microcapsules. Smaller particle size microcapsules are more easily dispersed, reducing the possibility of precipitation and agglomeration, thereby improving stability during storage and enhancing the protective effect. Good encapsulation properties help prevent damage to active probiotics during storage. As shown in Table 1, there is no significant difference in particle size between the unencapsulated group and the microcapsules produced by the present invention, both being less than 2 mm.

[0066] In summary, the co-embedded microcapsules prepared by the technical solution of the present invention not only have good storage stability, high temperature resistance, resistance to osmotic stress and other properties, but can also effectively promote the proliferation of bacteria.

[0067] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing bacterial cell-exopolysaccharide co-encapsulated membrane-state microcapsules, characterized in that: The following steps are involved: (1) Dissolve sodium alginate, pectin, and whey protein isolate, stir for 3-4 hours, and then sterilize to prepare a composite material solution; (2) The activated bacterial solution is inoculated into the composite wall material solution, and then the membrane matrix extracellular polysaccharide is added. After stirring and mixing, calcium chloride solution is added to form a gel. The excess gel is then removed and washed to obtain a membrane-state microcapsule co-encapsulated with bacteria and extracellular polysaccharide.

2. The preparation method according to claim 1, characterized in that In step (1), the mass ratio of sodium alginate, pectin and whey protein isolate is 1.5~3:1.5~3:2~4.

3. The preparation method according to claim 1, characterized in that In step (2), the extracellular polysaccharide of the membrane matrix is ​​derived from Lactobacillus plantarum LR-1, and its inoculation amount is 1-1.5%.

4. The preparation method according to claim 3, characterized in that The preparation process of the membrane matrix extracellular polysaccharide is as follows: S1. Strain culture The activated Lactobacillus plantarum LR-1 was inoculated into the liquid culture medium, then dispersed into the titer plate and cultured at 37°C for 48 h. S2. Extraction and separation of extracellular polysaccharides from the membrane matrix After centrifugation, the supernatant was collected and neutral protease was added to a concentration of 2 g / L. The reaction was allowed to proceed for 2-3 hours. The supernatant was then centrifuged and collected. Anhydrous ethanol was added to the supernatant and the polysaccharides were precipitated at 4°C. The precipitate was collected by further centrifugation and then reconstituted with deionized water to isolate the exopolysaccharide from the membrane matrix.

5. The preparation method according to claim 4, characterized in that The liquid culture medium in S1 is skim milk or mMRS liquid culture medium, and the cultured strain is an encapsulated strain or a planktonic strain.

6. The preparation method according to claim 5, characterized in that The isolated extracellular polysaccharide of the membrane matrix is ​​TBEPS0 or mBEPS0.

7. The preparation method according to claim 1, characterized in that The final concentration of the extracellular polysaccharide of the membrane matrix in step (2) in the composite wall material solution is 200-800 μg / mL.

8. The preparation method according to claim 1, characterized in that The concentration of the calcium chloride solution added in step (2) is 700-750 mmol / L.

9. A bacterial-exopolysaccharide co-encapsulated membrane-state microcapsule, characterized in that: The method according to any one of claims 1 to 7 is used to prepare the product.

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