Microcapsules embedding lactobacillus plantarum, preparation method and aronia melanocarpa beverage

Abstract

The application provides a microcapsule embedding lactobacillus plantarum, a preparation method and an aronia melanocarpa beverage, and relates to the technical field of food microcapsule embedding. The method is a complex coagulation-crosslinking method for preparing the microcapsule embedding lactobacillus plantarum by taking citrus pectin and chitosan as composite wall materials and calcium chloride as an ionic crosslinking agent. The application also provides an aronia melanocarpa beverage containing the microcapsule embedding lactobacillus plantarum. The beverage is prepared by dispersing the microcapsule in an aronia melanocarpa beverage base, and then performing blending, homogenization and mild sterilization. The application can significantly improve the tolerance of lactobacillus plantarum to gastric acid, bile salts, heat, light and oxygen, realize gastric tolerance and intestinal target release, effectively improve the survival rate and bioavailability of probiotics, protect the stability of aronia melanocarpa anthocyanins, and prolong the shelf life of the beverage system. The beverage system is uniform, not stratified, soft in taste and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of food microcapsule encapsulation technology, specifically to a microcapsule encapsulating Lactobacillus plantarum, a preparation method thereof, and a black chokeberry beverage. Background Technology

[0002] Developing functional beverages by combining black chokeberry with Lactobacillus plantarum can achieve synergistic effects of antioxidants and probiotics, representing an important direction for the development of high-end health drinks. However, direct blending presents significant technical challenges: black chokeberry juice has a low pH and high polyphenol content, which strongly inhibits the growth of Lactobacillus plantarum and accelerates bacterial death. Simultaneously, bacterial metabolism and inactivation accelerate anthocyanin degradation, leading to poor beverage stability, short shelf life, and significantly reduced functionality. Microencapsulation is a key technology for achieving probiotic stabilization. Currently, commonly used wall materials such as starch, alginate, gelatin, and guar gum generally suffer from insufficient mechanical strength, low encapsulation rate, poor controlled release, and weak resistance to gastric acid. Citrus pectin, as a natural anionic polysaccharide, has good film-forming properties, is resistant to gastric acid, and can be degraded by intestinal microorganisms, making it an ideal targeted release wall material. Chitosan, as a natural cationic polysaccharide, has excellent biocompatibility, strong mucosal adhesion, and certain antibacterial effects, which can significantly enhance… The density and stability of the wall material are enhanced by electrostatic recombination to form a core-shell structure, which is then cross-linked with calcium chloride ions. This significantly improves the mechanical strength, density, and acid and bile salt resistance of the wall material, enabling the construction of a highly efficient microcapsule system that is gastric-tolerant and intestinal-targeted. While there are many studies on encapsulating probiotics using single alginate, pectin, or chitosan, problems such as low encapsulation rates, rapid gastrointestinal release, and poor storage stability remain. A technical solution for encapsulating *Lactobacillus plantarum* using a citrus pectin-chitosan composite with calcium chloride and then applying it to a black chokeberry functional beverage has not yet been reported. Systematic research on the synergistic encapsulation of related composite wall materials, probiotic protection, anthocyanin stabilization, and beverage system compatibility is still lacking. Therefore, developing a microcapsule probiotic beverage with high encapsulation rate, high survival rate, gastric tolerance, intestinal-targeted release, and simultaneous stabilization of the active ingredients of black chokeberry has significant theoretical and market application value. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing technologies, such as low survival rate of probiotics in black chokeberry beverage systems, easy degradation of anthocyanins, poor beverage stability, and short shelf life. This invention provides a microcapsule encapsulating Lactobacillus plantarum, a preparation method, and a black chokeberry beverage. The microcapsule of this invention can achieve a highly efficient synergistic effect between probiotic function and antioxidant activity.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: This invention first provides a method for preparing microcapsules encapsulating Lactobacillus plantarum, comprising the following steps: Step 1: Disperse the Lactobacillus plantarum sludge in a protective agent solution to obtain a bacterial suspension; Step 2: Mix the citrus pectin solution and chitosan solution to obtain the composite wall material solution; Step 3: Add the bacterial suspension from Step 1 to the composite wall material solution obtained in Step 2, and stir to form a complex coagulation system; Step 4: Slowly add the complex coagulation system obtained in Step 3 to calcium chloride solution for ionic cross-linking to obtain microcapsules encapsulating Lactobacillus plantarum.

[0005] Preferably, the viable count of *Lactobacillus plantarum* in step one is 10-1. 9 -10 11 CFU / mL.

[0006] Preferably, the volume ratio of the citrus pectin solution and the chitosan solution in step two is 1:1.

[0007] Preferably, the concentration of the citrus pectin solution in step two is 2% (w / v), and the concentration of the chitosan solution is 1% (w / v).

[0008] Preferably, in step three, the pH of the system is adjusted to 4 with citric acid, the stirring speed is 500 rpm, the temperature is 25℃, and the time is 20 min.

[0009] Preferably, the core-to-wall ratio of the bacterial suspension to the composite wall material solution in step three is 5:1.

[0010] Preferably, the concentration of the calcium chloride solution in step four is 3% (w / v), the crosslinking temperature is 20°C, and the crosslinking time is 30 min.

[0011] The present invention also provides microcapsules containing *Lactobacillus plantarum* prepared by the above method.

[0012] The present invention also provides a black chokeberry beverage comprising the above-mentioned microcapsules encapsulating Lactobacillus plantarum.

[0013] The present invention also provides a method for preparing the above-mentioned black chokeberry beverage, comprising: mixing microcapsule suspension with black chokeberry beverage base, blending, homogenizing and sterilizing to obtain black chokeberry beverage.

[0014] Beneficial effects of the present invention This invention provides microcapsules encapsulating *Lactobacillus plantarum*, a preparation method, and a *Sorbus spp.* (black chokeberry) beverage. The microcapsule wall material, formed by cross-linking citrus pectin with chitosan and calcium chloride ions, provides comprehensive physical protection for *Lactobacillus plantarum*, significantly enhancing the bacteria's tolerance to gastric acid, bile salts, high temperatures, oxidation, and high-polyphenol environments, achieving targeted delivery with low gastric release and high intestinal release. Compared to Comparative Example 1 (free *Lactobacillus plantarum*), the microcapsules encapsulated probiotics prepared in this invention exhibit a survival rate ≥70% after 2 hours of simulated gastric juice treatment, an improvement of over 40%; and the viable bacterial count remains at 10 after 28 days of storage at 25°C in the dark. 7 With a concentration of CFU / mL or higher, it is 5.2 times that of control group 1; the microcapsule encapsulation rate is ≥85%, effectively solving the key problems of easy inactivation and low intestinal colonization rate of probiotics during processing, storage and oral administration, and significantly improving the bioavailability and functional stability of Lactobacillus plantarum.

[0015] Meanwhile, the microcapsule system isolates the polyphenols of black chokeberry from direct contact with probiotics, avoiding the inhibitory effects of acidity and polyphenols on the bacteria, and also reducing the oxidative degradation of anthocyanins by bacterial metabolism, thus forming a two-way protection. Compared with Comparative Examples 1 and 2, the beverage system prepared by this invention is uniform and stable, without precipitation or stratification. During the 28-day storage period, the fluctuations in turbidity, viscosity, and pH are minimal, and the system stability is significantly improved. The anthocyanin retention rate during storage is ≥85%, which is more than 30% higher than that of Comparative Example 1, effectively improving the product's color and flavor, masking the sour and astringent taste, and significantly extending the shelf life.

[0016] Furthermore, the wall materials used in this invention are all food-grade natural biodegradable polysaccharides, exhibiting good biocompatibility, safety, and non-toxicity. The process is mild and easily industrialized, achieving a highly efficient synergistic effect between antioxidant activity and probiotic function. In comparison with Comparative Example 1 and Comparative Example 2, this invention simultaneously solves three major technical challenges: low probiotic survival rate, easy degradation of anthocyanins, and instability of the beverage system. The resulting product combines probiotic activity with the antioxidant function of black chokeberry, possessing significant market application value and technological advantages. Attached Figure Description

[0017] Figure 1 The swelling rate variation curves of the probiotic microcapsules prepared in this invention in simulated gastric juice (SGF) and simulated intestinal juice (SIF); Figure 2 Release rate curve of the probiotic microcapsules prepared by this invention in simulated gastrointestinal digestion in vitro; Figure 3 The turbidity (OD) of the black chokeberry beverage prepared by this invention and the comparative example during a 28-day storage period. 600 Comparison chart of changes; Figure 4A comparison of the viscosity changes of the black chokeberry beverage prepared in this invention and the comparative example during a 28-day storage period. Figure 5 A comparison of pH changes between the black chokeberry beverage prepared in this invention and a comparative example during a 28-day storage period. Figure 6 A comparison chart of the changes in the number of viable Lactobacillus plantarum bacteria during the 28-day storage period between the black chokeberry beverage prepared in this invention and the comparative example. Figure 7 A comparison chart of the changes in viable bacteria count between the microcapsule group and the free probiotic group under different acidic pH conditions; Figure 8 A comparison of the changes in viable bacteria count between the microcapsule group and the free probiotic group under different bile salt concentrations in this invention; Figure 9 A comparison chart of the changes in viable bacteria count between the microcapsule group and the free probiotic group under different temperature treatment conditions. Detailed Implementation

[0018] This invention first provides a method for preparing microcapsules encapsulating Lactobacillus plantarum, comprising the following steps: Step 1: Disperse the Lactobacillus plantarum sludge in the protective agent solution, mix well, and adjust the viable bacteria concentration to preferably 10. 9 -10 11 CFU / mL, preferably 1.0 × 10⁻⁶ 10 CFU / mL, stored at low temperature and protected from light to obtain a bacterial suspension; the preferred protectant is trehalose solution. Step 2: Add citrus pectin to deionized water, stir to dissolve, and prepare a citrus pectin solution. The concentration of the citrus pectin solution is preferably 2% (w / v). Add chitosan to a citric acid aqueous solution, stir until transparent, and prepare a chitosan solution. The concentration of the citric acid aqueous solution is preferably 1% (w / v), and the concentration of the chitosan solution is preferably 1% (w / v). Mix the two solutions evenly to obtain a composite wall material solution. The volume ratio of the citrus pectin solution to the chitosan solution is preferably 1:1. Step 3: Slowly add the Lactobacillus plantarum suspension to the composite wall material solution at a core-to-wall ratio of 5:1, adjust the pH of the system to 3.5–4.5, preferably 4.0, with citric acid solution; stir at 20–30℃, preferably 25℃, and 400–600 rpm, preferably 500 rpm for 15–25 min, preferably 20 min, to form uniformly aggregated microdroplets; Step 4: Slowly add the complex coagulation system obtained in Step 3 to a calcium chloride solution for ionic crosslinking, preferably at 15–25°C, more preferably at 20°C, for static crosslinking for 20–40 min, preferably 30 min, so that the citrus pectin and chitosan can react in the calcium chloride solution.2+ Under the action of [a process described in the original text], a dense cross-linked structure is formed, resulting in microcapsules encapsulating *Lactobacillus plantarum*. The concentration of the calcium chloride cross-linking solution is preferably 3% (w / v).

[0019] The present invention also provides microcapsules containing *Lactobacillus plantarum* prepared by the above method.

[0020] The present invention also provides a black chokeberry beverage comprising the above-mentioned microcapsules encapsulating Lactobacillus plantarum.

[0021] The present invention also provides a method for preparing the above-mentioned black chokeberry beverage, comprising: mixing microcapsule suspension with black chokeberry beverage base, blending, homogenizing and sterilizing to obtain black chokeberry beverage.

[0022] According to the present invention, the black chokeberry beverage base comprises black chokeberry juice, deionized water, sweetener, acidity regulator, and suspending agent. The preferred mass ratio of the black chokeberry juice, deionized water, sweetener, acidity regulator, and suspending agent is 60:64.45:5:0.05:0.5. The amount of microcapsules added is 3%–8% of the total beverage mass, preferably 5%. The homogenization conditions are preferably 15–25 MPa, more preferably 20 MPa, for 1–3 cycles. Sterilization is preferably performed at 80–90℃ / 10–20 s, more preferably 85℃ / 15 s using pasteurization. The preferred sweetener is erythritol, the preferred acidity regulator is sodium citrate, and the preferred suspending agent is xanthan gum.

[0023] The microcapsules and black chokeberry beverage were characterized using the following tests: 1. Determination of microcapsule swelling properties The water absorption and swelling property is the core basis for the pH-responsive release of pectin-based microcapsules. The swelling rate of the microspheres in simulated gastric fluid (SGF, pH 1.8) and simulated intestinal fluid (SIF, pH 6.8) was evaluated using a gravimetric method: 50 mg of dried microcapsules were accurately weighed and placed in 50 mL of the corresponding medium. After timed intervals, the capsules were removed, surface moisture was absorbed, and the weight was calculated using the formula: Swelling rate (g / g) = (S1 – S0) / S0; In the formula: S1 is the weight after swelling, and S0 is the initial weight after drying. 2. In vitro simulated gastrointestinal digestion and release test Free probiotics and microcapsules were mixed with enzyme-containing SGF at a ratio of 1:9 (w / v), shaken at 37°C for 2 h, and then transferred into enzyme-containing SIF. Samples were taken every 30 min to count the viable bacteria and evaluate the release rate.

[0024] 3. Beverage stability testing pH measurement: The beverage was stored at 4℃ for 21 days, and samples were taken on days 1, 3, 7, 14 and 21. After being warmed to 2 hours, the pH was measured with a pH meter.

[0025] Turbidity determination: The turbidity of beverages stored at 4℃ was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0026] Viscosity measurement: The viscosity of beverages stored at 4℃ was measured using a viscometer.

[0027] 4. Storage stability test The beverage was stored at 4℃ and 25℃ for 28 days, and the number of viable Lactobacillus plantarum bacteria was counted every 7 days.

[0028] 5. Acid tolerance test The sample was mixed with PBS at pH 1.5 or 2.0 at a ratio of 1:9, shaken at 37°C and 200 rpm, and viable bacteria were counted every 30 min.

[0029] 6. Bile salt tolerance test The sample was mixed with 1.2% and 2.2% bile salt solutions at a ratio of 1:9, incubated at 37°C, and viable bacteria were counted at regular intervals.

[0030] The technical solution of the present invention will be fully described below with reference to the accompanying drawings and specific embodiments.

[0031] Example 1 Step 1: Centrifuge the Lactobacillus plantarum fermentation broth to collect the bacterial sludge, disperse it in trehalose preservative, and adjust the concentration to 1.0 × 10⁻⁶. 10 CFU / mL was used to obtain the composite wall material.

[0032] Step 2: Prepare a 2% citrus pectin solution and a 1% chitosan solution, mix them in a 1:1 volume ratio to obtain a composite wall material; prepare a 3% calcium chloride solution.

[0033] Step 3: Mix the bacterial suspension and the composite wall material at a core-to-wall ratio of 5:1, adjust the pH to 4.0, and stir at 25℃ and 500 rpm for 20 min.

[0034] Step 4: Add calcium chloride solution dropwise, crosslink at 20℃ for 30 min to obtain microcapsules.

[0035] Step 5: Add microcapsules at 5% of the total beverage mass, mix with black chokeberry juice (30%), deionized water (64.45%), erythritol (5%), sodium citrate (0.05%), and xanthan gum (0.5%); homogenize twice at 20 MPa; sterilize at 85℃ for 15 s; fill to obtain black chokeberry beverage.

[0036] Comparative Example 1 Free *Lactobacillus plantarum* was directly added to the *Sorbus nigra* (black chokeberry) beverage without microencapsulation. The amount of bacteria added was equivalent to the bacterial content within the microcapsules in Example 1 (i.e., a viable count of 1.0 × 10⁻⁶). 10 (CFU / mL beverage), the remaining components and steps are the same as in Example 1.

[0037] Comparative Example 2 The only blank beverage of black chokeberry was prepared without any added probiotics (free or microcapsules), and the remaining components and steps were the same as in Example 1.

[0038] Figure 1. Swelling rate variation curves of the probiotic microcapsules prepared in Example 1 of the present invention in simulated gastric juice (SGF) and simulated intestinal juice (SIF); it can be seen that the swelling rate of the microcapsules is low in SGF (pH 1.8), maintaining a dense structure; while the swelling rate increases significantly in SIF (pH 6.8), showing good pH-responsive swelling behavior.

[0039] Figure 2. Release rate curve of the probiotic microcapsules prepared in Example 1 of the present invention during simulated gastrointestinal digestion in vitro; wherein Figure 2 'a' represents the release rate curve. Figure 2 b shows the digestion characteristic curve. It can be seen that the probiotic release rate in SGF was less than 20% within 2 hours, but the release rate increased rapidly after transfer to SIF, exceeding 85% cumulatively within 4 hours, achieving gastric tolerance and intestinal-targeted release. After 2 hours of culture, the number of free probiotic live cells rapidly decreased to 1.98 lg CFU / g, and after another 1 hour of culture, no live cells were detected. After 3 hours of continuous digestion, the cell count of the microcapsules was 8.72, and remained at 6.21 lg CFU / g after 6 hours. The microcapsules provide better protection.

[0040] Figure 3. Turbidity (OD) of the black chokeberry beverage prepared in Example 1 of the present invention and Comparative Examples 1 and 2 during a 28-day storage period. 600 The comparison chart shows that the turbidity of the beverages in Example 1 and Comparative Example 2 changed very little, indicating that the system was stable; while the turbidity of Comparative Example 1 decreased significantly, and precipitation and stratification occurred.

[0041] Figure 4 compares the viscosity changes of the black chokeberry beverage prepared in Example 1 of this invention with those of Comparative Examples 1 and 2 during a 28-day storage period. It can be seen that Example 1 has a high initial viscosity, which remains stable during the 28-day storage period without significant fluctuations. Comparative Example 2 maintains the lowest and most stable viscosity throughout. In the early stages of storage (within 7 days), the viscosity of Comparative Example 1 increases significantly to 31.24 mPa·s.

[0042] Figure 5. A comparison of pH changes of the black chokeberry beverage prepared in Example 1 of the present invention with those of Comparative Examples 1 and 2 during the 28-day storage period; it can be seen that the pH of the blank control group remained stable; the pH of Comparative Example 1 (free probiotic group) decreased to 2.97 ± 0.01 after 28 days; although the pH of Example 1 (microcapsule probiotic group) decreased, the degree of decrease was much smaller than that of the free probiotic group.

[0043] Figure 6. Comparison of the changes in viable Lactobacillus plantarum count during 28 days of storage between the black chokeberry beverage prepared in Example 1 of the present invention and Comparative Example 1; wherein Figure 6 'a' represents the cell survival rate of free probiotics and microcapsules stored at 4°C for 28 days. Figure 6 b represents the cell viability of free probiotics and microcapsules stored at 25℃ for 28 days. It can be seen that at both 4℃ and 25℃, the viable count of both free probiotics and microcapsules decreased over time. Storage temperature has a significant impact on free probiotics. Microcapsules can protect *Lactobacillus plantarum* from adverse environmental damage and exhibit better storage stability at low temperatures.

[0044] Figure 7. Comparison of viable bacteria count changes between the microcapsule group of Example 1 and Comparative Example 1 under different acidic pH conditions; wherein Figure 7 'a' represents the cell viability of free probiotics and microcapsules at pH 1.5. Figure 7 b represents the cell viability of free probiotics and microcapsules at pH 2. It can be seen that the cell viability of Comparative Example 1 decreased rapidly with decreasing pH and prolonged time. After culturing for 2 hours at pH 1.5 and 2.0, Example 1 showed no significant changes.

[0045] Figure 8. Comparison of viable bacteria count changes between the microcapsule group of Example 1 and Comparative Example 1 under different bile salt concentrations; wherein Figure 8 'a' represents the cell survival rate of free probiotics and microcapsules under 1.5% bile salt conditions. Figure 8 b represents the cell viability of free probiotics and microcapsules under 2.2% bile salt conditions. It can be seen that the cell viability of both Example 1 and Comparative Example 1 decreased with increasing bile salt concentration and time. After 2 hours of treatment with 2.2% bile salt, the number of viable probiotic cells in Comparative Example 1 decreased significantly.

[0046] Figure 9. Comparison of viable cell count changes between the microcapsule group of Example 1 and Comparative Example 1 under different temperature treatment conditions. Figure 9 'a' represents the cell survival rate of free probiotics and microcapsules at 63°C. Figure 9b represents the cell viability of free probiotics and microcapsules at 71°C. It can be seen that the number of viable cells in both Example 1 and Comparative Example 1 decreased with increasing temperature and time. After treatment at 71°C for 30 min, the number of viable cells in Example 1 was significantly higher than that in Comparative Example 1.

[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0048] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are meant to be within the scope of the invention and form different embodiments. For example, in the foregoing claims, any of the claimed embodiments can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of the invention and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A method for preparing microcapsules encapsulating Lactobacillus plantarum, characterized in that, Includes the following steps: Step 1: Disperse the Lactobacillus plantarum sludge in a protective agent solution to obtain a bacterial suspension; Step 2: Mix the citrus pectin solution and chitosan solution to obtain the composite wall material solution; Step 3: Add the bacterial suspension from Step 1 to the composite wall material solution obtained in Step 2, and stir to form a complex coagulation system; Step 4: Slowly add the complex coagulation system obtained in Step 3 to calcium chloride solution for ionic cross-linking to obtain microcapsules encapsulating Lactobacillus plantarum.

2. The method for preparing microcapsules encapsulating *Lactobacillus plantarum* according to claim 1, characterized in that, The viable count of *Lactobacillus plantarum* in step one is 10. 9 -10 11 CFU / mL.

3. The method for preparing microcapsules encapsulating *Lactobacillus plantarum* according to claim 1, characterized in that, The volume ratio of the citrus pectin solution and the chitosan solution mentioned in step two is 1:

1.

4. The method for preparing microcapsules encapsulating *Lactobacillus plantarum* according to claim 1, characterized in that, The concentration of the citrus pectin solution in step two is 2% (w / v), and the concentration of the chitosan solution is 1% (w / v).

5. The method for preparing microcapsules encapsulating *Lactobacillus plantarum* according to claim 1, characterized in that, Step 3: Adjust the pH of the system to 4 with citric acid, stirring at 500 rpm, at 25°C, for 20 min.

6. The method for preparing microcapsules encapsulating *Lactobacillus plantarum* according to claim 1, characterized in that, The core-to-wall ratio of the bacterial suspension to the composite wall material solution in step three is 5:

1.

7. The method for preparing microcapsules encapsulating *Lactobacillus plantarum* according to claim 1, characterized in that, In step four, the calcium chloride solution concentration is 3% (w / v), the crosslinking temperature is 20℃, and the crosslinking time is 30 min.

8. Microcapsules containing *Lactobacillus plantarum* obtained by the preparation method according to claim 1.

9. A black chokeberry beverage, characterized in that, Including the microcapsules containing Lactobacillus plantarum as described in claim 8.

10. A method for preparing a black chokeberry beverage according to claim 9, characterized in that, include: The microcapsule suspension was mixed with the base material of black chokeberry beverage, then blended, homogenized, and sterilized to obtain the black chokeberry beverage.

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