Cucurbita pepo gel beads embedded with probiotics and preparation method thereof

Abstract

The invention discloses a probiotic-embedded cucumber fruit hydrogel bead and a preparation method thereof, and belongs to the technical field of embedding of probiotics. The preparation method of the probiotic-embedded cucumber pectin hydrogel beads comprises the following steps: 1) extracting cucumber pectin; (2) preparing a cucumber pectin-sodium alginate-gelatin gel solution; (3) preparing freeze-dried bacterial powder of lactobacillus rhamnosus and lactobacillus plantarum; and 4) uniformly mixing the freeze-dried bacterial powder with the gel solution, and dropwise adding the obtained mixed solution into CaCl2 to prepare the hydrogel beads. According to the preparation method disclosed by the invention, the protected lactobacillus rhamnosus and lactobacillus plantarum freeze-dried bacterial powder are embedded by the hydrogel beads prepared from the extracted cucumber pectin, the sodium alginate and the gelatin, and the hydrogel beads and the strain protective agent perform double protection on the lactic acid bacteria, so that the lactic acid bacteria have good acid resistance, digestion resistance and storability.

Description

Technical Field

[0001] This invention relates to the field of probiotic encapsulation technology, specifically to a cucumber pectin hydrogel bead encapsulating probiotics and its preparation method. Background Technology

[0002] Probiotics, such as *Lactobacillus plantarum* and *Lactobacillus rhamnosus*, have attracted much attention due to their numerous health benefits. Studies have shown that *Lactobacillus plantarum* can regulate the balance of gut microbiota, enhance immunity, and possess antioxidant and anti-inflammatory effects. *Lactobacillus rhamnosus* has been shown to inhibit the growth of pathogenic bacteria, alleviate diarrhea symptoms, and improve intestinal barrier function. However, the low survival rate of probiotics during processing, storage, and passage through the gastrointestinal tract limits their effectiveness.

[0003] Probiotic encapsulation technology protects probiotics and controls their release by selecting suitable wall materials. Encapsulation forms mainly include gels, emulsions, and powders. Among the many encapsulation systems, hydrogels, hydrophilic polymers with a three-dimensional network structure formed by physical or chemical cross-linking, have attracted widespread attention due to their simple preparation, good adjustability, and high biocompatibility. Natural polysaccharides and proteins are widely used to construct probiotic gel encapsulation systems. However, proteins are easily degraded in the stomach and small intestine, while polysaccharides have advantages such as good gelling properties, acid resistance, and high biocompatibility, making them the commonly used materials for constructing hydrogel encapsulation systems.

[0004] Pectin is a natural plant polysaccharide with amphiphilic properties. It can be used as a gelling agent, stabilizer, thickener, film-forming agent, and as a prebiotic to stimulate the growth of gut microbiota. There are reports of using pectin to encapsulate probiotics; however, some pectins may dissolve prematurely under acidic conditions, leading to the exposure of probiotics. Furthermore, pectin is relatively expensive, making the encapsulation of probiotics using pectin costly. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide cucumber pectin hydrogel beads encapsulating probiotics and a method for preparing the same.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A first aspect of the present invention provides the use of cucumber pectin in the preparation of probiotic encapsulation products; said cucumber pectin is prepared by the following method:

[0008] Cucumber powder is prepared by drying, grinding, and sieving cucumbers or cucumber processing by-products. The cucumber powder is mixed evenly with deionized water to obtain a cucumber solution. The pH of the cucumber solution is adjusted to 2.0 with citric acid, stirred in a water bath at 65-75℃ for 1.5-2.5 hours, and then sonicated for 0.5-1.5 hours. The solution is filtered, the liquid is collected, and the supernatant is collected by centrifugation. Ethanol is added to the supernatant, and the precipitate is collected by centrifugation for 10-14 hours. The precipitate is then dialyzed for 40-50 hours, and after dialysis, it is freeze-dried to prepare cucumber pectin.

[0009] The cucumber processing by-products include: cucumber peel, cucumber stems, etc.

[0010] This invention extracts cucumber pectin from cucumbers and cucumber processing byproducts, exhibiting good biocompatibility, biodegradability, and gelling properties. Its molecular structure contains abundant galacturonic acid, which can react with divalent cations (such as Ca²⁺, Ca²⁺, and Ca²⁺). 2 + Cross-linking forms a stable gel network, providing a protective microenvironment for probiotics.

[0011] Compared to other pectins, cucumber-derived pectin (CDP) is widely available and inexpensive. Cucumbers are a common vegetable, and their processing byproducts can be used to extract pectin, achieving resource utilization. Cucumber pectin is a natural macromolecular pectin; its longer molecular chains help it form solutions with higher viscosity and greater stability. Even in low pH environments, it maintains its intact bead-like shape, effectively resisting stomach acid and digestive enzymes, and enhancing its protective properties against lactobacilli. When used as an encapsulation wall material, it provides better protection for the core material, enhancing its environmental resilience. Furthermore, cucumber pectin has potential prebiotic effects; it can be fermented and utilized by gut microbiota, promoting the growth and reproduction of probiotics and exerting a synergistic effect.

[0012] Therefore, utilizing the advantageous gel structure of cucumber pectin to encapsulate *Lactobacillus plantarum* and *Lactobacillus rhamnosus* can effectively protect lactobacilli, improve their survival rate, and enhance their acid resistance, digestibility, and storage stability. It can also exert the prebiotic effect of cucumber pectin, showing broad application prospects.

[0013] In a second aspect, the present invention provides a hydrogel solution composed of cucumber pectin, sodium alginate and gelatin; wherein the mass ratio of cucumber pectin, sodium alginate and gelatin is (0.5-2):1:0.5.

[0014] Preferably, the mass ratio of cucumber pectin, sodium alginate, and gelatin in the hydrogel solution is 2:1:0.5.

[0015] In the hydrogel solution of this invention, cucumber pectin, sodium alginate, and gelatin work synergistically to form a dense gel network structure, significantly improving the mechanical strength and stability of the hydrogel beads. Compared with using cucumber pectin, sodium alginate, and gelatin alone, the effect is greater than the sum of its parts (1+1>2). This gel network structure formed by the three components can effectively protect the embedded lactobacilli from mechanical shearing forces, acidic environments, and digestive enzymes during processing, storage, and passage through the gastrointestinal tract.

[0016] A third aspect of the present invention provides a method for preparing the above-mentioned hydrogel solution, comprising the following steps:

[0017] Dissolve cucumber pectin in sterile deionized water, stir well, and let stand overnight to obtain a cucumber pectin solution; add sodium alginate and gelatin to the cucumber pectin solution, stir well, and let stand overnight to prepare a hydrogel solution.

[0018] A fourth aspect of the present invention provides the use of the above-described hydrogel solution in the preparation of probiotic encapsulation products.

[0019] In a fifth aspect, the present invention provides cucumber pectin hydrogel beads encapsulating probiotics, prepared by the following method:

[0020] Add the lyophilized probiotic powder to the above hydrogel solution, stir evenly, and let stand to obtain a hydrogel solution containing probiotics.

[0021] A probiotic-containing hydrogel solution was added dropwise to a cross-linking agent and left to stand overnight to prepare cucumber pectin hydrogel beads encapsulated with probiotics.

[0022] Preferably, (1-3)g of lyophilized probiotic powder is added to every 100mL of hydrogel solution.

[0023] Preferably, the probiotics are Lactobacillus plantarum and Lactobacillus rhamnosus.

[0024] Preferably, the crosslinking agent is a 0.3M CaCl2 solution; the volume ratio of the hydrogel solution containing probiotics to the crosslinking agent is 1:20.

[0025] The beneficial effects of this invention are:

[0026] (1) This invention is the first to use cucumber processing by-products as raw materials to extract and prepare cucumber pectin, which greatly reduces the preparation cost of pectin; moreover, the cucumber pectin of this invention is a natural macromolecular pectin with a long molecular chain, which helps to form a gel with greater viscosity and stronger stability, so that it can still maintain an intact bead shape in a low pH environment, thereby effectively resisting gastric acid and digestive enzymes.

[0027] (2) By utilizing the excellent gelling and stability of cucumber pectin and reasonably adjusting its application ratio, the cucumber pectin hydrogel beads obtained by this invention can effectively improve the survival rate of lactobacillus, giving it strong acid resistance, digestibility and storage resistance. The cucumber pectin hydrogel beads of this invention have an encapsulation rate as high as 97.64%. After treatment in simulated gastric fluid for 120 min, the survival rate of Lactobacillus in the cucumber pectin hydrogel beads is still as high as 85.82%, while the survival rate of Lactobacillus in hydrogel beads without cucumber pectin is 63.80%, and almost all free Lactobacillus is inactivated. After continuous gastrointestinal simulation for 6 h, the survival rate of Lactobacillus in the cucumber pectin hydrogel beads is 73.49%, while the survival rate of Lactobacillus in hydrogel beads without cucumber pectin is 37.07%, and almost all free Lactobacillus is inactivated. After storage at 4 °C for 60 d, the number of viable Lactobacillus in the cucumber pectin hydrogel beads decreases by only 0.5533 Log, while the number of viable Lactobacillus in hydrogel beads without cucumber pectin decreases by 5.95 Log, and the number of viable free Lactobacillus decreases by 7.28 Log. Attached Figure Description

[0028] Figure 1 Photo of cucumber pectin.

[0029] Figure 2 Fourier transform infrared spectrum of CDP

[0030] Figure 3 X-ray diffraction pattern of CDP.

[0031] Figure 4 Turbidity graphs of CDP solutions at different concentrations.

[0032] Figure 5 Apparent viscosity diagram of CDP solutions at different concentrations.

[0033] Figure 6 Photo of freeze-dried bacterial powder.

[0034] Figure 7 Photographs of the basic colony morphology of Lactobacillus plantarum and Lactobacillus rhamnosus (the right image is a magnified view of a portion of the left image).

[0035] Figure 8 Particle size distribution of CAG and SAG at different concentrations.

[0036] Figure 9 Turbidity graphs of CAG and SAG at different concentrations.

[0037] Figure 10 Apparent viscosity diagrams of CAG and SAG at different concentrations.

[0038] Figure 11 Morphological images of LCAGB and LSAGB hydrogel beads; from left to right: LSAGB, LCAGB0.5％ LCAGB 1％ LCAGB 1.5％ LCAGB 2％ .

[0039] Figure 12 Swelling rate diagram of LCAGB and LSAGB hydrogel beads.

[0040] Figure 13 : Encapsulation efficiency of LCAGB and LSAGB hydrogel beads.

[0041] Figure 14 : Hydrogel beads LCAGB 2％ SEM images of the surface morphology of LSAGB.

[0042] Figure 15 : Hydrogel beads LCAGB 2％ SEM images of the LSAGB cross-section.

[0043] Figure 16 : Hydrogel beads LCAGB 2％ Fourier transform infrared spectra of LSAGB and LSAGB.

[0044] Figure 17 : Hydrogel beads LCAGB 2％ X-ray diffraction patterns of LSAGB and LSAGB.

[0045] Figure 18 : Gastric juice tolerance graph.

[0046] Figure 19 : Digestive tolerance diagram.

[0047] Figure 20 Storage stability diagram. Detailed Implementation

[0048] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0049] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0050] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0051] The composition of MRS liquid culture medium is as follows: glucose 20 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, Tween 1 mL / L; pH is 6.0 (±0.1).

[0052] The composition of MRS solid medium is as follows: glucose 20 g / L, sodium acetate 5 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, peptone 10 g / L, yeast extract 5 g / L, beef extract 10 g / L, Tween 1 mL / L, and agar 2.5%; pH is 6.0 (±0.1).

[0053] Lactobacillus plantarum (LPL) and Lactobacillus rhamnosus (LGG) are currently commercially available probiotics.

[0054] Example 1: Preparation and structural properties study of cucumber pectin

[0055] 1. Preparation of cucumber pectin:

[0056] The processing waste of cucumbers (cucumber stems and cucumber peels) is dried, ground, and sieved to make cucumber powder; it is then stored in a cool, dry place for later use.

[0057] Cucumber powder and deionized water were mixed evenly at a ratio of 1g:15ml to obtain a cucumber solution. The pH of the cucumber solution was adjusted to 2.0 using 1M citric acid, and the solution was stirred in a 70℃ water bath for 2 hours, followed by sonication at 70℃ for 1 hour. After sonication, the solution was filtered through four layers of gauze. The liquid fraction was centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected. Three volumes of 95% ethanol were added, and the solution was precipitated for 12 hours. The precipitate was then centrifuged at 5000 rpm for 15 minutes, and the precipitate was transferred to a dialysis bag with a flow rate of 12000 rpm. The dialysis bag was immersed in deionized water and dialyzed for 48 hours (with water changes every 4 hours). After dialysis, the solution was pre-frozen for 24 hours and then freeze-dried to obtain cucumber-derived pectin (CDP).

[0058] The prepared cucumber pectin is shown in the following image. Figure 1 As shown, it is pale yellow and has a flocculent appearance.

[0059] 2. Study on the structural properties of cucumber pectin:

[0060] 2.1 Molecular weight determination:

[0061] The molecular weight (Mw) of cucumber pectin was determined by gel permeation chromatography (GPC). A Shimadzu LC20 high-performance liquid chromatography pump and a RID-20 differential refractive index detector system were used. The chromatographic column was a TOSOH TSKgel GMPWXL aqueous gel permeation column. Injection was performed using a Rheodyne 7725i manual six-way valve (20 μL quantitative loop). Data acquisition and processing were performed using an HW-2000 GPC workstation. The mobile phase was an aqueous solution containing 0.1 mol / L NaNO3 and 0.06% (w / v) NaN3, with a flow rate set at 0.6 mL / min and a constant column temperature of 35 °C. Molecular weight was determined using a narrow-distribution polyethylene glycol (PEG) standard set provided by TOSOH, and a standard curve was established based on the relative calibration method for quantitative analysis.

[0062] The molecular weight of cucumber pectin was determined as shown in Table 1.

[0063] Table 1: CDP Molecular Weight

[0064]

[0065] The weight-average molecular weight (Mw) of cucumber pectin was 134.397 ± 0.351 kDa, the number-average molecular weight (Mn) was 43.942 ± 0.462 kDa, and the Mw / Mn ratio was 3.05851 ± 0.001 kDa. This indicates that the CDP prepared in this example is a high molecular weight pectin, and the mild citric acid extraction process better preserved the pectin structure. The higher the molecular weight of the pectin, the higher the viscosity and the better the stability of the solution, thus providing better protection for the core material when used as an embedding wall material.

[0066] 2.2 Fourier Transform Infrared Spectroscopy (FT-IR) Measurement:

[0067] Fourier transform infrared spectroscopy was used for determination. Cucumber pectin was mixed with dry potassium bromide (KBr) at a ratio of 1:100 and thoroughly ground in an agate mortar until the cucumber pectin and potassium bromide were fully mixed. An appropriate amount of the mixture was placed in a tablet press, and the pressure was set to 1t for 1 minute to prepare a uniform, translucent thin film. The film was then placed in the instrument for detection, and the wavelength range of the scanning beam was set to 4000-400cm. -1 The resolution is 4cm. -1 The number of scans was 32.

[0068] The FT-IR spectrum of CDP is as follows: Figure 2 As shown, it exhibits typical FT-IR spectral characteristics of pectin. At 3404.2 cm⁻¹ -1The broad absorption band at 2935.2 cm⁻¹ indicates the stretching vibration of the hydroxyl-OH functional groups in the galacturonic acid backbone, which is caused by the interaction of gel molecules. -1 The weaker absorption peaks nearby are caused by the asymmetric stretching vibration of the methylene (CH) group in the sugar ring, which is a characteristic absorption peak of polysaccharides. (1234.5 cm⁻¹) -1 The peak at 1000-1100 cm⁻¹ reflects the stretching vibration of the COC glycosidic bond or the symmetric vibration of the ester group. -1 This is the characteristic absorption region of the pectin backbone polysaccharide, reflecting the chain conformation and monosaccharide composition of the polysaccharide, 1021.1 cm. -1 1073.9cm -1 The peak at 700-900 cm⁻¹ reflects the presence of pyranose in CDP. -1 The absorption peak at 708.4 cm⁻¹ is a fingerprint region, typically used to distinguish the cyclic configuration of polysaccharides (such as α- or β-type). -1 The absorption band at this location reflects the skeletal vibration of the pyran ring or the configurational characteristics of the α-glycosidic bond. 634.5 cm⁻¹ -1 The peak at 1737.3 cm⁻¹ may belong to the deformation vibration of the pyran ring or the weak absorption peak of the β-glycosidic bond. Absorption peaks in this region are usually related to the ring conformation of the polysaccharide or the stereochemistry of the substituents. -1 The peak at 1652.8 cm⁻¹ represents the C=O stretching vibration of the methyl ester group (-COOCH₃). -1 The peak at 1540-1560 cm⁻¹ corresponds to the C=O stretching vibration of the carboxylic acid group (-COOH). -1 The peak at 1540.8 cm⁻¹ is a characteristic peak of the amide group. -1 The peak at the peak indicates the NH bending of the amide III band protein, and its intensity reflects the degree of amidation. Amideation reduces charge density, which can improve calcium ion sensitivity and gel stability, making cucumber pectin less sensitive to changes in calcium ion concentration, providing a wider calcium ion window, and resulting in gels formed from cucumber pectin that are more heat-resistant, less prone to dehydration and shrinkage, and offer better protection for lactobacilli.

[0069] 2.3 XRD Measurement:

[0070] X-ray diffraction (XRD) was used to further measure the structural characteristics of the samples and to further analyze cucumber pectin. The scanning angle (2θ) ranged from 5° to 120°. The results are as follows: Figure 3 As shown.

[0071] XRD is commonly used to assess the crystallinity of biopolymers or compounds. The CDP peaks show two broad diffraction peaks, indicating a typical pectin crystal structure. The peak centered around 2θ = 20° is typical of amorphous polymers, suggesting that pectin chains in the solid state are primarily randomly coiled or partially ordered short-range arranged. These chains exhibit high flexibility and disorder in the solid state, which is precisely what is necessary for pectin to form a three-dimensional network gel structure in aqueous solution. A rigid crystalline structure would be detrimental to the formation of interchain interactions and network construction. Therefore, cucumber pectin has good gel-forming potential, consistent with the FT-IR spectral analysis results.

[0072] 2.4 Turbidity determination of cucumber pectin solution:

[0073] Mix cucumber pectin samples with deionized water to prepare cucumber pectin solutions with concentrations of 0.5%, 1%, 1.5%, and 2% (w / v); (w / v) represents the percentage concentration by weight and volume, and represents the number of grams (g) of solute contained in 100 ml (mL) of solution.

[0074] The turbidity of the solution was measured at 25°C. The solution was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 600 nm. The absorbance was then converted to turbidity using the following formula.

[0075] τ=-(1 / L)ln(I / I0)

[0076] Where L is the optical path length, I is the transmitted radiation intensity, and I0 is the incident radiation intensity.

[0077] The turbidity measurement results are shown in Figure 4 The turbidity of a CDP solution is positively correlated with its solute concentration; the higher the CDP content, the greater the turbidity. At low concentrations, pectin molecules are fully hydrated, with large intermolecular distances and weak interactions. As the concentration increases, the intermolecular distance decreases, the density of pectin molecules in the solution increases, and the molecular chains are closer together. Furthermore, the presence of hydrophobic regions on the pectin molecular chains (such as methoxylated galacturonic acid residues) encourages these regions to aggregate and associate to reduce contact with water. This results in more intermolecular hydrophobic microdomains, causing pectin molecules to form larger, loose, or irregular hydrated aggregates or micelles. These aggregates (micelles, microgel precursors) exceed the wavelength of the incident light and strongly scatter it. Therefore, the higher the CDP concentration, the more and larger the aggregates formed, the stronger their ability to scatter light, and the higher the turbidity. This result demonstrates that cucumber pectin possesses gelling ability and provides direct evidence that CDP molecules undergo a crucial molecular aggregation process when their concentration increases. This aggregation process forms the basis and precursor for building a gel network structure through intermolecular forces such as hydrophobic interactions and hydrogen bonds.

[0078] 2.5 Rheological property determination of cucumber pectin solution:

[0079] Cucumber pectin samples were used to prepare pectin solutions with concentrations of 0.5%, 1%, 1.5%, and 2% (w / v), respectively. These solutions were magnetically stirred at 300 rpm for 5 hours and then hydrated overnight at 4°C. The apparent viscosity of the pectin solutions was measured using a modular intelligent rheometer (equipped with a 50mm clamp). The pectin solutions were placed on the instrument clamp with a clamp gap of 1mm and a shear frequency of 0.1–100 rad / s.

[0080] The rheological test results are shown in Figure 5 As the shear rate increases, the apparent viscosity of solutions at different concentrations decreases, exhibiting a "shear thinning" phenomenon. This indicates that pectin solutions are pseudoplastic fluids (non-Newtonian fluids), and this phenomenon is further enhanced with increasing pectin concentration within a lower concentration range. The viscosity of the pectin solution increases with increasing CDP content. This is because pectin is a hydrophilic polymer; as the solution concentration increases, the distance between pectin molecular chains decreases, leading to entanglement and increased weak intermolecular forces (such as hydrogen bonds), thus increasing fluid flow resistance and consequently increasing the viscosity of the pectin solution. This is consistent with the molecular weight determination results of cucumber pectin. Utilizing the characteristic that cucumber pectin viscosity increases with concentration, within a certain range, increasing the density of the solution gel network can provide stronger protection for Lactobacillus, effectively improving its resistance to adverse conditions, such as acid resistance, digestibility, and storage stability.

[0081] Example 2: Preparation of probiotic powder:

[0082] 0.5g each of *Lactobacillus plantarum* (LPL) and *Lactobacillus rhamnosus* (LGG) were added to 50ml of MRS liquid medium and activated for 24h. Then, 0.56ml of the activated LPL and 0.94ml of LGG were added to 50ml of MRS liquid medium for a second activation, which lasted 22h. 1ml of the second-activated *Lactobacillus* culture was centrifuged at 5000r / min for 10min, the supernatant was discarded, and the culture was washed with sterile physiological saline 2-3 times until the culture turned milky white. The culture was then diluted 10 times with sterile physiological saline to prepare a solution of 10... -1 The bacterial suspension was stored at 4℃ for later use. Trehalose, fructooligosaccharides, and skim milk powder were each prepared as lyophilization protectants at 5% (w / v) with deionized water. After sterilization, the lyophilization protectants were mixed with the bacterial suspension at a volume ratio of 2:1. 3 mL of the mixture was dispensed into vials, pre-frozen for 24 hours, and then freeze-dried to obtain a lyophilized bacterial powder of *Lactobacillus plantarum* and *Lactobacillus rhamnosus* mixed culture. Figure 6 Place at 4℃ for later use.

[0083] The viable count in the freeze-dried bacterial powder was determined using the coating method, and the viable count was 1.16 × 10⁻⁶. 12 cfu / ml.

[0084] Example 3: Preparation and structural properties study of cucumber pectin-sodium alginate-gelatin hydrogel solution

[0085] 1.1 Preparation of the hydrogel system:

[0086] (1) Preparation of cucumber pectin-sodium alginate-gelatin hydrogel solution:

[0087] The cucumber pectin prepared in Example 1 was dissolved in sterile deionized water, magnetically stirred for 5 hours, and then allowed to stand overnight at 4°C. The cucumber pectin solutions were prepared at mass fractions of 0.5%, 1%, 1.5%, and 2% (w / v, g / 100mL), with a magnetic stirring speed of 300 rpm. The standing time was to remove air bubbles. A homogeneous cucumber pectin solution (CDP) was obtained. (0.5％、1％、1.5％、2％) .

[0088] Accurately weigh sodium alginate (SA) and gelatin (Gel) and add them to the cucumber pectin solution. Stir magnetically at 50°C for 3 hours, then let stand overnight at 4°C. A homogeneous cucumber pectin-sodium alginate-gelatin hydrogel solution (CAG) is obtained. (0.5％、1％、1.5％、2％) The sodium alginate and gelatin had a mass fraction of 1% (w / v, g / 100mL) and 0.5% (w / v, g / 100mL), respectively. The magnetic stirring speed was 300 rpm, and the mixture was allowed to stand to remove air bubbles.

[0089] (2) Preparation of sodium alginate-gelatin hydrogel solution:

[0090] Sodium alginate (SA) and gelatin (Gel) were accurately weighed and added to deionized water. The mass fractions of sodium alginate and gelatin were 1% (w / v, g / 100mL) and 0.5% (w / v, g / 100mL), respectively. The mixture was magnetically stirred at 300 rpm at 50℃ for 3 hours and then allowed to stand overnight at 4℃. A homogeneous sodium alginate-gelatin hydrogel solution (SAG) was obtained as a control group.

[0091] 1.2 Hydrogel Structure Analysis:

[0092] (1) Determination of particle size of hydrogel solution:

[0093] The particle size of CAG at 25℃ was measured using a laser particle size analyzer, with SAG used as a comparison. The samples were measured in parallel three times.

[0094] The particle size results of CAG and SAG hydrogels are as follows: Figure 8As shown, the particle size of CAG is significantly larger than that of SAG, and the particle size of CAG increases significantly with increasing CDP concentration. At high concentrations, the gaps between CDP molecules decrease, the cross-linking between molecular chains becomes tighter, forming a denser gel network with higher structural stability and significantly increased viscosity, leading to an increase in particle size.

[0095] (2) Measurement of Zeta potential of hydrogel solution:

[0096] The zeta potential of CAG at 25℃ was measured using a laser particle size analyzer, with SAG used as a comparison. The samples were measured in triplicate. The results are shown in Table 2.

[0097] Table 2: Results of Zeta potential measurements

[0098]

[0099] The results showed that the absolute potential value of the CAG gel solution with low CDP addition was lower than that of SAG, while the absolute potential value of the 2% CAG gel solution was significantly higher than that of SAG and low-concentration CAG gel solutions. Within a certain range, as the absolute potential value increased, the gel strength also increased, indicating that CAG has higher stability than SAG within a certain range.

[0100] (3) Turbidity determination of hydrogel solution:

[0101] Turbidity of CAG was measured, and SAG was used as a comparison. The samples were measured in triplicate. The solution was allowed to stand at room temperature for 1 hour, and the absorbance was measured at 600 nm at 25℃. The absorbance was then converted to turbidity using the following formula.

[0102] τ=-(1 / L)ln(I / I0)

[0103] Where L is the optical path length, I is the transmitted radiation intensity, and I0 is the incident radiation intensity.

[0104] Turbidity can reflect the aggregation of complexes in a system to a certain extent and is an important parameter reflecting the quality of a solution. For example... Figure 9 As shown, the turbidity of CAG was significantly greater than that of SAG (P<0.05), and the turbidity of the hydrogel increased with increasing CDP content. 2.0％ It exhibits the highest turbidity value. The addition of CDP results in a tighter cross-linking with sodium alginate and gelatin molecules, forming a denser gel network. This leads to a significant increase in hydrogel particle size, and the higher the CDP concentration, the higher the absolute value of the Zeta potential. Consequently, the complex in the hydrogel system aggregates, thus increasing the turbidity of the hydrogel.

[0105] (4) Hydrogel solution rheological determination

[0106] The apparent viscosity of CAG was determined using a modular intelligent rheometer (equipped with a 50mm clamp), with SAG used as a comparison. Samples were measured in triplicate. The solution was placed on the instrument clamp, with a clamp gap of 1mm, a shear frequency of 0.1-100 rad / s, and a temperature of 25℃.

[0107] The viscosity versus shear frequency curve is shown in [reference needed]. Figure 10 The apparent viscosity of CAG with different CDP concentrations was greater than that of SAG, and this phenomenon was more pronounced with higher CDP concentrations. On the one hand, increasing pectin concentration increases the density of polymer chains in the solution, leading to increased intermolecular entanglement and the formation of a denser network structure, thus increasing the initial viscosity. On the other hand, the mixture of sodium alginate and gelatin has a synergistic effect; the addition of pectin can further enhance the rigidity of the overall structure by forming a complex network with gelatin or sodium alginate through neutral sugar side chains, thus increasing viscosity. With increasing shear rate, the apparent viscosity of CAG and SAG with different CDP mass fractions decreased, exhibiting a "shear thinning" phenomenon. Pectin, sodium alginate, and gelatin are all high-molecular-weight polysaccharides, and their mixed solutions exhibit pseudoplastic fluid characteristics. CAG and SAG with low CDP mass fractions showed increased viscosity at shear rates below 20 s⁻¹. -1 At this time, the apparent viscosity of the solution decreases significantly, especially at shear rates greater than 20 s⁻¹. -1 At this time, the apparent viscosity of the solution tends to decrease slowly; while for CAG with a high CDP mass fraction, the decrease is slower at shear rates below 20 s⁻¹. -1 At this time, the apparent viscosity of the solution decreases sharply, especially at shear rates of 20-60 s. -1 At this time, the apparent viscosity of the solution decreases significantly, especially at shear rates greater than 60 s⁻¹. -1 At this point, the apparent viscosity of the solution decreases more slowly. The flow of pectin molecules is achieved through segmental transitions; the molecular chains have good flexibility, so when the shear rate increases, pectin molecules easily orient themselves through segmental movement, significantly reducing the shear viscosity. In summary, the cucumber pectin hydrogel system has a denser, more stable, and stronger gel network than the sodium alginate hydrogel system. Utilizing these properties, it can effectively protect lactobacilli from damage caused by adverse environmental factors such as strong acids and high temperatures.

[0108] Example 4: Preparation method, structure and performance study of cucumber pectin-sodium alginate-gelatin hydrogel beads (LCAGB)

[0109] 1. Preparation method of cucumber pectin-sodium alginate-gelatin hydrogel beads:

[0110] The freeze-dried bacterial powder prepared in Example 2 was added to the cucumber pectin-sodium alginate-gelatin hydrogel solution (CAG) prepared in Example 3. 0.5％、1％、1.5％、2％In the mixture, the ratio of freeze-dried bacterial powder to cucumber pectin-sodium alginate-gelatin hydrogel solution was 2% (w / v, g / 100mL). The mixture was magnetically stirred for 30 min, then allowed to stand at 4℃ for 30 min to remove air bubbles. This prepared a cucumber pectin-sodium alginate-gelatin viscous solution (LCAG) containing Lactobacillus. (0.5％、1％、1.5％、2％) .

[0111] The lyophilized bacterial powder prepared in Example 2 was added to the sodium alginate-gelatin hydrogel solution SAG prepared in Example 3. The ratio of the lyophilized bacterial powder to the sodium alginate-gelatin hydrogel solution was 2% (w / v, g / 100mL). The sodium alginate-gelatin viscous solution LSAG containing lactobacillus was prepared in the same manner.

[0112] Use a 5ml syringe to draw up a cucumber pectin-sodium alginate-gelatin viscous solution (LCAG) containing lactobacillus. 0.5％、1％ , 1.5％、2％ Add the mixture dropwise to 100 mL of 0.3 M CaCl2 solution, stir to promote cross-linking, and let stand overnight at 4 °C. This yields LCAGB hydrogel beads of *Lactobacillus plantarum* and *Lactobacillus rhamnosus* encapsulated in cucumber pectin, sodium alginate, and gelatin. (0.5％、1％、1.5％、2％ ).

[0113] The same procedure was used to prepare LSAGB hydrogel beads of Lactobacillus plantarum and Lactobacillus rhamnosus embedded in sodium alginate-gelatin.

[0114] Hydrogel Beads LCAGB (0.5％、1％、1.5％、2％) Photos of LSAGB hydrogel beads as shown Figure 11 As shown.

[0115] 2. Study on the structure and properties of cucumber pectin-sodium alginate-gelatin hydrogel beads:

[0116] (1) Determination of the diameter of hydrogel beads:

[0117] At LCAGB (0.5％、1％、1.5％、2％) Thirty hydrogel beads were randomly selected from each group of LSAGB beads and kept moist by immersing them in a liquid with the same permeability. A vernier caliper was calibrated for later use. Using flat-tipped toothless tweezers, the hydrogel beads were picked up and placed on moistened filter paper. The vernier caliper was used to accurately measure and read the values. Each bead was measured 2-3 times (rotating at different angles), and the average value was taken as the diameter of a single bead. At least 30 beads were measured in each group to ensure the accuracy of the statistical data. The results are shown in Table 3.

[0118] Table 3: Diameter of hydrogel beads

[0119]

[0120] The results showed that the diameters of different LCAGBs were all higher than those of LSAGBs, indicating that LCAGBs can better protect the lactobacilli loaded with lactobacilli from environmental factors.

[0121] (2) Determination of water absorption and swelling:

[0122] To measure the swelling ratio, freeze-dried hydrogel beads LCAGB were used. (0.5％、1％、1.5％、2％) Thirty beads were randomly selected from each group of LSAGB samples. The original diameter of the sample was recorded as D0. The samples were then immersed in deionized water for 3 hours at 25°C. During sampling and measurement, excess surface water was removed with filter paper, and the diameter of the sample was recorded as D1. The swelling rate of the gel beads was calculated using the formula:

[0123] Swelling rate (%) = (D1 - D0) / D0 × 100%

[0124] Swelling ratio reflects the ability of a hydrogel to absorb water and swell, and is generally related to the crosslinking density of the material. Results are provided by... Figure 12 As shown, the swelling ratio of LSAGB is significantly higher than that of LCAGB, and this phenomenon is more pronounced with higher CDP mass fractions. This is because the addition of high concentrations of CDP can effectively increase cross-linking with sodium alginate and gelatin, limiting water absorption during swelling and resulting in a lower swelling ratio. A lower swelling ratio indicates a compact hydrogel structure that maintains high cross-linking integrity even after water absorption. This structure of cucumber pectin effectively limits the diffusion and leakage of its contents, thus providing better protection for Lactobacillus and increasing its resistance to acid, digestion, and storage.

[0125] (3) Determination of the embedding efficiency of hydrogel beads:

[0126] Take 1g of LCAGB respectively (0.5％、1％、1.5％、2％) LSAGB was lysed in 9 ml of sodium citrate buffer to release the lactic acid bacteria embedded in the hydrogel beads. This buffer was then serially diluted to appropriate concentrations in 9 ml of sterile physiological saline. The diluted solutions were spread onto MRS solid medium, with each gradient performed in triplicate. The medium was incubated upside down at 37 (±0.5) °C for 48 h, and the bacteria counts were recorded. The experiment was performed in triplicate. The embedding efficiency (EE) was calculated using the following equation:

[0127] EE (%) = (N / N0) × 100%

[0128] Where EE is the encapsulation rate (%), N is the number of viable lactobacilli released from the hydrogel beads (Log CFU / mL), and N0 is the initial number of viable cells in the lactic acid bacteria stock solution used for encapsulation (Log CFU / mL).

[0129] The embedding efficiency of hydrogel beads is as follows Figure 13 As shown, LCAGB (0.5％、1％、1.5％、2％)The encapsulation rates were 43.23%, 67.61%, 80.68%, and 97.64%, respectively, while the encapsulation rate of LSAGB was 49.14%. Therefore, LSAGB... 2％ The encapsulation efficiency of the group was significantly higher than that of other groups, possibly due to the following reasons: 1) Cucumber pectin molecules have more free carboxyl groups, which bind more fully with calcium ions, forming a three-dimensional network structure through ionic cross-linking. As the pectin concentration increases, the number of cross-linking points between molecular chains increases, making the gel network denser and thus more effectively encapsulating Lactobacillus. 2) The viscosity of pectin increases with increasing concentration, which can better fix Lactobacillus and reduce its loss during gelation. 3) When pectin is combined with proteins (such as gelatin), it can enhance gel strength and improve encapsulation efficiency through hydrogen bonding and hydrophobic interactions. 4) High concentrations of pectin can reduce the pore size of the hydrogel, limiting the diffusion and escape of Lactobacillus.

[0130] Within a certain range, as the concentration of cucumber pectin increases, the cross-linking with sodium alginate and gelatin intensifies, forming a denser hydrogel network. The reduced pore size of this hydrogel network effectively decreases bacterial escape, thereby improving encapsulation efficiency. This is consistent with the results regarding particle size, potential, and rheology of the cucumber pectin hydrogel system. (LCAGB) 2％ The embedding rate was significantly higher than that of other groups, therefore LCAGB was subsequently selected. 2％ As a sample group, it is denoted as LCAGB 2％ LSAGB was set up as the control group for subsequent experimental investigation.

[0131] (4) SEM determination of hydrogel beads

[0132] The freeze-dried hydrogel beads LCAGB were examined using a ZEISS Sigma 500 field emission scanning electron microscope (SEM). 2％ The surface and cross-section of the LSAGB were photographed. The sample was fixed to the aluminum rod using double-sided tape, coated with gold by sputtering, and then scanned at 30 kV.

[0133] Figure 14 For LCAGB 2％ Surface topography images of LSAGB (from top to bottom: LSAGB, LCAGB) 2％ (From left to right, the charts show multiples of 50×, 1K×, and 20K×)

[0134] Figure 15 For LCAGB 2％ Cross-sectional view of LSAGB (from top to bottom: LSAGB, LCAGB) 2％ (From left to right, the charts show multiples of 50×, 5K×, and 20K×)

[0135] The surface morphology and cross-sectional structure of the lyophilized gel beads were observed using scanning electron microscopy. Figure 14 It can be seen that, structurally, both types of gel beads are spherical, but exhibit some shrinkage due to moisture loss during the freeze-drying process. (The sentence is incomplete and requires further context to translate accurately.) Figure 15 It can be seen that the pores in the cross-section of LSAGB gel beads are relatively loose, while the pores in the cross-section of LCAGB gel beads are denser and more uniform, indicating that the density of the gel bead structure can be improved. Furthermore, LCAGB... 2％ The interior is a uniform porous network, indicating that the embedded material is uniformly loaded; while the interior of LSAGB has stratification and large pores, which may lead to uneven embedding or burst release effects.

[0136] (5) Mid-infrared spectroscopy determination of hydrogel beads:

[0137] Measured using Fourier transform infrared spectroscopy. The freeze-dried hydrogel beads LCAGB... 2％ LSAGB and dry potassium bromide (KBr) are mixed at a ratio of 1:100 and thoroughly ground in an agate mortar until freeze-dried hydrogel beads of LSAGB are formed. 2％ Mix LSAGB and potassium bromide thoroughly. Take an appropriate amount of the mixture and place it in a tablet press. Set the pressure to 1t and the time to 1min to prepare a uniform, translucent thin film. Place the film in the machine for testing and set the wavelength range of the scanning beam to 4000-400cm. -1 The resolution is 4cm. -1 The scan was performed 32 times. The results are as follows: Figure 16 As shown.

[0138] LCAGB 2％ The similar infrared spectra of LCAGB and LSAGB indicate that both embedding systems possess similar composite gel network structures and intermolecular interactions during embedding. However, compared to LSAGB, LCAGB... 2％ It has two at 2340.1cm -1 and 2362.2cm -1 The absorption peak at that location may be due to the sample's high hygroscopicity during sample preparation, which leads to the appearance of the CO2 absorption peak.

[0139] (6) XRD determination of hydrogel beads:

[0140] X-ray diffraction (XRD) was used to further measure the structural characteristics of the samples and to further analyze the LCAGB hydrogel beads. 2％ LSAGB. Scanning angle (2θ) is 5°-120°. Results are as follows: Figure 17 As shown.

[0141] The position and intensity of diffraction peaks in XRD patterns can indicate the crystallinity and structure of polysaccharides. (LCAGB)2％ Similar to the overall trend of the LSAGB XRD pattern, the addition of pectin produced identifiable differences in the position and shape of the characteristic peaks. The similarities lie in the presence of broad peaks between 5° and 25°, a very sharp peak around 35°, a relatively sharp peak around 45°, and a few less distinct small sharp peaks after 60°, but the overall trend gradually smooths out. The sharp peaks at 35° and 45° reflect the retained calcium cross-linked regions or the crystalline structure of gelatin in the system.

[0142] (7) Determination of the tolerance of hydrogel beads in gastric juice

[0143] Preparation of in vitro simulated gastric juice (SGF): Sodium chloride 8.00 g / L, potassium dihydrogen phosphate 0.20 g / L, disodium hydrogen phosphate 1.15 g / L, pepsin 3.5 g / L, in sterile distilled water, stir until fully dissolved, adjust the pH of the solution to 2.5 with 1 mol / L dilute HCl, stir thoroughly and mix well, prepare fresh before use.

[0144] Weigh out a certain number of LCAGB hydrogel beads respectively 2％ The bacteria were divided into 5 groups and placed in conical flasks for subsequent experimental procedures. 9 mL of simulated gastric fluid was added to each group, and the mixture was thoroughly shaken and placed on a shaker. The flasks were incubated at 37℃ and 180 rpm for 2 hours. Samples were taken from one group at 0, 30, 60, 90, and 120 minutes of incubation, and the samples were lysed in sodium citrate buffer. The lysate was serially diluted, and appropriate gradients were selected for plating, with each gradient performed in triplicate. The flasks were incubated upside down at 37℃ for 48 hours, and colony counts were performed. The lyophilized bacterial powder suspension (La) served as the blank group, and LSAGB served as the control group.

[0145] Gastric juice tolerance is expressed as survival rate, calculated using the formula:

[0146] Survival rate (%) = (N / N0) × 100%

[0147] N represents the number of viable bacteria (CFU / mL) at different times when the sample is coated, and N0 represents the initial number of viable bacteria (CFU / mL) in the sample.

[0148] The results are as follows Figure 18 As shown, LCAGB 2％ After gastric digestion, the survival rate decreased from 100% at 0 min to 85.82% at 120 min; LSAGB's survival rate decreased from 100% to 63.80%, representing decreases of 14.18% and 36.2% respectively. 2％ The survival rate decline was less severe than with LSAGB; however, most free La was inactivated due to poor resistance, with the survival rate decreasing from 100% to 36.25%. Compared to LSAGB, LCAGB...2％ The survival rate remained high even after treatment with simulated gastric fluid, indicating that LCAGB 2％ It provides better protection. Compared to the La group, LCAGB... 2％ It exhibits good protective effects against Lactobacillus. The results show that cucumber pectin hydrogel beads have good acid resistance because cucumber pectin has strong gelling properties, enabling it to form a denser and more stable gel network. Furthermore, its interaction with sodium alginate and gelatin increases the stability of the hydrogel cross-linking network and reduces LCAGB. 2％ The pores present in cucumber pectin help to prevent gastric juice from entering, as can be seen from SEM results. Furthermore, the pH buffering capacity of cucumber pectin can protect lactobacilli from gastric acid damage to some extent.

[0149] (8) Continuous digestion assay of hydrogel beads:

[0150] Preparation of simulated gastric juice (SGF): The preparation method is the same as (7).

[0151] Preparation of simulated intestinal fluid (SIF): 8.00 g / L sodium chloride, 0.20 g / L potassium dihydrogen phosphate, 1.15 g / L disodium hydrogen phosphate, 1.00 g / L trypsin, sterile distilled water. Adjust the pH of the simulated intestinal fluid to 6.8 with 1 mol / L NaOH solution, stir well, and prepare fresh before use.

[0152] Weigh out a certain number of LCAGB hydrogel beads respectively 2％ The samples were divided into 5 groups and placed in Erlenmeyer flasks for subsequent experimental procedures. 9 mL of simulated gastric fluid was added to each group, and the mixture was thoroughly shaken and incubated on a shaker at 37℃ and 180 rpm for 2 h. The hydrogel bead mixtures from the 5 groups treated with gastric fluid were centrifuged, and the hydrogel beads were collected and added to Erlenmeyer flasks containing 9 mL of SIF. The flasks were incubated on a shaker at 37℃ and 180 rpm for 240 min. Samples were taken from one group at 0, 60, 120, 180, and 240 min of incubation, and the beads were lysed in sodium citrate buffer. The lysate was serially diluted, and appropriate gradients were selected for plating, with each gradient performed in triplicate. The flasks were incubated upside down at 37℃ for 48 h, and colony counts were performed. The lyophilized bacterial powder suspension (La) was used as the blank group, and LSAGB as the control group.

[0153] Experimental results are as follows Figure 19 As shown, after simulated continuous digestion, LCAGB 2％ Survival rates decreased to 73.49%, while LSAGB survival rates decreased to 37.07%, and most free La cells were inactivated due to poor resistance. Compared to LSAGB, LCAGB... 2％Even after treatment with simulated gastrointestinal fluid, the survival rate remained at a high level, reaching the order of magnitude of the beneficial effects of lactobacilli in the intestine. This indicates that cucumber pectin hydrogel beads have a better protective effect on lactobacilli and good digestibility.

[0154] (9) Determination of storage stability of hydrogel beads

[0155] Weigh out a certain number of LCAGB hydrogel beads respectively 2％ The beads were placed in test tubes containing 9 mL of sodium citrate buffer, with lyophilized bacterial powder suspension La as a blank and LSAGB as a control. The tubes were placed at 4 °C, and the beads were broken at 0, 7, 15, 30, and 60 days. The beads were then diluted and spread with the bacterial suspension, and incubated upside down at 37 °C for 48 hours to determine the number of viable bacteria and to analyze the storage stability.

[0156] LCAGB was studied. 2％ Storage stability at 4℃, experimental results are as follows: Figure 20 As shown. When the storage period reaches 30 days, according to LCAGB 2％ The number of viable *Lactobacillus plantarum* encapsulated increased by 0.2967 Log CFU, likely due to a decrease in the metabolic rate at low temperatures. However, during long-term storage, the bacteria may utilize slowly released nutrients to maintain basic metabolism or even achieve limited proliferation. After 60 days of storage, the number of viable free *La* decreased by 7.28 Log CFU, according to LCAGB. 2％ The viable counts of *Lactobacillus plantarum* encapsulated in cucumber pectin hydrogel beads decreased by 0.5533 Log CFU and 5.95 Log CFU, respectively. These results indicate that cucumber pectin hydrogel beads can effectively improve the storage stability of *Lactobacillus* and enhance its storage tolerance.

[0157] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of cucumber pectin in the preparation of probiotic encapsulation products.

2. The application according to claim 1, characterized in that, The cucumber pectin is prepared by the following method: Cucumber powder is prepared by drying, grinding, and sieving cucumbers or cucumber processing by-products. The cucumber powder is mixed evenly with deionized water to obtain a cucumber solution. The pH of the cucumber solution is adjusted to 2.0 with citric acid, stirred in a water bath at 65-75℃ for 1.5-2.5 hours, and then sonicated for 0.5-1.5 hours. The solution is filtered, the liquid is collected, and the supernatant is collected by centrifugation. Ethanol is added to the supernatant, and the precipitate is collected by centrifugation for 10-14 hours. The precipitate is then dialyzed for 40-50 hours, and after dialysis, it is freeze-dried to prepare cucumber pectin.

3. A hydrogel solution, characterized in that, The hydrogel solution is composed of cucumber pectin, sodium alginate and gelatin as described in claim 1; the mass ratio of cucumber pectin, sodium alginate and gelatin is (0.5-2):1:0.

5.

4. The hydrogel solution according to claim 3, characterized in that, In the hydrogel solution, the mass ratio of cucumber pectin, sodium alginate, and gelatin is 2:1:0.

5.

5. The method for preparing the hydrogel solution according to claim 3 or 4, characterized in that, Includes the following steps: Dissolve cucumber pectin in sterile deionized water, stir well, and let stand overnight to obtain cucumber pectin solution; Sodium alginate and gelatin were added to a cucumber pectin solution, stirred evenly, and allowed to stand overnight to prepare a hydrogel solution.

6. The use of the hydrogel solution according to claim 3 or 4 in the preparation of probiotic encapsulation products.

7. A cucumber pectin hydrogel bead encapsulating probiotics, characterized in that, It is prepared by the following method: Add the freeze-dried probiotic powder to the hydrogel solution described in claim 3 or 4, stir evenly, and let stand to obtain a hydrogel solution containing probiotics. A probiotic-containing hydrogel solution was added dropwise to a cross-linking agent and left to stand overnight to prepare cucumber pectin hydrogel beads encapsulated with probiotics.

8. The cucumber pectin hydrogel beads according to claim 7, characterized in that, Add (1-3)g of lyophilized probiotic powder to every 100mL of hydrogel solution.

9. The cucumber pectin hydrogel beads according to claim 7, characterized in that, The probiotics are Lactobacillus plantarum and Lactobacillus rhamnosus.

10. The cucumber pectin hydrogel beads according to claim 7, characterized in that, The crosslinking agent is a 0.3M CaCl2 solution; the volume ratio of the probiotic-containing hydrogel solution to the crosslinking agent is 1:20.

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