Preparation method of glycosylated bamboo shoot peptide chelated calcium

Abstract

The invention discloses a preparation method of glycosylated bamboo shoot peptide chelated calcium. Combining the bamboo shoot cleaning solution with the filtrate, carrying out alkali dissolution and acid precipitation, collecting precipitates, dispersing the precipitates in ultrapure water, and carrying out spray drying to obtain bamboo shoot protein powder; taking the bamboo shoot protein, adding alkaline protease, carrying out enzymolysis reaction, and carrying out ultrafiltration and freeze drying; the preparation method comprises the following steps: mixing BSH with six different saccharides, and dissolving in deionized water to prepare a solution; preparing BSH with similar grafting degrees and same time; cooling, filtering, and freeze-drying trapped fluid to obtain a BSH sample; mixing the glycosylated BSH solution with calcium chloride, and adding absolute ethyl alcohol to obtain glycosylated BSH-Capowder; the calcium binding capacity of the bamboo shoot peptide is effectively improved, an experimental basis is provided for development of BSH-Ca and XY-BSH-Ca as calcium supplements and functional peptide additives, the high-value utilization approach of bamboo shoot resources is expanded, and a new thought is provided for research and development of novel efficient calcium supplements.

Description

Technical Field

[0001] This invention relates to the field of preparation of glycosylated bamboo shoot peptide chelated calcium, and more particularly to a method for preparing glycosylated bamboo shoot peptide chelated calcium. Background Technology

[0002] Calcium is one of the most important minerals for the human body, accounting for approximately 1.5%–2.2% of body weight. Of this, 99% is stored as calcium phosphate in bones and teeth, while the remaining 1% participates in various physiological processes such as muscle contraction, blood clotting, cell metabolism, and heart function. However, due to changes in dietary structure, faster pace of life, and an aging population, insufficient calcium intake is becoming increasingly prominent. In China, over 90% of adults suffer from varying degrees of calcium deficiency, significantly increasing the risk of osteoporosis, rickets, hypertension, and colorectal cancer. Furthermore, anti-nutritional factors commonly found in plant-based foods (such as phytic acid, oxalic acid, and phosphates) can form insoluble precipitates with calcium ions, reducing intestinal absorption and bioavailability of calcium.

[0003] Therefore, developing novel calcium supplements that can improve absorption efficiency and bioavailability has become a key research direction in the field of nutritional science.

[0004] In recent years, food-derived peptide-calcium chelates have attracted widespread attention due to their excellent stability and bioavailability. Studies have shown that in calcium-deficient animal models, these complexes are more effective than traditional inorganic calcium salts in improving bone quality. Furthermore, they can simultaneously provide available calcium and functional peptides, offering dual nutritional benefits and demonstrating good potential for clinical application. Research reports that short peptides not only prevent calcium from forming insoluble complexes with tannic acid, oxalate, and phytate, but also significantly promote calcium absorption; animal and cell model experiments further confirm that peptide-calcium chelates can improve calcium absorption, bone mineral density, and bone mechanical strength. However, peptide-calcium chelates exhibit poor stability under strongly acidic conditions and are easily affected by pH changes, which limits their practical application.

[0005] To improve the stability and bioavailability of peptide-calcium chelates, glycosylation technology is widely used in protein modification. Common methods include Maillard reaction, enzymatic catalysis, and ionogel methods. Glycosylation can enhance protein solubility, hydrophobicity, and conformational flexibility, while also endowing them with antioxidant and prebiotic properties. Recent studies have shown that glycosylated peptide-calcium chelates have stronger anti-interference ability and structural stability, with calcium transport efficiency in the Caco-2 cell model reaching 3.54 times that of the control group, and can promote osteogenic activity and gut health. Other studies have indicated that COS-AERGVLYR (a chitosan oligosaccharide-modified peptide) can counteract the inhibitory effect of antinutritional factors on Ca²⁺ absorption, and glycosylation can significantly enhance the proliferation and mineralization activity of peptide-calcium chelates in MC3T3-E1 osteoblasts.

[0006] However, current research on the regulatory role of sugar structure and molecular weight in the performance of peptide-calcium chelates is still relatively limited. Most reports focus only on their overall effects on improving stability and bioavailability, lacking systematic research on structure-activity relationships. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing glycosylated bamboo shoot peptide chelated calcium.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] A method for preparing glycosylated bamboo shoot peptide chelated calcium includes the following steps:

[0010] Step 1: Crude extraction of bamboo shoot protein

[0011] First, the bamboo shoots are washed, cut, cell wall broken, alkali dissolved, filtered, and washed again. Then, the washing liquid and filtrate are combined, the pH value is adjusted, and acid precipitation is performed. After obvious separation, the lower layer of precipitate is removed, centrifuged, and the precipitate is collected. Finally, it is dispersed in ultrapure water, the pH value is adjusted, and it is spray-dried to obtain bamboo shoot protein powder.

[0012] Step 2: Preparation of bamboo shoot peptides

[0013] Bamboo shoot protein was added to alkaline protease, the pH value was adjusted, and enzymatic hydrolysis was carried out under magnetic stirring. Ultrafiltration was performed using 3KDa, 5KDa, and 10KDa ultrafiltration membranes to obtain <3KDa, 3-5KDa, 5-10KDa, and >10KDa peptide fractions, which were then freeze-dried.

[0014] Step 3: Preparation of different glycosylated BSH

[0015] Glycosylated BSH was mixed with six different sugars in a certain mass ratio and dissolved in deionized water to prepare a solution. The pH value was adjusted, and the reaction was stirred in a water bath to optimize the reaction time and prepare glycosylated BSH with similar grafting degree. After the reaction was completed, the solution was cooled, filtered, and the retentate was freeze-dried to obtain the glycosylated BSH sample.

[0016] Step 4: Preparation of glycosylated BSH-Ca²⁺

[0017] Take a glycosylated BSH solution, mix it with calcium chloride in a certain mass ratio, stir and react, add anhydrous ethanol, let the mixture stand, centrifuge, collect the precipitate and freeze dry to obtain glycosylated BSH-Ca²⁺ powder.

[0018] Preferably, in step 1, the cell wall is broken at a material-to-liquid ratio of 1:1 for 1 minute; and the pH is adjusted to 9.0 at a material-to-liquid ratio of 1:4 for 1 hour at 45°C.

[0019] Preferably, in step 1, the sample is filtered through a 300-mesh sieve, and the pH is adjusted to 4.2 with 2 mol / L HCl for acid precipitation. The lower precipitate is centrifuged at a rate of 4500 r / min for 20 min.

[0020] Preferably, in step 1, the bamboo shoot protein powder is dispersed in ultrapure water at a mass concentration ratio of 1:3, the pH is adjusted to 7.0, and then spray-dried to obtain bamboo shoot protein powder.

[0021] Preferably, in step 2, the substrate concentration of bamboo shoot protein is 2%, and an alkaline protease with a protein content of 9000 U / g is added; the enzymatic hydrolysis conditions are: pH value of 8, enzymatic hydrolysis temperature of 55℃, and enzymatic hydrolysis time of 2h.

[0022] Preferably, in step 3, glycosylated BSH is mixed with six different sugars at a mass ratio of 1:0.8 to prepare a solution with a final concentration of 50 mg / mL; wherein the six different sugars are: glucose, fructose, xylose, 1 kDa glucan, 5 kDa glucan, and 10 kDa glucan.

[0023] Preferably, in step 3, the pH is adjusted to 9, the reaction is stirred in a water bath at 90 °C for 3 h, and then filtered by centrifugation for 20 min using a 5 kDa ultrafiltration centrifuge tube with a centrifugal force of 10,000 × g to remove unreacted components.

[0024] Preferably, in step 4, the amount of glycosylated BSH solution added is 15 mL, with a mass-to-volume ratio of 36 mg / mL, and it is mixed with calcium chloride at a mass ratio of 11:1.

[0025] Preferably, in step 4, after stirring the reaction at 50 °C and pH 7 for 2 h, 10 times the volume of anhydrous ethanol is added.

[0026] Preferably, in step 4, the mixture is placed in a 4 °C environment and allowed to stand for 3 h to promote precipitation, followed by centrifugation at 10,000 × g for 20 min.

[0027] The present invention achieves the following technical effects compared to the prior art:

[0028] (1) This invention uses bamboo shoots as raw material, which broadens the source of raw materials for glycosylated peptide chelated calcium. It not only improves the level of deep processing and utilization of bamboo shoots, but also solves the bottleneck problem of insufficient conversion capacity in the bamboo shoot industry that restricts farmers' income and industrial upgrading.

[0029] (2) This invention uses six monosaccharides and polysaccharides with different structures and molecular weights to glycosylate bamboo shoot peptides, and systematically explores their modification effect on the calcium chelation performance of bamboo shoot peptides and their structure-activity relationship, providing a scientific basis for improving the calcium binding capacity of bamboo shoot peptides.

[0030] (3) This invention combines Caco-2 cell monolayer transport experiment and cytotoxicity evaluation to verify the safety and calcium absorption-promoting effect of glycosylated bamboo shoot peptide chelate calcium, laying an experimental foundation for the application of BSH-Ca²⁺ and XY-BSH-Ca²⁺ as novel calcium supplements and functional peptide additives.

[0031] (4) This invention not only expands the new path for the high-value utilization of bamboo shoots, but also provides new technical ideas for the development of efficient and safe new calcium supplements. Attached Figure Description

[0032] Figure 1 Grafting degree of different glycosylated BSH under the same reaction conditions (A) and grafting degree when there is no significant difference in grafting degree (B). Browning intensity (C), intermediate product content (D) and calcium binding capacity (E) of different glycosylated BSH.

[0033] Figure 2 The zeta potential (A) of monosaccharide glycosylated BSH and the zeta potential (B) of polysaccharide glycosylated BSH; the surface hydrophobicity (C) of monosaccharide glycosylated BSH and the surface hydrophobicity (D) of polysaccharide glycosylated BSH.

[0034] Figure 3 Fluorescence spectra of monosaccharide glycosylated BSH (A) and polysaccharide glycosylated BSH (B); Fourier transform infrared (FTIR) spectra of monosaccharide glycosylated BSH (C) and polysaccharide glycosylated BSH (D); X-ray diffraction (XRD) patterns of monosaccharide glycosylated BSH (E) and polysaccharide glycosylated BSH (F); Circular dichroism (CD) spectra of monosaccharide glycosylated BSH (G) and polysaccharide glycosylated BSH (H).

[0035] Figure 4 Scanning electron microscope (SEM) images of BSH with different glycosylation;

[0036] Figure 5Transepithelial resistance (TEER) of Caco-2 cell monolayers (A); cytotoxicity of calcium chloride (CaCl2), bamboo shoot protein hydrolysate-calcium chelate (BSH-Ca²⁺), xylose-modified bamboo shoot protein hydrolysate-calcium chelate (XY-BSH-Ca²⁺) (B), nimodipine (C), methyl-β-cyclodextrin (mβCD) (D), gadolinium chloride (Gd³⁺) (E), and cytochalasin D (F); calcium transport rates across Caco-2 cell monolayers of calcium chloride (CaCl2), bamboo shoot protein hydrolysate-calcium chelate (BSH-Ca²⁺), and xylose-modified bamboo shoot protein hydrolysate-calcium chelate (XY-BSH-Ca²⁺) (G);

[0037] Figure 6 A schematic diagram of the calcium ion transport mechanism of xylose-modified bamboo shoot protein hydrolysate-calcium chelate (XY-BSH-Ca²⁺);

[0038] Figure 7 The calcium ion transport rate of bamboo shoot protein hydrolysate-calcium chelate (BSH-Ca²⁺) after treatment with nimodipine (A), methyl-β-cyclodextrin (B), gadolinium chloride (C), and cytochalasin D (D) is given; the calcium ion transport rate of xylose-modified bamboo shoot protein hydrolysate-calcium chelate (XY-BSH-Ca²⁺) after treatment with nimodipine (E), methyl-β-cyclodextrin (F), gadolinium chloride (G), and cytochalasin D (H) is given. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] This invention discloses a method for preparing glycosylated bamboo shoot peptide chelated calcium, comprising the following steps:

[0041] Step 1: Crude extraction of bamboo shoot protein

[0042] Bamboo shoots were washed, cut, and blended in a blender at a ratio of 1:1 for 1 minute. The blender was then run on the juice program 4 times, and the mixture was dissolved in an alkaline solution at a ratio of 1:4 at 45℃ and pH 9 for 1 hour. The mixture was then filtered through a 300-mesh sieve. The filter residue was washed 3 times with an appropriate amount of ultrapure water to dissolve as much protein as possible. The washing liquid and filtrate were combined, and the pH was adjusted to 4.2 with 2 mol / L HCl for acid precipitation. After clear separation, the lower precipitate was centrifuged at 4500 r / min for 20 minutes. The precipitate was collected and dispersed in ultrapure water at a ratio of 1:3 (w / v). The pH was adjusted to 7.0, and the mixture was spray-dried to obtain bamboo shoot protein powder.

[0043] Step 2: Preparation of bamboo shoot peptides

[0044] Take bamboo shoot protein with an appropriate substrate concentration of 2%, add 9000 (U / g protein content) of alkaline protease, and enzymatically hydrolyze for 2 hours under the conditions of pH 8, 55℃ and magnetic stirring. Use ultrafiltration membranes with 3KDa, 5KDa and 10KDa to obtain peptide fractions with <3KDa, 3-5KDa, 5-10KDa and >10KDa cutoffs, and freeze-dry them.

[0045] Step 3: Preparation of different glycosylated BSH

[0046] BSH was mixed with six different sugars at a mass ratio of 1:0.8 and dissolved in deionized water to prepare a solution with a final concentration of 50 mg / mL. The pH was adjusted to 9.0, and the mixture was stirred in a 90℃ water bath for 3 h. By optimizing the reaction time, glycosylated BSH with similar grafting degrees was prepared. After the reaction, the solution was cooled to room temperature and centrifuged for 20 min using an ultrafiltration centrifuge tube with a centrifugation force of 10,000 × g at 5 kDa. The solution was filtered to remove unreacted components, and the retentate was freeze-dried to obtain the glycosylated BSH sample.

[0047] Step 4: Preparation of glycosylated BSH-Ca²⁺

[0048] Take 15 mL of glycosylated BSH solution with a mass ratio of 36 mg / mL and mix it with an appropriate amount of calcium chloride at a mass ratio of 11:1. After stirring and reacting for 2 h at 50 °C and pH 7, add 10 times the volume of anhydrous ethanol. Place the mixture in a 4 °C environment and let it stand for 3 h to promote precipitation. Then centrifuge at 10,000 × g for 20 min. Collect the precipitate and freeze-dry it to obtain glycosylated BSH-Ca²⁺ powder.

[0049] Example 1:

[0050] 1. Determination of browning value and intermediate products

[0051] Monitoring the formation of intermediate products and changes in browning intensity during peptide glycosylation: Dissolve the sample in deionized water to prepare a solution with a concentration of 1 mg / mL; use a UV-Vis spectrophotometer to measure the absorbance at 294 nm (to detect intermediate products) and 420 nm (to detect browning intensity); each sample was measured in triplicate.

[0052] 2. Determination of free amino acid content (grafting degree)

[0053] This invention uses the o-phthalaldehyde (OPA) method to determine the free amino group content in BSH before and after the Maillard reaction, and then calculates the grafting degree of six sugars. Freshly prepared OPA reagent contains OPA, methanol, sodium tetraborate buffer, sodium dodecyl sulfate (SDS), and β-mercaptoethanol, and should be stored protected from light. For detection, 1 mg / mL of sample is mixed with the OPA reagent and incubated in a 35°C water bath for 3 min; deionized water is used as a blank control, and absorbance is measured at 340 nm; each sample is measured in triplicate.

[0054] The formula for calculating grafting degree is as follows:

[0055] Grafting degree (%) = (A0 – A) t ) / A0 × 100 (1)

[0056] In the formula, A0 represents the free amino content (mM) before heating (i.e., before the reaction of BSH with monosaccharides / polysaccharides); A t The free amino content (mM) after heating for time t.

[0057] 3. Determination of the chelating capacity of glycosylated BSH-Ca²⁺ calcium

[0058] The calcium binding capacity was determined by EDTA titration: 5 mL of glycosylated BSH-Ca²⁺ mixture was added to 30 mL of ultrapure water, followed by 1 mL of triethanolamine, 1 mL of 1 mg / mL sodium hydroxide solution, and 0.01 g of Eriochrome Black T indicator. After thorough mixing, titration was performed using a Mettler Toledo T50 automatic potentiometric titrator with 0.025 mol / L EDTA standard solution. The solution was continuously stirred during titration until the color changed from purple-red to bright blue, which was the titration endpoint. The volume of EDTA consumed at the endpoint was recorded.

[0059] Based on the EDTA consumption, the chelation efficiency and the actual calcium content in the sample are calculated using the following formula:

[0060] Calcium chelation capacity (%) = V1 / V0 × 100% (2)

[0061] In the formula, V0 is the volume (mL) of EDTA solution required to titrate the total calcium in the mixture; V1 is the volume (mL) of EDTA solution required to titrate the chelated calcium in the peptide sample.

[0062] 4. Potential Measurement

[0063] The zeta potential of BSH and glycosylated BSH was determined as follows: The samples were dispersed in ultrapure water to prepare a solution with a concentration of 1 mg / mL; the zeta potential was measured using a Malvern Zetasizer Nano ZS90 potentiometer at 25 °C and a relative refractive index of 1.095; the samples were equilibrated for 2 min before measurement; each sample was measured in triplicate and the average value was taken as the final zeta potential result.

[0064] 5. Surface hydrophobicity determination

[0065] BSH and glycosyl BSH were prepared into solutions with a concentration of 2 mg / mL, and then gradually diluted to final concentrations of 0, 0.25, 0.5, 1, and 2 mg / mL. 2 mL of each concentration sample solution was taken, and 20 μL of 8 mM 8-anilino-1-naphthalenesulfonic acid (ANS) was added. The solutions were incubated in the dark for 15 min. Using a Hitachi F-4700 fluorescence spectrophotometer, the fluorescence emission intensity of each sample was measured under the conditions of an excitation wavelength of 390 nm, an emission wavelength of 470 nm, and an excitation and emission slit width of 5 nm. A standard curve was plotted with protein concentration on the x-axis and fluorescence intensity on the y-axis. The slope of the linear regression curve represents the hydrophobicity of the protein surface.

[0066] 6. Fluorescence spectroscopy determination

[0067] Fluorescence spectra of BSH and glycosylated BSH were determined: The samples were prepared into solutions with a concentration of 1 mg / mL and measured using a Hitachi F-4700 fluorescence spectrophotometer; the excitation wavelength was set to 290 nm, the excitation and emission slit widths were both 10 nm, the scan rate was 2400 nm / min, and the photomultiplier tube voltage was 400 V; the emission spectra were recorded in the range of 300–600 nm; background correction was performed using distilled water as a blank.

[0068] 7. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

[0069] Sample preparation: BSH and glycosylated BSH were thoroughly dried and then mixed with dried potassium bromide (KBr) powder at a ratio of 1:100 (w / w). The mixture was ground evenly and pressed into tablets. Pure KBr tablets were used as blank controls. The analysis was performed using a Tianjin Gangdong FT1R-650 Fourier transform infrared spectrometer at 25 °C. The scanning parameters were set as follows: wavenumber range 400–4000 cm⁻¹, resolution 4 cm⁻¹, and 32 scans per sample. The functional group characteristics and molecular structure changes of the samples were analyzed by spectral analysis.

[0070] 8. X-ray diffraction (XRD) analysis

[0071] The crystal structures of BSH and glycosylated BSH were analyzed using X-ray diffraction: The samples were scanned using a Bruker D2 PHASER X-ray diffractometer from Germany, within the range of 5°–90° at the 2θ angle, at a scanning speed of 4° / min, to obtain the X-ray diffraction patterns.

[0072] 9. Circular dichroism (CD) spectral analysis

[0073] The secondary structures of BSH and glycosylated BSH were analyzed by circular dichroism spectroscopy: the samples were prepared into solutions with a concentration of 0.25 mg / mL (pH 7) and analyzed using a Jasco J-1500 circular dichroism spectroscopy instrument (Japan); the scan range was set to 190 nm–260 nm and the scan rate was 100 nm / min; the secondary structure composition (α-helix, β-sheet, β-turn, random coil) of BSH and glycosylated BSH was calculated using the SELCON3 program in CDPro software.

[0074] 10. Scanning Electron Microscopy (SEM) Analysis

[0075] The microstructure of BSH and glycosylated BSH was observed using scanning electron microscopy: freeze-dried sample powder was fixed on the sample stage and a thin gold film was sputtered on it; the surface morphology of the sample was analyzed using a German Zeiss Sigma 360 scanning electron microscope under the conditions of accelerating voltage of 15 kV, working distance of 12.8 mm, test temperature of 25 °C, and magnification of 10,000×.

[0076] 11. Amino acid composition analysis

[0077] Amino acid composition analysis was performed using acid hydrolysis. Subsequently, 17 amino acids in the filtrate were determined using an external standard method with a Hitachi LA8080 amino acid analyzer (proline was detected at 440 nm, and other amino acids were detected at 570 nm). The amino acid content was calculated based on the linear standard curves plotted using known concentrations of amino acids.

[0078] 12. Establishment of the Caco-2 cell model

[0079] Establishing a Caco-2 cell monolayer model: Caco-2 cells were layered at a density of 1×10⁻⁶. 5 Cells were seeded at a density of 1.5 mL cells / mL into the top (AP) chamber of a 6-well Transwell plate, and 3 mL of complete culture medium was added to the bottom (BL) chamber. The cells were then cultured in a humidified incubator at 37 °C and 5% CO2. During the initial culture period, the culture medium in both chambers was changed every two days. From day 8 onwards, the medium was changed daily until day 21. The integrity of the monolayer was verified by measuring transepithelial electrical resistance (TEER). A TEER value ≥300 Ω・cm² ​​was considered to indicate the formation of a confluent and well-differentiated monolayer, which could be used for subsequent experiments.

[0080] Transepithelial resistance (Ω·cm²) = [Measured resistance (Ω) - Blank resistance (Ω)] × Membrane area (cm²)

[0081] 13. MTT cytotoxicity assay

[0082] The cytotoxicity of CaCl2, BSH-Ca²⁺, XY-BSH-Ca²⁺, and calcium channel inhibitors was assessed using the MTT assay: Caco-2 cells were seeded into 96-well plates and cultured to approximately 80% confluence, followed by exposure to different treatment solutions for 24 h; 100 μL of MTT solution (0.1 mg / mL) was added to each well, and incubation was performed for 4 h; the supernatant was removed, and formazan crystals were dissolved in DMSO; absorbance was measured at 570 nm using a microplate reader; cell viability was calculated using the control group (set as 100%) as a reference, as shown in the following formula:

[0083] V = (Od – Ob) / (On – Ob) × 100%

[0084] In the formula, V represents cell viability; Od represents the absorbance of the well containing calcium preparation, inhibitor, cells, and culture medium; On represents the absorbance of the well containing cells and culture medium; and Ob represents the absorbance of the blank control (containing only culture medium).

[0085] 14. Caco-2 monolayer cell calcium transport experiment

[0086] To investigate the transport of calcium preparations: After culturing Caco-2 cells for 21 days, the top and bottom chambers were rinsed three times with HBSS to remove cell debris and dead cells. Fresh HBSS was added to both chambers, and the cells were incubated at 37 °C for 40 min to equilibrate the cells and remove residual culture medium. Subsequently, the HBSS was removed, and the calcium preparation (final calcium concentration approximately 0.28 mg / mL) was prepared with HBSS. 1.5 mL of calcium solution was added to the top chamber, and 3 mL of HBSS was added to the bottom chamber. The culture plate was incubated at 37 °C for 2 h, and 1 mL of sample was collected from the bottom chamber at 30, 60, 90, and 120 min for calcium content determination. An equal amount of fresh HBSS was added after each sampling to maintain a constant volume.

[0087] 15. Research on calcium absorption pathways

[0088] To investigate the potential pathways of calcium uptake mediated by XY-BSH-Ca²⁺ and BSH-Ca²⁺, Caco-2 monolayer cells were pre-incubated for 30 min with HBSS containing different concentrations of transport activators or inhibitors (1.5 mL in the top chamber and 3 mL in the bottom chamber). The solution was then removed, and calcium preparations containing the corresponding regulators (1 mg / mL) were added to the top chamber. The Transwell chambers were then transferred to the bottom chambers of a 6-well plate containing 3 mL of HBSS and incubated at 37 °C and 5% CO₂ for 120 min. After incubation, 1 mL of solution was collected from the bottom chamber for calcium content analysis.

[0089] All experiments were performed in triplicate. Data were analyzed using Origin Pro and SPSS software (SPSS Inc., Chicago, USA). Duncan's multiple range test was used to analyze statistical differences between samples, with a significance level set at p < 0.05.

[0090] Example 2:

[0091] 1. Binding of six different sugars with BSH

[0092] Six glycosylated BSH derivatives were prepared under the same reaction conditions, and their grafting degrees are shown in Figure 1A. The results showed that the grafting degree of the glycosylated BSH samples ranged from 7.82±2.25% to 38.85±1.68%. Among the three monosaccharides, the grafting degree of pentoses (xylose, XY) was significantly higher than that of hexoses (glucose, GL; fructose, FR), while there was no significant difference in the grafting degree between aldoses (glucose) and ketoses (fructose). This result is consistent with the grafting degree patterns of different sugars modifying whey protein hydrolysates in previous studies, i.e., pentoses exhibit higher grafting reactivity with BSH, while the type of functional group (aldehyde or ketone) has a relatively small impact on reaction efficiency. This may be because the two sugars mainly exist in cyclic form in aqueous solution, thus weakening the reaction differences of their functional groups in the open-chain state. Furthermore, the grafting degree of high molecular weight polysaccharides such as dextran (DE) with BSH is significantly lower than that of monosaccharides. This phenomenon is similar to previous research results, possibly because the larger molecular size of polysaccharides creates steric hindrance, and their lower reaction site density limits the binding efficiency of peptides in the Maillard reaction. Based on the preliminary analysis of the reaction behavior of different sugars with BSH, the reaction conditions were further optimized by adjusting the Maillard reaction time, and glycosylated peptides with similar grafting degrees (36.86±0.58%–38.99±0.79%) were successfully prepared, as shown in Figure 1B.

[0093] Subsequently, the browning intensity and intermediate product content of 12 glycosylated BSH samples were determined (Figures 1C-D). The results showed that the trends in browning value and intermediate product content were generally consistent with the grafting degree. Notably, although the grafting degrees of glucose and fructose were similar, the browning degree and intermediate product formation of the fructose group were significantly higher. This may be because ketoses (fructose) are more prone to dehydration during the reaction, generating typical intermediates such as hydroxymethylfurfural (HMF); in addition, ketoses are more prone to 1,2-enolization in aqueous solution, forming enol intermediates and diketone structures, which in turn promotes the formation of brown high molecular weight products with conjugated double bonds, enhancing the browning reaction. Meanwhile, glycosylated peptides with similar grafting degrees had higher browning values ​​and intermediate product contents than glycosylated peptides prepared under the same conditions, indicating that extending the reaction time can enhance their binding with BSH. Furthermore, the absorbance at 294 nm (A294) of all samples was higher than that at 420 nm (A420), indicating that the concentration of compounds in the early and intermediate stages was significantly higher than that in the late glycosylation end products (AGEs), meaning that less harmful substances were generated in glycosylated BSH.

[0094] 2. Effects of different glycosylated BSH on chelation efficiency

[0095] After preparing glycosylated BSH, glycosylated BSH-Ca²⁺ was further synthesized under the chelation conditions optimized by previous single-factor experiments, and its calcium chelation efficiency was measured, as shown in Figure 1E. The chelating ability of BSH was improved after modification with different sugars, consistent with previous research (xylo-oligosaccharide modification of red snapper scale protein hydrolysate can improve its calcium chelation ability). This may be attributed to the high carbonyl content in the six sugars; in addition, the hydrophilicity of numerous hydroxyl groups and the flexible structure of the sugars may also enhance their affinity for calcium ions. Notably, increasing the glycopeptide grafting degree did not significantly improve the chelation efficiency. This may be because prolonged heating during the reaction caused partial denaturation of the protein structure, hindering the effective exposure and binding of the coordinating groups (such as carboxyl and amino groups) required for chelation. Further analysis of the performance of different glycosylated peptides under the same chelation conditions revealed no significant difference in chelation efficiency between aldose (glucose) and ketose (fructose) glycosylated peptides, while the chelation efficiency of pentose (xylose) glycosylated peptides was generally higher than that of hexose (glucose, fructose) glycosylated peptides; the chelation efficiency of monosaccharide glycosylated peptides was also significantly better than that of polysaccharide (glucan) glycosylated peptides. This indicates that the type and structural complexity of sugars may play a key regulatory role in chelation ability. To further explore the mechanism behind the differences, this study selected glycosylated peptide samples with similar grafting degrees to 10 kDa glucan glycosylated peptides and prepared under the same conditions, analyzing their structural characteristics and chelation performance. The aim was to elucidate the specific influence of sugar source structure on the chelation behavior of glycosylated products and to provide a theoretical basis for the design and optimization of glycopeptide mineral carriers.

[0096] 3. Effects of different glycosylated BSH on calcium chelation mechanisms

[0097] The zeta potential (ζ-potential) is a commonly used indicator to characterize the stability and surface charge properties of proteins or peptides in solution. As shown in Figure 2(A), the ζ-potential of BSH increases after glycosylation with different monosaccharides. This may be because the reducing sugar modifies the amino group of BSH in the Maillard reaction, reducing the positive charge and thus increasing its net negative charge. The ζ-potential of pentose (xylose) is higher than that of hexose (glucose, fructose), and the ζ-potential of ketose (fructose) is higher than that of aldose (glucose). This difference may be due to the different monosaccharide structures, leading to differences in the early Maillard reaction products (Schiff bases) and late glycosylation end products (AGEs), which in turn affect the ζ-potential. This phenomenon is consistent with the ζ-potential changes of different sugar-glycosylated whey protein hydrolysates in previous studies. In addition, comparing monosaccharides and polysaccharides (Figure 2(B)), it can be seen that, except for 10 kDa dextran, the ζ-potential of monosaccharides is generally higher than that of polysaccharides. This may be because the large molecular weight of dextran leads to a slower Maillard reaction rate and a higher degree of oxidation modification in the resulting advanced glycosylation products (AGEs), which then form carboxylic acids. This result is consistent with the chelation experiments described earlier, indicating that the difference in zeta potential of glycosylated peptides may affect their binding ability to calcium ions; 10 kDa dextran, due to its greater steric hindrance, may be less conducive to calcium binding. Furthermore, studies have shown that calcium binding capacity is positively correlated with the absolute value of the negative zeta potential.

[0098] Figure 2 (CD) illustrates the surface hydrophobicity of seven glycosylated BSHs. Glycosylation increased the surface hydrophobicity of all BSHs, and the trend of hydrophobicity change was highly consistent with the trend of calcium chelation capacity change. However, the opposite was true for 10 kDa dextran-glycosylated BSHs with the same grafting degree. This may be because the accumulation of glycans formed hydrophobic microregions. The increased hydrophobicity after glycosylation may be due to the reduction of glycans inducing a conformational change in bamboo shoot peptides, exposing previously buried hydrophobic groups. This phenomenon is consistent with the surface hydrophobicity changes observed in glycosylated tropomyosin (α-TM) from *Discochaeta rubiginica* in previous studies.

[0099] Fluorescence spectroscopy is often used to assess changes in the microenvironment of aromatic amino acids (such as tryptophan), thereby reflecting structural changes in peptides. As shown in Figure 3 (AB), after glycosylation, the characteristic fluorescence peaks of all seven glycosylated peptides showed a slight red shift, accompanied by fluorescence quenching. This is consistent with the findings of Shi et al., possibly because sugar grafting enhances the hydrophilicity of BSH, alters its spatial structure, and improves conformational flexibility. In addition, intermediate products generated during glycosylation (such as furfural derivatives (HMF) and α-dicarbonyl compounds) may also lead to fluorescence quenching. Among the three monosaccharides, aldoses showed higher fluorescence intensity than ketoses, and hexoses showed higher fluorescence intensity than pentoses. The fluorescence intensity of monosaccharides was also higher than that of polysaccharides, and there were differences in fluorescence intensity among polysaccharides with different degrees of polymerization (10 kDa < 1 kDa < 5 kDa < 10 kDa). The differences in fluorescence intensity may stem from the different degrees of Maillard reaction between BSH and sugars with different structures or degrees of polymerization. Smaller sugar molecules may diffuse more easily into the internal cavity of BSH, thereby increasing the consumption of aromatic amino acids such as tyrosine and tryptophan. These results are consistent with the previous analysis of Maillard reaction products and intermediates. Furthermore, the study shows that the degree of fluorescence quenching increases with increasing sugar grafting amount. These results indicate that small-molecule sugar modification of BSH may significantly alter its spatial structure and flexibility, thus making it more conducive to calcium ion binding.

[0100] Figure 3 (CD) shows the Fourier transform infrared (FTIR) spectra of seven different glycosylated BSH groups. The results show that glycosylation modification retains the characteristic absorption peaks of BSH, but the peak intensities are weakened and a slight red shift occurs. This may be because the reaction between amino and carbonyl groups in the Maillard reaction forms a large molecular compound, consuming the corresponding functional groups and thus reducing the intensity of bending or stretching vibrations. This phenomenon is consistent with the FTIR spectral changes of different glycosylated sheep hoof collagen in previous studies. Furthermore, characteristic absorption peaks of carbohydrates were observed at 920 cm⁻¹, 895 cm⁻¹, and 760 cm⁻¹, indicating that the glycosylation reaction was complete. Numerous studies have shown that calcium ions primarily bind to NH (3200-3700 cm⁻¹), -OH (2900-3000 cm⁻¹), C=O (1600-1700 cm⁻¹), and -COO⁻ (1400-1600 cm⁻¹) residues in peptides. Among the three monosaccharides, GL-BSH and FR-BSH exhibit similar absorption peaks for these four functional groups, while XY-BSH shows relatively stronger characteristic absorption peaks, suggesting that XY-BSH may possess more potential calcium ion binding sites. This is consistent with the results of the chelation experiments described earlier. Further analysis of glycosylated peptides with different degrees of polymerization revealed no significant difference in the characteristic absorption peaks of DE(1 kDa)-BSH and DE(5 kDa)-BSH, and these peaks were lower than those of DE(10 kDa)-BSH and XY-BSH. Interestingly, the characteristic absorption peak of DE(10 kDa)-BSH at -COO⁻ was significantly higher than that of other glycosylated BSHs. This may be because the larger molecular weight of dextran slows down the Maillard reaction rate, making the advanced glycosylation products (AGEs) more easily oxidized and generate carboxylic acids. This is consistent with the previous hypothesis about the increased ζ potential of DE(10 kDa)-BSH.

[0101] X-ray diffraction (XRD) is one of the most commonly used methods for determining protein crystal structure, and it can determine the protein crystal structure based on the distribution of X-ray scattering intensity. Figure 3 (EF) shows the XRD patterns of seven glycosylated BSHs. The results show that the XRD patterns of the seven glycosylated BSHs are similar, with broad diffraction peaks appearing around 20°. After glycosylation, these diffraction peaks become narrower and their intensity increases. This may be because glycosylation modification changes the protein crystal structure, and the way the glycans connect to the protein and their interaction may affect the protein recrystallization process and crystal orientation.

[0102] Figure 3 (G–H) shows the secondary structure composition (including α-helices, β-sheets, β-turns, and random coils) of seven glycosylated BSH samples, with random coils being the main structural element. After glycosylation, the content of α-helices decreased, while the proportions of β-sheets, β-turns, and random coils generally increased, a trend consistent with previous research (ovalbumin glycosylation favors the conversion of α-helices to β-sheets). Furthermore, the increased proportions of β-turns and random coils indicate that glycosylation promotes the unfolding of the BSH spatial structure, transforming some ordered structures into disordered conformations. A comparison of BSH modified with three different monosaccharides revealed α-helice / β-sheet ratios of: XY-BSH (0.125), FR-BSH (0.136), and GL-BSH (0.250). Previous studies have shown that this ratio reflects the flexibility of protein molecules; a lower ratio generally indicates greater flexibility and is more conducive to improving protein functional properties. Therefore, among the three monosaccharide-modified BSHs, XY-BSH exhibits the highest molecular flexibility, suggesting it may be more readily chelating with calcium ions. Regarding the effect of molecular weight, the α-helix / β-sheet ratio of DE-BSH shows that DE(10 kDa)-BSH (0.285) is higher than that of the lower molecular weight DE(1 kDa)-BSH (0.174) and DE(5 kDa)-BSH (0.136), indicating that the lower molecular weight DE-BSH is more flexible. Furthermore, comparing grafting density reveals that the α-helix / β-sheet ratio of JDE(10 kDa)-BSH (0.136) is lower than that of DE(10 kDa)-BSH (0.285), indicating that higher grafting density leads to more significant structural changes and greater flexibility. In summary, monosaccharide-modified BSH generally exhibits higher molecular flexibility than dextran-modified BSH, and the flexibility of lower molecular weight samples is often higher than that of higher molecular weight samples. This pattern is highly consistent with the differences in calcium chelating ability.

[0103] The surface morphology of seven glycosylated BSH samples was observed using scanning electron microscopy (SEM) (Figure 4). Natural BSH exhibits a smooth, loose, irregular sheet-like structure; after glycosylation, the complex gradually forms a smoother, more uniform, denser, and more regular surface feature. This transformation may be because the electrostatic repulsion and steric hindrance introduced by glycosylation promote peptide chain extension, thereby driving the BSH structure to unfold and improving its conformational flexibility and solubility. Furthermore, previous studies have shown that polysaccharides may attach to the surface of casein phosphopeptides (CPP), protecting their active sites and thus improving structural stability. Interestingly, compared to DE (10 kDa)-BSH, JDE (10 kDa)-BSH exhibits a relatively loose morphology, with strip-like protrusions, small gaps, and fibrous structures. This phenomenon may be related to the inhibitory effect of high-concentration polysaccharides on the complete unfolding and cross-linking of protein molecules.

[0104] Table 1–2 shows the amino acid composition of the seven glycosylated peptides, revealing an overall decrease in amino acid content after glycosylation. Previous studies have reported that calcium ions preferentially bind to aspartic acid, glutamic acid, cysteine, and lysine, and may also interact with arginine and alanine. Among the three monosaccharide-BSH samples, XY-BSH had the highest aspartic acid content, while GL-BSH contained no aspartic acid, only glutamic acid, which may weaken its calcium-binding capacity. FR-BSH had relatively high cysteine ​​and lysine content, but a low total amino acid content, and both GL-BSH and FR-BSH lacked alanine and arginine. These results suggest that XY-BSH may have a stronger calcium-binding potential compared to GL-BSH and FR-BSH. Furthermore, the amino acid content of monosaccharide-BSH was consistently higher than that of DE-BSH at different molecular weights, indicating that monosaccharide-BSH can provide more potential binding sites for calcium ions, consistent with the previous experimental results.

[0105] Table 1: Amino acid composition of monosaccharide glycosylated bamboo shoot protein hydrolysate (BSH)

[0106] Name BSH(w(g / L)) GL-BSH(w(g / L)) FR-BSH(w(g / L)) XY-BSH(w(g / L)) BSH(%) GL-BSH(%) FR-BSH(%) XY-BSH(%) Asp 0.11 - 0.02 0.06 8.18 - 3.43 6.93 Thursday 0.05 0.02 - 0.03 3.79 1.80 0.54 3.28 Sir 0.06 0.05 0.01 0.03 4.26 4.83 1.17 3.83 Glu 0.17 0.33 0.02 0.10 13.00 31.65 3.43 11.04 Gly 0.09 0.07 0.05 0.06 6.97 6.48 7.21 6.22 Ala 0.05 - - 0.03 4.08 0.00 0.00 3.51 Cys 0.07 0.06 0.06 0.06 5.08 6.19 9.76 7.19 Shaft 0.09 0.05 0.04 0.04 7.02 4.80 6.75 4.97 Met 0.05 0.05 0.04 0.04 3.59 4.50 6.32 4.87 With 0.05 - 0.03 0.03 3.66 0.00 3.89 3.09 Leo 0.09 0.06 0.05 0.05 6.66 5.77 8.02 5.82 Tyre 0.10 0.11 0.12 0.11 7.58 10.93 17.56 12.55 Phew 0.02 0.05 0.02 0.03 1.41 4.38 3.55 2.79 Dirty 0.23 0.18 0.17 0.17 17.46 17.49 26.51 18.65 Feeling 0.03 0.02 0.01 0.01 1.88 1.49 1.99 1.55 Arg 0.07 - - 0.03 5.03 0.00 0.00 3.86 Total amino acid content 1.33 1.04 0.66 0.90 99.65 100.30 100.12 100.16

[0107] Table 2: Amino acid composition of polysaccharide-glycosylated bamboo shoot protein hydrolysate (BSH)

[0108] Name DE(1KDa)-BSH(w(g / L)) DE(5KDa)-BSH(w(g / L)) DE(10KDa)-BSH(w(g / L)) JDE(10KDa)-BSH(w(g / L)) DE(1KDa)-BSH(%) DE(5KDa)-BSH(%) DE(10KDa)-BSH(%) JDE(10KDa)-BSH(%) Asp 0.01 - 0.01 0.01 1.66 - 1.50 1.37 Thursday 0.01 - - - 1.70 - 0.64 0.00 Sir 0.03 - 0.02 - 4.94 - 3.64 0.45 Glu 0.01 - 0.02 0.03 1.75 - 2.72 5.29 Gly 0.02 0.02 0.06 0.01 2.48 2.75 8.91 1.46 Ala 0.01 - 0.02 - 1.86 - 2.72 Cys 0.06 0.06 0.06 0.06 10.48 11.45 10.19 10.78 Shaft 0.04 0.04 0.04 0.04 6.47 7.44 5.86 6.44 Met 0.04 0.03 0.04 0.04 6.62 6.24 6.22 6.60 With 0.02 0.02 0.02 0.02 3.73 3.76 3.49 3.57 Leo 0.05 0.05 0.04 0.04 7.52 8.22 6.63 7.19 Tyre 0.12 0.12 0.12 0.11 19.05 21.22 18.50 20.11 Phew 0.02 0.03 0.03 0.03 4.00 4.61 4.29 4.73 Dirty 0.14 0.14 0.13 0.13 23.41 24.58 20.86 22.56 Feeling 0.01 0.01 0.01 0.01 2.32 2.22 1.76 1.87 Arg 0.02 0.03 0.03 0.02 3.84 5.16 4.09 4.24 Total amino acid content 0.61 0.55 0.63 0.57 99.98 99.51 99.30 99.38

[0109] .

[0110] 4. Mechanism by which glycosylated peptide-calcium chelates promote calcium absorption

[0111] Caco-2 cells are widely used to establish in vitro human intestinal epithelial models. To obtain a monolayer of cells with intestinal epithelial characteristics, barrier formation was assessed by monitoring transepithelial electrical resistance (TEER) (Figure 5A). The results showed that the TEER value gradually increased with prolonged culture time, exceeding 300 Ω·cm² on day 21, indicating the formation of a complete monolayer of cells with tight intercellular connections, which can be used for calcium absorption studies.

[0112] Before conducting transepithelial transport experiments, the cytotoxicity of CaCl2, BSH-Ca²⁺, XY-BSH-Ca²⁺, and four calcium uptake regulators was assessed using the MTT assay (Figures 5B–F). The results showed that CaCl2 had no significant effect on Caco-2 cell viability, while BSH-Ca²⁺ and XY-BSH-Ca²⁺ both promoted cell proliferation to some extent. Therefore, 1 mg / mL BSH-Ca²⁺ (equivalent to approximately 0.28 mg / mL Ca²⁺) was selected as the optimal treatment concentration for subsequent experiments, while CaCl2 and XY-BSH-Ca²⁺ were adjusted to the same Ca²⁺ concentration. Conversely, concentrations higher than 15 μM of nimodipine, higher than 3 mM of mβCD, or higher than 1 mM of Gd³⁺ significantly reduced cell viability, while 1 μM of cytochalasin D showed no significant cytotoxicity. Based on this, nimodipine (15 μM), mβCD (3 mM), Gd³⁺ (1 mM) and cytochalasin D (1 μM) were selected as experimental conditions to explore the potential mechanism of calcium transmembrane absorption.

[0113] As shown in Figure 5G, calcium transport in Caco-2 monolayers increased over time in all treatment groups. XY-BSH-Ca²⁺ showed the highest transport efficiency, followed by BSH-Ca²⁺, while CaCl₂ showed the lowest. This is consistent with previous findings (COS-AERGVLYR significantly enhances calcium transport in Caco-2 monolayers). The increased transport efficiency of peptide-mineral chelates may be due to their interaction with the cell membrane and / or activation of specific calcium channels. Numerous studies have confirmed that calcium uptake in Caco-2 cells mainly involves CAV1.3, TRPV6 channels, endocytosis, and paracellular pathways. Figure 7 ).

[0114] To further elucidate the transport mechanisms of BSH-Ca²⁺ and XY-BSH-Ca²⁺, pathway-specific inhibitors or modulators were employed, including nimodipine (CAV1.3 blocker, 15 μM), Gd³⁺ (TRPV6 blocker, 1 mM), mβCD (endocytosis inhibitor, 3 mM), and cytochalasin D (paracellular pathway enhancer, 1 μM). As shown in Figure 6, treatment with nimodipine, Gd³⁺, and mβCD reduced the transport rate of BSH-Ca²⁺ by 2.97%, 4.12%, and 3.31%, respectively, while treatment with cytochalasin D increased the transport rate by 2.10%. This indicates that the uptake of BSH-Ca²⁺ is achieved through CAV1.3, TRPV6 channels, and endocytosis, and that the paracellular pathway further promotes its transport. Previous studies have indicated that peptide-calcium chelates may act as calcium carriers by interacting with the extracellular domains of TRPV6 / CAV1.3 channels, promoting local Ca²⁺ release at the channel inlet. This is consistent with our findings. Similarly, in the XY-BSH-Ca²⁺ group (Figure 6), treatment with nimodipine, Gd³⁺, and mβCD reduced calcium transport by 2.88%, 5.35%, and 3.51%, respectively, while treatment with cytochalasin D increased calcium transport by 3.91%. Compared to BSH-Ca²⁺, XY-BSH-Ca²⁺ showed a stronger dependence on TRPV6 channel-mediated transport and paracellular pathways. Furthermore, previous studies have shown that the introduction of xylo-oligosaccharides (XOS) can significantly improve the calcium uptake efficiency of red snapper scale protein hydrolysate-calcium complex (CSPHs-Ca) in the Caco-2 cell model. Notably, CSPHs-Ca is not dose-dependent, while XOS-CSPHs-Ca-TG is significantly dose-dependent. This suggests that CSPHs-Ca-mediated calcium transport may be saturated and mainly depends on transcellular pathways, while the mechanism by which XOS-CSPHs-Ca-TG promotes calcium uptake may be different, involving more paracellular pathways.

[0115] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing glycosylated bamboo shoot peptide chelated calcium, characterized in that, Includes the following steps: Step 1: Crude extraction of bamboo shoot protein First, the bamboo shoots are washed, cut, cell wall broken, alkali dissolved, filtered, and washed again. Then, the washing liquid and filtrate are combined, the pH value is adjusted, and acid precipitation is performed. After obvious separation, the lower layer of precipitate is removed, centrifuged, and the precipitate is collected. Finally, it is dispersed in ultrapure water, the pH value is adjusted, and it is spray-dried to obtain bamboo shoot protein powder. Step 2: Preparation of bamboo shoot peptides Bamboo shoot protein was added to alkaline protease, the pH value was adjusted, and enzymatic hydrolysis was carried out under magnetic stirring. Ultrafiltration was performed using 3KDa, 5KDa, and 10KDa ultrafiltration membranes to obtain <3KDa, 3-5KDa, 5-10KDa, and >10KDa peptide fractions, which were then freeze-dried. Step 3: Preparation of different glycosylated BSH Glycosylated BSH was mixed with six different sugars in a certain mass ratio and dissolved in deionized water to prepare a solution. The pH value was adjusted, and the reaction was stirred in a water bath to optimize the reaction time and prepare glycosylated BSH with similar grafting degree. After the reaction was completed, the solution was cooled, filtered, and the retentate was freeze-dried to obtain the glycosylated BSH sample. Step 4: Preparation of glycosylated BSH-Ca²⁺ Take a glycosylated BSH solution, mix it with calcium chloride in a certain mass ratio, stir and react, add anhydrous ethanol, let the mixture stand, centrifuge, collect the precipitate and freeze dry to obtain glycosylated BSH-Ca²⁺ powder.

2. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 1, the cell wall is broken at a material-to-liquid ratio of 1:1 for 1 minute; then, at a material-to-liquid ratio of 1:4, the pH is adjusted to 9.0 at 45°C for 1 hour.

3. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 1, the sample is filtered through a 300-mesh sieve, and the pH is adjusted to 4.2 with 2 mol / L HCl for acid precipitation. The lower precipitate is centrifuged at a rate of 4500 r / min for 20 min.

4. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 1, the bamboo shoot protein powder is dispersed in ultrapure water at a mass concentration ratio of 1:3, the pH is adjusted to 7.0, and then spray-dried to obtain bamboo shoot protein powder.

5. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 2, the substrate concentration of bamboo shoot protein is 2%, and an alkaline protease with a protein content of 9000 U / g is added; the enzymatic hydrolysis conditions are: pH value of 8, enzymatic hydrolysis temperature of 55℃, and enzymatic hydrolysis time of 2h.

6. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 3, glycosylated BSH is mixed with six different sugars at a mass ratio of 1:0.8 to prepare a solution with a final concentration of 50 mg / mL; wherein the six different sugars are: glucose, fructose, xylose, 1 kDa glucan, 5 kDa glucan, and 10 kDa glucan.

7. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 3, the pH is adjusted to 9, and the reaction is stirred in a water bath at 90 °C for 3 h. The mixture is then centrifuged for 20 min using a 5 kDa ultrafiltration centrifuge tube with a centrifugal force of 10,000 × g to remove unreacted components.

8. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 4, 15 mL of glycosylated BSH solution was added, with a mass-to-volume ratio of 36 mg / mL, and mixed with calcium chloride at a mass ratio of 11:

1.

9. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 4, after stirring the reaction at 50 °C and pH 7 for 2 h, 10 times the volume of anhydrous ethanol is added.

10. The method for preparing glycosylated bamboo shoot peptide chelated calcium according to claim 1, characterized in that, In step 4, the mixture is placed in a 4 °C environment and left to stand for 3 h to promote precipitation, and then centrifuged at 10,000 × g for 20 min.

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