Preparation method of bamboo shoot peptide chelated calcium

Abstract

The invention discloses a preparation method of bamboo shoot peptide chelated calcium. The preparation method comprises the following steps: step 1, preparing bamboo shoot protein powder; step 2, preparing bamboo shoot peptide; and step 3, preparing the bamboo shoot peptide chelated calcium. According to the preparation method, bamboo shoots are used as raw materials, the raw material source of peptide chelated calcium is expanded, the bottleneck problems that bamboo farmer income is increased and bamboo shoot industry upgrading and development are restricted due to the fact that the deep processing conversion capacity of bamboo shoot products is insufficient are solved to a certain extent, meanwhile, 3KDa, 5KDa and 10KDa ultrafiltration membrane bags are adopted to obtain intercepted peptide components with different molecular weights, and the peptide chelated calcium is prepared. The bottleneck problem that the peptide chelated calcium is difficult to separate is solved, the prepared intercepted peptide component chelated calcium with different molecular weights has higher use efficiency and pertinence, in addition, the existence form of the bamboo shoot peptide chelated calcium is identified by adopting dithizone and ninhydrin, and the problem of incomplete alcohol precipitation is solved.

Description

Technical Field

[0001] The present invention relates to the field of preparation of bamboo shoot peptide chelated calcium, and particularly to a method for preparing bamboo shoot peptide chelated calcium. Background Art

[0002] Calcium is the fifth most abundant element in the human body, after carbon, hydrogen, oxygen, and nitrogen, accounting for 1.5% - 2.2% of the total body weight and playing a key role in physiological functions, especially in bone development and mineralization. As an essential micronutrient for maintaining structural integrity and metabolic regulation, calcium is mainly ingested through diet. Long-term insufficient intake poses risks because calcium deficiency can lead to bone diseases such as rickets, osteopenia, osteoporosis, and osteomalacia. In addition, studies have shown that insufficient calcium levels are also associated with systemic diseases, including hypertension, and in women, calcium deficiency is also associated with an increased risk of breast cancer, ovarian cancer, and premenstrual syndrome. Maintaining adequate calcium supplementation is crucial not only for bone health but also for the normal functioning of many physiological processes.

[0003] To address the problem of calcium deficiency, various calcium supplements have been developed. Inorganic calcium salts, such as calcium carbonate and calcium chloride, are inexpensive and have a high calcium content, but their poor solubility and gastrointestinal irritation often cause adverse reactions such as abdominal discomfort, arteriosclerosis, and kidney stones. Organic calcium salts, such as calcium citrate, have improved solubility and absorption rate, but their calcium content is lower, and excessive intake may still cause gastrointestinal discomfort. Although scientists have developed amino acid chelated calcium, which partially solves the problems of solubility and bioavailability, it is costly, has poor stability, and may pose a risk of negative nitrogen balance with long-term use.

[0004] In contrast, peptide chelated calcium is formed by the binding of calcium to bioactive peptides (derived from protein hydrolysis), and has higher stability and bioavailability. The neutral charge of the peptide - calcium chelate usually facilitates efficient intestinal absorption. Studies have shown that peptide chelated calcium is more effective than inorganic calcium salts in restoring bone mass in calcium - deficient animal models. In addition, this form has dual nutritional advantages by providing bioavailable calcium and functional peptides, highlighting its clinical potential.

[0005] Many studies have explored the use of food-derived peptides to synthesize calcium-chelating peptides or peptide-calcium chelates, indicating their potential as next-generation supplements. Xixi Cai et al. found that adding short peptides could effectively protect calcium ions from dietary inhibitors (such as tannic acid, oxalate, phytate, and metal ions), preventing their precipitation. At the same time, Rérat et al. perfused short peptides or free amino acids with the same amino acid composition into the duodenum of six non-anesthetized pigs to evaluate the production rates of insulin and glucagon. They found that short peptides had advantages over free amino acids, such as lower energy consumption, faster transport, and less carrier saturation. This indicates that peptide-chelated calcium, mainly absorbed through peptides, has obvious advantages in calcium absorption. In rat experiments, skipjack protein peptide-chelated calcium not only promoted the weight gain of rats and inhibited the activity of alkaline phosphatase (ALP), but also increased calcium absorption rate, enhanced bone density, improved the trabecular bone microstructure, and promoted bone growth and development. In addition, studies have shown that peptide-chelated calcium not only improves the absorption of calcium ions, but also has antioxidant, antibacterial, probiotic effects, and angiotensin-converting enzyme (ACE) inhibitory activity.

[0006] With the increasing interest in plant protein sources, more and more studies have begun to explore forest-based proteins to produce high-quality protein and peptide components, such as calcium delivery materials. Bamboo shoots are rich in resources in China and India, but they are an underutilized high-quality protein source. Bamboo shoots contain 2.6 grams of protein per 100 grams and provide all eight essential amino acids required for human health, and their amino acid composition meets the FAO / WHO recommended standards. In addition, bamboo shoots are particularly rich in amino acids such as aspartic acid, glutamic acid, and serine, which have a strong affinity for calcium, making bamboo shoots an ideal raw material for producing peptide-chelated calcium.

[0007] In the study, it was found that by using commercial protein extraction methods combined with ethanol-enhanced solvent extraction, the extraction process of bamboo shoot protein could be optimized, and these improvements could significantly support the development of calcium-chelating peptides using bamboo shoot protein. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing bamboo shoot peptide-chelated calcium.

[0009] To solve the above technical problems, the present invention adopts the following technical solutions: A method for preparing bamboo shoot peptide-chelated calcium, comprising the following steps: Step 1: Preparation of bamboo shoot protein powder Wash, cut, break the wall, dissolve with alkali, and filter the bamboo shoots. Add ultrapure water to the filter residue for washing, mix the washing liquid and the filtered liquid, adjust the pH value for acid precipitation. After obvious stratification, take the lower layer precipitate for centrifugation, collect the precipitate, disperse it in ultrapure water, adjust the pH value again, and dry to obtain bamboo shoot protein powder; Step 2: Preparation of bamboo shoot peptides Take bamboo shoot protein powder, add alkaline protease, adjust the pH value, and carry out enzymatic hydrolysis under magnetic stirring. Ultrafiltration is carried out using ultrafiltration membrane packages with molecular weights of 3KDa, 5KDa, and 10KDa to obtain peptide fractions retained by <3KDa, 3 - 5KDa, 5 - 10KDa, and >10KDa. Then, freeze-drying is carried out to obtain bamboo shoot peptides; Step 3: Preparation of calcium chelated bamboo shoot peptides Take the peptide solution and add it to anhydrous calcium chloride with a peptide-to-calcium mass ratio of 8:1. Adjust the pH value and carry out the reaction. Then, add an anhydrous ethanol solution, let it stand, centrifuge, precipitate, and freeze-dry to obtain calcium chelated bamboo shoot peptides.

[0010] Preferably, in step 1, the material-liquid ratio for cell wall breaking is 1:1, the cell wall breaking time is 1 min, and the number of cell wall breaking times is 4 times.

[0011] Preferably, in step 1, the material-liquid ratio for alkali dissolution is 1:4, the alkali dissolution temperature is 45 °C, the alkali dissolution time is 1 h, the pH value for alkali dissolution is 9.0; the filter screen is 300 mesh.

[0012] Preferably, in step 1, the pH value is adjusted to 4.2 with 2 mol / L HCl for acid precipitation.

[0013] Preferably, in step 1, the centrifugation rate is 4500 r / min and the centrifugation time is 20 min.

[0014] Preferably, in step 1, the precipitate is dispersed in ultrapure water at a ratio of 1:3 (w / v); the pH value is adjusted to 7.0 for the second time.

[0015] Preferably, in step 2, the protein content of the alkaline protease is 9000 U / g, the pH value is adjusted to 8, the enzymatic hydrolysis temperature is 55 °C, and the enzymatic hydrolysis time is 2 h.

[0016] Preferably, in step 3, take 15 ml of the peptide solution with a mass concentration of 36 mg / ml; adjust the pH value to 7, the reaction temperature is 50 °C, and the reaction time is 2 h.

[0017] Preferably, in step 3, add 10 times the volume of anhydrous ethanol solution and let it stand at 4 °C for 3 h.

[0018] Preferably, in step 3, the centrifugation speed is 5000 rpm and the centrifugation time is 20 min.

[0019] The present invention has achieved the following technical effects compared with the prior art: (1) The preparation method of the present invention uses bamboo shoots as raw materials, expanding the raw material sources of peptide chelated calcium, and to a certain extent solving the bottleneck problem that the income increase of bamboo farmers and the upgrading and development of the bamboo shoot and bamboo industry are restricted due to the insufficient deep processing and transformation ability of bamboo shoot products; (2) The present invention uses ultrafiltration membrane packages with molecular weights of 3KDa, 5KDa, and 10KDa to obtain peptide components with different molecular weight cutoffs. This not only solves the bottleneck problem of the difficult separation of peptide chelated calcium, but also the chelated calcium of peptide components with different molecular weight cutoffs prepared has higher usage efficiency and pertinence; (3) The present invention uses dithizone and ninhydrin to identify the existence form of bamboo shoot peptide chelated calcium, solving the problem of incomplete alcohol precipitation. Description of the Drawings

[0020] Figure 1 is a flow chart of a preparation method of a bamboo shoot peptide chelated calcium of the present invention; Figure 2 is a graph showing the influence results of temperature, pH value, peptide-calcium ratio, and reaction time on the preparation method of a bamboo shoot peptide chelated calcium of the present invention; Among them, Figure A is a graph showing the influence result of temperature, Figure B is a graph showing the influence result of pH value, Figure C is a graph showing the influence result of peptide-calcium ratio, and Figure D is a graph showing the influence result of reaction time; Figure 3 is a graph showing the influence on the chelating ability of a bamboo shoot peptide calcium chelate, the protein content and Zeta potential of different molecular weight components, the scanning electron microscope images of BSH and bamboo shoot peptide calcium chelate, the X-ray diffraction pattern of BSH and bamboo shoot peptide calcium chelate, and the circular dichroism spectrum of the present invention's preparation method of a bamboo shoot peptide chelated calcium; Among them, Figure A is a graph showing the influence on the chelating ability of a bamboo shoot peptide calcium chelate; Figure B is a graph showing the influence of the protein content and Zeta potential of different molecular weight components; Figure C is a scanning electron microscope (SEM) image of BSH; Figure D is a scanning electron microscope image of a bamboo shoot peptide calcium chelate; Figure E is an X-ray diffraction pattern of BSH and bamboo shoot peptide calcium chelate; Figure F is a circular dichroism (CD) spectrum; Figure 4 is a graph showing the fluorescence spectrum, Fourier transform infrared spectrum, and particle size potential curve of BSH and bamboo shoot peptide chelated calcium of the present invention's preparation method of a bamboo shoot peptide chelated calcium; Among them, Figure A is a fluorescence spectrum; Figure B is a Fourier transform infrared spectrum; Figure C is a graph of particle size potential curve; Figure 5Thermogravimetric analysis (TG) results of bamboo shoot peptide and calcium chelated bamboo shoot peptide, differential scanning calorimetry (DSC) analysis results of bamboo shoot peptide and calcium chelated bamboo shoot peptide, pH stability, phosphate stability and gastrointestinal digestion stability of calcium chelated bamboo shoot peptide; experimental result graphs of scavenging rates of calcium chelated bamboo shoot peptide with different concentrations on DPPH free radicals, ABTS free radicals and hydroxyl free radicals in the preparation method of a kind of calcium chelated bamboo shoot peptide of the present invention Among them, Figure A is the thermogravimetric analysis (TG) result graph of bamboo shoot peptide; Figure B is the thermogravimetric analysis (TG) result graph of calcium chelated bamboo shoot peptide; Figure C is the differential scanning calorimetry (DSC) analysis result graph of bamboo shoot peptide and calcium chelated bamboo shoot peptide; Figure D is the pH stability experimental result graph of calcium chelated bamboo shoot peptide; Figure E is the phosphate stability experimental result graph; Figure F is the gastrointestinal digestion stability experimental result graph; Figure G is the experimental result graph of DPPH free radicals of calcium chelated bamboo shoot peptide with different concentrations; Figure H is the experimental result graph of ABTS free radicals; Figure I is the experimental result graph of scavenging rate of hydroxyl free radicals. Specific embodiments

[0021] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] As Figure 1 shown, the present invention discloses a preparation method of calcium chelated bamboo shoot peptide, including the following steps: Step 1: Preparation of bamboo shoot protein powder Wash, cut, break the wall, dissolve with alkali, and filter the bamboo shoots. Among them, the material-liquid ratio for breaking the wall is 1:1, the wall-breaking time is 1 min, the number of wall-breaking times is 4 times, the material-liquid ratio for alkali dissolution is 1:4, the temperature for alkali dissolution is 45 °C, the time for alkali dissolution is 1 h, the pH value for alkali dissolution is 9.0, the filter screen is 300 meshes, add ultrapure water to the filter residue for washing, mix the washing liquid and the filtered liquid, adjust the pH value to 4.2 with 2 mol / L HCl for acid precipitation. After obvious stratification, take the lower layer precipitate for centrifugation, the centrifugation rate is 4500 r / min, the centrifugation time is 20 min, collect the precipitate, disperse the precipitate in ultrapure water at a ratio of 1:3 (w / v), adjust the pH value to 7.0 again, and dry to obtain bamboo shoot protein powder; Step 2: Preparation of bamboo shoot peptide Take bamboo shoot protein powder, add alkaline protease with a protein content of 9000 U / g, adjust the pH value to 8, the enzymatic hydrolysis temperature to 55 °C, and the enzymatic hydrolysis time to 2 h. Conduct enzymatic hydrolysis under magnetic stirring, and perform ultrafiltration using ultrafiltration membrane packages with molecular weights of 3 KDa, 5 KDa, and 10 KDa to obtain peptide fractions retained by <3 KDa, 3 - 5 KDa, 5 - 10 KDa, and >10 KDa. Then conduct freeze-drying to obtain bamboo shoot peptides. Step 3: Preparation of bamboo shoot peptide chelated calcium Take 15 ml of peptide solution with a mass concentration of 36 mg / ml, add it to anhydrous calcium chloride with a peptide-to-calcium mass ratio of 8:1, adjust the pH value to 7, and conduct the reaction at a reaction temperature of 50 °C for 2 h. Add 10 times the volume of anhydrous ethanol solution, let it stand at 4 °C for 3 h, and then centrifuge at a rotation speed of 5000 rpm for 20 min. Precipitate and conduct freeze-drying to obtain bamboo shoot peptide chelated calcium.

[0023] Example 1: Identification of bamboo shoot peptide chelated calcium Take a certain amount of bamboo shoot peptide chelated calcium solution and add a few drops of dithizone. If it turns green, there is no free calcium ion. If it is red or yellow, continue alcohol precipitation until the color turns green.

[0024] Take a certain amount of bamboo shoot peptide chelated calcium solution, add a few drops of ninhydrin reagent and heat it to boiling. If the solution turns blue-violet, it indicates the presence of free polypeptide or amino acid in the solution, and continue washing with anhydrous ethanol until the color does not change. Then take another certain amount of bamboo shoot peptide chelated calcium solution, add sodium sulfide. If a precipitate appears, it indicates that calcium ions are displaced. After centrifugation, add a few drops of ninhydrin reagent and heat it to boiling. If the solution turns blue-violet, it indicates that the polypeptide and calcium ions exist as a conjugate.

[0025] Example 2: Determination of chelation rate of bamboo shoot peptide chelated calcium Take an appropriate amount of peptide-calcium chelate sample for reaction, and measure the total calcium content in the reaction solution and the calcium content in the peptide-calcium chelate after the reaction; successively add 30 mL of ultrapure water, 1 mL of triethanolamine, 1 mL of sodium hydroxide (1 mg / ml), and 0.01 g of eriochrome black T indicator, mix well, and then use a Swiss Mettler-Toledo automatic potentiometric titrator T50 for EDTA titration. The end point of the titration is marked by the appearance of pure blue, and record the volume of EDTA used.

[0026] Calculate the chelation rate according to the following formula:

[0027] 。

[0028] To study the effect of temperature on the formation of bamboo shoot peptide-calcium chelate, 15 mL of bamboo shoot peptide solution was mixed with anhydrous calcium chloride at a mass ratio of 8:1; the pH value of the solution was adjusted to 7.0, and chelation reactions were carried out at different temperatures of 30 °C, 40 °C, 50 °C, 60 °C, and 70 °C while keeping other treatment conditions unchanged.

[0029] To explore the effect of pH value, the pH value of the solution was adjusted to 5, 6, 7, 8, and 9 before the chelation process; in addition, to evaluate the effect of the peptide-calcium mass ratio, 15 mL of peptide solution (concentration of 36 mg / mL) was treated with anhydrous calcium chloride at mass ratios of 2:1, 5:1, 8:1, 11:1, and 14:1.

[0030] To evaluate the effect of reaction time, the conditions with the highest chelating ability under the conditions of mass ratio, pH value, and temperature were selected, and the chelation reaction times were set to 0.5 h, 1 h, 1.5 h, 2 h, and 2.5 h, respectively. All other steps were consistent with the protocol described in Section 3.2.3.

[0031] Example 3: Structural Characterization 1. Analysis of the composition and amino acid composition of bamboo shoot peptide-calcium chelate The protein content of bamboo shoot peptide-calcium chelate was determined using an elemental analyzer (Vario Max cube CNS, Elementar, Germany), and the moisture content was determined according to the AOAC official method.

[0032] Amino acid composition analysis was carried out by acid hydrolysis method; 200 μL of bamboo shoot peptide-calcium chelate solution with a concentration of 0.25 mg / mL was placed in a hydrolysis tube, and 200 μL of analytical grade hydrochloric acid was added; nitrogen was introduced using a nitrogen blower for 15 minutes to remove oxygen, and then it was sealed; the sealed sample was hydrolyzed in an oven at 110 °C for 24 hours, cooled and opened, and diluted to 50 mL; 1 mL of the diluted sample was accurately pipetted, deacidified using a rotary evaporator, and dried at 60 °C. Finally, 1.0 mL of 0.02 mol / L hydrochloric acid was added to dissolve the sample, and it was filtered through a 0.22 μm aqueous filter membrane; the filtrate was analyzed for 17 amino acids using an amino acid analyzer (LA8080, Hitachi, Japan); The chromatographic column was a cation exchange column with an inner diameter of 4.6 mm × 60 mm, the mobile phase was a citrate buffer system, and the flow rate was 0.2 mL / min; the column temperature was maintained at 55 °C, and the reaction chamber temperature was set at 135 °C. The free amino acid content was determined by the external standard method, proline was detected at 440 nm, and other amino acids were detected at 570 nm; the amino acid content was calculated based on the amino acid standard curve with a known concentration.

[0033] 2. Fluorescence spectrum The fluorescence spectra of bamboo shoot hydrolysate (BSH) and bamboo shoot peptide-calcium chelate at a concentration of 1 mg / mL were recorded using a fluorescence spectrophotometer (F-4700, Hitachi, Japan); the excitation wavelength was set at 290 nm, and the emission wavelength range was 300 - 600 nm; the excitation and emission slit widths were both set at 10 nm, the scanning speed was 2400 nm / min, the voltage was 400 V, and the background fluorescence was calibrated using distilled water.

[0034] 3. Fourier transform infrared spectroscopy After thoroughly drying the bamboo shoot hydrolysate (BSH) and bamboo shoot peptide-calcium chelate powders, they were mixed with dry KBr powder in a ratio of 1:100; the mixture was ground into a fine powder and pressed into tablets; the KBr tablets were used as blank controls; the FTIR spectra were measured using a Fourier transform infrared spectrometer (FT1R-650, Tianjin Gangdong, China) at 25 °C with a resolution of 4 cm⁻¹, 32 scans, and a wavelength range of 400 - 4000 cm⁻¹.

[0035] 4. Circular dichroism The circular dichroism spectra of BSH and bamboo shoot peptide-calcium chelate at pH 7 and a concentration of 0.25 mg / mL were recorded using a J-1500 circular dichroism spectrometer from Jasco Corporation, Japan.

[0036] The scanning range was 190 - 260 nm, and the scanning rate was 100 nm / min; then, the secondary structure compositions of BSH and bamboo shoot peptide-calcium chelate were calculated using CDPro software equipped with the SELCON3 program.

[0037] 5. SEM The microstructures of BSH and bamboo shoot peptide-calcium chelate were observed using a scanning electron microscope (SEM) (Sigma 360 model from Carl Zeiss AG, Germany); the freeze-dried sample powders were fixed on the sample stage and coated with a thin layer of gold; The tests were carried out under the following conditions: the acceleration voltage was 15 kV, the working distance was 12.8 mm, the test temperature was 25 °C; the magnification factors were set at 2000 times and 10000 times respectively.

[0038] 6. XRD The crystallinity of calcium chloride (CaCl2), BSH, and bamboo shoot peptide-calcium chelate was analyzed using an X-ray diffractometer (D2 PHASER model from Bruker Corporation, Germany); the samples were continuously scanned at a speed of 4 ° / min within the 2θ angle range of 5° to 90°.

[0039] 7. Particle size distribution and zeta potential Dissolve BSH and bamboo shoot peptide-calcium chelate in ultrapure water to prepare a sample solution with a concentration of 1 mg / mL; use a Malvern Zetasizer Nano ZS90 instrument to measure the particle size distribution and zeta potential of the sample at 25 °C and a relative refractive index of 1.095; before measurement, the sample needs to be equilibrated for 2 minutes.

[0040] 8. In vitro digestion Simulated gastric digestion: Adjust 16 mL of the sample with a concentration of 10 mg / mL to a pH of 2.0 with 2 M hydrochloric acid; then, add 1.2 mL of simulated gastric juice, incubate the mixture in a water bath at 37 °C, and stir gently; at specific time points (0 h, 0.5 h, 1 h, 1.5 h, and 2 h), collect 2 mL of the sample, heat it in a water bath at 100 °C for 10 minutes to inactivate the enzyme, and then add 10 times the volume of absolute ethanol; let the mixture stand at 4 °C for 3 hours; finally, centrifuge the sample at 8000 × g for 20 minutes and measure the calcium content in the precipitate.

[0041] Simulated intestinal digestion: After simulated gastric digestion, adjust the pH of the sample to 7.0 with 1 mol / L sodium hydroxide. Then, add 6 mL of the prepared simulated intestinal juice, and oscillate the mixture in a water bath at 37 °C; as before, collect 2 mL of the sample at 0 h, 0.5 h, 1 h, 1.5 h, and 2 h, heat it in a water bath at 100 °C for 5 minutes to inactivate the enzyme; the remaining steps are carried out in the same manner as above.

[0042] The calculation method of calcium retention rate is as follows: Calcium ion retention rate (%) = m1 / m2 × 100% (3) Where m1 is the calcium ion content (mg) in the precipitate after treatment; m2 is the calcium ion content (mg) in the sample before treatment.

[0043] 9. Stability analysis Use a Mettler-Toledo thermogravimetric / differential scanning calorimeter (STAR model) to evaluate the thermal stability of the sample; weigh each sample and put it into a sealed crucible (10 mg); set the program to increase the temperature from 25 °C to 600 °C at a heating rate of 10 °C / min, and set the nitrogen gas flow rate to 30 mL / min; record the mass change of the sample in real time.

[0044] Dissolve the bamboo shoot peptide-calcium chelate in ultrapure water to prepare a sample solution with a concentration of 2 mg / mL; add different concentrations of phosphate (0, 25, 50, 75, 100, 125, 150 mmol / L) to this solution at 50 °C under different pH conditions (5, 6, 7, 8, 9) and react for 1 hour; after the reaction, perform a centrifugation operation (10,000×g, 15 minutes), and measure the calcium retention rate to evaluate the pH stability of BSH and the bamboo shoot peptide-calcium chelate.

[0045] 10. Antioxidant activity Dilute the freeze-dried sample with ultrapure water to prepare solutions with concentration gradients of 1, 2, 3, 4, and 5 mg / mL, with a volume of 3 mL for each solution; use the following kits from Shanghai Yuanye Bio-Technology Co., Ltd. to evaluate the DPPH radical scavenging ability, ABTS radical scavenging ability, and hydroxyl radical scavenging ability of the sample: DPPH radical scavenging ability detection kit (product number: R27138), ABTS radical scavenging ability detection kit (product number: R32103), and hydroxyl radical scavenging ability detection kit (product number: R32869).

[0046] Results and analysis 1. Optimization of the preparation conditions of bamboo shoot peptide calcium chelate Research shows that the preparation conditions of the peptide-calcium chelate have a significant impact on its calcium chelating ability; to obtain BSH with the best calcium-carrying capacity, various factors such as chelation temperature, pH value, peptide-calcium ratio, and reaction time were studied for their effects on calcium chelating ability and calcium content; as Figure 2 shown in A, the chelating ability and calcium content initially increase with the increase in temperature and then start to decline after reaching a certain level. The optimal reaction temperature is determined to be 50 °C; this indicates that within a specific temperature range, heating helps molecular collisions and promotes the formation of coordination bonds; however, too high a temperature will destroy the structure of the peptide, hinder the establishment of coordination bonds, and cause partial dissociation of the peptide-calcium chelate.

[0047] As the pH value increases, the chelating ability and calcium content show a similar trend of first increasing and then decreasing, and the optimal pH value is 7, as Figure 2 shown in B. Under acidic conditions, the protonation of carboxyl groups may reduce the availability of calcium binding sites; under alkaline conditions, hydroxide ions may interact with calcium ions to form precipitates, thereby reducing the chelating ability and calcium content.

[0048] As the peptide-calcium mass ratio increases, as Figure 2As shown in C, the chelating ability and calcium content first increase and then decrease; when the mass ratio is 11:1, the chelating ability and calcium content reach the highest; further increasing the content of BSH may lead to a decrease in chelating ability, which may be due to the reduced collision required for the formation of chelates between calcium ions and peptides in the mixture; after reacting for 2 hours, the chelating ability reaches the maximum value, which may be attributed to the dynamic characteristics of the binding between calcium and BSH; if the time is too long, the peptide-calcium chelate will dissociate, as Figure 2 shown in D.

[0049] Generally speaking, the optimal preparation conditions for bamboo shoot peptide-calcium chelate are as follows: mixing with calcium chloride at a mass ratio of 11:1, reacting for 2 hours under the conditions of pH value of 7 and temperature of 50 °C.

[0050] 2. Structural characteristics of bamboo shoot peptide-calcium chelate In order to more deeply understand the role of BSH with different molecular weights in the chelation process, the present invention studied the chelating ability of BSH with different molecular weights, as Figure 3 shown in A. The BSH component with a molecular weight of 5-10 KDa showed the highest chelating ability and calcium content, which were 54.73% and 46.01 mg respectively. This is probably because this component was moderately hydrolyzed, improving its solubility and increasing the possibility of interaction between calcium ions and peptides. In addition, more chelating sites may be exposed, thus enhancing the chelating ability and calcium content. Moreover, the protein content of the BSH components with molecular weights of 5-10 KDa, 3-5 KDa and <3 KDa was higher than that of the BSH component with a molecular weight >10 KDa and the unseparated BSH. The Zeta potential of the BSH component with a molecular weight of 5-10 KDa was also higher than that of other BSH components, which enhanced its binding ability with calcium ions, as Figure 3 shown in B.

[0051] Based on the chelating ability and calcium ion content, the BSH components with molecular weights of 5-10 KDa, 3-5 KDa and <3 KDa were selected for preliminary composition analysis, and the change trend of the protein content of these chelates was consistent with the previous analysis results of BSH components with different molecular weights. In addition, for the BSH components with molecular weights of 5-10 KDa and 3-5 KDa, their protein content, calcium ion content and moisture content all exceeded 80%. Therefore, the present invention selected the BSH component with a molecular weight of 5-10 KDa for further characterization analysis because this BSH component showed higher chelating ability, more calcium ion content and higher component purity.

[0052] 3. Scanning electron microscope (SEM) and X-ray diffraction (XRD) analysis As Figure 3 shown in C and Figure 3As shown in Figure D, scanning electron microscope images of the surface morphology of BSH and its calcium chelate at magnification factors of 2000 times and 10000 times are presented respectively. It can be observed that the surface of BSH appears relatively smooth, showing a layered and plate-like structure, and is relatively fluffy in texture. After adding calcium ions, many smaller, denser and more uniform rough spherical structures are formed. This may be due to the cross-linking interaction induced by calcium ions.

[0053] To further explore the effect of calcium ions on the spatial structure of BSH, X-ray diffraction (XRD) analysis was carried out on BSH, bamboo shoot peptide calcium chelate and anhydrous calcium chloride. As Figure 3 shown in Figure E, anhydrous calcium chloride shows distinct crystal diffraction peaks, while such diffraction peaks are not observed for BSH or bamboo shoot peptide calcium chelate. It is worth noting that the addition of calcium ions results in new, broader and stronger diffraction peaks in the BSH sample, indicating that calcium ions have changed the crystal structure of BSH. This means that calcium ions do not simply mix mechanically with BSH, but form a new chemical chelate.

[0054] 4. Circular dichroism spectroscopy analysis Circular dichroism (CD) spectroscopy is a well-established method for studying the secondary structure of proteins or peptides in aqueous solutions, which can provide crucial information on conformational changes and structural stability. As Figure 3 shown in Figure F, the secondary structure of BSH consists of α-helix, β-sheet, β-turn and random coil structures, among which random coil is the main component, and the contents of other structures increase in turn. After adding calcium ions, the contents of α-helix, β-turn and random coil structures decrease, while the content of β-sheet increases. This indicates that calcium ions may promote the transformation of BSH from a disordered structure to a more ordered structure, which is beneficial to the formation of the β-sheet secondary structure. In addition, this result is consistent with the scanning electron microscope (SEM) analysis, further confirming that calcium ions have a significant impact on the structural properties of BSH.

[0055] 5. Fluorescence spectroscopy analysis The fluorescence spectra of BSH and bamboo shoot peptide calcium chelate are as Figure 4As shown in Figure A; when the excitation wavelength is 290 nm, the maximum fluorescence emission peak appears at 356.2 nm for BSH. After adding calcium ions, this peak undergoes a red shift to 357.6 nm, accompanied by an obvious fluorescence quenching phenomenon. The observed red shift and the decrease in fluorescence intensity are likely due to the coordination interaction between calcium ions and specific functional groups (such as carboxyl, carbonyl, and amino groups) in BSH. This interaction triggers the folding and conformational rearrangement of the peptide molecules, forming a more compact structure, partially shielding the fluorescent groups, and thus reducing the fluorescence intensity. In addition, the densification of this structure is consistent with the surface morphological changes observed during the scanning electron microscopy (SEM) analysis of the calcium chelate of bamboo shoot peptide, further demonstrating that the binding of calcium ions has a significant impact on the molecular structure of BSH.

[0056] 6. Fourier Transform Infrared Spectroscopy To further explore the potential calcium binding sites in BSH, Fourier transform infrared spectroscopy (FTIR) was used for analysis. The FTIR results are shown in Figure 4 Figure B. There are significant differences between BSH and calcium chelated bamboo shoot peptide at multiple characteristic absorption peaks, mainly located at 3292.98 cm⁻¹, 2961.97 cm⁻¹, 1651.58 cm⁻¹, 1584.46 cm⁻¹, and 1454.38 cm⁻¹. The N-H stretching vibration peak shifts from 3292.98 cm⁻¹ to 3381.93 cm⁻¹, accompanied by an increase in peak width and intensity, which may be attributed to the coordination of Ca²⁺ with N-H, inducing stretching vibration and hydrogen ion substitution, thereby enhancing the electron cloud density. The -OH stretching vibration peak shifts from 2961.97 cm⁻¹ to 2935.4 cm⁻¹ and the peak band becomes narrower, indicating that Ca²⁺ replaces the hydrogen bond in -OH. The amide I band mainly related to the C=O stretching vibration shifts from 1651.58 cm⁻¹ to 1654.84 cm⁻¹ with a decrease in intensity; while the amide II band corresponding to the N-H deformation vibration also weakens significantly, indicating that Ca²⁺ may interact with the C=O and N-H groups. In addition, the disappearance of the -COO⁻ in-plane vibration peak at 1454.38 cm⁻¹ indicates that Ca²⁺ chelates with the -COO⁻ group. Further shifts and intensity changes observed in the region of 1300 - 1000 cm⁻¹ are attributed to the C-N and C-O stretching vibrations, indicating their participation in calcium coordination. These findings suggest that calcium ions lead to structural rearrangement and stabilization by interacting with the N-H, C=O, -COO⁻, and -OH residues in BSH and involving the contributions of C-N and C-O bonds.

[0057] 7. Particle Size and Zeta Potential Analysis Particle size and Zeta potential are often used to characterize the size distribution, stability, and surface charge characteristics of proteins or polypeptides in solution. Studies have shown that calcium ions have a charge shielding effect, which may be due to the binding of calcium ions to free amino and carboxyl groups on the polypeptide chain. Therefore, Zeta potential analysis can be used to reflect the binding between calcium ions and BSH. As Figure 4 shown in C, the Zeta potentials of BSH and calcium bamboo shoot peptide chelate are both negative. The Zeta potential of BSH is -18.7 mV. After adding calcium ions, the Zeta potential decreases to -10.57 mV. This change indicates that electron migration occurs during the binding of BSH and calcium ions, and new compounds may be formed. In addition, the average particle sizes of BSH and calcium bamboo shoot peptide chelate are 250.17 ± 9.40 nm and 666.77 ± 13.93 nm, respectively, and the polydispersity indices (PDI) are 0.49 ± 0.05 and 0.21 ± 0.01, respectively. This shows that the addition of calcium ions significantly increases the particle size of BSH, but at the same time makes the distribution of BSH in the solution more uniform.

[0058] 8. Thermal Stability The results of thermogravimetric analysis (TG) and differential scanning calorimetry (DSC) curves of BSH and calcium bamboo shoot peptide chelate are shown in Figure 5 A, 5B, 5C. As can be seen from the TG curve Figure 5 A of BSH, the weight loss process is divided into three stages: 45.27 - 109.09 °C, with a weight loss of 9.66%; 150.56 - 203.64 °C, with a weight loss of 7.14%; 247.97 - 367.32 °C, with a weight loss of 47.01%, and finally 34.38% remains. The weight loss process of calcium bamboo shoot peptide chelate Figure 5 B is also divided into three stages: 52.73 - 143.91 °C, with a weight loss of 15.62%; 225.58 - 281.04 °C, with a weight loss of 15.64%; 325.15 - 436.44 °C, with a weight loss of 16.94%, and finally 47.56% remains. Studies have shown that the weight loss in the range of 50 - 120 °C may be related to the volatilization of water, while the weight loss in the range of 120 - 359 °C may be due to the breaking of hydrogen bonds or certain chemical bonds. Compared with BSH, the addition of calcium ions increases the weight loss temperature in the first weight loss stage of BSH, decreases the weight loss rate and increases the weight loss temperature in the second weight loss stage, indicating that calcium ions significantly enhance the thermal stability of BSH.

[0059] From the DSC curves of BSH and calcium bamboo shoot peptide chelate Figure 5It can be seen that BSH has two endothermic peaks at 73.71 °C and 512.49 °C respectively. After adding calcium ions, the endothermic peaks shifted to 90.75 °C and 514.09 °C respectively, and a new endothermic peak appeared at 251.82 °C, indicating that calcium ions enhanced the bond energy of relevant chemical bonds and more energy was required to break these bonds. Thus, it can be seen that bamboo shoot peptide chelated calcium has a more stable structure than BSH and the possibility of thermal denaturation is reduced.

[0060] 9. pH Stability The calcium retention rate of bamboo shoot peptide chelated calcium under different pH conditions is as Figure 5 shown in E. As the pH value increases, the calcium retention rate shows a trend of first increasing and then decreasing. In the pH range of 6 - 7, the calcium retention rate is 71.223% - 85.132%, showing good stability. Under acidic conditions, the calcium retention rate decreases significantly, probably due to the competition between excessive hydrogen ions and calcium ions, resulting in the dissociation of calcium ions from the chelate. In an alkaline environment, excessive hydroxide ions combine with the dissociated calcium ions to form a precipitate, thus reducing the calcium retention rate. Generally speaking, bamboo shoot peptide chelated calcium has good stability in the pH range of 6 - 7. When used as a nutritional fortifier, an overly acidic or overly alkaline environment should be avoided. In addition, the pH values of most foods are in the range of 5 - 9. Therefore, theoretically, bamboo shoot peptide calcium chelate can be added as a calcium supplement to most foods and remain stable.

[0061] 10. Phosphate Stability During human digestion, substances such as phytates, phosphates, and oxalates will significantly affect the bioavailability of calcium ions due to their strong binding ability to calcium ions. Therefore, in this experiment, phosphate buffer solutions (PBS) with different concentrations were used to explore the phosphate stability of bamboo shoot peptide chelated calcium. The experimental results are as Figure 5 shown in D. When the phosphate concentration is 50 mmol / L, the calcium retention rate of bamboo shoot peptide chelated calcium is 87.53%, indicating that it has good stability in a low-concentration phosphate environment. As the phosphate concentration increases, the calcium retention rate of bamboo shoot peptide chelated calcium gradually decreases. When the phosphate concentration reaches 150 mmol / L, the calcium retention rate drops significantly to 5.28%. This result shows that high-concentration phosphate has a strong complexation effect with the calcium ions in bamboo shoot peptide chelated calcium, resulting in the dissociation of calcium ions from the bamboo shoot peptide chelate, thus affecting its stability and bioavailability. Generally speaking, bamboo shoot peptide chelated calcium shows good stability in a low-concentration phosphate buffer solution and can effectively resist the influence of phosphate on calcium retention.

[0062] 11. Gastrointestinal Digestion Stability The digestion and absorption process of daily foods in the body mainly includes two stages: first, initial digestion occurs in the stomach, and then the absorption of nutrients is completed in the small intestine. For peptide chelated calcium, its absorption in the small intestine mainly enters the small intestinal cells in an integral form, and this structure helps to stabilize calcium ions, thereby improving its bioavailability. However, during the gastrointestinal digestion process, peptide chelated calcium may be affected by digestive enzymes, resulting in hydrolysis of the peptide chain or cleavage of the chelation bond, thereby affecting the stability and absorption efficiency of calcium. Therefore, the calcium retention rate in the gastrointestinal system is an important indicator for evaluating the effectiveness of peptide chelated calcium as a calcium supplement. The experimental results are as Figure 5 shown in Figure F. During the simulated gastric digestion process of bamboo shoot peptide chelated calcium, its calcium retention rate significantly decreased to 71.22% after 0.5 hours of digestion. With the extension of digestion time, the final calcium retention rate decreased to 44.20%. This phenomenon may be related to the poor stability of the chelate in an acidic environment or the action of pepsin. However, based on the analysis of the pH stability experimental results mentioned above, the sensitivity of bamboo shoot peptide chelated calcium to pepsin is not significant. When further simulating the intestinal digestion process, the results showed that the calcium retention rate of bamboo shoot peptide chelated calcium only decreased slightly during the whole digestion process, indicating that trypsin has a relatively small impact on bamboo shoot peptide chelated calcium. Therefore, during the digestion process of bamboo shoot peptide chelated calcium in the gastrointestinal tract, its stability is mainly affected by the pH environment, while the action of digestive enzymes is relatively weak.

[0063] 12. DPPH and ABTS radical scavenging abilities DPPH radicals and ABTS radicals are relatively stable organic radicals with a clear electron structure and a long half-life. They mainly react with antioxidants through the electron transfer (ET, Electron Transfer) mechanism. Antioxidants donate electrons to reduce the radicals, thereby neutralizing their activity and causing a fading phenomenon. Therefore, the radical scavenging ability can be evaluated by the change in absorbance. The experimental results showed that the scavenging abilities of bamboo shoot peptide chelated calcium against DPPH and ABTS radicals, as Figure 5 shown in Figures G and 5H, are positively correlated with the increase in concentration, and the highest scavenging rates are 60.36% and 59.47% respectively. This antioxidant property may be attributed to the relatively high content of hydrophobic amino acids in bamboo shoot peptide chelated calcium (Pol, et al. 2023) and the addition of calcium ions, as shown in Table 1. And research has pointed out that the introduction of metal ions may promote the increase in the content of hydrophobic amino acids in bamboo shoot peptides, thereby enhancing their antioxidant ability. This indicates that bamboo shoot peptide chelated calcium exhibits good antioxidant potential through its specific amino acid composition and metal chelation characteristics.

[0064] 13. Hydroxyl radical scavenging ability Hydroxyl radical (·OH) is a highly reactive and short-lived radical that usually reacts with antioxidants through hydrogen atom transfer (HAT) or electron transfer (ET) mechanisms. The scavenging ability of bamboo shoot peptide chelated calcium on hydroxyl radicals is as follows Figure 5 shown in I. The experimental results show that the scavenging ability of bamboo shoot peptide chelated calcium on hydroxyl radicals is significantly weaker than that on DPPH and ABTS radicals. With the increase of concentration, its scavenging effect shows a trend of increasing first and then decreasing, and the highest scavenging rate is about 14.98%. This phenomenon may be related to the low content of histidine (His) in bamboo shoot peptide chelated calcium, because histidine residues have a strong ability to capture free radicals. In addition, high-concentration proteins may cause some side reactions, such as protein polymerization or hydroxylation, and then generate chain by-products. These products may compete with free radicals for reaction resources, thereby further reducing its free radical scavenging efficiency. In addition, the protein structural characteristics of bamboo shoot peptide chelated calcium and its reaction kinetics with hydroxyl radicals may also limit its rapidity and efficiency in scavenging hydroxyl radicals. In summary, bamboo shoot peptide chelated calcium shows certain selectivity in scavenging different types of free radicals, and its weak scavenging ability on hydroxyl radicals may be the result of the combined action of amino acid composition and structural characteristics.

[0065] Table 1 Amino acid composition and content of bamboo shoot calcium chelated peptide Amino acid species Bamboo shoot peptide chelated calcium (%) Asp 10.16 Thr 2.79 Ser 3.74 Glu 9.86 Gly 5.55 Ala 5.37 Cys 24.40 Val 6.52 Met 3.64 Ile 3.33 Leu 2.34 Tyr 3.51 Phe 4.42 His 1.54 Lys 6.61 Arg 2.19 Pro 4.03

[0066] The above is only a preferred embodiment of the present invention, and does not limit the technical scope of the present invention. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for preparing bamboo shoot peptide chelated calcium, characterized in that: The steps include: Step 1: Preparation of bamboo shoot protein powder The bamboo shoots are washed, cut, broken, alkali-dissolved, and filtered, ultrapure water is added to the filter residue for washing, the washing liquid is mixed with the filtrate, the pH value is adjusted for acid precipitation, after obvious stratification, the lower layer of precipitate is taken and centrifuged, the precipitate is collected, dispersed in ultrapure water, the pH value is adjusted again, and dried to obtain bamboo shoot protein powder; Step 2: Preparation of bamboo shoot peptide Take bamboo shoot protein powder, add alkaline protease, adjust the pH value, perform enzymatic hydrolysis under magnetic stirring, use 3KDa, 5KDa and 10KDa ultrafiltration membrane packages for ultrafiltration, obtain <3KDa, 3-5KDa, 5-10KDa, >10KDa retained peptide components, perform freeze drying, and obtain bamboo shoot peptides; Step 3: Preparation of bamboo shoot peptide chelated calcium The peptide solution was added to anhydrous calcium chloride with a peptide-calcium mass ratio of 8:1, the pH value was adjusted, the reaction was carried out, anhydrous ethanol solution was added, the mixture was allowed to stand, centrifuged, precipitated, and freeze-dried to obtain bamboo shoot peptide chelated calcium.

2. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In the step 1, the material-liquid ratio of the wall breaking is 1:1, the wall breaking time is 1 min, and the number of wall breaking times is 4 times.

3. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In the step 1, the material-liquid ratio of the alkali dissolution is 1:4, the temperature of the alkali dissolution is 45° C., the time of the alkali dissolution is 1 h, the pH value of the alkali dissolution is 9.0, and the filter screen is 300 mesh.

4. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In the step 1, the pH value is adjusted to 4.2 with 2 mol / L HCl for acid precipitation.

5. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In the step 1, the centrifugal speed is 4500 r / min and the centrifugal time is 20 min.

6. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In the step 1, the precipitate is dispersed in ultrapure water at a ratio of 1:3 (w / v); the pH value is adjusted to 7.0 for the second time.

7. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In the step 2, the protein content of alkaline protease is 9000 U / g, the pH value is adjusted to 8, the enzymolysis temperature is 55° C., and the enzymolysis time is 2 h.

8. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In step 3, 15 ml of a peptide solution with a mass concentration of 36 mg / ml was taken; the pH value was adjusted to 7, the reaction temperature was 50° C., and the reaction time was 2 h.

9. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In the step 3, 10 times the volume of anhydrous ethanol solution is added and the mixture is allowed to stand at 4° C. for 3 hours.

10. The method for preparing bamboo shoot peptide chelated calcium according to claim 1, characterized in that: In step 3, the centrifugal speed is 5000 rpm and the centrifugal time is 20 min.

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