A method for preparing high-purity amino acid chelated calcium using mussel shells

Abstract

The present invention discloses a method for preparing high-purity amino acid chelated calcium using mussel shells. After the mussel shells are treated with an alkaline aqueous solution, they are fermented by acid-producing bacteria to obtain mussel shells with surface impurities removed, ball milled to obtain mussel shell ultrafine powder; amino acids and distilled water are added to the obtained mussel shell ultrafine powder to chelate to obtain amino acid chelated calcium. The present invention adopts a method of alkali-acid-producing bacteria fermentation combined treatment to purify mussel shells for the first time, which greatly reduces environmental pollution problems. The lightness of the mussel shell powder after alkali-acid-producing bacteria fermentation combined purification treatment is increased from 67.28 to 91.67, the calcium content on the shell surface is increased from 0.39% to 37.46%, and the heavy metal content such as Pb content is reduced from 0.9mg / kg to undetected. The yield and chelation rate of amino acid chelated calcium prepared using the purified mussel shell powder as raw material are significantly improved.

Description

(I) Technical field

[0001] The invention relates to the field of high-value utilization of aquatic product by-products, and in particular to a method for preparing high-purity amino acid chelated calcium by utilizing mussel shells. (II) Background technology

[0002] Mussels are a common edible shellfish, which are farmed in large quantities in coastal provinces such as Shandong, Liaoning, and Zhejiang. Mussels are rich in nutrients, high in protein, and the meat is rich in a variety of physiologically active substances. At present, the use of mussels in my country is mainly limited to the edible meat, but it is difficult to develop and utilize the shells that account for more than 60% of the total mass. Shells discarded as solid waste on the seashore not only occupy land resources, but also cause serious environmental pollution, and have become a major environmental hazard in mussel farming areas.

[0003] The main component of mussel shells is calcium carbonate. As an animal calcium, it is superior to inorganic minerals in terms of absorption rate and safety, and is a potential new calcium source. Calcium is the fifth important element in animals after carbon, hydrogen, oxygen, and nitrogen, and plays a vital role in maintaining the normal structure and function of organisms. However, a layer of natural melanin is attached to the surface of mussel shells, which has poor solubility and is not easy to separate. At the same time, melanin usually contains a certain amount of impurities, which will affect the preparation efficiency and product quality of amino acid chelated calcium. However, in the prior art, when preparing calcium supplements using mussel shells as a calcium source, the impurities on the shell surface are not removed, so the product quality is not high. Therefore, those skilled in the art are in urgent need of a method for preparing high-quality amino acid chelated calcium using mussel shells as raw materials. (III) Summary of the invention

[0004] The invention aims to provide a method for preparing high-purity amino acid chelated calcium by using mussel shells. The amino acid chelated calcium prepared by using mussel shells as a calcium source and chelating with amino acids has higher absorption rate, good bioavailability and low side effects, good solubility and thermal stability. The invention is a novel calcium supplement, which solves the defects of high impurity content and poor product quality of the calcium supplement currently prepared by using mussel shells as raw materials.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a method for preparing high-purity amino acid chelated calcium using mussel shells, and the method is carried out according to the following steps:

[0007] (1) Alkali treatment: The mussel shells were dried and crushed into small pieces and then added to an alkaline aqueous solution and soaked at room temperature for 24-72 hours. 40-70 kHz ultrasound was used for synergistic treatment in the first hour and the last hour of soaking respectively. The shells were rinsed with clean water until the surface melanin was completely detached and dried to obtain alkali-treated mussel shells.

[0008] (2) Fermentation treatment: Weigh the mussel shells treated with alkali in step (1) and add them to a synthetic culture medium, inoculate acid-producing bacteria, and ferment at a speed of 100-300 r / min and 20-40°C (preferably 200 r / min and 30°C) for 48-72 hours, and then add glucose once every 36-48 hours to perform batch feeding fermentation to maintain the glucose concentration at 0.5-1.5 g / L; after the fermentation is completed, rinse the shells with clean water until the water is colorless and clear, and dry them (preferably place them in a drying oven at 65°C for 3 hours) to obtain a product with surface impurities removed. Mussel shells; the acid-producing bacteria include acetic acid bacteria or lactic acid bacteria; the synthetic culture medium for acetic acid bacteria fermentation comprises: 10 g / L glucose, 10 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, and the solvent is water; the synthetic culture medium for lactic acid bacteria fermentation comprises: 20 g / L glucose, 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.3 g / L manganese sulfate, 2 g / L ammonium citrate, 5 g / L sodium acetate, 1 mL / L Tween 80, and the solvent is water;

[0009] (3) adding the mussel shells from which surface impurities have been removed in step (2) into a ball mill, adding a ball milling medium and water, and performing wet ball milling. After removing the ball milling medium, drying (preferably drying at 65° C. for 3 h and passing through a 400-mesh sieve) is performed to obtain mussel shell ultrafine powder; the ball milling medium is a stainless steel ball with a diameter of 5-15 mm;

[0010] (4) adding amino acids and distilled water to the mussel shell ultrafine powder obtained in step (3) and adjusting the pH to 3-7, chelating for 50-150 min (preferably 90 min) at a temperature of 40-60° C. and a stirring speed of 10-20 rpm (preferably 12 rpm), quickly filtering with a filter cloth, and rotating and concentrating the filtrate at 40-60° C. and 50-100 rpm (preferably 60 rpm) until it becomes viscous, adding anhydrous ethanol to the concentrate to remove free calcium ions and precipitate, centrifuging (preferably centrifuging at 1000 rpm for 10 min), and drying the precipitate (preferably vacuum freeze-drying at -40° C. for 24 h) to obtain amino acid chelated calcium.

[0011] Preferably, in step (1) the mussel shells are dried and crushed by placing the mussel shells in a drying oven for drying (preferably at 65° C.), and crushing the dried mussel shells into small pieces, preferably by using a 17-40 type double-roll crusher.

[0012] Preferably, the alkali in the alkaline aqueous solution in step (1) comprises sodium hydroxide, potassium hydroxide, barium hydroxide or ammonia water; the concentration of the alkaline aqueous solution is 0.05-0.1 mol / L, and the volume of the alkaline aqueous solution is 5-10 mL / g based on the mass of the mussel shells.

[0013] Preferably, the alkali-treated mussel shells in step (2) are added to the synthetic culture medium in an amount of 0.1-0.25 g / mL (preferably 0.2 g / mL); the acid-producing bacteria are added in the form of seed solution, and the accession amount is 10-15% based on the volume of the synthetic culture medium. The glucose is added by pump flow, and the flow addition amount each time is 0.1-5 g / L (preferably 1 g / L) based on the volume of the synthetic culture medium, and the feed is supplemented 2-4 times.

[0014] Preferably, the acid-producing bacteria in step (2) include acetic acid bacteria (Acetobacter sp.) GIM1.152 or lactic acid bacteria (Lactobacillus sp.) bio-089504.

[0015] Preferably, the seed liquid of the acid-producing bacteria in step (2) is prepared by one of the following methods: (1) when the acid-producing bacteria is acetic acid bacteria, the acid-producing strain is inoculated into a seed culture medium, and cultured at 30°C and 200rpm for 24h to obtain a seed liquid; the seed culture medium is composed of: 10g / L glucose, 10g / L yeast extract, 0.5g / L magnesium sulfate, 0.5g / L potassium dihydrogen phosphate, and the solvent is water; (2) when the acid-producing bacteria is lactic acid bacteria, the acid-producing strain is inoculated into a seed culture medium, and cultured at 37°C and 200rpm for 24h to obtain a seed liquid; the seed culture medium is composed of: 20g / L glucose, 10g / L peptone, 8g / L beef extract, 4g / L yeast extract, 0.5g / L magnesium sulfate, 0.3g / L manganese sulfate, 2g / L ammonium citrate, 5g / L sodium acetate, 1mL / L Tween 80, and the solvent is water.

[0016] Preferably, the ball milling speed in step (3) is 400-600 r / min, and the ball milling time is 10-30 min; the ball-to-material ratio (the mass ratio of the ball milling medium to the mass ratio of the shell powder) is 2-10:1 (the ball-to-material ratio affects the particle size of the shell powder, and thus affects the yield and chelation rate of amino acid chelated calcium, preferably 8.8:1), and the slurry concentration (the relative water content of mussel shells) is 20-60% (preferably 42%).

[0017] Preferably, the amino acids in step (4) include one or more of aspartic acid, glutamic acid, valine, isoleucine, phenylalanine or lysine; the mass ratio of the superfine mussel shell powder to the amino acid is 1:1-4; the mass ratio of the total mass of the superfine mussel shell powder and the amino acid to the mass of distilled water is 1:4-6 (preferably 1:5); the volume ratio of the anhydrous ethanol to the concentrate is 9:1.

[0018] Compared with the existing methods, the beneficial effects of the present invention are mainly reflected in:

[0019] The present invention adopts the method of alkali-acid-producing bacteria fermentation combined treatment for the first time to purify mussel shells, which greatly reduces the environmental pollution problem compared with the traditional strong acid treatment. In addition, the fermentation method of acid-producing bacteria adopts continuous feeding fermentation treatment. By continuously adding materials to the culture system, the concentration of nutrients in the culture solution is continuously maintained in a more suitable range, which not only ensures the nutritional needs of microbial growth, but also avoids adverse effects, thereby achieving the purpose of improving the yield. The lightness of the mussel shell powder after the alkali-acid-producing bacteria fermentation combined purification treatment increased from 67.28 to 91.67, the calcium content on the shell surface increased from 0.39% to 37.46%, and the heavy metal content such as Pb content decreased from 0.9 mg / kg to undetectable. The yield and chelation rate of amino acid chelated calcium prepared with purified mussel shell powder as raw material are significantly improved. As one of the raw materials for the final product amino acid chelated calcium, the brightness of the chelated calcium is increased by 0.99, the yield is increased from 54.05% to 59.18%, and the chelation rate is increased from 76.97% to 78.43% compared with the chelated calcium prepared from unpurified mussel shells. (IV) Description of the drawings

[0020] Figure 1 The process flow chart of preparing high-purity amino acid chelated calcium using mussel shells as raw materials.

[0021] Figure 2 This is a picture of amino acid chelated calcium.

[0022] Figure 3 This is the EDS spectrum of purified L-aspartate chelated calcium.

[0023] Figure 4 This is the EDS spectrum of unpurified L-aspartate chelated calcium. (V) Specific implementation methods

[0024] The present invention is further described below in conjunction with specific examples, but the protection scope of the present invention is not limited thereto: The mussel shells used in the examples of the present invention refer to the shells of mussels (scientific name: Mytilus edulis) that have been cleaned and fleshed. The room temperature refers to 25-30°C.

[0025] Example 1: Alkali treatment to remove the cuticle on the surface of mussel shells

[0026] Pretreatment: Wash the mussels, soak them in 80℃ water bath for 100s to remove the flesh, scrub the outer surface of the shells, put them into a drying oven, and dry them at 65℃. The dried mussel shells are crushed into small pieces with a 17-40 double-roll crusher, which are the unpurified mussel shell pieces.

[0027] (1) Sodium hydroxide treatment: 1.25g of sodium hydroxide was placed in a beaker, 250mL of distilled water was added and the pH was adjusted to 13.0 to prepare a 0.1mol / L sodium hydroxide aqueous solution, and then 25g of unpurified mussel shells were added and soaked at room temperature for 36h. 40kHz ultrasonic co-treatment was used in the first and last hours of soaking. The soaked shells were rinsed with clean water until the surface melanin was completely removed and placed in a 65℃ drying oven for 3h to obtain 24.12g of alkali-treated mussel shells. The lightness of the mussel shell powder treated with sodium hydroxide solution was 90.64 using the Color Quest colorimeter of HunterLab, USA.

[0028] (2) Potassium hydroxide treatment: 5.0 g of potassium hydroxide was placed in a beaker, 500 mL of distilled water was added and the pH was adjusted to 13.0 to prepare a 0.06 mol / L potassium hydroxide aqueous solution, and then 50 g of unpurified mussel shells were added and soaked at room temperature for 36 h. 40 kHz ultrasonic co-treatment was used in the first and last hours of soaking. The soaked shells were rinsed with clean water until the surface melanin was completely removed and placed in a 65°C drying oven for 3 h to obtain 48.16 g of alkali-treated mussel shells. The lightness of the mussel shell powder treated with potassium hydroxide solution was 91.03 as detected by the method of Example 1.

[0029] (3) Ammonia treatment: 450 ml of 6 mol / L ammonia solution was prepared and placed in a beaker, and the pH was adjusted to 13.0 with concentrated ammonia solution to 500 ml, and then 50 g of unpurified mussel shells were added and soaked at room temperature for 36 h. 40 kHz ultrasound was used for synergistic treatment in the first and last hours of soaking. The shells were rinsed with clean water until the surface melanin was completely removed and placed in a 65°C drying oven for 3 hours to obtain 48.47 g of alkali-treated mussel shells. The lightness of the mussel shell powder treated with ammonia solution was 88.71 when tested using the method in Example 1.

[0030] Considering that the alkalinity of potassium hydroxide is higher than that of sodium hydroxide, and the difference in brightness is not much, sodium hydroxide solution was chosen to remove the cuticle on the surface of mussel shells.

[0031] Table 1 Lightness and darkness of mussel shell powder after different alkali treatments

[0032]

[0033] Example 2: Removal of low-purity calcium carbonate by microbial fermentation

[0034] (1) Acetic acid bacteria fermentation treatment to remove low-purity calcium carbonate: Weigh 25g of mussel shells treated with NaOH solution in step (1) of Example 1 and add them to 125mL of acetic acid bacteria synthetic culture medium, sterilize at 115°C for 20min, then inoculate acetic acid bacteria seed liquid at a volume concentration of 10%, and ferment and culture at a speed of 200r / min and 30°C for 72h, then add glucose once every 48h for batch feeding fermentation, and add glucose twice in total, each time adding 1g / L based on the volume of synthetic culture medium to maintain glucose at 1g / L. After fermentation for 168h, filter, rinse the shells with clean water until the water flowing out is colorless and clear, and then place them in a drying oven at 65°C for 3h to obtain mussel shells with surface impurities removed, i.e., purified mussel shells.

[0035] Determination of calcium content: Take 1mL of the filtrate filtered after fermentation in a centrifuge tube, add 1 drop of 0.1% methyl red indicator, 2mL of 4% ammonium oxalate aqueous solution and 0.5mL of 20% acetic acid aqueous solution, shake well, adjust to slightly blue with 20% ammonia aqueous solution, and then adjust to slightly red with 20% acetic acid aqueous solution, let it settle for 1h, centrifuge at 6000r / min for 15min, pour off the supernatant, add a small amount of 2% ammonia aqueous solution, flick the centrifuge tube with your fingers to loosen the precipitate, add 10mL of 2% ammonia aqueous solution, centrifuge at 6000r / min for 20min, pour off the supernatant, add 2mL of 2mol / L sulfuric acid aqueous solution, shake well, heat in a water bath at 70℃~80℃ to completely dissolve the precipitate. Titrate with calibrated potassium permanganate to slightly red. The results show that the final concentration and final conversion rate of calcium ions are 33.99mg / mL and 37.33%, respectively. It was proved that the batch feeding operation effectively controlled the substrate (glucose) concentration in the fermentation broth, increased the production of metabolites, and promoted the conversion of inorganic calcium to ionic calcium in the shells. The lightness of the mussel shell powder obtained by the sodium hydroxide-acetic acid bacteria fermentation was 91.67.

[0036] Acetobacter seed solution: inoculate acetic acid bacteria (Acetobacter sp., GIM1.152) into a seed culture medium, culture at 30°C and 200 rpm for 1 day, and obtain seed solution when the acidity reaches 1.5-2% and the culture medium is composed of 10 g / L glucose, 10 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, and the solvent is water.

[0037] The composition of the acetic acid bacteria synthetic culture medium is as follows: glucose 10 g / L, yeast extract 10 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, and the solvent is water.

[0038] (2) Lactic acid bacteria fermentation treatment to remove low-purity calcium carbonate: Weigh 50g of mussel shells treated with NaOH solution in step (1) of Example 1 and add them to 250mL of lactic acid bacteria synthetic culture medium, sterilize at 115℃ for 20min, then inoculate lactic acid bacteria seed liquid at a volume concentration of 10%, ferment and culture at a speed of 400r / min and 37℃ for 72h, add glucose once every 48h for batch feeding fermentation, add glucose twice in total, add 1g / L of synthetic culture medium volume each time, and maintain glucose at 1g / L. After the fermentation is completed, filter, rinse the shells with clean water until the water flowing out is colorless and clear, and place them in a drying oven at 65℃ for 3h to obtain mussel shells with surface impurities removed, i.e., purified mussel shells.

[0039] Determination of calcium content (same method as above): The results showed that the final concentration and final conversion rate of calcium ions were 32.17 mg / mL and 35.53%, respectively. This proved that the batch feeding operation effectively controlled the substrate (glucose) concentration in the fermentation broth, increased the production of metabolites, and promoted the conversion of inorganic calcium to ionic calcium in shells.

[0040] Lactic acid bacteria seed liquid: lactic acid bacteria (Lactobacillus., bio-089504) were inoculated into the seed culture medium, cultured at 37°C and 200rpm for 1 day, and the acidity reached 1.5-2% when it was mature, and the seed liquid was obtained; the composition of the seed culture medium was: 20g / L glucose, 10g / L peptone, 8g / L beef extract, 4g / L yeast extract, 0.5g / L magnesium sulfate, 0.3g / L manganese sulfate, 2g / L ammonium citrate, 5g / L sodium acetate, 1mL / L Tween 80, and the solvent was water. The lightness of the mussel shell powder obtained by the sodium hydroxide-lactic acid bacteria fermentation was 90.89.

[0041] The composition of the synthetic culture medium for lactic acid bacteria: 20 g / L glucose, 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.3 g / L manganese sulfate, 2 g / L ammonium citrate, 5 g / L sodium acetate, 1 mL / L Tween 80, and the solvent is water.

[0042] Table 2 Calcium ion concentration, final conversion rate and brightness after different microbial fermentation treatments

[0043]

[0044] Example 3: Preparation of amino acid chelated calcium from mussel shell ultrafine powder

[0045] (1) Preparation of amino acid chelated calcium from purified mussel shell ultrafine powder: 25 g of mussel shells obtained in step (1) of Example 2 with surface impurities removed were added to a ball mill (YXQM-2L), and then 24 ball milling media (MITR stainless steel balls with a diameter of 10 mm) and 60 mL of water were added for wet grinding, so that the ball-to-material ratio (the mass ratio of the ball milling media to the mass ratio of the shell powder wetted with water) was 8.8:1, the slurry concentration (the total dry weight of the shell powder relative to the water content) was 42%, the ball milling speed was 400 r / min, and the ball milling time was 28.5 min. After the ball milling was completed, the ball milling media was removed, dried at 65° C. for 3 h, and crushed through a 400 mesh sieve to obtain 24.35 g of purified mussel shell ultrafine powder. The average particle size of the mussel shell ultrafine powder was 2.41 μm as measured by a Mastersizer 3000 laser particle size analyzer (0.05 g of shell powder sample was added to the sample tank, the shading rate was 10%, and the ultrasonic time was 5 min in the laser particle size analyzer).

[0046] Preparation of chelated calcium: Weigh 11.788g of mussel shell ultrafine powder, 20.002g of aspartic acid and 160mL of distilled water and adjust the pH to 4.0 with 2mol / L hydrochloric acid, chelate for 90min at a constant temperature of 50°C and a stirring speed of 12r / s, quickly filter with a filter cloth while hot, and concentrate the filtrate under the conditions of rotary evaporation temperature of 60°C and rotation speed of 60rpm until it becomes viscous. Obtain a concentrate and add 9 times the volume of the concentrate of anhydrous ethanol to dissolve and precipitate, centrifuge at 10000rpm for 10min, and freeze-dry the precipitate at -40°C for 24h to finally obtain 21.293g of aspartic acid chelated calcium, with a yield of 66.98%, a chelation rate of 81.12%, and a lightness measurement of 99.76 (the chelated calcium powder is placed in a PE ziplock bag and pressed, and the color of the sample is measured with a colorimeter). EDS was used to determine the surface elements of chelated calcium. No bromine was detected in the chelated calcium made from purified mussel shell powder, and the chlorine content was 4.95%. See the sample photo Figure 2 , EDS spectrum see Figure 3 .

[0047] (2) Preparation of amino acid chelated calcium from unpurified mussel shell ultrafine powder: 25 g of unpurified mussel shell pieces from Example 1 were added to a ball mill (YXQM-2L), and then 24 ball milling media (MITR stainless steel balls with a diameter of 10 mm) and 60 mL of water were added for wet grinding, so that the ball-to-material ratio (the mass ratio of the ball milling media to the mass ratio of the shell powder wetted with water) was 8.8:1, the slurry concentration (the total dry weight of the shell powder relative to the water content) was 42%, the ball milling speed was 400 r / min, and the ball milling time was 28.5 min. After the ball milling was completed, the ball milling media was removed, dried at 65° C. for 3 h, and crushed through a 400 mesh sieve to obtain 24.28 g of unpurified mussel shell ultrafine powder. The average particle size of the mussel shell ultrafine powder was 2.78 μm as measured by a Mastersizer 3000 laser particle size analyzer (0.05 g of unpurified mussel shell ultrafine powder sample was added to the sample tank, the shading rate was 10%, and the ultrasonic time was 5 min).

[0048] Preparation of chelated calcium: 13.971 g of unpurified mussel shell ultrafine powder was weighed, 20.002 g of aspartic acid and 170 mL of distilled water were added, and the pH was adjusted to 4.0 with 2 mol / L hydrochloric acid. The mixture was chelated for 90 min at a constant temperature of 50° C. and a stirring speed of 12 r / s. The mixture was quickly filtered while hot with a filter cloth. The filtrate was concentrated to a viscous state under the conditions of a rotary evaporation temperature of 60° C. and a rotation speed of 60 rpm. Anhydrous ethanol in an amount of 9 times its volume was added to the concentrate to remove free calcium ions and precipitate. The mixture was centrifuged at 10,000 rpm for 10 min, and the precipitate was freeze-dried at -40° C. for 24 h to finally obtain 18.808 g of aspartic acid chelated calcium, with a yield of 59.18%, a chelation rate of 78.43%, and a lightness of 98.65 (the chelated calcium powder was placed in a PE ziplock bag and pressed tightly, and the color of the sample was measured using a colorimeter). EDS was used to determine the surface elements of chelated calcium. The chelated calcium prepared from unpurified mussel shell powder was found to contain 1.44% bromine and 7.80% chlorine. Figure 4 .

[0049] Table 3 Differences in the preparation of amino acid chelated calcium from purified and unpurified mussel shell powder

[0050]

[0051] Example 4: Preparation of different types of amino acid chelated calcium

[0052] (1) Aspartic acid chelated calcium: Weigh 12.5 g of the purified mussel shell ultrafine powder prepared by the method of step (1) of Example 3, add 32.3 g of aspartic acid (CaCO3: amino acid molar ratio = 1:2) and 224.0 mL of distilled water, and adjust the pH to 4.0 with 2 mol / L hydrochloric acid. Chelate for 90 min at a temperature of 50° C. and a stirring speed of 12 r / s. Quickly filter with a filter cloth while hot, and concentrate the filtrate by rotary evaporation at a temperature of 60° C. and a rotation speed of 60 rpm until it becomes viscous. Add 9 times the volume of anhydrous ethanol to the concentrate to remove free calcium ions and precipitate. Centrifuge at 10,000 rpm for 10 min. The precipitate is freeze-dried at -40° C. for 24 h to obtain 29.2 g of aspartic acid chelated calcium, with a yield of 65.18% and a chelation rate of 80.94%.

[0053] (2) Glutamic acid chelated calcium: Weigh 12.5 g of the purified mussel shell ultrafine powder prepared by the method of step (1) of Example 3, add 35.7 g of glutamic acid (CaCO3: amino acid molar ratio = 1:2) and 240 mL of distilled water, and adjust the pH to 4.0 with 2 mol / L hydrochloric acid. Chelate for 90 min at a temperature of 50° C. and a stirring speed of 12 r / s. Quickly filter the filtrate while hot with a filter cloth. Rotate and concentrate the filtrate to a viscous state under the conditions of a rotary evaporation temperature of 60° C. and a rotation speed of 60 rpm. Add 9 times the volume of anhydrous ethanol to the concentrate to remove free calcium ions and precipitate. Centrifuge at 10,000 rpm for 10 min. The precipitate is freeze-dried at -40° C. for 24 h to obtain 28.13 g of glutamate chelated calcium, with a yield of 58.36% and a chelation rate of 63.88%.

[0054] (3) Lysine chelated calcium: Weigh 12.5 g of the purified mussel shell ultrafine powder prepared by the method of step (1) of Example 3, add 35.5 g of lysine (CaCO3: amino acid molar ratio = 1:2) and 240.0 mL of distilled water, and adjust the pH to 4.0 with 2 mol / L hydrochloric acid. Chelate for 90 min at a temperature of 50° C. and a stirring speed of 12 r / s. Quickly filter the filtrate while hot with a filter cloth. Rotate and concentrate the filtrate to a viscous state under the conditions of a rotary evaporation temperature of 60° C. and a rotation speed of 60 rpm. Add 9 times the volume of anhydrous ethanol to the concentrate to remove free calcium ions and precipitate. Centrifuge at 10,000 rpm for 10 min. The precipitate is freeze-dried at -40° C. for 24 h to obtain 28.92 g of lysine chelated calcium with a yield of 60.24% and a chelation rate of 63.70%.

[0055] (4) Valine chelated calcium: Weigh 12.5 g of the purified mussel shell ultrafine powder prepared by the method of step (1) of Example 3, add 28.4 g of valine (CaCO3: amino acid molar ratio = 1:2) and 204.5 mL of distilled water, and adjust the pH to 4.0 with 2 mol / L hydrochloric acid. Chelate for 90 min at a temperature of 50°C and a stirring speed of 12 r / s, and quickly filter with a filter cloth while hot. The filtrate is rotary concentrated to a viscous state under the conditions of a rotary evaporation temperature of 60°C and a rotation speed of 60 rpm. Add 9 times the volume of anhydrous ethanol to the concentrate to remove free calcium ions and precipitate. Centrifuge at 10,000 rpm for 10 min. The precipitate is freeze-dried at -40°C for 24 h to finally obtain 20.40 g of valine chelated calcium with a yield of 49.87% and a chelation rate of 51.07%.

[0056] (5) Isoleucine chelated calcium: 12.5 g of the purified mussel shell ultrafine powder prepared by the method of step (1) of Example 3 was weighed, and 31.8 g of isoleucine (CaCO3: amino acid molar ratio = 1:2) and 221.5 mL of distilled water were added, and the pH was adjusted to 4.0 with 2 mol / L hydrochloric acid. The mixture was chelated at a temperature of 50°C and a stirring speed of 12 r / s for 90 min, and the mixture was quickly filtered with a filter cloth while hot. The filtrate was concentrated by rotary evaporation at a temperature of 60°C and a rotation speed of 60 rpm until it became viscous. Anhydrous ethanol in an amount of 9 times its volume was added to the concentrate to remove free calcium ions and precipitate. The concentrate was centrifuged at 10,000 rpm for 10 min, and the precipitate was freeze-dried at -40°C for 24 h to finally obtain 22.76 g of isoleucine chelated calcium with a yield of 51.37% and a chelation rate of 68.09%.

[0057] (6) Phenylalanine chelated calcium: Weigh 12.5 g of the purified mussel shell ultrafine powder prepared by the method of step (1) of Example 3, add 40.1 g of phenylalanine (CaCO3: amino acid molar ratio = 1:2) and 263.0 mL of distilled water, and adjust the pH to 4.0 with 2 mol / L hydrochloric acid. Chelate for 90 min at a temperature of 50° C. and a stirring speed of 12 r / s. Quickly filter the mixture while hot with a filter cloth. Rotate and concentrate the filtrate to a viscous state under the conditions of a rotary evaporation temperature of 60° C. and a rotation speed of 60 rpm. Add 9 times the volume of anhydrous ethanol to the concentrate to remove free calcium ions and precipitate. Centrifuge at 10,000 rpm for 10 min. The precipitate is freeze-dried at -40° C. for 24 h to finally obtain 25.24 g of phenylalanine chelated calcium with a yield of 47.98% and a chelation rate of 65.50%.

[0058] Table 4 Differences in the preparation of chelated calcium by different types of amino acids

[0059]

Claims

1. A method for preparing high-purity amino acid chelated calcium using mussel shells, characterized in that: The method is carried out according to the following steps: (1) Alkali treatment: The mussel shells were dried and crushed into small pieces and then added to an alkaline aqueous solution and soaked at room temperature for 24-72 hours. 40-70 kHz ultrasound was used for synergistic treatment in the first hour and the last hour of soaking respectively. The shells were rinsed with clean water until the surface melanin was completely detached and dried to obtain alkali-treated mussel shells. (2) Fermentation treatment: weigh the mussel shells treated with alkali in step (1), add them to a synthetic culture medium, inoculate acid-producing bacteria, and ferment at a speed of 100-300 r / min and 20-40° C. for 48-72 hours, then add glucose once every 36-48 hours to perform batch feeding fermentation, and maintain the glucose concentration at 0.5-1.5 g / L; after the fermentation is completed, rinse the shells with clean water until the water is colorless and clear, and dry them to obtain mussel shells with surface impurities removed; the acid-producing bacteria are acetic acid bacteria or lactic acid bacteria. bacteria; the synthetic culture medium for acetic acid bacteria fermentation is composed of: 10g / L glucose, 10g / L yeast extract, 0.5g / L magnesium sulfate, 0.5g / L potassium dihydrogen phosphate, and the solvent is water; the synthetic culture medium for lactic acid bacteria fermentation is composed of: 20g / L glucose, 10g / L peptone, 8g / L beef extract, 4g / L yeast extract, 0.5g / L magnesium sulfate, 0.3g / L manganese sulfate, 2g / L ammonium citrate, 5g / L sodium acetate, 1mL / L Tween 80, and the solvent is water; (3) adding the mussel shells from which surface impurities have been removed in step (2) into a ball mill, adding a ball milling medium and water, performing wet ball milling, removing the ball milling medium and drying to obtain mussel shell ultrafine powder; the ball milling medium is a stainless steel ball with a diameter of 5-15 mm; (4) adding amino acids and distilled water to the mussel shell ultrafine powder obtained in step (3) and adjusting the pH to 3-7, chelating for 50-150 min at a temperature of 40-60° C. and a stirring speed of 10-20 rpm, quickly filtering with a filter cloth, and rotating and concentrating the filtrate at 40-60° C. and 50-100 rpm until it becomes viscous, adding anhydrous ethanol to the concentrate to remove free calcium ions and precipitate, centrifuging, and drying the precipitate to obtain amino acid chelated calcium.

2. The method according to claim 1, characterized in that Step (1) mussel shell drying and crushing is to put the mussel shells into a drying oven for drying at 65° C. and crush the dried mussel shells into small pieces.

3. The method according to claim 1, characterized in that The alkali in the alkaline aqueous solution in step (1) is sodium hydroxide, potassium hydroxide, barium hydroxide or ammonia water; the concentration of the alkaline aqueous solution is 0.05-0.1 mol / L, and the volume of the alkaline aqueous solution is 5-10 mL / g based on the mass of the mussel shells.

4. The method according to claim 1, characterized in that In step (2), the alkali-treated mussel shells are added to the synthetic culture medium in an amount of 0.1-0.25 g / mL; the acid-producing bacteria are added in the form of seed liquid, and the inoculation amount is 10-15% of the volume of the synthetic culture medium.

5. The method according to claim 1, characterized in that In step (2), the glucose is added by pumping, and the amount of each addition is 0.1-5 g / L based on the volume of the synthetic culture medium, and the feed is supplemented 2-4 times.

6. The method according to claim 1, characterized in that The acid-producing bacteria in step (2) are acetic acid bacteria (Acetobacter sp.) GIM1.152 or lactic acid bacteria (Lactobacillus sp.) bio-089504.

7. The method according to claim 1 or 4, characterized in that The seed liquid of the acid-producing bacteria in step (2) is prepared according to one of the following methods: (1) when the acid-producing bacteria is acetic acid bacteria, the acid-producing strain is inoculated into a seed culture medium, and cultured at 30° C. and 200 rpm for 24 hours to obtain a seed liquid; the seed culture medium is composed of: 10 g / L glucose, 10 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.5 g / L potassium dihydrogen phosphate, and the solvent is water; (2) when the acid-producing bacteria is lactic acid bacteria, the acid-producing strain is inoculated into a seed culture medium, and cultured at 37° C. and 200 rpm for 24 hours to obtain a seed liquid; the seed culture medium is composed of: 20 g / L glucose, 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 0.5 g / L magnesium sulfate, 0.3 g / L manganese sulfate, 2 g / L ammonium citrate, 5 g / L sodium acetate, 1 mL / L Tween 80, and the solvent is water.

8. The method according to claim 1, characterized in that In step (3), the ball milling speed is 400-600 r / min, and the ball milling time is 10-30 min; the mass ratio of the ball milling medium to the mussel shell is 2-10:1, and the concentration of the mussel shell relative to water is 20-60%.

9. The method according to claim 1, characterized in that The amino acid in step (4) is one or more of aspartic acid, glutamic acid, valine, isoleucine, phenylalanine or lysine.

10. The method according to claim 1, characterized in that In step (4), the mass ratio of the superfine mussel shell powder to the amino acid is 1:1-4; the mass ratio of the total mass of the superfine mussel shell powder and the amino acid to the distilled water is 1:4-6.

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