Method for preparing water-soluble oyster powder through enzyme-acid hydrolysis

Abstract

The invention discloses a method for preparing water-soluble oyster powder through enzyme-acid hydrolysis. The method comprises the following steps: step 1, cleaning and drying oyster shells, and grinding and sieving the oyster shells to prepare oyster shell powder; step 2, carrying out two times of enzymolysis on the oyster shell powder by using neutral protease to prepare enzymatic hydrolysate; 3, adding acetic acid into the enzymatic hydrolysate for reaction, and filtering to obtain filtrate; and 4, refrigerating the filtrate at an ultralow temperature, and performing freeze-drying treatment to obtain the water-soluble oyster powder. The water-soluble oyster powder is prepared by adopting a safe and efficient enzyme-acid hydrolysis method, so that the absorption rate and the bioavailability of calcium in oyster shells by a human body are effectively improved. Meanwhile, optimal preparation process parameters of the preparation method are obtained through a large number of experiments, the calcium content of the obtained water-soluble oyster powder solid powder reaches 90.12%, the amino nitrogen content is 20.0 mg / g, the solubility is improved to 42.56 g / 100 ml, and the preparation method has very important significance on further expanding resource utilization of waste oyster shells.

Description

Technical Field

[0001] The present invention relates to the technical field of water-soluble oyster powder preparation, and particularly relates to a method for preparing water-soluble oyster powder by enzyme-acid hydrolysis. Background Art

[0002] Oyster shells have been used as traditional marine Chinese medicinal materials in previous editions of the pharmacopoeia. Their source is the shells of oyster family animals such as Ostrea gigas Thunberg, Ostrea talienwhanensis Crosse, or Ostrea rivularis Gould; oyster shells contain more than 94% calcium carbonate, and also contain more than 20 trace elements such as copper, iron, zinc, manganese, strontium, chromium, nickel, and 17 amino acids such as glycine, cystine, and methionine. It has the effects of calming the mind, subduing yang and nourishing yin, softening hardness and dissipating nodules, etc. It can be used alone or in combination with other drugs to treat convulsions, insomnia, dizziness, tinnitus, spontaneous sweating, night sweats, stomachache, acid regurgitation and other diseases.

[0003] Except for using part of oysters as medicinal materials, soil conditioners and for lime production, most oyster shells are landfilled, composted and dumped into the ocean, causing environmental pollution problems.

[0004] In recent years, the research on the high-value utilization of preparing food and drug grade calcium preparations from oyster shells has attracted much attention from researchers. Using oyster shells as a calcium source, calcium acetate, calcium lactate, calcium citrate, glutamic acid chelated calcium, L-aspartic acid chelated calcium, etc. have been made respectively; in order to prepare a new type of food and drug grade calcium supplement suitable for people with digestive tract ulcers, our research group has also developed a preparation process for ultrafine vaterite calcium carbonate using oyster shells; the above single calcium preparations cannot retain all the components of oysters and cannot exert the comprehensive curative effect of traditional Chinese medicine; for this reason, Chen Weimin et al. developed a preparation process for oyster shell calcium chewable tablets using water milling and pulverization technology, which can ensure that the effective components in the calcium tablets are not lost and can effectively improve the absorption and utilization of calcium elements by the human body; however, the disadvantage of the water milling and pulverization technology is that the water-soluble components of oyster shells will be lost during the pulverization process, and the prepared oyster shell calcium chewable tablets have poor water solubility and are difficult for the human body to fully absorb; therefore, it is particularly important to develop all-natural water-soluble oyster powder; so far, there has been no relevant report on the preparation process of water-soluble oyster powder. Therefore, the present invention proposes a method for preparing water-soluble oyster powder by enzyme-acid hydrolysis to solve the problems existing in the prior art. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to propose a method for preparing water-soluble oyster powder by enzyme-acid hydrolysis. The method for preparing water-soluble oyster powder by enzyme-acid hydrolysis mainly uses the enzyme-acid hydrolysis method to explore the optimal process conditions of water-soluble oyster powder; then, the structure of the water-soluble oyster powder is characterized; finally, the components of the water-soluble oyster powder are determined and analyzed to evaluate the preparation quality. The research results of this method are of great significance for further expanding the resource utilization of waste oyster shells and have broad application prospects in the aspects of raw materials or additives for medicines, health foods, cosmetics, etc.

[0006] To achieve the object of the present invention, the present invention is realized through the following technical solutions: A method for preparing water-soluble oyster powder by enzyme-acid hydrolysis, comprising the following steps:

[0007] Step 1: Wash the oyster shells and then perform a drying treatment, and then crush, grind and sieve them to obtain oyster shell powder.

[0008] Step 2: Weigh 4.5 g of oyster shell powder and put it into a beaker, add distilled water to 90 ml, boil and cool down, then add 0.6 g of neutral protease and react for 180 min for primary enzymatic hydrolysis, and then add 0.2 g of neutral protease again and react for 180 min for secondary enzymatic hydrolysis to obtain an enzymatic hydrolysate.

[0009] Step 3: Heat the enzymatic hydrolysate to 90 °C, add 6.0 ml of acetic acid, then boil and keep warm for 2 h and then filter to obtain a filtrate.

[0010] Step 4: Extract the filtrate and transfer it to a vial, store it in a -80 °C refrigerator for ultra-low temperature preservation overnight, and finally freeze-dry it in a freeze dryer for 30 h to obtain a solid powder of water-soluble oyster powder.

[0011] Further improvement lies in: In the specific step 1, the oyster shells are washed clean with tap water and then rinsed once with distilled water, and then placed in an oven at 80 °C for drying for 6 h, and then crushed by a swing-type high-speed universal pulverizer and a variable-speed high-speed pulverizer, and then the obtained powder with smaller particles is sieved through a vibrating sieve with 140 meshes to obtain oyster shell powder with a particle size less than 106 μm.

[0012] Further improvement lies in: The boiling and heat preservation temperature in step 3 is controlled at 95 °C - 100 °C.

[0013] Further improvement lies in: The calcium content of the solid powder of water-soluble oyster powder obtained in step 4 is 90.12%, the amino nitrogen content is 20.0 mg / g, and the solubility is 42.56 g / 100 ml.

[0014] The beneficial effects of the present invention are as follows: The present invention uses a safe and efficient enzyme-acid hydrolysis method to prepare water-soluble oyster powder, effectively improving the calcium absorption rate and bioavailability of oyster shells by the human body, converting calcium carbonate into calcium acetate, increasing the solubility of calcium, and obtaining the optimal parameters of this preparation method through a large number of experiments. The calcium content of the prepared water-soluble oyster powder solid powder reaches 90.12%, the amino nitrogen content reaches 20.0 mg / g, and the solubility is increased to 42.56 g / 100 ml. It has very important significance for further expanding the resource utilization of waste oyster shells and has broad application prospects in aspects such as raw materials or additives for medicines, health foods, and cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a comparison chart of the calcium content and nitrogen content of oyster powder at different enzymatic hydrolysis temperatures in Example 1 of the present invention.

[0016] Figure 2 It is a comparison chart of the calcium content and nitrogen content of oyster powder with different first enzyme addition amounts in Example 1 of the present invention.

[0017] Figure 3 It is a comparison chart of the calcium content and nitrogen content of oyster powder with different second enzyme addition amounts in Example 1 of the present invention.

[0018] Figure 4 It is a comparison chart of the calcium content and nitrogen content of oyster powder at different enzymatic hydrolysis times in Example 1 of the present invention.

[0019] Figure 5 It is a comparison chart of the calcium content and nitrogen content of oyster powder with different particle sizes of oyster shell powder in Example 1 of the present invention.

[0020] Figure 6 It is a comparison infrared spectrum chart of calcium acetate standard product and water-soluble oyster powder in Example 1 of the present invention.

[0021] Figure 7 It is a comparison infrared spectrum chart of calcium carbonate standard product and oyster shell powder in Example 1 of the present invention.

[0022] Figure 8 It is a flow chart of the preparation method in Example 2 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] To deepen the understanding of the present invention, the following will further elaborate on the present invention in combination with embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0024] Example 1

[0025] According to Figure 1-7As shown in the figure, this embodiment provides the single-factor experimental design and result analysis of process parameter optimization in a method for preparing water-soluble oyster powder by enzyme-acid hydrolysis, specifically including:

[0026] I. Experimental Design

[0027] 1. Influence of Enzymolysis Temperature on Water-Soluble Oyster Powder

[0028] The enzymolysis temperatures are 29°C, 37°C, and 45°C respectively.

[0029] Take three identical beakers, labeled 1, 2, and 3 respectively. Add 4.5 g of oyster shell powder passed through a 200-mesh sieve, weighed accurately to 0.0001 g. Add distilled water to 90 ml, boil, cool to room temperature, add 0.6 g of neutral protease, and place them in water baths at 29°C, 37°C, and 45°C respectively for 3 h. Then add 0.3 g of neutral protease and place them in water baths at 29°C, 37°C, and 45°C respectively for another 3 h. Raise the temperature to 90°C, add 6 ml of acetic acid, keep warm for 2 h, filter by suction to obtain the filtrate, transfer the filtrate to a vial, place it in an ultra-low temperature (-80°C) storage box and store it overnight, and then freeze-dry it in a freeze-drying system for 30 h to obtain a solid powder. Measure the calcium content and nitrogen content of the water-soluble oyster powder at different enzymolysis temperatures, and obtain the optimal enzymolysis temperature based on the calcium content and nitrogen content as indicators.

[0030] Measure the calcium content and nitrogen content of the solid powder obtained from the enzymolysis temperature optimization experiment for three consecutive days, three times a day. The results are as shown in the attached Figure 1 As shown in the figure, taking the calcium content (a in the figure) and nitrogen content (b in the figure) as reference indicators, compare the calcium content and nitrogen content at three enzymolysis temperatures, and select the optimal enzymolysis temperature. By comparing the calcium content at three temperatures, no obvious difference is found. However, when comparing the nitrogen content at three temperatures, it is found that the nitrogen content is the highest at 45°C, followed by that at 37°C, and the lowest at 29°C. According to the calcium content and nitrogen content at different enzymolysis temperatures, 45°C is obtained as the optimal enzymolysis temperature.

[0031] 2. Influence of Enzyme Dosage on Water-Soluble Oyster Powder

[0032] The first enzyme dosages are 0.5 g, 0.6 g, and 0.7 g respectively.

[0033] Take three identical beakers, labeled 1, 2, and 3 respectively. Add 4.5 g of oyster shell powder that has passed through a 200-mesh sieve, weighed accurately to 0.0001 g. Add distilled water to 90 ml, boil, and let it cool to room temperature. Then add 0.5 g, 0.6 g, and 0.7 g of neutral protease respectively, and place them in a water bath at 37°C for 3 h. Then add 0.3 g of neutral protease and place it in a water bath at 37°C for 3 h. Raise the temperature to 90°C, add 6 ml of acetic acid, keep warm for 2 h, filter by suction to obtain the filtrate, transfer the filtrate to a vial, place it in an ultra-low temperature (-80°C) storage box and store it overnight, and then freeze-dry it in a freeze-drying system for 30 h to obtain a solid powder. Measure the calcium content and nitrogen content of the water-soluble oyster powder under different first enzyme addition amounts, and obtain the optimal first enzyme addition amount based on the calcium content and nitrogen content.

[0034] Measure the calcium content and nitrogen content of the solid powder obtained from the optimization experiment of the first enzyme addition amount. Measure it continuously for three days, three times a day. The results are as shown in the appendix of the specification Figure 2 As shown, taking the calcium content (a in the figure) and nitrogen content (b in the figure) as reference indicators, compare the calcium content and nitrogen content under different first enzyme addition amounts, and select the optimal first enzyme addition amount.

[0035] By comparing the calcium content under different first enzyme addition amounts, it is found that the calcium content under the condition of 0.5 g is better than that under the conditions of 0.6 g and 0.7 g; by comparing the nitrogen content under different first enzyme addition amounts, it is found that the nitrogen content under the condition of 0.5 g is significantly lower than that under the conditions of 0.6 g and 0.7 g, while there is no significant difference in the nitrogen content between the conditions of 0.6 g and 0.7 g. It is concluded that the optimal first enzyme addition amount is 0.6 g;

[0036] The ratios of the second enzyme addition amounts to the optimal first enzyme addition amount are 1:3, 1:2, and 1:1 respectively.

[0037] Take three identical beakers, labeled 1, 2, and 3 respectively. Add 4.5 g of oyster shell powder that has passed through a 200-mesh sieve, weighed accurately to 0.0001 g. Add distilled water to 90 ml, boil, and let it cool to room temperature. Add 0.6 g of neutral protease, place it in a water bath at 37°C for 3 h. Then add 0.2 g, 0.3 g, and 0.6 g of neutral protease respectively, and place them in a water bath at 37°C for 3 h. Raise the temperature to 90°C, add 6 ml of acetic acid, keep warm for 2 h, filter by suction to obtain the filtrate, transfer the filtrate to a vial, place it in an ultra-low temperature (-80°C) storage box and store it overnight, and then freeze-dry it in a freeze-drying system for 30 h to obtain a solid powder. Measure the calcium content and nitrogen content of the water-soluble oyster powder under different second enzyme addition amounts, and obtain the optimal second enzyme addition amount based on the calcium content and nitrogen content.

[0038] Measure the calcium content and nitrogen content of the solid powder obtained from the optimization experiment of the second enzyme addition amount for three consecutive days, with three measurements per day. The results are shown in the appendix of the specification. Figure 3 As shown in the figure. Taking the calcium content (a in the figure) and nitrogen content (b in the figure) as reference indicators, compare the calcium content and nitrogen content under different second enzyme addition amounts, and select the optimal second enzyme addition amount.

[0039] By comparing the calcium content under different second enzyme addition amounts, it is found that the calcium content under the 1:1 condition is better than that under the 1:2 condition and 1:3 condition, and there is no significant difference in the calcium content between the 1:2 condition and the 1:3 condition; by comparing the nitrogen content under different second enzyme addition amounts, it is found that there is no significant difference in the nitrogen content among the 1:3 condition, 1:2 condition, and 1:1 condition. It is concluded that the optimal second enzyme addition amount is 1:3 (i.e., 0.2 g).

[0040] 3. Influence of Enzymolysis Time on Water-Soluble Oyster Powder

[0041] The enzymolysis times are 160 min, 180 min, and 200 min respectively.

[0042] Take three identical beakers, labeled 1, 2, and 3 respectively. Add 4.5 g of oyster shell powder passed through a 200-mesh sieve, weighed accurately to 0.0001 g. Add distilled water to 90 ml, boil, and cool to room temperature. Add 0.6 g of neutral protease and place it in a water bath at 37 °C to react for 160 min, 180 min, and 200 min respectively. Add 0.3 g of neutral protease and place it in a water bath at 37 °C to react for 160 min, 180 min, and 200 min respectively. Heat up to 90 °C, add 6 ml of acetic acid, keep warm for 2 h, filter by suction to obtain the filtrate, transfer the filtrate to a vial, place it in an ultra-low temperature (-80 °C) storage box and store it overnight, and then freeze-dry it in a freeze-drying system for 30 h to obtain a solid powder. Measure the calcium content and nitrogen content of the water-soluble oyster powder at different enzymolysis times, and obtain the optimal enzymolysis time based on the calcium content and nitrogen content.

[0043] Measure the calcium content and nitrogen content of the solid powder obtained from the enzymolysis time optimization experiment for three consecutive days, with three measurements per day. The results are shown in the appendix of the specification. Figure 4 As shown in the figure. Taking the calcium content (a in the figure) and nitrogen content (b in the figure) as reference indicators, compare the calcium content and nitrogen content at different enzymolysis times, and select the optimal enzymolysis time.

[0044] By comparing the calcium content under different enzymatic hydrolysis times, it was found that the calcium content under the condition of 180 min was better than that under the conditions of 160 min and 200 min, and there was no significant difference in the calcium content between the 200 min condition and the 160 min condition; by comparing the calcium content under different enzymatic hydrolysis times, it was found that the nitrogen content under the condition of 180 min and the nitrogen content under the condition of 200 min were better than the nitrogen content under the condition of 160 min, and there was no significant difference in the nitrogen content between the 200 min condition and the 180 min condition. It was concluded that the optimal enzymatic hydrolysis time was 180 min.

[0045] 4. Influence of Oyster Shell Powder Particle Size on Water-Soluble Oyster Powder

[0046] The particle sizes of the shell powder were 50 mesh (300 μm), 100 mesh (150 μm), 140 mesh (106 μm), and 200 mesh (75 μm) respectively.

[0047] Take four identical beakers, labeled 1, 2, 3, and 4 respectively. Add 4.5 g of oyster shell powder passing through 50 mesh (300 μm), 100 mesh (150 μm), 140 mesh (106 μm), and 200 mesh (75 μm) respectively, weigh accurately to 0.0001 g, add distilled water to 90 ml, boil, cool to room temperature, add 0.6 g of neutral protease, place it in a water bath at 37 °C and react for 3 h, add 0.3 g of neutral protease, place it in a water bath at 37 °C and react for 3 h, raise the temperature to 90 °C, add 6 ml of acetic acid, keep warm for 2 h, filter by suction to obtain the filtrate, transfer the filtrate to a vial, place it in an ultra-low temperature (-80 °C) storage box and store it overnight, and freeze-dry it in a freeze-drying system for 30 h to obtain a solid powder. Measure the calcium content and nitrogen content of the water-soluble oyster powder under different shell powder particle sizes, and obtain the optimal shell powder particle size with the calcium content and nitrogen content as indicators.

[0048] Measure the calcium content and nitrogen content of the solid powder obtained through the oyster shell powder particle size optimization experiment for three consecutive days, and measure it three times a day. The results are as shown in the attached Figure 5 Instructions. Taking the calcium content (a in the figure) and nitrogen content (b in the figure) as reference indicators, compare the calcium content and nitrogen content under different shell powder particle sizes, and select the optimal shell powder particle size.

[0049] By comparing the calcium content under different shell powder particle sizes, it was found that as the particle size decreased, the calcium content increased. The calcium content was the highest under the condition of 150 μm. After 150 μm, as the particle size became smaller and smaller, the calcium content also decreased; by comparing the nitrogen content under different shell powder particle sizes, it was found that as the particle size decreased, the nitrogen content increased. The nitrogen content was the highest under the condition of 106 μm. After 106 μm, as the particle size became smaller and smaller, the nitrogen content also decreased. It was concluded that the optimal shell powder particle size was 106 μm, that is, passing through a 140-mesh sieve.

[0050] Summarize the results of the above optimal process parameters for the preparation of water-soluble oyster powder: Weigh 4.5286 g of oyster shell powder that has passed through a 140-mesh sieve (106 μm) and put it into a beaker. Add distilled water to 90 ml, boil it, cool it down, add 0.6013 g of neutral protease, react for 180 min, then add 0.2005 g of neutral protease, react for 180 min, heat up to 90 °C, add 6.00 ml of analytical pure acetic acid, keep warm for 2 h (95 °C - 100 °C), filter by suction to obtain the filtrate, transfer the filtrate to a vial, put it in an ultra-low temperature storage box and store it overnight, and then freeze-dry it in a freeze-drying system for 30 h to obtain a solid powder. The calcium content and nitrogen content under the optimal process parameters are shown in Table 1 and Table 2 below.

[0051] Table 1 Calcium content table under optimal process parameters

[0052]

[0053] Table 2 Nitrogen content table under optimal process parameters

[0054]

[0055] II. Orthogonal experiment

[0056] According to the results of the single-factor experiment, design a four-factor and three-level table L9(4 3 ). According to the orthogonal experiment scheme table (Table 3 and Table 4 below), investigate the influence of each factor on the calcium content and amino nitrogen content of oyster shells, and determine the optimal factor level combination under the two indexes respectively, so as to determine the best process conditions for extracting calcium ions and amino acids from oyster shells.

[0057] Table 3 Orthogonal experiment scheme table with calcium content as the index

[0058]

[0059]

[0060] Table 4 Orthogonal experiment scheme table with nitrogen content as the index

[0061] Number Enzymolysis temperature First enzyme dosage Enzymolysis time Particle size of oyster shell powder 1 1 1 1 1 2 1 2 3 2 3 1 3 2 3 4 2 1 3 3 5 2 2 2 1 6 2 3 1 2 7 3 1 2 2 8 3 2 1 3 9 3 3 3 1

[0062] Results of the orthogonal experiment: According to the better reaction conditions determined by the single-factor method, conduct an orthogonal experiment with four factors of A (enzymolysis temperature / °C), B (enzyme addition amount / g), C (enzymolysis time / min), and D (shell powder particle size / μm). Select three levels near the optimal conditions and refer to the L9(4 3 ) orthogonal table (Table 3 and Table 4) to conduct the experiment. With the calcium content as the inspection index, its design is shown in Table 5 below.

[0063] According to the orthogonal experiment scheme designed in Table 3, the calcium content of each group of water-soluble oyster powder was investigated according to the experimental method to determine the optimal process conditions. The experimental results are shown in Table 6. Among the four factors listed in Table 5, the first level of enzymatic hydrolysis temperature is the best, the first level of enzyme dosage is the best, the third level of enzymatic hydrolysis time is the best, and the second level of shell powder particle size is the best. Thus, the optimal process conditions for preparing water-soluble oyster powder by enzyme-acid hydrolysis method with calcium content as the index are as follows: the enzymatic hydrolysis temperature is 29 °C, the enzyme dosage is 0.5 g, the enzymatic hydrolysis time is 200 min, and the shell powder particle size is passing through a 100-mesh sieve (150 μm). Under these optimal conditions, the calcium content of the water-soluble oyster powder is 91.02%. By comparing the R values, it can be found that the primary and secondary order of the influence of these four factors on the calcium content is: enzymatic hydrolysis time > shell powder particle size > enzymatic hydrolysis temperature > enzyme dosage.

[0064] Table 5 Orthogonal experiment table with calcium content as the index

[0065]

[0066] Table 6 Calcium content table of water-soluble oyster powder under different process parameter conditions

[0067]

[0068] According to the better reaction conditions determined by the single-factor method, an orthogonal experiment was carried out with four factors: A (enzymatic hydrolysis temperature / °C), B (enzyme dosage / g), C (enzymatic hydrolysis time / min), and D (shell powder particle size / μm). Three levels were selected near the better conditions, and the experiment was carried out with reference to the L9(3 4 ) orthogonal table, with the nitrogen content as the investigation index. The design is shown in Table 7 below.

[0069] According to the orthogonal experiment scheme designed in Table 4, the calcium content of each group of water-soluble oyster powder was investigated according to the experimental method to determine the optimal process conditions. The experimental results are shown in Table 8 below. Among the four factors listed in Table 7, the third level of enzymatic hydrolysis temperature is the best, the third level of enzyme dosage is the best, the second level of enzymatic hydrolysis time is the best, and the first level of shell powder particle size is the best. Thus, the optimal process conditions for preparing water-soluble oyster powder by enzyme-acid hydrolysis method with nitrogen content as the index are as follows: the enzymatic hydrolysis temperature is 45 °C, the enzyme dosage is 0.7 g, the enzymatic hydrolysis time is 180 min, and the shell powder particle size is passing through a 100-mesh sieve (150 μm). Under these optimal conditions, the nitrogen content of the water-soluble oyster powder is 23.3 mg / g. By comparing the R values, it can be found that the primary and secondary order of the influence of these four factors on the calcium content is: enzyme dosage > enzymatic hydrolysis time > enzymatic hydrolysis temperature = shell powder particle size.

[0070] Table 7 Orthogonal experiment table with nitrogen content as the index

[0071]

[0072] Table 8 Nitrogen content of water-soluble oyster powder under different process parameters

[0073]

[0074]

[0075] III. Characterization of water-soluble oyster powder

[0076] 1. Determination of calcium content

[0077] Take 0.2 g of the sample, weigh accurately to 0.0001 g, record as m, add water to 100 ml to dissolve. After complete dissolution, add 5 ml of 20% triethanolamine, then add 1 mol / L sodium hydroxide to adjust the pH to 12 - 13, add 0.1 g of calconcarboxylic acid indicator, and the solution turns purple-red; titrate with 0.05 mol / L EDTA solution until the solution changes from purple-red to pure blue and does not change color within half an hour, which is the titration end point. Stop titration and record the volume of the consumed EDTA standard solution (V). Titrate each sample solution in parallel three times and take the average value to calculate the calcium ion concentration according to the following formula.

[0078] c = c1×V1 / V0

[0079] Where c1 is the concentration of the disodium EDTA solution, and V0 is the volume of the sample, in ml.

[0080] Calcium content (%) = 17.62×c×V / m×100%

[0081] Where c is the concentration of the disodium EDTA solution.

[0082] 2. Determination of nitrogen content

[0083] Blank titration: Add water to 30 ml, add 10 ml of 40% neutral formaldehyde solution, measure the pH after 2 hours, and titrate with 0.01 mol / L NaOH solution until the pH is about 9.2, stop titration and record the volume of the consumed NaOH standard solution (V0);

[0084] Take 0.2 g of the sample, weigh accurately to 0.0001 g, record as m, add water to 30 ml to dissolve. After complete dissolution, add 10 ml of 40% neutral formaldehyde solution, measure the pH after reacting for 2 hours, and titrate with 0.01 mol / L NaOH solution until the pH is about 9.20, stop titration and record the volume of the consumed NaOH standard solution (V1). Titrate each sample solution in parallel three times and take the average value to calculate the nitrogen content according to the following formula.

[0085] Amino nitrogen (mg / g) = (V1 - V0)×c×140.08 / m

[0086] Wherein, V1 is the volume of NaOH standard solution consumed in the sample, and V0 is the volume of NaOH standard solution consumed in the blank titration.

[0087] 3. Solubility determination

[0088] Take a sufficient amount of water-soluble oyster shell powder, dry it in an oven to constant weight, weigh it (accurate to 0.0001g), record it as m, accurately transfer 1.00ml of pure water, add the sample until it no longer dissolves and solid precipitates, accurately weigh the remaining solid mass, record it as m1, filter and dry the undissolved solid to constant weight, record the dried solid as m2, determine the mass of the sample that can be dissolved in 100ml of water when the water-soluble oyster shell powder reaches saturation, measure three times and take the average value.

[0089] Solubility (g / 100ml) = m-(m1+m2) / 1×100

[0090] Experimental results: The solubility of the water-soluble oyster powder prepared with the optimal process parameters was measured in parallel three times, and the average value was taken. The results are shown in Table 9 below. The solubility of the water-soluble oyster powder is 42.56 g / 100 ml, and the solubility of the calcium acetate standard at 20°C is 34.7 g / 100 ml. It can be considered that the water-soluble oyster powder has good solubility properties.

[0091] Table 9 Solubility test results

[0092]

[0093] 4. Product infrared absorption spectrum detection

[0094] Take 100 mg KBr in a mortar, add the dried sample to be tested, grind thoroughly, transfer the powder to a mold and evenly distribute it, press it on a tablet press to obtain a transparent sheet, and use a KBr blank sheet as a control at a wavelength of 400-4000 cm -1 Scan inside to obtain a spectrum, and analyze the spectrum.

[0095] Take a small amount of water-soluble oyster powder and measure its infrared spectrum, as shown in the instructions. Figure 6 By comparing the infrared spectrum of water-soluble oyster powder with that of calcium acetate standard, the water-soluble oyster powder has a -1 , 1613cm -1 There is a strong and narrow absorption peak at 1453cm, which is the C=O stretching vibration absorption peak of the carboxyl salt; -1 , 1024cm -1 They are the CH in-plane bending vibration absorption peak and the CC single bond skeleton vibration absorption peak; 665cm -1The absorption peak at this position is the O-H bending vibration absorption peak. By comparing the characteristic absorption peak positions of water-soluble oyster powder and calcium acetate standard, it can be preliminarily determined that water-soluble oyster powder contains acetate ions.

[0096] Take a small amount of oyster shell powder and measure its infrared spectrum as shown in the attached instructions. Figure 7 As shown, by comparing the infrared spectrum of oyster shell powder with that of calcium carbonate standard, oyster shell powder has a strong and narrow absorption peak at 1412 cm -1 which is the C-O stretching vibration absorption peak; at 882 cm -1 is the out-of-plane bending vibration region of C-H. By comparing the infrared spectra of oyster shell powder and calcium carbonate standard, it can be seen that their characteristic absorption peaks are similar, and the positions and intensities of each peak are close, so it can be preliminarily judged that oyster shell powder contains carbonate ions.

[0097] 5. Determination of product scanning electron microscopy

[0098] For the sample, the direct dispersion method is used. Cut a small piece of conductive adhesive and stick it on the copper sheet. Directly disperse the fully dried sample to be tested on the conductive adhesive, and gently blow the sample with an ear bulb to remove the sample that is not adhered to the conductive adhesive. Since the conductivity of the sample is poor, it needs to be sputter-coated with gold before being placed in the scanning electron microscope for observation to observe the crystal form and morphology of the sample at different magnifications.

[0099] Perform electron microscopy scanning on the calcium carbonate standard at 100 times and 340 times to observe the morphological characteristics of the calcium carbonate standard. It is found that the morphology of the calcium carbonate standard is blocky and evenly dispersed at 100 times; at 340 times, the morphology of the calcium carbonate standard is cubic.

[0100] Perform electron microscopy scanning on oyster shell powder at 100 times and 340 times. It is found that the morphology of oyster shell is irregular and unevenly dispersed, and some are in an aggregated state at 100 times; at 340 times, oyster shell powder has a porous structure and the overall structure is loose.

[0101] Perform electron microscopy scanning on water-soluble oyster powder at 100 times and 340 times. It is found that the morphology of water-soluble oyster powder is needle-like or rod-like, and some are in a blocky structure at both 100 times and 340 times.

[0102] Perform electron microscopy scanning on calcium acetate standard at 100 times and 340 times. It is found that the morphology of calcium acetate standard is spherical at 100 times; at 340 times, the morphology of calcium acetate standard is irregular and has many pores inside.

[0103] Example 2

[0104] According to Figure 8 shown, this example provides a method for preparing water-soluble oyster powder by enzyme-acid hydrolysis, including the following steps:

[0105] Step 1: Scrub the oyster shells clean with tap water, then rinse them once with distilled water, and place them in an oven at 80 °C for 6 h of drying. Then, use a swing-type high-speed universal pulverizer and a variable-speed high-speed pulverizer for pulverization treatment. Subsequently, pass the obtained powder with smaller particles through a 140-mesh sieve using a vibrating sieve shaker to obtain oyster shell powder with a particle size less than 106 μm.

[0106] Step 2: Weigh 4.5 g of oyster shell powder and put it into a beaker, add distilled water to 90 ml, boil and cool down, then add 0.6 g of neutral protease and react for 180 min for primary enzymatic hydrolysis. Then, add another 0.2 g of neutral protease and react for 180 min for secondary enzymatic hydrolysis to obtain an enzymatic hydrolysate.

[0107] Step 3: Heat the enzymatic hydrolysate to 90 °C, then add 6.0 ml of acetic acid, then boil and keep warm for 2 h (the holding temperature is 95 °C - 100 °C), and then filter to obtain a filtrate.

[0108] Step 4: Extract the filtrate and transfer it to a vial, place it in a -80 °C refrigerator for ultra-low temperature storage overnight, and finally put it into a freeze dryer for freeze-drying treatment for 30 h to obtain a water-soluble oyster powder solid powder. The calcium content in the water-soluble oyster powder solid powder is 90.12%, the amino nitrogen content is 20.0 mg / g, and the solubility is 42.56 g / 100 ml.

[0109] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing water-soluble oyster powder by enzyme-acid hydrolysis, characterized in that: The following steps are involved: Step 1, washing and drying the oyster shells, and then crushing, grinding and sieving them to obtain oyster shell powder; Step 2: weigh 4.5 g of oyster shell powder and put it into a beaker, add distilled water to 90 ml and boil to cool, then add 0.6 g of neutral protease and react for 180 min for primary enzymolysis, then add 0.2 g of neutral protease and react for 180 min for secondary enzymolysis to obtain an enzymolysis solution; Step 3, heating the enzymatic hydrolyzate to 90°C, adding 6.0 ml of acetic acid, then boiling and keeping warm for 2 hours and filtering to obtain a filtrate; Step 4: extract the filtrate and transfer it to a vial, place it in a -80°C refrigerator for ultra-low temperature storage overnight, and finally place it in a freeze dryer for freeze drying for 30 hours to obtain a water-soluble oyster powder solid powder.

2. The method for preparing water-soluble oyster powder by enzyme-acid hydrolysis according to claim 1, characterized in that: In the step 1, the oyster shells are specifically cleaned with tap water and then rinsed with distilled water, and placed in an oven at 80° C. for drying for 6 hours, and then crushed using a swing-type high-speed universal grinder and a variable-speed high-speed grinder, and then the obtained powder with smaller particles is sieved through a 140-mesh sieve by a vibrating sieving machine to obtain oyster shell powder with a particle size of less than 106 μm.

3. The method for preparing water-soluble oyster powder by enzyme-acid hydrolysis according to claim 1, characterized in that: In the step 3, the boiling and heat preservation temperature is controlled at 95° C.-100° C.

4. The method for preparing water-soluble oyster powder by enzyme-acid hydrolysis according to claim 1, characterized in that: The water-soluble oyster powder solid powder obtained in step 4 has a calcium content of 90.12%, an amino nitrogen content of 20.0 mg / g, and a solubility of 42.56 g / 100 ml.

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