CN108354183A - The method for preparing the compound calcium powder of high-quality ocean source amino acid - Google Patents

Abstract

The invention discloses a kind of methods preparing the compound calcium powder of high-quality ocean source amino acid, include the following steps：（1）Aquatic product leftovers clean；（2）It is broken；（3）Desalination；（4）Preliminary acidolysis；（5）Microbial fermentation is degraded；（6）Amino acid tentatively compounds；（7）Enzymolysis；（8）The secondary compounding of amino acid；（9）It is dry；（10）Ultramicro grinding.Using living marine resources such as shrimp shell, crab shell, fish-bones marine organisms calcium powder is made, bioavailability is high, has a good application prospect by the preparation process of enzymolysis, organic acid, amino acid compounding in the present invention.The present invention increases fermenting step, while the step of alternateing progress using enzymolysis and acidolysis, can greatly improve the recovery rate and bioavailability of biological calcium powder in preparation process.

Description

Method for preparing high-quality marine source amino acid compound calcium powder

technical field

The invention relates to a method for preparing high-quality marine amino acid compound calcium powder, which belongs to the field of biotechnology.

Background technique

Calcium is an essential element for the human body. The nutritional status of calcium in the diet is closely related to human growth and development, cardiovascular diseases and chronic diseases. Sufficient calcium intake plays an important role in improving the symptoms of osteoporosis. At present, calcium products are mainly divided into They are inorganic calcium, organic acid calcium, amino acid calcium and other categories. Studies have found that different sources of calcium and different forms of calcium have great differences in the utilization of calcium. For example, Wang Xiaofeng (2010) et al. The comparison of absorption shows that the apparent absorption rate of fish bone calcium preparations can reach 93%, which is better than that of animal calcium products; Zhang Wenjing (2015) found that PHC-Ca prepared from shrimp shells has the effect of promoting calcium absorption. The bioavailability is better than that of calcium citrate and shrimp shell powder with the same calcium content. Therefore, it has become a development trend to use marine biological resources to prepare calcium products.

The coast of Zhejiang is rich in marine resources, especially shrimps, crabs, and fishes. However, the leftovers such as shrimp shells, crab shells, and fish bones are not effectively comprehensively utilized every year, resulting in a huge waste of resources. At present, there are few bioactive calcium products on the market, and there is no scale, and the utilization of marine biological calcium is even worse.

There are many shrimp, crab, and fish aquatic product processing enterprises in Zhejiang that have a large number of leftover shrimp shells, crab shells, and fish bones. Currently, the leftovers are mainly processed in the form of waste or low-value fish meal, resulting in a huge waste of resources and even pollution. environment, the present invention carries out research on the extraction of marine biological calcium from shrimp and crab shells, product development, etc., which is of great significance for Zhejiang Province to build a "blue granary", improve the utilization rate of marine resources and sustainable development of aquatic product processing: (1) It can improve marine Comprehensive utilization of resources; (2) Enrich the types of marine biological calcium products and optimize the calcium consumption market; (3) Improve the high value of calcium source waste from aquatic product processing. This project is in line with the strategy of the marine deep processing industry, and is of great significance to the construction of the current aquatic product processing industry structure. economic and social benefits.

Contents of the invention

In the prior art, calcium powder is prepared from marine raw materials by enzymatic hydrolysis, but the enzymatic hydrolysis efficiency is low, the yield of calcium powder obtained is low, and the bioavailability is not high. The technical problem to be solved by the present invention is to provide a method for preparing high-quality marine amino acid compound calcium powder.

The method for preparing high-quality marine source amino acid compound calcium powder provided by the present invention is characterized in that it comprises the following steps:

(1) Cleaning of the leftovers of aquatic products: wash the leftovers of aquatic products 2-3 times with clean water, and then dry them.

(2) Crushing: Crushing the dried aquatic product leftovers to obtain 10-30 mesh powder.

(3) Desalting: Soak the product powder obtained in step (2) in a hydrochloric acid solution with a mass ratio of 1:3-5 and a concentration of 0.5-1mol/L for 20-30 minutes, and the soaking process is assisted by ultrasound.

In marine raw materials, the salt content is relatively high, so it is necessary to remove the salt as much as possible to enhance the efficiency of enzymatic hydrolysis and reduce the salt content of the final product.

(4) Preliminary acid hydrolysis: put 100 parts by weight of the powder into 3-5 times the weight of water, and add 10-15 parts by weight of organic acid at the same time, boil for 30 minutes, cool to 30-35 °C, and maintain for 3-5 hours .

The invention adopts the step of preliminary acid hydrolysis, and after the marine raw materials are cooked, the preliminary acid hydrolysis is carried out, which is beneficial to the microbial fermentation in the subsequent steps.

(5) Degradation by microbial fermentation: heat up the raw materials after initial acid hydrolysis to 35-40°C, add 0.03-0.06 parts of Bacillus licheniformis, yeast and Bacillus subtilis, mix well, and ferment for 1-2 days.

The present invention applies microbial fermentation to the preparation of biological calcium powder, and perfects its function and efficacy. On the one hand, it utilizes microorganisms to separate out the calcium element in the raw materials, that is, to achieve the purpose of decalcifying the raw materials. At the same time, it utilizes related microorganisms in the During the fermentation process, various proteases and hydrolytic enzymes are produced to further enzymolyze the raw materials. For example, both Bacillus licheniformis and Bacillus subtilis can produce various proteases, and the proteases produced during the fermentation process and the various enzymes added in the following step (6) can better enzymolyze the raw materials.

(6) Preliminary compounding of amino acids: Add 1-2 times the citric acid-sodium citrate buffer solution and 1-3 times the amino acid complex acid aqueous solution to the fermented materials, raise the temperature to 40-60°C, and ultrasonically treat 1- 2h.

In order to increase the chelation rate of amino acids and calcium sources, the present invention adopts a two-step compounding step, one time of amino acid compounding after microbial fermentation, and one more compounding after enzymatic hydrolysis, so as to increase the chelation probability of amino acids and precipitated calcium .

(7) Enzymolysis: Add alkaline protease, cathepsin and lysozyme to the material after the initial compounding of amino acids, and the weight of the three enzymes is 1.5-3%, 0.5-2% and 0.05-0.5% of the material weight respectively %; maintain the temperature at 30-40°C for 2-5 hours, then raise the temperature to 80-90°C to inactivate the enzyme activity, then centrifuge and collect the supernatant.

The three enzymes selected in the present invention can enzymolyze the protein in the leftovers of aquatic products into polypeptides and amino acids, and at the same time, the lysozyme can enzymolyze the microorganisms in the material to release more proteolytic enzymes.

(8) Secondary compounding of amino acids: Add 3-5 times the citric acid-sodium citrate buffer solution and 3-5 times the amino acid complex acid aqueous solution to the obtained supernatant, raise the temperature to 40-70°C, and ultrasonically treat at the same time 2-3h.

(9) Drying: The material in step (8) is concentrated and dried in vacuum at an absolute pressure of 0.06-0.2 MPa and a temperature of 95-100°C for 1-2 hours.

(10) Superfine grinding: crushed into 200-mesh particles, and then sieved and packaged.

Preferably, the leftovers of aquatic products are selected from shrimp shells, shrimp heads, fish bones, crab shells and the like.

Preferably, (4) the organic acid in the preliminary acidolysis is selected from compound acids of citric acid, tartaric acid and malic acid, and the weight ratio of the three acids is 5:2-3:1.

Preferably, the compound acids used in amino acid compounding in step (6) and step (8) include tryptophan, phenylalanine, L-aspartic acid and glycine, and the weight ratio of the four acids is 1:2 -5:3-7:3-4, the mass percentage of amino acid in the aqueous solution is 20-35%.

Further, in the preliminary acidolysis process of step (4), in order to enhance the effect of acidolysis, a certain amount of guar gum solution can be added, and guar gum can be used as a thickener in the food field. In the present invention, adding guar gum It can combine the cooked raw material and organic acid more closely to achieve better acidolysis effect.

The beneficial effects of the present invention are:

The invention utilizes marine biological resources such as shrimp shells, crab shells, and fish bones, and prepares marine biological calcium powder through a preparation process of enzymatic hydrolysis, organic acid, and amino acid compounding. The calcium powder has high bioavailability and has good application prospects.

In the preparation process of the present invention, the fermentation link is added, and the steps of enzymatic hydrolysis and acid hydrolysis are alternately adopted at the same time, so that the extraction rate and bioavailability of the biological calcium powder can be greatly improved.

Detailed ways

Example 1

This embodiment provides a method for preparing high-quality marine source amino acid compound calcium powder using shrimp shells, comprising the following steps:

(1) Cleaning of the leftovers of aquatic products: Wash the leftovers of aquatic products 3 times with clean water, and then dry them.

(2) Crushing: Crushing the dried aquatic product leftovers to obtain 30-mesh powder.

(3) Desalting: Soak the product powder obtained in step (2) in a 1:5 mass ratio hydrochloric acid solution with a concentration of 1mol/L for 30 minutes, and the soaking process is assisted by ultrasound.

In marine raw materials, the salt content is relatively high, so it is necessary to remove the salt as much as possible to enhance the efficiency of enzymatic hydrolysis and reduce the salt content of the final product.

(4) Preliminary acid hydrolysis: put 100 parts by weight of powder into 5 times the weight of water, and add 15 parts by weight of organic acid at the same time, boil for 30 minutes, cool to 35°C, and maintain for 5 hours.

The invention adopts the step of preliminary acid hydrolysis, and after the marine raw materials are cooked, the preliminary acid hydrolysis is carried out, which is beneficial to the microbial fermentation in the subsequent steps.

(5) Degradation by microbial fermentation: heat up the raw materials after initial acid hydrolysis to 40°C, add 0.06 parts each of Bacillus licheniformis, yeast and Bacillus subtilis, mix well, and ferment for 2 days.

The present invention applies microbial fermentation to the preparation of biological calcium powder, and perfects its function and efficacy. On the one hand, it utilizes microorganisms to separate out the calcium element in the raw materials, that is, to achieve the purpose of decalcifying the raw materials. At the same time, it utilizes related microorganisms in the During the fermentation process, various proteases and hydrolytic enzymes are produced to further enzymolyze the raw materials. For example, both Bacillus licheniformis and Bacillus subtilis can produce various proteases, and the proteases produced during the fermentation process and the various enzymes added in the following step (6) can better enzymolyze the raw materials.

(6) Preliminary compounding of amino acids: Add 2 times the citric acid-sodium citrate buffer solution and 2 times the amino acid complex acid aqueous solution to the fermented materials, raise the temperature to 60°C, and ultrasonicate for 2 hours at the same time.

In order to increase the chelation rate of amino acids and calcium sources, the present invention adopts a two-step compounding step, one time of amino acid compounding after microbial fermentation, and one more compounding after enzymatic hydrolysis, so as to increase the chelation probability of amino acids and precipitated calcium .

(7) Enzymolysis: Add alkaline protease, cathepsin and lysozyme to the material after the initial compounding of amino acids, and the weight of the three enzymes is 3%, 1% and 0.5% of the material weight respectively; the temperature is maintained at 40 °C for 5 hours, then raised to 90 °C to inactivate the enzyme activity, then centrifuged to collect the supernatant.

The three enzymes selected in the present invention can enzymolyze the protein in the leftovers of aquatic products into polypeptides and amino acids, and at the same time, the lysozyme can enzymolyze the microorganisms in the material to release more proteolytic enzymes.

(8) Secondary compounding of amino acids: Add 5 times of citric acid-sodium citrate buffer solution and 5 times of amino acid complex acid aqueous solution to the obtained supernatant, raise the temperature to 60°C, and ultrasonicate for 3 hours at the same time.

(9) Drying: The material in step (8) was concentrated and dried in vacuum for 1.5 hours at an absolute pressure of 0.2 MPa and a temperature of 100°C.

(10) Superfine grinding: crushed into 200-mesh particles, and then sieved and packaged.

(4) The organic acid in the preliminary acid hydrolysis is selected from compound acids of citric acid, tartaric acid and malic acid, and the weight ratio of the three acids is 2-1.

The compound acids used in amino acid compounding in step (6) and step (8) include tryptophan, phenylalanine, L-aspartic acid and glycine, and the weight ratio of the four acids is 1:3:5: 4. The mass percentage of amino acid in the aqueous solution is 35%.

In the preliminary acidolysis process of step (4), in order to enhance the effect of acidolysis, a certain amount of guar gum solution was added, and 5 parts by weight of guar gum solution with a mass concentration of 12% was added. Guar gum can be used as a thickener in the food field. In the present invention, the addition of guar gum can combine the cooked raw materials and organic acids more closely to achieve better acidolysis effect.

Utilize the flame atomic absorption method to measure the calcium content of the product of this embodiment, the calcium content in the main component of the composite calcium powder reaches 51.4%, and the total amino acid content reaches 43.8%.

Example 2

This embodiment provides a method for preparing high-quality marine source amino acid compound calcium powder using fish bones, comprising the following steps:

(1) Cleaning of the leftovers of aquatic products: Wash the leftovers of aquatic products twice with clean water, and then dry them.

(2) Crushing: Crushing the dried aquatic product leftovers to obtain 20-mesh powder.

(3) Desalting: Soak the product powder obtained in step (2) in a hydrochloric acid solution with a mass ratio of 1:4 and a concentration of 1mol/L for 20-30 minutes, and the soaking process is assisted by ultrasound.

In marine raw materials, the salt content is relatively high, so it is necessary to remove the salt as much as possible to enhance the efficiency of enzymatic hydrolysis and reduce the salt content of the final product.

(4) Preliminary acid hydrolysis: put 100 parts by weight of powder into 4 times the weight of water, and add 15 parts by weight of organic acid at the same time, boil for 30 minutes, cool to 35°C, and maintain for 5 hours.

The invention adopts the step of preliminary acid hydrolysis, and after the marine raw materials are cooked, the preliminary acid hydrolysis is carried out, which is beneficial to the microbial fermentation in the subsequent steps.

(5) Degradation by microbial fermentation: heat up the raw materials after initial acid hydrolysis to 40°C, add 0.06 parts each of Bacillus licheniformis, yeast and Bacillus subtilis, mix well, and ferment for 2 days.

The present invention applies microbial fermentation to the preparation of biological calcium powder, and perfects its function and efficacy. On the one hand, it utilizes microorganisms to separate out the calcium element in the raw materials, that is, to achieve the purpose of decalcifying the raw materials. At the same time, it utilizes related microorganisms in the During the fermentation process, various proteases and hydrolytic enzymes are produced to further enzymolyze the raw materials. For example, both Bacillus licheniformis and Bacillus subtilis can produce various proteases, and the proteases produced during the fermentation process and the various enzymes added in the following step (6) can better enzymolyze the raw materials.

(6) Preliminary compounding of amino acids: add 2 times of citric acid-sodium citrate buffer solution and 3 times of amino acid complex acid aqueous solution to the fermented materials, heat up to 60°C, and ultrasonically treat for 2 hours at the same time.

In order to increase the chelation rate of amino acids and calcium sources, the present invention adopts a two-step compounding step, one time of amino acid compounding after microbial fermentation, and one more compounding after enzymatic hydrolysis, so as to increase the chelation probability of amino acids and precipitated calcium .

(7) Enzymolysis: Add alkaline protease, cathepsin and lysozyme to the material after the preliminary compounding of amino acids, and the weight of the three enzymes is 3%, 2% and 0.5% of the material weight respectively; the temperature is maintained at 40 °C for 4 hours, then raised to 890 °C to inactivate the enzyme activity, then centrifuged to collect the supernatant.

The three enzymes selected in the present invention can enzymolyze the protein in the leftovers of aquatic products into polypeptides and amino acids, and at the same time, the lysozyme can enzymolyze the microorganisms in the material to release more proteolytic enzymes.

(8) Secondary compounding of amino acids: Add 5 times of citric acid-sodium citrate buffer solution and 5 times of amino acid complex acid aqueous solution to the obtained supernatant, raise the temperature to 70°C, and ultrasonicate for 2-3 hours at the same time.

(9) Drying: The material in step (8) was concentrated and dried in vacuum for 2 hours at an absolute pressure of 0.2 MPa and a temperature of 100°C.

(10) Superfine grinding: crushed into 200-mesh particles, and then sieved and packaged.

(4) The organic acid in the preliminary acid hydrolysis is selected from the compound acids of citric acid, tartaric acid and malic acid, and the weight ratio of the three acids is 5:2-3:1.

The compound acids used in amino acid compounding in step (6) and step (8) include tryptophan, phenylalanine, L-aspartic acid and glycine, and the weight ratio of the four acids is 1:5:3: 3. The mass percentage of amino acid in the aqueous solution is 35%.

Utilize the flame atomic absorption method to measure the calcium content of the product of this example, the calcium content in the main component of the composite calcium powder reaches 43.6%, and the total amino acid content reaches 39.8%.

Embodiment 3 The bioabsorption and utilization determination of the biological complex calcium prepared by the present invention:

Utilize the composite calcium powder prepared in Example 1 to measure. The bioabsorption utilization rate of the present invention is measured according to the report in the prior art, for strengthening the contrast effect of the present invention and the prior art, according to Zhang Wenjing etc. published in food science " preparation process optimization and large-scale analysis of composite calcium powder in shrimp shells Rat evaluation of its absorption effect".

The specific experimental steps are as follows:

3.1 Rat groups

4-week-old weaned rats, with an average weight of 83 g, were adaptively fed with basal feed for 1 week, and 100 rats with no significant difference in body weight were selected after weighing. According to the calcium content calculation in the present invention's composite calcium powder and calcium carbonate, each group of calcium dosage is divided into 5 groups at random: low calcium control group, calcium carbonate control group, composite calcium powder low dosage group, middle dosage group and high dosage group, 20 in each group.

3.2 Rat feeding methods

The low-calcium control group was fed with low-calcium feed (low-calcium feed formula: 19.4% casein, 39.4% corn starch, 10% soybean oil, 13.6% maltodextrin, 8.7% sucrose, 5% cellulose, 1% mixed vitamins, DL-methionine 0.1%, choline bitartrate 0.25%, tert-butyl hydroquinone 0.002%, mineral mixture 2.5%, calcium 0.1%).

Rats in low, medium and high dose groups were given low, medium and high doses of calcium compound calcium powder by intragastric administration every day on the basis of low calcium feed.

Oral administration dose reference: 800 mg (adult body weight 60 kg) is converted to 13.3 mg/(kg·d), and the low, medium and high doses are 2, 5, and 10 times the human dose. That is to say, the corresponding calcium doses for rats were 26.6, 66.5, and 133 mg/(kg·d) (based on calcium content and rat body weight). Rats in each group were fed under the same environment, free to eat, and weighed once a week. Rats in each group were fed under the same environment, free to eat, and weighed once a week.

3.3 Evaluation of growth and calcium absorption effects of rats ingesting compound calcium powder

During the rearing period, no disease or death occurred in the experimental animals. At the initial stage of the experiment, there was no significant difference in the body weight and length of the rats in each group. After feeding for 3 weeks, the rats with different doses of compound calcium powder grew well. The body weight of rats in each dose group of compound calcium powder was significantly higher than that of the low calcium control group. The growth index of the rats in the low-calcium control group was significantly lower than that of the other dose groups. At this time, the rats' coat color was dull, and their actions were sluggish. Specifically, the effect of compound calcium powder on the growth of rats is shown in Table 1.

The effect of table 1 compound calcium powder on the growth of rats

Claims (4)

1. the method for preparing the compound calcium powder of high-quality ocean source amino acid, which is characterized in that include the following steps：

（1）Aquatic product leftovers clean：Aquatic product leftovers are cleaned 2-3 times with clear water, are then dried；

（2）It is broken：The aquatic product leftovers of drying are subjected to break process, obtain 10-30 mesh powders；

（3）Desalination：By step（2）The product powder of acquisition is with 1:The hydrochloric acid solution of a concentration of 0.5-1mol/L of 3-5 mass ratioes It is impregnated, impregnates 20-30min, soaking process assisting ultrasonic；

（4）Preliminary acidolysis：The powder of 100 parts by weight is put into the water of 3-5 times of weight, while 10-15 parts by weight are added Organic acid, boil 30min, be cooled to 30-35 DEG C, maintain 3-5h；

（5）Microbial fermentation is degraded：Raw material after preliminary acidolysis is warming up to 35-40 DEG C, bacillus licheniformis, saccharomycete is added It with 0.03-0.06 parts of Bacillus subtilis strain, fully mixes thoroughly, ferments 1-2 days；

（6）Amino acid tentatively compounds：1-2 times of citric acid-sodium citrate buffer and 1-3 is added in the material that fermentation is obtained The compound aqueous acid of amino acid again, is warming up to 40-60 DEG C, while ultrasonication 1-2h；

（7）Enzymolysis:Addition alkali protease, cathepsin and lysozyme in the material after amino acid tentatively compounds, three kinds The weight of enzyme is respectively 1.5-3%, 0.5-2% and 0.05-0.5% of weight of material；Temperature maintains 30-40 DEG C, holds time Then 2-5h is warming up to 80-90 DEG C of enzyme deactivation activity, is then centrifuged for, collect supernatant；

（8）The secondary compounding of amino acid：By obtained supernatant be added 3-5 times citric acid-sodium citrate buffer and 3-5 times The compound aqueous acid of amino acid, be warming up to 40-70 DEG C, while ultrasonication 2-3h；

（9）It is dry：By step（8）Material using be concentrated in vacuo drying, absolute pressure 0.06-0.2MPa, temperature 95- It is 100 DEG C, 1-2 hours dry；

（10）Ultramicro grinding：It is ground into the particle of 200 mesh, then screening packaging.

2. the preparation method of the compound calcium powder of ocean source according to claim 1 amino acid, which is characterized in that aquatic products are got a foothold Material is selected from shrimp shell, shrimp head, fish-bone, crab shell etc..

3. the preparation method of the compound calcium powder of ocean source according to claim 1 amino acid, which is characterized in that（4）Preliminary acid Organic acid in solution is selected from the Compound-acid of citric acid, tartaric acid, malic acid, three kinds of sour weight ratio 5:2-3:1.

4. the preparation method of the compound calcium powder of ocean source according to claim 1 amino acid, which is characterized in that step（6）With Step（8）Compound-acid in middle amino acid compounding includes tryptophan, phenylalanine, ASPARTIC ACID and glycine, four kinds The weight ratio of acid is 1:2-5:3-7:The mass percent of 3-4, Ammonia In Aqueous Solution base acid are 20-35%.