阅读说明：本技术 一种利用微生物发酵从虾壳中提取甲壳素的方法 (Method for extracting chitin from shrimp shells by utilizing microbial fermentation ) 是由 李永成 张巧 黄兴 于 2019-10-29 设计创作，主要内容包括：本发明公开了利用微生物发酵从虾壳中提取甲壳素的方法。本发明以虾壳为原料,洗净干燥,研磨成粉,然后加入适当浓度的葡萄糖,灭菌后首先接种枯草芽孢杆菌,然后流加适当浓度乙醇并接种醋酸杆菌继续发酵。枯草芽孢杆菌生长产生的蛋白酶降解虾壳中的蛋白质。醋酸杆菌则以乙醇为碳源,上述被枯草芽孢杆菌降解的虾壳蛋白为氮源,生长产生醋酸,溶解虾壳中的矿物质使其变成可溶性的钙等金属离子。本发明公开的甲壳素制备方法将虾壳脱蛋白与脱盐两工艺过程耦合起来,合二为一,操作简单可行,脱蛋白和脱盐效果好,不仅实现了对虾壳的高值化利用,且简化了甲壳素的生产工艺,降低生产成本,减少对环境的污染。(The invention discloses a method for extracting chitin from shrimp shells by utilizing microbial fermentation. The invention takes shrimp shell as raw material, which is cleaned, dried, ground into powder, then added with glucose with proper concentration, firstly inoculated with bacillus subtilis after sterilization, and then fed with ethanol with proper concentration and inoculated with bacillus acetate for continuous fermentation. Protease produced by the growth of bacillus subtilis degrades proteins in shrimp shells. The acetobacter uses ethanol as a carbon source, the shrimp shell protein degraded by the bacillus subtilis is used as a nitrogen source, acetic acid is generated by growth, and mineral substances in the shrimp shells are dissolved to be changed into soluble metal ions such as calcium and the like. The chitin preparation method disclosed by the invention couples the two technological processes of shrimp shell deproteinization and desalination, is simple and feasible to operate, has good deproteinization and desalination effects, realizes high-value utilization of shrimp shells, simplifies the production technology of chitin, reduces the production cost and reduces the pollution to the environment.)

1. A method for extracting chitin from shrimp shells by utilizing microbial fermentation is characterized by comprising the following steps:

step S1: crushing the shrimp shell to obtain shrimp shell powder;

step S2: adding glucose, yeast extract and water into shrimp shell powder, uniformly stirring, and sterilizing to obtain a shrimp shell culture medium;

step S3: inoculating bacillus subtilis in a shrimp shell culture medium, and fermenting for 48-52 hours at 35-38 ℃ and 160-200 rpm to obtain a bacillus subtilis fermentation medium;

step S4: directly adding 5-7% absolute ethyl alcohol into the bacillus subtilis fermentation substrate without replacing the culture medium after the bacillus subtilis fermentation is finished, and uniformly stirring to obtain an ethanol fermentation substrate;

step S5: after ethanol is fed into the bacillus subtilis fermentation substrate, inoculating acetic acid bacillus, and fermenting for 60-72 hours at 30-35 ℃ and 160-200 rpm to obtain acetic acid bacillus fermentation liquor;

step S6: carrying out solid-liquid separation on the acetic acid bacillus fermentation liquor, washing the precipitate with water to be neutral, and mixing the precipitate with water according to a solid-liquid weight ratio of 1: 10 adding 10% hydrogen peroxide solution for decoloring, and soaking for 2h at room temperature;

step S7: and washing and drying the decolored solid matter to obtain the white solid chitin.

2. The method for extracting chitin from shrimp shells by microbial fermentation as claimed in claim 1, wherein the step S1 is specifically: and grinding the dried shrimp shell raw material, and screening the ground shrimp shell raw material through a 60-80-mesh screen to obtain the shrimp shell powder.

3. The method for extracting chitin from shrimp shells by microbial fermentation as claimed in claim 1, wherein: in the step S2, the adding amounts of the shrimp shell powder, the glucose and the yeast extract are respectively 4-6%, 5-8% and 0.2-0.5% of the volume of the water.

4. The method for extracting chitin from shrimp shells by microbial fermentation as claimed in claim 1, wherein: in the step S3, the inoculation amount of the bacillus subtilis is 1-3% of the volume of the shrimp shell culture medium.

5. The method for extracting chitin from shrimp shells by microbial fermentation as claimed in claim 1, wherein: in the step S5, the inoculation amount of the acetobacter is 3-5% of the volume of the ethanol fermentation substrate.

Technical Field

The invention belongs to the technical field of food industry, relates to a method for extracting chitin, and particularly relates to a method for preparing chitin by removing proteins and minerals in shrimp shells through microbial fermentation.

Background

At present, the shrimp processing is mainly performed on shrimp meat, the heads and the shells of the shrimps are removed during processing, and the heads and the shells of the shrimps account for about 30 to 40 percent of the weight of the whole shrimps. Due to the technical condition limitation, the processing wastes are difficult to treat, occupy land after being discarded, and cause environmental pollution. The main components of the shrimp shell are chitin (also called chitin) (10-20%), protein (20-40%) and inorganic salt (30-40%). Chitin is a biological macromolecule with important application value, and after chemical or biological deproteinization and inorganic salt removal, shrimp shells can be converted into chitin with high added value and derivatives thereof. Therefore, how to comprehensively utilize the shrimp shells is not only an urgent environmental protection problem in the shrimp processing industry, but also an effective way for realizing industrial upgrading.

The preparation method of the shrimp shell chitin comprises a chemical method, an enzymatic method and a fermentation method. The chemical method is obtained by removing protein, calcium carbonate and other components in the shrimp shells by using strong acid and strong alkali. Because of the application of strong acid and strong alkali, the environmental pollution is serious. The focus of the current research is to produce chitin by using organic acid produced by microbial fermentation and protease treatment of shrimp shells. In contrast, the biological method for preparing the chitin has the advantages of mild reaction, little environmental pollution, little damage to the chitin structure, capability of keeping the biological characteristics of the chitin and the like, and is the current main production method.

The traditional fermentation method shrimp shell chitin production process adopts a two-stage fermentation process, takes bacillus as protease-producing microorganism and lactic acid bacteria as acid-producing bacteria. In the first stage of fermentation, firstly culturing lactic acid bacteria to ferment the shrimp shell, producing acid and removing salt, then separating the shrimp shell, replacing the culture medium, sterilizing again, inoculating the microorganism producing protease, and performing secondary fermentation denitrification. Because the desalination and the deproteinization in the process are separated, after the desalination is finished in the first fermentation stage, the shrimp shells need to be separated, and the culture medium needs to be replaced, so that the second stage fermentation of the deproteinization can be carried out, therefore, the operation process flow is complex, and the production cost is higher.

Disclosure of Invention

The invention aims to provide a method for extracting chitin from shrimp shells by microbial fermentation, which has the advantages of simple process, mild reaction and low cost, combines the traditional two-stage microbial process of the shrimp shells into one, has better effects of removing minerals and proteins, and ensures that the obtained chitin has high quality and the chitin yield is high.

In order to achieve the purpose, the technical scheme of the invention is as follows: provides a method for extracting chitin from shrimp shells by utilizing microbial fermentation, which comprises the following steps:

step S1: crushing the shrimp shell to obtain shrimp shell powder;

step S2: adding glucose, yeast extract and water into shrimp shell powder, uniformly stirring, and sterilizing to obtain a shrimp shell culture medium;

step S3: inoculating bacillus subtilis in a shrimp shell culture medium, and fermenting for 48-52 hours at 35-38 ℃ and 160-200 rpm to remove proteins of shrimp shells to obtain a bacillus subtilis fermentation medium;

step S4: directly adding 5-7% (V/V) absolute ethyl alcohol into the bacillus subtilis fermentation substrate without replacing the culture medium after the bacillus subtilis fermentation is finished, and uniformly stirring to obtain an ethanol fermentation substrate;

step S5: after ethanol is fed into the bacillus subtilis fermentation substrate, inoculating acetobacter, and fermenting for 60-72 hours at 30-35 ℃ and 160-200 rpm to remove minerals to obtain an acetobacter fermentation broth;

step S6: carrying out solid-liquid separation on the acetic acid bacillus fermentation liquor, washing the precipitate with water to be neutral, and mixing the precipitate with water according to a solid-liquid weight ratio of 1: 10 adding 10% hydrogen peroxide solution for decoloring, and soaking for 2h at room temperature;

step S7: and washing and drying the decolored solid matter to obtain the white solid chitin.

Preferably, the step S1 specifically includes: and (3) grinding the dried (45-50 ℃) shrimp shell raw material, and screening the ground material through a 60-80-mesh screen to obtain the shrimp shell powder.

Preferably, in step S2, the addition amounts of the shrimp shell powder, the glucose and the yeast extract are respectively 4% -6%, 5% -8% and 0.2% -0.5% of the volume of the water.

Preferably, in the step S3, the inoculation amount of the bacillus subtilis is 1-3% (V/V) of the volume of the shrimp shell culture medium.

Preferably, in the step S5, the inoculation amount of the acetobacter is 3-5% (V/V) of the volume of the ethanol fermentation substrate.

Preferably, the culturing and preparing of the bacillus subtilis strain: taking 2ml LB broth culture medium (composition g/L: tryptone 10, yeast extract 5, NaCl, pH 7.0) in a 10ml test tube, sterilizing at 121 ℃ for 15min, cooling, inoculating 2-3 ring Bacillus subtilis test tube slant strain, and culturing at 37 ℃ and 180rpm to OD₆₀₀And (5) 0.8 for standby. Then placing 500ml triangular flask into LB broth culture medium 100ml, sterilizing at 121 deg.C for 15min, cooling, inoculating 1ml of above strain, culturing at 37 deg.C and 180rpm to OD₆₀₀0.8-1.0 for standby.

Preferably, the culture and preparation of the acetobacter species: acetobacter culture medium (w/v): 1% yeast extract, 1% glucose, pH 5.5, and 3% (v/v) absolute ethanol before use. 2% agar is added into the solid culture medium, and the culture process and conditions are the same as those of the bacillus subtilis, but the culture temperature is 30 ℃.

The method for extracting the chitin from the shrimp shells by utilizing microbial fermentation has the following beneficial effects:

the invention takes bacillus subtilis as protease producing bacteria and acetic acid bacillus as acid producing acid. Firstly, the bacillus subtilis is denitrified, then ethanol is fed, and the bacillus aceticus is directly inoculated for desalination. The advantages are that: firstly, the acetic acid desalting capacity is stronger than that of lactic acid, secondly, the carbon source required by the fermentation of the acetobacter is ethanol, which is inconsistent with the glucose required by the bacillus subtilis, the competition of the carbon source and the interference of the growth do not exist in the two bacteria, and simultaneously, after the ethanol is fed, the growth of the bacillus subtilis can be inhibited, the influence on the growth of the acetic acid bacteria is reduced, so that the fermentation of the two microorganisms can be combined into one, the preparation process of the chitin is simplified, and the production cost is saved; the invention can make the protein removing rate in the shrimp shell reach 93-98%, and the mineral removing rate reach 95-98%.

Detailed Description