阅读说明：本技术 一种饲用缬氨酸及蛋白肽的生产方法 (Production method of feeding valine and protein peptide ) 是由 徐庆阳 李志华 李国华 张宁 杨柳 于 2020-06-09 设计创作，主要内容包括：本发明属于氨基酸及蛋白肽生产技术领域,公开了一种饲用缬氨酸及蛋白肽的生产方法,步骤如下：将产缬氨酸的谷氨酸棒杆菌活化、种子培养、发酵获得含缬氨酸的发酵液；加氢氧化钠调节pH为11,使得发酵液中的谷氨酸棒杆菌体裂解为变性的菌体蛋白；加入碱性蛋白酶,初步酶解菌体蛋白；然后加盐酸调节菌体裂解液pH,并向其中加入复合蛋白酶,对菌体蛋白进行二次酶解,获得含有蛋白肽的酶解液；⑷酶解结束后,酶解液进行减压浓缩,同时灭酶,获得浓缩液；将浓缩液进行喷雾干燥。本发明方法过程简单,污染小,低成本,容易操作,过程中几乎不产生废料,是一种低成本高效的生产方法。(The invention belongs to the technical field of amino acid and protein peptide production, and discloses a production method of feeding valine and protein peptide, which comprises the following steps: activating, seed culturing and fermenting corynebacterium glutamicum producing valine to obtain fermentation liquor containing valine; adding sodium hydroxide to adjust the pH value to 11, so that the glutamic acid rod thalli in the fermentation liquor are cracked into denatured mycoprotein; adding alkaline protease to carry out primary enzymolysis on the mycoprotein; then adding hydrochloric acid to adjust the pH value of the thallus lysate, adding compound protease into the thallus lysate, and performing secondary enzymolysis on the thallus protein to obtain an enzymolysis solution containing protein peptide; after enzymolysis is finished, carrying out reduced pressure concentration on the enzymatic hydrolysate, and simultaneously carrying out enzyme deactivation to obtain a concentrated solution; and (4) carrying out spray drying on the concentrated solution. The method has the advantages of simple process, little pollution, low cost, easy operation and little waste generation in the process, and is a low-cost and high-efficiency production method.)

1. A production method of feeding valine and protein peptide is characterized by comprising the following steps: 1) preparing valine fermentation liquor, 2) preparing thallus lysate, 3) carrying out preliminary enzymolysis, 4) carrying out compound enzymolysis, 5) concentrating and inactivating enzyme, and 6) carrying out spray drying.

2. The production method according to claim 1, characterized in that it comprises the steps of:

1) preparing valine fermentation liquor: inoculating the seed liquid of the strain which produces valine by fermentation to a fermentation culture medium for culture for 60h, wherein the culture parameters are as follows: 15 percent of inoculation amount, the control temperature of 32 +/-0.5 ℃, 30 +/-10 percent of dissolved oxygen, the pH value of 7.0 +/-0.3 and the concentration of glucose of not less than 5 g/L;

2) preparing a thallus lysate: slowly adding sodium hydroxide solid into valine fermentation liquor, adjusting pH to 11, heating to 80-90 deg.C, and maintaining the temperature for 60min to obtain thallus lysate;

3) preliminary enzymolysis: adding alkaline protease into the thallus lysate for enzymolysis, controlling the temperature at 45 ℃ and the enzymolysis time at 6h to obtain a primary enzymolysis liquid;

4) compound enzymolysis: adding hydrochloric acid into the preliminary enzymolysis liquid to adjust the pH to 8.0-9.0, and adding compound protease into the preliminary enzymolysis liquid for enzymolysis to obtain enzymolysis liquid rich in amino acid and protein peptide;

5) concentrating and inactivating enzyme: after enzymolysis, carrying out reduced pressure concentration on the enzymolysis liquid, and inactivating enzyme to obtain a concentrated solution;

6) spray drying: and (4) carrying out spray drying on the concentrated solution to obtain a product with the water content of less than 5%.

3. The production method according to claim 2, wherein the fermentation medium has a composition of: on the basis of conventional components, methionine, oleic acid and choline chloride were added.

4. The method for producing the compound protease according to claim 2, wherein the compound protease is 1 or a combination of 2 or more of trypsin, papain and subtilisin.

5. The production method according to claim 2, wherein the reduced pressure concentration adopts a four-effect falling film evaporator and is carried out to 30-40% of the volume of the raw material liquid.

6. The production method according to claim 2, wherein the seed liquid of the valine-producing strain is prepared by the following steps:

subculturing the activated strain in a eggplant-shaped bottle, culturing at 32 ℃ for 12h until the strain is covered with bacterial lawn, then inoculating the strain in a seeding tank for expanded culture, controlling the temperature to be 32 +/-0.5 ℃, the dissolved oxygen to be 30 +/-10% and the pH to be 7.0 +/-0.3, culturing for 12h, carrying out microscopic examination on the strain without mixed bacteria, preparing to inoculate the strain in a fermentation medium, and starting fermentation culture; the culture medium used for subculture and expanded culture comprises: 25g/L of glucose, 20g/L of corn steep liquor dry powder, 10mL/L of soybean meal hydrochloric acid hydrolysate, 1.5g/L of dipotassium hydrogen phosphate, 0.4g/L of magnesium chloride, 10mg/L of manganese sulfate, and vitamin B₁0.3mg/L, biotin 0.2mg/L, methionine 1g/L, yeast powder 5g/L, adjusting pH to 7.0.

7. The production method according to claim 3, wherein the fermentation medium has a composition of: 80g/L of glucose, 16g/L of corn steep liquor dry powder, 20mL/L of soybean meal hydrochloric acid hydrolysate, 2.5g/L of dipotassium phosphate, 2g/L of magnesium chloride, 10mg/L of manganese sulfate, 0.7g/L of methionine, 5g/L of oleic acid and 1g/L of choline chloride, and the pH value is adjusted to 7.0.

8. The method for producing a peptide of claim 4, wherein the complex protease is a combination of three enzymes selected from the group consisting of trypsin, papain and subtilisin.

9. The production method according to claims 6 to 7, wherein the preparation process of the soybean meal hydrochloric acid hydrolysate is as follows: the hydrolysis time is 5h, the temperature is 90 ℃, the hydrochloric acid concentration is 3mol/l, the material-liquid ratio is 1 g: 5 ml.

10. The production method according to claim 8, wherein the enzymolysis reaction condition of the compound protease is as follows: the reaction temperature is controlled at 45-55 ℃ and the reaction time is 4-8 h.

Technical Field

The invention belongs to the technical field of production of amino acid and protein peptide, and particularly relates to a production method of feeding valine and protein peptide.

Background

Valine is an essential amino acid for mammals and is widely applied to the animal feed industry. Has important effects on promoting the development of mammary gland of the sow, improving the milk performance and improving the weight and the immunity of piglets in the weaning period.

Currently, most of valine for feed production is produced by fermentation of corynebacterium glutamicum, 98.5% of valine is obtained by gradually separating, extracting and refining the valine from the fermentation through processes such as membrane filtration, ion exchange, decoloration and the like, and the valine is compounded with other components to produce the feed. The valine extraction process is complex and tedious, generates a large amount of waste water to cause environmental pollution, and also has the defects that a large amount of nutrient substances (such as nucleic acid, amino acid, polypeptide, vitamin, inorganic salt and the like) wastes contained in the fermentation liquor are difficult to thoroughly treat, thereby increasing the production cost of enterprises. However, these materials can be used in feed to provide necessary nutrients and improve performance for animal growth and development.

Through searching, no patent publication related to the present invention patent is found.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a method for producing valine and protein peptide for feed, which has the advantages of simple process, little pollution, low cost, easy operation and almost no waste generation in the process, and is a method for efficiently producing valine-rich protein peptide feed at low cost.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a production method of feeding valine and protein peptide is characterized by comprising the following steps: 1) preparing valine fermentation liquor, 2) preparing thallus lysate, 3) carrying out preliminary enzymolysis, 4) carrying out compound enzymolysis, 5) concentrating and inactivating enzyme, and 6) carrying out spray drying.

Further, the production method comprises the following steps:

1) preparing valine fermentation liquor: inoculating the seed liquid of the strain which produces valine by fermentation to a fermentation culture medium for culture for 60h, wherein the culture parameters are as follows: the inoculation amount is 15 percent, the temperature is controlled to be 32 +/-0.5 ℃, the dissolved oxygen is 30 +/-10 percent, the pH is 7.0 +/-0.3, and the glucose concentration is controlled to be not lower than 5 g/L;

2) preparing a thallus lysate: slowly adding sodium hydroxide solid into valine fermentation liquor, adjusting pH to 11, heating to 80-90 deg.C, and maintaining the temperature for 60min to obtain thallus lysate;

3) preliminary enzymolysis: adding alkaline protease into the thallus lysate for enzymolysis to obtain a primary enzymolysis liquid; (the addition amount is 2000U/g dry thallus), the temperature is controlled at 45 ℃, and the enzymolysis time is 6 h;

4) compound enzymolysis: adding hydrochloric acid into the primary enzymolysis liquid to adjust the pH to 8.0-9.0, adding compound protease into the primary enzymolysis liquid, and performing second-step enzymolysis to obtain enzymolysis liquid rich in amino acid and protein peptide;

5) concentrating and inactivating enzyme: after enzymolysis, carrying out reduced pressure concentration on the enzymolysis liquid, and inactivating enzyme at the same time to obtain a concentrated solution;

6) spray drying: and (4) carrying out spray drying on the concentrated solution to obtain a product with the water content of less than 5%.

Preferably, the components of the fermentation medium are: on the basis of conventional components, methionine, oleic acid and choline chloride were added.

Preferably, the compound protease is 1 or a combination of more than 2 of trypsin, papain and subtilisin.

Preferably, a four-effect falling film evaporator is adopted during the reduced pressure concentration, and the reduced pressure concentration is carried out until the volume of the raw material liquid is 30-40%.

Preferably, the preparation process of the seed liquid of the valine-producing strain by fermentation comprises the following steps: activating strains:

subculturing the activated strain in a eggplant-shaped bottle, culturing at 32 ℃ for 12h until the strain is covered with bacterial lawn, then inoculating the strain in a seeding tank for expanded culture, controlling the temperature to be 32 +/-0.5 ℃, the dissolved oxygen to be 30 +/-10% and the pH to be 7.0 +/-0.3, culturing for 12h, carrying out microscopic examination on the strain without mixed bacteria, preparing to inoculate the strain in a fermentation medium, and starting fermentation culture; the culture medium used for subculture and expanded culture comprises: 25g/L of glucose, 20g/L of corn steep liquor dry powder, 10mL/L of soybean meal hydrochloric acid hydrolysate, 1.5g/L of dipotassium hydrogen phosphate, 0.4g/L of magnesium chloride, 10mg/L of manganese sulfate, and vitamin B₁0.3mg/L, biotin 0.2mg/L, methionine 1g/L, yeast powder 5g/L, adjusting pH to 7.0.

More preferably, the components of the fermentation medium are: 80g/L of glucose, 16g/L of corn steep liquor dry powder, 20mL/L of soybean meal hydrochloric acid hydrolysate, 2.5g/L of dipotassium phosphate, 2g/L of magnesium chloride, 10mg/L of manganese sulfate, 0.7g/L of methionine, 5g/L of oleic acid and 1g/L of choline chloride, and the pH value is adjusted to 7.0.

Most preferably, the complex protease is a combination of three enzymes, trypsin, papain and subtilisin.

Preferably, the preparation process of the soybean meal hydrochloric acid hydrolysate comprises the following steps: the hydrolysis time is 5h, the temperature is 90 ℃, the hydrochloric acid concentration is 3mol/l, the material-liquid ratio is 1 g: 5 ml.

Preferably, the enzymolysis reaction conditions of the compound protease are as follows: the reaction temperature is controlled at 45-55 ℃ and the reaction time is 4-8 h.

Moreover, the obtained feeding valine containing the protein peptide is used in the following method:

the feeding valine containing the protein peptide is compounded with other feed components and used for feeding animals.

The invention has the advantages and positive effects that:

1. the method adopts the low-ammonium culture medium for fermentation culture, reduces ammonium ions in the fermentation liquor, reduces the salt content of the product, and improves the product quality; the corynebacterium glutamicum (valine producing strain) is subjected to enzymolysis by adopting the compound protease, so that the high molecular protein in the strain is further degraded into low (small) molecular protein, polypeptide, amino acid and the like which are suitable for animals to absorb, and the utilization rate and the conversion rate of the feed are improved.

2. According to the invention, through optimizing the fermentation medium, the growth promotion factor combination mode of methionine, oleic acid and choline chloride is adopted to improve the biomass of the strain to the maximum extent, and the biomass is improved by more than 50% compared with a control group.

3. The invention adopts the sequence of alkaline lysis, preliminary enzymolysis and composite enzymolysis, the cell breakage rate is highest, the obtained low molecular weight protein (micromolecular peptide) is the most, and the variety and the content of amino acid are greatly improved.

4. All nutrient components (nitrogen source, carbon source, phosphorus source and other trace elements) in the valine fermentation liquor are almost completely converted into the feed, no other carbon source is wasted except carbon dioxide generated by respiratory metabolism of bacteria, and the conversion rate is high and the waste is less in the aspects of material input and output.

5. The method has the advantages of simple process, short process flow, little pollution, low cost, easy operation and almost no waste generated in the process. The conventional production of feeding valine requires that a product with the purity of 98.5 percent is obtained by gradually separating, extracting and refining the valine from the fermentation liquor with the mass concentration of 8-12 percent of the valine and then is compounded with other components, so that the process is complicated, the product waste loss is serious, and the energy and material cost is high.

6. The feed produced by the method has obvious effects of improving the reproductive performance of the sows and the health of piglets in the lactation period, and has high practical application value.

Drawings

FIG. 1 fermenter valine fermentation data.

FIG. 2: distributing the total protein of the fermentation liquor; m is protein Mark; sample No. 1: 1; sample No. 2: 2.

FIG. 3: distributing single enzyme enzymolysis protein; m is protein Mark; 1, total protein of fermentation liquor; 2, performing enzymolysis on the subtilisin; 3, carrying out enzymolysis by trypsin; 4, carrying out enzymolysis on the papain.

FIG. 4: influence of three enzymes on fermentation liquor by separate enzymolysis.

FIG. 5: distributing the supernatant protein after the enzymolysis of the compound enzyme.

FIG. 6: the influence of the enzymolysis of the complex enzyme on the protein content and solid residue of the supernatant.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be described more clearly and completely below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.