Enzymolysis process of amino acid fermentation thallus
阅读说明：本技术 氨基酸发酵菌体的酶解工艺 (Enzymolysis process of amino acid fermentation thallus ) 是由 赵兰坤 时夫龙 王伟涛 张婷婷 于 2019-12-13 设计创作，主要内容包括：本发明属于生物技术领域,公开了氨基酸发酵菌体的酶解工艺,包括如下步骤：步骤1)分离菌体,步骤2)高压匀浆,步骤3)离心,步骤4)超声处理,步骤5)酶解,步骤6)蒸发浓缩。本发明不采用酸或碱水解的方式,减少了环境污染的压力,避免了对设备的腐蚀,而且仅仅通过酶解的方式,条件比较温和,不会对营养物质造成破坏,而且酶解产物成分较为稳定,质量可控,利于规模化发酵体系的质量控制。(The invention belongs to the technical field of biology, and discloses an enzymolysis process of amino acid fermentation thalli, which comprises the following steps: step 1) separating thalli, step 2) high-pressure homogenizing, step 3) centrifuging, step 4) ultrasonic processing, step 5) enzymolysis, and step 6) evaporating and concentrating. The method does not adopt an acid or alkali hydrolysis mode, reduces the pressure of environmental pollution, avoids corrosion to equipment, is mild in condition and free from damage to nutrient substances only by an enzymolysis mode, has stable components of enzymolysis products and controllable quality, and is beneficial to quality control of a large-scale fermentation system.)
1. The enzymolysis process of the amino acid fermentation thallus comprises the following steps: step 1) separating thalli, step 2) high-pressure homogenizing, step 3) centrifuging, step 4) ultrasonic processing, step 5) enzymolysis, and step 6) evaporating and concentrating.
2. The enzymatic hydrolysis process according to claim 1, wherein the enzymatic hydrolysis process comprises the steps of:
step 1) separation of thalli: separating the amino acid fermentation liquor by a disc separator, and collecting thallus precipitate;
step 2) high-pressure homogenization: adding a potassium dihydrogen phosphate aqueous solution into the thallus sediment to prepare a thallus cell suspension with the concentration of 100-;
step 3) centrifugation: stopping homogenizing, centrifuging at 1000rpm for 3-5min, and collecting upper layer liquid and precipitate;
step 4), ultrasonic treatment: adding 5-10 times of purified water into the precipitate, stirring, and treating with ultrasonic wave for 20-30 min;
step 5) enzymolysis: adjusting the temperature to 45 ℃, hydrolyzing by adopting neutral protease for 6-9 h;
step 6) evaporation and concentration: heating to 90-100 ℃, inactivating enzyme for 3-5min, mixing with the upper layer liquid obtained in step 3), and finally evaporating and concentrating to obtain an extract with the dry weight part of 60-70%.
3. The enzymolysis process as claimed in claim 2, wherein the rotation speed of the disk centrifuge is 4000-5000rpm, and the centrifugation time is 3-5 min.
4. The enzymatic hydrolysis process of claim 2, wherein the concentration of the aqueous solution of monopotassium phosphate is 5-10 g/L.
5. The enzymatic hydrolysis process of claim 2, wherein said homogenization parameters are: homogenizing at 25 deg.C under 90Mpa for 3 times.
6. The enzymatic hydrolysis process of claim 2, wherein the ultrasonic waves have a frequency of 20 kHz.
7. The enzymatic hydrolysis process according to claim 2, wherein the neutral protease is added in an amount of 500u enzyme activity/g dry matter.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an enzymolysis process of amino acid fermentation thalli.
Background
The main fermentation body in the amino acid biological fermentation is microorganism, glutamic acid, lysine, threonine, leucine and the like are amino acid types with larger yield, and a large amount of microorganism thalli can be generated in the amino acid fermentation process. Taking glutamic acid fermentation as an example, about 200kg of dry bacteria can be extracted from fermentation liquor per 1 ton of glutamic acid produced, and the protein content of the bacteria is about 70 percent, so that a large amount of waste bacteria can be produced every year only by using glutamic acid which is one amino acid.
The Fufeng group is a leading enterprise for producing amino acids such as glutamic acid, threonine, lysine, tryptophan, leucine and the like and derivative products globally, and is an aircraft carrier of the first monosodium glutamate production enterprise globally, the first xanthan gum production enterprise globally and the biological fermentation industry of China. The waste thallus produced by yearly fermentation of Fufeng group is more than dozens of million tons and is increased by more than 10% every year, and how to treat thallus protein and improve added value is the focus of research in recent years.
Glutamic acid bacteria is a single-cell protein and is rich in protein and other nutrient substances. At present, the fermentation waste liquid of monosodium glutamate factories in China contains a large amount of glutamic acid thalli, and a large amount of glutamic acid thalli are directly discharged by a plurality of factories, so that serious environmental pollution is caused, and a large amount of protein is wasted. Many foreign monosodium glutamate plants recover glutamic acid bacteria as feed protein. In China, the consumption in the aspect is not much at present, but with the improvement of the living standard of people, the protein hydrolysate has a wide market. The existing hydrolysis method of glutamic acid mycoprotein mainly comprises a chemical method and an enzymatic method. Although the chemical method is simple, the reaction requirement is high, the pollution is serious, and the method is very limited, and the organic acid or alkali can damage the structure of the amino acid and the components such as vitamins, and the nutritional value is reduced. The enzymatic hydrolysis is to hydrolyze protein by using protease, and has the advantages of mild reaction conditions, low energy consumption, less side reactions and wider application. The test utilizes the compound protease method to hydrolyze the glutamic acid mycoprotein, can improve the enzymolysis efficiency and obtain the compound amino acid hydrolysate, but the optimal reaction conditions of different enzymes are different, and effective compatibility is difficult to carry out.
Regarding enzymatic hydrolysis of mycoprotein, there are many reports in the prior art, such as "optimization of enzymatic hydrolysis conditions of glutamic acid mycoprotein, zhou da wei, etc., chinese brewing" optimization of enzymatic hydrolysis conditions of glutamic acid mycoprotein by using a response surface method, taking a degree of proteolysis as a reference index, determining that an optimal enzymatic hydrolysis process is that an acid protease and β -glucanase are added in a ratio of 3: L, a total enzyme addition amount is 2% (relative to a substrate), a pH value is 4.0, a hydrolysis temperature is 50 ℃, a substrate concentration is 15%, a hydrolysis time L is 0h, under the above conditions, a degree of proteolysis reaches about 18%, study of complex enzymatic hydrolysis of monascus mycoprotein, a lugdong process, etc., chinese "complex enzymatic hydrolysis of monascus purpureus by using a neutral protease and an alkaline protease, tests determine appropriate complex enzymatic hydrolysis conditions, under the optimal conditions, a degree of complex enzymatic hydrolysis reaches 10.60%, the above documents optimize an optimal enzymatic hydrolysis process, but a degree of proteolysis is low, an optimal hydrolysis process of a neutral protease and an optimal hydrolysis process of alpha-alkaline protease, a fermentation process of a neutral protease and alkaline protease, a fermentation process of a neutral protease.
The compound amino acid obtained by enzymolysis of the thalli has complete components, also contains rich vitamins and the like, is very suitable for being used as the components of an amino acid fermentation culture medium, can replace expensive yeast extract, vitamins and other components, and reduces the fermentation cost; however, how to obtain an enzymolysis thallus product with high nutritive value, and the quality is controllable, and the stability of fermentation efficiency is maintained is a technical problem that needs to be solved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the enzymolysis process of the amino acid fermentation thalli, the process is simple to operate, an acid or alkali hydrolysis mode is not adopted, the pollution is reduced, the corrosion to equipment is avoided, the condition is mild only by an enzymolysis mode, the nutrient substances are not damaged, the components of an enzymolysis product are stable, the quality is controllable, and the quality control of a large-scale fermentation system is facilitated.
The invention is realized by the following scheme:
the enzymolysis process of the amino acid fermentation thallus comprises the following steps: step 1) separating thalli, step 2) high-pressure homogenizing, step 3) centrifuging, step 4) ultrasonic processing, step 5) enzymolysis, and step 6) evaporating and concentrating.
The enzymatic hydrolysis process according to claim 1, wherein the enzymatic hydrolysis process comprises the steps of:
step 1) separation of thalli: separating the amino acid fermentation liquor by a disc separator, and collecting thallus precipitate;
step 2) high-pressure homogenization: adding a potassium dihydrogen phosphate aqueous solution into the thallus sediment to prepare a thallus cell suspension with the concentration of 100-;
step 3) centrifugation: stopping homogenizing, centrifuging at 1000rpm for 3-5min, and collecting upper layer liquid and precipitate;
step 4), ultrasonic treatment: adding 5-10 times of purified water into the precipitate, stirring, and treating with ultrasonic wave for 20-30 min;
step 5) enzymolysis: adjusting the temperature to 45 ℃, hydrolyzing by adopting neutral protease for 6-9 h;
step 6) evaporation and concentration: heating to 90-100 ℃, inactivating enzyme for 3-5min, mixing with the upper layer liquid obtained in step 3), and finally evaporating and concentrating to obtain an extract with the dry weight part of 60-70%.
The enzymolysis process as claimed in
The enzymatic hydrolysis process of
The enzymatic hydrolysis process of
The enzymatic hydrolysis process of
The enzymatic hydrolysis process according to
Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:
the invention does not adopt an acid or alkali hydrolysis mode, reduces pollution, avoids corrosion to equipment, only adopts an enzymolysis mode, has mild conditions, does not damage nutrient substances, has stable components of enzymolysis products and controllable quality, and is beneficial to the quality control of a large-scale fermentation system.
The invention adopts high-pressure homogenate treatment, can fully break cell walls and release intracellular protein, adopts monopotassium phosphate aqueous solution as buffer solution, can improve the breaking rate, and monopotassium phosphate can also be used as a component of a fermentation medium, thereby killing two birds with one stone;
the operation pressure of the homogenization treatment is crucial to cell disruption and protein release, the disruption dynamics and the disruption process effect homogenization pressure are integrated, and multiple tests are carried out, preferably 90 MPa; the cell breakage rate is rapidly increased 2 times before homogenization, the cell breakage rate is not obviously improved after the homogenization again, but after the homogenization for the 3 rd time, macromolecular protein is also cracked into micromolecular short peptide or free amino acid under the action of the homogenization, the content of amino acid nitrogen is increased by a certain amount, the degree of proteolysis is increased, the number of times of homogenization is continuously increased, the degree of proteolysis is not obviously increased, and certain damage is caused to equipment;
the supernatant liquid is centrifuged after homogenization, the level of amino acid nitrogen contained in the supernatant liquid is higher, and the partial product is not required to be subjected to enzymolysis treatment, so that the use amount of enzyme is reduced, and the cost is reduced; the precipitate is cell fragment and macromolecular protein, still contains a certain amount of protein in the cell wall fragment moreover, and some protein can adhere on the cell wall fragment in addition, through carrying out ultrasonic treatment to the precipitate for protein and polysaccharide on the cell fragment release, and the polysaccharide is utilized as nutrient by the bacterial strain, and after ultrasonic treatment, and then adopt neutral protease to carry out enzymolysis, the use amount and the action time of enzyme reduce by a wide margin, have practiced thrift the cost.
The paste prepared by the invention and the conventionally used nitrogen source yeast paste (the dry matter content is 65-70 percent), the amino acid nitrogen content is higher, the nutrient content is more comprehensive, and the utilization of microorganisms is facilitated.
Drawings
FIG. 1: influence of high-pressure homogenization times on the cell breakage rate of thalli;
FIG. 2: the effect of the number of high pressure homogenizations on the degree of hydrolysis.
Detailed Description
Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products and methods described herein may be made and utilized without departing from the spirit, scope, and spirit of the invention. For a further understanding of the present invention, reference will now be made in detail to the following examples.
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