Single-cell protein extraction and hydrolysis process

Abstract

The invention discloses a single-cell protein extraction and hydrolysis process. The invention discloses a single-cell protein extraction and hydrolysis process which comprises the following steps: S1, adding a chemical reagent into single-cell protein thalli to lyse cells to obtain a cell lysis solution; s2, adding a protein denaturation reagent into the cell lysis solution, and centrifuging to obtain a protein denaturation precipitate; s3, adding a protein dissolving reagent into the protein denaturation precipitate to obtain a protein solution; and S4, adding enzyme into the protein solution, and carrying out protein hydrolysis to obtain a hydrolysate. The method has a wide application prospect, and is expected to promote technical progress and industrial development in related fields.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and relates to a single cell protein extraction and hydrolysis process. Background Art

[0002] In the fields of bioengineering, food science and chemical engineering, single-cell protein is an important resource. Single-cell protein refers to the conversion of single-cell organisms such as microorganisms, algae or plant cells into protein-rich biomass through biotechnology means such as fermentation. As a high-protein, low-fat, easily digestible and absorbable food raw material, single-cell protein is widely used in food, feed, medicine and chemical industries. In the process of utilizing single-cell protein, how to efficiently extract and utilize the protein in it is an important research direction. Traditional extraction methods mainly use physical and chemical means, such as heating, high pressure, acid and alkali treatment, to break the cell wall and release the protein in the cell.

[0003] There are still some problems with the existing process. First, although cells can be effectively lysed by low-temperature repeated freezing and thawing and ultrasonic treatment, some cells are still difficult to completely lyse, which will lead to a low protein extraction rate. Secondly, even if protein can be converted into soluble amino acids or small peptides by protease hydrolysis, the content of free amino acids is still low, and most proteins still exist in the form of polypeptides, which will affect its application effect in food and feed. Due to the presence of cell walls, its contents are difficult to directly extract and utilize. Traditional methods often use physical crushing (such as ball milling, ultrasound) or chemical reagent treatment (such as acid and alkali treatment) to achieve cell wall breaking, but these methods often have problems such as high energy consumption, high cost, large pollution or destruction of protein biological activity. Traditional single-cell protein production often ignores the cell wall crushing process, resulting in incomplete protein extraction. Therefore, how to further improve the protein extraction rate and the content of free amino acids is a problem that needs to be solved in this field. Summary of the invention

[0004] The invention improves the release and hydrolysis efficiency of protein through cell wall breaking technology, protein purification and hydrolysis technology, thereby increasing the content of free amino acids.

[0005] Specifically, the present invention provides a single cell protein extraction and hydrolysis process, comprising the following steps:

[0006] S1, adding chemical reagents to the single-cell protein bacteria to lyse the cells to obtain a cell lysate;

[0007] S2, adding a protein denaturing agent to the cell lysate, and centrifuging to obtain a protein denatured precipitate;

[0008] S3, adding a protein solubilizing agent to the protein denatured precipitate to obtain a protein solution;

[0009] S4, adding enzyme to the protein solution to hydrolyze the protein to obtain a hydrolyzate.

[0010] In some embodiments, in the above process, in step S1, the chemical reagent is sodium dodecyl sulfate and / or Triton X-100;

[0011] Preferably, sodium dodecyl sulfate and / or Triton X-100 with a final concentration of 1-2.5 wt% is added to the single-cell protein bacteria to lyse the cells.

[0012] In some embodiments, step S1 comprises the following steps:

[0013] Sodium dodecyl sulfate and / or Triton X-100 at a final concentration of 1-2.5wt% (e.g., 1, 1.5, 2, 2.5wt%, or a range or any value between these values) is added to the single-cell protein bacteria, and stirred (by destroying the cell membrane and cell wall to release the intracellular substances including the target product (protein)) to obtain a cell lysate.

[0014] In some embodiments, in any of the above processes, in step S2, the protein denaturing agent is chloroform and / or methanol;

[0015] Preferably, chloroform and / or methanol at a final concentration of 3-8 vol% is added to the cell lysate, and the cell lysate is centrifuged (eg, at a low speed of 1000-2000 rpm for 5-10 minutes) to obtain a protein denatured precipitate.

[0016] In some embodiments, step S2 comprises the following steps:

[0017] Add chloroform and / or methanol with a final concentration of 3-8 vol% (e.g., 3, 4, 5, 6, 7, 8 vol%, or a range or any value between these values) to the cell lysate, stir to denature the protein, and centrifuge (e.g., centrifuge at a low speed of 1000-2000 rpm for 5-10 minutes) to obtain a denatured protein precipitate.

[0018] In some embodiments, in any of the above processes, in step S3, the protein solubilizing reagent is urea and / or guanidine hydrochloride;

[0019] Preferably, urea and / or guanidine hydrochloride with a final concentration of 1.5-3 wt % is added to the protein denatured precipitate to obtain a protein solution.

[0020] In some embodiments, step S3 comprises the following steps:

[0021] Urea and / or guanidine hydrochloride with a final concentration of 1.5-3 wt % (eg, 1.5, 2, 2.5, 3 wt %, or ranges or any values ​​therebetween) are added to the protein denatured precipitate, and the protein is dissolved by stirring to obtain a protein solution.

[0022] In some embodiments, in any of the above processes, in step S4, the enzyme is selected from one or more of serine carboxypeptidase, pepsin, and trypsin;

[0023] Preferably, an enzyme having a final concentration of 1-1.5 mg / mL is added to the protein solution to hydrolyze the protein to obtain a hydrolyzate.

[0024] In some embodiments, step S4 comprises the following steps:

[0025] An enzyme having a final concentration of 1-1.5 mg / mL is added to the protein solution, and the solution is stirred at a pH of 6.0-7.0 and a temperature of 30-37° C. to hydrolyze the protein to obtain a hydrolyzate.

[0026] In some embodiments, in any of the above-mentioned processes, there is also a pretreatment step before step S1: the single-cell protein bacteria fermentation broth is centrifuged (for example, at 7800-8800 rpm for 15-30 minutes), the precipitate after centrifugation is dissolved in pure water, and centrifuged again (for example, at 7800-8800 rpm for 15-30 minutes) for washing to obtain the washed precipitate, which is the single-cell protein bacteria in step S1.

[0027] In some embodiments, in any of the above processes, the following steps are further included after step S4:

[0028] S5, filtering the hydrolyzate to obtain a hydrolyzed protein supernatant;

[0029] Preferably, the filtration is performed using a filter membrane, more preferably a 0.22-0.45 μm filter membrane.

[0030] In some embodiments, step S5 comprises the following steps:

[0031] The hydrolyzate was filtered using a 0.22-0.45 μm filter membrane to remove insoluble matter and obtain a hydrolyzed protein supernatant.

[0032] In some embodiments, in any of the above processes, the following steps are further included after step S5:

[0033] S6, concentrating and drying the protein supernatant after hydrolysis to obtain a single-cell protein product rich in free amino acids;

[0034] Preferably, the concentration and drying are carried out by distillation under reduced pressure.

[0035] In some embodiments, step S6 comprises the following steps:

[0036] S6. The protein supernatant after hydrolysis is subjected to reduced pressure distillation (condition is 1-10 MPa, 12-18 hours) to remove water, thereby obtaining a single cell protein product rich in free amino acids.

[0037] In some embodiments, in the above process, the free amino acid content of the single-cell protein product rich in free amino acids is ≥62wt%, and the degree of hydrolysis (DH) is ≥84%.

[0038] In some embodiments, in any of the above processes, the single-cell protein bacteria is Saccharomyces cerevisiae and / or Yarrowia lipolytica.

[0039] The beneficial effects of the present invention are:

[0040] 1. Improve protein extraction rate: The present invention adopts surfactant cell lysis technology, which can more thoroughly lyse single-cell organisms, thereby improving protein extraction rate. Compared with the prior art, the cell lysis efficiency of the present invention is higher, which can release the protein in the cell to the maximum extent, and provide more raw materials for subsequent protein hydrolysis.

[0041] 2. Increase the content of free amino acids: In the process of protein hydrolysis, the present invention adds protease, which can convert protein into soluble amino acids or small peptides under mild conditions. Compared with the prior art, the protein hydrolysis efficiency of the present invention is higher, and more protein can be converted into free amino acids, thereby increasing the content of free amino acids. This is of great significance for improving the application effect of single-cell protein in food and feed.

[0042] 3. After enzymatic hydrolysis, part of the protein is degraded into proteins or polypeptide products with smaller molecular weight, while more is completely hydrolyzed into amino acids, thereby effectively improving the solubility of the protein.

[0043] 4. Simple process and easy operation: The process of the present invention is simple and easy to operate, does not require complicated equipment and technical requirements, and is suitable for large-scale industrial production. Compared with the prior art, the process of the present invention is simpler, can reduce production costs and improve production efficiency.

[0044] In summary, compared with the prior art, the present invention has the advantages of high protein extraction rate, high free amino acid content, simple process and easy operation, and is a more ideal single-cell protein extraction and hydrolysis process.

[0045] The present invention has a wide range of applications in the fields of bioengineering, food science, and chemical engineering. First, in the field of bioengineering, the present invention can greatly improve the protein extraction rate and the content of free amino acids in single-cell protein. This will greatly promote the application of single-cell protein in the fields of medicine and chemical industry, especially in the production of high-protein, low-fat healthy food and feed, to meet the huge market demand. Secondly, in the field of food science, the present invention provides an effective method for improving the taste and nutritional value of food. By increasing the content of free amino acids in single-cell protein, the freshness and nutritional value of food can be enhanced to meet consumers' demand for healthy food. In addition, the present invention can also provide a new source of protein for the food industry, reduce dependence on traditional protein resources, and promote the sustainable development of the food industry. Finally, in the field of chemical engineering, the present invention can be applied to the extraction and purification of various proteins. In general, the present invention has broad application prospects and is expected to promote technological progress and industrial development in related fields. DETAILED DESCRIPTION

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0047] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0048] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0049] (1) Detection method of free amino acid content

[0050] The mass percentage of free amino acids was tested according to the conventional acid hydrolysis method in the national standard GB / T 18246-2019 "Determination of Amino Acids in Feed".

[0051] (2) Determination of hydrolysis degree (%)

[0052] Degree of hydrolysis (Degree of Hydrolysis (DH) represents the degree of cleavage of protein peptide bonds during hydrolysis, and is usually expressed as a percentage. The commonly used method for determining the degree of hydrolysis is the ninhydrin method, which uses the complete hydrolyzate of the protein sample to be tested as the standard sample for ninhydrin colorimetry to determine the degree of hydrolysis of the protein:

[0053] DH=h / htot×100%

[0054] In the above formula, h is the number of millimoles of peptide bonds cleaved per gram of protein after hydrolysis (mmol / g); htot is the number of millimoles of peptide bonds in each gram of original protein (mmol / g). As long as the number of millimoles of peptide bonds cleaved after hydrolysis is measured, that is, the h value, the corresponding DH value can be calculated.

[0055] Saccharomyces cerevisiae was purchased from China Industrial Microbiological Culture Collection Center, with the collection number: CICC 1388.

[0056] Yarrowia lipolytica was purchased from China Industrial Microbiological Culture Collection Center, with the collection number: CICC 32291.

[0057] Triton X-100 is a product of Sigma-Aldrich, catalog number 92046-34-9.

[0058] Example 1

[0059] 1. Take the single-cell protein bacterial fermentation liquid of Saccharomyces cerevisiae (OD600 is 200) for pretreatment: centrifuge the bacterial liquid at 8800rpm for 15 minutes, and add pure water to dissolve the precipitate after centrifugation.

[0060] 2. The bacteria dissolved in pure water were centrifuged again at 8800 rpm for 15 minutes to wash, and the precipitate was obtained. The free amino acid content of the precipitate was detected to be 4 wt%.

[0061] 3. Add sodium dodecyl sulfate with a final concentration of 1 wt% to the washed precipitate and stir for 30 minutes to obtain a cell lysis solution.

[0062] 4. Add chloroform with a final concentration of 3 vol% to the cell lysate, stir for 1 hour to denature the protein, and centrifuge at 1000 rpm for 5 minutes to obtain the denatured protein precipitate.

[0063] 5. Add urea with a final concentration of 1.5 wt% to the denatured protein precipitate, stir for 1 hour to dissolve the protein, and obtain a high-purity protein solution.

[0064] 6. Add trypsin at a final concentration of 1 mg / mL to the high-purity protein solution, stir for 2 hours at a pH of 7.0 (adjust the pH with a 0.5 mol / L aqueous hydrochloric acid solution or a 2 mol / L aqueous sodium hydroxide solution) and a temperature of 37°C to obtain a hydrolyzate.

[0065] 7. Filter the hydrolyzate using a 0.22 μm filter membrane to remove insoluble matter and obtain the hydrolyzed protein supernatant.

[0066] 8. The protein supernatant after hydrolysis is subjected to reduced pressure distillation (condition: 10 MPa, 18 hours) to remove water, thereby obtaining a single-cell protein product rich in free amino acids.

[0067] After testing, the free amino acid content of the obtained single-cell protein product was 62wt%, and the degree of hydrolysis (DH) was 84%.

[0068] Example 2

[0069] Repeat the steps of Example 1, except that step 3 is replaced by:

[0070] 3. Add Triton X-100 with a final concentration of 2.5 wt% to the washed precipitate and stir for 2 hours to obtain a cell lysate.

[0071] After testing, the free amino acid content of the obtained single-cell protein product was 64wt%, and the degree of hydrolysis (DH) was 88%.

[0072] Example 3

[0073] Repeat the steps of Example 1, except that step 4 is replaced by:

[0074] 4. Add methanol with a final concentration of 8 vol% to the cell lysate, stir for 1 hour to denature the protein, and centrifuge at 2000 rpm for 10 minutes to obtain the denatured protein precipitate.

[0075] After testing, the free amino acid content of the obtained single-cell protein product was 63wt%, and the degree of hydrolysis (DH) was 85%.

[0076] Example 4

[0077] Repeat the steps of Example 1, except that step 5 is replaced by:

[0078] 5. Add guanidine hydrochloride with a final concentration of 3 wt% to the denatured protein precipitate, stir for 1 hour to dissolve the protein, and obtain a high-purity protein solution.

[0079] After testing, the free amino acid content of the obtained single-cell protein product was 65wt%, and the degree of hydrolysis (DH) was 87%.

[0080] Example 5

[0081] Repeat the steps of Example 1, except that step 6 is replaced by:

[0082] 6. Add serine carboxypeptidase, pepsin and trypsin at a concentration of 0.5 mg / mL to the high-purity protein solution, stir for 2 hours at a pH of 7.0 (adjust the pH with a 0.5 mol / L aqueous hydrochloric acid solution or a 2 mol / L aqueous sodium hydroxide solution) and a temperature of 37°C to obtain a hydrolyzate.

[0083] After testing, the free amino acid content of the obtained single-cell protein product was 69wt%, and the degree of hydrolysis (DH) was 89%.

[0084] Example 6

[0085] Repeat the steps of Example 1, except that steps 1 and 2 are replaced by:

[0086] 1. The single-cell protein bacterial fermentation liquid of Yarrowia lipolytica (OD600 is 200) was pretreated: the bacterial liquid was centrifuged at 8800 rpm for 15 minutes, and the precipitate after centrifugation was dissolved in pure water.

[0087] 2. The bacteria dissolved in pure water were centrifuged again at 8800 rpm for 15 minutes to wash, and the precipitate was obtained. The free amino acid content of the precipitate was detected to be 3.5 wt%.

[0088] After testing, the free amino acid content of the obtained single-cell protein product was 65wt%, and the degree of hydrolysis (DH) was 87%.

[0089] Comparative Example 1

[0090] 1. Take the single-cell protein bacterial fermentation liquid of Saccharomyces cerevisiae (OD600 is 200) for pretreatment: centrifuge the bacterial liquid at 8800 rpm for 15 minutes, and add pure water to dissolve the precipitate after centrifugation.

[0091] 2. The bacteria dissolved in pure water were centrifuged again at 8800 rpm for 15 minutes to wash, and the precipitate was obtained. The free amino acid content of the precipitate was detected to be 4 wt%.

[0092] 3. Add chloroform with a final concentration of 3 vol% to the washed precipitate, stir for 1 hour to denature the protein, and centrifuge at 1000 rpm for 5 minutes to obtain a denatured protein precipitate.

[0093] 4. Add urea with a final concentration of 1.5 wt% to the denatured protein precipitate, stir for 1 hour to dissolve the protein, and obtain a high-purity protein solution.

[0094] 5. Add trypsin at a final concentration of 1 mg / mL to the high-purity protein solution, stir for 2 hours at a pH of 7.0 (adjust the pH with a 0.5 mol / L aqueous hydrochloric acid solution or a 2 mol / L aqueous sodium hydroxide solution) and a temperature of 37°C to obtain a hydrolyzate.

[0095] 6. Filter the hydrolyzate using a 0.22 μm filter membrane to remove insoluble matter and obtain the hydrolyzed protein supernatant.

[0096] 7. The protein supernatant after hydrolysis is subjected to reduced pressure distillation (condition is 10MPa, 18 hours) to remove water to obtain a single cell protein product.

[0097] After testing, the free amino acid content of the obtained single-cell protein product was 10wt%, and the degree of hydrolysis (DH) was 18%.

[0098] Comparative Example 2

[0099] The steps of Example 1 were repeated, except that in step 4, chloroform was replaced with an equal volume of water.

[0100] After testing, the free amino acid content of the obtained single-cell protein product was 11wt%, and the degree of hydrolysis (DH) was 21%.

[0101] Comparative Example 3

[0102] The steps of Example 1 were repeated, except that in step 5, urea was replaced with an equal weight of water.

[0103] After testing, the free amino acid content of the obtained single-cell protein product was 12wt%, and the degree of hydrolysis (DH) was 24%.

[0104] Comparative Example 4

[0105] The steps of Example 1 were repeated, except that in step 6, trypsin was replaced with an equal weight of water.

[0106] After testing, the free amino acid content of the obtained single-cell protein product was 5wt%, and the degree of hydrolysis (DH) was 7%.

Claims

1. A single cell protein extraction and hydrolysis process, comprising the following steps: S1, adding chemical reagents to the single-cell protein bacteria to lyse the cells to obtain a cell lysate; S2, adding a protein denaturing agent to the cell lysate, and centrifuging to obtain a protein denatured precipitate; S3, adding a protein solubilizing agent to the protein denatured precipitate to obtain a protein solution; S4, adding enzyme to the protein solution to hydrolyze the protein to obtain a hydrolyzate.

2. The process according to claim 1, characterized in that: In step S1, the chemical reagent is sodium dodecyl sulfate and / or Triton X-100; Preferably, sodium dodecyl sulfate and / or Triton X-100 with a final concentration of 1-2.5 wt% is added to the single-cell protein bacteria to lyse the cells.

3. The process according to claim 1 or 2, characterized in that: In step S2, the protein denaturing agent is chloroform and / or methanol; Preferably, chloroform and / or methanol at a final concentration of 3-8 vol% is added to the cell lysate, and the mixture is centrifuged to obtain a protein denatured precipitate.

4. A process as claimed in any one of claims 1 to 3, characterized in that: In step S3, the protein dissolving reagent is urea and / or guanidine hydrochloride; Preferably, urea and / or guanidine hydrochloride with a final concentration of 1.5-3 wt % is added to the protein denatured precipitate to obtain a protein solution.

5. The process according to any one of claims 1 to 4, characterized in that: In step S4, the enzyme is selected from one or more of serine carboxypeptidase, pepsin, and trypsin; Preferably, an enzyme having a final concentration of 1-1.5 mg / mL is added to the protein solution to hydrolyze the protein to obtain a hydrolyzate.

6. A process as claimed in any one of claims 1 to 5, characterized in that: There is also a pretreatment step before step S1: the single-cell protein bacteria fermentation liquid is centrifuged, the precipitate after centrifugation is dissolved in pure water, and centrifuged again for washing to obtain the washed precipitate, which is the single-cell protein bacteria in step S1.

7. The process according to any one of claims 1 to 6, characterized in that: After step S4, the following steps are further performed: S5, filtering the hydrolyzate to obtain a hydrolyzed protein supernatant; Preferably, the filtration is performed using a filter membrane, more preferably a 0.22-0.45 μm filter membrane.

8. The process according to claim 7, characterized in that: After step S5, the following steps are further performed: S6, concentrating and drying the protein supernatant after hydrolysis to obtain a single-cell protein product rich in free amino acids; Preferably, the concentration and drying are carried out by distillation under reduced pressure.

9. The process according to claim 8, characterized in that: The free amino acid content of the single-cell protein product rich in free amino acids is ≥62wt%, and the hydrolysis degree is ≥84%.

10. The process according to any one of claims 1 to 9, characterized in that: The single-cell protein bacteria are Saccharomyces cerevisiae and / or Yarrowia lipolytica.