CN107513548B - Method for preparing peptone by using fish meal squeezed liquid - Google Patents
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses a method for preparing peptone by using fish meal squeezed liquid, which comprises the following steps: (I) standing the fish meal squeezed liquid to remove fish oil; (II) concentrating the fish meal squeezed liquid obtained in the step I at low temperature under negative pressure; (III) heating the concentrate obtained in step II and adding citric acid to the concentrate; (IV) adding acid protease into the mixture obtained in the step III for enzymolysis; (V) adding disodium hydrogen phosphate and bean dregs into the mixture obtained in the step IV, adding trypsin for enzymolysis, and then inactivating; (VI) filtering the fermentation mixture obtained in the step V by using a plate-and-frame filter press; (VII) concentrating the filtrate and obtaining a finished peptone powder product by spray drying. According to the invention, the bean dregs are added for mixed enzymolysis, and the enzymolysis conditions are optimized, so that the prepared peptone has particularly excellent characteristics of a bacterial culture medium. The method has reasonable process, advanced technology and strong operability, and can be widely used for manufacturing the culture medium of bacteria.
Description
Technical Field
The invention relates to preparation of peptide, in particular to a method for preparing peptone by using fish meal squeezed liquid.
Background
It is known that fish meal is a high-protein feed material obtained by using one or more low-value fishes as raw materials, cooking, squeezing, deoiling, dehydrating, drying and crushing. The fish meal wastewater formed after squeezing is directly discharged without treatment. Because the fish meal wastewater contains solid matters such as dissolved fish protein and the like, and components such as fish oil, amino acid, trace elements and the like are extremely easy to decay, the eutrophication of offshore seawater in China is caused, the serious pollution is caused to the ecological environment, and the coastal beach and offshore culture in China is influenced.
Therefore, how to prepare peptone by using fish meal processing waste liquid is an urgent problem to be solved.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide the method for preparing the peptone by using the fish meal squeezed liquid, which has the advantages of reasonable process, advanced technology and strong operability.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for preparing peptone by using fish meal squeezed liquid is characterized by comprising the following steps: which comprises the following steps:
(I) selecting fresh, pollution-free and normal-smell fish meal squeezed liquid, putting the fish meal squeezed liquid into a standing tank, and removing fish oil through standing;
(II) carrying out negative pressure low-temperature concentration on the fish meal squeezed liquid obtained in the step I until the solid content is 16-18% in terms of wet weight, and the weight of the concentrated mixture is 10000 parts in weight;
(III) heating the concentrated solution obtained in the step (II) to 40-45 ℃, and adding 45-70 parts by weight of citric acid into the concentrated solution;
(IV) adding acid protease to the mixture obtained in the step III to 2000-4500U/mL, and carrying out enzymolysis for 2-4 hours at 40-45 ℃;
(V) adding 60-180 parts by weight of disodium hydrogen phosphate, 600-1200 parts by weight of bean dregs and trypsin to 3000-5000U/mL into the mixture obtained in the step IV, continuing enzymolysis for 8-16 hours at 40-45 ℃, and inactivating at 80-90 ℃;
(VI) filtering the fermentation mixture obtained in the step V by using a plate-and-frame filter press, and removing impurities to obtain peptone filtrate; wherein the filter cloth of the plate-and-frame filter press is 100-200 meshes, and the plate-and-frame pressure is 0.3-0.5 MPa;
(VII) feeding the filtrate obtained in the step VI into a vacuum concentrator for concentration, and obtaining a finished peptone powder product through spray drying.
In a preferred aspect of the invention, in step II, said concentration is carried out at 5-10 degrees celsius.
In a preferred aspect of the present invention, in step III, the concentrate is heated to 42 degrees Celsius, and 55 parts by weight of citric acid is added to the concentrate.
In a preferred aspect of the invention, in step IV, the mixture obtained in step III is subjected to enzymatic hydrolysis with acid protease to 3000U/mL for 3 hours at 42 ℃.
In a preferred aspect of the present invention, in step V, 125 parts by weight of disodium hydrogen phosphate and 800 parts by weight of okara are added to the mixture obtained in step IV, trypsin is added to 4000U/mL, and the enzymatic hydrolysis is continued at 42 ℃ for 12 hours.
In a preferred aspect of the invention, in step VI, the fermentation mixture obtained in step V is filtered by a plate and frame filter press to remove impurities, peptone filtrate; wherein, the filter cloth of the plate-and-frame filter press is 160 meshes, and the pressure of the plate frame is 0.4 MPa.
The method realizes the high-efficiency production of the peptone by adding the two proteases in time and step. And the bean dregs are added in the second step of enzymolysis for mixed enzymolysis, and the enzymolysis conditions are optimized, so that the prepared peptone has particularly excellent bacterial culture medium characteristics. The method has reasonable process, advanced technology and strong operability, and can be widely used for manufacturing the culture medium of bacteria, especially brevibacillus brevis.
Detailed Description
Unless otherwise indicated, "fish meal press" refers to the liquid waste obtained in the process of producing fish meal, after the fish have been comminuted, in the pressing process. After pressing, the obtained solid matter is further dried to obtain fish meal, which is used as feed additive and the like.
Unless otherwise indicated, both acid protease and trypsin in various embodiments of the present invention are available from biosciences, Inc., Yongcheng Huadao. Both enzymes were 80 ten thousand U/g dry powder. When adding, firstly, adding water into the dry powder to prepare 10 ten thousand U/mL enzyme solution, and then adding a certain volume of enzyme solution into the seafood mixture to reach the expected amount.
Comparative examples 1-5 share the product of step III of example 1, unless otherwise stated, and thus the material composition of the test of example 1 and the comparative example can be considered as parallel tests before step IV begins.
Example 1
A method for preparing peptone by using fish meal squeezed liquid comprises the following steps:
(I) selecting fresh, pollution-free and normal-smell fish meal squeezed liquid, putting the fish meal squeezed liquid into a standing tank, and removing fish oil through standing;
(II) concentrating the pressed fish meal obtained in the step I under negative pressure at 5-10 ℃ until the solid content is 16-18% by wet weight, wherein the weight of the concentrated mixture is 10000 parts by weight (the solid weight of each concentration result is different because the concentration equipment is limited, and the concentration cannot be completely concentrated at one time, so that the solid weight of each concentration result is different, and the measurement result is sampled and measured because the complete solid separation test cannot be carried out, so that the measurement result has large uncertainty and only one range value can be obtained. likewise, the concentration temperature is controlled within the range of 5-10 ℃, and the temperature of all areas of the equipment is difficult to ensure to be consistent and not to fluctuate in large-scale production;
(III) heating the concentrated solution obtained in the step (II) to 42 ℃, and adding 55 parts by weight of citric acid into the concentrated solution;
(IV) adding acid protease into the mixture obtained in the step III to 3000U/mL, and carrying out enzymolysis for 3 hours at 42 ℃;
(V) adding 125 parts by weight of disodium hydrogen phosphate and 800 parts by weight of bean dregs into the mixture obtained in the step IV, adding trypsin to 4000U/mL, continuing enzymolysis for 12 hours at 42 ℃, and inactivating at 85 ℃;
(VI) filtering the fermentation mixture obtained in the step V by using a plate-and-frame filter press, and removing impurities to obtain peptone filtrate; wherein the filter cloth of the plate-and-frame filter press is 160 meshes, and the pressure of the plate frame is 0.4 MPa;
(VII) feeding the filtrate obtained in the step VI into a vacuum concentrator for concentration, and obtaining a finished peptone powder product through spray drying. The 100 kg of the concentrated mixture finally produced 6.26 kg of finished peptone powder.
Example 2
A method for preparing peptone by using fish meal squeezed liquid comprises the following steps:
(I) selecting fresh, pollution-free and normal-smell fish meal squeezed liquid, putting the fish meal squeezed liquid into a standing tank, and removing fish oil through standing;
(II) concentrating the fish meal squeezed liquid obtained in the step I under negative pressure at 5-10 ℃ until the solid content is 16-18% by wet weight, and the weight of the concentrated mixture is 10000 parts by weight;
(III) heating the concentrated solution obtained in the step (II) to 40 ℃, and adding 45 parts by weight of citric acid into the concentrated solution;
(IV) adding acid protease to the mixture obtained in the step III to 4500U/mL, and carrying out enzymolysis for 4 hours at 40 ℃;
(V) adding 60 parts by weight of disodium hydrogen phosphate and 600 parts by weight of bean dregs into the mixture obtained in the step IV, adding trypsin to 5000U/mL, continuing enzymolysis for 16 hours at 40 ℃, and inactivating at 80 ℃;
(VI) filtering the fermentation mixture obtained in the step V by using a plate-and-frame filter press, and removing impurities to obtain peptone filtrate; wherein the filter cloth of the plate-and-frame filter press is 100 meshes, and the pressure of the plate frame is 0.3 MPa;
(VII) feeding the filtrate obtained in the step VI into a vacuum concentrator for concentration, and obtaining a finished peptone powder product through spray drying. The 100 kg of the concentrated mixture finally produced 5.88 kg of finished peptone powder.
Example 3
A method for preparing peptone by using fish meal squeezed liquid comprises the following steps:
(I) selecting fresh, pollution-free and normal-smell fish meal squeezed liquid, putting the fish meal squeezed liquid into a standing tank, and removing fish oil through standing;
(II) concentrating the fish meal squeezed liquid obtained in the step I under negative pressure at 5-10 ℃ until the solid content is 16-18% by wet weight, and the weight of the concentrated mixture is 10000 parts by weight;
(III) heating the concentrated solution obtained in the step (II) to 45 ℃, and adding 70 parts by weight of citric acid into the concentrated solution;
(IV) adding acid protease into the mixture obtained in the step III to 2000U/mL, and carrying out enzymolysis for 2 hours at the temperature of 45 ℃;
(V) adding 180 parts by weight of disodium hydrogen phosphate and 1200 parts by weight of bean dregs into the mixture obtained in the step IV, adding trypsin to 3000U/mL, continuing enzymolysis for 8 hours at 45 ℃, and inactivating at 90 ℃;
(VI) filtering the fermentation mixture obtained in the step V by using a plate-and-frame filter press, and removing impurities to obtain peptone filtrate; wherein the filter cloth of the plate-and-frame filter press is 200 meshes, and the pressure of the plate frame is 0.5 MPa;
(VII) feeding the filtrate obtained in the step VI into a vacuum concentrator for concentration, and obtaining a finished peptone powder product through spray drying. The 100 kg of the concentrated mixture finally produced 5.76 kg of finished peptone powder.
Comparative example 1
In this comparative example, 125 parts by weight of disodium hydrogenphosphate and 800 parts by weight of okara were not added in step V, but the procedure was otherwise the same as in example 1. The 100 kg of the concentrated mixture finally produced 4.51 kg of finished peptone powder.
Comparative example 2
In this comparative example, 125 parts by weight of disodium hydrogenphosphate and 800 parts by weight of bean dregs were not added in step V; the trypsin in step V was added in step IV together with the acid protease for a total of 14 hours, and the rest was the same as in example 1. The 100 kg of the concentrated mixture finally produced 4.28 kg of finished peptone powder.
Comparative example 3
In this comparative example, step V was not performed and the enzymatic time in step VI was extended to 14 hours, otherwise the same as in example 1. The 100 kg of the concentrated mixture finally produced 3.19 kg of finished peptone powder.
Comparative example 4
In this comparative example, step IV was not performed, and step V was directly performed, the rest being the same as in example 1. The 100 kg of the concentrated mixture finally produced 2.96 kg of finished peptone powder.
Comparative example 5
In this comparative example, 125 parts by weight of disodium hydrogenphosphate in step V was replaced with 78 parts by weight of sodium carbonate, and the rest was the same as in example 1. The pH of the enzymatic mixture was measured to be 7.9 after 78 parts by weight of sodium carbonate was added. In example 1, the pH of the enzyme mixture after addition of disodium hydrogen phosphate was 7.8. Both have similar pH values. The 100 kg of the concentrated mixture finally produced 4.92 kg of finished peptone powder.
Measurement of peptone molecular weight
The obtained peptone powder finished products of examples 1-3 and comparative examples 1-5 are light yellow and have better physical properties. The molecular weight distributions of the peptone powder products of examples 1-3 and comparative examples 1-5 were measured using SDS-PAGE method. The results showed that the peptones of the above groups all had a molecular weight distribution of 600-3000 Da. When the aqueous peptone powder solution described above was dropped into saturated sodium sulfate, no precipitate appeared. This indicates that the peptone products above are per se compliant with the general peptone requirements.
Second, peptone quality test
The brevibacillus brevis used in this test may be a brevibacillus brevis QSYL which is actually a brevibacillus brevis 7316, and QSYL stands for chinese character 7316. The strain is purchased from Shanghai-research Biotechnology Co., Ltd. The quality of each group of peptone products is evaluated by the following method that (1) after recovery culture, slant culture is ground into physiological saline bacterial suspension containing 10 hundred million brevibacillus QSYL; (2) preparing an aqueous solution containing 0.1% by mass of sodium chloride, about 0.015% by mass of sodium dihydrogen phosphate and 1% by mass of peptone, and subpackaging the aqueous solution into 5 ml/tube (examples 1-3 and comparative example 4 do not need to add sodium dihydrogen phosphate because the peptone contains sodium dihydrogen phosphate close to the content); (3) adding 1ml of bacterial suspension into 1 tube of peptone water, uniformly mixing, and marking as dilution 1 and simultaneously making three parallel samples; taking 1ml from a tube with the dilution of 1, adding into 1 tube of peptone water, uniformly mixing, and marking as the dilution of 2 and simultaneously making three parallel samples; and so on until 1ml bacterial suspension is diluted to the dilution 9; taking 5 to 9 serial dilutions, wherein 15 tubes are counted for each peptone, putting the peptone into an incubator at 36 +/-1 ℃ for 96 hours, observing every 12 hours, and recording the number of tubes with positive (mycelial clusters) and judging the quality of the peptone.
The results of the tests in the peptones obtained in example 1 and comparative examples 1-5 are reported in tables 1-2. Wherein 3/3 indicates that all three parallel samples were positive, 2/3 indicates that two parallel samples were positive, and so on. All positive results were summed to give an integral of the panel. The higher the score, the earlier a positive result appears.
Table 1: culture results of Brevibacillus sp.of peptone in example 1 and comparative examples 1-2
Table 2: comparative examples 3-5 Bacillus brevis culture results of peptone
As can be seen from the test results of tables 1-2 above, example 1 has the best bacterial culture performance, followed by comparative example 2. Comparative examples 1 and 5 also performed better and closer together. The worst are comparative examples 3 and 4. In comparative example 5, replacing sodium hydrogen phosphate with sodium carbonate, the culture performance of peptone was found to be decreased. Considering that each group of culture medium contains similar content of sodium dihydrogen phosphate, the sodium dihydrogen phosphate in the culture process is excluded, which shows that the sodium dihydrogen phosphate has influence on the composition of peptone except the working pH of the pH regulating enzyme, so that the peptone has better bacterial culture performance. As can be seen from the comparison of example 1 with comparative examples 1-3, the addition of disodium hydrogen phosphate and okara gives peptone with better culture properties. As can be seen from the comparison of example 1 with comparative example 4, the enzymolysis using only trypsin and okara does not result in good cultivation performance of peptone.
In summary, the experiment proves that the peptone has good bacterial culture performance by combining the bean dregs with fermentation and using the disodium hydrogen phosphate as the enzymolysis auxiliary agent.
Third, peptone dilution test
Examples 2-3 were also tested at dilutions 7-9 to account for problems and to save test materials. The results showed substantially the same results as in the test of example 1 with the dilution of 7 to 9. Examples 2-3 were all three tubes positive after 12 hours at a dilution of 7. Three tubes were positive at 24 hours at 8 dilution, 2 tubes at 12 hours and 8 dilution in example 2, and 1 tube in example 3. Example 2 was 1-tube positive at 12 hours, 2-tube positive at 24 hours, and 3-tube positive after 36 hours at dilution 9. Example 3 and example 1 performed identically at dilution 9. It can be seen that examples 2-3 have similar culture performance compared to example 1.
Claims (6)
1. A method for preparing peptone by using fish meal squeezed liquid is characterized by comprising the following steps: which comprises the following steps:
(I) selecting fresh, pollution-free and normal-smell fish meal squeezed liquid, putting the fish meal squeezed liquid into a standing tank, and removing fish oil through standing;
(II) carrying out negative pressure low-temperature concentration on the fish meal squeezed liquid obtained in the step I until the solid content is 16-18% in terms of wet weight, and the weight of the concentrated mixture is 10000 parts in weight;
(III) heating the concentrated solution obtained in the step (II) to 40-45 ℃, and adding 45-70 parts by weight of citric acid into the concentrated solution;
(IV) adding acid protease to the mixture obtained in the step III to 2000-4500U/mL, and carrying out enzymolysis for 2-4 hours at 40-45 ℃;
(V) adding 60-180 parts by weight of disodium hydrogen phosphate, 600-1200 parts by weight of bean dregs and trypsin to 3000-5000U/mL into the mixture obtained in the step IV, continuing enzymolysis for 8-16 hours at 40-45 ℃, and inactivating at 80-90 ℃;
(VI) filtering the fermentation mixture obtained in the step V by using a plate-and-frame filter press, and removing impurities to obtain peptone filtrate; wherein the filter cloth of the plate-and-frame filter press is 100-200 meshes, and the plate-and-frame pressure is 0.3-0.5 MPa;
(VII) feeding the filtrate obtained in the step VI into a vacuum concentrator for concentration, and obtaining a finished peptone powder product through spray drying.
2. The method for preparing peptone by using fish meal squeezed liquid as claimed in claim 1, characterized in that: in step II, the concentration is carried out at 5-10 ℃.
3. The method for preparing peptone by using fish meal squeezed liquid as claimed in claim 1, characterized in that: in step III, the concentrate was heated to 42 degrees celsius, and 55 parts by weight of citric acid was added to the concentrate.
4. The method for preparing peptone by using fish meal squeezed liquid as claimed in claim 1, characterized in that: in step IV, adding acid protease to the mixture obtained in step III to 3000U/mL, and performing enzymolysis at 42 ℃ for 3 hours.
5. The method for preparing peptone by using fish meal squeezed liquid as claimed in claim 1, characterized in that: in the step V, 125 parts by weight of disodium hydrogen phosphate and 800 parts by weight of bean dregs are added into the mixture obtained in the step IV, trypsin is added to 4000U/mL, and enzymolysis is continued for 12 hours at 42 ℃.
6. The method for preparing peptone by using fish meal squeezed liquid as claimed in claim 1, characterized in that: in the step VI, filtering the fermentation mixture obtained in the step V by using a plate-and-frame filter press, and removing impurities to obtain peptone filtrate; wherein, the filter cloth of the plate-and-frame filter press is 160 meshes, and the pressure of the plate frame is 0.4 MPa.
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Denomination of invention: A method for preparing peptone from fish meal pressing liquid Effective date of registration: 20211110 Granted publication date: 20210122 Pledgee: Shandong Rongcheng Rural Commercial Bank Co.,Ltd. Pledgor: RONGCHENG RIXIN AQUATIC PRODUCTS CO.,LTD. Registration number: Y2021980012232 |
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