CN108586727B - A method of utilizing aqueous two-phase separation and Extraction epsilon-polylysine - Google Patents

Abstract

The invention discloses a kind of methods using aqueous two-phase separation and Extraction epsilon-polylysine, belong to extraction and separation technology field.The present invention uses organic solvent/inorganic salts double-aqueous phase system, waste water generation can either be reduced, simplify operating procedure, reduce extraction cost by providing one kind, epsilon-polylysine (ε-PL) extracting method high with extraction recovery, extracting the advantages such as finished product purity is high again, using the method separation and Extraction epsilon-polylysine, the rate of recovery may be up to 98% or more, have significant advantage compared to conventional ion exchange extracting method.

Description

A method for extracting ε-polylysine by two-phase separation

technical field

The invention relates to a method for extracting ε-polylysine by using two-phase separation and belongs to the technical field of extraction and separation.

Background technique

ε-polylysine (ε-PL) is a homotype amino acid polymer produced by extracellular secretion of microorganisms such as Streptomyces, filamentous fungi or Bacillus, which generally consists of 25-35 L-lysine monomers through It is formed by dehydration condensation of α-COOH and ε-NH2, and its molecular weight is usually 2500-4500Da.

Due to the advantages of good water solubility, strong thermal stability, and broad antibacterial spectrum, ε-PL is currently mainly used as a biological food preservative and widely used in the food industry in Japan, South Korea, Europe, America and other countries and regions. In 2014, my country also officially approved the application of ε-PL and its hydrochloride in the food fields such as fruits and vegetables, rice and flour products, meat products, condiments, beverages and baking.

In fact, ε-PL has significant complementarity with other biological food preservatives, such as nisin and natamycin, in terms of antibacterial spectrum, that is, the scope of use. ε-PL can significantly inhibit Gram-positive and Gram-negative bacteria, nisin can only significantly inhibit Gram-positive bacteria and spores, and natamycin can only have a good inhibitory effect on mold and yeast , if they are used in combination in the food industry, the antibacterial effect can be guaranteed to the greatest extent.

Therefore, the development and promotion of the application of ε-PL and its salt in the food industry is of great significance for solving the food safety problems caused by the current chemical food preservatives.

However, the high production of ε-PL has become the main factor limiting its wide application in the food industry.

The preparation of ε-PL mainly includes two parts: microbial fermentation, extraction and refining. In terms of microbial fermentation, domestic scholars have increased the fermentation level of 5L-scale ε-PL from about 10g/L to more than 30g/L (ZL201410156360.0), and the highest breakthrough is above 50g/L (ZL201510021744.6, ZL20091003033.0) . In fact, ε-PL fermentation units have reached the fermentation level of most industrial fermentation products, indicating that the cost of ε-PL fermentation is close to the acceptable level of large-scale industrial production.

In terms of extraction and purification, since the disclosure of ZL200910152931.2, ion exchange technology has been the core method of ε-PL extraction and has attracted the attention of many researchers. Based on this core operation unit, the ε-PL extraction process has also been improved. For example, CN107164417A changes the ion type of ion exchange resin from hydrogen type to ammonium type, which saves the subsequent desalination operation and simplifies the operation process; CN106380592A establishes Two-step ion exchange method, used to separate and extract ε-PL from fermentation broth with high ε-PL concentration and high impurity environment; ZL201110053004.2 uses ion exchange resin to extract ε-PL from fermentation broth, and also extracts by-products γ-polydiaminobutyric acid was isolated.

However, compared with the progress made in the fermentation level, the ε-PL post-extraction technology has not made substantial progress, mainly because the ion exchange process has not been eliminated.

As we all know, although ion exchange technology has the advantages of high product recovery rate, strong impurity removal ability and mature operation, its defects of large acid and alkali consumption and large amount of wastewater in the activation and regeneration process are still unavoidable.

Therefore, in the context of increasingly stringent environmental protection requirements, the establishment of an ion-exchange-free ε-PL extraction process is of great significance for the establishment and future development of the green ε-PL industry.

Contents of the invention

In order to overcome the shortcomings of the traditional ion exchange technology, the present invention uses an organic solvent/inorganic salt two-phase system to provide a method that can reduce waste water production, simplify operation steps, reduce extraction costs, and has high extraction recovery rate and high purity of extracted products. Advanced ε-polylysine (ε-PL) extraction method.

Technical scheme of the present invention is as follows:

The invention provides a method for separating and extracting ε-polylysine. The method is to process ε-polylysine fermentation liquid, remove bacteria in the ε-polylysine fermentation liquid, and obtain the fermentation broth clear liquid ; Treat the fermented broth so that its pH value is 4.0 to 11.0 to obtain ε-polylysine feed liquid; add inorganic salt to the ε-polylysine feed liquid, so that the mass ratio of the inorganic salt to the final concentration is 10-30%, and then add an organic solvent, so that the mass ratio of the organic solvent to a final concentration of 10-30%, to obtain a two-phase system; after the obtained two-phase system is left to stand for phase separation, the upper and lower phases are separated, and the upper and lower phases are obtained. phase and the lower phase; the obtained upper phase is processed, the organic solvent is recovered and the ε-polylysine is extracted;

The ε-polylysine fermentation broth is obtained by fermentation with ε-polylysine-producing bacteria.

In one embodiment of the present invention, the method is to treat the ε-polylysine fermentation broth by centrifugation or filtration, remove the bacteria in the ε-polylysine fermentation broth, and obtain the fermentation broth clear liquid; , hydrochloric acid or cationic resin to treat the fermentation broth clear liquid, so that its pH value is 4.0 to 11.0, and obtain the ε-polylysine feed liquid; add inorganic salt to the ε-polylysine feed liquid first, so that the quality of the inorganic salt The specific final concentration is 10-30%, fully mixed and dissolved, and then an organic solvent is added so that the mass ratio of the organic solvent is 10-30% final concentration, and after sufficient shaking or stirring, a two-phase aqueous system is obtained; the obtained The upper and lower phases are obtained by separating the upper and lower phases of the aqueous two-phase system after static phase separation; the upper phase is processed to recover the organic solvent and extract ε-polylysine.

In one embodiment of the present invention, the method is to treat the ε-polylysine fermentation broth by centrifugation or filtration, remove the bacteria in the ε-polylysine fermentation broth, and obtain the fermentation broth clear liquid; , hydrochloric acid or cationic resin to treat the fermentation broth clear liquid, so that its pH value is 9.0, and obtain ε-polylysine feed liquid; first add inorganic salt in ε-polylysine feed liquid, so that the mass ratio of inorganic salt is finally The concentration is 20%, and fully mixed and dissolved, and then add an organic solvent, so that the mass ratio of the organic solvent to a final concentration of 20%, after sufficient shaking or stirring, a two-phase system is obtained; the obtained two-phase system is left to stand After phase separation, upper and lower phases are separated to obtain an upper phase and a lower phase; the obtained upper phase is processed, the organic solvent is recovered and ε-polylysine is extracted.

In one embodiment of the present invention, the inorganic salt is one or more of NaCl, Na ₂ CO ₃ , CaCl ₂ , (NH ₄ ) ₂ SO ₄ , KH ₂ PO ₄ or K ₂ HPO ₄ .

In one embodiment of the present invention, the inorganic salt is (NH ₄ ) ₂ SO ₄ .

In one embodiment of the present invention, the organic solvent is one or more of methanol, ethanol, n-propanol, isopropanol or acetone.

In one embodiment of the present invention, the organic solvent is ethanol.

In one embodiment of the present invention, the time for standing still for phase separation is 1-3 hours.

In one embodiment of the present invention, the time for the static phase separation is 2.0 h.

In one embodiment of the present invention, the upper and lower phases are separated by natural separation or centrifugal separation of the upper and lower phases.

In one embodiment of the present invention, the processing of the upper phase is to distill the obtained upper phase, recover the organic solvent and collect the ε-polylysine concentrate for subsequent refining treatment to obtain ε-polylysine acid.

In one embodiment of the present invention, the temperature for distilling the upper phase is 40-60°C.

In one embodiment of the present invention, the temperature for distilling the upper phase is 50°C.

In one embodiment of the present invention, the subsequent refining treatment of the ε-polylysine concentrate includes decolorization, desalination and drying.

In one embodiment of the present invention, the upper phase is treated by adding an inorganic salt solution to the upper phase obtained, so that the mass ratio of the final concentration of the inorganic salt is 20-30%, and the ε-polylysine After multiple extractions, recovery of the organic solvent and extraction of ε-polylysine are carried out.

In one embodiment of the present invention, the upper phase is treated as adding an inorganic salt solution to the obtained upper phase, so that the mass ratio of the final concentration of the inorganic salt is 20%, and ε-polylysine is subjected to 3 After the second extraction, the recovery of the organic solvent and the extraction of ε-polylysine were carried out.

In one embodiment of the present invention, the method further includes processing the obtained lower phase to recover the organic solvent and the inorganic salt.

In one embodiment of the present invention, the processing of the lower phase is to distill the lower phase, recover the organic solvent and obtain the concentrated solution of the inorganic salt, concentrate and crystallize the obtained concentrated solution of the inorganic salt, and recover the inorganic salt;

Or add an organic solvent to the lower phase, filter and recover the precipitated inorganic salt to obtain a filtrate, and distill the obtained filtrate to recover the organic solvent.

In one embodiment of the present invention, the lower phase is treated as distilling the lower phase at 40-60°C, recovering the organic solvent and obtaining the concentrated solution of inorganic salts, and distilling the concentrated solution of the obtained inorganic salts at 55- Concentrate at 80°C and crystallize at 4-10°C to recover inorganic salts;

Or add an organic solvent 0.5-3 times the volume of the lower phase to the lower phase, filter and recover the precipitated inorganic salt to obtain a filtrate, and distill the obtained filtrate at 50-80° C. to recover the organic solvent.

In one embodiment of the present invention, the processing of the lower phase is to distill the lower phase at 50°C, recover the organic solvent and obtain the concentrated solution of the inorganic salt, and then carry out the process of distilling the concentrated solution of the obtained inorganic salt at 50°C. Concentrate and crystallize at 10°C to recover inorganic salts;

Or add an organic solvent 5 times the volume of the lower phase to the lower phase, filter and recover the precipitated inorganic salt to obtain a filtrate, and distill the obtained filtrate at 50° C. to recover the organic solvent.

The present invention provides ε-polylysine obtained by separating and extracting ε-polylysine by applying the above method for separating and extracting ε-polylysine.

The present invention provides the application of the above-mentioned method for separating and extracting ε-polylysine in the production of ε-polylysine.

The present invention provides the above method for separating and extracting ε-polylysine or the application of the ε-polylysine obtained by the above separation and extraction in the food field.

Beneficial effect:

(1) The present invention directly constructs an organic solvent/inorganic salt two-phase system in the feed liquid after sterilization for the separation and extraction of ε-PL. Compared with the traditional ion exchange extraction method, the generation of waste water is basically eliminated. and acid-base consumption;

(2) The organic solvent and inorganic salt used in the present invention are all reused, therefore, the implementation of the present invention will greatly reduce the extraction cost of ε-PL;

(3) The technical process of the present invention is simple, the operation time is short, the investment cost is low, it is convenient for industrial scale-up, and it has high practical application value and obvious advantages;

(4) Adopting the method of the present invention to separate and extract ε-polylysine, the recovery rate can be as high as more than 98%.

Detailed ways

Below in conjunction with embodiment, the present invention is further elaborated.

The ε-PL fermented liquid in the following examples is prepared by the following method, and the specific operation steps are:

The seed liquid of Streptomyces albus CGMCC NO.10480 was inoculated into a 5L fermenter equipped with a fermentation medium according to an inoculation amount of 6%, and the pH value of the medium was adjusted to 7.5 with ammonia water or NaOH solution to start fermentation. During the fermentation process, the temperature is controlled at about 30°C, the stirring speed is controlled at 200-800rpm, the ventilation rate is 0.5-2vvm, and the dissolved oxygen concentration is controlled at about 30%; when the pH value drops spontaneously to 5.0, ammonia water or NaOH solution controls the pH value at about 5.0 and keeps it for 10 hours; then lowers the pH value to about 3.0 and keeps it for about 24 hours, then raises the pH value to about 4.5 and keeps it until the end of fermentation. When the residual glycerin or glucose concentration in the fermentation broth drops to 10g/L, automatically add sterilized pure glycerin or 500g/L glucose solution to control the concentration in the fermentation broth at about 10g/L; When the concentration of NH ⁴⁺ -N in the fermentation broth drops to 1g/L, ammonium sulfate solution is automatically added to keep the concentration at 1g/L.

According to the above-mentioned fermentation control method, after 192 hours of fed-batch fermentation, the yield of ε-PL can reach 50g/L.

The above-mentioned Streptomyces albus CGMCC NO.10480 was disclosed in the article "Genome Shuffling and Gentamicin-Resistance to Improveε-Poly-L-Lysine Productivity of Streptomyces albulus W-156" in Appl Biochem Biotechnol magazine on June 15, 2016.

The detection methods involved in the following examples are as follows:

Recovery and purity detection method references: Kahar P., Iwata T., Hiraki J., Park E.Y., Okabe M., Enhancement ofε-polylysine production by Streptomyces albulus strain410using pH control, J.Biosci.Bioeng.91(2001) 190–194.

Example 1

1) Bacteria removal from the fermentation broth: after the fermentation broth is diluted, use a centrifuge to centrifuge the fermentation dilution to obtain the fermentation broth;

2) Adjustment of pH value and preparation of inorganic salt solution: Dissolve 10 g of NaCl (10%) in 90 mL of ε-PL fermentation broth, and use 1.0 M NaOH to adjust the pH of the mixed solution to 4.0;

3) Construction of the two-phase aqueous system: Add 10ml of ethanol (10%) to the ε-PL fermentation broth containing NaCl at pH 4.0 above, shake and mix well, then let stand at room temperature for 1.0h, and then reuse The liquid funnel removes the lower phase and collects the upper phase;

4) Separation and extraction of ε-PL from the upper phase: the upper phase was subjected to vacuum distillation and concentration at 40° C. to recover ethanol, and the ε-PL concentrated solution was collected for vacuum drying to obtain ε-PL samples.

In this example, the recovery rate of ε-PL extracted from the fermentation supernatant was 98.5%.

Example 2

1) Bacterial removal of the fermentation broth: after the fermentation broth is filtered through a plate frame, the fermentation clear liquid is obtained;

2) Adjustment of pH value and preparation of inorganic salt solution: 30g K ₂ HPO ₄ (30%) was dissolved in 90mL ε-PL fermentation broth, and 1.0M NaOH was used to adjust the pH of the mixed solution to 11.0;

3) Construction of the two-phase aqueous system: Add 30ml of acetone (30%) to the above-mentioned ε-PL fermentation broth containing K ₂ HPO ₄ at pH 11.0, shake and mix well, then let stand at room temperature for 3.0 h, then Use a separatory funnel to remove the lower phase and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: the upper phase was subjected to vacuum distillation and concentration at 60° C. to recover ethanol, and the ε-PL concentrated solution was collected for vacuum drying to obtain ε-PL samples.

In this example, the recovery rate of ε-PL extracted from the fermentation supernatant was 94.6%.

Example 3

1) Bacteria removal from the fermentation broth: after the fermentation broth is diluted, use a centrifuge to centrifuge the fermentation dilution to obtain the fermentation broth;

2) pH value adjustment and preparation of inorganic salt solution: 200g (NH ₄ ) ₂ SO ₄ (20%) was dissolved in 900mLε-PL fermentation broth, and 1.0M NaOH was used to adjust the pH of the mixed solution to 9.0;

3) Construction of the two-phase aqueous system: Add 200ml of ethanol (20%) to the above-mentioned ε-PL fermentation broth containing (NH ₄ ) ₂ SO ₄ at pH 9.0, shake and mix well, and then let it stand at room temperature for 1.5 h, then use centrifugation to remove the lower phase and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: add 900mL 20% (NH ₄ ) ₂ SO ₄ solution to the collected upper phase, oscillate and mix well, let stand at room temperature for 1.0h, and remove the lower phase by centrifugation. phase, collect phase;

5) Separating and extracting ε-PL from the upper phase: Distilling and concentrating the upper phase under reduced pressure at 50°C, recovering ethanol, and collecting the ε-PL concentrate;

6) Purification of ε-PL: After decolorizing the ε-PL concentrated solution with activated carbon, the filtrate was desalted and concentrated by nanofiltration membrane, and then vacuum-dried to obtain ε-PL samples.

In this example, the recovery rate of ε-PL extracted from the fermentation supernatant was 98.3%.

Example 4

1) Bacterial removal of the fermentation broth: after the fermentation broth is filtered through a plate frame, the fermentation clear liquid is obtained;

2) Adjustment of pH value and preparation of inorganic salt solution: 200g (NH ₄ ) ₂ SO ₄ (20%) was dissolved in 900mLε-PL fermentation broth, and 1.0M NaOH was used to adjust the pH of the mixed solution to 10.0;

3) Construction of a two-phase aqueous system: Add 200ml of n-propanol (20%) to the ε-PL fermentation broth containing (NH ₄ ) ₂ SO ₄ at pH 10.0 above, shake and mix thoroughly, and then stand at room temperature. Set aside for 2.0 h, remove the lower phase with a separatory funnel, and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: Add 900mL of 10% Na ₂ CO ₃ solution to the upper phase collected for the first time, shake and mix well, let stand at room temperature for 2.0h, and remove by centrifugation Lower phase, collect upper phase;

5) Re-separation and extraction of ε-PL from the upper phase: add 900mL 25% KH ₂ PO ₄ solution to the upper phase collected for the second time, shake and mix well, let stand at room temperature for 1.5h, and remove by centrifugation Lower phase, collect upper phase;

6) Separating and extracting ε-PL from the upper phase: Distilling and concentrating the upper phase under reduced pressure at 60° C., recovering n-propanol, and collecting the ε-PL concentrate;

7) Purification of ε-PL: After decolorizing the ε-PL concentrated solution with activated carbon, the filtrate was desalted and concentrated by nanofiltration membrane, and then vacuum-dried to obtain ε-PL sample.

In this example, the recovery rate of ε-PL extracted from the fermentation supernatant was 99.1%.

Example 5

1) Bacterial removal of the fermentation broth: after the fermentation broth is filtered through a plate frame, the fermentation clear liquid is obtained;

2) pH value adjustment and preparation of inorganic salt solution: 200g Na ₂ CO ₃ (20%) was dissolved in 900mLε-PL fermentation broth, and 1.0M NaOH was used to adjust the pH of the mixed solution to 10.0;

3) Construction of the two-phase aqueous system: Add 200ml of n-propanol (20%) to the ε-PL fermentation broth containing Na ₂ CO ₃ at pH 10.0, shake and mix well, and then stand at room temperature for 2.0 h , using a separatory funnel to remove the lower phase and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: Add 900mL of 10% Na ₂ CO ₃ solution to the upper phase collected for the first time, shake and mix well, let stand at room temperature for 2.0h, and remove by centrifugation Lower phase, collect upper phase;

5) Separation and extraction of ε-PL from the upper phase: add 900mL 25% Na ₂ CO ₃ solution to the upper phase collected for the second time, shake and mix well, then let stand at room temperature for 1.5h, and remove by centrifugation Lower phase, collect upper phase;

6) Separating and extracting ε-PL from the upper phase: Distilling and concentrating the upper phase under reduced pressure at 60° C., recovering n-propanol, and collecting the ε-PL concentrate;

7) Purification of ε-PL: After decolorizing the ε-PL concentrated solution with activated carbon, the filtrate was desalted and concentrated by nanofiltration membrane, and then vacuum-dried to obtain ε-PL sample.

In this example, the recovery rate of ε-PL extracted from the fermentation supernatant was 98.1%.

Example 6

1) Bacterial removal of the fermentation broth: after the fermentation broth is filtered through a plate frame, the fermentation clear liquid is obtained;

2) pH value adjustment and preparation of inorganic salt solution: 200g CaCl ₂ (20%) was dissolved in 900mLε-PL fermentation broth, and 1.0M NaOH was used to adjust the pH of the mixed solution to 7.0;

3) Construction of the two-phase aqueous system: Add 200ml of n-propanol (20%) to the above pH 7.0, ε-PL fermented liquid containing CaCl ₂ , shake and mix well, then let stand at room temperature for 2.0h, use Remove the lower phase from the separatory funnel and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: Add 900mL of 10% CaCl ₂ solution to the upper phase collected for the first time, shake and mix well, then let it stand at room temperature for 2.0h, and remove the lower phase by centrifugation , collect photograms;

5) Separation and extraction of ε-PL from the upper phase: add 900mL 25% CaCl ₂ solution to the upper phase collected for the second time, shake and mix well, then let stand at room temperature for 1.5h, and remove the lower phase by centrifugation , collect photograms;

6) Separating and extracting ε-PL from the upper phase: Distilling and concentrating the upper phase under reduced pressure at 60° C., recovering n-propanol, and collecting the ε-PL concentrate;

7) Purification of ε-PL: After decolorizing the ε-PL concentrated solution with activated carbon, the filtrate was desalted and concentrated by nanofiltration membrane, and then vacuum-dried to obtain ε-PL sample.

In this example, the recovery rate of ε-PL extracted from the fermentation supernatant was 97.1%.

Example 7

1) Bacterial removal of the fermentation broth: after the fermentation broth is filtered through a plate frame, the fermentation clear liquid is obtained;

2) pH value adjustment and preparation of inorganic salt solution: 200g KH ₂ PO ₄ (20%) was dissolved in 900mLε-PL fermentation broth, and 1.0M NaOH was used to adjust the pH of the mixed solution to 9.0;

3) Construction of the two-phase aqueous system: add 200ml of n-propanol (20%) to the above pH 9.0 ε-PL fermentation broth containing KH ₂ PO ₄ , shake and mix well, and then stand at room temperature for 2.0h , using a separatory funnel to remove the lower phase and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: add 900mL 10% K ₂ HPO ₄ solution to the upper phase collected for the first time, shake and mix well, let stand at room temperature for 2.0h, and remove by centrifugation Lower phase, collect upper phase;

5) Separation and extraction of ε-PL from the upper phase: add 900mL 25% K ₂ HPO ₄ solution to the upper phase collected for the second time, shake and mix well, let stand at room temperature for 1.5h, and remove by centrifugation Lower phase, collect upper phase;

6) Separating and extracting ε-PL from the upper phase: Distilling and concentrating the upper phase under reduced pressure at 60° C., recovering n-propanol, and collecting the ε-PL concentrate;

7) Purification of ε-PL: After decolorizing the ε-PL concentrated solution with activated carbon, the filtrate was desalted and concentrated by nanofiltration membrane, and then vacuum-dried to obtain ε-PL sample.

In this example, the recovery rate of ε-PL extracted from the fermentation supernatant was 98.7%.

Comparative example 1

1) Bacteria removal from the fermentation broth: after the fermentation broth is diluted, use a centrifuge to centrifuge the fermentation dilution to obtain the fermentation broth;

2) Adjustment of pH value and preparation of inorganic salt solution: Dissolve 10 g of NaCl (10%) in 90 mL of ε-PL fermentation broth, and use 1.0 M NaOH to adjust the pH of the mixed solution to 5.0;

3) Construction of the two-phase aqueous system: Add 10ml of ethanol (10%) to the ε-PL fermentation broth containing NaCl at pH 5.0 above, shake and mix well, let stand at room temperature for 1.0h, and reuse The liquid funnel removes the lower phase and collects the upper phase;

4) Separation and extraction of ε-PL from the upper phase: the upper phase was subjected to vacuum distillation and concentration at 40° C. to recover ethanol, and the ε-PL concentrated solution was collected for vacuum drying to obtain ε-PL samples.

In this comparative example, the recovery rate of ε-PL extracted from the fermentation serum was 72.8%.

Comparative example 2

1) Bacteria removal from the fermentation broth: after the fermentation broth is diluted, use a centrifuge to centrifuge the fermentation dilution to obtain the fermentation broth;

2) Adjustment of pH value and preparation of inorganic salt solution: Dissolve 10 g of NaCl (10%) in 90 mL of ε-PL fermentation broth, and use 1.0 M NaOH to adjust the pH of the mixed solution to 3.0;

3) Construction of the two-phase aqueous system: Add 10ml of ethanol (10%) to the ε-PL fermentation broth containing NaCl at pH 3.0, shake and mix well, then let stand at room temperature for 1.0h, and then reuse The liquid funnel removes the lower phase and collects the upper phase;

4) Separation and extraction of ε-PL from the upper phase: the upper phase was subjected to vacuum distillation and concentration at 40° C. to recover ethanol, and the ε-PL concentrated solution was collected for vacuum drying to obtain ε-PL samples.

In this comparative example, the recovery rate of ε-PL extracted from the fermentation serum was 81.5%.

Comparative example 3

1) Bacterial removal of the fermentation broth: after the fermentation broth is filtered through a plate frame, the fermentation clear liquid is obtained;

2) pH value adjustment and preparation of inorganic salt solution: 200g CaCl ₂ (20%) was dissolved in 900mLε-PL fermentation broth, and 1.0M NaOH was used to adjust the pH of the mixed solution to 9.0;

3) Construction of a two-phase aqueous system: Add 200ml of n-propanol (20%) to the above pH 9.0, ε-PL fermentation broth containing CaCl ₂ , shake and mix well, then let stand at room temperature for 2.0h, use Remove the lower phase from the separatory funnel and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: Add 900mL of 10% CaCl ₂ solution to the upper phase collected for the first time, shake and mix well, then let it stand at room temperature for 2.0h, and remove the lower phase by centrifugation , collect photograms;

5) Separation and extraction of ε-PL from the upper phase: add 900mL 25% CaCl ₂ solution to the upper phase collected for the second time, shake and mix well, then let stand at room temperature for 1.5h, and remove the lower phase by centrifugation , collect photograms;

6) Separating and extracting ε-PL from the upper phase: Distilling and concentrating the upper phase under reduced pressure at 60° C., recovering n-propanol, and collecting the ε-PL concentrate;

7) Purification of ε-PL: After decolorizing the ε-PL concentrated solution with activated carbon, the filtrate was desalted and concentrated by nanofiltration membrane, and then vacuum-dried to obtain ε-PL sample.

In this comparative example, the recovery rate of ε-PL extracted from the fermentation serum was 87.4%.

Comparative example 4

1) Bacterial removal of the fermentation broth: after the fermentation broth is filtered through a plate frame, the fermentation clear liquid is obtained;

2) Adjustment of pH value and preparation of inorganic salt solution: Dissolve 200g Na ₂ CO ₃ (20%) in 900mLε-PL fermentation broth, and adjust the pH of the mixed solution to 7.0 with 1.0M NaOH;

3) Construction of a two-phase aqueous system: Add 200ml of n-propanol (20%) to the ε-PL fermentation broth containing Na ₂ CO ₃ at pH 7.0, shake and mix well, and then let stand at room temperature for 2.0 hours , using a separatory funnel to remove the lower phase and collect the upper phase;

4) Separation and extraction of ε-PL from the upper phase: Add 900mL of 10% Na ₂ CO ₃ solution to the upper phase collected for the first time, shake and mix well, let stand at room temperature for 2.0h, and remove by centrifugation Lower phase, collect upper phase;

5) Separation and extraction of ε-PL from the upper phase: add 900mL 25% Na ₂ CO ₃ solution to the upper phase collected for the second time, shake and mix well, then let stand at room temperature for 1.5h, and remove by centrifugation Lower phase, collect upper phase;

6) Separating and extracting ε-PL from the upper phase: Distilling and concentrating the upper phase under reduced pressure at 60° C., recovering n-propanol, and collecting the ε-PL concentrate;

7) Purification of ε-PL: After decolorizing the ε-PL concentrated solution with activated carbon, the filtrate was desalted and concentrated by nanofiltration membrane, and then vacuum-dried to obtain ε-PL sample.

In this comparative example, the recovery rate of ε-PL extracted from the fermentation serum was 79.8%.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (9)

1. A method for separating and extracting ε-polylysine, characterized in that, the method is to process ε-polylysine fermentation broth, remove the thallus in the ε-polylysine fermentation broth, and obtain fermentation broth Clear liquid; treating the fermented liquid clear liquid so that its pH value is 4.0 to obtain ε-polylysine feed liquid; first adding NaCl to the ε-polylysine feed liquid so that the mass ratio of NaCl to a final concentration of 10% , and then add ethanol so that the mass ratio of ethanol to the final concentration is 10%, to obtain a two-phase system; after the obtained two-phase system is left to stand for phase separation, the upper and lower phases are separated to obtain an upper phase and a lower phase; the obtained The upper phase is processed, the organic solvent is recovered and ε-polylysine is extracted; Alternatively, the method is to process the ε-polylysine fermentation liquid, remove the bacterium in the ε-polylysine fermentation liquid, and obtain the fermentation broth liquid; treat the fermentation liquid liquid so that its pH value is 11.0, and obtain ε-polylysine feed solution; first add K ₂ HPO ₄ to the ε-polylysine feed solution, so that the mass ratio of K ₂ HPO ₄ to a final concentration of 30%, and then add acetone, so that the mass ratio of acetone The final concentration is 30%, and the two-phase aqueous system is obtained; the upper and lower phases are separated after the obtained two-phase aqueous system is statically separated to obtain the upper phase and the lower phase; the obtained upper phase is processed, the organic solvent is recovered and the ε - polylysine; Alternatively, the method is to process the ε-polylysine fermentation liquid, remove the bacterium in the ε-polylysine fermentation liquid, and obtain the clear liquid of the fermented liquid; treat the clear liquid of the fermented liquid so that its pH value is 10.0, and obtain ε-polylysine feed solution; add (NH ₄ ) ₂ SO ₄ or Na ₂ CO ₃ to the ε-polylysine feed solution first, so that the mass ratio of (NH ₄ ) ₂ SO ₄ or Na ₂ CO ₃ Final concentration is 20%, then add n-propanol, make the mass ratio final concentration of n-propanol be 20%, obtain two-phase system; After the two-phase system obtained is left to stand for phase separation, upper and lower phases are separated to obtain The upper phase and the lower phase; the upper phase obtained is processed, the organic solvent is recovered and ε-polylysine is extracted; Alternatively, the method is to process the ε-polylysine fermentation broth, remove the bacterium in the ε-polylysine fermentation broth, and obtain the fermentation broth clear liquid; treat the fermentation broth clear liquid so that its pH value is 7.0, and obtain ε-polylysine feed solution; add CaCl ₂ to the ε-polylysine feed solution first, so that the mass ratio of CaCl ₂ to a final concentration of 20%, and then add n-propanol, so that the mass ratio of n-propanol The final concentration is 20%, and the two-phase aqueous system is obtained; the upper and lower phases are separated after the obtained two-phase aqueous system is statically separated to obtain the upper phase and the lower phase; the obtained upper phase is processed, the organic solvent is recovered and the ε - polylysine; Alternatively, the method is to process the ε-polylysine fermentation broth, remove the bacteria in the ε-polylysine fermentation broth, and obtain the fermentation broth clear liquid; treat the fermentation broth clear liquid so that its pH value is 9.0, and obtain ε-polylysine feed solution; first add KH ₂ PO ₄ to the ε-polylysine feed solution, so that the mass ratio of KH ₂ PO ₄ to a final concentration of 20%, and then add n-propanol to make n-propanol The mass ratio of the alcohol to the final concentration is 20%, to obtain a two-phase aqueous system; after the obtained two-phase aqueous system is left to stand for phase separation, the upper and lower phases are separated to obtain an upper phase and a lower phase; the obtained upper phase is processed, and the organic phase is recovered. Solvent and extract ε-polylysine; The ε-polylysine fermentation broth is obtained by fermentation with ε-polylysine-producing bacteria. 2. a kind of method for separating and extracting ε-polylysine as claimed in claim 1, is characterized in that, described method is to process ε-polylysine fermented liquid by centrifugation or filtration, removes ε-polylysine Sour the bacteria in the fermentation broth to obtain the fermentation broth clear liquid; treat the fermentation liquid clear liquid with sulfuric acid, hydrochloric acid or cationic resin, so that its pH value is 4.0, and obtain the ε-polylysine feed liquid; in the ε-polylysine First add NaCl to the acid feed solution so that the mass ratio of NaCl to a final concentration of 10%, and fully mix and dissolve, and then add ethanol so that the mass ratio of ethanol to a final concentration of 10%, fully shake or stir to obtain dihydrate Phase system; the obtained two-phase system is left to stand for phase separation, and then the upper and lower phases are separated to obtain the upper phase and the lower phase; the obtained upper phase is processed, the organic solvent is recovered and ε-polylysine is extracted; Alternatively, the method is to process the ε-polylysine fermentation broth by centrifugation or filtration, remove the bacterium in the ε-polylysine fermentation broth, and obtain the serum of the fermentation broth; treat the fermentation broth with sulfuric acid, hydrochloric acid or cationic resin Clear solution, so that its pH value is 11.0, to obtain ε-polylysine feed solution; first add K ₂ HPO ₄ to ε-polylysine feed solution, so that the mass ratio of K ₂ HPO ₄ final concentration is 30% , and fully mixed and dissolved, and then add acetone, so that the mass ratio of acetone to a final concentration of 30%, fully shaken or stirred, to obtain a two-phase system; the obtained two-phase system is statically separated and then the upper and lower phases are separated. Separation to obtain an upper phase and a lower phase; process the obtained upper phase, recover the organic solvent and extract ε-polylysine; Alternatively, the method is to process the ε-polylysine fermentation broth by centrifugation or filtration, remove the bacterium in the ε-polylysine fermentation broth, and obtain the serum of the fermentation broth; treat the fermentation broth with sulfuric acid, hydrochloric acid or cationic resin Clear solution, so that its pH value is 10.0, to obtain ε-polylysine feed solution; first add (NH ₄ ) ₂ SO ₄ or Na ₂ CO ₃ to ε-polylysine feed solution, so that (NH ₄ ) The final mass ratio of ₂ SO ₄ or Na ₂ CO ₃ concentration is 20%, and fully mixed and dissolved, and then add n-propanol, so that the mass ratio final concentration of n-propanol is 20%, after fully shaking or stirring, get Aqueous two-phase system; the obtained two-phase aqueous system is left to stand for phase separation, and the upper and lower phases are separated to obtain an upper phase and a lower phase; the obtained upper phase is processed, the organic solvent is recovered and ε-polylysine is extracted; Alternatively, the method is to process the ε-polylysine fermentation broth by centrifugation or filtration, remove the bacterium in the ε-polylysine fermentation broth, and obtain the serum of the fermentation broth; treat the fermentation broth with sulfuric acid, hydrochloric acid or cationic resin Clear solution, so that its pH value is 7.0, to obtain ε-polylysine feed solution; add CaCl ₂ to the ε-polylysine feed solution first, so that the mass ratio of CaCl ₂ to a final concentration of 20%, and fully mix and dissolve, and then add n-propanol, so that the final mass ratio of n-propanol is 20%. After fully shaking or stirring, a two-phase system is obtained; the obtained two-phase system is left to stand for phase separation, and then the upper and lower phases are carried out. Separation to obtain an upper phase and a lower phase; process the obtained upper phase, recover the organic solvent and extract ε-polylysine; Alternatively, the method is to process the ε-polylysine fermentation broth by centrifugation or filtration, remove the bacterium in the ε-polylysine fermentation broth, and obtain the serum of the fermentation broth; treat the fermentation broth with sulfuric acid, hydrochloric acid or cationic resin Clear solution, so that its pH value is 9.0, to obtain ε-polylysine feed solution; first add KH ₂ PO ₄ to ε-polylysine feed solution, so that the final mass ratio of KH ₂ PO ₄ concentration is 20% , and fully mixed and dissolved, and then add n-propanol, so that the mass ratio of n-propanol to a final concentration of 20%, fully shake or stir to obtain a two-phase system; the obtained two-phase system is left to separate phases Afterwards, upper and lower phases are separated to obtain an upper phase and a lower phase; the obtained upper phase is processed, the organic solvent is recovered and ε-polylysine is extracted. 3. A method for separating and extracting ε-polylysine as claimed in claim 1 or 2, characterized in that the time for standing still for phase separation is 1-3h. 4. A kind of method for separating and extracting ε-polylysine as claimed in claim 1, characterized in that, the upper phase is processed as distilling the obtained upper phase, reclaiming the organic solvent and collecting ε-polylysine. The lysine concentrate is subjected to subsequent refining treatment to obtain ε-polylysine. 5. a kind of method for separating and extracting ε-polylysine as claimed in claim 2, is characterized in that, described upper phase is processed as the upper phase that obtains is distilled, reclaims organic solvent and collects ε-polylysine The lysine concentrate is subjected to subsequent refining treatment to obtain ε-polylysine. 6. a kind of method for separating and extracting ε-polylysine as claimed in claim 3, is characterized in that, described upper phase is processed as the upper phase that obtains is distilled, reclaims organic solvent and collects ε-polylysine The lysine concentrate is subjected to subsequent refining treatment to obtain ε-polylysine. 7. A method for separating and extracting ε-polylysine as claimed in claim 4, characterized in that the subsequent refining treatment of the ε-polylysine concentrate comprises decolorization, desalting and drying. 8. A method for separating and extracting ε-polylysine as claimed in claim 5, characterized in that the subsequent refining treatment of the ε-polylysine concentrate comprises decolorization, desalting and drying. 9. A method for separating and extracting ε-polylysine as claimed in claim 6, characterized in that the subsequent refining treatment of the ε-polylysine concentrate comprises decolorization, desalting and drying.