CN111440167A

CN111440167A - Method for extracting pyrroloquinoline quinone from fermentation liquor

Info

Publication number: CN111440167A
Application number: CN202010410870.1A
Authority: CN
Inventors: 孙荣; 刘建民; 任明; 张雷
Original assignee: Shandong Huishilai Biotechnology Co ltd
Current assignee: Shandong Huishilai Biotechnology Co ltd
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2020-07-24

Abstract

A method for extracting pyrroloquinoline quinone from fermentation liquor is mainly obtained by the following steps of pretreating the fermentation liquor to obtain filtrate, adjusting the pH value of the filtrate to 2.5-3.5 by using acid, adding an adsorbent, and carrying out primary filtration to obtain filtrate; adjusting the pH value of the filtrate to 7.5-8, adding an adsorbent, and carrying out secondary filtration to obtain a filtrate; and (3) carrying out ultrafiltration and nanofiltration on the filtrate to obtain nanofiltration concentrated solution, carrying out reduced pressure concentration on the nanofiltration concentrated solution, adjusting the pH value to 3-3.5, and carrying out low-temperature crystallization to obtain the product. The purity of the finished product PQQ is more than or equal to 98 percent, the total yield is more than or equal to 85 percent, and the extraction process adopts multi-stage membrane equipment, so that the extraction operation is simpler, the automatic operation is easier, the equipment maintenance cost is reduced, the use amount of organic solvents in the production is reduced, and the production cost of enterprises is reduced.

Description

Method for extracting pyrroloquinoline quinone from fermentation liquor

Technical Field

The invention relates to the technical field of biological fermentation engineering, in particular to a method for extracting pyrroloquinoline quinone from fermentation liquor.

Background

Pyrroloquinoline quinone (PQQ) is a water-soluble, episomal compound, a third oxidoreductase coenzyme found after pyridine nucleotides (NAD, NADP) and riboflavin (FMN, FAD). PQQ is widely present in organisms, has the functions of stimulating the growth of nerve factors, eliminating free radicals and the like, and has important development prospects in the fields of foods and medical health-care products. PQQ was primarily produced by chemical synthesis in the early days, but the synthesis has the disadvantages of complicated steps, high cost, high pollution, etc., and is gradually being replaced by microbial fermentation.

The main strains in nature capable of producing PQQ by fermentation are Methylobacterium (Methylobacterium), Klebsiella (Klebsiella), Microbacterium mycelii (Hyphomicrobium), Gluconobacter (Gluconobacter), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas) and the like, and the yield of PQQ can reach 2 g/L after optimized fermentation conditions are disclosed in patent CN 103224965A.

The extraction research of PQQ in fermentation liquor is numerous, the extraction process is different, patent CN1334084A discloses a method for extracting PQQ in fermentation liquor, the technical route is fermentation liquor → centrifugation → supernatant → FP L C-Q column → adsorption → elution → crystallization → product, the purity of PQQ is more than 90%, the yield is 56%, patent CN1329004A discloses a method for extracting PQQ in fermentation liquor, the main technical route is fermentation liquor → centrifugation → supernatant → anion exchange adsorption column → elution → crystallization → Seppak C18 reverse adsorption column → elution → crystallization → product, the recovery rate of PQQ product is improved by about 30%, the cost is reduced by 30-40. patent CN107056782A discloses a method for separating and purifying PQQ in fermentation liquor, the main technical route is fermentation liquor → supernatant → macroporous resin → adsorption → hydrophilic silica gel → alkaline solution → acid precipitation → product, the purity of PQQ product is increased by two times of salting out and salting out, the operation → separation of the resin → the crude product, the process of the product is high purity of the product, the product obtained by salting out → the salting out → adsorption → the resin → the process of the product, the process of the product is high purity of the product, the product obtained by salting out → the resin → the process of the product, the product is high purity of the product, the product obtained by the salting out → the resin → the process of the resin → the process of the crude product, the crude product is high purity of the crude product, the crude product obtained by the crude product, the crude product is increased by the crude product, the secondary salting out → the secondary operation of the crude product, the crude product.

Disclosure of Invention

In order to solve the technical problems, the invention provides the method for extracting pyrroloquinoline quinone from the fermentation liquor, and the method for extracting PQQ is simple in process and more suitable for industrial operation.

A method for extracting pyrroloquinoline quinone from fermentation liquor is mainly obtained by the following steps:

1. pretreating fermentation liquor to obtain filtrate a;

2. adjusting the pH value of the filtrate a obtained in the step 1 to 2.5-3.5 by using acid, adding an adsorbent, and carrying out primary filtration to obtain a filtrate b;

3. adjusting the pH value of the filtrate b obtained in the step 2 to 7.5-8, adding an adsorbent, and carrying out secondary filtration to obtain a filtrate c;

4. respectively carrying out ultrafiltration and nanofiltration on the filtrate c in the step 3 to obtain nanofiltration concentrated solution d,

5. and (4) concentrating the nanofiltration concentrated solution d in the step (4) under reduced pressure, adjusting the pH to 3-3.5, crystallizing at low temperature, and the like to obtain the product.

In the method, the fermentation medium in the step 1 is fermentation liquor taking methanol as a sole carbon source;

in the above method, the fermentation liquid in step 1 is specifically: producing a fermentation broth by using hyphomicrobiumdentificans (Hyphomicrobiumdentificans) as a production strain;

in the method, the acid for adjusting the pH value in the step 1 is as follows: one or more of hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid, and preferably 10% dilute hydrochloric acid solution;

in the above method, the pretreatment of the fermentation liquid in step 1 is: and (3) carrying out conventional solid-liquid separation, and collecting filtrate, wherein the solid-liquid separation mode is as follows: centrifuging, plate-frame filtering or ceramic membrane filtering, wherein the preferred centrifugation is centrifugal filtering, and the further preferred centrifugation condition is 8000-10000r/min, and centrifuging for 30 min;

in the above method, the adsorbent in step 2 is: one or more of diatomite and activated carbon, wherein the preferable addition amount of the diatomite is 1-2%, the addition amount of the activated carbon is 2-3%, the treatment time is 1.5-2.5h, and the treatment temperature is 10-20 ℃;

in the above method, the first filtering in step 2 is: one of plate-frame filtration and centrifugal separation;

in the above method, the adsorbent in step 3 is: one or more of diatomite and activated carbon, wherein the preferable addition amount of the diatomite is 2-3%, the addition amount of the activated carbon is 3-5%, the treatment time is 1-2h, and the treatment temperature is 10-20 ℃;

in the above method, the second filtering in step 3 is: filtering by a ceramic membrane, wherein the preferred pore diameter of the ceramic membrane is 30-40 nm;

in the method, the ultrafiltration in the step 4 is carried out, the interception flow of the adopted ultrafiltration membrane is 500-1000Da, the filtrate is collected, 1 volume of the ultrafiltration membrane is washed, and the loss of PQQ is controlled to be less than 5%;

in the method, the interception flow of the nanofiltration membrane adopted in the nanofiltration in the step 4 is 200-300Da, the intercepted liquid is collected and concentrated until the content of PQQ is 4-6 g/L;

in the method, the temperature of the material is controlled to be 20-30 ℃ by decompressing and concentrating in the step 5, and the material is concentrated until the content of PQQ is 10-20 g/L;

in the method, the crystallization temperature in the step 5 is 0-4 ℃, and the crystallization time is 24-48 h.

The preparation method and the obtained product have the following advantages and beneficial effects:

the method adopts multistage membrane separation and purification to PQQ fermentation liquor, the purity of the final product PQQ is more than or equal to 98 percent, the total yield is more than or equal to 85 percent, and multistage membrane equipment is adopted in the extraction process, so that the extraction operation is simpler, the automatic operation is easier, the equipment maintenance cost is reduced, the use amount of organic solvents in production is reduced, and the sewage treatment cost of enterprises is reduced.

The specific implementation mode is as follows:

the following further describes the embodiments of the present invention with reference to examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

5L fermentation liquor with the PQQ content of 10g, centrifuging to collect filtrate 4.91L, adjusting the pH value to 3 by using 10% hydrochloric acid, adding 50g of diatomite, stirring for 1.5h at 15 ℃, filtering to collect filtrate 5L, adjusting the pH value to 8 by using 10% NaOH solution, adding 250g of activated carbon, stirring for 2h at 15 ℃, filtering by using a ceramic membrane with the pore diameter of 30-40nm, collecting filtrate 8L, treating the filtrate by using an ultrafiltration membrane with the cut-off quantity of 1000Da to obtain filtrate 16L, continuously treating the filtrate by using a nanofiltration membrane with the cut-off molecular weight of 200Da, concentrating to the PQQ content of 6 g/L, collecting concentrated solution 1.5L, continuously concentrating the concentrated solution under reduced pressure, controlling the material temperature to 20 ℃, concentrating to the PQQ content of 20 g/L, adjusting the pH value to 3.5 by using 10% dilute hydrochloric acid, crystallizing for 36h at 2 ℃ to obtain the PQQ product with the purity of 98.5%, and the.

Example 2

5L fermentation liquor with the PQQ content of 10g, centrifuging to collect filtrate 4.93L, adjusting the pH value to 3.5 by using 10% hydrochloric acid, adding 100g of diatomite, stirring at 15 ℃ for 1.5h, filtering to collect filtrate 5L, adjusting the pH value to 7.5 by using 10% NaOH solution, adding 150g of active carbon, stirring at 15 ℃ for 1h, filtering by using a ceramic membrane with the pore diameter of 30-40nm, collecting filtrate 8L, treating the filtrate by using an ultrafiltration membrane with the cut-off value of 800Da to obtain filtrate 16L, continuously treating the filtrate by using a nanofiltration membrane with the molecular weight cutoff of 300Da, concentrating to the PQQ content of 5 g/L, collecting concentrated solution 2L, continuously concentrating the concentrated solution under reduced pressure, controlling the material temperature to 20 ℃, concentrating to the PQQ content of 10 g/L, adjusting the pH value to 3 by using 10% dilute hydrochloric acid, crystallizing at 4 ℃ for 24h to obtain the PQQ product with the purity of 98.6%, and the.

Example 3

5L fermentation liquor with the PQQ content of 10g, centrifuging to collect filtrate 4.8L, adjusting the pH value to 3.3 by using 10% hydrochloric acid, adding 80g of diatomite, stirring for 1.5h at 15 ℃, filtering to collect filtrate 5L, adjusting the pH value to 7.5 by using 10% NaOH solution, adding 200g of active carbon, stirring for 1.5h at 15 ℃, filtering by using a ceramic membrane with the pore diameter of 30-40nm, collecting filtrate 8L, treating the filtrate by using an ultrafiltration membrane with the cut-off value of 900Da to obtain filtrate 16L, continuously treating the filtrate by using a nanofiltration membrane with the molecular weight cutoff of 250Da, concentrating to the PQQ content of 5.5 g/L, collecting concentrated solution 1.8L, continuously concentrating the concentrated solution under reduced pressure, controlling the material temperature to 25 ℃, concentrating to the PQQ content of 15 g/L, adjusting the pH value to 3 by using 10% dilute hydrochloric acid, crystallizing at 4 ℃ for 48h to obtain the PQQ product with the purity of 98.9% and the extraction yield of 87.

Claims

1. A method for extracting pyrroloquinoline quinone from fermentation liquor is characterized by comprising the following steps:

(1) pretreating fermentation liquor to obtain filtrate a;

(2) adjusting the pH value of the filtrate a obtained in the step (1) to 2.5-3.5 by using acid, adding an adsorbent, and carrying out primary filtration to obtain a filtrate b;

(3) adjusting the pH value of the filtrate b obtained in the step (2) to 7.5-8, adding an adsorbent, and carrying out secondary filtration to obtain a filtrate c;

(4) respectively carrying out ultrafiltration and nanofiltration on the filtrate c in the step (3) to obtain nanofiltration concentrated solution d;

(5) and (4) concentrating the nanofiltration concentrated solution d in the step (4) under reduced pressure, adjusting the pH to 3-3.5, crystallizing at low temperature, and the like to obtain the product.

2. The method for extracting pyrroloquinoline quinone from fermentation broth according to claim 1, wherein the pretreatment of the fermentation broth in step (1) is: and (3) carrying out conventional solid-liquid separation, and collecting filtrate, wherein the solid-liquid separation mode is as follows: centrifugation, plate-frame filtration and ceramic membrane filtration.

3. The method for extracting pyrroloquinoline quinone from a fermentation broth as claimed in claim 1, wherein the adsorbent in step (2) is: one or more of diatomite and activated carbon, wherein the preferable addition amount of the diatomite is 1-2%, the addition amount of the activated carbon is 2-3%, and the first filtration is as follows: plate-frame filtration and centrifugal separation.

4. The method for extracting pyrroloquinoline quinone from a fermentation broth as claimed in claim 1, wherein the adsorbent in step (3) is: one or more of diatomite and activated carbon, wherein the preferable addition amount of the diatomite is 2-3%, the addition amount of the activated carbon is 3-5%, and the second filtration is as follows: ceramic membrane filtration, preferably with a pore size of 30-40 nm.

5. The method for extracting pyrroloquinoline quinone from fermentation broth as claimed in claim 1, wherein the pore size of the ultrafiltration membrane in step (4) is 500-1000Da, and the pore size of the nanofiltration membrane is 200-300 Da.

6. The method for extracting pyrroloquinoline quinone from a fermentation broth according to claim 1, wherein in the step (5), the material temperature is controlled to be 20-30 ℃, the crystallization temperature is controlled to be 0-4 ℃, and the crystallization time is controlled to be 24-48 h.