WO2011150775A1

WO2011150775A1 - Method for treating fermentation liquor of natamycin

Info

Publication number: WO2011150775A1
Application number: PCT/CN2011/074793
Authority: WO
Inventors: 鲁来政; 孙远功; 乔海玉
Original assignee: 郑州奇泓生物科技有限公司
Priority date: 2010-05-31
Filing date: 2011-05-27
Publication date: 2011-12-08
Also published as: CN101870716A; CN101870716B

Abstract

Disclosed is a method for treating a fermentation liquor of natamycin which is a fungus inhibitor. In this method, protease and lipase are added to the fermentation liquor for enzymolysis. Water insoluble proteins and lipid in filter cakes are hydrolyzed into water soluble amino acids as well as glycerol and fatty acids. The viscosity of the fermentation liquor is reduced considerably, fermentation liquor of autolysis is filtered and crystallized liquor of production is filtered rapidly. The amount of filter cakes is reduced and the impurities in the filter cakes are reduced correspondingly and then a product with high-purity is obtained. The producing period is reduced and the production efficiency and yield are raised, then the loss is reduced. The usage amount of an organic solvent in refining period of post product is reduced considerably due to the reduction of the amount of the filter cakes.

Description

FIELD OF THE INVENTION The present invention relates to a method for treating a fungal inhibitor natamycin fermentation broth.

BACKGROUND OF THE INVENTION The addition of food preservatives is an important means of prolonging the preservation period of foods. The preservatives currently used are mainly chemical preservatives, and common are sorbic acid (potassium), benzoic acid (sodium), calcium propionate and the like. Since chemical preservatives can remain in the human body, long-term consumption may affect human health. Therefore, the naturalization of preservatives has become a trend of anti-corrosion technology. Natural preservatives at home and abroad mainly focus on the inhibition of bacteria.

Natamycin is a natural, broad-spectrum, highly effective and safe fungal inhibitor of yeasts such as yeasts and molds. It not only inhibits fungi, but also prevents the production of mycotoxins. Natamycin is harmless to the human body, is difficult to be absorbed by the human digestive tract, and is difficult for microorganisms to develop resistance. At the same time, it is commonly used for the surface preservation of foods because of its low solubility. Natamycin is currently the only antifungal microbial preservative in the world. In 1997, the Ministry of Health officially approved natamycin as a food preservative. At present, the product has been widely used in more than 50 countries, and is widely used in the production and preservation of dairy products, meat products, fermented wine, beverages and other foods.

Natamycin is a multi-olefin macrolide antifungal agent whose molecule is an active cyclic tetraene compound containing more than 3 crystal waters, which is white (or creamy) and tasteless. The crystallized powder has a molecular formula of C ₃₃ H ₄₇ N0 ₁₃ and a molecular weight of 665. 7 3 . Natamycin is an amphoteric substance having an acidic group and a basic group in the molecule, and has an ionization constant pKa of 8.35 and 4.6, an isoelectric point of 6.5, and a melting point of 280 °C. Its structure has two configurations, an enol structure and a ketone structure, which determines its low solubility in many solvents. It has the lowest solubility at neutral pH and solubility in solvents. Increase in pH or decrease, and dissolve at pH below 3 or above 9. The degree of increase is very stable and is very stable in the pH range of most foods. It is usually a water insoluble substance under normal conditions. It can be obtained by fermentation, extraction and purification of Streptomyces.

The fermentation broth containing natamycin is obtained by fermentation of Streptomyces after fermentation. The extraction and refining methods disclosed in the foreign and domestic patent documents mostly filter the filter cake containing the natamycin and mycelium and the unused water-insoluble medium from the fermentation liquid, and then add the organic solvent. Soak the filter cake under alkaline or acidic conditions, filter to obtain the filtrate containing natamycin, adjust the pH of the filtrate to neutral, and crystallize to obtain a crude product. Since the crude product also contains a large amount of impurities such as protein and fat, it is necessary to carry out another step. Crystallization can get a qualified product.

Since the filter cake contains water-insoluble medium, which contains a large amount of protein, and some defoaming vegetable oil, the presence of these substances increases the viscosity of the filtrate, increases the resistance during filtration, and severely restricts filtration. Speed, prolonged production cycle and increased product loss. When the bacteria are autolyzed, the fermentation broth cannot be filtered or even the particles are not collected. Moreover, when the solvent is immersed in the filter cake, the water-insoluble protein and fat not only affect the filtration speed, but also partially enter the product, which seriously affects the purity of the product and reduces the filtration rate of the crystallization liquid. In order to speed up the filtration rate of the fermentation broth, it is usually necessary to add some filter aids such as diatomaceous earth, perlite, calcium carbonate and the like. Although these filter aids increase the filtration rate to some extent, they are eventually left in the filter cake, which reduces the content of the product in the filter cake and increases the post-treatment load.

SUMMARY OF THE INVENTION The present invention utilizes enzymatic chemistry to enzymatically hydrolyze a fermentation broth, which can well solve the above problems. That is, the protease and lipase are added to hydrolyze the water-insoluble protein and fat in the filter cake into water-soluble small peptide amino acids or glycerol and fatty acid, so that the viscosity of the fermentation liquid is greatly reduced, and the filtration can be carried out quickly and stably. The fermentation broth which dissolves the bacteria body is also filtered, which reduces the loss, and also avoids the use of the filter aid, reduces the amount of the filter cake, and reduces the impurity of the filter cake obtained, which can be easily crystallization by one step. A product with a purity of 4 艮 is obtained, which shortens the production cycle and improves production efficiency and product yield. The reduction of the amount of cake also greatly reduces the organic solvent in the later product refining. The amount of product used, the product crystallization solution can also be quickly filtered due to the removal of protein and fatty oil. Moreover, the enzymatic hydrolysis cost is low, the reaction conditions are mild, and the product titer is not lowered.

The present invention is achieved by a method for treating a natamycin fermentation broth, wherein the method comprises the following steps:

(1) Enzymatic hydrolysis in the natamycin fermentation broth; the enzymatic hydrolysis conditions are: protease weight-volume ratio 0.002-0.01% (enzyme weight / fermentation broth volume), temperature 35-55 ° C, pH = 3.0- 8.5, hydrolysis time 2-24h, reaction completion enzyme; lipase enzymatic hydrolysis conditions: lipase weight volume ratio of 0.002-0.01% (enzyme weight / fermentation broth volume), temperature 35-45 ° C, pH = 5.5 -10.5, hydrolysis time 2-24h;

(2) After the enzymatic hydrolysis is completed, the pH is adjusted to 5.0-10.5, filtered, and the filter cake is collected;

(3) The filter cake is soaked with decyl alcohol in an alkaline environment, filtered, and the filtrate is adjusted to have a pH of 5.0-10.5, and the crystallization is carried out;

(4) The crystallization liquid is subjected to solid-liquid separation to obtain a wet product, and after drying, a natamycin product is obtained. The present invention can also be achieved in that the protease used in the step (1) is either an acidic protease, a neutral protease, or an alkaline protease, or a combination of at least two proteases of the above proteases.

The step (1) neutral protease hydrolysis conditions are: protease dosage 0.002-0.01% (enzyme weight / fermentation broth volume), temperature 35-55 ° C, pH = 6.0-7.5, hydrolysis time 2-24h, the reaction is over Inactivated enzymes.

The step (1) alkaline protease enzymatic hydrolysis conditions are: protease dosage 0.002-0.01% (enzyme weight / fermentation broth volume), temperature 35-55 ° C, pH=7.0-8.5, hydrolysis time 2-24h, reaction end Inactivated enzymes.

The step (1) acid protease enzymatic hydrolysis conditions are: protease dosage 0.002-0.01% (enzyme weight / fermentation broth volume), temperature 35-55 ° C, pH=3.0-6.0, hydrolysis time 2-24h, reaction end Enzyme. 002-0. 01% , temperature 35-45 ° C, pH=6. 5-8 , hydrolysis time 2 - the temperature of the lipase is 0. 002-0. 01%, temperature 35-45 °C, pH=6. 8h;

The present invention can also be achieved in that the protease used in the step (1) is either an acidic protease, a neutral protease, or an alkaline protease, or a combination of at least two proteases of the above proteases.

The present invention can also be achieved in that the lipase used in the step (1) is a combination of one or more kinds of lipases for hydrolysis of vegetable oils and fats.

The present invention can also be achieved in that the protease and lipase used in the step (1) are simultaneously added with protein and lipolysis, or the protease is firstly subjected to protein enzymatic hydrolysis and then lipase is added for lipolysis, or Lipase is added for lipolysis and then protease is added for protein digestion. The present invention can also be carried out in such a manner that the pH of the step (2) and the step (3) is 6-7, preferably 6.5.

The present invention can also be achieved in this manner, the amount of the sterol in the step (3) is added according to the weight ratio of the wet cake: decyl alcohol = 1: 3-4, and the pH of the alkaline environment is 10-11;

BEST MODE FOR CARRYING OUT THE INVENTION The treatment method of the natamycin fermentation broth of the present invention will be described below by way of specific embodiments. law. Rather, the scope of the invention is to be construed as being limited by the scope of the invention. It will be apparent to those skilled in the art that various changes or modifications to the culture conditions, contents, enzymatic conditions, separation and extraction conditions in these embodiments are also within the scope of the present invention. protected range.

The invention utilizes enzymatic chemistry to enzymatically hydrolyze the fermentation broth, that is, adding protease and lipase to the fermentation broth to hydrolyze the water-insoluble proteins and fats entering the filter cake into water-soluble small peptides and amino acids, as well as glycerol and fatty acids, so that The viscosity of the fermentation broth is greatly reduced, so that the filtration energy Fast enough and stable.

The protease used in the present invention is either an acidic protease, or a neutral protease, or an alkaline protease, or a combination of one or two or three or one of the above three types of proteases or a combination of several proteases. . That is, the protease used in the present invention adopts one of three types of either alkaline protease, neutral protease, or acid protease, or any one of the three or two or three types or Several combinations.

Protease digestion conditions: Protease dosage 0.002-0.01% (enzyme weight / fermentation broth volume), temperature 35-55, pH = 3.0-8.5, hydrolysis time 2-6h, the reaction ends the enzyme.

Lipase enzymatic hydrolysis conditions: Lipase dosage 0.002-0.01% (enzyme weight / fermentation broth volume), temperature 35-45 °C, pH=5.5-10.5, hydrolysis time 2-6h

Embodiment 1:

Take nattomycin fermentation broth 20000L, first enzymatic hydrolysis, then lipase hydrolysis; (1), add alkaline protease 0.006% (w / v), control H = 7.2-7.8, hydrolysis at 40 ° C for 3h The reaction process is maintained at pH=7.2-7.8 with an alkali solution, and the enzyme is inactivated for 10 minutes after the reaction is completed;

(2), adding 95% (w/v) of lipase, controlling the temperature to be hydrolyzed at 40 ° C for 3 h; adjusting the pH to 6. 3-6.7 after completion of the hydrolysis; and killing the enzyme for 10 minutes after the reaction;

(3), rapidly filtering the fermentation broth to obtain a filter cake. According to the wet cake: sterol = 1: 4 ratio of sterol, adjust the pH to 10.2-10.7, extract 2h, filter, collect the filtrate;

(4), the filtrate was adjusted to pH ^6.4 _6.6, which crystallized on standing;

(5), filtering the crystallization liquid, collecting the product crystals, washing with water and drying to obtain a finished product. The product content is 97.5% (dry basis), and the yield is 90.1%.

Embodiment 2:

A natto fermentation broth 20000 L was taken and divided into two portions, one portion was treated in a conventional manner, and one portion was treated according to the present invention.

a. no enzymatic hydrolysis Adjust the pH of the fermentation broth to about 6.5, add 4% (w / v) diatomaceous earth filter aid, stir and hook, then filter with plate frame. Filtration time 10h;

The filter cake was collected, and decyl alcohol was added in the proportion of wet cake: decyl alcohol = 1:3, the pH was adjusted to about 10.5, extracted for 2 hours, filtered, and the filtrate was collected. Filtration time 3.5h;

Adjust the pH of the filtrate to about 6.5, and place the crystal;

The crystallization liquid was filtered, and the crude product was collected. The product content was 60.8% (dry basis), and the yield of the first two steps was 81.6%. The crude product was recrystallized once, filtered and washed to obtain a finished product, the content was 97.6% (dry basis), and the yield was 94.4%, the total yield was 77.0%.

b. Simultaneous use of protease and lipase enzymatic hydrolysis

(1) Adding alkaline protease 0.006% (w/v) and lipase to the natamycin fermentation broth

0.006% (w/v), control pH=7.3-7.7, hydrolysis at 40 °C for 4 h, the reaction process is maintained with lye pH = 7.3-7.7, and the enzyme is inactivated for 10 minutes after the reaction. The pH of the fermentation broth was adjusted to 6.3-6.7, and the fermentation broth was quickly filtered to obtain a filter cake. Filtration time 4h;

(2), collect the filter cake, according to the wet cake: sterol = 1: 3 ratio of sterol, adjust the pH to 10.2-10.7, extract 2h, filter, collect the filtrate. Filter time lh;

(3) The filtrate was adjusted to pH ^6.4 _6.6, which crystallized on standing;

(4), filtering the crystallization liquid, collecting the product crystals, washing with water and drying to obtain the finished product. The product content is 97.1% (dry basis), and the total yield is 89.7%.

Embodiment 3:

(1), adding alkaline protease 0.006% (w / v), neutral protease 0.004% (w / v) and lipase 0.006% (w / v) (plant fat hydrolysis lipase), control pH = 6.8 -7.2, hydrolysis at 45 ° C for 4 h, the reaction was maintained at pH = 6, 8-7.2 with an alkali solution, and the enzyme was inactivated for 10 minutes after completion of the reaction. The pH of the fermentation broth was adjusted to 6.3-6.7, and the fermentation broth was quickly filtered to obtain a filter cake. Filtration time 4h;

(2), collecting the filter cake, according to the wet cake: sterol = 1: 3 ratio of sterol, adjust the pH to 10.2-10.7, extract 2h, filter, collect the filtrate. Filtration time lh; (3), adjust the pH of the filtrate to about 6.3-6.7, and place the crystal;

(4), filtering the crystallization liquid, collecting the product crystals, washing with water and drying to obtain the finished product. The product content is 97.6% (dry basis), and the total yield is 90.0%.

Embodiment 4:

(1), adding alkaline protease 0.003% (w / v), neutral protease 0.005% (w / v) and lipase 0.01% (w / v) (plant fat hydrolysis lipase), control pH = 6.5 -7. ² , hydrolysis at 40 ° C for 4 h, the reaction process is maintained at pH = 6.5-7.2 with lye, and the enzyme is inactivated for 10 minutes after the reaction. The pH of the fermentation broth was adjusted to 6.3-6.7, and the fermentation broth was quickly filtered to obtain a filter cake. Filtration time 3h;

(2), collect the filter cake, according to the wet cake: sterol = 1: 4 ratio of sterol, adjust the pH to 10.3-10.7, extract 2h, filter, collect the filtrate. Filter time lh;

(3), adjust the pH of the filtrate to about 6.4-6.6, and place the crystal;

(4), filtering the crystallization liquid, collecting the product crystals, washing with water and drying to obtain the finished product. The product content is 97.8% (dry basis), and the total yield is 91.0%. Industrial Applicability The present invention can greatly reduce the viscosity of the fermentation broth, so that the filtration can be carried out quickly and stably, shortening the post-treatment cycle by 3-6 hours, shortening the production cycle, and improving the production efficiency of the enterprise.

The fermentation broth of the present invention in which the cells are autolyzed can also be filtered to reduce the loss. At the same time, the use of filter aid is avoided, the amount of filter cake is reduced, and the filter cake impurities are correspondingly reduced; the product with high purity can be obtained by simple one-step crystallization; the product loss caused by repeated treatment is reduced, The method of the invention increases the overall yield of the product by more than 16% over the control.

The invention reduces the amount of the filter cake, and the amount of the organic solvent decyl alcohol used in the later product refining is reduced by more than 50%. The product crystallization liquid can also be quickly filtered due to the removal of protein and fatty oil.

Due to the enzymatic hydrolysis method, the post-treatment time of the fermentation broth is shortened by 3-6 hours, effectively avoiding The problem of autolysis of the bacteria caused by the long storage time of the fermentation broth avoids the loss of natamycin caused by the autolysis of the bacteria, and indirectly increases the yield of natamycin.

The invention has low cost, mild reaction conditions and does not lower the product titer.

Claims

Claim

A method for treating a natamycin fermentation broth, characterized in that the method consists of the following steps:

(1) Natamycin fermentation broth is enzymatically hydrolyzed; the enzymatic hydrolysis conditions are: protease weight-volume ratio 0.002-0.01%, temperature 35-55 ° C, pH=3.0-8.5, hydrolysis time 2-24 h, end of reaction Inactivation of the enzyme; lipase enzymatic hydrolysis conditions: lipase weight-volume ratio of 0.002-0.01%, temperature 35-45 ° C, pH = 5.5-10.5, hydrolysis time 2-24h;

(2) After the enzymatic hydrolysis is completed, adjust the pH to 5.0-10.5, filter, and collect the filter cake;

(3) The filter cake is soaked with decyl alcohol in an alkaline environment, filtered, and the pH of the filtrate is adjusted to 5.0-10.5, and the crystallization is performed;

(4) The crystallization liquid is subjected to solid-liquid separation to obtain a wet product, which is dried to obtain a natamycin product.

The method for treating a natamycin fermentation broth according to claim 1, wherein the protease of the step (1) is either an acid protease, a neutral protease or an alkaline protease, or at least A combination of the two.

The method for treating a natamycin fermentation broth according to claim 2, wherein the neutral protease is hydrolyzed according to a protease dosage of 0.002-0.01%, a temperature of 35-55 ° C, and a pH of 6.0- 7.5, hydrolysis time 2-24h, the reaction ends to kill the enzyme.

The method for treating a natamycin fermentation broth according to claim 2, wherein the alkaline protease is hydrolyzed under the conditions of a protease dosage of 0.002-0.01%, a temperature of 35-55 ° C, and a pH of 7.0- 8.5, hydrolysis time 2-24h, the reaction ends to kill the enzyme.

The method for treating a natamycin fermentation broth according to claim 2, wherein the acid protease is hydrolyzed under the conditions of a protease dosage of 0.002-0.01%, a temperature of 35-55 ° C, and a pH of 3.0-6.0. , hydrolysis time 2-24h, the end of the reaction to kill the enzyme.

The method for treating a natamycin fermentation broth according to claim 1, wherein the step (1) lipase is a combination of one or more of lipases for hydrolysis of vegetable oils and fats.

The method for treating a natamycin fermentation broth according to claim 1, wherein the protease or lipase used in the step (1) is simultaneously subjected to protein and lipolysis, or the protease is first added to the protein. Enzymatic hydrolysis and then lipase for lipolysis, or first add lipase for lipolysis and then add protease for protein digestion.

The method for treating a natamycin fermentation broth according to claim 1 or 7, wherein the lipase enzymatic hydrolysis condition of the step (1) is: a lipase weight-to-volume ratio of 0.002-0.01%, a temperature of 35 -45 ° C, pH = 6.5-8, hydrolysis time 2-8h.

The method for treating a natamycin fermentation broth according to claim 1, wherein the weight ratio of the wet cake: sterol in the step (3) is 1: 3-4, and the alkaline environment pH is 10-11.

The method for treating a natamycin fermentation broth according to claim 1, wherein the step (1) comprises adding 0.006% of an alkaline protease and 0.006% of a lipase to control a pH of 7.3-7.7. , hydrolysis at 40 ° C for 4 h, the pH of the lye maintained at 7.3-7.7, after the reaction is completed, the enzyme is destroyed;

(2) adjusting the pH of the fermentation broth to 6.3-6.7, filtering the fermentation broth to obtain a filter cake, adding sterol according to the ratio of wet cake: sterol = 1:3, adjusting the pH to 10.2-10.7, extracting for 2 hours, filtering, collecting (3) adjust the pH of the filtrate to about 6.4-6.6, place the crystal; (4) filter the crystal solution, collect the product crystal, wash with water and dry to obtain the finished product.