CN104946709A - Method for fermenting and producing natamycin - Google Patents

Abstract

The invention discloses a method for fermenting and producing natamycin, which comprises the step of simultaneously feeding reducing sugar and propionate during the fermentation process. The highest fermentation unit of natamycin synthesized by the method of the invention can reach 8.13g/L. The invention has the advantages of short natamycin production cycle and low fermentation cost, and is beneficial to large-scale industrialization.

Description

A method for fermenting and producing natamycin

technical field

The invention relates to a process for fermenting and producing natamycin with Streptomyces natalensis , specifically a method for fermenting and producing natamycin.

Background technique

Natamycin is a polyene macrolide antibiotic, mainly fermented by Streptomyces chmanovgensis , Streptomyces gilvosporeus and Streptoyces natalensis produce. It is a high-efficiency, broad-spectrum, safe new biological preservative and medical antibacterial agent, which can specifically inhibit mold and fungi, but has no effect on bacteria, and has low toxicity to mammalian cells. It is widely used in medicine, food, and agriculture. Has good prospects. However, due to the low yield of natamycin, its industrial production as a food additive is limited.

Propionate is used as a precursor substance for natamycin synthesis, and its propionic acid unit is used as a synthesis unit of natamycin macrolide, which is beneficial to the synthesis of natamycin macrolide; on the other hand, it also It can be used as an inducer of acetyl-CoA and propionyl-CoA synthetase, thereby promoting the synthesis of natamycin and increasing the output of natamycin.

At present, the production of natamycin by fermentation at home and abroad mainly increases the yield of natamycin by adding short-chain fatty acids at one time or separately, or by adding a mixture of lower alcohol and reducing sugar. The concentration of reducing sugar in the water is maintained at about 20g/L. The present invention proposes to add propionate and reducing sugar mixed liquid flow into the fermented liquid, and the concentration of both in the fermented liquid is not more than 1g/L, this method not only provides enough carbon source for the generation of natamycin , and make the bacteria in a semi-starved state, relieve the repression of the secondary metabolic enzyme system caused by the excessive amount of reducing sugar, and also avoid the damage to the bacteria caused by the toxicity of propionic acid; this method shortens the fermentation cycle to 60- 90h, the cost is reduced, and the output of natamycin is improved, which is beneficial to industrial production.

Contents of the invention

The purpose of the present invention is to improve the output of natamycin in the fermenter.

A method for fermenting and producing natamycin, the method comprising preparing a culture medium, then inserting a seed culture of Streptomyces natalis into a fermenter with a culture medium, and fermenting under certain conditions, which is characterized in that , Feed propionate and reducing sugar at the same time during the fermentation process.

The ratio of reducing sugar to propionate is 5-30:3.

The propionate is one or both of sodium propionate and calcium propionate.

The reducing sugar is glucose, and the propionate is sodium propionate.

The reducing sugar concentration in the fermentation broth should not exceed 1g/L.

The concentration of propionate in the fermentation broth should not exceed 1g/L.

Reducing sugar and propionate start to feed from a certain point in time between 20-50h until the end of fermentation.

The volume of the used fermenter is 5-100L.

The feeding ratio of reducing sugar and propionate in the present invention is (5-30):3, preferably 25:3, when the feeding ratio of reducing sugar and propionate is less than 5:3 or greater than 30:3 , the fermentation yield was significantly reduced.

The highest fermentation unit of natamycin synthesized by the method of the invention can reach 8.13g/L.

The advantages of adding reducing sugar and propionate mixed solution to produce natamycin in the present invention are:

1. It is easy to operate and significantly increases the yield of natamycin;

2. The production cycle of natamycin is shortened, the production cost is reduced, and it is beneficial to large-scale industrialization.

Description of drawings

Fig. 1 is the fermented product natamycin production curve of embodiment 1, in Fig. 1, abscissa is fermentation time (h), and ordinate is the natamycin production (g/L) that measures in fermented liquid ;

Fig. 2 is the natamycin yield curve of the fermentation product of Example 2. In Fig. 2, the abscissa is the fermentation time (h), and the ordinate is the natamycin yield (g/L) measured in the fermentation broth ;

Fig. 3 is the natamycin yield curve of the fermentation product of Example 3. In Fig. 3, the abscissa is the fermentation time (h), and the ordinate is the natamycin yield (g/L) measured in the fermentation broth ;

Fig. 4 is the fermented product natamycin production curve of embodiment 4, and in Fig. 4, abscissa is fermentation time (h), and ordinate is the natamycin production (g/L) measured in fermented liquid ;

Figure 5 is the natamycin yield curve of the fermentation product of Comparative Example 1. In Figure 5, the abscissa is the fermentation time (h), and the ordinate is the natamycin yield (g/L) measured in the fermentation broth .

Detailed ways

The present invention will be described in detail below by way of examples.

Example 1

1) Shake flask seed culture: under aseptic conditions, spread the spores of Streptomyces natalis on the agar slant medium, culture them in the culture room at 28°C, and then configure the cultured Streptomyces natalis experimental strains Sporulation suspension, draw 1ml of this suspension and add it to the triangular flask of seed medium (glucose 10g/L, yeast powder 50g/L, yeast extract 5g/L, pH 7.2), shake the flask at 220rpm, 28°C, and cultivate 60h.

2) Fermentation culture in a 5L fermenter: Inoculate the cultured seed liquid with a 10% inoculum in the fermentation medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before elimination) In the fermenter, the liquid volume is 3L, the stirring speed is 200-700rpm, the culture temperature is 28°C, and the dissolved oxygen is maintained at 30%-40%. After 30h, the mixture of glucose and sodium propionate is added until the fermentation is completed. The mixing ratio is 25 : 3, the total concentration is 28%, the sugar and propionate concentration in the fermented liquid is no more than 1g/L, cultivates 70h and puts the tank, and the natamycin output in the fermented liquid when putting the tank reaches 8.13g/L by HPLC method measurement (See Figure 1).

3) HPLC detection method is as follows:

Take 0.5ml of natamycin fermentation broth, add 7ml of methanol, shake well, centrifuge at 6000rpm for 10min, take the supernatant and filter it with a 0.45μm microporous membrane to obtain the test solution.

The preparation of standard product: Accurately weigh 0.02g, 0.04g, 0.06g, 0.08g, 0.1g natamycin standard product, dissolve with methanol respectively and settle to 100ml, and prepare 0.2g/L, 0.4g/L L, 0.6g/L, 0.8g/L, 1.0g/L natamycin standard solution, and make a standard curve for use when testing samples.

HPLC conditions: the instrument is Shimadzu LC-15C; the mobile phase is methanol:water=70:30 (volume ratio to fermentation medium v/v); the chromatographic column is Shimadzu LC-C18 (5μm, 4.6mm×250mm); The column temperature is 25°C; the detection wavelength is 303nm; each injection volume is 20μl; the injection flow rate is 1.0ml/min.

Example 2

1) The shake flask seed cultivation method is the same as in Example 1;

2) Fermentation culture in a 5L fermenter: Inoculate the cultured seed liquid with a 10% inoculum in the fermentation medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before elimination) In the fermenter, the liquid volume is 3L, the stirring speed is 200-700rpm, the culture temperature is 28°C, and the dissolved oxygen is maintained at 30%-40%. After 30h, the mixture of glucose and calcium propionate is added until the end of fermentation. The mixing ratio is 15 : 3, the total concentration is 18%, the concentration of sugar and calcium propionate in the fermented liquid is no more than 1g/L, cultivates 70h and puts the tank, and the natamycin output in the fermented liquid reaches 7.26g/L by HPLC method measurement when putting the tank (See Figure 2).

3) The HPLC detection method is the same as in Example 1.

Example 3

1) The shake flask seed cultivation method is the same as in Example 1;

2) Fermentation culture in a 5L fermenter: Inoculate the cultured seed liquid with a 10% inoculum in the fermentation medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before elimination) In the fermenter, the liquid volume is 3L, the stirring speed is 200-700rpm, the culture temperature is 28°C, and the dissolved oxygen is maintained at 30%-40%. After 20h, the mixture of glucose and sodium propionate is added until the end of fermentation. The mixing ratio is 25 : 3, the total concentration is 28%, the sugar and calcium propionate concentration in the fermented liquid is no more than 1g/L, cultivates 70h and puts the tank, and the natamycin output in the fermented liquid when putting the tank reaches 6.94g/L by HPLC method measurement (See Figure 3).

3) The HPLC detection method is the same as in Example 1.

Example 4

1) The shake flask seed cultivation method is the same as in Example 1;

2) Fermentation culture in a 5L fermenter: Inoculate the cultured seed liquid with a 10% inoculum in the fermentation medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before elimination) In the fermenter, the liquid volume is 3L, the stirring speed is 200-700rpm, the culture temperature is 28°C, and the dissolved oxygen is maintained at 30%-40%. After 50h, the mixture of glucose and sodium propionate is added until the end of fermentation. The mixing ratio is 25 : 3, the total concentration is 28%, the sugar and calcium propionate concentration in the fermented liquid is no more than 1g/L, cultivates 70h and puts the tank, and the natamycin output reaches 5.57g/L in the fermented liquid when putting the tank by HPLC method (See Figure 4).

3) The HPLC detection method is the same as in Example 1. As can be seen from the above examples, reducing sugar and propionate can be fed from a certain time point between 20-50h until the end of fermentation to obtain a higher natamycin yield, wherein the ratio of feeding at 30h is 25 : 3 glucose and sodium propionate mixture obtained the highest natamycin yield.

Comparative example 1

1) The shake flask seed cultivation method is the same as in Example 1;

2) Fermentation culture in a 5L fermenter: Inoculate the cultured seed liquid with a 10% inoculum in the fermentation medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before elimination) In the fermenter, the liquid volume is 3L, the stirring speed is 200-700rpm, the culture temperature is 28°C, the dissolved oxygen is maintained at 30%-40%, and 25% glucose is added at 30 hours until the end of fermentation. No more than 1g/L, cultured for 70 hours and placed in tanks, the yield of natamycin in the fermentation broth was determined to reach 4.68g/L by HPLC (see Figure 5).

3) The HPLC detection method is the same as in Example 1.

As can be seen from the comparative examples and examples, the natamycin yield of adding reducing sugar and propionate mixed solution simultaneously is 74% higher than that of adding reducing sugar alone.

Claims (8)

1. A method for fermenting and producing natamycin, the method comprising preparing medium, then inserting the seed culture of Streptomyces natalis in a fermentor with medium, fermenting under certain conditions, its It is characterized in that propionate and reducing sugar are fed simultaneously during the fermentation process. 2. The method according to claim 1, characterized in that the ratio of the reducing sugar to propionate is 5-30:3. 3. The method according to claim 1, wherein the propionate is one or both of sodium propionate and calcium propionate. 4. The method according to claim 3, wherein the reducing sugar is glucose, and the propionate is sodium propionate. 5. The method according to claim 1, characterized in that the reducing sugar concentration in the fermentation broth is no more than 1g/L. 6. The method according to claim 1, characterized in that the concentration of propionate in the fermentation broth is no more than 1g/L. 7. The method according to claim 1, characterized in that, reducing sugar and propionate start to feed from a certain point in time between 20-50h until the end of fermentation. 8. The method according to claim, characterized in that the volume of the fermenter used is 5-100L.