CN104946709A

CN104946709A - Method for fermenting and producing natamycin

Info

Publication number: CN104946709A
Application number: CN201510346199.8A
Authority: CN
Inventors: 卢诗瑶; 孙立洁; 袁丽霞; 陈祥松; 吴金勇; 姚建铭; 朱薇薇; 凌瑞
Original assignee: Institute of Plasma Physics of CAS
Current assignee: Institute of Plasma Physics of CAS
Priority date: 2015-06-19
Filing date: 2015-06-19
Publication date: 2015-09-30

Abstract

The invention discloses a method for fermenting and producing natamycin, which comprises the steps that reducing sugars and propionate are feed at the same time during the fermentation process. The method for fermenting and producing the natamycin can achieve the highest fermentation unit of natamycin of 8.13g/L. The method for fermenting and producing the natamycin has the advantages of short production cycle of the natamycin and low fermentation cost, and is beneficial for large-scale industrialization.

Description

A kind of method of natamycin fermentation preparation

Technical field

The present invention relates to Streptomyces natalensis ( streptomyces natalensis) processing method of natamycin fermentation preparation, be specifically a kind of method of natamycin fermentation preparation.

Background technology

Tennecetin is a kind of polyenoid class macrolide antibiotic, primarily of streptomyces chatanoogensis ( streptomyces chmanovgensis), brown yellow spore streptomycete ( streptomyces gilvosporeus) and Streptomyces natalensis ( streptoyces natalensis) etc. fermentation produce.It is a kind of New Biological Preservatives and medical antibacterial agent of efficient, wide spectrum, safety, mould fungus inhibition that can be single-minded and fungi, but does not act on bacterium, and low to mammalian cell toxicity, medicine, food, agriculture in have good prospect.But because the output of tennecetin is lower, it is subject to a definite limitation as the industrialization production of foodstuff additive.

The precursor substance that propionic salt synthesizes as tennecetin, its propionic acid unit, as the synthesis unit of tennecetin macrolide, is conducive to the synthesis of tennecetin macrolide; On the other hand, it also as the inductor of acetyl-CoA, propionyl-CoA synthetase, thus can promote the synthesis of tennecetin, improves the output of tennecetin.

At present, domestic and international natamycin fermentation preparation, mainly by disposable interpolation or separately stream add short chain fatty acid, or add lower alcohol by stream and reducing sugar mixed solution improves tennecetin output, and the concentration of reduced sugar in fermenting process in fermented liquid maintains about 20g/L.The present invention proposes propionic salt and reducing sugar mixed solution stream to add in fermented liquid, and the two concentration in fermented liquid is all no more than 1g/L, this method is both for the generation of tennecetin provides enough carbon sources, thalline is made again to be in semistarvation state, relieve that reducing sugar amount is excessive checks effect to secondary metabolism enzyme system, the toxicity that simultaneously it also avoid propionic acid causes damage to thalline; Fermentation period is foreshortened to 60-90h by this method, reduces cost, turn improves the output of tennecetin, is conducive to industrialization and produces.

Summary of the invention

The object of the invention is to the output improving tennecetin in fermentor tank.

A kind of method of natamycin fermentation preparation, the method comprises preparation substratum, in the fermentor tank being placed with substratum, then accesses the inoculum of Streptomyces natalensis, ferment under certain condition, it is characterized in that, stream adds propionic salt and reducing sugar simultaneously during the fermentation.

The ratio of described reducing sugar and propionic salt is 5-30:3.

Described propionic salt is one or both in Sodium Propionate and calcium propionate.

Described reducing sugar is glucose, and described propionic salt is Sodium Propionate.

In fermented liquid, concentration of reduced sugar is no more than 1g/L.

In fermented liquid, propionic salt concentration is no more than 1g/L.

Reducing sugar and propionic salt stream from certain time point between 20-50h adds, until fermentation ends.

The fermenter volume used is 5-100L.

The stream of reducing sugar described in the present invention and propionic salt adds ratio for (5-30): 3, preferred 25:3, when the stream of reducing sugar and propionic salt add ratio be less than 5:3 or be greater than 30:3 time, fermentation yield obviously reduces.

The highest fermentation unit being synthesized tennecetin by the inventive method can reach 8.13g/L.

Stream of the present invention adds reducing sugar and propionic salt mixed solution produces tennecetin advantageously:

1. easy and simple to handle, significantly improve tennecetin output;

2. shorten the tennecetin production cycle, reduce production cost, be conducive to extensive industrialization.

Accompanying drawing explanation

Fig. 1 is the tunning tennecetin yield curve of embodiment 1, and in FIG, X-coordinate is fermentation time (h), and ordinate zou is tennecetin output (g/L) measured in fermented liquid;

Fig. 2 is the tunning tennecetin yield curve of embodiment 2, and in fig. 2, X-coordinate is fermentation time (h), and ordinate zou is tennecetin output (g/L) measured in fermented liquid;

Fig. 3 is the tunning tennecetin yield curve of embodiment 3, and in figure 3, X-coordinate is fermentation time (h), and ordinate zou is tennecetin output (g/L) measured in fermented liquid;

Fig. 4 is the tunning tennecetin yield curve of embodiment 4, and in the diagram, X-coordinate is fermentation time (h), and ordinate zou is tennecetin output (g/L) measured in fermented liquid;

Fig. 5 is the tunning tennecetin yield curve of comparative example 1, and in Figure 5, X-coordinate is fermentation time (h), and ordinate zou is tennecetin output (g/L) measured in fermented liquid.

Embodiment

Below by embodiment, the present invention is elaborated.

Embodiment 1

1) shake-flask seed is cultivated: under aseptic condition, Streptomyces natalensis spore is coated in agar slant culture-medium, be placed between 28 DEG C of cultivations and cultivate, again cultured Streptomyces natalensis experimental strain is configured to spore suspension, draw this suspension of 1ml and add seed culture medium (glucose 10g/L, yeast powder 50g/L, yeast extract 5g/L, pH 7.2) triangular flask in, shaking flask rotating speed 220rpm, 28 DEG C, cultivate 60h.

2) 5L ferment tank cultivate: by cultured seed liquor with 10% inoculum size be inoculated in fermention medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before disappearing) fermentor tank in, liquid amount is 3L, stirring velocity 200-700rpm, culture temperature 28 DEG C, dissolved oxygen maintains 30%-40%, 30h starts stream and adds glucose and Sodium Propionate mixed solution until fermentation ends, blending ratio is 25:3, total concn is 28%, sugar in fermented liquid and propionic salt concentration are no more than 1g/L, cultivate 70h and put tank, when putting tank in fermented liquid tennecetin output measured by HPLC method reach 8.13g/L(see Fig. 1).

3) HPLC detection method is as follows:

Get 0.5ml natamycin fermentation liquor, add 7ml methyl alcohol, after fully shaking up, the centrifugal 10min of 6000rpm, gets supernatant liquor 0.45 μm of filtering with microporous membrane and namely obtains liquid to be measured.

The preparation of standard substance: accurately take 0.02g, 0. 04g, 0.06g, 0.08g, 0.1g tennecetin standard substance, 100ml is settled to respectively with dissolve with methanol, be mixed with the tennecetin standardized solution of 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, 1.0g/L, and make typical curve, use during detected sample.

HPLC condition: instrument is Shimadzu LC-15C; Moving phase is methyl alcohol: water=70:30(and fermention medium volume ratio v/v); Chromatographic column is Shimadzu LC-C18(5 μm, 4.6mm × 250mm); Column temperature is 25 DEG C; Determined wavelength is 303nm; Each sample size is; 20 μ l sample introduction flow velocitys are 1.0ml/min.

Embodiment 2

1) shake-flask seed cultural method is with embodiment 1;

2) 5L ferment tank cultivate: by cultured seed liquor with 10% inoculum size be inoculated in fermention medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before disappearing) fermentor tank in, liquid amount is 3L, stirring velocity 200-700rpm, culture temperature 28 DEG C, dissolved oxygen maintains 30%-40%, 30h starts stream and adds glucose and calcium propionate mixed solution until fermentation ends, blending ratio is 15:3, total concn is 18%, sugar in fermented liquid and calcium propionate concentration are no more than 1g/L, cultivate 70h and put tank, when putting tank in fermented liquid tennecetin output measured by HPLC method reach 7.26g/L(see Fig. 2).

3) HPLC detection method is with embodiment 1.

Embodiment 3

1) shake-flask seed cultural method is with embodiment 1;

2) 5L ferment tank cultivate: by cultured seed liquor with 10% inoculum size be inoculated in fermention medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before disappearing) fermentor tank in, liquid amount is 3L, stirring velocity 200-700rpm, culture temperature 28 DEG C, dissolved oxygen maintains 30%-40%, 20h starts stream and adds glucose and Sodium Propionate mixed solution until fermentation ends, blending ratio is 25:3, total concn is 28%, sugar in fermented liquid and calcium propionate concentration are no more than 1g/L, cultivate 70h and put tank, when putting tank in fermented liquid tennecetin output measured by HPLC method reach 6.94g/L(see Fig. 3).

3) HPLC detection method is with embodiment 1.

Embodiment 4

1) shake-flask seed cultural method is with embodiment 1;

2) 5L ferment tank cultivate: by cultured seed liquor with 10% inoculum size be inoculated in fermention medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before disappearing) fermentor tank in, liquid amount is 3L, stirring velocity 200-700rpm, culture temperature 28 DEG C, dissolved oxygen maintains 30%-40%, 50h starts stream and adds glucose and Sodium Propionate mixed solution until fermentation ends, blending ratio is 25:3, total concn is 28%, sugar in fermented liquid and calcium propionate concentration are no more than 1g/L, cultivate 70h and put tank, when putting tank in fermented liquid tennecetin output measured by HPLC method reach 5.57g/L(see Fig. 4).

3) HPLC detection method is with embodiment 1.Can be found out by above embodiment, reducing sugar and propionic salt stream from certain time point between 20-50h adds until fermentation ends can obtain higher tennecetin output, and wherein 30h starts stream and adds glucose that ratio is 25:3 and the tennecetin output that Sodium Propionate mixed solution obtains is the highest.

Comparative example 1

1) shake-flask seed cultural method is with embodiment 1;

2) 5L ferment tank cultivate: by cultured seed liquor with 10% inoculum size be inoculated in fermention medium (glucose 20g/L, yeast extract 5g/L, yeast powder 30g/L, pH 7.7 before disappearing) fermentor tank in, liquid amount is 3L, stirring velocity 200-700rpm, culture temperature 28 DEG C, dissolved oxygen maintain 30%-40%, 30h starts the glucose that stream adds 25%, until fermentation ends, sugared concentration in fermented liquid is no more than 1g/L, cultivates 70h and puts tank, when putting tank in fermented liquid tennecetin output measured by HPLC method reach 4.68g/L(see Fig. 5).

3) HPLC detection method is with embodiment 1.

Can be found out by comparative example and embodiment, simultaneously stream add reducing sugar and propionic salt mixed solution than independent stream add reducing sugar tennecetin output increased 74%.

Claims

1. the method for a natamycin fermentation preparation, the method comprises preparation substratum, in the fermentor tank being placed with substratum, then accesses the inoculum of Streptomyces natalensis, ferment under certain condition, it is characterized in that, stream adds propionic salt and reducing sugar simultaneously during the fermentation.

2. the method for claim 1, is characterized in that, the ratio of described reducing sugar and propionic salt is 5-30:3.

3. the method for claim 1, is characterized in that, described propionic salt is one or both in Sodium Propionate and calcium propionate.

4. method as claimed in claim 3, it is characterized in that, described reducing sugar is glucose, and described propionic salt is Sodium Propionate.

5. the method for claim 1, is characterized in that, in fermented liquid, concentration of reduced sugar is no more than 1g/L.

6. the method for claim 1, is characterized in that, in fermented liquid, propionic salt concentration is no more than 1g/L.

7. the method for claim 1, is characterized in that, reducing sugar and propionic salt stream from certain time point between 20-50h adds, until fermentation ends.

8. method as claimed in claim, it is characterized in that, the fermenter volume used is 5-100L.