CN108329370B

CN108329370B - Preparation method of tartaric acid/tylosin phosphate

Info

Publication number: CN108329370B
Application number: CN201810356481.8A
Authority: CN
Inventors: 成祥兴; 范泽雷; 居亚东; 刘萍
Original assignee: Qilu Pharmaceutical Inner Mongolia Co ltd
Current assignee: Qilu Pharmaceutical Inner Mongolia Co ltd
Priority date: 2018-04-19
Filing date: 2018-04-19
Publication date: 2021-04-20
Anticipated expiration: 2038-04-19
Also published as: CN108329370A

Abstract

The invention relates to a preparation method of tartaric acid/tylosin phosphate, the special protein which is not filtered out in the fermentation liquor can be separated out from the filtered fermentation liquor through specific alkali liquor and specific pH, the special protein which is not filtered out in the fermentation liquor, fatty acid and partial grease which are generated by fermentation are thoroughly removed through centrifugation, and the butyl acetate serving as a solvent is purified, so that the extraction is efficiently carried out, the solvent is not affected while being purified, the extraction is ensured to be carried out smoothly, meanwhile, the treatment means of simple operation, low cost, simplicity and low cost can effectively and thoroughly reduce the impurities in the fermentation filtrate, and the purity and the solubility of the product are improved.

Description

Preparation method of tartaric acid/tylosin phosphate

Technical Field

The invention relates to a preparation method of tartaric acid/tylosin phosphate, belonging to the technical field of biological pesticide preparation.

Background

Tylosin was first isolated from thailand soil by Hamill et al, an american scholars producing strains of streptomyces fradiae (s.fradiae, NRR2702, 2703), streptomyces rimosus (s.rimosus) and streptomyces hygroscopicus (s.hygro-scopicus); the tylosin active ingredients include: tylosin A (eutylosin), tylosin B (decarburizing enzyme tylosin Desmosin), tylosin C (macromycin micosin) and tylosin D (ralomycin Relomycin), wherein the component A is more than or equal to 80%, the total component A + B + C + D is more than or equal to 95%, and the titer is more than 800 IU. The chemical structural formula of each effective component of tylosin is shown in the following table 1:

TABLE 1

The fermentation of tylosin uses soybean oil as a main carbon source. Glucose has obvious inhibition effect on the biosynthesis of tylosin, if excessive glucose exists in fermentation in a differentiation period, the increase of ATP level is promoted, so that the growth of tylosin is inhibited, and oleic acid and linoleic acid in soybean oil are methylated to form methyl oleic acid which is a precursor substance of tylosin and can promote the biosynthesis of the tylosin. Therefore, soybean oil is mainly supplemented in the fermentation process to meet the requirement of the producing bacteria on a carbon source. The strict control of the addition of the soybean oil is the key for determining the success or failure of the fermentation. Soybean oil content is low, which is not beneficial to hypha growth, and content is too high, which can not be fully utilized, and fatty acid accumulation is generated, thus leading to pH value reduction, thus inhibiting activity of colicin C methyltransferase and influencing component content. The temperature, PH, hypha production status and soybean oil addition amount in the tylosin fermentation process have characteristic index significance on the activity of the colicin C methyltransferase and are the key points for determining the yield of tylosin.

The fermentation of tylosin should strictly control the mixed bacteria pollution, if the condition is limited or unavoidable or the bacteria contamination is caused by the reasons of operation, equipment and the like, the tylosin fermentation liquor after the bacteria contamination is put into a tank with poor quality, peculiar smell exists, the filtration speed is slow, the filtrate is turbid, the titer is reduced, the residual oil is large, great difficulty is brought to the extraction production, and the yield and the quality of the tylosin finished product are directly influenced. If the fermentation liquor is infected with bacteria seriously, the fermentation liquor cannot be treated and is forced to be poured into a tank, thereby causing waste and loss. The reason for the deterioration of the fermentation liquid is that the growth and reproduction of the tylosin producing strain are abnormal due to the invasion of infectious microbes. The growth of other bacteria is dominant, and its metabolic products, such as various enzymes and toxins can greatly inhibit the normal growth of tylosin producing bacteria, and promote the autolysis of tylosin mycelium. The culture medium has a large amount of colloidal protein, the fermentation liquor becomes turbid, and the filtration rate is slow, so that the treatment is difficult.

The tylosin components have a weak alkaline, since they have an imino group on the carbomycin glucosamine in the side chain, in addition to a 16-membered cyclic lactone. Under alkaline condition, the tylosin hydrochloride exists in the form of tylosin alkali and is easily dissolved in organic solvent; and the salt form of the compound is combined with acid under acidic condition, and is easy to dissolve in water. According to the characteristic, the prior extraction process of tylosin generally adopts a solvent extraction method. Specifically, the fermentation liquor is subjected to solid-liquid separation to obtain filtrate, alkalized to pH value of 9-10, extracted by using a solvent to obtain an organic phase, then back extracted by using phosphoric acid or tartaric acid to obtain a water phase, and finally treated by Ca (OH)₂Neutralizing to remove excessive acid, decolorizing with activated carbon, air desolventizing, nano-filtering, and spray drying to obtain tylosin phosphate or tylosin tartrate.

In the extraction process, the protein which is not filtered and removed enters an organic phase in the extraction process of the filtered filtrate, and part of fatty acid generated by fermentation also enters an ester phase, so that impurities cannot be effectively separated.

Therefore, the method effectively removes impurities in the tylosin fermentation broth and improves the quality of the tylosin becomes a prominent problem to be urgently solved in the front of numerous pharmaceutical enterprises.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the preparation method of the tartaric acid/tylosin phosphate, which thoroughly removes impurities such as filtered filtrate protein, residual oil and the like, makes full preparation for extraction, improves the purity and solubility of the tylosin product, and has the advantages of simple process, low cost, short working procedure and easy popularization and utilization.

The technical scheme of the invention is as follows:

a preparation method of tylosin tartrate/phosphate comprises the following steps:

adding a flocculating agent or a filter aid into the tylosin fermentation liquor obtained by fermentation, stirring and pretreating, separating by a plate frame to obtain a filtrate, purifying the filtrate, extracting, carrying out back extraction, decoloring and drying to obtain a tylosin product; wherein, the purification is to add sodium hydroxide solution into the filtrate, then pump the filtrate into a mixer for mixing, control the pH value to be within the range of 7.5-8.0, centrifuge the mixed solution at the rotating speed of 4000-.

According to the invention, the flocculant is polyaluminium chloride, the weight ratio of the addition amount of the polyaluminium chloride to the fermentation liquor is 1.0 +/-0.3% (w/w), the filter aid is polyaluminium chloride solution with the mass concentration of 20-22%, and the mass volume ratio of the addition amount of the polyaluminium chloride solution to the fermentation liquor is 0.6% (w/v); the pretreatment time is 20-30 min.

According to the invention, the solid-liquid separation is preferably carried out by adopting a filter pressing or squeezing mode, and the filter pressing or squeezing pressure is less than or equal to 0.4 MPa.

According to the invention, the purification is preferably controlled to a pH of 7.5 to 7.9, preferably the purification is controlled to a pH of 7.5 to 7.7, and most preferably the purification is controlled to a pH of 7.54.

Preferably, according to the invention, the centrifugal speed is 4500-4800 r/min, and the centrifugal time is 0.2s-2 min.

According to the invention, the sodium hydroxide solution preferably has a concentration of 2 to 6 wt.%.

According to the method, the filtrate is mixed with liquid alkali and then centrifuged, protein which cannot be removed by solid-liquid separation, fatty acid and partial grease generated by fermentation are completely removed, and impurities in the filtrate during extraction are reduced. During extraction, protein and fat in the filtrate are obviously reduced, and emulsion is obviously reduced after extraction. Through the purification treatment of the invention, the treatment capacity of the extraction on the contamination tank is greatly enhanced, the pollution of the protein and the grease in the feed liquid to the butyl ester is obviously reduced, the protein and the fat in the feed liquid are further intercepted by increasing the filter and the filtering precision in the subsequent process, the solubility of the existing product is obviously improved after the improvement, and the product content is also improved to 934u/mg from 893u/mg of the original process.

The inventor of the invention unexpectedly finds that the special protein which is not filtered in the fermentation liquor can be separated out after the pH of the filtered fermentation liquor is adjusted by the specific alkali liquor, the special protein which is not filtered in the fermentation liquor, fatty acid and partial grease which are generated by fermentation are thoroughly removed by centrifugation, the solvent butyl acetate is purified, the extraction is promoted to be carried out efficiently, other influences on the solvent can not be caused while the solvent is purified, the extraction is ensured to be carried out smoothly, and meanwhile, the invention has the advantages of simple operation, low cost, simple and low-cost treatment means, namely, the impurities in the fermentation filtrate can be efficiently and thoroughly reduced, and the purity and the solubility of the product are improved.

The invention has the following beneficial effects:

1. the special protein which is not filtered in the fermentation liquor can be separated out and separated from the filtered fermentation liquor through the special alkali liquor and the special pH, the special protein which is not filtered in the fermentation liquor, fatty acid and partial grease which are generated by fermentation are thoroughly removed through centrifugation, the solvent butyl acetate is purified, the extraction is promoted to be carried out efficiently, the solvent is not influenced at the same time of purifying the solvent, the extraction is ensured to be carried out smoothly, and meanwhile, the method is simple in operation, low in cost, simple and low in cost, can achieve the purposes of efficiently and thoroughly reducing the impurities in the fermentation filtrate, and improves the purity and the solubility of the product.

The raw materials and equipment used in the invention are all the prior art.

Drawings

FIG. 1 is a high performance liquid chromatogram of a filtrate obtained by filter-pressing tylosin fermentation broth in example 1;

FIG. 2 is a high performance liquid chromatogram of a purified filtrate obtained by filter-pressing and alkali-adding purification of tylosin fermentation broth in example 1;

FIG. 3 is a high performance liquid chromatogram of a filtrate obtained by filter-pressing tylosin fermentation broth in example 2;

FIG. 4 is a high performance liquid chromatogram of the purified filtrate obtained by filter-pressing and alkali-adding purification of tylosin fermentation broth in example 2;

FIG. 5 is a high performance liquid chromatogram of a filtrate obtained by filter-pressing tylosin fermentation broth in example 3;

FIG. 6 is a high performance liquid chromatogram of the purified filtrate obtained by filter-pressing and alkali-adding purification of tylosin fermentation broth in example 3.

Detailed Description

The present invention is further illustrated by, but is not limited to, the following specific examples.

Sources of tylosin fermentation liquor: fermentation liquor obtained after fermentation in a tylosin fermentation workshop is finished.

Example 1

the volume of tylosin fermentation liquor is 10m³The titer was 12386U/mL, with a total billion of 123.9. Adding polyaluminium chloride, the addition amount of the polyaluminium chloride and the fermentation liquorThe weight ratio of (A) to (B) is 1.0 + -0.3% (w/w), and stirring is carried out for 25 + -5 minutes. Filtering the fermentation liquid with plate-and-frame filter, controlling feeding pressure at 0.3MPa, and filter-pressing to obtain filtrate (12 m in volume) of tylosin filtrate³The titer was 9621U/mL.

Pumping a sodium hydroxide solution with the mass concentration of 3% and the filtrate into a mixer through a pump, controlling the pH value to be 7.7, and pumping the mixed feed liquid into a centrifugal machine to centrifuge for 2s at the rotating speed of 4500 r/min to obtain purified filtrate; sending the purified filtrate, butyl acetate and caustic soda liquid into a mixer for mixing, adjusting the pH value to be within 9.8, sending the mixture into a centrifuge for centrifugation after the mixing is finished, and sending the ester phase into a water tank for cooling and waiting for treatment; cooling and standing the ester phase in the water tank, adding 2% phosphoric acid/tartaric acid, stirring (adjusting pH to 3.6), standing, separating, and collecting the lower water phase; adding calcium hydroxide solution to adjust pH to 6.1, adding active carbon to decolorize, adding into decolorization plate frame, filtering, squeezing, and feeding water phase into desolventizing tank. And (4) desolventizing the materials in the tank by using nitrogen until the materials have no butyl ester. Concentrating the water phase by using a nanofiltration membrane, and removing water to obtain a tylosin concentrated solution; and (3) drying the concentrated solution by using a spray centrifugal drying tower to obtain the tylosin phosphate/tartrate powder.

Example 2

the volume of tylosin fermentation liquor is 9.5m³The titer is 13194U/mL, and the total billion is 126.9. Adding 20-22% polyaluminium chloride solution, wherein the mass volume ratio of the addition amount of the polyaluminium chloride solution to the fermentation broth is 0.6% (w/v), and stirring for 40 min. Filtering the fermentation liquid with plate-and-frame filter, controlling the pressure of filter pressing/squeezing to be 4.0MPa, and filter pressing to obtain filtrate (11 m in volume) of tylosin filtrate³The titer was 10832U/mL.

Pumping a sodium hydroxide solution with the mass concentration of 4 wt% and the filtrate into a mixer through a pump, controlling the pH value to be 7.9, and pumping the mixed feed liquid into a centrifugal machine to centrifuge for 2s at the rotating speed of 4800 r/min to obtain purified filtrate; feeding the purified filtrate, butyl acetate and liquid caustic soda into a mixer for mixing, adjusting the pH value to be within 10.5, feeding the mixture into a centrifuge for centrifugation after the mixing is finished, and feeding the ester phase into a water tank for cooling and waiting for treatment; cooling and standing the ester phase in the water tank, adding 2% phosphoric acid/tartaric acid, stirring (adjusting pH to 3.7), standing, separating, and collecting the lower water phase; adding calcium hydroxide solution to adjust pH to 6.3, adding active carbon to decolorize, adding into decolorization plate frame, filtering, squeezing, and feeding water phase into desolventizing tank. And (4) desolventizing the materials in the tank by using nitrogen until the materials have no butyl ester. Concentrating the water phase by using a nanofiltration membrane, and removing water to obtain a tylosin concentrated solution; and (3) drying the concentrated solution by using a spray centrifugal drying tower to obtain the tylosin phosphate/tartrate powder.

Example 3

A method for preparing tylosin tartrate/phosphate, which is the same as that described in example 1, except that:

pumping the sodium hydroxide solution with the mass concentration of 3 wt% and the filtrate into a mixer simultaneously by a pump, controlling the pH value to be 7.54, centrifuging and extracting.

By comparing fig. 1 and fig. 2, it can be clearly seen that the impurity peaks in the chromatogram of the purified filtrate are significantly less than those in fig. 1, by comparing fig. 3 with fig. 4, it can be clearly seen that the impurity peaks in the chromatogram of the purified filtrate are significantly less than those in fig. 3, and by comparing fig. 5 with fig. 6, it can be clearly seen that the impurity peaks in the chromatogram of the purified filtrate are significantly less than those in fig. 5, therefore, after the purification treatment of the present invention, it can be confirmed by the high performance liquid phase comparison that the impurities in the filtrate are significantly reduced or decreased, and the number of the impurity peaks is changed from the original 16 impurity peaks to 10. During extraction, protein and fat in the filtrate are obviously reduced, and emulsion is obviously reduced after extraction.

Comparative example 1

A preparation method of tartaric acid/tylosin phosphate is carried out according to a traditional method, namely, fermentation liquor is subjected to solid-liquid separation to obtain filtrate, the pH of the filtrate is 5.63, the filtrate is alkalized to the pH value of 9-10, then solvent is used for extracting the filtrate into an organic phase, then phosphoric acid or tartaric acid is used for back extraction to a water phase, and finally Ca (OH) is carried out₂Neutralizing to remove excessive acidDecolorizing with charcoal, air desolventizing, nano-filtering and spray drying to obtain tylosin phosphate or tylosin tartrate.

Experimental example:

1. the method of example 3 and comparative example 1 is adopted to carry out 5 batches of production, the filtrate obtained after purification in example 3 and the filtrate obtained after filtration in comparative example 1 in each batch of production are taken, the transmittance of the filtrate is tested, and the test results are shown in the following table 2:

TABLE 2 comparison of the filtrate transmittances

	Comparative example 1%	Example 3%
			Batch 1	61.3	90.6
Batch 2	64.8	91.9
			Batch 3	67.1	94.2
Batch 4	62.5	93.7
			Batch 5	68.0	91.4

2. The method of example 3 and comparative example 1 was used to perform 5 batches of production, and 3g of the obtained product was taken and dissolved in 10ml of water, and the transmittance of the solution was measured, and the measurement results are shown in table 3 below:

TABLE 3 comparison of the transmittance of the product solutions

	Comparative example 1%	Example 3%
			Batch 1	81.7	97.8
Batch 2	76.1	96.9
			Batch 3	72.9	97.4
Batch 4	68.2	98.2
			Batch 5	84.3	95.5

The method of example 3 and comparative example 1 was used to perform 5 batches of production, and the product was taken and the potency (dry basis) of the product was measured, with the results shown in table 4 below:

TABLE 4 comparison of product potency

	Comparative example 1u/mg	Example 3u/mg
			Batch 1	891	935
Batch 2	886	934
			Batch 3	897	931
Batch 4	895	935
			Batch 5	894	936

Claims

1. A preparation method of tylosin tartrate/phosphate comprises the following steps:

adding a flocculating agent or a filter aid into the tylosin fermentation liquor obtained by fermentation, stirring and pretreating, separating by a plate frame to obtain a filtrate, purifying the filtrate, extracting, carrying out back extraction, decoloring and drying to obtain a tylosin product; wherein, the purification comprises adding sodium hydroxide solution into the filtrate, pumping into a mixer for mixing, controlling the pH within the range of 7.5-7.7, centrifuging the mixed solution at the rotation speed of 4500-4800 r/min for 0.2s-2min to obtain purified filtrate, and adding butyl acetate solvent into the purified filtrate for extraction; the mass concentration of the sodium hydroxide solution is 2-6 wt%.

2. The method for preparing tylosin tartrate/phosphate according to claim 1, wherein the flocculant is polyaluminium chloride, the weight ratio of the added polyaluminium chloride to the fermentation broth is 1.0 +/-0.3%, the filter aid is a polyaluminium chloride solution with the mass concentration of 20-22%, and the added mass of the polyaluminium chloride solution is 0.6% of the volume ratio of the fermentation broth; the pretreatment time is 20-30 min.

3. The method for preparing tylosin tartrate/phosphate according to claim 1, wherein the plate-and-frame separation is performed by a filter pressing or squeezing method, wherein the filter pressing or squeezing pressure is less than or equal to 0.4 Mpa.

4. The method for preparing tylosin tartrate/phosphate according to claim 1, wherein the purification is performed at a pH of 7.54.