CN1057860A - The liquid fermenting of zearalenone and refinement - Google Patents

Abstract

The invention provides the liquid fermenting of zearalenone and put forward the silk worm production technique.Processes such as Gibberella zeae bacterium D4-21 is activated, seed preparation, fermentation culture can produce a large amount of zearalenones.To ferment then the gained mycelia through alkali dissolution, Acid precipitation, solvent extraction or directly soak, concentrate, process such as crystallization, finally can obtain pure product.This material has the cattle and sheep of promotion protein synthesis, and the ability of improving food conversion ratio is arranged.

Description

The liquid fermenting of zearalenone and refinement

The present invention relates to a kind of biosynthesizing and refining technique thereof that can promote the material-zearalenone (zearale-none) of cattle and sheep growths, it is that Gibberella zeae bacterium (Gibberellazeae) by Fusarium generates through fermentation culture.

In the past aspect the body weight of utilizing medicine increase cattle and sheep, just microbiotic and hormone are mixed in the feed, or in the injection animal body, make it weightening finish, but do not notice the ratio of the protein that increased and fat, and the shared microbiotic of people and animals easily causes the enrichment of a large amount of resistance pathogeny microorganisms, in food chain simultaneously because the toxicity of hormone medicine and the side effect that causes thereof are too big, so this class promotes the material of growth to be eliminated gradually, forbidding.1962, the U.S. separates the Gibberella zeae bacterium that has obtained synthesizing zearalenone on mouldy corn, and obtained the crystallization product of zearalenone by fermentation culture, proved that it is that its chemical structural formula is by a kind of secondary metabolites of Gibberella zeae bacterium synthetic:

Thus structure as can be seen, it does not have the typical steroid nuclear structure of sexual hormoue, World Health Organization (WHO) is categorized as nonsteroidal metabolism assimilation material to it.It can significantly promote the protein assimilation of cattle and sheep, makes it weightening finish, and what increase is protein rather than fat, simultaneously, also can improve food conversion ratio.Afterwards, they have carried out mutagenesis to original strain, obtain several strains and can in solid or liquid nutrient medium, synthesize the bacterial strain of zearalenone in a large number, and to its biosynthesis condition and refining technique application United States Patent (USP), entered the suitability for industrialized production stage very soon, sixties Mo is released the reduzate of ZER (the zearalanol trade name is Ralgro)-a kind of zearalenone, approve through united states drug food control office (FDA), as cattle and sheep weightening finish medicine, promote the use of by many American-European countries are large-area, prove that it is a kind of novel, have no side effect, obvious results promotes the material of cattle and sheep growth-weight gain.

The present invention is by the domestic Gibberella zeae bacterium wild strain that is separated to is carried out mutagenesis, seed selection, obtain a strain can liquid medium within the bacterial strain of synthetic zearalenone, strain number D4-21.The present invention has filled up the blank of China aspect production and refinement zearalenone.

Characteristics of the present invention: the D4-21 bacterial strain is gone up growth rapidly at improvement Cha Shi (Czapek) substratum (prescription sees Table I), cultivates five days for 22 °～25 ℃, and colony diameter can reach 9 centimetres.Bacteria colony white is the fine hair shape, produces the red-purple soluble pigment, does not produce spore on this solid medium, (does not add agar) and can produce a small amount of Rainey's corpuscle in this liquid nutrient medium.

Table I improvement Cha Shi (Czapek) culture medium prescription

Kind	Percentage composition (%)
		Sucrose	3
SODIUMNITRATE	0.3
		Dipotassium hydrogen phosphate	0.1
Sal epsom	0.05
		Repone K	0.05
Yeast leaches cream	0.1
		Agar	1.5

The D4-21 bacterial strain is transferred to improvement czapek's solution inclined-plane activates from the refrigeration inclined-plane, cultivated 4-5 days for 22 °～25 ℃, a small amount of mycelium inoculation of picking is in liquid improvement czapek's solution, and last shaking table is cultivated 2-3 days (culture temperature is the same), makes seed liquor.Again seed liquor is inoculated in the fermention medium (prescription sees Table II) in triangular flask or fermentor tank, 22 °-25 ℃ substratum 15-18 days, can synthesize a large amount of zearalenones.

Table II fermentative medium formula

Kind	Percentage composition (%)
		Glucose	18
Urea	0.27
		Yeast leaches cream	0.1
Dipotassium hydrogen phosphate	0.05
		Sal epsom	0.025
Repone K	0.25

Will be through the filtering fermentation liquor of 15-18 days fermentation gained, get its mycelia filter cake, add the 0.25%NaOH solution soaking 1-2 hour, and refiltered, get its filtrate, filtrate transfers PH to 2-3 with dilute hydrochloric acid, add ethyl acetate extraction again, the ethyl acetate after the extraction obtains the zearalenone coarse crystallization through concentrating, with this coarse crystallization decolouring, recrystallization repeatedly can obtain the pure crystallization of zearalenone again.

Zearalenone is the pure white crystallization, is soluble in organic solvents such as methyl alcohol, ethanol, acetone, ethyl acetate, chloroform and the alkaline aqueous solution, is slightly soluble in hexane, sherwood oil equal solvent, is insoluble to neutrality or acidic aqueous solution.Molten point is 164 °-165 ℃.In the ultra-violet absorption spectrum, the highest absorption peak in addition, under the UV-irradiation of 253 nanometers, presents blue green fluorescence in 236,274 and 313 nanometers.

Zearalenone passes through hydrogenating reduction again, its reduzate α-Yu Michimeichun (Zearalanol) is made the bullet (every bullet content 12mg) of 2.5 millimeters of high 3 mm dias and is put into filling gun, can heeling-in use.The usage quantity of ox is 36 milligrams, and gross weight weightening finish comparison is according to high 15%-20%; The usage quantity of sheep is 12 milligrams, and gross weight weightening finish comparison is according to high 5%-10%.And in the feeding process of cattle and sheep, make feed conversion rate improve 10%.

Production instance of the present invention: mycelia takes a morsel from the refrigeration inclined-plane of Gibberella zeae bacterium D4-21 bacterial strain, be inoculated on the improvement czapek's solution inclined-plane and activate, cultivated 4-5 days for 22 °-25 ℃, a small amount of mycelium inoculation of getting again on the activated inclined plane is improved in the triangular flask of czapek's solution in liquid is housed, loading amount is a triangular flask volumetrical 1/5th, and last shaking table was cultivated 2-3 days, and culture temperature is the same, so far, make seed liquor.Seed liquor is inoculated in the triangular flask that fermention medium is housed, and inoculum size 1%-5% is put in rotating speed and is on 180 rev/mins the shaking table and cultivates 15-18 days (culture temperature is the same), and fermenting process finishes.As seed liquor is inoculated in the fermentor tank, fermention medium and culture temperature are the same, and stirring velocity is 200 rev/mins, and air flow is during the fermentation 1: 0.3(vvm) and 1: regulate 0.8(vvm), cultivated 15-18 days, stop fermenting.

The extractive process of zearalenone: get 1 liter of filtering fermentation liquor, gained mycelia filter cake refilters after 1-2 hour with 1000 milliliters of 0.25%NaOH solution soaking, gained filtrate is used the dilute hydrochloric acid acid adjustment, termination PH is 2-3, zearalenone can precipitate, and adds equal volume of ethyl acetate then; Also can be with the filter cake behind the filtering fermentation liquor directly with ethyl acetate or soaked in absolute ethyl alcohol extraction.To extract the gained ethyl acetate and concentrate, and can obtain 1.8 gram zearalenone coarse crystallization, this coarse crystallization is dissolved in ethyl acetate or the dehydrated alcohol again, decolouring back recrystallization 2-3 time can obtain the pure crystallization of 1.6 gram zearalenones.

Claims (5)

1, a kind of Gibberella zeae bacterium (Gibberellazeae) D4--21 bacterial strain that utilizes carries out the technology that liquid fermenting is produced zearalenone, and this technology comprises the refining technique of seed culture technology, fermentating culturing process and the zearalenone of D4--21 bacterial strain.

2, by the described seed culture technology of claim 1, be characterized in that the D4-21 bacterial strain is activated, be inoculated in the seed culture medium that contains 3% sucrose, 0.3% SODIUMNITRATE, 0.1% yeast leaching cream, 0.1% dipotassium hydrogen phosphate, 0.05% sal epsom, 0.05% Repone K, 22 °-25 ℃, shaking table was cultivated 2-3 days.

3, by the described fermentating culturing process of claim 1, be characterized in seed liquor is inoculated in and contain 18% glucose, 0.27% urea, 0.1% yeast and leach cream, 0.05% dipotassium hydrogen phosphate, in the fermention medium of 0.025% sal epsom, 0.025% Repone K, 22 °-25 ℃, shake the bottle or fermentor cultivation 15-18 days.

4, by the described refining technique of claim 1, be characterized in the mycelia of fermentation culture gained is soaked with 0.25% sodium hydroxide solution, the filtrate of filtering gained is regulated PH to 2-3 with dilute hydrochloric acid, use ethyl acetate extraction again, the ethyl acetate of extraction gained is concentrated, get coarse crystallization.

5, by claim 1 and 4 described refining techniques, be characterized in the mycelia filter cake is directly soaked with ethyl acetate or dehydrated alcohol, will filter the gained solvent and concentrate, get coarse crystallization.