CN101979630B

CN101979630B - Method for preparing insecticidal bioactivator

Info

Publication number: CN101979630B
Application number: CN2010102700240A
Authority: CN
Inventors: 吴吉安; 孙东昌; 竺利红; 桑金隆; 施跃峰
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2010-08-31
Filing date: 2010-08-31
Publication date: 2012-11-21
Anticipated expiration: 2030-08-31
Also published as: CN101979630A

Abstract

The invention relates to the technical field of microbiological fermentation engineering, and aims to provide a method for preparing an insecticidal bioactivator. In the method, piericidin A is prepared by adopting microbiological fermentation technology. The method is characterized in that: an adopted producing bacterium is streptomucespiomogeues var.hangzhouwanensis which is collected in China general microbiological culture collection center (CGMCC). In the method, a fermentation culture medium of the invention mainly comprises the components of ordinary agricultural products, and a fermentation cycle is relatively shorter, so the preparation cost is relatively lower.

Description

A kind of preparation method of desinsection biologically active substance

Technical field

The invention belongs to the microbial fermentation engineering technical field, particularly a kind of desinsection biologically active substance---the preparation method of piericidin A.

Background technology

Insect pest brings about great losses to agriculture prodn every year, and in order to realize high and stable yields, people mainly adopt insecticide pesticide to control always.Piericidin (Piericidin) is a kind of insecticide active substance of finding in the sixties in last century, equals report in 1963 by Tamura the earliest, and it produces bacterium is streptomyces mobaraensis (S.molaraensis).Initial assert that its active constituent mainly contains two, i.e. piericidin A and piericidin B, after report that successively it has a plurality of homologues, have 16 more than so far.Piericidin mainly has strong toxic action to crop pests such as housefly, white butterfly, tetranychid and aphids.

Goal of the invention

The technical problem that the present invention will solve is a kind of new desinsection biologically active substance---the preparation method of piericidin A to be provided, in the hope of reducing the preparation cost of piericidin.

Be the technical solution problem, technical scheme of the present invention is:

A kind of preparation method of desinsection biologically active substance is provided; Be to adopt microbial fermentation technology to produce piericidin A, it is characterized in that used generation bacterium is the mutation of ticket mycin streptomycete Hangzhou Wan; The Latin name is called Streptomucespiomogeues var.hangzhouwanensis; Culture presevation is in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and preserving number is CGMCC No.4029, preservation date on July 20th, 2010.

The character analysis of used generation bacterium ticket mycin streptomycete Hangzhou Wan mutation:

1, the classification that produces bacterium is identified:

(1) morphological specificity

Bacterial strain on synthetic medium, fibrillae of spores straight, gentle bent, spore is oval to cylindricality, spore surface is smooth, sees accompanying drawing 1, accompanying drawing 2.

(2) cultural characteristic

Gas silk light gray is to grey on most of synthetic mediums, and base silk bamboo shoot skin is brown not to have soluble pigment in early days to filbert, and later stage fermented bean drink Huang is to Ji Sise, and cultural characteristic is seen table 1.

The cultural characteristic of table 1 bacterial strain

(3) physiological and biochemical property

That milk peptonizes, do not solidify, gelatin does not liquefy, starch water is situated between is strong, do not grow on the Mierocrystalline cellulose, nitrate reduction is weak, do not produce H ₂A S and a type melanochrome.

(4) utilization of carbon source

Bacterial strain can utilize glucose, glycerine, starch, inositol, N.F,USP MANNITOL, Hydrocerol A, pectinose, synanthrin, seminose; Sucrose, fructose, sorbose are utilized suspicious, do not utilize L-rhamnosyl, D-wood sugar, SANMALT-S, raffinose, lactose, semi-lactosi etc.See table 2.

The utilization of carbon source of table 2 bacterial strain

Carbon source kind	Utilize situation	Carbon source kind	Utilize situation
				Glucose	+	N.F,USP MANNITOL	+
Sucrose	±	Glycerine	+
				The L-rhamnosyl	-	Starch	+
L-arabinose	+	Hydrocerol A	+

The D-wood sugar	-	SANMALT-S	-
				D-fructose	±	Lactose	-
Raffinose	-	Semi-lactosi	-
				Inositol	+	Sorbose	±
Synanthrin	+	Seminose	+

Notes: "+"---utilization, " ± "---utilize suspicious, "-"---do not utilize

(5) cell wall components analysis

Amino acid analysis: compare with 2,6 diaminopimelic acids and glycocoll strain cell wall hydrolyzate is carried out full cell amino acid analysis, this strain cell wall contains L-DAP and glycocoll as a result, shows that this bacterial strain belongs to cell walls I type.

The sugar analysis of components: the strain cell hydrolyzate is analyzed its sugared composition with chromatography, does not find to have principal character property sugar, and promptly pectinose, semi-lactosi and wood sugar belong to sugared C type.

(6) antagonism property

A little less than bacteriological action, but very strong restraining effect (seeing table 3) is arranged to many plant pathogenic fungis such as cotton seedling blight, watermelon anthrax, sclerotinia rot of colza, blue mould of citrus and to fungi such as Penicllium chrysogenum, black mold, terreus, aspergillus oryzae, gypsum tinea capitis and part yeast such as rhodotorula glutinis, Candida albicans etc.

The antimicrobial spectrum of table 3 bacterial strain

Measure the bacterium title	Antibacterial circle diameter (mm)
		Bacillus subtilus	13.2
Intestinal bacteria	0
		Proteus vulgaris	0
Shame posterior division bacillus	12.5
		Streptococcus aureus	12.0
Sarcina lutea	11.5
		Penicllium chrysogenum	29.0
Black mold	33.0
		Terreus	33.1
Aspergillus oryzae	33.0
		Gypsum tinea capitis	31.0
Rhodotorula glutinis	33.5
		Candida albicans	29.0
Torula	11.5
		Dark red tinea	28.0

(7) expert's conclusion:

This bacterial strain and ticket mycin streptomycete (Streptomuces piomogeues) are more closely similar; The two spore is straight; The aerial hyphae light gray is to grey, and the spore ellipse is to cylindricality, and spore surface is smooth; But some cultural characteristic, physiological and biochemical property, and utilization of carbon source on some difference (seeing table 4) are arranged again, so called after ticket mycin streptomycete Hangzhou Wan mutation (Streptomuces piomogeues var.hangzhouwanensis).

Table 4 bacterial strain and the two difference of allied species S.piomogenes

2. the separation of effective ingredient and evaluation:

(1) extraction separation of effective ingredient:

Fermented liquid promptly gets pure article through filtration, solvent extraction, high-efficient liquid phase chromatogram purification.Idiographic flow is:

Filtering fermentation liquor obtains that mycelium, alcohol lixiviate concentrating under reduced pressure obtain liquid concentrator, the preparative high performance liquid chromatography purifying obtains pure article; During chromatogram purification, stationary phase is Waters XTerra RP 18, and moving phase is methyl alcohol: water (80: 20).

(2) structure of effective ingredient is identified:

1. ESI-MS analyzes:

Sample is carried out ESI-MS negative ions mode scanning, do not have isotopic peaks such as Si, S, Cl, Br in the MS spectrogram.M/z 414 ions are [M-H] in the mass spectrum of negative source ^-Ion, m/z 416 is respectively [M+H] with m/z 438 in the mass spectrum of positive source ⁺Ion and [M+Na] ⁺Ion, the molecular weight that this shows compound is 415.According to the nitrogen rule of molecular weight, can confirm to contain in this compound molecule odd number N atom again.

At sample [M+H] ⁺In the CID mass spectrum of ion m/z 416, fragmention m/z 398 is the principal character ion, and it is that its forerunner's ion is lost a molecular water generation.Can release thus and contain hydroxyl-OH.

At [M+H] ⁺In the quasi-molecular ions, the intensity of first isotopic peak is 28%, ¹³The C natural abundance of isotopes is 1.1%, therefore, can confirm to contain in the compound the individual C of 25 (28/1.1=25).

2. ¹³C-NMR analyzes:

At sample ¹³Among the C-NMR, confirm altogether to contain 25 C in the compound structure, be consistent with the mass spectrometric measurement structure.Its specifying information such as table 5:

Table 5. sample ¹³The C-NMR data

By determined pulsating quality sum in the table 5 is 367; And this compound molecular weight is 415; Button removes a N atom again, and residue 34Da quality segment (415-367-14=34) can be confirmed as 2 hydroxyl segments; One of them goes up hydroxyl for rank oxygen methine carbon (δ 82ppm), the hydroxyl that another one can only be on aryl.Simultaneously, can infer that this molecular formula is C ₂₅H ₃₇NO ₄, do not have M such as Si, S, Cl among this and the MS ^{+ 2}The isotopic peak signal is consistent.

3. H-NMR analyzes:

In the H-NMR of sample, associative list 5 information, confirm altogether to contain 35 H, its specifying information such as table 6 in the compound structure:

The H-NMR data of table 6. sample

Can know do not have chemical shift at the fignal center more than 7.0 in the hydrogen spectrum by table 6, explain not contain aryl hydrogen in the compound structure.

4. expert's conclusion:

Information confirms that through analysis-by-synthesis the structure of this compound is to kill the plain A of powder dish per sample.Its chemical structure is seen accompanying drawing 3, and nuclear-magnetism is respectively composed the peak ownership and seen table 7:

Table 7.NMR data analysis:

Among the present invention, use to produce the microbial fermentation technology that bacterium prepares piericidin A, its detailed process can adopt habitual technique means to realize.

Examples of implementation provided by the invention; Its preparation process comprises successively: concentrate after slant strains, seed tank culture, fermentor cultivation, fermented liquid solid-liquid separation, the solvent extraction or further carry out chromatographic separation, promptly get piericidin A (PiericidinA) finished product (seeing accompanying drawing 4); Wherein:

(1) produce bacterial classification:

With ticket mycin streptomycete Hangzhou Wan mutation (Streptomuces piomogeues var.hangzhouwanensis) serves as to produce bacterium; Bacterial classification is preserved in CGMCC (China Committee for Culture Collection of Microorganisms common micro-organisms center) on July 20th, 2010, and the preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.

(2) seed slant medium:

KNO ₃0.1%, K ₂HPO ₄0.075%, MgSO ₄0.05%, NaCI 0.05%, Zulkovsky starch 2.0%, agar 2.0%, wheat bran 1.0% (boil 20 minutes after-filtration and get filtrating), pH 7.2～7.4; Said ratio is the degree that accounts for the substratum total mass;

(3) fermention medium:

Nutrition source in the substratum adopts carbon source and nitrogenous source, and wherein carbon source is at least a in Semen Maydis powder, starch, dextrin, sucrose, wheat bran, glucose or the maltose; Nitrogenous source is at least a in ammonium sulfate, dried silkworm chrysalis meal, urea, peptone, soybean cake powder, yeast powder or the steeping water; On the basis of carbon source and nitrogenous source, add at least a in phosphoric acid salt, vitriol, sodium salt, sylvite, magnesium salts or the calcium salt then again;

(4) fermentation culture conditions:

Culture temperature is 24～33 ℃, and cultivating the pH value is 5～9, and incubation time is 30～120 hours.

Among the present invention, said fermented liquid solid-liquid separation is meant carries out centrifugal or filtration treatment to fermented liquid, obtains mycelium.

Among the present invention, said solvent extraction concentrates and is meant use alcohol or acetone extraction, and lixiviate is after concentrating under reduced pressure except that desolvating, gets the piericidin A crude extract.

The present invention further provides a kind of preparation method of piericidin A preparation, is the piericidin A crude extract is diluted to packing behind the finite concentration, is piericidin A different concns formulation products.

The present invention also provides a kind of piericidin A former medicament preparation, is silicagel column on the piericidin A crude extract is carried out chromatographic separation, and substep is collected the back evaporate to dryness and promptly got the former medicine product of piericidin A; Eluent when silica gel column chromatography separates is a toluene: ETHYLE ACETATE (80～100: 20～0).

Beneficial effect of the present invention is:

The fermention medium staple is common agricultural byproducts, and fermentation period is also shorter, thereby preparation cost is lower.

Description of drawings:

Fig. 1 is the ripe fibrillae of spores photo of bacterial strain;

Fig. 2 is a bacterial strain spore photo;

Fig. 3 is for killing the chemical structural drawing of the plain A of powder dish;

Fig. 4 is a piericidin product preparation process schema.

Embodiment

Embodiment one: the preparation of piericidin 1% preparation

In 5 tons of fermentor tanks, be made into 3.7 tons of substratum (pH 7.4) by the proportioning of sweet potato starch 4.0%, soybean cake powder 1.5%, yeast powder 0.5%, ammonium sulfate 1.0%, lime carbonate 0.5%, sal epsom 0.05%, potassium primary phosphate 0.05%.By ordinary method sterilization postcooling to 28 ℃, the mutation of inoculation ticket mycin streptomycete Hangzhou Wan, promptly got the fermented liquid of about 1800 μ g/ml that tire in 50 hours through 28 ℃ of aerobic culture.This fermented liquid is through filter press, and thalline is with 95% industrial spirit lixiviate of 1.2 times of fermentating liquid volumes three times, and vat liquor is removed alcohol through concentrating under reduced pressure, be diluted to 10000～11000 μ g/ml that tire after, packing is 1% formulation products.

Embodiment two: the preparation of the former medicine of piericidin

In 5 tons of fermentor tanks, be made into 3.5 tons of substratum (pH 7.2) by the proportioning of Semen Maydis powder 5.0%, dried silkworm chrysalis meal 1.5%, yeast powder 0.5%, urea 0.5%, wheat bran 1.0%, saltpetre 0.5%, lime carbonate 0.5%, sal epsom 0.05%, potassium primary phosphate 0.05%.By ordinary method sterilization postcooling to 28 ℃, the mutation of inoculation ticket mycin streptomycete Hangzhou Wan, promptly got the fermented liquid of about 1900 μ g/ml that tire in 55 hours through 28 ℃ of aerobic culture.This fermented liquid is through filter press; Thalline is with the acetone extraction of 1.3 times of fermentating liquid volumes three times, and vat liquor is removed acetone through concentrating under reduced pressure, transfers PH3～5 to use ethyl acetate extraction; Extracting solution washs through the 0.5N sodium hydrogencarbonate; Go up silicagel column behind the concentrating under reduced pressure and carry out chromatographic separation, eluent is a toluene: ETHYLE ACETATE (98: 2), substep are collected the back evaporate to dryness and are promptly got the former medicine product of piericidin.

Claims

1. the preparation method of a desinsection biologically active substance; Be to adopt microbial fermentation technology to produce piericidin A; It is characterized in that used generation bacterium is the mutation of ticket mycin streptomycete Hangzhou Wan, the Latin name is called Streptomyces piomogeuse.var.hangzhouwanensis; Culture presevation is in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC No.4029;

The chemical structural formula of said piericidin A is: