CN101173229B - Spherosinin degradation bacterium calcium acetate fixed bacillus YLZZ-1 and method for producing the same - Google Patents

Abstract

The invention discloses a swainsonine degradation bacteria acetic acid calcium acinetobacter YLZZ-1, which is of short rod shape, 0.2 to 0.3Mum x 0.5 to 1.0Mum, gram-negative without capsule, germ andmotion; bacterial colony is circular, ivory-white, smooth on the surface, wet, elevated and regular in edge and not transparent; bacterial strain is artificially separated and screened out from locoweed growing soil in areas such as Ganshu, Tibet, Inner Mongolia and Qinghai and is preserved at the preservation center for typical culturing in China on Jul., 20, 2007 with preservation No. of CCTCCNO: M 207108. The invention has the advantages of degrading swainsonine which is main toxin of locoweed (Swainsonine, SW).

Description

A kind of spherosinin degradation bacterium calcium acetate fixed bacillus YLZZ-1-1 and preparation method thereof

Technical field

The invention belongs to microbial technology field, relate to a kind of new Acinetobacter calcoaceticus (Acinetobacter calcoaceticus), be specifically related to a kind of main toxin trihydroxyoctahydroindolizidine of loco weed (Swainsonine that can effectively degrade, SW) degradation bacterium calcium acetate fixed bacillus YLZZ-1-1 also relates to this degradation bacteria preparation and cultural method and application thereof.

Background technology

Loco weed (Locoweed) is the general name that the pulse family whin belongs to (Oxytropis) and Astragalus (Astragalus) poisonous plants, also is the most serious class poisonous weeds of harm pasture animal husbandry in the world wide.In China, loco weed is distributed widely in the vast grassland in northwest, North China and southwest, and hazard area reaches 1,100 ten thousand hm2, accounts for 2.8% of national grassland area.Because loco weed has very strong adaptive faculty, growth is vigorous on desert steppe, and turn green early, late withered, pastured animal is searched for food under the situation that other plant lacks or growing way is bad for a long time or in a large number, causes large quantities of domestic animals to be poisoned to death, or dam is infertile, miscarriage, fetal anomaly, male animal is sterile and the livestock product downgrade, causes enormous economic loss to pasture animal husbandry.

Trihydroxyoctahydroindolizidine (Swainsonine, SW) be that the water-soluble indoles that loco weed (Locoweed) class plant materials contains is decided alkaloid now, molecular formula is C8H15NO3, chemical name is 1,2, western pyridine (trihydroxyoctahydro-indolizidine) in the 8-trihydroxy-octahydro indoles is one of main toxic ingredient of loco weed class plant.Trihydroxyoctahydroindolizidine can suppress lysosome alpha-Mannosidase activity, make oligose accumulation in the cell, cause the cell vacuolation, thereby make cell lose normal function, even cause necrocytosis, can also suppress gorky's mannosidase II, influence synthesizing, handle and shifting of glycoprotein, these cause cell adhesion, hormone transhipment and cell surface receptor dysfunction.The clinical manifestation that animal poisons comprises that spirit is depressed, insensitive, oversensitive, myasthenia of the limbs, emaciation, reproduction function are impaired etc., and chronic poisoning often causes neural persistent expendable infringement.

Though loco weed is poisonous weeds, its nutritive value is also very abundant, and the crude protein content of some whin surpasses the clover of " king of feed " up to 20%.At China, the loco weed of the riotous growth of western grassland more than 30% is become available high quality forage, the forage grass resource that can nearly double can play powerful promoter action to local Developing of Animal Industry.

In recent years, both at home and abroad in harm, toxic ingredient, the toxicity mechanism of loco weed, prevent and kill off and the research of aspect such as utilization has obtained impressive progress, but also do not control the effect method that loco weed spreads so far, cause loco weed poisoning problem to be on the rise, become the great trouble of pasture animal husbandry.Domestic scholars once attempted handling loco weed with water seaoning, sour water infusion method and pyroprocessing method etc., was removing toxic palatability and the nutritive value that has destroyed loco weed simultaneously of loco weed, and the expense height, was not suitable for promoting.Wangkais etc. are developed serial toxinicides such as " anti-E number of sour jujubes "; can effectively postpone the time that the loco weed toxicity symptom occurs; virgin German etc. are connected synthetic SW-BSA with SW with macromolecular carrier protein B SA; with SW-BSA immunogen inoculation animal; make animal obtain immunizing power and when searching for food loco weed, be protected; these two researchs have obtained certain progress, bring hope for preventing and kill off animal loco weed toxonosis.

At present, using microbe degraded toxic substance is the focus of studying both at home and abroad always, the field that relates to is very extensive, but the research aspect the microbiological deterioration plant poison is less, but also obtained certain achievement, be separated to multiple microorganism strain more than 70 of the sinigrin of can degrading in the Zhou Bincong rapeseed cake, through identifying that these bacterium are white torulopsis (Torulopsis candida), zhizopchin (Rhizopus chinensis), Mucor racemosus (Mucor racemosus), aspergillus oryzae (Aspergillus oryzae) determines that by fermentation test its virus elimination rate is 80%.Tan Beiying etc. are from the ox cud of Weizhou Island, Guangxi; isolate 4 strain anaerobic bacteriums; through being accredited as lactobacillus (Lactobacillus) (2 strain), streptococcus bovis (Streptococcus bovis) and clostridium sporogenes (Clostridium sporogenes); above-mentioned bacterial strains has degrading activity to mimosine and dihydroxy-pyridine toxoid; these detoxification bacterium live in the cud, can protect host animal to avoid the murder by poisoning of Leucaena leucocephala (L.).Locoism toxin biological degradation research yet there are no report, but these research methods can be for using for reference, explore a biodegradable approach of SW, filter out the microorganism of the SW that to degrade, and the enzyme gene of this toxin of will degrading changes on animal gastrointestinal tract normal microflora or the animal body, can not only fundamentally solve animal loco weed poisoning problem, can also bring good economic benefits and environmental benefit, active effect is played in the biology control and the range ecology research of grassland poisonous weeds.

Summary of the invention

At problems of the prior art and defective, the object of the present invention is to provide a kind of spherosinin degradation bacterium calcium acetate fixed bacillus YLZZ-1-1, its main toxin of loco weed----trihydroxyoctahydroindolizidine of effectively degrading makes it animal toxicity is reduced.

For achieving the above object, technical scheme of the present invention is as follows:

A kind of trihydroxyoctahydroindolizidine (Swainsonine, SW) degradation bacterium calcium acetate fixed bacillus YLZZ-1-1 (Acinetobacter calcoaceticus YLZZ-1), be preserved in Chinese typical culture collection center on July 20th, 2007, preserving number is: CCTCC NO:M 207108, preservation address: China. Wuhan. and Wuhan University.

Another object of the present invention provides the preparation method of calcium acetate fixed bacillus YLZZ-1-1, comprises the following steps:

1) gathers careless sample: pick up from loco weeds vegetatively such as Gansu, Tibet, Inner Mongol, Qinghai;

2) soil sample is cultivated: the 1kg loco weed is embedded in the soil, and the degree of depth is 10～20cm, takes out careless sample soil on every side after 3～6 months, with the soil sample thorough mixing of gathering;

3) separation screening of bacterial strain: get the well-mixed soil sample adding of 10g and be equipped with in the triangular flask of 90mL sterilized water and sterile glass beads, place on the shaking table, the 200r/min 10min that fully vibrates, the preparation bacteria suspension, getting the 10mL bacteria suspension then adds in the 100m L enrichment culture liquid, and at 30 ℃, 180r/min, overnight incubation under pH 7.0 conditions, and then be inoculated in the fresh domestication substratum that contains trihydroxyoctahydroindolizidine (SW) 30mg/L with 10% inoculum size and cultivate, later on once every the 4d culture transferring, and increase trihydroxyoctahydroindolizidine (SW) content gradually and be respectively 50,80,100,120,150,180,200mg/L, when SW concentration reaches 200mg/L, be inoculated in the fresh minimal medium that contains trihydroxyoctahydroindolizidine (SW) 200mg/L with 10% inoculum size again and cultivate 7d, then with on nutrient solution dilution-plate method coating trihydroxyoctahydroindolizidine (SW) the inorganic salt solid medium, single bacterium colony bacterial strain that choosing colony comes in every shape, be stored in behind the purifying on the ordinary nutrient agar medium slant, obtain an Acinetobacter calcoaceticus YLZZ-1.

The component of described enrichment culture liquid and proportioning are: extractum carnis 5g, peptone 10g, NaCl5g, KH ₂PO ₄1g, distilled water 1L, pH are 7.0～7.2,121 ℃ of autoclaving 20min.

Described domestication substratum is trihydroxyoctahydroindolizidine (SW) solution that adds filtration sterilization in enrichment culture liquid, and making the concentration of SW in the domestication substratum is 0～200mg/L.

The component of described minimal medium and proportioning are: NH ₄NO ₃1.0g, MgSO ₄0.15g, (NH ₄) ₂SO ₄0.5g, KH ₂PO ₄0.5g, NaCl 0.5g, K ₂HPO ₄1.5g, distilled water 100mL, pH 7.0～7.2,121 ℃ of sterilization 20min, the sterilization back adds trihydroxyoctahydroindolizidine (SW) solution of filtration sterilization, and the concentration that makes SW in the minimal medium is 0～200mg/L.

Described inorganic salt solid medium is to add agar in minimal medium, and making agar concentration therein is 20g/L.

The component of described ordinary nutrient agar substratum and proportioning are: extractum carnis 5g, peptone 10g, NaCl5g, KH ₂PO ₄1.0g, agar 20g, distilled water 1L, pH7.0～7.2,121 ℃ autoclaving 20min.Calcium acetate fixed bacillus YLZZ-1-1 bacterial strain has following character:

1, form and physiological and biochemical property

The thalline of YLZZ-1 bacterial strain is the club shape, 0.2～0.3 μ m * 0.5～1.0 μ m, Gram-negative, no pod membrane, no gemma, no mobility; Bacterium colony is rounded, oyster white, and smooth surface, moistening, protuberance, neat in edge is opaque; Glucose fermentation produces small amount of acid, and aerogenesis is not grown under the anaerobic condition, edwardsiella hoshinae, M.R are tested negative, and V.P tests positive, hydrolyzed starch does not reduce nitrate, can not utilize Citrate trianion, utilize malonate, do not produce H2S, liquefy gelatin, the catalase positive, oxidase negative, urease-positive, in 5%NaCl, grow, but slower, do not grow among the 7%NaCl.Concrete physiological and biochemical property sees Table 1.

The physiological and biochemical property of table 1.YLZZ-1 bacterial strain

Experimental project	The result	Experimental project	The result
				Carbohydrate produces acid		Nitrate reduction	-
Glucose	+	Citrate trianion utilizes	-
				Lactose	+	Malonate utilizes	+
Sucrose	-	Produce H 2S	-
				Aerobic	+	Gelatine liquefication	+
Anaerobism	-	Catalase	+
				Indoles produces	-	Oxydase	-
The M.R test	-	Urase	+
				The V-P test	+	The 5%NaCl growth	+
The starch hydrolysis	-	The 7%NaCl growth	-

Annotate: "+" expression growth or reacting positive; "-" expression is not grown or reaction negative.

YLZZ-1 bacterial strain 16S rDNA sequence is seen sequence table SEQ ID NO.1, and its GenBank accession number is EU022688 (sequence is unexposed).

Through 16S rDNA sequence homology analysis (seeing embodiment 2), according to principle of classification and the combining form, physiological and biochemical property of homology in phylogeny, with reference to " uncle's outstanding bacteriology handbook (the 8th edition) ", determine that finally the YLZZ-1 bacterial strain is Acinetobacter calcoaceticus (Acinetobacter calcoaceticus).

This SW degradation bacteria is measured and 16S rDNA sequence homology analysis through form, physiological and biochemical property, and this bacterium can be only carbon source with trihydroxyoctahydroindolizidine, and the degradable trihydroxyoctahydroindolizidine is for the murder by poisoning that thoroughly solves loco weed lays the foundation.

Description of drawings

Fig. 1 is the form of YLZZ-1 bacterial strain under microscope lens

Fig. 2 is the single colonial morphology of YLZZ-1 bacterial strain

Fig. 3 is the control group gas chromatogram of YLZZ-1 degrading swainsonine (SW)

Fig. 4 is the test group gas chromatogram in mid-term of YLZZ-1 degrading swainsonine (SW)

Fig. 5 is the test group later stage gas chromatogram of YLZZ-1 degrading swainsonine (SW)

Fig. 6 is the degradation curve of YLZZ-1 degrading swainsonine (SW);

Fig. 7 utilizes DNAStar software building systematic evolution tree.

Embodiment

Embodiment 1: the separation screening of trihydroxyoctahydroindolizidine (SW) degradation bacteria (YLZZ-1 bacterial strain)

1) gathers careless sample: pick up from loco weeds vegetatively such as Gansu, Tibet, Inner Mongol, Qinghai;

2) soil sample is cultivated: the 1kg loco weed is embedded in the soil, and the degree of depth is 10～20cm, takes out careless sample soil on every side after 3～6 months, with the soil sample thorough mixing of gathering;

3) separation screening of bacterial strain: get the well-mixed soil sample adding of 10g and be equipped with in the triangular flask of 90mL sterilized water and sterile glass beads, place on the shaking table, the 200r/min 10min that fully vibrates, the preparation bacteria suspension, getting the 10mL bacteria suspension then adds in the 100m L enrichment culture liquid, and at 30 ℃, 180r/min, overnight incubation under pH 7.0 conditions, and then be inoculated in the fresh domestication substratum that contains trihydroxyoctahydroindolizidine (SW) 30mg/L with 10% inoculum size and cultivate, later on once every the 4d culture transferring, and increase trihydroxyoctahydroindolizidine (SW) content gradually and be respectively 50,80,100,120,150,180,200mg/L, when trihydroxyoctahydroindolizidine (SW) when concentration reaches 200mg/L, be inoculated in the fresh minimal medium that contains trihydroxyoctahydroindolizidine (SW) 200mg/L with 10% inoculum size again and cultivate 7d, then with on nutrient solution dilution-plate method coating trihydroxyoctahydroindolizidine (SW) the inorganic salt solid medium, single bacterium colony bacterial strain that choosing colony comes in every shape, be stored in behind the purifying on the ordinary nutrient agar medium slant, obtain an Acinetobacter calcoaceticus YLZZ-1.

The component of described enrichment culture liquid and proportioning are: extractum carnis 5g, peptone 10g, NaCl 5g, KH ₂PO ₄1g, distilled water 1L, pH7.0～7.2,121 ℃ autoclaving 20min.

The component of described ordinary nutrient agar substratum and proportioning are: extractum carnis 5g, peptone 10g, NaCl5g, KH ₂PO ₄1.0g, agar 20g, distilled water 1L, pH7.0～7.2,121 ℃ autoclaving 20min.

The component of described minimal medium and proportioning are: NH ₄NO ₃1.0g, MgSO ₄0.15g, (NH ₄) ₂SO ₄0.5g, KH ₂PO ₄0.5g, NaCl 0.5g, K ₂HPO ₄1.5g, distilled water 100mL, pH7.0～7.2,121 ℃ sterilization 20min, the sterilization back adds trihydroxyoctahydroindolizidine (SW) solution of filtration sterilization, and the concentration that makes SW in the minimal medium is 200mg/L.

Described inorganic salt solid medium is to add agar in minimal medium, and making agar concentration therein is 20g/L.

Described domestication substratum: as required, add the SW solution of filtration sterilization in the enrichment culture liquid after sterilization, making its concentration is 50,80,100,120,150,180,200mg/L.

The homology analysis of the 16S rDNA sequence of embodiment 2YLZZ-1 bacterial strain

The 16S rDNA sequence (sequence table SEQ ID NO.1) of YLZZ-1 bacterial strain is carried out sequence homology analysis by the Blast retrieval system of NCBI, utilize DNAStar software building systematic evolution tree, see Fig. 7.Draw by Fig. 7, the genetic evolution distance of YLZZ-1 bacterial strain is nearest with acinetobacter, it and Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) are in the branch of a minimum together, reach 100% with the homology of known strains A cinetobacter calcoaceticus strain CAI-13 (DQ257421), illustrate that this bacterium belongs to Acinetobacter calcoaceticus on the Molecular Phylogeny taxonomy.

Test example 1: use degradation bacteria (YLZZ-1 bacterial strain) degrading swainsonine (SW)

Adopt the degradation rate of this bacterium of gas chromatography determination to SW.Inoculum size with 5% is inoculated into degradation bacteria in the minimal medium that contains 50mg/L SW, 30 ℃, the 180r/min wave and culture is contrast with the SW minimal medium that does not connect bacterium, takes a sample every 2h, content (Fig. 3 with the gas chromatography determination trihydroxyoctahydroindolizidine, Fig. 4 Fig. 5), the results are shown in Table 2, calculate degradation rate, draw degradation curve (Fig. 6).

The degradation rate (%) of the different incubation time SW of table 2.

Sum up: the YLZZ-1 bacterial strain is complete with the spherosinin degradation of 50mg/L basically in 14h, and the control group trihydroxyoctahydroindolizidine does not have degraded.This bacterium optimum growing condition is: pH is 6～8, and temperature is 25 ℃～35 ℃.

Sequence table 1

＜110〉Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology

＜120〉a kind of spherosinin degradation bacterium calcium acetate fixed bacillus YLZZ-1-1 and preparation method thereof

<160>1

<210>1

<211>1436

<212>DNA

<213>ACINETOBACTER CALCOACETICUS YLZZ-1

<400>1

AATGCAAGTC GAGCGGAGAG AGGTAGCTTG CTACTGATCT TAGCGGCGGA CGGGTGAGTA 60

ATGCTTAGGA ATCTGCCTAT TAGTGGGGGA CAACATTTCG AAAGGAATGC TAATACCGCA 120

TACGTCCTAC GGGAGAAAGC AGGGGATCTT CGGACCTTGC GCTAATAGAT GAGCCTAAGT 180

CGGATTAGCT AGTTGGTGGG GTAAAGGCCT ACCAAGGCGA CGATCTGTAG CGGGTCTGAG 240

AGGATGATCC GCCACACTGG GACTGAGACA CGGCCCAGAC TCCTACGGGA GGCAGCAGTG 300

GGGAATATTG GACAATGGGC GCAAGCCTGA TCCAGCCATG CCGCGTGTGT GAAGAAGGCC 360

TTATGGTTGT AAAGCACTTT AAGCGAGGAG GAGGCTACTT TAGTTAATAC CTAGAGATAG 420

TGGACGTTAC TCGCAGAATA AGCACCGGCT AACTCTGTGC CAGCAGCCGC GGTAATACAG 480

AGGGTGCAAG CGTTAATCGG ATTTACTGGG CGTAAAGCGC GCGTAGGCGG CTAATTAAGT 540

CAAATGTGAA ATCCCCGAGC TTAACTTGGG AATTGCATTC GATACTGGTT AGCTAGAGTG 600

TGGGAGAGGA TGGTAGAATT CCAGGTGTAG CGGTGAAATG CGTAGAGATC TGGAGGAATA 660

CCGATGGCGA AGGCAGCCAT CTGGCCTAAC ACTGACGCTG AGGTGCGAAA GCATGGGGAG 720

CAAACAGGAT TAGATACCCT GGTAGTCCAT GCCGTAAACG ATGTCTACTA GCCGTTGGGG 780

CCTTTGAGGC TTTAGTGGCG CAGCTAACGC GATAAGTAGA CCGCCTGGGG AGTACGGTCG 840

CAAGACTAAA ACTCAAATGA ATTGACGGGG GCCCGCACAA GCGGTGGAGC ATGTGGTTTA 900

ATTCGATGCA ACGCGAAGAA CCTTACCTGG CCTTGACATA GTAAGAACTT TCCAGAGATG 960

GATTGGTGCC TTCGGGAACT TACATACAGG TGCTGCATGG CTGTCGTCAG CTCGTGTCGT 1020

GAGATGTTGG GTTAAGTCCC GCAACGAGCG CAACCCTTTT CCTTATTTGC CAGCGAGTAA 1080

TGTCGGGAAC TTTAAGGATA CTGCCAGTGA CAAACTGGAG GAAGGCGGGG ACGACGTCAA 1140

GTCATCATGG CCCTTACGGC CAGGGCTACA CACGTGCTAC AATGGTCGGT ACAAAGGGTT 1200

GCTACCTAGC GATAGGATGC TAATCTCAAA AAGCCGATCG TAGTCCGGAT TGGAGTCTGC 1260

AACTCGACTC CATGAAGTCG GAATCGCTAG TAATCGCGGA TCAGAATGCC GCGGTGAATA 1320

CGTTCCCGGG CCTTGTACAC ACCGCCCGTC ACACCATGGG AGTTTGTTGC ACCAGAAGTA 1380

GCTAGCCTAA CTGCAAAGAG GGCGGTTACC ACGGTGTGGC CGATGACTGG GGGAAG 1436

Claims (2)

1. a spherosinin degradation bacterium Acinetobacter calcoaceticus (Acinetobacter calcoaceticus) YLZZ-1 is preserved in Chinese typical culture collection center on July 20th, 2007, and preserving number is: CCTCCNO:M 207108.

2. claim 1 described calcium acetate fixed bacillus YLZZ-1-1 application in the trihydroxyoctahydroindolizidine in the degraded loco weed.

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