Background technology
Deoxynivalenol (deoxynivalnol, DON), chemical name is 3 α, 7 α, 15-trihydroxy--12, the single-ended spore of 13-epoxy is mould-9-alkene-8-ketone, it is mainly to infect by Fusarium graminearum (Fusarium graminearum), fusarium culmorum (Fusarium culmorum) the Trichothecenes toxin that the cereal such as wheat, barley, oat, corn produce, because this material can cause the symptoms of emesis of animal, therefore have another name called vomitoxin (vomitoxin).In worldwide, DON is one of main pollution mycotoxin of grain, feed, food, has a strong impact on the health of people and livestock.People and animals have taken in after the food being polluted by vomitoxin, can cause apocleisis, vomiting, diarrhoea, fever, astasia, the acute poisoning symptom such as slow in reacting, when serious, damage hemopoietic system and cause death, its serious harm has caused the generally attention of various countries.
Vomitoxin is quite general to the pollution of China's cereals raw material, its recall rate and detected level are all one the highest in mycotoxin, investigate demonstration according to people such as Zhen Yangguang, the ratio of exceeding standard of China's feed and raw material vomitoxin approaches 70%, in corn, the exceeding standard rate of vomitoxin is 57.1%, toxin average content is 1.01mg/kg, and its high-content is 2.13mg/kg.Due to universal existence, high-content characteristic and the acute toxicity and chronic toxicity of vomitoxin in cereal, feed, reduce or remove its toxicity and seem particularly important and urgent.At present, vomitoxin poison-removing method mainly contains Physical, chemical treatment and biological method three major types both at home and abroad.Although physics, chemical process detoxification have obtained success to a certain degree, but still there is the shortcomings such as detoxification efficiency loss, cost limited, that may cause important nutrient are higher.Biological method has can make the virulence of toxin reduce under gentle condition, and sensory properties, the palatability etc. of raw material are affected to the advantages such as minimum, and is considered to best poison-removing method.Because microbe species is many, wide material sources, nearly all organic pollutant can be decomposed by microorganism, and has degraded thoroughly, and the advantage of non-secondary pollution, so the microbial detoxification of vomitoxin is a kind of effective poison-removing method.Along with the continuous appearance of drawback of physics, chemical detoxication means and the understanding of the biotechnology advantage to fast development increase, utilizing microorganism to carry out detoxicated research both at home and abroad progressively launches, although also few about the achievement in research with microbial detoxification, on going result has all showed good development and application prospect.The people such as Yoko in 2011 screen a strain promise Ka Shi and belong to bacterial strain in soil, this bacterial strain can utilize vomitoxin for sole carbon source and energy growth under aerobic condition, its degraded toxin concentration scope is 10 – 1000 μ g/mL(Nocardioides sp.strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epideoxynivalenol, Ikunaga Y, Sato I, Grond S, et al. (2011), Appl Microbiol Biotechnol89:419 – 427).
In sum, for solving toxin pollution problem in feed, feedstuff raw material, be necessary that from natural resources separation screening is efficient, the microorganism strains of safe disposal vomitoxin, further its biological nature of research, toxin degradation characteristic and mechanism of degradation, research and development are applicable to the mycotoxin microbial detoxification agent of feedstuff industry, improve grain utilization ratio, ensure the safety in production of livestock industry, improve animal husbandry economy benefit.
Summary of the invention
The technical problem to be solved in the present invention is to provide a strain can degrade De Wosi Salmonella and the application thereof of vomitoxin.
For solving the problems of the technologies described above, the present invention adopts following technical scheme:
The invention provides a strain De Wosi Salmonella (Devosia sp.), called after DDB001, belong to the novel species in De Wosishi genus, to separate and obtain from the soil of Nanyang Prefecture plantation wheat, (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica to be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 22nd, 2013, postcode 100101), its deposit number is CGMCC No.7185.The situation biological with other is the same, and the bacterial strain DDB001 that the present invention has degraded vomitoxin still easily morphs.Therefore, can utilize physics and chemistry method known in the art to obtain the mutant strain of this bacterial strain.For example, can be by with chemical agent, as N-methyl-N'-nitro-N-nitrosoguanidine, processing obtains its mutant strain, these mutagenic mutants, as long as retained the such feature of vomitoxin degradation capability, also belong to a part of the present invention.
The present invention also provides a kind of microbial inoculum, and its activeconstituents is De Wosi Salmonella (Devosia sp.) DDB001, and its deposit number is CGMCC No.7185.This microbial inoculum can be that liquid-type microbial inoculum can be also solid type microbial inoculum, and prepares by published preparation method in prior art.Particularly, the invention provides a kind of preparation method of above-mentioned microbial inoculum, the method comprises:
(1) be that De Wosi Salmonella (Devosia sp.) DDB001 of CGMCC No.7185 activates on solid medium by deposit number;
(2) bacterial classification after activation is inoculated in seed culture medium and is cultured to logarithmic phase, make seed liquor;
(3) described seed liquor is inoculated in fermention medium and is cultured to stationary phase, make liquid-type microbial inoculum.
Further, the method also comprises mixes aforesaid liquid type microbial inoculum with sorbent material, after being dried, makes solid type microbial inoculum.Wherein, the weight ratio of described liquid-type microbial inoculum and sorbent material is 1:2 – 10, and described sorbent material can be one or more in corn cob meal, wheat bran, starch, diatomite, vermiculite, light calcium carbonate or peat.
Further, in above-mentioned preparation method, the substratum that is used for cultivating De Wosi Salmonella culture is diversified, but economy, cellular biomass, detoxification active angle, preferably some substratum from producing.For example, preferred carbon source is glucose, but also can use maltose, glycerine, fructose, semi-lactosi, seminose, alpha-lactose etc.Preferred nitrogenous source is yeast extract paste, casein peptone, but also can use corn steep liquor, yeast extract paste, extractum carnis etc.The nutrition inorganic salt that can mix in substratum have the conventional soluble salt that can produce following ion: zine ion, sodium ion, magnesium ion, calcium ion, iron ion, chlorion, carbanion, sulfate ion, nitrate ion etc.In an embodiment of the invention, described seed culture medium is made up of according to volume ratio following component: yeast extract paste 0.8%, peptone 0.4%, glucose 0.2%, sodium-chlor 0.4%, PH7.2 – 7.4; Described fermention medium is made up of according to volume ratio following component: yeast extract paste 0.8%, peptone 0.4%, glucose 0.2%, NaCl0.4%, K
2hPO
43H
2o0.3%, KH
2pO
40.075g/L, PH7.2 – 7.4.
Further, in above-mentioned preparation method, the bacterial classification after described activation is inoculated into seeding tank by 1 – 10% inoculum size of seed culture medium in seeding tank; Described seed liquor is inoculated into fermentor tank by 1 – 10% inoculum size of fermentation cylinder for fermentation substratum.
Further, step (3) fermentation culture conditions is: the air flow of sterile air is 1:0.5 – 1.2, and stirring velocity is 240 revs/min of 80 –, 37 DEG C of culture temperature approximately 28 –, and in liquid-type microbial inoculum, viable cell quantity at least reaches 10
9more than CFU/ml.
The present invention also provides described De Wosi Salmonella (Devosia sp.) DDB001 or the application of its microbial inoculum in degraded vomitoxin.
Particularly, in the time of degraded vomitoxin, any in employing following (a) or (b) mode:
(a). liquid-type microbial inoculum is sprayed in cereal or feed and the feedstuff raw material for the treatment of detoxification, degraded vomitoxin, the viable cell quantity of described liquid-type microbial inoculum at least reaches 10
7cFU/ml;
(b). the addition that is 0.01 – 5% according to quality percentage composition by solid type microbial inoculum joins in the cereal or feedstuff raw material and feed for the treatment of detoxification, mixes degraded vomitoxin.
In above-mentioned application, described cereal is corn, wheat, barley, paddy or jowar.Described feedstuff raw material and feed are the feed that the feedstuff raw materials such as corn, wheat, barley, paddy or jowar and they process.
The invention has the advantages that: preserving number provided by the invention is that the De Wosi Salmonella strain of CGMCC No.7185 has good degradation effect to vomitoxin.The advantages such as the solid type or the liquid-type microbial preparation that use the bacterial strain production degraded vomitoxin that preserving number is CGMCC No.7185, have production and application cost low, simple, easy to operate, and vomitoxin degraded is safe, efficient.Bacterial strain provided by the invention and microbial inoculum can be for the removals of vomitoxin in cereal, feed, feedstuff raw material, for solving toxin pollution problem in feed and raw material thereof, improve grain utilization ratio, ensure that the safety in production of livestock industry, raising animal husbandry economy benefit have great importance.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but be not to limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is routine biochemistry reagent suppliers and buys and obtain.
Embodiment 1 moral Butterworth Salmonella (Devosia sp.) DDB001 obtains and qualification
From Nanyang, henan wheat field, gather soil sample, taking 96 microwell plates as culture carrier, first by the enrichment culture method of 6 continuous toxin concentration gradients, acquisition has the bacteria suspension of degraded vomitoxin, then bacteria suspension is coated on to TY agar plate (yeast extract 4g/L by method of dilution butteron on plate with suitable extent of dilution, Tryptones 8g/L, agar powder 15g/L, pH7.2, 121 DEG C of sterilizing 30min) on, good and the colonial morphology of picking separation degree, the bacterium colony that color is different, be TY liquid nutrient medium (the yeast extract 4g/L of 200 μ g/mL in DON concentration, Tryptones 8g/L, pH7.2, 121 DEG C of sterilizing 30min) in carry out detoxification test, ethyl acetate is extracted residual toxin and is carried out HPLC detection, verify the toxin degradation effect of each pure growth, the final bacterial strain DDB001 that successfully obtains the vomitoxin of can degrading, the mono-bacterium colony of picking DDB001 is in TY liquid nutrient medium, while being cultured to logarithmic phase mid-term, glycerine with 50% mixes with culture equal-volume and is placed on-80 DEG C of preservations.
Adopt the classification such as 16S rRNA sequential analysis, Physiology and biochemistry, Chemical Characteristics to the degradation bacteria strains qualification of classifying, result shows that the De Wosishi that the classification position of this bacterial strain belongs in Hyphomicrobiaceae (Hyphomicrobiaceae) belongs to the novel species member in (Devosia), and the Genbank accession number of its 16S rRNA sequence is JX392051.On January 22nd, 2013 Devsoia sp.DDB001 is preserved in to China Committee for Culture Collection of Microorganisms's common micro-organisms center and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), its deposit number is CGMCC No.7185.
Cultural characteristic and morphological specificity: this bacterial strain is on TY solid medium, at 30 DEG C of temperature, through its colonial morphology of the cultivations of 3 days: ivory white, circle and neat in edge, smooth surface and projection, big or small 0.5 – 1.5 μ m; Microscopic morphology: bar-shaped, big or small 0.7 – 1.3 μ m × 0.5 – 0.8 μ m, single polar flagella (as shown in Figure 1), without sporulation, Gram-negative, strictly aerobic.
Physiological characteristic: catalase and oxydase are all positive, can grow having in salt or salt-free TY substratum, still can grow, but have no growth phenomenon under 5% concentration at the TY substratum that contains 3% sodium-chlor; PH tolerance range is 5.0 – 9.0, has surveyed 40 DEG C of growth temperature range 15 –.The carbon source that can utilize has L-arabinose, D-cellobiose, L-trehalose, D-semi-lactosi, alpha-D-glucose, maltose, D-MANNOSE, D-melibiose, gluconic acid, α-hydroxybutyric acid, D, Pfansteihl, D-Glucose diacid, N-ACETYL-D-GLUCOSAMINE, ribitol, gentiobiose, α-D-lactose, L-rhamnosyl, D-glucitol, sucrose, D-trehalose, turanose, Xylitol, monomethyl succinate, glucuronamide, glycerine, D-Glucose-6-phosphoric acid, D-R alcohol, D-Fructose, PEARLITOL 25C, lactulose; Unavailable carbon source has dextrin, acetic acid, Serine, D-Ser, L-PROLINE, L-Leu, raffinose, N-acetyl-D-galactosamine, cis equisetic acid, D-Glucosaminic acid, D-Glucose aldehydic acid.
16S rRNA sequential analysis: utilize primers F 9 (5'-GAGTTTGATCCTGGCTCAG-3') and R1544 (5'-AGAAAGGAGGTGATCCA-3') to carry out pcr amplification, length is that in the 16SrRNA sequence of 1479bp and Genbank database, existing sequence is carried out Blast and compared, it is higher that this sequence and Devosia belong to 16S rRNA similarity, and similarity scope is between 94.6 – 96.0%.Belong in 16 all kinds of generally acknowledging at De Wosishi, similarity is the highest is Devosia insulae DS-56
t.Phylogenetic tree show (Fig. 2), DDB001 by cluster in this group of Devosia, DDB001 and Devosia insulae DS-56
t(EF012357) be in a branch, but their 16S rRNA sequence similarity only reaches 96%(<97%), can not be attributed to same kind.
The main fatty acid of chemical constitution: DDB001 comprises 11-methyl ω 7c 18 carbon monounsaturated fatty acids (28.16%), saturated 16 carbon fatty acids (21.72%), ω 8c type ring type 19 saturated fatty acids (16.69%) and saturated 18 carbon fatty acids (14.95%) (in table 1); Breathing quinone main Types is Coenzyme Q10 99.0; Polar lipid main Types is phosphatidyl glycerol (PG) and diphosphatidylglycerol (DPG); G+C mol% is 64.6%.
Table 1
Show according to above all results, DDB001 is different from the typical strain of 16 kinds of generally acknowledging in the past, be enough to become a novel species in De Wosishi genus, this classification is the retrieval of comparing and the description of the similar species of having delivered is done based on direct laboratory.
The detoxification efficiency of embodiment 2 moral Butterworth Salmonella (Devosia sp.) DDB001 in TY substratum
DDB001 after activation is seeded to 1% inoculum size in the TY liquid nutrient medium of the 50mL that contains 200 μ g/mL vomitoxins, the detoxification of carrying out under the oscillating condition of 200 revs/min two days is cultivated, the remaining vomitoxin of ethyl acetate extracting, in final cultures, the residue content of vomitoxin shows through HPLC detected result: the vomitoxin in TY is degradable, degradation rate reaches 100%, and produces the toxin metabolite (as shown in Figure 3) that retention time is about 9min.
The preparation of embodiment 3 moral Butterworth Salmonella (Devosia sp.) DDB001 liquid-type microbial inoculums
Solid medium component and proportioning: yeast extract paste 0.8%, peptone 0.4%, NaCl0.5%, agar powder 1.5%, high-temp steam sterilizing 30min at 7.4,121 DEG C of pH7.2 –.
Seed culture medium component and proportioning: yeast extract paste 0.8%, peptone 0.4%, glucose 0.2%, sodium-chlor 0.5%, pH7.2 – 7.4; Fermention medium component and proportioning: yeast extract paste 0.8%, peptone 0.4%, glucose 0.2%, NaCl0.5%, K
2hPO
43H
2o0.3%, KH
2pO
40.075g/L, pH7.2 – 7.4.
(1) actication of culture: the De Wosi Salmonella DDB001 that is CGMCC No.7185 by preserving number is inoculated on solid medium, cultivates 3 days under 30 DEG C of conditions, and measures its toxin degradation property, is inoculated on test tube slant for subsequent use.
(2) seed culture: in picking list bacterium colony access seed culture medium, be cultured to logarithmic phase under 30 DEG C of conditions from slant medium, make bacterial classification; Then, with the seeding tank of 500 liters, 350 liters of seed culture medium charging capacitys, 121 DEG C of high pressure moist heat sterilizations after feeding intake, are cooled to after 30 DEG C, by above-mentioned cultured bacterial classification with 10%(volume percent) inoculum size inoculate into seeding tank, stirring velocity is 200 revs/min, 30 DEG C of culture temperature, sterile air intake is 1:0.8(volume ratio), approximately 48 – 60 hours are cultured to logarithmic phase, obtain seed liquor.
(3) fermentation culture: employing volume is the production tank of 5000 liters, and 3500 liters of fermention medium charging capacitys, at 1.1kg/cm
2pressure, the temperature of 121 DEG C under, carry out high pressure moist heat sterilization, after sterilizing cooling 30 DEG C, seed liquor is inoculated into fermentor tank by 10% inoculum size, fermentation condition: the air flow of sterile air is 1:0.8 – 1.2, stirring velocity is 240 revs/min, 30 DEG C of culture temperature, incubation time approximately 48 – 60 hours, puts and forms the liquid-type microbial inoculum with vomitoxin degraded after tank.In this liquid-type microbial inoculum, viable cell quantity at least reaches 10
9more than CFU/ml.
The preparation of embodiment 4 moral Butterworth Salmonella (Devosia sp.) DDB001 solid type microbial inoculums
After the DDB001 liquid-type microbial inoculum that embodiment 3 is produced mixes according to the weight ratio of 1:4 with corn cob meal, 40 DEG C of following cryodrying to moisture are below 8%, form solid state, thereby make the solid type microbial inoculum of DDB001.
The degradation effect of embodiment 5 moral Butterworth Salmonella (Devosia sp.) DDB001 liquid-type microbial inoculums to vomitoxin in cereal
DDB001 liquid-type microbial inoculum prepared by embodiment 3 first dilutes, and the liquid-type bacterial strain viable cell quantity after dilution at least reaches 10
7individual/ml, be sprayed onto in the Semen Maydis powder that pollutes DON as test group by weight the ratio of 1:1, be sprayed onto and pollute in the Semen Maydis powder of toxin as a control group with same ratio without bacteria fermentation culture medium, every group of three repetitions, detoxification after 3 days at the temperature of 30 DEG C after mixing, accurately weigh 5g sample in centrifuge tube from control group and test group respectively, add the acetonitrile of 20ml85% to detoxification sample, vibration makes the abundant extracting of toxin, 4000 revs/min, centrifugal 15 minutes, supernatant was transferred in new centrifuge tube; Again add 85% acetonitrile solution of 12ml, vibration mixes extracting again, similarity condition is centrifugal, merging supernatant spontaneous evaporation to acetonitrile volatilizees completely, add the saturated sodium-chloride of 12ml and mix, then use twice of the ethyl acetate extracting of 12ml, after merging twice ethyl acetate and naturally volatilizing mutually, 1ml methanol constant volume is dissolved, detect the content of vomitoxin with high performance liquid chromatography, result shows the detoxification through 3 days to vomitoxin pollution Semen Maydis powder of DDB001 liquid-type microbial inoculum, and its degradation rate can reach 92.4%, and control group is without any signs of degradation (the results are shown in Figure 4).
The degradation effect of embodiment 6 moral Butterworth Salmonella (Devosia sp.) DDB001 solid type microbial inoculums to vomitoxin in cereal
Solid type microbial inoculum prepared by embodiment 4 adds to according to 4% weight ratio in the test group Semen Maydis powder that pollutes vomitoxin, fermention medium and corn cob meal (mixing with 1:4 ratio) mixture joins in the control group Semen Maydis powder that pollutes vomitoxin with 4% weight ratio equally, control group and test group are all added to distilled water in the ratio of 1:1, every group of three repetitions, detoxification 3 days at the temperature of 30 DEG C after mixing, accurately weigh 5g sample from control group and test group respectively and put into 50ml centrifuge tube, add the acetonitrile of 20ml85% to sample, vibration makes the abundant extracting of toxin, 4000 revs/min, centrifugal 15 minutes, supernatant is transferred in new centrifuge tube, solid substance adds 85% acetonitrile solution of 12ml again, vibration mixes extracting again, centrifugal under similarity condition, merging supernatant spontaneous evaporation to acetonitrile volatilizees completely, add the saturated sodium-chloride of 12ml and mix, then use twice of the ethyl acetate extracting of 12ml, after merging twice ethyl acetate and naturally volatilizing mutually, 1ml methanol constant volume is dissolved, detect the content of vomitoxin with high performance liquid chromatography, result shows the detoxification through 3 days to pollution Semen Maydis powder of DDB001 solid type microbial inoculum, and its degradation rate can reach 84.9%, and control group is without any signs of degradation (the results are shown in Figure 4).
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give exhaustive to all embodiments.Everyly belong to apparent variation or the still row in protection scope of the present invention of variation that technical scheme of the present invention extends out.