CN106520593B - A kind of microorganism formulation that can reduce zearalenone and vomitoxin in ensiling - Google Patents
A kind of microorganism formulation that can reduce zearalenone and vomitoxin in ensiling Download PDFInfo
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Abstract
The invention belongs to microbial preparation fields, and in particular to a kind of lactobacillus plantarum preparation and its application that can reduce zearalenone and vomitoxin in ensiling.Preparation provided by the present invention is prepared by lactobacillus plantarum CGMCC No.11932, it can be effectively reduced vomitoxin reduction and the zearalenone in ensilage, every kilogram of corn Silage adds 0.05 gram of microorganism formulation, it is sealed by fermentation 30 days, vomitoxin reduces by 36.5%, and zearalenone reduces by 49.38%；Every kilogram of alfalfa silage adds 0.10 gram of microorganism formulation, is sealed by fermentation 35 days, and vomitoxin concentration is reduced to 18.88 μ g/Kg by the 24.14 μ g/Kg originated, and zearalenone concentration reduces by 26.83%；Meanwhile ensilage is after lactobacillus plantarum SCI-03 fermentation, and it is soft, there is sour-sweet fragrant, livestock is fond of, and digestibility can be improved 20%.
Description
Technical field:
The invention belongs to microbial preparation fields, and in particular to a kind of to reduce zearalenone and vomiting poison in ensiling
The lactobacillus plantarum preparation of element and its application.
Background technique:
It rises along with the continuous development of China's livestock and the continuous of high-quality feed price, ensilage is with it
Easy to operate, storage tolerance and the characteristic having a wide range of application have been widely used.
But during the growth process due to the ensilings such as corn and clover raw material, harmful mould of surface attachment can produce vomiting poison
The mycotoxins such as element and zearalenone, mould early period of ensiling, which breeds rapidly, in addition can also generate a large amount of mycotoxin,
Certain influence may be generated to the health of domestic animal after leading to ensiling, situation seriously may result in the death of livestock.Wherein,
The mycotoxin zearalenone for being widely present ensiling may act on the reproductive system of livestock, so that domestic animal is generated female hormone high
Into disease, gravidic animal miscarriage, stillborn foetus and monster etc., it is also possible to the poisoning symptom for causing central nervous system, as mind presses down
Strongly fragrant and incoordination etc..Vomitoxin has very strong toxicity to animal, can cause animal vomiting, diarrhea, skin irritatin, food refusal,
Neurological disorders, miscarriage, stillborn foetus etc..
Relatively broad about above-mentioned toxin Study on degradation at present, the Chinese invention if publication No. is CN 105771147A is special
" zearalenone degradation agent and its application " is bright discloses a kind of zearalenone (ZON) degradation agent for benefit comprising ingredient
A, or ingredient a and ingredient b, wherein the volume ratio of ingredient a and ingredient b are 1:3-3:1；The ingredient a is obtained through following step:
Candida utili, bacillus subtilis K3 and bacillus subtilis K4 obtain each bacterium solution after individually cultivating 16-24h, then with
The volume ratio of 1:1:1,1:2:2 or 1:3:3 mix to obtain the final product；The ingredient b is rice song obtained after the fermented culture of aspergillus oryzae
Mould crude enzyme liquid.It is found through experiment that: when the ratio of candida utili, bacillus subtilis K3, bacillus subtilis K4 are 1:2:2
When, ZON degradation rate reaches 92.21%；When the proportion of three kinds of compound probiotics and aspergillus oryzae is 2:1, ZON degradation rate reaches
95.15%.Chinese invention patent ZL201110459937.1 discloses a kind of " microbial degradation method of zearalenone ",
It is with bacillus cereus and/or lactobacillus plantarum bacterium powder come degrading zearalenone.The present invention utilizes bacillus cereus
And/or lactobacillus plantarum bacterium powder degrades to zearalenone, by being measured respectively under Imitative gastroenteric environments in vitro
Above-mentioned bacterium powder finds out its best degradation condition to ZEN to the degradation effect of ZEN, is biological solution further to be developed
Toxic agent, the degradation for ZEN in cereal and feed provide strong guidance.
But at present not yet occur can simultaneously degrading zearalenone and vomitoxin probiotics, therefore, screening
With can degrading zearalenone but also vomitoxin function of degrading microorganism formulation, have for livestock-raising
Significance.
Summary of the invention:
To achieve the goals above, the present invention provide it is a kind of can be with the microorganism of degrading zearalenone and vomitoxin
Preparation, the microorganism formulation include lactobacillus plantarum, and preparation method is specific as follows:
(1) lactobacillus plantarum high density fermentation culture
Lactobacillus plantarum seed liquor is accessed into high density fermentation culture medium by 5-10% inoculum concentration, initial pH6.5-7.0 leads to
Nitrogen keeps pressure 0.03-0.05MPa, revolving speed 60-80r/min, permanent pH 5.5-5.9,37 DEG C of fermentation 5-8h, lactobacillus plantarum
Viable count reaches 1010Fermentation liquid is made in CFU/mL or more；
High density fermentation culture medium: soyabean protein powder 60-80g, glucose 40-80g, anhydrous sodium acetate 10-20g, K2HPO4
7-10g, sodium citrate 9-12g, yeast powder 7-15g, Tween-80 0.1-0.2g, pH6.5-7.0；
(2) protective agent is added
Protective agent dry powder is directly added to fermentation liquid, is uniformly mixed, the mass ratio composition of protective agent each component and fermentation liquid
Are as follows: skimmed milk 3.0-8.0% (m/m), trehalose 1.5-5.0% (m/m), soyabean protein powder 10-15% (m/m), vitamin C
0.4-0.6% (m/m)；
(3) dry
It is freeze-dried to obtain freeze-dried vaccine powder microorganism formulation by the above-mentioned fermentation liquid being uniformly mixed with protective agent, it obtains
Bacterium powder lactobacillus plantarum viable count can achieve 2 × 1011CFU/g or more；
Further, the lactobacillus plantarum is specially lactobacillus plantarum (Lactobacillus plantarum) SCI-
03, which is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 24th, 2015
(address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101), preservation are compiled
Number CGMCC No.11932；
The strain lactobacillus plantarum SCI-03 (initial inoculum 1 × 106CFU/mL) containing 100 μ g/L Gibberella zeaes
In the MRS fluid nutrient medium of ketenes, 25 DEG C after constant temperature incubation 24 hours, zearalenone concentration is reduced to 58 μ g/L, plant cream
Bacillus SCI-03 viable count is 8 × 106CFU/mL significantly reduces zearalenone content；Lactobacillus plantarum SCI-03 is (initial
Inoculum concentration 1 × 106CFU/mL) in the MRS fluid nutrient medium containing 100 μ g/L vomitoxins, 25 DEG C constant temperature incubation 24 hours
Afterwards, zearalenone concentration is reduced to 68 μ g/L, and lactobacillus plantarum SCI-03 viable count is 7 × 106CFU/mL, degradation effect are aobvious
It writes.
It is described can be as follows with the application method of degrading zearalenone and the microorganism formulation of vomitoxin:
Every kilogram of ensilage adds 0.05-0.10 grams of microorganism formulation, is sealed by fermentation 30-35 days, can make ensilage
In vomitoxin reduce by 40% or so, zearalenone reduce by 50% or so.
The utility model has the advantages that
1, microorganism formulation provided by the invention can be effectively reduced the reduction of the vomitoxin in ensilage and corn is red
Mould ketenes, every kilogram of corn Silage add 0.05 gram of microorganism formulation, are sealed by fermentation 30 days, and vomitoxin concentration reduces
36.5%, zearalenone concentration reduces by 49.38%；Every kilogram of alfalfa silage adds 0.10 gram of microorganism formulation, close
Seal ferment 35 days, vomitoxin concentration was reduced to 18.88 μ g/Kg, zearalenone concentration drop by the 24.14 μ g/Kg originated
Low 26.83%；
2, the carbohydrate in ensiling raw material can be converted to organic acid by lactobacillus plantarum SCI-03, and phenyllactic acid resists
Bacterium polypeptide, bacteriocin, carbon dioxide reduce pH, inhibit the growth of harmful bacteria, improve storage-stable, and ammonia in ensilage
State nitrogen concentration is reduced, and butyric acid density reduces, and lactic acid concn increases, to improve ensilage quality and palatability, makes dry matter
Loss reduces.Ensilage is soft after lactobacillus plantarum SCI-03 fermentation, has sour-sweet fragrant, and livestock is fond of, disappears
Rate can be improved 20%.
Specific embodiment:
1 lactobacillus plantarum SCI-03 of embodiment degradation toxin ability measurement
(1) activation of strain: by the lactobacillus plantarum (Lactobacillus plantarum) of -80 DEG C of freezen protectives
SCI-03 is inoculated in 5ml MRS fluid nutrient medium (MRS culture medium: peptone 10g, beef extract 10g, yeast extract 5g, grape
Sugared 20g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, trisodium citrate 2g, Tween-80 1g, magnesium sulfate 200mg, manganese sulfate 54mg steam
Distilled water 1000mL, pH6.5 121 DEG C of 15min sterilizing are spare), 37 DEG C are cultivated 18 hours, are activated for such secondary culture 2 times
Strain；
(2) seed culture: above-mentioned activated strain is inoculated in seed culture medium by 2%, 37 DEG C of stationary cultures 10
Hour；
Seed culture medium: soyabean protein powder 60g, glucose 40g, anhydrous sodium acetate 10g, K2HPO47g, sodium citrate 9g,
Yeast powder 7g, Tween-80 0.1g, distilled water 1L, pH7.0；
(3) lactobacillus plantarum high density fermentation culture
Lactobacillus plantarum seed liquor is accessed into high density fermentation culture medium by 5% inoculum concentration, initial pH7.0 leads to nitrogen and protects
Pressure 0.03MPa, 5.9,37 DEG C of fermentation 5h of revolving speed 60r/min, permanent pH are held, lactobacillus plantarum viable count reaches 1010CFU/mL with
On；
High density fermentation culture medium: soyabean protein powder 60g, glucose 40g, anhydrous sodium acetate 10g, K2HPO47g, citric acid
Sodium 9g, yeast powder 7g, Tween-80 0.1g, distilled water 1L, pH7.0；
(4) zearalenone degradation capability measures: after adding 100 μ g/L zearalenones in MRS fluid nutrient medium,
By 1 × 106CFU/mL concentration access step (3) made from lactobacillus plantarum SCI-03 culture solution, 25 DEG C constant temperature incubation 24 hours
Afterwards, the concentration for measuring zearalenone is 58 μ g/L, significantly reduces zearalenone content；
(5) vomitoxin degradation capability measures: after adding 100 μ g/L vomitoxins in MRS fluid nutrient medium, by 1 ×
106CFU/mL concentration accesses lactobacillus plantarum SCI-03 culture solution made from step (3), and 25 DEG C after constant temperature incubation 24 hours, are surveyed
Determine vomitoxin concentration and be reduced to 68 μ g/L, degradation effect is significant；
MRS fluid nutrient medium composition is as follows: 10g peptone, 5g beef extract, 4g yeast extract, 20g glucose, 2g phosphoric acid
1000mL distillation is added in hydrogen dipotassium, 5g sodium acetate, 2g trisodium citrate, 1mL Tween 80,0.2g magnesium sulfate, 0.05g manganese sulfate
Water adjusts pH to 6.5,121 DEG C of sterilizing 15min.
The film that zearalenone and vomitoxin pass through 0.22 μm is filtered degerming.
Embodiment 2: a kind of preparation of microorganism formulation and its application in corn silage
(1) activation of strain: by the lactobacillus plantarum (Lactobacillus plantarum) of -80 DEG C of freezen protectives
SCI-03 is inoculated in 5ml MRS fluid nutrient medium (MRS culture medium: peptone 10g, beef extract 10g, yeast extract 5g, grape
Sugared 20g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, trisodium citrate 2g, Tween-80 1g, magnesium sulfate 200mg, manganese sulfate 54mg steam
Distilled water 1000mL, pH6.5 121 DEG C of 15min sterilizing are spare), 37 DEG C are cultivated 18 hours, are activated for such secondary culture 2 times
Strain；
(2) seed culture: above-mentioned activated strain 2% is inoculated in seed culture medium, 37 DEG C of stationary cultures 10 are small
When；
Seed culture medium: soyabean protein powder 60g, glucose 40g, anhydrous sodium acetate 10g, K2HPO47g, sodium citrate 9g,
Yeast powder 7g, Tween-80 0.1g, distilled water 1L, pH7.0；
(3) lactobacillus plantarum high density fermentation culture
Lactobacillus plantarum SCI-03 seed liquor is accessed into high density fermentation culture medium by 8% inoculum concentration, initial pH6.5 leads to
Nitrogen keeps pressure 0.04MPa, 5.5,37 DEG C of fermentation 6h of revolving speed 70r/min, permanent pH, and lactobacillus plantarum viable count reaches
1010CFU/mL or more；
High density fermentation culture medium: soyabean protein powder 70g, glucose 60g, anhydrous sodium acetate 15g, K2HPO48g, citric acid
Sodium 10g, yeast powder 10g, Tween-80 0.15g, distilled water 1L, pH6.5；
(4) protective agent is added
Fermentation liquid directly adds that protective agent dry powder blend is uniform, the mass ratio composition of protective agent each component and fermentation liquid are as follows:
Skimmed milk 3.0% (m/m), trehalose 1.5% (m/m), soyabean protein powder 10% (m/m), vitamin C 0.4% (m/m)；
(5) dry
Freeze-dried vaccine powder is obtained by the above-mentioned fermentation liquid being uniformly mixed with protective agent is freeze-dried, obtains bacterium powder plant cream bar
Bacterium viable count can achieve 5 × 1011CFU/g；
(6) in corn silage zearalenone and vomitoxin degradation
Every kilogram of ensilage adds 0.05 gram of microorganism formulation, be sealed by fermentation 30 days, and vomitoxin concentration is by originating
44.98 μ g/Kg, are reduced to 28.54 μ g/Kg, and zearalenone concentration is reduced to 51.94 μ by the 102.61 μ g/Kg originated
g/Kg。
Embodiment 3: a kind of preparation of microorganism formulation and its application in alfalfa ensilage
(1) activation of strain: by the lactobacillus plantarum (Lactobacillus plantarum) of -80 DEG C of freezen protectives
SCI-03 is inoculated in 5ml MRS fluid nutrient medium (MRS culture medium: peptone 10g, beef extract 10g, yeast extract 5g, grape
Sugared 20g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, trisodium citrate 2g, Tween-80 1g, magnesium sulfate 200mg, manganese sulfate 54mg steam
Distilled water 1000mL, pH6.5 121 DEG C of 15min sterilizing are spare), 37 DEG C are cultivated 18 hours, are activated for such secondary culture 2 times
Strain；
(2) seed culture: above-mentioned activated strain is inoculated in seed culture medium by 2%, 37 DEG C of stationary cultures 10
Hour；
Seed culture medium: soyabean protein powder 60g, glucose 40g, anhydrous sodium acetate 10g, K2HPO47g, sodium citrate 9g,
Yeast powder 7g, Tween-80 0.1g, distilled water 1L, pH7.0；
(3) lactobacillus plantarum high density fermentation culture
Lactobacillus plantarum SCI-03 seed liquor is accessed into high density fermentation culture medium by 10% inoculum concentration, initial pH7.0 leads to
Nitrogen keeps pressure 0.05MPa, 5.7,37 DEG C of fermentation 8h of revolving speed 80r/min, permanent pH, and lactobacillus plantarum viable count reaches
1010CFU/mL or more；
High density fermentation culture medium: soyabean protein powder 80g, glucose 80g, anhydrous sodium acetate 20g, K2HPO410g, lemon
Sour sodium 12g, yeast powder 15g, Tween-80 0.2g, distilled water 1L, pH7.0；
(4) protective agent is added
Fermentation liquid directly adds that protective agent dry powder blend is uniform, the mass ratio composition of protective agent each component and fermentation liquid are as follows:
Skimmed milk 8.0% (m/m), trehalose 5.0% (m/m), soyabean protein powder 15% (m/m), vitamin C 0.6% (m/m)；
(5) dry
The above-mentioned fermentation liquid being uniformly mixed with protective agent is freeze-dried to obtain freeze-dried vaccine powder, obtains bacterium powder lactobacillus plantarum
Viable count can achieve 4 × 1011CFU/g；
(6) in alfalfa ensilage zearalenone and vomitoxin degradation
Every kilogram of ensilage adds 0.10 gram of microorganism formulation, be sealed by fermentation 35 days, and vomitoxin concentration is by originating
24.14 μ g/Kg, are reduced to 18.88 μ g/Kg, and zearalenone concentration is reduced to 45.92 μ g/ by the 62.76 μ g/Kg originated
Kg。
The detection method of zearalenone:
Mobile phase: A water, B (V (acetonitrile): V (water)=36:64), flow velocity 0.5mL/min, analytical column: ZORBAX
Elipse AAA C18 (3.5um, 4.6 × 150mm)；Detector: FLD (fluorescence detector)；Excitation wavelength: 235nm；Transmitted wave
It is long, 460nm.
Mass Spectrometry Conditions: mass spectrum mode: ESI cation；Capillary voltage: 2.5KV；Orifice potential: 40V；Taper hole throughput:
0L/Hr；Desolventizing gas flow: 600L/Hr；Source temperature: 100 DEG C, desolventizing temperature: 600 DEG C.
The detection method of vomitoxin:
Mobile phase: in A (V water: V methanol: V acetonitrile=96:2:2), every 1000mL A plus 5mg sodium azide, B acetonitrile flow
Speed: 0.5ml/min, analytical column: C18 (5um, 4.6 × 100mm), detector: VWD, Detection wavelength: 220nm.
Mass Spectrometry Conditions: mass spectrum mode: ESI cation；Capillary voltage: 2.5KV；Orifice potential: 40V；Taper hole throughput:
0L/Hr；Desolventizing gas flow: 600L/Hr；Source temperature: 100 DEG C, desolventizing temperature: 600 DEG C.
Claims (4)
1. one kind can be by that will plant with the microorganism formulation of degrading zearalenone and vomitoxin, the microorganism formulation
Object lactobacillus prepares freeze-dried vaccine powder and forms by high density fermentation, addition protective agent, which is characterized in that the lactobacillus plantarum tool
Body is lactobacillus plantarum (Lactobacillus plantarum) SCI-03, deposit number CGMCC No.11932.
2. one kind as described in claim 1 can be with the microorganism formulation of degrading zearalenone and vomitoxin, feature
It is, preparation method is specific as follows:
(1) lactobacillus plantarum high density fermentation culture
Lactobacillus plantarum seed liquor is accessed into high density fermentation culture medium by 5-10% inoculum concentration, initial pH6.5-7.0 leads to nitrogen
Keep pressure 0.03-0.05MPa, revolving speed 60-80r/min, permanent pH 5.5-5.9,37 DEG C of fermentation 5-8h, lactobacillus plantarum viable bacteria
Number reaches 1010Fermentation liquid is made in CFU/mL or more；
The lactobacillus plantarum is specially lactobacillus plantarum (Lactobacillus plantarum) SCI-03, deposit number
CGMCC No.11932；
(2) protective agent is added
Protective agent dry powder is directly added to fermentation liquid, is uniformly mixed,
(3) dry
Freeze-dried vaccine powder is obtained by the above-mentioned fermentation liquid being uniformly mixed with protective agent is freeze-dried, and it is living to obtain bacterium powder lactobacillus plantarum
Bacterium number reaches 2 × 1011CFU/g or more.
3. one kind as claimed in claim 2 can be with the microorganism formulation of degrading zearalenone and vomitoxin, feature
It is, the high density fermentation culture medium composition are as follows: soyabean protein powder 60-80g, glucose 40-80g, anhydrous sodium acetate 10-
20g, K2HPO47-10g, sodium citrate 9-12g, yeast powder 7-15g, Tween-80 0.1-0.2g, distilled water 1L, pH6.5-
7.0。
4. one kind as claimed in claim 2 can be with the microorganism formulation of degrading zearalenone and vomitoxin, feature
It is, the mass ratio composition of the protective agent each component and fermentation liquid are as follows: skimmed milk 3.0-8.0%, trehalose 1.5-5.0%,
Soyabean protein powder 10-15%, vitamin C 0.4-0.6%.
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| CN109007544A (en) * | 2018-08-04 | 2018-12-18 | 苏州登高生物科技有限公司 | The method for reducing vomitoxin in bagasse |
| CN110122734A (en) * | 2019-05-19 | 2019-08-16 | 河南工业大学 | A kind of preparation method for the probiotics for protecting kidney and releasing zearalenone |
| CN111996151B (en) * | 2020-09-17 | 2021-02-09 | 黑龙江省农业科学院畜牧兽医分院 | Microbial preparation containing multiple lactic acid bacteria and application of microbial preparation in corn silage |
| CN116083296A (en) * | 2022-11-30 | 2023-05-09 | 吉林中粮生化有限公司 | Lactobacillus plantarum, microbial inoculum containing same and method for degrading vomitoxin |
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| CN102550893A (en) * | 2011-12-31 | 2012-07-11 | 青岛蔚蓝生物集团有限公司 | Micro-biological degradation method for zearalenone |
| CN102864096A (en) * | 2012-04-18 | 2013-01-09 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum and preparation method thereof for high-density culture and freeze-drying bacteria powder |
| CN104099251A (en) * | 2014-04-11 | 2014-10-15 | 江南大学 | New aspergillus niger strain and application thereof in degradation of a plurality of kinds of fungaltoxin |
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| CN102550893A (en) * | 2011-12-31 | 2012-07-11 | 青岛蔚蓝生物集团有限公司 | Micro-biological degradation method for zearalenone |
| CN102864096A (en) * | 2012-04-18 | 2013-01-09 | 北京和美科盛生物技术有限公司 | Lactobacillus plantarum and preparation method thereof for high-density culture and freeze-drying bacteria powder |
| CN104099251A (en) * | 2014-04-11 | 2014-10-15 | 江南大学 | New aspergillus niger strain and application thereof in degradation of a plurality of kinds of fungaltoxin |
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