CN102061271A - Lactobacillus casei bacteriocin and use thereof in feed - Google Patents
Lactobacillus casei bacteriocin and use thereof in feed Download PDFInfo
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- CN102061271A CN102061271A CN2010102775571A CN201010277557A CN102061271A CN 102061271 A CN102061271 A CN 102061271A CN 2010102775571 A CN2010102775571 A CN 2010102775571A CN 201010277557 A CN201010277557 A CN 201010277557A CN 102061271 A CN102061271 A CN 102061271A
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Abstract
The invention discloses lactobacillus casei bacteriocin and use thereof in feed. The lactobacillus casei provided by the invention is separated from Tibet plateau traditional fermented yak yogurt, and the produced bacteriocin is obtained by ammonium sulfate precipitation, Sephadex G-100 gel filtration and reversed-phase high-performance liquid chromatography (RP-HPLC). The bacteriocin has thermal and acid stability, can be degraded by protease and has a wide antibacterial spectrum. When the bacteriocin is added into feed, and the growth of bacteria can be inhibited. The bacteriocin has a bright prospect in use as a feed additive.
Description
Technical field
The present invention relates to the bacteriocin of strain lactobacterium casei product and the application in feed thereof.
Background technology
Bacteriocin lab (Bacteriocins of LAB) is meant that milk-acid bacteria is had protein, polypeptide or the precursor polypeptide (Klaenhammer of bacteriostatic activity by rrna synthetic one class in metabolic process, TR.Genetics of bacteriocins produced by lactic acid bacteria.FEMS.Microbiology Review[J] .1993.12:39～85), aspects such as multiple pathogenic micro-organism and food spoilage bacterium are significant suppressing.
China's livestock industry sustainable and stable development has become mainstay of the national economy industry over past ten years.China's meat, eggs rank first in the world; Milk occupies the world the 5th.Yet veterinary drug and fodder additives are when having promoted the aquaculture production development, and the negative impact that brings also is quite serious, and be too high and have a strong impact on human health as the residual quantity of fodder additives in animal product.In recent years, incidents such as " beta-stimulants " poisoning, red-yolk duck egg, how precious fish and the trimeric cyanamide of domestic a large amount of generations, show invariably and guarantee food and feed safety, the extreme importance of green non-pollution, this not only is related to the sound development of aquaculture, and direct relation the significant problem of the stable and development of human physical and mental health, society.
Along with to the research of natural microbial antibiont progressively deeply, Chinese scholars finds that bacteriocin is a kind of good antibiotic substitute.Bacteriocin has good bacteriostatic activity owing to it has to multiple pathogenic micro-organism, there is not the antigen activity again, nontoxic, have no side effect, noresidue, no resistance, do not produce the accumulation of the property accumulated in animal body, therefore before 2000, just bacteriocin is widely used in food, makeup, feed etc. abroad, has obtained extraordinary effect.
Because bacteriocin itself has many advantageous properties: have selectivity sterilization effect preferably, thereby be not only applicable to general feed, also applicable to biological fodder etc.; Easily degraded, can not accumulate in animal body and cause untoward reaction by some proteolytic enzyme (as trypsinase) in the human body alimentary canal, simultaneously also free from environmental pollution; Have thermostability, and acidproof, low temperature resistant storage; The mouthfeel of feed, flavor etc. are had no adverse effects; Its use simultaneously can reduce the sterilization temperature of feed, reduces heat treatment time, thereby keeps feed original nutritive value and local flavor.Bacteriocin not only has the beneficial effect similar to antibiotic feed additive in a word, and nontoxic, have no side effect, noresidue, no resistance and have good diffustivity, simultaneously also free from environmental pollution, so bacteriocin will progressively develop, and in feed, be widely used.
Application in feed has huge application space to bacteriocin as antibiotic substitute, but present scholar both domestic and external also is in the starting stage to the applied research of bacteriocin, and commercially produced product is also very limited, therefore demands large-scale development and use urgently.In today of food safety, bacteriocin has become possibility as a kind of fodder additives of highly effective and safe, and along with going deep into of research and development, it is the human being's production green food that bacteriocin the most really substitutes microbiotic.
Summary of the invention
The lactobacterium casei that the purpose of this invention is to provide a strain bacteriocinogeny.This lactobacterium casei on August 9th, 2010 by being positioned at China. Wuhan. the Chinese typical culture collection center preservation of Wuhan University, its deposit number is: CCTCC M 2010197, the classification called after: lactobacterium casei lyT-7(
Lactobacillus caseiLyT-7).
Lactobacterium casei lyT-7 provided by the present invention separates from the sour yak Ruzhong of Qinghai-Tibet traditional zymotic to obtain.On the MRS solid medium, bacterium colony is circular, milky smooth colony.Thalline is a rod-short, catenation.According to following physiological and biochemical property: can utilize glucose, fructose, sucrose, maltose, semi-lactosi, sorbyl alcohol and N.F,USP MANNITOL, glucose fermentation produces not aerogenesis of acid, and the MR experiment is positive, H
2S experiment, indoles experiment, V-P test, gelatin liquification test, starch hydrolysis experiment, arginine produce the ammonia experiment and nitrate reduction is tested all negative.Be accredited as lactobacterium casei (Lactobacillus casei) through morphology physiological and biochemical property and 16S rDNA sequential analysis.
Second purpose of the present invention provides a kind of bacteriocin.
Bacteriocin provided by the present invention is the bacteriocin that fermentation lactobacterium casei (Lactobacillus casei) lyT-7 obtains.
The substratum that is used to cultivate lactobacterium casei (Lactobacillus casei) lyT-7 in the described method is a modified MRS culture medium, and composition consists of: soy peptone 10.0g, extractum carnis 10.0g, yeast extract 5g, glucose 20.0g, K
2HPO
42.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, MgSO
4.7H
2O 0.58g, MnSO
4.4H
2O 0.25g, tween 80 1ml, agar 15.0g, pH nature, distilled water 1000mL.(Ling Daiwen, eastern elegant pearl. the lactic-acid-bacterium classification is identified and experimental technique [M]. the .1999 of China Light Industry Press)
Culture condition in the described method is 30 ℃, static cultivation 48h.
Concrete, lactobacterium casei bacteriocin of the present invention can obtain by the following method, but is not limited thereto.
The bacteriocin that obtains in the described method can carry out the substep purifying by the following method:
The first step will be centrifugal by the fermented liquid that described cultural method obtains, and get supernatant liquor, add ammonium sulfate to whole saturation ratio 50% in this supernatant liquor, saltout; The centrifuging and taking precipitation obtains the bacteriocin throw out then; With the bacteriocin throw out amount of damming of packing into is in 1000 the dialysis tubing, dialyses in pure water, concentrates with polyoxyethylene glycol, obtains the bacteriocin crude extract.
Second step, the bacteriocin that obtains as stated above is further purified in accordance with the following methods: further adopt Sephadex G-100 gel filtration chromatography to carry out purifying this bacteriocin crude extract, described chromatography condition is: the Tris-HCl damping fluid with 0.01mol/L pH5.0 carries out wash-out, flow velocity is 3min/mL, and the collection retention time is that the protein peak of 180mim-460min is the active peak of bacteriocin; The bacteriocin sample of collecting is carried out RP-HPLC to be separated, preparation condition is: with water (containing 0.1% trifluoroacetic acid): the elution system that second fine (containing 0.1% trifluoroacetic acid) constitutes is carried out gradient elution, adopt the condition of gradient elution to be: the concentration at the 0-35min acetonitrile progressively is increased to 45% by 10%, and the collection retention time is that the protein peak of 20min-25min is the active peak of bacteriocin.
Lactobacterium casei bacteriocin of the present invention also can pass through other ordinary method purifying, as absorption method (Yang, R.G., Monty, C., Johnson, E.Novel method extract amountsof bacteriocins from Lactic acid bacteria, Appl Environ.Microbiol[J] .1992,58 (10): 3353～3359); Organic solvent method (Cheeseman, G.C.and Berridge, N.J.An improved meehod of preparingnisin, biochem J[J] .1957 65:603～608); Column chromatography (Wilimowska-pelc, A.The use of Gel-filtratlon for the isolation of purc Nisin from commercial products, Microbiological Polonica[J] .1976 (1): 71～77) etc.
By the bacteriocin crude extract that described method obtains, heat, acid are stablized, under 121 ℃ of conditions, handle 20min, still can keep bacteriostatic activity preferably; In the scope of pH2.0-11.0, keep stable; Can be by proteasome degradation, behind Proteinase K and trypsin acting, the bacteriostatic activity completely dissolve after the stomach en-effect, actively descends 85%.This bacteriocin antimicrobial spectrum is wide, can suppress intestinal bacteria (E.coli), Salmonella typhimurium (S.typhim uriu), streptococcus aureus (S.aureus), subtilis (B.subtili), bacillus cereus (B.cereus), lactobacillus bulgaricus (L.bulgaricus), Lactobacterium acidophilum (L..acidoph ilus), flavus (A.flavus).
The 3rd purpose of the present invention is that lactobacterium casei (Lactobacillus casei) bacteriocin is used in feed.
It is 13095IU/mL that the bacteriocin crude extract that will obtain by the first step of described method is measured its inhibitory potency, it is added in the feed, when adding in every 1000g feed in the scope of amount at 2.5mg-10.0mg of effective antibacterial substance, can obviously suppress the growth of bacterium, and along with the increase of additive capacity, fungistatic effect strengthens.
Description of drawings
To be feed store fungistatic effect under the condition in routine to Fig. 1
To be feed store fungistatic effect under the condition in reinforcement to Fig. 2
Embodiment
The separation and purification of embodiment 1 lactobacterium casei (Lactobacillus casei) lyT-7 bacteriocinogeny
1, the bacteriostatic activity analysis of lactobacterium casei (Lactobacillus casei) lyT-7
Bacteriostatic activity analytical procedure: under the incident light condition of 600 λ, the optical density value of indicator (intestinal bacteria and streptococcus aureus) bacteria suspension is adjusted to 0.120, be poured into 15mL LB solid medium, with diameter is that the punch tool of 7mm punches on substratum, the fermented supernatant fluid that adds lactobacterium casei (Lactobacillus casei) lyT-7, at room temperature spread 30min, under 30 ℃ of conditions, cultivated 16-18 hour, observe inhibition zone.
Lactobacterium casei (Lactobacillus casei) lyT-7 is inserted in the modified MRS nutrient solution, and its composition is: soy peptone 10.0g, extractum carnis 10.0g, yeast extract 5g, glucose 20.0g, K
2HPO
42.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, MgSO
4.7H
2O 0.58g, MnSO
4.4H
2O 0.25g, tween 80 1ml, agar 15.0g, pH nature, distilled water 1000mL.At 30 ℃ of static cultivation 48h, the centrifugal 4min of 8000rpm obtains fermented supernatant fluid.Fermented supernatant fluid is carried out following experiment:
(1) gets rid of the influence of acid: with the NaOH solution of 1M/L the pH of fermented supernatant fluid is transferred to 5.0, do bacteriostatic experiment.
(2) influence of eliminating hydrogen peroxide: fermented supernatant fluid at 80 ℃ of following water bath processing 10min, is done bacteriostatic test.
(3) proteolytic enzyme susceptibility experiment: will get rid of and add proteolytic enzyme k processing 1h in the fermented supernatant fluid after acid and hydrogen peroxide influence, and do bacteriostatic test.
After above-mentioned 1-2 step processing, fermented supernatant fluid still has activity; Handle the back bacteriostatic activity through above-mentioned the 3rd step and disappear, judge that tentatively the antibacterial substance that lactobacterium casei (Lactobacillus casei) lyT-7 produces belongs to bacteriocin.
2, the separation of bacteriocin, purifying
Its optimal culture condition that produces bacteriocin is 30 ℃ of static cultivation 48h, and the fermented liquid that obtains through the centrifugal 4min of 8000rpm, is obtained fermented supernatant fluid.In this supernatant liquor, add ammonium sulfate to whole saturation ratio 50%, saltout; The centrifuging and taking precipitation obtains the bacteriocin throw out then; With the bacteriocin throw out street flow of packing into is in 1000 the dialysis tubing, dialyses in pure water, concentrates with polyoxyethylene glycol, obtains the bacteriocin crude extract.Further adopt Sephadex G-100 gel filtration chromatography to carry out purifying this bacteriocin crude extract, described chromatography condition is: the Tris-HCl damping fluid with 0.01mol/L pH5.0 carries out wash-out, flow velocity is 3min/mL, and the collection retention time is that the protein peak of 180mim-460min is the active peak of bacteriocin; The bacteriocin sample of collecting is carried out RP-HPLC to be separated, preparation condition is: with water (containing 0.1% trifluoroacetic acid): the elution system that second fine (containing 0.1% trifluoroacetic acid) constitutes is carried out gradient (Teixeira, M.L., Cladera-Olivera, F., Santos, J., Brandelli, A.Purification and characterization of a peptide from Bacillus licheniformis showing dual antimicrobial and emulsifying activities.Food Research International[J], 2008,10:1064-1070) wash-out, adopt the condition of gradient elution to be: the concentration at the 0-35min acetonitrile progressively is increased to 45% by 10%, and the collection retention time is that the protein peak of 20-25min is the active peak of bacteriocin.
3, the antimicrobial spectrum of bacteriocin
Test strain is intestinal bacteria (E.coli), Salmonella typhimurium (S.typhim uriu), streptococcus aureus (S.aureus), subtilis (B.subtill), bacillus cereus (B.cereus), lactobacillus bulgaricus (L.bulgaricus), Lactobacterium acidophilum (L.acidoph ilus), flavus (A.flavus), Penicllium chrysogenum (P.chrysogenum), Mucor racemosus (M.racemosus), geotrichum candidum (G.candidum).
Get fermented supernatant fluid and adopt step 2 bacteriostatic activity analytical procedure, 11 strain indicators have been carried out antibacterial test.Experimental result is as shown in table 1.
The antimicrobial spectrum of table 1 lactobacterium casei lyT-7 fermented supernatant fluid
4, bacteriocin antagonistic property research
(1) thermostability experiment: with the bacteriocin crude extract 60 ℃ (water-baths), 80 ℃ (water-bath), 100 ℃ (water-bath) keeps 20min under 121 ℃ of (high pressure) conditions, measures bacteriostatic activity.The result is as shown in table 2, and under different treatment temp conditions, the bacteriostatic activity of crude extract changes hardly, and fungistatic effect slightly descends after handling 20min under 121 ℃ of conditions, but still has tangible bacteriostatic activity.Illustrate that the antibacterial substance that lyT-7 produces has good thermostability.
Table 2 different treatment temperature is to the influence of crude extract bacteriostatic activity
The diameter in hole is 7mm
(2) ph stability experiment: with 1mol/L NaOH and 1mol/L HCl the pH of sample is transferred to 2.0,3.0,4.0,5.0 respectively, 6.0,7.0,8.0,9.0,10.0 and 11.0, keep 1h at ambient temperature.The pH value being recalled to the variation of measuring bacteriostatic activity behind the pH5.0, is contrast with the fungistatic effect of handling under the pH6.0 condition behind the 1h again, measures bacteriostatic activity, and it is as shown in the table for the result.The result is as shown in table 3, and bacteriostatic activity remains unchanged substantially crude extract is handled 1h between pH3.0 to 9.0 after, and fungistatic effect goes down to some extent after handling 1h under the condition of pH2.0 and 11.0.
Table 3 different treatment pH is to the influence of crude extract bacteriostatic activity
The diameter in hole is 7mm
(3) enzyme susceptibility experiment: in sample, add Proteinase K (1.5mg/mL) respectively, trypsin 1.5mg/mL) and stomach en-(1.5mg/mL), regulate the suitableeest action pH value (the Proteinase K pH value 7.6 of pH value to each proteolytic enzyme, trypsinase, papoid pH value 7.0), 37 ℃ of following water-bath 1h, again pH being transferred to 5.0, is contrast with the sample of handling under the same conditions that does not add proteolytic enzyme, measures the variation of bacteriostatic activity.After the result showed that crude extract process Proteinase K and trypsinase are cleared up, bacteriostatic activity completely lost; After the process stomach en-was cleared up, bacteriostasis rate only was 15% of a fermented supernatant fluid.
Crude extract is carried out bacteriostatic test plate, measure antibacterial circle diameter and converse according to the inhibition zone typical curve that tiring of effective constituent is 13095IU/mL in the crude extract.
Formula for pig forage: powder feed prescription: Semen Maydis powder 58%, wheat bran 17%, soya-bean cake 20%, fish meal 4%, bone meal 0.5%, salt 0.5%
By adding the effective antipathogenic composition 2.5mg of crude extract (Tc1), 5mg (Tc2), 10mg (Tc3) in every 1000g feed.Store at (room temperature, humidity are 85%) and impaired condition (37 ℃ of high temperature, high humiditys, humidity is more than 90%) under physical environment respectively, do blank (CKc), sample is carried out bacterial count every 7 days with the feed that does not add any antibacterial substance.Found that the bacteriocin crude extract can effectively suppress microbial growth under the described conditions.
Claims (9)
1. a strain lactobacterium casei (Lactobacillus casei), the preservation name is called lactobacterium casei lyT-7, depositary institution: Chinese typical culture collection center, preserving number is: CCTCC M2010197.It is characterized in that: bacterium colony is circle, milky smooth colony on the MRS solid medium.
2. a method of producing the lactobacterium casei bacteriocin is fermentation lactobacterium casei (Lactobacillus casei) lyT-7, obtains bacteriocin.
3. method according to claim 2 is characterized in that: the substratum that is used to cultivate lactobacterium casei (Lactobacillus casei) lyT-7 has the subordinate to become to be grouped into: soy peptone 10.0g, extractum carnis 10.0g, yeast extract 5g, glucose 20.0g, K
2HPO
42.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, MgSO
4.7H
2O 0.58g, MnSO
4.4H
2O0.25g, tween 80 1ml, agar 15.0g, pH nature, distilled water 1000mL.
4. method according to claim 3 is characterized in that: the culture condition in the described method is 30 ℃, static cultivation 48h.
5. according to arbitrary described method in the claim 2 to 5, it is characterized in that: the bacteriocin that obtains in the described method carries out preliminary purification in accordance with the following methods: will be centrifugal by the fermented liquid that described cultural method obtains, get supernatant liquor, in this supernatant liquor, add ammonium sulfate to whole saturation ratio 50%, saltout; The centrifuging and taking precipitation obtains the bacteriocin throw out then; With the bacteriocin throw out street flow of packing into is in 1000 the dialysis tubing, dialyses in pure water, concentrates with polyoxyethylene glycol, obtains the bacteriocin crude extract.
6. according to arbitrary described method in the claim 2 to 6, it is characterized in that: the bacteriocin that obtains in the described method is further purified in accordance with the following methods: further adopt Sephadex G-100 gel filtration chromatography to carry out purifying this bacteriocin crude extract, described chromatography condition is: the Tris-HCl damping fluid with 0.01mol/L pH5.0 carries out wash-out, flow velocity is 3min/mL, and the collection retention time is that the protein peak of 180mim-460min is the active peak of bacteriocin; The bacteriocin sample of collecting is carried out RP-HPLC separate, preparation condition is: with water (containing 0.1% trifluoroacetic acid): the elution system that second fine (containing 0.1% trifluoroacetic acid) constitutes is carried out gradient
[10]Wash-out, the condition of employing gradient elution
[6,11,12]For: the concentration at the 0-35min acetonitrile progressively is increased to 45% by 10%, and adjusting back by 45% in the concentration of 35-45min acetonitrile is 10%, and the collection retention time is that the protein peak of 20min-25min is the active peak of bacteriocin.
7. this bacteriocin that obtains by arbitrary described method and known conventional method in the claim 2 to 7 or the lactobacterium casei fermented liquid that contains this bacteriocin.
8. the bacteriocin that obtains of the method for claim 2 to 5 and known conventional method or the application of lactobacterium casei fermented liquid in feed that contains this bacteriocin.
9. described bacteriocin of claim 7 or the application of lactobacterium casei fermented liquid in the preparation fodder additives that contain this bacteriocin.
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| CN102367428A (en) * | 2011-10-24 | 2012-03-07 | 甘肃农业大学 | Low temperature fermenting Lactobacillus casei and method for preparing hard yark milk cheese from Lactobacillus casei |
| CN103202386A (en) * | 2013-05-06 | 2013-07-17 | 四川大学 | Application of Lactobacillus casei fermentation liquor to feeds |
| CN103320370A (en) * | 2013-07-17 | 2013-09-25 | 北京农学院 | Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method |
| CN105218643A (en) * | 2015-10-30 | 2016-01-06 | 四川大学 | A kind of synthesis of lactobacterium casei antibacterial peptide and antibody preparation |
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| CN105801896A (en) * | 2016-04-21 | 2016-07-27 | 北京农学院 | Preparation method of bacteriocin biological fresh-keeping film |
| CN106676036A (en) * | 2016-12-09 | 2017-05-17 | 中国农业科学院蔬菜花卉研究所 | Lactobacillus casei bacterial strain and application thereof |
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| CN102367428A (en) * | 2011-10-24 | 2012-03-07 | 甘肃农业大学 | Low temperature fermenting Lactobacillus casei and method for preparing hard yark milk cheese from Lactobacillus casei |
| CN103202386A (en) * | 2013-05-06 | 2013-07-17 | 四川大学 | Application of Lactobacillus casei fermentation liquor to feeds |
| CN103320370A (en) * | 2013-07-17 | 2013-09-25 | 北京农学院 | Bacillus subtilis C3 and its anti-Listeria monocytogenes bacteriocin preparation method |
| CN103320370B (en) * | 2013-07-17 | 2015-07-29 | 北京农学院 | The preparation method of a kind of subtilis C3 and anti-Listeria monocytogenes bacteriocin thereof |
| CN105218643A (en) * | 2015-10-30 | 2016-01-06 | 四川大学 | A kind of synthesis of lactobacterium casei antibacterial peptide and antibody preparation |
| CN105801896B (en) * | 2016-04-21 | 2019-02-15 | 北京农学院 | A kind of preparation method of bacteriocin preservative film |
| CN105733008A (en) * | 2016-04-21 | 2016-07-06 | 北京农学院 | Preparation method of novel antibacterial biological preservative film |
| CN105801896A (en) * | 2016-04-21 | 2016-07-27 | 北京农学院 | Preparation method of bacteriocin biological fresh-keeping film |
| CN105733008B (en) * | 2016-04-21 | 2019-02-12 | 北京农学院 | A kind of preparation method of antibacterial biological preservation film |
| CN106676036A (en) * | 2016-12-09 | 2017-05-17 | 中国农业科学院蔬菜花卉研究所 | Lactobacillus casei bacterial strain and application thereof |
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| CN110205266A (en) * | 2019-05-31 | 2019-09-06 | 上海应用技术大学 | The lactobacillus paracasei of one plant of bacteriocinogeny and its application |
| CN111286474A (en) * | 2019-12-24 | 2020-06-16 | 浙江工商大学 | Lactobacillus paracasei and application thereof |
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| CN112438998B (en) * | 2020-11-27 | 2023-01-10 | 四川大学 | Probiotics agent PAPH for preventing and treating helicobacter pylori infection and preparation method thereof |
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