CN102061271B - Lactobacillus casei bacteriocin and use thereof in feed - Google Patents
Lactobacillus casei bacteriocin and use thereof in feed Download PDFInfo
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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 one type of rrna synthetic in metabolic process; TR. Genetics of bacteriocins produced by lactic acid bacteria. FEMS.Microbiology Review [J]. 1993. 12:39～85), aspects such as several diseases pathogenic microorganism 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 like 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 the several diseases pathogenic microorganism; 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.; Be prone to be degraded by some proteolytic enzyme (like trypsinase) in the human body alimentary canal, can not accumulate in animal body and cause untoward reaction, the while is 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 with 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.
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, SANMALT-S, 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, l-arginine produce the ammonia experiment and nitrate reduction is tested all negative.Through morphology physiological and biochemical property and 16S rDNA sequential analysis be accredited as lactobacterium casei (
Lactobacillus casei).
Second purpose of the present invention provides a kind of bacteriocin.
Bacteriocin provided by the present invention be the fermentation lactobacterium casei (
Lactobacillus casei) bacteriocin that obtains of lyT-7.
Be used to cultivate in the said method lactobacterium casei (
Lactobacillus casei) substratum of lyT-7 is modified MRS culture medium, composition consists of: soy peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5g, glucose 20.0g, K
2HPO
42.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, tween 80 1ml, agar 15.0g, pH nature, zero(ppm) water 1000mL.(Ling Daiwen, eastern elegant pearl. the lactic-acid-bacterium classification is identified and experimental technique [M]. China Light Industry Press. 1999)
Culture condition in the said method is 30 ℃, static cultivation 48h.
Concrete, lactobacterium casei bacteriocin of the present invention can obtain through following method, but is not limited thereto.
The bacteriocin that obtains in the said method can carry out the substep purifying by following method:
The first step will be centrifugal by the fermented liquid that said cultural method obtains, and get supernatant, in this supernatant, add ammonium sulfate to whole saturation ratio 50%, saltout; The centrifuging and taking deposition obtains the bacteriocin throw out then; With the bacteriocin throw out amount of damming of packing into is in 1000 the dialysis tubing, in pure water, dialyses, and concentrates with polyoxyethylene glycol, obtains the bacteriocin crude extract.
Second step; The bacteriocin that obtains as stated above is further purified according to following method: further adopt Sephadex G-100 gel filtration chromatography to carry out purifying this bacteriocin crude extract; Said 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 RT 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 eyeball (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 RT 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, like 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 said 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), lactobicillus bulgaricus (
L.bulgaricus), Lactobacterium acidophilum (
L.acidoph ilus), flavus (
A. flavus).
The 3rd purpose of the present invention be with lactobacterium casei (
Lactobacillus casei) bacteriocin uses in feed.
It is 13095IU/mL that the bacteriocin crude extract that will obtain by the first step of said 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
Fig. 1 is that feed stores the fungistatic effect under the condition in routine
Fig. 2 is that feed stores the fungistatic effect under the condition in reinforcement
Embodiment
1, lactobacterium casei (
Lactobacillus casei) the bacteriostatic activity analysis of lyT-7
Bacteriostatic activity analytical procedure: under the incident light condition of 600 λ; The OD value of indicator (intestinal bacteria and streptococcus aureus) bacteria suspension is adjusted to 0.120; Be poured into 15mL LB solid medium, use diameter on substratum, to punch as the punch tool of 7mm, the adding lactobacterium casei (
Lactobacillus casei) fermented supernatant fluid of lyT-7, at room temperature spread 30min, under 30 ℃ of conditions, cultivated 16-18 hour, observe inhibition zone.
With lactobacterium casei (
Lactobacillus casei) lyT-7 inserts in the modified MRS nutrient solution, its composition is: soy peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5g, glucose 20.0g, K
2HPO
42.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, MgSO
47H
2O 0.58g, MnSO
44H
2O 0.25g, tween 80 1ml, agar 15.0g, pH nature, zero(ppm) 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, tentatively judge lactobacterium casei (
Lactobacillus casei) antibacterial substance that produces of lyT-7 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, add ammonium sulfate to whole saturation ratio 50%, saltout; The centrifuging and taking deposition obtains the bacteriocin throw out then; With the bacteriocin throw out amount of damming of packing into is in 1000 the dialysis tubing, in pure water, dialyses, and 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; Said 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 RT 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 (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 RT is that the protein peak of 20-25min is the active peak of bacteriocin.
3, the antimicrobial spectrum of bacteriocin
Test strain be intestinal bacteria (
E. coli), Salmonella typhimurium (
S.typhim uriu), streptococcus aureus (
S. aureus), subtilis (
B.subtili), bacillus cereus (
B.cereus), lactobicillus 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 shown in table 1.
The antimicrobial spectrum of table 1 lactobacterium casei lyT-7 fermented supernatant fluid
Fungistatic effect is represented with antibacterial circle diameter :-, do not have bacteriostatic activity; ±, 7-8 mm; +, 8-10 mm; ++,＞10mm;
[0030]4, bacteriocin antagonistic property research
(1) thermostability experiment: with the bacteriocin crude extract 60 ℃ (water-baths), 80 ℃ (water-bath), 100 ℃ (water-bath) keeps 20 min under 121 ℃ of (high pressure) conditions, measure 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.Explain 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
| T(℃) | 60 | 80 | 100 | 121 | Contrast |
| Average diameter of inhibition zone (mm) | 13.69±0.57 | 13.58±0.62 | 13.09±0.61 | 12.14±0.74 | 13.73±0.66 |
| Active decline percentage (%) | 0.29 | 1.09 | 4.66 | 11.58 | 0 |
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.Again 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, 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 basically 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
| pH | Average diameter of inhibition zone (mm) | Active decline percentage (%) |
| 2.0 | 13.06±0.41 | 5.70 |
| 3.0 | 13.69±0.77 | 1.16 |
| 4.0 | 13.70±0.49 | 1.08 |
| 5.0 | 13.85±0.32 | 0 |
| 6.0 | 13.74±0.64 | 0.79 |
| 7.0 | 13.65±0.58 | 1.44 |
| 8.0 | 13.62±0.62 | 1.66 |
| 9.0 | 13.57±0.71 | 2.02 |
| 10.0 | 13.10±0.57 | 5.42 |
| 11.0 | 12.59±0.66 | 9.10 |
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 righttest action pH value (Proteinase K pH value 7.6, trypsinase, papoid pH value 7.0) of pH value to each proteolytic enzyme; 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 was merely 15% of 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), 5 mg (Tc2), 10 mg (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), whenever sample is carried out bacterial count at a distance from 7 days with the feed that does not add any antibacterial substance.The result finds that the bacteriocin crude extract can effectively suppress microbial growth under the described conditions.
Claims (8)
1.
One 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 is characterized in that: bacterium colony is circle, milky smooth colony on the MRS solid medium.
2.
Lactobacterium casei described in a kind of fermentation claim 1 (
Lactobacillus casei) lyT-7 produces the method for bacteriocin, it is characterized in that: be used to cultivate lactobacterium casei (
Lactobacillus casei) substratum of lyT-7 is made up of following compositions: soy peptone 10.0g, Carnis Bovis seu Bubali cream 10.0g, yeast extract 5g, glucose 20.0g, K
2 HPO 4 2.0g, dibasic ammonium citrate 2.0g, sodium acetate 5.0g, MgSO 4 7H 2 O 0.58g, MnSO 4 4H 2 O 0.25g, tween 80 1ml, agar 15.0g, pH nature, zero(ppm) water 1000mL.
3.
method according to claim 2, it is characterized in that: the culture condition in the said method is 30 ℃, static cultivation 48h.
4.
are according to claim 2 or 3 described methods; It is characterized in that: the bacteriocin that obtains in the said method carries out preliminary purification according to following method: will be centrifugal by the fermented liquid that said cultural method obtains; Get supernatant; In this supernatant, add ammonium sulfate to whole saturation ratio 50%, saltout; The centrifuging and taking deposition obtains the bacteriocin throw out then; With the bacteriocin throw out amount of damming of packing into is in 1000 the dialysis tubing, in pure water, dialyses, and concentrates with polyoxyethylene glycol, obtains the bacteriocin crude extract.
5.
method according to claim 2; It is characterized in that: the bacteriocin that obtains in the said method is further purified according to following method: further adopt Sephadex G-100 gel filtration chromatography to carry out purifying this bacteriocin crude extract; Said 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 RT 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: to contain the water of 0.1% trifluoroacetic acid: the elution system that contains the acetonitrile formation of 0.1% trifluoroacetic acid 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%; Concentration at the 35-45min acetonitrile is 10% by 45% readjustment, and the collection RT is that the protein peak of 20min-25min is the active peak of bacteriocin.
6.
this bacteriocin that obtains by claim 2 or 5 described methods or the lactobacterium casei fermented liquid that contains this bacteriocin.
7. this bacteriocin that obtains of
claim 2 or 5 method or the application of lactobacterium casei fermented liquid in feed that contains this bacteriocin.
8.
described bacteriocin of claim 6 or the application of lactobacterium casei fermented liquid in the preparation fodder additives that contain this bacteriocin.
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| CN102367428B (en) * | 2011-10-24 | 2012-10-03 | 甘肃农业大学 | Low temperature fermenting Lactobacillus casei and method for preparing hard yark milk cheese from Lactobacillus casei |
| CN103202386B (en) * | 2013-05-06 | 2014-09-10 | 四川大学 | Application of Lactobacillus casei fermentation liquor to feeds |
| 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 |
| CN105733008B (en) * | 2016-04-21 | 2019-02-12 | 北京农学院 | A kind of preparation method of antibacterial biological preservation film |
| CN106676036B (en) * | 2016-12-09 | 2019-11-01 | 中国农业科学院蔬菜花卉研究所 | A kind of lactobacillus casei bacterial strain and its application |
| CN110205266B (en) * | 2019-05-31 | 2022-06-10 | 上海应用技术大学 | Lactobacillus paracasei capable of producing bacteriocin and application thereof |
| CN111286474B (en) * | 2019-12-24 | 2022-03-22 | 浙江工商大学 | Lactobacillus paracasei and application thereof |
| 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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