CN103173372B - Lactococcus lactis with high bacteriostatic activity and application thereof - Google Patents
Lactococcus lactis with high bacteriostatic activity and application thereof Download PDFInfo
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- CN103173372B CN103173372B CN201210240190.5A CN201210240190A CN103173372B CN 103173372 B CN103173372 B CN 103173372B CN 201210240190 A CN201210240190 A CN 201210240190A CN 103173372 B CN103173372 B CN 103173372B
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Abstract
The invention relates to Lactococcus lactis vscc-4 CGMCC No.6056 separated from fresh milk, and a powdery microecological preparation prepared by utilizing the Lactococcus lactis through high-density liquid submerged fermentation, centrifugal enrichment and vacuum freeze drying. The lactobacillus has wide antibacterial spectrum, and has good antibacterial effect on gram-positive bacteria and gram-negative bacteria; has high antibacterial activity. The microbial ecological agent prepared by the bacteria has simple process, short production period and low cost, and each gram of dry powder contains more than trillion cfu of viable bacteria.
Description
Technical field
The present invention relates to a kind of Lactococcus lactis, particularly relate to Lactococcus lactis and the application of a plant height bacteriostatic activity.
Background introduction
The growth promotion health-care agent such as abuse of antibiotics, chemical synthetic drug, hormone, beta-stimulants, heavy metal, tranquilizer in current feed and livestock and poultry cultivation, cause series of problems and consequence, comprise: animal products drug residue is serious, directly be detrimental to health, even produce " three cause ", and the animal products quality of China is declined, lack the market competitiveness and trust; Bacterial drug resistance strengthens, Resistant strain increases, and result medication is invalid, threatens human security etc.World Health Organization expert thinks, the various resistance germs that livestock and poultry produce, and can pass to people by food, and produces the disease being difficult to cure, and harm humans is healthy.Simultaneously, the microbiotic that duomycin, terramycin, paraxin, olaquindox, sulfonamide, Maduramicin etc. easily remain and toxicity is larger and a large amount of uses of synthesising bacteria anti-reflecting medicine, drug residue is caused to increase, Resistant strain spreads, animal flora imbalance, the immunizing power of animal is suppressed, secondary superinfection.
In view of the problems existed in above-mentioned feed and livestock and poultry cultivation, China starts " feed safety engineering " and have disabled a collection of toxicity and residual larger medicine.Country's " fodder industry " 12 " development program " explicitly points out the exploitation and production that will accelerate novel fodder additive, the especially production of the fodder additives of substitute antibiotics.Therefore, probiotics becomes the novel focus of industrialized development as a green fodder additives, is one of main products of substitute antibiotics.
As autochthonous flora in digestive tube, milk-acid bacteria has regulating intestinal canal colony balance, suppresses the effects such as harmful bacteria growth, strengthening immunity.Probiotics adopts milk-acid bacteria or milk-acid bacteria in the composite form of other probiotic bacteriums mostly on the market, in raising livestock and poultry food consumption, prevents there is remarkable effect in diarrhoea etc.But milk-acid bacteria also exists following defects as probiotics product: the viable count of dry powder of lactic acid bacteria is relatively low, and the viable bacteria survival time is short, the while of has a broad antifungal spectrum, the bacterial classification of good antimicrobial effect is few, and production cost is relatively high.
Summary of the invention
The object of this invention is to provide Lactococcus lactis (Lactococcus lactis) the vscc-4CGMCC No.6056 of a strain wide spectrum high bacteriostatic activity.
The Lactococcus lactis obtaining a strain wide spectrum high bacteriostatic activity is separated from cow's milk, Lactococcus lactis vscc-4 (Lactococcus lactis) is accredited as through 16S rDNA, and in Song China Microbial Culture Preservation Commission common micro-organisms center preservation April 26 in 2012, be numbered CGMCC No.6056; Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of Chinese Academy of Sciences postcode: 100101 phones: 86-10-64807596.
1, the Lactococcus lactis of a plant height bacteriostatic activity and application, is characterized in that: this bacterium is Lactococcus lactis (Lactococcus lactis) vscc-4CGMCC No.6056, has stronger bacteriostatic activity.
2, the Lactococcus lactis of a plant height bacteriostatic activity according to claim 1 and application, is characterized in that described scope of restraining fungi activity refers to all have bacteriostatic action to intestinal bacteria, Salmonellas, micrococcus luteus, streptococcus aureus.
3, the Lactococcus lactis of a plant height bacteriostatic activity according to claim 1 and application, is characterized in that described bacteriostatic activity refers to that the antibacterial circle diameter of indicator reaches 25mm-42mm.
4, containing a probiotics for the Lactococcus lactis of high bacteriostatic activity, it is characterized in that described probiotics is take Lactococcus lactis as main raw material, and its concrete production technique is as follows:
1) bacterial classification is selected: bacterial classification used is Lactococcus lactis (Lactococcus lactis) vscc-4CGMCC No.6056;
2) slant culture: Lactococcus lactis (Lactococcus lactis) vscc-4CGMCCNo.6056 of-80 DEG C of glycerine pipes preservation is inoculated in solid slant culture base, cultivates 24-30h for 30 DEG C;
3) seed culture: by the strain inoculation of slant culture in liquid nutrient medium, 30 DEG C of quiescent culture 8-12h;
4) fermentor cultivation: primary seed solution is inoculated in fermentor cultivation liquid by 1% inoculum size, 30 DEG C, 100-200rpm ferments 10-20h;
Above-mentioned medium component is: sucrose 4-6g/L, Pepsin peptone 12-14g/L, yeast extract paste 4-8g/L, potassium primary phosphate 5-9g/L, sodium-chlor 2-7g/L, bitter salt 0.2-1.0g/L, and pH is adjusted to 7.0-7.35; Slant medium separately adds 1.5% agar powder.
5) thalline enrichment: fermented liquid is passed in tubular-bowl centrifuge, 15000-20000rpm collected by centrifugation thalline.
6) lyophilize: after being mixed with isopyknic 10%D-Sorbitol Solution USP by the bacterium mud after centrifugal, be placed in freeze-drying in freeze drier.
7) aftertreatment of freeze-dried vaccine powder: take glucose as carrier, mix with freeze-dried vaccine powder, makes mixed probiotics viable count reach 1,000,000,000,000 cfu/g.
5, a kind of Lactococcus lactis containing high bacteriostatic activity is as follows as the consumption in fodder additives at probiotics:
For normally planting fowl, addition be feed heavy 0.1%;
For diarrhoea kind of a fowl, addition be feed heavy 0.2%;
For normal piglets, addition be feed heavy 0.1%;
For diarrhoea piglet, addition be feed heavy 0.3%.
The present invention has the following advantages: described bacterial classification has a broad antifungal spectrum, bacteriostatic activity are high; Described probiotics viable count is the highest in commercially available at present Strains of lactobacillus; Production technique is simple, and the cycle is short, and production cost is low.
Accompanying drawing explanation
Fig. 1 be Lactococcus lactis (Lactococcus lactis) vscc-4CGMCC No.6056 to the fungistatic effect of micrococcus luteus, what wherein diameter was less is control group is aseptic culture medium; What diameter was larger is experimental group, is the supernatant liquor of the nutrient solution of Lactococcus lactis (Lactococcuslactis) vscc-4CGMCC No.6056.
Embodiment
Embodiment one: the high bacteriostatic activity of Lactococcus lactis (Lac tococcus lactis) vscc-4CGMCC No.6056
1. the preparation of nutrient solution supernatant
The Lactococcus lactis of slant culture is inoculated in liquid nutrient medium, 30 DEG C of quiescent culture 16h, gets the centrifugal 1min of 1mL bacterium liquid 12000rpm, get supernatant liquor.
2. bacteriostatic activity detects dull and stereotyped preparation
The LB nutrient agar dissolved completely by 15mL is poured in culture dish, after it solidifies, vertically put Oxford cup; Separately get a LB nutrient agar dissolved completely, be cooled to add when 55 DEG C indicator mixing until it, get the substratum that 10mL is mixed with indicator and pour in the culture dish being placed with Oxford cup, after solidifying completely, extract Oxford cup, make detect dull and stereotyped.
3. the mensuration of bacteriostatic activity
Getting 200 μ L nutrient solution supernatants adds in the hole detecting flat board, puts into 37 DEG C of incubators and cultivates 20h, measure inhibition zone size 25mm-42mm after 4 DEG C of placement 24h.
Embodiment two: high viable count probiotics preparation technology
1) bacterial classification is selected: bacterial classification used is Lactococcus lactis (Lactococcus lactis) vscc-4CGMCC No.6056.
2) slant culture: Lactococcus lactis (Lactococcus lactis) vscc-4CGMCCNo.6056 of-80 DEG C of glycerine pipes preservation is inoculated in solid slant culture base, cultivates 24-30h for 30 DEG C.
3) seed culture: by the strain inoculation of slant culture in liquid nutrient medium, 30 DEG C of quiescent culture 8h.
4) fermentor cultivation: be inoculated in fermentor cultivation liquid by 1% inoculum size by seed liquor, 30 DEG C, 100rpm ferments 20h.Above-mentioned medium component is (g/L): sucrose 4, Pepsin peptone 12, yeast extract paste 4, potassium primary phosphate 5, sodium-chlor 2, and bitter salt 0.2, pH is adjusted to 7.0; Slant medium separately adds 1.5% agar powder
5) thalline enrichment: fermented liquid is passed in tubular-bowl centrifuge, 20000rpm collected by centrifugation thalline.
6) lyophilize: after being mixed with isopyknic 10%D-Sorbitol Solution USP by the bacterium mud after centrifugal, be placed in freeze-drying in freeze drier.
7) aftertreatment of freeze-dried vaccine powder: take glucose as carrier, mix with freeze-dried vaccine powder, makes mixed probiotics viable count reach 1,000,000,000,000 cfu/g.
Claims (2)
1. the Lactococcus lactis of a plant height bacteriostatic activity, is characterized in that: this bacterium be Lactococcus lactis (
lactococcus lactis) vscc-4 CGMCC No.6056, there is stronger bacteriostatic activity.
2., containing a probiotics for the Lactococcus lactis of high bacteriostatic activity, it is characterized in that described probiotics is take Lactococcus lactis as main raw material, and its concrete production technique is as follows:
1) bacterial classification select: bacterial classification used be Lactococcus lactis (
lactococcus lactis) vscc-4 CGMCC No.6056;
2) slant culture: by the Lactococcus lactis of-80 DEG C of glycerine pipes preservation (
lactococcus lactis) vscc-4 CGMCC No.6056 is inoculated in solid slant culture base, cultivates 24-30h for 30 DEG C;
3) seed culture: by the strain inoculation of slant culture in liquid nutrient medium, 30 DEG C of quiescent culture 8-12h;
4) fermentor cultivation: seed liquor is inoculated in fermentor cultivation liquid by 1% inoculum size, 30 DEG C, 100-200rpm ferments 10-20h;
Above-mentioned medium component is: sucrose 4-6g/L, Pepsin peptone 12-14g/L, yeast extract paste 4-8g/L, potassium primary phosphate 5-9g/L, sodium-chlor 2-7g/L, bitter salt 0.2-1.0g/L, and pH is adjusted to 7.0-7.35; Slant medium separately adds 1.5% agar powder;
5) thalline enrichment: fermented liquid is passed in tubular-bowl centrifuge, 15000-20000rpm collected by centrifugation thalline;
6) lyophilize: after being mixed with isopyknic 10%D-Sorbitol Solution USP by the bacterium mud after centrifugal, be placed in freeze-drying in freeze drier;
7) aftertreatment of freeze-dried vaccine powder: take glucose as carrier, mix with freeze-dried vaccine powder, makes mixed probiotics viable count reach 1,000,000,000,000 cfu/g.
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| CN102286393A (en) * | 2011-03-01 | 2011-12-21 | 安徽农业大学 | Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide |
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| CN102286393A (en) * | 2011-03-01 | 2011-12-21 | 安徽农业大学 | Lactococcus lactis subsp.lactis, antibacterial peptide produced by lactococcus lactis subsp.lactis and application of antibacterial peptide |
Non-Patent Citations (4)
| Title |
|---|
| 一株抗G-菌和酵母菌的乳酸乳球菌的分离鉴定与抗菌活性;黄新凤 等;《微生物学通报》;20090920;第36卷(第9期);1356-1362 * |
| 乳酸乳球菌L9 所产类细菌素Lactococcin GJ-9 的初步研究;荆谷 等;《食品与发酵工业》;20031030;第29卷(第9期);19页，表4；20页，表5 * |
| 传统乳制品中一产细菌素乳酸乳球菌的筛选;周雨霞 等;《乳业科学与技术》;20061231(第2期);59-61 * |
| 陈沛 等.产细菌素乳酸菌的筛选及其纯化和稳定性研究.《天津师范大学报(自然科学版)》.2011,第31卷(第2期),82-85. * |
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