CN102726637A - Application of lactobacillus johnsonii in lowering pH value of intestinal tract of weaning piglet - Google Patents
Application of lactobacillus johnsonii in lowering pH value of intestinal tract of weaning piglet Download PDFInfo
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- CN102726637A CN102726637A CN2012102302543A CN201210230254A CN102726637A CN 102726637 A CN102726637 A CN 102726637A CN 2012102302543 A CN2012102302543 A CN 2012102302543A CN 201210230254 A CN201210230254 A CN 201210230254A CN 102726637 A CN102726637 A CN 102726637A
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Abstract
The invention discloses an application of lactobacillus johnsonii CGMCC No.4926 in lowering a pH value of intestinal tract of a weaning piglet. Addition of proper dosage of the lactobacillus johnsonii to forage can lower pH values of ileum and cecum of the weaning piglet. A low pH value of the intestinal tract is not benefit for growth of intestinal tract pathogen, so that with the addition of the lactobacillus johnsonii, effect of prevention of diseases caused by the pathogen can be reached.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the application of Yue Shi lactobacillus in reducing weanling pig enteron aisle pH value.
Background technology
Yue Shi lactobacillus (Lactobacillus johnsonii) belongs to Firmicutes (Firmicutes) bacillus guiding principle (Bacilli) lactobacillus order (Lactobacillales) Lactobacillaceae (Lactobacillaceae) lactobacillus (Lactobacillus); 1980 separated first, yet on taxology, it is divided into lactobacillus acidophilus (Lactobacillus acidophilus) always.Fujisawa in 1992 etc. name the lactobacillus into Yue Shi through biochemical test and DNA hybrid experiment with it.The Yue Shi lactobacillus mainly is present in animal and human's the alimentary canal, and is dominant microflora.
Food security office of European Union (EFSA) can issue to the health of Yue Shi lactobacillus respectively at 2009 and 2011 twice and declare; Statement points out that the Yue Shi lactobacillus has the microorganism that reduces pathogen in the enteron aisle, strengthens natural defence and immune system and keeps skin health.And European Union and the Yue Shi lactobacillus had been carried out safe recognition of qulifications in 2010 (qualified presumption of safety QPS) ratifies it and in feed and food, uses.Comprehensive present bibliographical information about the Yue Shi lactobacillus; Its benefit to animal is given birth to function and is mainly reflected in following several respects: 1. the Yue Shi lactobacillus has regulating action to natural animal system of defense and immunity function, and its concrete regulatory site is like TGF β; TLR9; NF-κ B, IgA, the initial acceptor of animal body immune response often; Therefore the mechanism of action of its prevention Animal diseases possibly be to stimulate animal immune system in the specific period, activates the downstream immune response in early days and then the generation that wards off disease; 2. the Yue Shi lactobacillus shows the growth inhibited effect to enteric pathogenic bacteria.3. the Yue Shi lactobacillus is to the exclusiveness competition effect of pathogen.The Yue Shi lactobacillus is grown some pathogens such as salmonella, staphylococcus aureus, Escherichia coli deciding on intestinal epithelial cell and is produced sterically hinderedly, can prevent that pathogen to the infecting of intestinal epithelial cell, reducing disease and taking place.
General office of China Ministry of Public Health was indexed to the Yue Shi lactobacillus in " the bacterial classification list that can be used for food " in 2010, ratified it and gave birth to the food additives use of function as having benefit.Do not see as yet at home at present to have the Yue Shi lactobacillus is emerged as the product that additive adds food and feed additive; The also rarely seen report of its application on the animal livestock and poultry cultivation; The Yue Shi lactobacillus is developed to the animal microecological feed addictive,, screens the Yue Shi lactobacillus that beneficial natural disposition can be good for filling the domestic gaps; To promoting China's livestock and poultry cultivation level significant, can in livestock and poultry cultivation, play a greater role.
Summary of the invention
In order to address the above problem, the present invention provides Yue Shi lactobacillus CGMCC No.4926 application in reducing weanling pig enteron aisle pH value.
Yue Shi lactobacillus of the present invention (Lactobacillus johnsonii); Be all to have potential benefit to give birth to the Yue Shi lactobacillus of function through the external acidproof strain that screening obtains with bile tolerance; Called after Yue Shi lactobacillus (Lactobacillus johnsonii) YS-12; And having carried out preservation at China Committee for Culture Collection of Microorganisms common micro-organisms preservation center on June 8th, 2011, preserving number is CGMCC No.4926.
Wherein, the screening process of described Yue Shi lactobacillus YS-12 is following:
(1) collects bacterial classification
The intercepting health pig colon second half section, with normal saline flushing three times, repeatedly hang the attachment of getting on the gut epithelium with toothpick, put into the physiological saline of 10mL, as sample stoste, stoste is carried out 10 times of gradient dilutions.
(2) bacterial classification is cultivated
With each dilution factor, get 100 microlitres and coat on the MRS flat board, be inverted for 37 ℃ and cultivated 24 hours.
(3) bacterial classification is identified
Single bacterium colony on the picking flat board carries out that its 16SrRNA is carried out bacterium colony PCR to be identified; Through in database, carrying out sequence alignment; Find its 16S sequence (the GenBank accession number: HQ641209) with database in the similarity of 16S sequence of known Yue Shi lactobacillus reach 99%, further be confirmed that it is the Yue Shi lactobacillus through physical and chemical experiment.
(4) bacterial screening
1) thalline is resuspended in PBS liquid after, gradient dilution to 10
-2, draw 10 with pipettor
-2Dilution 100 microlitres add respectively in the cholate solution and PBS (contrast) of 900 microlitres 0.3%, and 37 ℃ are incubated 3 hours.
2) thalline is resuspended in PBS liquid after, gradient dilution to 10
-2, draw 10 with pipettor
-2Dilution 100 microlitres add respectively in the buffer solution and PBS (contrast) of 900 microlitre pH3.0, and 37 ℃ are incubated 3 hours.
3) the bacterium liquid with acid treatment continues to be diluted to 10
-5, get 10
-3, 10
-4, 10
-5(control group is diluted to 10 to three dilution factors
-6, get 10
-4, 10
-5, 10
-6) each 0.1ml coating MRS flat board, be inverted for 37 ℃ and cultivated 48 hours.Picking tolerance bacterial strain carries out repeated screening, obtains acidproof and the Yue Shi lactobacillus bile tolerance function admirable.
(5) culture presevation
Yue Shi lactobacillus (Lactobacillus johnsonii) YS-12 that screening is obtained is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and obtaining preserving number is CGMCC No.4926.
Can significantly reduce the pH value of weanling pig ileum and caecum after feeding with Yue Shi lactobacillus CGMCC No.4926 separately or be prepared in the basal diet that preparation adds dissimilar weanling pigs to.
In one embodiment of the invention, in the basal diet of weanling pig, add described Yue Shi Lemonal, make the content of described Yue Shi lactobacillus in every gram basal diet reach 1.0 * 10
5~ 10
8Cfu has significantly reduced the pH value in ileum and the caecum.
In a preferred embodiment, described Yue Shi Lemonal can be this area conventional formulation, and it is freeze-dried to be preferably the Yue Shi lactobacillus.
Wherein, said Yue Shi lactobacillus is freeze-dried also can to contain freeze drying protectant, and preferred freeze drying protectant is that mass ratio is skimmed milk, trehalose, soluble starch, sodium glutamate and the glycerine of 2:2:4:1:2.
Wherein, the freeze-dried preparation method of said Yue Shi lactobacillus is following:
1) with the Yue Shi lactobacillus CGMCC No.4926 of freezing preservation MRS agar plate activation three times; Picking list colony inoculation is in 10mL deep layer MRS liquid tube; Cultivate after 10 hours for 37 ℃, transfer in 250mL MRS fluid nutrient medium, cultivate after 8 ~ 10 hours as seed liquor with 1% inoculum concentration;
2) seed liquor of step 1) gained is inoculated in the fermentation tank of 5L with 5%～10% inoculum concentration and cultivates;
3) keeping zymotic fluid pH value in the sweat is 6.0～6.5, when pH surpasses setting value, adds feed liquid; When pH is lower than setting value, any among auto-feeding antalkali ammoniacal liquor, KOH, the NaOH; At 37～39 ℃ of temperature, rotating speed 100rpm condition bottom fermentation 10～14 hours, sweat stuffiness or feeding N
2, keep dissolved oxygen less than 1%;
4) when adding that material liquid pH no longer reduces or the light absorption value (be OD600) of zymotic fluid under the 600nm wavelength when no longer increasing, stop fermentation, promptly get Yue Shi living preparation of lactobacillus number and reach 10
11The zymotic fluid that cfu/ml is above;
5) 4000-8000 rev/min centrifugal 5-10 minute, obtain Yue Shi lactobacillus bacterium mud;
6) product that obtains is according to bacterium mud (g): protective agent (mL) mass volume ratio 1:5 adds freeze drying protectant, carries out vacuum freeze drying, and it is freeze-dried to obtain the Yue Shi lactobacillus.
Yue Shi lactobacillus CGMCC No.4926 of the present invention adds in the feed with the suitable dosage of suitable dosage has significant effect to the pH value that reduces weanling pig ileum and caecum.Since contain in ileum and the caecum a large amount of to the responsive pathogenic bacteria of low pH, like salmonella, staphylococcus aureus etc., so the low pH environment in ileum and the caecum can effectively suppress the growth of pathogenic bacteria, prevents the Animal diseases generation.
Yue Shi lactobacillus among the present invention (Lactobacillus johnsonii) YS-12 is preserved in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center; Institute of Microorganism, Academia Sinica; 100101) preservation; Preservation date is on June 8th, 2011, and preserving number is CGMCC No.4926.
The specific embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.The freeze-dried preparation of embodiment 1 Yue Shi lactobacillus CGMCC No.4926
1) with the Yue Shi lactobacillus CGMCC No.4926 of freezing preservation MRS agar plate activation three times; Picking list colony inoculation is in 10mL deep layer MRS liquid tube; Cultivate after 10 hours for 37 ℃, transfer in 250mL MRS fluid nutrient medium, cultivate after 10 hours as seed liquor with 1% inoculum concentration;
2) seed liquor of step 1) gained is inoculated in the fermentation tank of 5L with 5% inoculum concentration and cultivates;
3) keeping zymotic fluid pH value in the sweat is 6.0, when pH surpasses setting value, adds feed liquid; When pH is lower than setting value, auto-feeding antalkali ammoniacal liquor; At 37 ℃ of temperature, rotating speed 100rpm condition bottom fermentation 14 hours, sweat feeds N
2, keep dissolved oxygen less than 1%;
4) when adding that material liquid pH no longer reduces or the light absorption value (be OD600) of zymotic fluid under the 600nm wavelength when no longer increasing, stop fermentation, promptly get Yue Shi living preparation of lactobacillus number and reach 10
11The zymotic fluid that cfu/ml is above;
5) 8000 rev/mins centrifugal 10 minutes, obtain Yue Shi lactobacillus bacterium mud;
6) product that obtains is according to bacterium mud (g): it is skimmed milk, trehalose, soluble starch, sodium glutamate and the glycerine of 2:2:4:1:2 that protective agent (mL) mass volume ratio 1:5 adds mass ratio; Carry out vacuum freeze drying; It is freeze-dried to obtain the Yue Shi lactobacillus; Wherein, the viable count of Yue Shi lactobacillus can reach 3.7 * 10
11Cfu/g.
Embodiment 2
(1) animal used as test and experimental design
The single factor experiment design is adopted in test; Select the wean of 180 28 ages in days, healthy DLY three way cross weanling pig, average initial body weight is 8.03kg, by consistent, public female half and half the principle of body weight; Be divided into 5 processing at random, feed respectively and in basal diet, add 0cfu/g, 1.0 * 10
5Cfu/g, 1.0 * 10
6Cfu/g, 1.0 * 10
7Cfu/g and 1.0 * 10
8Cfu/g Yue Shi lactobacillus, each handles 6 repetitions, and each repeats 6 pigs.Experimental period 28d.Basal diet prescription and nutritional labeling are seen table 1.
Table 1 basal diet prescription and nutritional labeling
Annotate:
a4% premix provides the per kilogram mixed feed: vitamin A, 300 ~ 400KIU; Vitamin D
3, 50 ~ 250KIU; Vitamin E, 500IU; Vitamin K
3, 75mg; Cobastab
1, 35mg; Cobastab
2, 100mg; Cobastab
6, 75mg; Cobastab
12, 0.5mg; Nicotinic acid, 1000mg; Pantothenic acid, 350mg; Folic acid, 20mg; Biotin, 1.1mg; Choline Chloride, 10g; Fe, 2.3 ~ 13g; Cu, 2 ~ 5g; Zn, 1.5 ~ 15g; Mn, 0.5 ~ 2g; I, 10 ~ 30mg; Se, 5 ~ 12mg; Ca, 100 ~ 250g; P, 20g.
bME is a calculated value, and other are measured value.
(2) animal experiment method
Before the test pig house is carried out thorough disinfection, and clean hopper, tank, and test pig is carried out routine immunization and expelling parasite.The pig house temperature is controlled at about 25 ℃, free choice feeding, freely drinks water.Sterilization is undertaken by conventional program.Strictness follows that raise on the pig farm and management system, notices observing that pig searches for food every day, ight soil and mental status, finds that disease in time handles.
(3) enteron aisle pH pH-value determination pH
The 28th day of experiment, each repeats after 1 pig of picked at random (each handles 6 pigs, public mother half and half) carries out bloodletting, butchers and measure respectively with acidometer the pH value of jejunum, ileum, caecum and colon chyme.
(4) statistical method
Test data adopts the SAS8.0 statistical software to carry out one-way analysis of variance, Deng Kenshi multiple ratio.< 0.05 is significant difference to P, and < 0.01 to be regarded as difference extremely remarkable for P.
(5) interpretation of result
Interpolation varying level Yue Shi lactobacillus is seen table 2 to the result that influences of weanling pig enteron aisle pH value in the daily ration.Can find out from table 2, feed and add the weanling pig of varying level Yue Shi lactobacillus daily ration processed group, its test the 28th day the time, the daily ration of feeding adds 1.0 * 10
6Cfu/g, 1.0 * 10
7The weanling pig of cfu/g Yue Shi lactobacillus bacterium powder, its ileum pH value significantly are lower than control group and add 1.0 * 10 with the daily ration of feeding
5Weanling pig (the P of cfu/g Yue Shi lactobacillus bacterium powder<0.05); The daily ration of feeding adds 1.0 * 10
5Cfu/g, 1.0 * 10
6Cfu/g, 1.0 * 10
7Cfu/g and 1.0 * 10
8The weanling pig of the Yue Shi lactobacillus bacterium powder of cfu/g, its caecum pH value significantly is lower than the weanling pig (P of control group<0.05); The daily ration of feeding adds 1.0 * 10
8The weanling pig of the Yue Shi lactobacillus bacterium powder of cfu/g, its colon pH value have the region daily ration of feeding and add 1.0 * 10
5The trend (P=0.06) of the weanling pig of cfu/g Yue Shi lactobacillus bacterium powder.This result of the test shows that daily ration adds 1.0 * 10
5Cfu/g, 1.0 * 10
6Cfu/g, 1.0 * 10
7Cfu/g and 1.0 * 10
8During cfu/g Yue Shi lactobacillus bacterium powder, can not influence the pH value of jejunum and the colon of weanling pig, but can significantly reduce the ileum of weanling pig and the pH value (P of caecum<0.05).Since contain in ileum and the caecum a large amount of to the responsive pathogenic bacteria of low pH, like salmonella, staphylococcus aureus etc., so the low pH environment in ileum and the caecum can effectively suppress the growth of pathogenic bacteria, prevents the Animal diseases generation.
Table 2 daily ration of feeding adds the influence of varying level Yue Shi lactobacillus to weanling pig enteron aisle pH value
: with significant difference between the different lowercases of shoulder mark in the line data (P < 0.05) or extremely significantly (P 0.01); Do not indicate that letter or same letter represent difference not significantly (P>0.05).
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (5)
1. the application of Yue Shi lactobacillus (Lactobacillus johnsonii) CGMCC No.4926 in reducing weanling pig enteron aisle pH value.
2. application according to claim 1 is characterized in that the application of Yue Shi lactobacillus CGMCC No.4926 in reducing weanling pig ileum and caecum pH value.
3. application according to claim 1 is characterized in that, in the basal diet of weanling pig, adds described Yue Shi Lemonal, makes the content of described Yue Shi lactobacillus in every gram basal diet reach 1.0 * 10
5~ 10
8Cfu.
4. like each described application of claim 1 ~ 3, it is characterized in that described Yue Shi Lemonal is that the Yue Shi lactobacillus is freeze-dried.
5. application as claimed in claim 4 is characterized in that, the freeze-dried freeze drying protectant of said Yue Shi lactobacillus is that mass ratio is skimmed milk, trehalose, soluble starch, sodium glutamate and the glycerine of 2:2:4:1:2.
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Cited By (1)
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| CN110878266A (en) * | 2019-11-21 | 2020-03-13 | 中国农业科学院兰州兽医研究所 | Lactobacillus johnsonii and application thereof |
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