CN101768559A - Lactobacillus, paracasei subsp. paracasei strain, bacteriostatic composition and applications thereof - Google Patents
Lactobacillus, paracasei subsp. paracasei strain, bacteriostatic composition and applications thereof Download PDFInfo
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Abstract
The invention discloses a Lactobacillus, paracasei subsp. paracasei strain, a bacteriostatic composition and applications thereof. The preservation number of the SG96 strain microorganism is CGMCC2697. The strain can inhibit the growth of pathogenic bacteria of Escherichia coli, Salmonella typhimurium and the like, and has bacteria killing effect. The invention also discloses a bacteriostatic composition containing the Lactobacillus, paracasei subsp. paracasei strain SG96. The composition can be prepared into animal water-feeding additives, animal fodder additives, animal and human medical compositions, food additives, beverage additives, foods, beverages, health foods, and the like.
Description
Technical field
The present invention relates to secondary cheese subspecies (Lactobacillus paracaseisubsp.paracasei) the SG96 bacterial strain of a kind of lactobacillus paraceasi, particularly relate to a kind of secondary cheese subspecies of lactobacillus paraceasi microbial strain that can suppress intestinal bacteria (Escherichiacoli) and Salmonellas pathogenic bacterias such as (Salmonella typhimurium), and contain the bacteria inhibiting composition of the secondary cheese subspecies of this lactobacillus paraceasi and the purposes of said composition, belong to microorganism field.
Background technology
In recent decades, because population increases fast, the material life level significantly promotes, people are heightened to the demand of meat, in order to meet the need of market, scale is had in the raising of many economic animals gradually, especially backward country is lower for the ability of food import, thereby a large amount of high-density or big area raising economic animal, causes numerous disease to disseminate fast between animal.In order to improve the sick problem of economic animal or to allow economic animal grow fast, the practice for a long time is to add microbiotic or to animal injection microbiotic, the result causes the generation of a large amount of Resistant strain in feed.
Nineteen sixty, antibiotic appearance made the human unprecedented triumph that obtains on the field of treatment bacterial infection for latter stage, and the transmissible disease that many scripts are fatal is no longer fearful.Yet in recent years, the appearance of tool antibiotic-resistant bacteria, human worry and fear to communicable disease raises again once again, because microbiotic no longer has been the cure-all medicine of disease controlling, the tool antibiotic-resistant bacteria disseminates speed and exceeds our imagination especially.The decline of being not only curative effect that bacterial drug resistance is brought even invalid, and badly influenced human beings'health.The number that infectious diseases is died from the 40 years preceding whole world every year is about 7,000,000, and has arrived now, and medical science has been made significant headway, and the number of dying from infectious diseases every year but rises to 2,000 ten thousand.When people are for a long time edible when containing the animal foods such as meat, egg, milk of antibiotic remains, these antibiotic remainss can produce deleterious effect to human body, and accumulate in human body, cause drug-fast strain constantly to take place.
The problem last decade of meat safety is subjected to human consumer, the agricultural fishing herding producer and government most and pays close attention to, the human consumer to meat safety continue show great attention to, terrified meat security incident is made us in several of the generations both at home and abroad in the past few years that result from, and for example incidents such as H5N1 bird flu take place for mad cow syndrome, South East Asia and the China's Mainland of Britain's generation.In order to take the edible security of meat into account, countries in the world cause the also quite attention of adverse consequences problem to antibiotic remains.In order to delay the generation of tool drug-fast strain problem, in mid-term nineteen ninety, some European countries begin to hope and reduce the speed that drug-fast strain produces in this way reaching constraint on the agricultural, the use of control microbiotic in the medical treatment.European Union is since nineteen ninety-five, forbid some microbiotic successively and be used for feed, and receive good effect, for example, the Denmark veterinary laboratories researchist in Copenhagen represents, behind the microbiotic of forbidding peace Ba Su (avoparcin), the faecalis (Enterococcus faecium) in the tender chicken digestive tube of Denmark is to the resistance of peace Ba Su, from 73% of nineteen ninety-five be reduced to 2000 6%.Forbidding peace Ba Suhou in 5 years of Germany and Dutch phase at the same time, the drug-fast strain that also is shown in animal and the human body has all reduced, so European Union begins to forbid adding microbiotic in feed in January, 2006.
Adding antibiotic purpose in feed is to promote growth, reduces roughage, can improve the growth efficiency of livestock and poultry, but because the growth cycle of livestock and poultry shortens, and the precipitation of nutritive substance must reduce, makes distinctive local flavor of animal products and quality descend; Even more serious is the residual to human beings'health of medicine, particularly the health of children in vegetative period is caused serious harm.Therefore, forbidding microbiotic or reduce microbiotic gradually and use kind, consumption trend in the feed of economic animal as the world.But after microbiotic is progressively forbidden, many problems of also can deriving, for making livestock industry energy ordinary production, the dealer now can accept to utilize the idea of probiotic agent (Probiotics) as the microbiotic surrogate, and then adopts as fodder additives (Feed additives).
Probiotic bacterium mainly is a milk-acid bacteria, because the colony balance of livestock and poultry alimentary tract can be kept and recover to milk-acid bacteria, eliminates the stress situation (stress) of livestock and poultry, improves the immune disease-resistance ability of livestock and poultry, reduces sickness rate.Advocate the silage and the liquid fermenting feed that use lactobacillus-fermented, because the lactobacillus-fermented feed both can effectively keep the nutritive ingredient of feed, improve digestive utilization ratio, can eliminate during the fermentation again and can cause the microorganism of zoonosis with some by morbific enteric pathogenic bacteria.Milk-acid bacteria has been acknowledged as very ideal substitute of microbiotic.
Probiotic bacterium comprises at the intravital beneficial effect of livestock and poultry:
1. keeping and recovering livestock and poultry alimentary tract interior is the colony balance of dominant microflora with the lactobacillus.Environmental disorder in the digestive tube of eliminating livestock and poultry to be in stress situation (stress is as wean, replacing feed, high temperature, cold, transportation, scaring, disease etc.) and causing.Suppress the invasion of the abnormality proliferation of harmful bacterium and pathogenic bacterium and grow surely, digestive tract diseases such as prevention and treatment diarrhoea, diarrhea reduce or eliminate the generation of vivotoxin.For the still unfounded cub of microorganism system, young fowl, the effect that replenishes milk-acid bacteria is especially showing.
2. produce the non-specific immunity regulatory factor, improve the fowl poultry immune resistance against diseases, reduce disease infection rate and mortality ratio.Effectively substitute microbiotic and chemicals, reduce the probability of drug residue, promote human food quality.
3. supplement the nutrients, promote growth of animals or poultry.Produce digestive ferment, strengthen and digest and assimilate ability, showing the output and the benefit that improve aquaculture.
4. stink substance in the minimizing feces of livestock and poultry improves the environment of plant.
5. show the output that improves milk, meat, egg, increasing the benefit of aquaculture.
Food-safety problem has caused the great attention of countries in the world government.National governments just actively promote green livestock industry at present, support the development of green product on policy, financial resources, material resources.Set up the demonstration base of green cultivation, foster the brand of green livestock products, the manager of green product can be received more tangible benefits, allow the human consumer can access high-quality, non-harmful safety, green livestock and poultry food, bring benefit to the mankind.
Summary of the invention
One of the object of the invention promptly is to provide the secondary cheese subspecies of a kind of lactobacillus paraceasi SG96 bacterial strain, the kind system relation of the secondary cheese subspecies of this bacterial strain and existing lactobacillus paraceasi BCRC910220 (Lactobacillus paracasei) and lactobacillus paraceasi BCRC14001, BCRC16100 (Lactobacillus paracasei subsp.paracasei) has notable difference, is the secondary cheese subspecies of new lactobacillus paraceasi microbial strain.
Two of the object of the invention is to provide a kind of bacteria inhibiting composition that contains the above-mentioned new secondary cheese subspecies of lactobacillus paraceasi SG96, and said composition has can suppress the effect that intestinal bacteria (Escherichia coli) and Salmonellas (Salmonella typhimurium) pathogenic bacteria of etc.ing grow.
Three of the object of the invention is with containing the additive of the bacteria inhibiting composition of the secondary cheese subspecies of above-mentioned new lactobacillus paraceasi SG96 as animal-feed or drinking-water, to promote the ability of animal opposing pathogenic bacteria; This bacteria inhibiting composition more can be further used as human food prods, drink additive or food, to promote the ability of human body opposing pathogenic bacteria.
Can reach the secondary cheese subspecies of the lactobacillus paraceasi SG96 of foregoing invention purpose, contain bacteria inhibiting composition of this SG96 bacterial strain and uses thereof, include: the secondary cheese subspecies of new lactobacillus paraceasi SG96 bacterial strain, this bacterial strain screening is from a pig enteron aisle movement, through the gramstaining analysis, API50CHL analyzes, 16S rDNA sequencing is analyzed, confirm as secondary cheese subspecies (the Lactobacillus paracasei subsp.paracasei) microbial strain of lactobacillus paraceasi of a novelty behind the pedigree analysis, the secondary cheese subspecies of this lactobacillus paraceasi SG96 bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (China General Microbiological Culture Collection Center, CGMCC), deposit number is CGMCC2697, and preservation date is on October 9th, 2008.
With the secondary cheese subspecies of the lactobacillus paraceasi SG96 bacterial strain that the present invention screened, carry out respectively that filter paper agar diffusion method (disc-agar diffusion) is analyzed and with pathogenic bacteria mixed culture analysis of experiments, the result shows, SG96 bacterial strain of the present invention can suppress intestinal bacteria (E.coli) and Salmonellas growth of pathogenic bacteria such as (S.typhimurium), even sterilization effect is arranged.
The secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain is mixed with aqueous solution feeding not from the sucking pig son, the result shows that SG96 bacterial strain of the present invention can obviously promote not from the suckling piglet weight increase, improve the meat change rate of pig starter feed, and has a good anti-diarrhea effect, and effect is better than the general microbiotic that uses, and proves that SG96 bacterial strain of the present invention has the ability of promoting animal opposing pathogenic bacteria.
The antimicrobial compound that contains the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain, except that being formulated as the aqueous solution as the animal drinking water, the conventional feed additive of also can arranging in pairs or groups is made technology, to be prepared into other form, comprising that animal-feed additive, animal are used and human medical composition, food additives, beverage additives, food, drink, the heath food etc. used, is animal and human the picked-up via oral way.In a preferred embodiment, this antimicrobial compound can be made into lactic acid lozenge, powder or particulate state, and can make an addition to (as: 1 ton feed 1 kilogram of powdery of interpolation or the secondary cheese subspecies of granular lactobacillus paraceasi SG96 bacterial strain (10 in animal-feed or the food
7CFU/g); Or can separately or with other milk-acid bacterias milk-product such as Yogurt or yogurt be made in the dairy products fermentation.
The secondary cheese subspecies of lactobacillus paraceasi provided by the present invention SG96, contain bacteria inhibiting composition of this SG96 bacterial strain and uses thereof, mainly have following advantage mutually relatively the time with other prior aries:
1. behind the analysis of the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain process gramstaining, API50CHL analysis, the analysis of 16S rDNA sequencing, the pedigree analysis, confirming to have notable difference with the kind system relation of existing lactobacillus paraceasi BCRC910220 and the secondary cheese subspecies of lactobacillus paraceasi BCRC14001, BCRC16100, is the new secondary cheese subspecies of lactobacillus paraceasi.
2. with the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain, carry out respectively that the filter paper agar diffusion method is analyzed and with pathogenic bacteria mixed culture analysis of experiments, the result shows, SG96 bacterial strain of the present invention not only can suppress growth of pathogenic bacteria such as intestinal bacteria and Salmonellas, and its bacteriostatic action is strong than the bacteriostatic action of microbiotic streptomycin, even sterilization effect is arranged.
3. the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain is mixed with aqueous solution feeding not from the sucking pig son, the result shows that SG96 bacterial strain of the present invention can obviously promote not from the suckling piglet weight increase, improve the meat change rate of pig starter feed, and has a good anti-diarrhea effect, and effect is better than the general microbiotic that uses, and proves that SG96 bacterial strain of the present invention has the ability of promoting animal opposing pathogenic bacteria.
Description of drawings
Figure 1A is the gramstaining result of the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain; Figure 1B is the kenel figure of the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain;
Fig. 2 is the agargel electrophoresis figure after the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 16S rDNA fragment is amplified with PCR;
Fig. 3 is the 16S rDNA sequence alignment figure of the secondary cheese subspecies of lactobacillus paraceasi of the present invention bacterial strain SG96, BCRC910220, BCRC14001, BCRC16100;
Fig. 4 is an analysis chart for the kind pedigree of the secondary cheese subspecies of lactobacillus paraceasi of the present invention bacterial strain SG96, BCRC910220, BCRC14001, BCRC16100 and the secondary cheese subspecies of other lactobacillus paraceasis bacterial strain;
Fig. 5 analyzes the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain for colibacillary restraining effect with the filter paper agar diffusion method;
Fig. 6 analyzes the restraining effect of the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain for Salmonellas with the filter paper agar diffusion method;
Fig. 7 is the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
8The result of bacterium number and the test of pathogenic bacteria mixed culture;
Fig. 8 is the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
7The result of bacterium number and the test of pathogenic bacteria mixed culture;
Fig. 9 is the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
6The result of bacterium number and the test of pathogenic bacteria mixed culture;
Figure 10 is the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
5The result of bacterium number and the test of pathogenic bacteria mixed culture;
Figure 11 is the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
4The result of bacterium number and the test of pathogenic bacteria mixed culture;
Figure 12 for the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain to not from the influence of suckling piglet body weight change; And
Figure 13 for the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain to not from the influence of suckling piglet diarrhea.
Embodiment
Below explanation partly and graphic intention, only have an exemplary illustration and non-limiting.Other specific exampless of the present invention can partly be understood from this explanation of reading by knowing well in this skill person.
The screening of the secondary cheese subspecies of embodiment one lactobacillus paraceasi of the present invention SG96 bacterial strain with separate
Collect a pig enteron aisle movement, insert MRS broth culture (Difco
TM, REF288130) in, after cultivating 24 hours under 37 ℃ of anaerobic environments, this culture is coated on MRS agar plate (Difco
TM, REF288210) on, cultivated 3 days at 37 ℃ then.After the cultivation, be collected in the bacterium colony that occurs on the nutrient agar, obtain the acid-producing bacteria of strain more than 1000 altogether, filter out with filter paper agar diffusion method (disc-agar diffusion) again and have inhibition intestinal bacteria (E.coli) (BCRC11634) and Salmonellas (S.typhimurium) milk-acid bacteria (BCRC129407), again from wherein picking out secondary cheese subspecies (the Lactobacillus paracasei subsp.paracasei) SG96 of active best function lactobacillus paraceasi, this spawn culture feature is as follows: growing up to diameter on the MRS agar plate is the grey bacterium colony of 2-3mm, and optimum culturing temperature is at 37 ℃.The secondary cheese subspecies of described lactobacillus paraceasi SG96 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (China General Microbiological Culture Collection Center, CGMCC), deposit number is CGMCC2697, and preservation date is on October 9th, 2008.
The gramstaining of the secondary cheese subspecies of embodiment two lactobacillus paraceasis of the present invention SG96 bacterial strain
Utilize gram staining method to detect the feature of bacterial strain of the present invention.The result is shown in Figure 1A, and bacterial strain of the present invention is gram-positive microorganism (Gram-positive bacteria), and no brood cell anerobe (non-sporinganaerobes), and no mobility bacterium (non-mobility) are the characteristic feature of Bacterium lacticum.
The mycology feature of the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain is as follows:
(a) morphological feature:
(1) cell shape and size: when cell places the MRS broth culture, after cultivating 24 hours under 37 ℃ of anaerobic environments, can observe in microscopically and present shaft-like bacillus (shown in Figure 1B).
(2) motility: no mobility
(3) flagellum: do not have
(4) sporulation: no sporulation
(5) gramstaining: the positive
(b) cultural characteristic:
(1) substratum: MRS broth culture, pH=6.25
(2) culture condition: 37 ℃ of anaerobic environments
(c) physiologic character:
(1) catalase: feminine gender
(2) oxydase: feminine gender
(3) API50CHL test: see also shown in the embodiment three.
The 16SrDNA sequencing of the secondary cheese subspecies of embodiment three lactobacillus paraceasis of the present invention SG96 bacterial strain is analyzed
1.DNA extraction
Utilize FavorPrep
TMBlood Genomic DNA Extraction Mimi Kit purify DNA is cultivated this bacterium overnight, gets 200 μ L bacterium liquid and adds 20 μ L proteinase K (proteinase K adds 110 μ LddH before using
2O vibration mixes 5min, and to make concentration be 11mg/mL, ice be stored in-20 ℃ standby), add 200 μ LFABG damping fluids, vibration mixed 5 seconds, 60 ℃ of heating are of short duration centrifugal after 15 minutes.Add 200 μ L95% alcohol, vibration mixed 10 seconds, and is of short duration centrifugal, add in the FABG tubing string (FABG column), and FABG column puts into CollectionTube, and 10, centrifugal 2 minutes of 000rpm.The FABG column of centrifugal back is taken out, to descend clear liquid to pour out after the back blots with toilet paper, FABG column is put back among the Collection Tube, add 500 μ L W1 damping fluids (must add 8mL95% alcohol when taking for the first time), 10, centrifugal 2 minutes of 000rpm is with the FABG column taking-up of centrifugal back, to descend clear liquid to pour out the back and blot, FABG column will be put back among the Collection Tube with toilet paper.Add 750 μ L WB (must add 40mL 95% alcohol when taking for the first time), 10, centrifugal 2 minutes of 000rpm will descend clear liquid to pour out the back and blot with toilet paper, FABG column put back among the Collection Tube, 10, centrifugal 6 minutes of 000rpm.FABG column with centrifugal back puts into the 1.5ml centrifuge tube again, adds Elution damping fluid 150 μ L, left standstill 3 minutes, 10, centrifugal 4 minutes of 000rpm, the DNA that this is purifying, place-20 ℃ standby.
2.16S rDNA PCR fragment is amplified
Get the DNA of the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain, and the DNA of lactobacillus paraceasi GMNL-32 bacterial strain (this bacterial strain deposit number is BCRC910220, this bacterial strain has obtained TaiWan, China patent of invention I284149, for having the lactobacillus paraceasi bacterial strain of the irritated correlation function of treatment), carrying out 16SrDNA (ribosomal DNA) fragment amplifies, used introduction (primers) is the general introduction of prokaryotic organism 16S rDNA PCR (the William G.Weisburg according to E.coli 16S rDNA gene 8-27 bit base and 1510-1492 bit base sequences Design, Susan M.Barns, Dale A.Pelletier, and DavidJ.Lane.16S Ribosomal DNA Amplification for Phylogenetic Study.J.Bacteriol.1991 January; 173 (2): 697-703), the introduction sequence is as follows:
Forward introduction fD1:
5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQIDNo：1)
Reverse introduction rP1:
5′-ACGGTTACCTTGTTACGACTT-3′ (SEQIDNo：2)
This 16S rDNA PCR fragment amplification program is as follows: (1) 95 ℃ following 5 minutes; (2) 94 ℃ following 1 minute; (3) 60 ℃ following 30 seconds; (4) 72 ℃ following 1.5 minutes; (5) 72 ℃ following 10 minutes, step (2) to (4) repeats 30 circulations.
In addition, get the secondary cheese subspecies BCRC14001 (Lactobacillus paracasei subsp.paracasei) of lactobacillus paraceasi,
And the DNA of the secondary cheese subspecies BCRC16100 of lactobacillus paraceasi (Lactobacillus paracaseisubsp.paracasei) carries out 16S rDNA fragment amplification, used introduction is according to lactobacillus paraceasi 16S rRNA V1 zone, and conservative (conserved) sequence (Ward of Bacterium lacticum 16S rRNA, L.J.H., and Timmins, M.J.1999.Differentiation of Lactobacilluscasei, Lactobacillus paracasei and Lactobacillus rhamnosus bypolymerase chain reaction.Lett.Appl.Microbiol.29:90-92.), the introduction sequence is as follows:
The forward introduction:
5′-CACCGAGATTCAACATGG-3′ (SEQIDNo：3)
Reverse introduction:
5′-CCCACTGCTGCCTCCCGTAGGAGT-3′ (SEQIDNo：4)
This 16S rDNA PCR fragment amplification program is as follows: (1) 95 ℃ following 5 minutes; (2) 94 ℃ following 1 minute; (3) 60 ℃ following 30 seconds; (4) 72 ℃ following 1.5 minutes; (5) 72 ℃ following 10 minutes, step (2) to (4) repeats 30 circulations.
3.16S rDNA PCR fragment sequencing, comparison
The PCR product of the 16S rDNA of the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain is carried out agargel electrophoresis, and the result as shown in Figure 2; Learnt that by Fig. 2 this PCR product fragment is about 1, (length of standard substance fragment from top to bottom is 3k to 500bp in regular turn, 2k, 1.5k, 1K, 900,800,700,600,500,400,300,200,100bp), and carry out sequencing work, the sequence of the 16S rDNA of the secondary cheese subspecies of lactobacillus paraceasi SG96 bacterial strain is shown in SEQIDNo:5; Sequence is contrasted multiplexed sequence comparison data collection (NCBI blastn, http://www.ncbi.nlm.nih.gov/BLAST) carries out sequence alignment, the result as shown in Figure 3, the sequence alignment result is 98% tolerance range lactobacillus paraceasi (Lactobacillusparacasei).
The API50CHL of the secondary cheese subspecies of embodiment four lactobacillus paraceasis of the present invention SG96 bacterial strain analyzes
Utilize the carbohydrate metabolism performance of API50CHL reagent test lactic bacterium strains of the present invention, API50CHL reagent can be used to identify that bacterial strain is in the difference that belongs to or plant, after testing, the API50CHL analytical results as shown in Table 1, the carbohydrate metabolic activity that shows the secondary cheese subspecies of bacterial strain of the present invention and lactobacillus paraceasi (Lactobacillusparacasei subsp.paracasei 1) has 98% similarity, therefore analyzes with API50CHL and confirms that further bacterial strain of the present invention ranges the secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) of secondary lactobacillus johnsonii.
Table one API50 CHL analytical results
The secondary cheese subspecies of embodiment five lactobacillus paraceasis of the present invention SG96 pedigree analysis
With NCBI Nucleotide database (htt p: //the 45 strain lactobacillus paraceasis (Lactobacillus paracasei) announced in www.ncbi.nlm.nih.gov/), the sequence of secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) of secondary lactobacillus johnsonii and lactobacterium casei (Lactobacillus casei), with the two strain BCRC14001 that announced in the BCRC database, the sequence of BCRC16100, and TaiWan, China patent and United States Patent (USP) are checked and approved the sequence of each strain GMNL-32 (BCRC910220) and CSK01, utilize EMBL-EBI ClustalW2 (http://www.ebi.ac.uk/clustalw) to carry out pedigree analysis relatively, the pedigree analysis result as shown in Figure 4, SG96 only belongs to same group with five strain bacterial classifications wherein, but can divide inconsistent another strain independently with this five strain, on behalf of SG96, this have uniqueness on categorizing system, and therefore proof SG96 is secondary cheese subspecies (the Lactobacillus paracasei subsp.paracasei) microbial strain of a novel secondary lactobacillus johnsonii really.
The secondary cheese subspecies of embodiment six lactobacillus paraceasis of the present invention SG96 bacterial strain is to the restraining effect of intestinal bacteria and Salmonellas
1. filter paper agar diffusion method (disc-agar diffusion)
The bacterium amount of intestinal bacteria (deposit number BCRC11634) and Salmonellas (deposit number BCRC12947) being got a platinum loop respectively is inoculated in TSA substratum (Soybean-Casein Digest Agar Medium, Difco
TM, REF236950) inclined-plane was cultivated 17-24 hour through 35-37 ℃, made bacterial strain reach the growth stage of stable development, and used spectrophotometer to carry out turbidity measurement, adjust suitable thalline turbidity (turbidity 80% of about 600nm, 80%T).Draw respectively in these two kinds of pathogenic bacteria bacterium liquid 0.5ml are about 48 ℃ to temperature the flat-plate solid substratum, pour in the culture dish again, place waited to solidify in 1 hour after, cut-off footpath 8mm aseptic filter paper ingot picks the bacterium liquid of the secondary cheese subspecies of lactobacillus paraceasi SG96 bacterial strain, put down the aseptic filter paper ingot gently on above-mentioned culture dish, cultivate 17-24 hour observations down in 35-37 ℃, measure and suppress loop diameter, use microbiotic Streptomycin sulphate (streptomycin) 15g/mL to do positive control group (positive control) simultaneously.
Result such as Fig. 5, shown in Figure 6, the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain is 11.8mm (as shown in Figure 5) to the inhibition loop diameter of intestinal bacteria (BCRC11634), and the inhibition figure diameter of microbiotic streptomycin is 11.0mm, shows that the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain has stronger restraining effect.And bacterial strain of the present invention is 12.5mm (as shown in Figure 6) to the inhibition figure diameter of Salmonellas (BCRC12947), and the inhibition loop diameter of microbiotic streptomycin is 11.2mm, shows that the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain has stronger restraining effect.
2. secondary cheese subspecies SG96 bacterial strain of lactobacillus paraceasi and the test of pathogenic bacteria mixed culture
In this experiment, select for use intestinal bacteria (BCRC11634) and Salmonellas (BCRC12947) as pathogenic bacteria.With pathogenic bacteria and the secondary cheese subspecies of lactobacillus paraceasi SG96 bacterial strain mixed culture (cultivating based on 37 ℃) with the TSA cultivation, and between incubation period, take a sample, the sampling sample is coated with and coils on MRS and TSA culture dish through getting 100uL after the serial dilution suitable multiple respectively, the MRS culture dish places under the 35-37 ℃ of anaerobic environment and cultivates, leave standstill and cultivated 24-48 hour, the TSA culture dish places under the 35-37 ℃ of aerobic environment and cultivates, leave standstill and cultivated 24 hours, treat to count respectively after bacterium colony generates the bacterium number (MRS culture dish) and pathogenic bacteria bacterium number (TSA culture dish) of the secondary cheese subspecies of lactobacillus paraceasi SG96 bacterial strain.
As shown in Figure 7, get the secondary cheese subspecies of lactobacillus paraceasi SG96 bacterial strain 10
8Bacterium number (CFU/ml) respectively with intestinal bacteria (BCRC11634) and Salmonellas (BCRC12947) mixed culture, the result be intestinal bacteria in 8 hours the bacterium number by 10
8Be reduced to 10
1, Salmonellas in 6 hours the bacterium number by 10
8Be reduced to 10
1, it is strong that the secondary cheese subspecies of visible lactobacillus paraceasi of the present invention SG96 bacterial strain suppresses effect, even sterilization effect is arranged.
As shown in Figure 8, get the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
7The bacterium number respectively with intestinal bacteria (BCRC11634) and Salmonellas (BCRC12947) mixed culture, intestinal bacteria in 22 hours the bacterium number by 10
8Be reduced to 0, Salmonellas in 16 hours the bacterium number by 10
8Be reduced to 0.
As shown in Figure 9, get the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
6The bacterium number respectively with intestinal bacteria (BCRC11634) and Salmonellas (BCRC12947) mixed culture, intestinal bacteria in 24 hours the bacterium number by 10
8Be reduced to 10
2, Salmonellas in 18 hours the bacterium number by 10
8Be reduced to 0.
As shown in figure 10, get the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
5The bacterium number respectively with intestinal bacteria (BCRC11634) and Salmonellas (BCRC12947) mixed culture, intestinal bacteria bacterium number is by 10 after 32 hours
8Be reduced to 0, and Salmonellas in 24 hours the bacterium number by 10
8Be reduced to 0.
As shown in figure 11, get the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain 10
4The bacterium number respectively with intestinal bacteria (BCRC11634) and Salmonellas (BCRC12947) mixed culture, intestinal bacteria bacterium number is by 10 after 38 hours
8Be reduced to 0, and Salmonellas in 24 hours the bacterium number by 10
8Be reduced to 0.
Embodiment seven animal experiments
1. the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain to nascent not from the influence of suckling piglet body weight
Each peasant household selects nascent not from suckling piglet by what produce same sow same period, is divided into two groups and feeds and raise, and one group is only to bestow every day to contain 1 * 10
7The secondary cheese subspecies of the lactobacillus paraceasi of the CFU/mL SG96 bacterial strain aqueous solution, another group is control group, feeds to raise microbiotic (OTC, oxytetracycline), feeding the time of raising is 21 days, notes down weekly the body weight change of piglet, and carries out statistical study with T-test.Result such as Figure 12 feed and to raise the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain after two weeks, and its weight increase rate is raised control group and shown (p＜0.05) than feeding, and its weight average increases the 0.7-0.8 kilogram weekly; Feed and raise the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain after three weeks, piglet is always put on weight also many than control group, show to feed and to raise the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain and can obviously promote, and improve the meat change rate of pig starter feed not from the suckling piglet weight increase.
2. the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain reduces nascent not from the effect of suckling piglet diarrhea
Because nascent not from the common diarrhea of suckling piglet, this diarrhea situation causes because of sow lactation or environmental factors usually, and piglet often early dies young because of the diarrhea situation is serious, as if improving diarrhea in early days, benefit will be arranged in the piglet normal growth.
Each peasant household select by produce same sow same period not from the sucking pig son, be divided into two groups and feed and raise, one group is only to bestow every day to contain 1 * 10
7The SG96 milk-acid bacteria aqueous solution of CFU/mL, another group is control group, feed and raise microbiotic (OTC, oxytetracycline), feeding the time of raising is 21 days, note down baby pig diarrhoea situation weekly, marked, the scoring of diarrhea mark is ordered (Underdahl NR according to documentation standards that the people delivered such as Underdahl, Torres-Medina A, Dosten AR.1982.Effect of Streptococcus faeciumC-68 in control of Escherichia coli-induced diarrhea in gnotobioticpigs.Am.J.Vet.Res.1982.12:2227-2232), scoring explanation:
0 minute=normal;
Dry around 1 minute=soft stool, anus;
Moist around the 2 minutes=anus;
3 minutes=water dysentery;
4 minutes=standard 1-3+ is off one's feed, loses weight and becomes thin
And carry out statistical study with T-test.Result such as Figure 13 feed and raise the effect that the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain can reach the minimizing diarrhea, and its effect is similar to the control group effect when second week.Through feed raised for three weeks after, feed and to raise the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain to reduce the diarrhea effect better than control group (feed and raise the microbiotic group), and apparent difference (p＜0.05) is arranged, represent that the secondary cheese subspecies of lactobacillus paraceasi of the present invention SG96 bacterial strain has good anti-diarrhea effect, and effect is better than the microbiotic that general junket peasant household uses, and can allow piglet not produce the problem of antibiotic remains.
Though just special specific examples and application note are crossed the present invention, but those of ordinary skills can produce additional specific examples from the disclosed scope of this specification sheets, and revise and do not disobey from the order of being advocated of the present invention or surpass the scope that the present invention advocated.Therefore, graphic and explanation that understanding person this paper is partly through proposing as an example to help understanding of the present invention and should not be considered as limiting its scope.
Above-listed detailed description system specifies at one of the present invention possible embodiments, and only this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implements or change, all should be contained in the claim of this case.
The PB20080475 sequence table
＜110〉Syngen Biotech Co., Ltd. (SyngenbiotechCO., LTD.)
＜120〉the secondary cheese subspecies bacterial strain of lactobacillus paraceasi and bacteria inhibiting composition and purposes
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<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward introduction
<400>1
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<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse introduction
<400>2
acggttacct?tgttacgact?t?21
<210>3
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉forward introduction
<400>3
caccgagatt?caacatgg?18
<210>4
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reverse introduction
<400>4
cccactgctg?cctcccgtag?gagt?24
<210>5
<211>1007
<212>DNA
＜213〉the secondary cheese subspecies SG96 bacterial strains (Lactobacillu sparacaseisubsp.paracasei SG96 strain) of lactobacillus paraceasi
<400>5
ggggcgcgtg?ctatacatgc?aagtcgaacg?agttctcgtt?gatgatcggt?gcttgcaccg 60
agattcaaca?tggaacgagt?ggcggacggg?tgagtaacac?gtgggtaacc?tgcccttaag 120
tgggggataa?catttggaaa?cagatgctaa?taccgcatag?atccaagaac?cgcatggttc 180
ttggctgaaa?gatggcgtaa?gctatcgctt?ttggatggac?ccgcggcgta?ttagctagtt 240
ggtgaggtaa?tggctcacca?aggcgatgat?acgtagccga?actgagaggt?tgatcggcca 300
cattgggact?gagacacggc?ccaaactcct?acgggaggca?gcagtaggga?atcttccaca 360
atggacgcaa?gtctgatgga?gcaacgccgc?gtgagtgaag?aaggctttcg?ggtcgtaaaa 420
ctctgttgtt?ggagaagaat?ggtcggcaga?gtaactgttg?tcggcgtgac?ggtatccaac 480
cagaaagcca?cggctaacta?cgtgccagca?gccgcggtaa?tacgtaggtg?gcaagcgtta 540
tccggattta?ttgggcgtaa?agcgagcgca?ggcggttttt?taagtctgat?gtgaaagccc 600
tcggcttaac?cgaggaagcg?catcggaaac?tgggaaactt?gagtgcagaa?gaggacagtg 660
gaactccatg?tgtagcggtg?aaatgcgtag?atatatggaa?gaacaccagt?ggcgaaggcg 720
gctgtctggt?ctgtaactga?cgctgaggct?cgaaagcatg?ggtagcgaac?aggattagat 780
accctggtag?tccatgccgt?aaacgatgaa?tgctaggtgg?ttgggagggt?ttccgccctt 840
cagtgccgca?gctaacgcat?taagcattcc?gcctggggga?gtacgaccgc?aaggttgaac 900
tcaaggaatt?gacgggggcc?cgcacaagcg?gtggagcatg?tggtttattc?gaagcaacgc 960
gagagcttac?cagtcttgac?atcttttgat?cactgagaga?tcagttt 1007
Claims (9)
1. secondary cheese subspecies (Lactobacillus paracasei subsp.paracasei) the SG96 bacterial strain of lactobacillus paraceasi, its microbial preservation number are: CGMCC2697.
2. the secondary cheese subspecies of the described lactobacillus paraceasi of claim 1 SG96 bacterial strain suppresses the medicine of intestinal bacteria (Escherichia coli) growth or the purposes in the reagent in preparation.
3. the secondary cheese subspecies of the described lactobacillus paraceasi of claim 1 SG96 bacterial strain suppresses the medicine of Salmonellas (Salmonella typhimurium) growth or the purposes in the reagent in preparation.
4. the secondary cheese subspecies of the described lactobacillus paraceasi of claim 1 SG96 bacterial strain suppresses the medicine of diarrhea or the purposes in the reagent in preparation.
5. composition is characterized in that being made up of the secondary cheese subspecies SG96 bacterial strain of the described lactobacillus paraceasi of the claim 1 of significant quantity and carrier or auxiliary material.
6. the described composition of claim 5 suppresses the medicine of intestinal bacteria (Escherichia coli) growth or the purposes in the reagent in preparation.
7. the described composition of claim 5 suppresses the medicine of Salmonellas (Salmonella typhimurium) growth or the purposes in the reagent in preparation.
8. the described composition of claim 5 suppresses the medicine of diarrhea or the purposes in the reagent in preparation.
9. composition as claimed in claim 5 is characterized in that said composition is prepared into animal drinking-water additive, animal-feed additive, animal usefulness medical composition, human with medical composition, human food prods's additive, human beverage additives, human food prods, human drink or human health food.
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| CN102373162A (en) * | 2010-08-09 | 2012-03-14 | 生展生物科技股份有限公司 | Lactobacillus salivarius M6 and antibacterial composition containing same |
| CN103070287A (en) * | 2013-02-26 | 2013-05-01 | 湖南省微生物研究所 | Lactobacillus paracasei fermented feed and preparation method thereof, fermenting equipment and application of lactobacillus paracasei fermented feed |
| CN104254599A (en) * | 2012-04-09 | 2014-12-31 | 科.汉森有限公司 | Bioprotection using lactobacillus paracasei strains |
| CN107164269A (en) * | 2017-06-06 | 2017-09-15 | 北京市农林科学院 | A kind of lactobacillus paracasei, preparation and its application in pig feed |
| CN108373984A (en) * | 2018-04-09 | 2018-08-07 | 北京博锦元生物科技有限公司 | A kind of Lactobacillus paracasei and its application |
| CN110878270A (en) * | 2019-12-21 | 2020-03-13 | 福建省农业科学院农业工程技术研究所 | Lactobacillus paracasei subspecies paracasei and application thereof |
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| CN117778278A (en) * | 2024-02-27 | 2024-03-29 | 山东中科嘉亿生物工程有限公司 | Compound lactobacillus silage additive and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1364586A1 (en) * | 2002-05-24 | 2003-11-26 | Nestec S.A. | Probiotics and oral tolerance |
| WO2006091103A2 (en) * | 2005-02-28 | 2006-08-31 | N.V. Nutricia | Nutritional composition with probiotics |
| CN1285722C (en) * | 2005-03-07 | 2006-11-22 | 黑龙江大学 | Peptides natural microbial antiseptic agent producing strain, its use and preparation method for antiseptic agent |
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| CN102373162A (en) * | 2010-08-09 | 2012-03-14 | 生展生物科技股份有限公司 | Lactobacillus salivarius M6 and antibacterial composition containing same |
| CN104254599A (en) * | 2012-04-09 | 2014-12-31 | 科.汉森有限公司 | Bioprotection using lactobacillus paracasei strains |
| CN104254599B (en) * | 2012-04-09 | 2018-01-23 | 科.汉森有限公司 | Using the biological protection of Lactobacillus paracasei bacterial strain |
| CN103070287A (en) * | 2013-02-26 | 2013-05-01 | 湖南省微生物研究所 | Lactobacillus paracasei fermented feed and preparation method thereof, fermenting equipment and application of lactobacillus paracasei fermented feed |
| CN107164269A (en) * | 2017-06-06 | 2017-09-15 | 北京市农林科学院 | A kind of lactobacillus paracasei, preparation and its application in pig feed |
| CN107164269B (en) * | 2017-06-06 | 2020-03-24 | 北京市农林科学院 | Lactobacillus paracasei, preparation and application of lactobacillus paracasei in pig feed |
| CN108373984A (en) * | 2018-04-09 | 2018-08-07 | 北京博锦元生物科技有限公司 | A kind of Lactobacillus paracasei and its application |
| CN110959865A (en) * | 2018-09-30 | 2020-04-07 | 内蒙古伊利实业集团股份有限公司 | New application of lactobacillus paracasei K56 capable of adjusting gastrointestinal flora balance |
| US11666613B2 (en) | 2018-09-30 | 2023-06-06 | Inner Mongolia Yili Industrial Group Co., Ltd. | Use of Lactobacillus paracasei subsp. paracasei K56 capable of regulating gastrointestinal flora balance |
| CN110878270A (en) * | 2019-12-21 | 2020-03-13 | 福建省农业科学院农业工程技术研究所 | Lactobacillus paracasei subspecies paracasei and application thereof |
| CN117778278A (en) * | 2024-02-27 | 2024-03-29 | 山东中科嘉亿生物工程有限公司 | Compound lactobacillus silage additive and preparation method and application thereof |
| CN117778278B (en) * | 2024-02-27 | 2024-05-03 | 山东中科嘉亿生物工程有限公司 | Compound lactobacillus silage additive and preparation method and application thereof |
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