JP2010161944A - Lactobacillus paracasei subsp. paracasei (sg96) of new type, microbe-inhibiting composition containing the same and application thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microbial strain of Lactobacillus paracasei subsp. paracasei of a new type, having evident difference from the conventional one. <P>SOLUTION: The Lactobacillus paracasei subsp. paracasei (SG96) of the new type, the microbe-inhibiting composition containing the subsp. paracasei, and the application thereof are provided. The strain including the Lactobacillus paracasei subsp. paracasei (SG96) is deposited in China General Microbiological Culture Collection Center (CGMCC) as an accession number of CGMCC 2697, and has effects for inhibiting or sterilizing the growth of a germ such as Escherichia coli and Salmonella. The microbe-inhibiting composition is used as a form of an additive for animal drinking water, an additive for animal feed, a medicinal composition for animals and human beings, a food additive, a beverage additive, a food, a beverage, a healthy food or the like. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to Lactobacillus paracasei subsp. The present invention relates to a fungus-suppressing composition containing a subspecies of casei, a combination thereof, and use thereof.

  In recent decades, the population has grown rapidly, the level of material life has greatly improved, and people's demand for meat has increased significantly. Due to the large scale, especially in the undeveloped countries, the ability to import food is relatively low, and as a result of breeding economic animals in large quantities, in high density or in large areas, various diseases have spread rapidly among animals. In order to improve the disease problem of economic animals, or to grow economical animals quickly, the method of adding antibiotics to the feed for a long time or the method of injecting antibiotics into animals is taken, resulting in drug-resistant strains. Is incurred.

  In the late 1960s, with the advent of antibiotics, mankind had an unprecedented victory in the field of treating gonococcal infections and no longer had to be afraid of many deadly infectious diseases. But in recent years, the emergence of drug-resistant gonorrhea has raised concerns and fears of human infectious diseases again, because antibiotics are no longer a panacea for controlling disease states, and the diffusion speed of drug-resistant gonorrhea is even more of our imagination. There is something that goes away. The fact that the koji mold has chemical resistance not only reduces or eliminates the therapeutic effect, but also has a serious impact on human health. Forty years ago, the number of deaths from infectious diseases worldwide was approximately 7 million per year. Today, medical science has made tremendous progress, while deaths from infectious diseases have reached 2,000 per year. The number has risen to 10,000. When people eat meat, eggs, milk and other poultry foods that contain and retain antibiotics for a long period of time, these antibiotic residues cause harmful effects on the human body, accumulate in the human body, and are resistant to chemicals. This has led to the constant outbreak of sex strains.

  Meat safety issues have been of most interest to consumers, agricultural and livestock producers and governments over the last decade. The reason why consumers have continued to have a high level of concern for meat safety has been the result of several amazing meat safety incidents at home and abroad over the past few years, such as the mad cow disease that occurred in the UK. H5N1 bird sense in Southeast Asia and Mainland China. In order to protect the safety of edible meat, countries around the world attach great importance to the effects caused by antibiotic residues, and in order to delay the development of drug-resistant strains, In the medical and agricultural fields, we started restricting the use and control of antibiotics, and tried to suppress the generation of drug-resistant strains in this way. The European Union has banned the use of certain antibiotics one after another since 1995 and has been doing well, for example, researchers in the Danish veterinary lab in Copenhagen have avoparcin After the ban on the use of antibiotics, the resistance of Enterococcus faecium to Avoparsin in the Danish chick digestive tract has declined from 73% in 1995 AD to 6% in 2000. Germany and the Netherlands banned the use of avopalcin during the same period of five years, resulting in a decrease in drug-resistant strains in animals and the human body, so the European Union will add antibiotics to the feed from January 2006 Began to ban.

  The purpose of adding antibiotics to the feed is to increase growth efficiency of the poultry by promoting growth and reducing the roughage, while the growth cycle of the poultry is shortened, Precipitation is inevitably reduced and the peculiarity and quality of poultry products declines; more importantly, drug residues are a serious hazard to human health, especially in childhood. Effect. Therefore, prohibiting the use of antibiotics in the feed of economic animals or reducing the types and doses of antibiotics has become a global trend. However, even after the ban on the use of antibiotics, many problems have arisen, and in order for livestock farming to be able to produce normally, contractors have accepted the idea of using Probiotics as a substitute for antibiotics. It goes one step further and adopts it as feed additives.

  Probiotics are mainly lactic acid bacteria, lactic acid bacteria can maintain and restore the balance of the gut flora of poultry, eliminate stress, increase immune anti-disease capacity, reduce disease incidence . The reason for advocating the green-cutting feed and liquid fermented feed fermented using lactic acid bacteria is that the lactic acid bacteria fermented feed effectively retains the nutritional components of the feed, increases the digestive utilization rate, and further causes illness in the fermentation process This is because it is possible to eliminate microorganisms that cause pathogenic bacteria and human-cause common diseases. Lactic acid bacteria are already recognized as an ideal substitute for antibiotics.

The beneficial effects of probiotics in poultry are
(1) Maintain and restore the bacterial group equilibrium with lactic acid bacteria as the dominant bacterial group in the digestive tract of poultry. Eliminate environmental disorders in the gastrointestinal tract due to stress (eg, weaning, changing feed, high temperature, cold, transportation, frightening, disease, etc.) of poultry. It suppresses abnormal growth of harmful bacteria and invasion and colonization of lethal bacteria, prevents and treats gastrointestinal diseases such as diarrhea, and reduces or eliminates endotoxin generation. The effect of supplementing lactic acid bacteria on young animals and chicks whose microbial system has not yet been established is particularly remarkable.
(2) Generate non-specific immune regulators, increase the immune anti-disease capacity of poultry, and reduce the disease infection rate and mortality rate. It becomes an effective substitute for antibiotics and chemical drugs, reduces the approximate rate of drug residues, and improves human food quality.
(3) Replenish nutritional components, promote the growth of poultry, produce digestive enzymes, enhance digestive absorption capacity, and significantly improve aquaculture production and efficiency.
(4) To reduce the odorous substance in the feces faeces and improve the environment of the farm.
(5) Significantly increase milk, meat and egg production, and improve the effects and profits of the aquaculture industry.

  Food safety issues have already been highly valued by governments around the world. Currently, governments are actively promoting green livestock farming, supporting the development of green products in terms of policy, financial strength and physical strength. Build a green aquaculture model base, cultivate a brand of green livestock products, the green product managers get real benefits, and provide consumers with high quality, pollution-free and safe green poultry foods , Trying to bring happiness to mankind.

  As can be seen from the above, the conventional method of injecting antibiotics into an economical animal still has many drawbacks, and it is difficult to say that it is a good design, and improvement has been awaited.

  The inventor of the present application tried to make new improvements as much as possible in view of the disadvantages of the respective items derived from the use of the above-mentioned antibiotics, and after many years of hard work, finally the subtype of the new casei (Lactobacillus paracasei subsp. Paracasei) SG96, a fungus-suppressing composition containing the same, and its use were completed.

  The object of the present invention is to provide a subspecies of a new type of casei, which is clearly different from the species relationship of the conventional casei BCRC 910220 (Lactobacillus paracasei) and subtype BCRC14001, BCRC16100 (Lactobacillus paracasei subsp. Paracasei). It is to provide a microbial strain.

  Another object of the present invention is to provide a fungus-suppressing composition containing a new strain of casei SG96 and having an effect of inhibiting the growth of pathogenic bacteria such as Escherichia coli and Salmonella typhimurium. is there.

  Still another object of the present invention is to provide a new strain of bacterial casei, subspecies SG96, and to provide an application of the fungus-suppressing composition. The fungus-suppressing composition comprises an additive for animal feed and drinking water. By doing so, the ability of the animal to resist pathogenic bacteria can be enhanced; the fungus-suppressing composition can be further advanced to enhance the ability of human body to resist pathogenic bacteria as a food, food additive or food. Can do.

  In order to achieve the above-mentioned object, the subtype SG96 of the new casei bacterium according to the present invention, the fungus-suppressing composition containing the same, and the use thereof include the subspecies SG96 strain of the new casei. After selecting from intestinal excrement, after performing Gram staining analysis, API 50 CHL analysis, 16S rDNA sequence analysis, phylogenetic analysis, etc., it was confirmed as a microbial strain of a new type of Lactobacillus paracasei subsp. The strain of the new casei subspecies SG96 has been deposited with the China General Microbiological Culture Collection Center (CGMCC), the accession number is CGMCC 2697, and the accession date is October 9, 2008. Day.

  As a result of carrying out analysis by paper disc agar diffusion method (disc-agar diffusion) and pathogen mixed culture test analysis, respectively, the strain of casei subsp. SG96 screened according to the present invention, the SG96 strain according to the present invention, It has been shown to suppress the growth of pathogenic bacteria such as E. coli and S. typhimurium and to further have a sterilizing effect.

  As a result of preparing an aqueous solution of a strain of the casei subsp. SG96 according to the present invention and feeding it to the piglet before weaning, the SG96 strain according to the present invention significantly increases the weight of the pre-weaning piglet, Improves feed efficiency of feed and has a good anti-diarrheal effect, and the results are superior to commonly used antibiotics, and the strain of SG96 according to the present invention has the ability to enhance the resistance to animal pathogens Proven to be.

The antibacterial composition containing the strain of casei subsp. SG96 according to the present invention is prepared by mixing an aqueous solution into animal drinking water, and in addition to conventional feed additive manufacturing technology, other forms such as animal feed Additives, veterinary and human medical compositions, food additives, beverage additives, foods, drinks, health foods, etc. can be taken by animals and humans in an oral dosage manner. In one preferred embodiment, the antimicrobial composition is made as a lactic acid tablet / powder or granule and can be added to animal feed or food (eg 1 kilogram of powder or granule per ton of feed). Jo casei subspecies SG96 strain of bacteria (10 ⁷ CFU / g) added); also individually or in other fermented dairy product with lactic acid bacteria can also make dairy products such as yogurt.

Subtype SG96 of the new casei bacterium according to the present invention, and a fungus-suppressing composition containing the same, and uses thereof have the following advantages compared to other conventional techniques,
(1) As a result of Gram staining analysis, API 50 CHL analysis, 16S Rdna sequence analysis, phylogenetic analysis, etc. for the subsp. SG96 strain of casei according to the present invention, the conventional casei BCRC 910220 and subtypes of casei There was a clear difference in the species relationship with the species BCRC14001 and BCRC16100, confirming that it was a new variant of casei.
(2) As a result of paper filter paper agar diffusion method analysis and pathogen mixed culture test analysis for the subsp. SG96 strain of casei according to the present invention, respectively, the SG96 strain according to the present invention is a pathogen such as Escherichia coli and Salmonella. Not only can growth be suppressed, but also the fungus-suppressing effect is stronger than that of the antibiotic streptomycin, indicating that it has a sterilizing effect.
(3) As a result of preparing a water-soluble liquid using the subspecies SG96 strain of casei according to the present invention and administering it to a piglet before weaning, the SG96 strain according to the present invention clearly increases the weight of the pre-weaning piglet And improve the feed efficiency of piglet feed, have a good diarrhea suppression effect, and the results are superior to commonly used antibiotics, the SG96 strain according to the present invention can enhance the ability of animals to resist pathogens Proved.

It is a Gram stain result of subspecies SG96 strain of casei according to the present invention. It is a form figure of subspecies SG96 strain of casei according to the present invention. It is the agar gel electrophoretic diagram which carried out PCR expansion of the 16S rDNA fragment | piece of the subspecies SG96 strain of the said casei. It is a 16S rDNA sequence comparison diagram of subtype strains SG96, BCRC 910220, BCRC14001, BCRC16100 of Bacillus casei. It is a species-related systematic analysis diagram with the subspecies strains SG96, BCRC 910220, BCRC14001, BCRC16100, and other subspecies strains of casei. This is an analysis of the inhibitory effect of Escherichia coli subsp. SG96 strain on Escherichia coli by paper filter paper agar diffusion method. This is an analysis of the inhibitory effect of subtype SG96 strain of casei on Salmonella by paper filter paper agar diffusion method. It is a result of the mixed culture test of the subspecies SG96 strain 10 < ⁸ > bacteria number and pathogenic bacteria of casei. Casei subspecies SG96 strain 10 ⁷ Results of mixed culture test bacteria count and pathogenic bacteria. Casei subspecies SG96 strain ¹⁰⁶ results in mixed culture test bacteria count and pathogenic bacteria. It is the result of the mixed culture test of the subspecies SG96 strain 10 < ⁵ > bacteria and pathogenic bacteria of casei. It is a result of the mixed culture test of subspecies SG96 strain 10 ⁴ bacteria number and pathogenic bacteria of casei. It is the influence with respect to the body weight change of the weanling piglet of the subspecies SG96 strain of casei. It is the influence with respect to diarrhea of the preweaning piglet of the subspecies SG96 strain of casei. It is an analysis result of API50CHL of subspecies SG96 strain of casei.

  The present invention will be described with reference to the following examples. However, the present invention is not limited to the following examples.

Example 1 Selection and Isolation of Casei subsp. SG96 strain Porcine intestinal excreta were collected, placed in MRS meat soup medium (Difco ^™ , REF288130) and cultured in an anaerobic environment at 37 ° C for 24 hours. Thereafter, the culture was applied to an MRS agar plate (Difco ^™ , REF288210) and cultured at 37 ° C. for 3 days. After culture, collect colonies of bacteria that appeared on the agar medium, obtain a total of about 1,000 living acid bacteria, and then use E. coli (BCRC11634) and paper disc agar diffusion method. A lactic acid bacterium capable of suppressing S. typhimurium (BCRC129407) was selected, and among them, the most active subtype of Lactobacillus paracasei subsp. Paracasei SG96 was selected. The culture characteristics of this species are as follows: grow to gray colonies with a diameter of 2-3 mm on MRS agar plates, optimal culture temperature is 37 ° C. The above-mentioned casei subspecies SG96 is deposited with the China General Microbiological Culture Collection Center (CGMCC), the accession number is CGMCC 2697, and the date is October 9, 2008 It is.

Example 2 Gram stain of casei subsp. SG96 strain The characteristics of the strain were examined by the Gram stain method. As shown in FIG. 1, the strain of the present invention is a Gram-positive bacteria,
Non-sporing anaerobes and non-mobility, typical characteristics of lactic acid bacteria.
The bacteriological characteristics of the casei strain SG96 are as follows:

(A) Morphological features Morphological features (1) Shape and size of cells: When cells are placed in an MRS meat soup culture medium and cultured in an anaerobic environment at 37 ° C. for 24 hours, they appear in the shape of a rod under a microscope. Aspergillus is observed (as shown in FIG. 1B).
(2) Activity: non-motility (3) Flagella: None (4) Sporulation: Not formed (5) Gram staining: Positive (b) Culture characteristics:
(1) Culture medium: MRS meat soup culture medium, pH = 6.25
(2) Culture conditions: 37 ° C anaerobic environment (c) Physiological characteristics:
(1) Hydrogen peroxide enzyme: negative (2) Oxidase: negative (3) API 50 CHL test: See Example 3.

Example 3 16S rDNA sequence analysis of subsp. SG96 strain of casei DNA extraction
FavorPrep ^TM Blood Genomic DNA Extraction Mimi Kit for purifying DNA by the placing cultures overnight, 200 [mu] L of 110 [mu] L ddH ₂ O prior to the bacteria solution that uses proteinase K (proteinase K of 20 [mu] L addition of 5 Add the concentration at 11 mg / Ml and stock at -20 ° C), add 200 μL FABG buffer, shake for 5 seconds, heat at 60 ° C for 15 minutes, and centrifuge briefly . Add 200 μL of 95% alcohol, shake and mix for 10 seconds, centrifuge briefly, place in FABG column (FABG column), place FABG column in Collection Tube, and centrifuge at 10,000 rpm for 2 minutes. Remove the centrifuged FABG column, discard the subnatant, blot with toilet paper and dry, return the FABG column into the Collection Tube, add 500 μL of W1 buffer (the first time add 8 mL of 95% alcohol), Centrifuge at 10,000 rpm for 2 minutes, remove the centrifuged FABG column, discard the subnatant, blot with toilet paper and dry, and place the FABG column back into the Collection Tube. Add 750 μL of WB (first time with 40 mL of 95% alcohol) and centrifuge at 10,000 rpm for 2 minutes. Discard the subnatant, blot with toilet paper and dry, return the FABG column into the Collection Tube, and centrifuge at 10,000 rpm for 6 minutes. Next, put the centrifuged FABG column into a 1.5 ml centrifuge tube, add 150 μL of Elution Buffer, leave it for 3 minutes, and centrifuge at 10,000 rpm for 4 minutes. This is purified DNA. Stockpile at ℃.

2. Enlargement of 16S rDNA PCR fragment Take the DNA of subtype SG96 of Casei and the DNA of GMNL-32 of casei (the accession number of the strain is BCRC 910220, the strain has already obtained the Chinese invention patent I284149) The primer used to expand the fragment is a base sequence at positions 8-27 and 1510-1492 in the E. coli 16S rDNA gene. Prokaryotic 16S rDNA PCR primer designed based on (William G. Weisburg, Susan M. Barns, Dale A. Pelletier, and David J. Lane.16S Ribosomal DNA Amplification for Phylogenetic Study.J. Bacteriol. 1991 January; 173 (2): 697-703), Primer sequence Is as follows,

Forward primer fD1:
5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID No: 1)
Reverse primer rP1:
5'-ACGGTTACCTTGTTACGACTT-3 '(SEQ ID No: 2)

  The 16S rDNA PCR fragment expansion procedure is as follows: (1) 5 minutes at 95 ° C; (2) 1 minute at 94 ° C; (3) 30 seconds at 60 ° C; (4) 72 ° C 1.5 minutes below; (5) Steps (2) to (4) are repeated 30 cycles at 72 ° C. for 10 minutes.

  In addition, when the 16S rDNA PCR fragment was used to expand the DNA of the casei subsp.BCRC14001 (Lactobacillus paracasei subsp.paracasei) and the casei subsp.CRC16100 (Lactobacillus paracasei subsp.paracasei) And according to the rank of conserved chyle 16S rRNA. (Ward, L. J. H., and Timmins, M. J. 1999. Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction.Lett. Appl. Microbiol. 29: 90-92.)

Forward primer:
5'-CACCGAGATTCAACATGG-3 '(SEQ ID No: 3)
Reverse primer:
5'-CCCACTGCTGCCTCCCGTAGGAGT-3 '(SEQ ID No: 4)

The 16S rDNA PCR fragment expansion procedure is as follows: (1) 95 ° C. for 5 minutes; (2) 94 ° C. for 1 minute; (3) 60 ° C. for 30 seconds; (4) 72 ° C. Repeat for 1.5 minutes; (5) Repeat for 10 cycles at 72 ° C and repeat steps (2) to (4) for 30 cycles.

3. Sequence analysis and comparison of 16S rDNA PCR fragments The results of agar gel electrophoresis on the PCR products of subtype SG96 strain 16S rDNA of casei are as shown in FIG. 2; The PCR product fragment is approximately 1,500 bp (the length of the standard fragment is 3k, 2k, 1.5k, 1K, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp in order from top to bottom) The sequence of Lactobacillus paracasei 16S rDNA, which has been subjected to sequence analysis work, is as shown in SEQ ID No: 5; the sequence is a composite sequence comparison data collection (NCBI blastn, http : //www.ncbi.nlm.nih.gov/BLAST), and the results are shown in FIG. 3, and the results of the order comparison are 98% accurate Lactobacillus paracasei (Lactobacillus paracasei) Met.

Example 4 API 50 CHL analysis of casei subsp. SG96 strain
API 50 CHL reagent used for the inspection of carbonic acid compound metabolism performance of lactic acid bacteria strains according to the present invention, API 50 CHL reagent can be used to identify differences in the genus or species of strains, and commissioned to Hsinchu Food Industry Development Laboratory However, as shown in Table 1 and FIG. 14, the analysis results of API 50 CHL show that the strain according to the present invention and the subspecies of Lactobacillus paracasei subsp. Therefore, the strain according to the present invention was confirmed as a subspecies of Lactobacillus paracasei subsp. Paracasei by one step by API 50 CHL analysis.

Example 5 Phylogenetic analysis of subtype SG96 of casei
45 strains of Lactobacillus paracasei, Lactobacillus paracasei subsp. Paracasei and Lactobacillus paracasei published in the NCBI Nucleotide database (http://www.ncbi.nlm.nih.gov/) The rank order of casei (Lactobacillus casei) published in the BCRC database, the rank order of two BCRC14001 and BCRC16100, the Chinese patent and the US patent share GMNL-32 (BCRC910220) and the order of CSK01, EMBL-EBI Phylogenetic analysis was performed by ClustalW2 (http://www.ebi.ac.uk/clustalw). The results of the phylogenetic analysis are as shown in FIG. 4. SG96 belongs to the same family group as five of them. However, it is also possible to diverge another independent line from these five strains, which represents the uniqueness of SG96 on the taxonomic lineage, so SG96 is indeed a new type of Lactobacillus paracasei subsp paracasei) Proving that it is a microbial strain.

Example 6 Inhibitory Action of Kasei Bacilli SG96 on E. coli and Salmonella Paper-disc agar diffusion method
Escherichia coli (Accession No. BCRC 11634) and Salmonella (Accession No. BCRC 12947) are each inoculated on the slope of TSA culture medium (Soybean-Casein Digest Agar Medium, Difco ^™ , REF236950), After culturing at 37 ° C for 17-24 hours, the strain was brought to a stable growth stage, and the turbidity was measured using a spectrophotometer, and the turbidity of the cells was appropriately adjusted (turbidity of about 600 nm). Degree 80%, 80% T). Draw 0.5 ml of each of these two pathogens into a plate solid culture medium at about 48 ° C, then transfer to a culture dish and let stand for 1 hour to solidify, then use a sterile filter paper tablet with a diameter of 8 mm Inoculate the bacterial solution of the strain SG96 of the fungus, place this sterile filter paper tablet lightly on the above culture dish, incubate at 35-37 ° C for 17-24 hours, observe the results, The diameter was measured and a positive control group was made using 15 g / mL of the antibiotic streptomycin.

  As a result, as shown in FIGS. 5 and 6, the diameter of the inhibitory rod against the Escherichia coli (BCRC 11634) of the subsp. SG96 strain of casei according to the present invention is 11.8 mm (see FIG. 5), whereas the suppression of streptomycin The diameter of the cocoon is 11.0 mm, which indicates that the casei subsp. SG96 has a relatively strong inhibitory action. The diameter of the inhibition zone for Salmonella (BCRC 12947) of the strain according to the present invention is 12.5 mm (see FIG. 6), while the inhibition diameter of the antibiotic streptomycin is 11.2 mm, This shows that the strain has a relatively strong inhibitory action.

2. Mixed culture test of casei subsp. SG96 and pathogen In this experiment, E. coli (BCRC 11634) and Salmonella (BCRC 12947) were selected as pathogens. Pathogen and casei subsp. SG96 strains were mixed (cultured at 37 ° C with TSA medium) and sampled during the culture period. Samples were serially diluted to an appropriate multiple and then 100 μL each. Take MRS and TSA culture dish and apply to MRS and TSA culture dishes. MRS culture dishes were allowed to stand for 24 hours in an anaerobic environment at 35-37 ° C. TSA culture dishes were in an aerobic environment at 35-37 ° C. After the colony formation of the fungi that had been allowed to stand for 24 hours, the number of subsp. SG96 strains of M. casei (MRS culture dish) and the fungus of the pathogens (TSA culture dish) were counted.

As shown in FIG. 7, 10 ⁸ subtype SG96 strains (CFU / ml) were taken and mixed with Escherichia coli (BCRC 11634) and Salmonella (BCRC 12947), respectively. number of bacteria was reduced from 10 ⁸ to 10 ¹ within, Salmonella bacteria count in the 6 hours was reduced from 10 ⁸ to 10 ^1, this inhibiting effect is stronger, it can be seen that there is a further sterilization effect.

As shown in FIG. 8, a subspecies SG96 strain 10 ⁷ bacteria count of casei were respectively mixed culture with E. coli (BCRC 11634) and Salmonella (BCRC 12947), Escherichia coli bacteria count in the 22 hours from 0 to 10 ⁸ The reduced Salmonella decreased from 10 ⁸ to 0 within 16 hours.

As shown in FIG. 9, casei respectively coli subspecies SG96 strain 10 ⁶ bacteria count of bacteria (BCRC 11,634) and salmonella (BCRC 12947) and mixed and incubated, Escherichia coli 10 ² bacteria count from 10 ⁸ to 24 hours The number of Salmonella decreased from 10 ⁸ to 0 within 18 hours.

As shown in FIG. 10, a subspecies SG96 strain 10 ⁵ number of bacteria casei were respectively mixed culture with E. coli (BCRC 11,634) and salmonella (BCRC 12947), after 32 hours, E. coli is the number of bacteria from 10 ⁸ 0 The reduced Salmonella decreased from 10 ⁸ to 0 within 18 hours.

As shown in FIG. 11, a subspecies SG96 strain 10 ⁴ cell count casei were respectively mixed culture with E. coli (BCRC 11,634) and salmonella (BCRC 12947), after 38 hours, E. coli zero bacteria count from 10 ⁸ The reduced Salmonella was reduced from 10 ⁸ to 0 within 24 hours.

Example 7 Animal Test Select the synchronization birth was pre-weaning piglets from the same parent pig in subspecies SG96 impact dairy farmers on the body weight of the pre-weaning piglet strains of Lactobacillus casei bacteria, were bred divided into two sets, one set is open daily from 1 * 10 ⁷ Only the aqueous solution of subtype SG96 strain of casei containing CFU / mL was given, another set was the control group, antibiotics (OTC, oxytetracycline) were given, the breeding time was 21 days, and the change in the weight of the piglet every week Recorded and statistical analysis was performed with T-test. The results are as shown in FIG. 12. As a result of giving the casei subsp. SG96 strain for 2 weeks, the weight gain rate was more remarkable than the control group (p <0.05), and the weight averaged 0.7-0.8 kg weekly. Three weeks after giving the casei subsp. SG96 strain, the piglet also gained more weight than the control group, and the casei subsp. SG96 strain clearly promoted the weight gain of the piglet before weaning, It was also found that the feed efficiency of piglet feed can be improved.

2. The effect of reducing the diarrhea of weanling piglets by subtype SG96 of casei bacteria Diarrhea is common in piglets before weaning, but this diarrhea is usually caused by suckling or environmental factors of the parent pig, If the diarrhea can be improved early or early, it can help normal growth of the piglet.
A dairy farmer selected pre-weaned piglets that had been delivered from the same parent pig in synchrony and raised them in two groups. One group was given only an aqueous solution of casei subsp. SG96 containing 1 * 10 ⁷ CFU / mL daily. Another group was a control group, which gave antibiotics (OTC, oxytetracycline), raised the diarrhea situation every week for 21 days and recorded the diarrhea status of piglets every week, and the assessment score of diarrhea was announced by Underdahl et al. (Underdahl NR, Torres-Medina A, Dosten AR. 1982. Effect of Streptococcus faecium C-68 in control of Escherichia coli-induced diarrhea in gnotobiotic pigs. Am. J. Vet. Res. 1982. 12: 2227-2232), description of grade points:
0 points = normal;
1 point = soft stool, dry around the anus;
2 points = wet around the anus;
3 points = aqueous diarrhea;
4 points = standard 1-3 + loss of appetite, weight loss

  Furthermore, statistical analysis was performed with T-test. The results are shown in FIG. Administration of casei subsp. Strain SG96 has the effect of reducing diarrhea, and its efficacy is similar to that of the second week control group. When administered for 3 weeks, the reduction effect of diarrhea by the subsp. SG96 strain of casei was better than the control group (antibiotic administration) and there was a significant difference (p <0.05). Has a good diarrhea-inhibiting effect, and the result is superior to antibiotics used in general dairy farmers, and can eliminate the problem of antibiotic residue in piglets.

  The above detailed description is a specific description of the feasible embodiments of the present invention. However, the embodiments do not limit the scope of claims of the present invention, and do not depart from the technical spirit of the present invention. Equivalent implementation or change made, for example, application of a fungus-suppressing composition containing the subsp. SG96 strain of casei, and production of a fungus-suppressing composition containing the subsp. All examples are intended to be within the scope of the claims of this application.

Name: SG96, accession number: CGMCC NO.2697

Claims (6)

A new strain of Lactobacillus paracasei subsp. Paracasei SG96, which is deposited with the General Microbiology Center of the Chinese microbial species storage management committee and has the accession number CGMCC 2697. A composition comprising the subsp. SG96 strain of casei according to claim 1. The composition according to claim 2, wherein the composition is used to inhibit the growth of Escherichia coli. The composition according to claim 2, wherein the composition is used for inhibiting the growth of Salmonella typhimurium. The composition according to claim 2, wherein the composition is used for suppressing diarrhea. The composition is in the form of an animal drinking water additive, an animal feed additive, an animal and human medical composition, a food additive, a beverage additive, a food, a drink, a health food, or the like. The composition according to claim 2.