CN114085789A - Pediococcus pentosaceus MA.WTPQJ01 and application thereof - Google Patents
Pediococcus pentosaceus MA.WTPQJ01 and application thereof Download PDFInfo
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- CN114085789A CN114085789A CN202111295174.1A CN202111295174A CN114085789A CN 114085789 A CN114085789 A CN 114085789A CN 202111295174 A CN202111295174 A CN 202111295174A CN 114085789 A CN114085789 A CN 114085789A
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- pediococcus pentosaceus
- wtpqj01
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Abstract
The invention relates to the technical field of probiotics for feeding, in particular to pediococcus pentosaceus MA.WTPQJ01 and application thereof. The Pediococcus pentosaceus (Pediococcus pentosaceus) MA.WTPQJ01 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No. 21825. The strain has the characteristics of acid resistance and bile salt resistance, and has excellent probiotic, antibacterial activity and vomitoxin degradation function. The strain has safe and reliable effect after being fed to animals, can improve the utilization rate of the feed, promote the digestion and absorption of nutrient substances in the feed, improve the daily gain, reduce the feed conversion ratio, enhance the immune function of the animals, has remarkable effects on promoting the growth of the animals and improving the weight of the animals, and has the advantages of no pollution, no residue, biological environmental protection and the like.
Description
Technical Field
The invention relates to the technical field of probiotics for feeding, in particular to pediococcus pentosaceus MA.WTPQJ01 and application thereof.
Background
In the livestock breeding industry, the microecological preparation as a green environment-friendly feed additive can improve the microecological environment of intestinal tracts of livestock and poultry, promote the proliferation of beneficial bacteria in the intestinal tracts of the livestock and poultry, prevent or inhibit the propagation of harmful bacteria, adjust the balance of intestinal flora, obviously relieve the ammonia odor of animal excrement, reduce mosquito, fly and insect pests, improve and optimize the ecological environment of livestock and poultry breeding, reduce environmental pollution, improve the anti-stress capability and immunity of animals, improve the conversion rate of animal feed, reduce the production cost and increase the economic benefit.
The ideal strain which can be directly fed in the microecological preparation has the following characteristics: firstly, people and animals cannot be pathogenic, and hybrid seeds cannot be generated between the pathogenic microorganisms and the pathogenic microorganisms; secondly, the propagation is easy in vitro and in vivo, and the in vitro propagation speed is high; ③ the drug can survive in low pH and bile and can be implanted into intestinal mucosa; fourthly, substances such as lactic acid, hydrogen peroxide and the like can be generated in the fermentation process; can synthesize the inhibitors for enteropathogenic bacteria such as escherichia coli, salmonella, staphylococcus, clostridium and the like without influencing the activity of the inhibitors; sixthly, the survival rate of the live bacteria is high after processing, and the stability at high temperature is good after the live bacteria is mixed into the feed; preferably from the animal's own intestinal tract; is favorable for promoting the growth and development of hosts and improving the disease resistance.
Lactic acid bacteria, which are important members of probiotics, have long been recognized by the animal nutrition community for their probiotic effect and safety, and are the first probiotics to be published and used directly as feed additives. The lactobacillus can form flora dominance in intestinal tracts of livestock and poultry, and the lactobacillus serving as the dominant flora can achieve a probiotic effect in modes of producing acid, lactobacillin and the like. The lactobacillus preparation is fed to the livestock and poultry in the young period to enhance the disease resistance of the livestock and poultry and facilitate the permanent planting in the intestinal tract. The growth promoting effect of lactic acid bacteria is mainly realized by adjusting the pH value of intestinal tracts and providing bacterial nutrition.
Mycotoxins (mycotoxins) are mainly toxic metabolites produced by fungi in food or feed polluted by the fungi, and can enter animal bodies through the feed to cause acute or chronic toxicity of the animals and damage liver, kidney, nerve tissue, hematopoietic tissue, skin tissue and the like of the organisms. The degradation of mycotoxins in the feed is of great significance to the safety of the feed and the health of animals. In the prior art, the growth promotion effect and the immunity enhancement effect on lactic acid bacteria are reported more, but few lactic acid bacteria with the effect of degrading mycotoxin are reported.
Disclosure of Invention
One of the purposes of the invention is to provide pediococcus pentosaceus MA.WTPQJ01 and a product thereof. The invention also aims to provide the application of the strain and the product thereof.
Specifically, the invention provides the following technical scheme:
the invention provides Pediococcus pentosaceus MA.WTPQJ01, which is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 of West Lu No. 1 of the sunward area of Beijing, China academy of sciences, the code of postal code 100101) 2.25.2021.2.25.4.2.4.4.1.A classified name is Pediococcus pentosaceus, and the preservation number is CGMCC No. 21825.
Pediococcus pentosaceus MA.WTPQJ01 is obtained by separating and screening bovine rumen fluid of a certain beef cattle experimental base in Beijing, performing ultraviolet mutagenesis. The strain MA.WTPQJ01 is identified as Pediococcus pentosaceus (Pediococcus pentosaceus) by analyzing 16S rRNA gene sequence and the like.
The microbiological characteristics of pediococcus pentosaceus ma.wtpqj01 are: gram-positive bacteria, the cell morphology is spherical; the size of a single colony is less than or equal to 1mm, and the single colony is circular, milky, smooth, convex, neat in edge and opaque; the strain can grow in an acid environment with the pH value of more than 3.0, and has strong bile salt resistance; and has certain bacteriostatic ability, and especially has obvious effect of inhibiting aeromonas hydrophila, citrobacter freundii and salmonella.
The invention provides a microbial inoculum containing the pediococcus pentosaceus MA.WTPQJ01.
The microbial inoculum can be a solid microbial inoculum or a liquid microbial inoculum. The microbial inoculum may also contain adjuvants commonly used in the field of microbial preparations, such as: lyoprotectants, and the like.
The probiotic effect of pediococcus pentosaceus MA.WTPQJ01 is identified, and the result shows that the pediococcus pentosaceus MA.WTPQJ01 has the characteristics of acid resistance and cholate resistance, can resist the internal environment of gastrointestinal tracts, can improve the daily feed intake and daily weight gain of animals after being fed with the strain, improves the disease resistance of the animals, and has the potential of probiotics.
Based on the functions, the invention provides a feed additive containing the pediococcus pentosaceus MA.WTPQJ01.
Preferably, the content of viable bacteria of the pediococcus pentosaceus MA.WTPQJ01 in the feed additive is 1 × 106CFU/g～1×1010CFU/g; more preferably 1X 108CFU/g～1×109CFU/g。
The invention also provides a feed containing the pediococcus pentosaceus MA.WTPQJ01.
Preferably, the content of viable bacteria of the pediococcus pentosaceus MA.WTPQJ01 in the feed is 1 × 105CFU/kg～8×108CFU/kg, more preferably 5X 107CFU/kg～6×108CFU/kg。
The pediococcus pentosaceus MA.WTPQJ01 also has outstanding capability of degrading mycotoxin, particularly vomitoxin.
Based on the above, the present invention provides a mycotoxin degrading agent containing the pediococcus pentosaceus ma.wtpqj01.
The antibacterial function of pediococcus pentosaceus MA.WTPQJ01 is analyzed, and the result shows that the strain has outstanding antibacterial property, and especially has excellent antibacterial effect on gram-negative bacteria such as aeromonas hydrophila, Citrobacter freundii, salmonella and the like.
Based on the above-mentioned functions, the present invention provides a preservative containing the pediococcus pentosaceus ma.wtpqj01.
The invention also provides a medicament containing the pediococcus pentosaceus MA.WTPQJ01.
Preferably, the medicament is a bacteriostatic medicament or a medicament for improving the disease resistance and the immunity of animals.
Based on the above-mentioned functions of pediococcus pentosaceus ma.wtpqj01, the present invention provides the following uses of the strain:
the invention provides application of pediococcus pentosaceus MA.WTPQJ01 or a microbial inoculum containing the strain or a mycotoxin degradation agent containing the strain in mycotoxin degradation.
In particular, the application is the degradation of mycotoxin in crop products, feeds and foods.
The invention provides application of pediococcus pentosaceus MA.WTPQJ01 or a microbial inoculum containing the strain or feed additive containing the strain in improving animal feed intake, promoting animal growth, promoting animal weight gain or improving feed utilization rate.
The invention provides application of pediococcus pentosaceus MA.WTPQJ01 or a microbial inoculum containing the strain in preparing feed, feed additives or medicines for improving the disease resistance of animals.
The disease resistance of the animals is preferably improved, and diarrhea of the animals is relieved.
The invention provides application of pediococcus pentosaceus MA.WTPQJ01 or a microbial inoculum containing the strain or a preservative or a medicament containing the strain in bacteriostasis and preservation.
In particular, the application may be in vitro bacteriostatic, preservative, or for non-therapeutic purposes inhibiting bacteria in the animal.
Preferably, the bacteriostatic, preservative is against gram negative bacteria.
Further preferably, the gram-negative bacteria are bacteria of the genera Aeromonas (Aeromonas), Citrobacter (Citrobacter), or Salmonella (Salmonella), more preferably Aeromonas hydrophila, Citrobacter freundii, or Salmonella, including but not limited to Aeromonas hydrophila ATCC7966, Citrobacter freundii ATCC43864, Salmonella pullorum CVCC 1791.
The invention also provides a preparation method of a microbial inoculum containing pediococcus pentosaceus MA.WTPQJ01, which comprises the following steps: culturing Pediococcus pentosaceus MA.WTPQJ01 to obtain a culture, centrifuging the culture, taking a precipitate to obtain bacterial sludge, mixing the bacterial sludge with a freeze-drying protective agent, and freeze-drying.
In the above method, the medium used preferably comprises the following components: 30-35g/L of whey powder, 30-35g/L of soybean meal, 4-6g/L of glucose, 3-5g/L of sodium chloride, 0.1-0.2g/L of zinc sulfate, 0.6-0.7g/L of manganese sulfate, 0.8-1.2g/L of magnesium sulfate, 1-2g/L of diammonium hydrogen citrate and 0.01-0.02% of defoaming agent by volume/v.
In the above method, the culture is preferably performed under the following conditions: introducing mixed gas with the volume ratio of nitrogen to hydrogen of 9:1 under the stirring conditions of 37 ℃ and the rotating speed of 180-220rpm, and carrying out fermentation culture for 20-26 h.
In the above method, the lyoprotectant preferably comprises the following components: 10% of skimmed milk powder and 6% of lactose. The ratio of the bacterial sludge to the freeze-drying protective agent is preferably 3: 1.
The invention has the beneficial effects that: the pediococcus pentosaceus MA.WTPQJ01 provided by the invention has the characteristics of acid resistance and bile salt resistance, and has excellent probiotic and antibacterial properties and the function of degrading vomitoxin. The strain has safe and reliable effect after being fed to animals, can improve the utilization rate of the feed, promote the digestion and absorption of nutrient substances in the feed, improve the daily gain, reduce the feed conversion ratio, enhance the immune function of the animals, has remarkable effects on promoting the growth of the animals and improving the weight of the animals, and has the advantages of no pollution, no residue, biological environmental protection and the like.
Drawings
FIG. 1 is a colony morphology of Pediococcus pentosaceus MA. WTPQJ01 in example 1 of the present invention.
FIG. 2 is a gram stain of Pediococcus pentosaceus MA. WTPQJ01 of example 1 of the present invention.
FIG. 3 shows the results of acid resistance measurement of Pediococcus pentosaceus MA. WTPQJ01 in example 2 of the present invention.
FIG. 4 shows the results of the detection of the bile salt resistance of Pediococcus pentosaceus MA. WTPQJ01 of the present invention in example 2.
Fig. 5 shows the results of the measurement of the bacteriostatic ability of pediococcus pentosaceus ma.wtpqj01 in example 4 of the present invention.
FIG. 6 is a growth curve of Pediococcus pentosaceus MA. WTPQJ01 according to example 6 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The formulation method of MRS medium used in the following examples was as follows: weighing 10g of peptone, 5g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 801.08 g of tween-801.08 g, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triammonium citrate and magnesium sulfate (MgSO)4·7H2O)0.2g, manganese sulfate (MnSO)4·4H2O)0.05g and agar 15g, adding distilled water to a constant volume of 1L, adjusting pH to 6.2, and sterilizing at 121 deg.C for 20 min.
Example 1 isolation and characterization of Pediococcus pentosaceus (Pediococcus pentosaceus) MA. WTPQJ01
Separation of strain MA.WTPQJ01
1. Isolation culture of strains
Taking 1ml of a bovine rumen fluid sample from a certain beef cattle experimental base in Beijing, putting the bovine rumen fluid sample into a test tube containing 9ml of normal saline, shaking and uniformly mixing the bovine rumen fluid sample by a vortex machine to obtain a 1:10 diluent, taking the diluent for 10 times of incremental dilution, and then selecting 1ml of each diluent with 3 proper gradients to coat the diluent on an MRS culture medium. Culturing at 37 deg.C for 48-72 hr, observing and recording colony morphology, selecting single colony with good growth condition, streaking, separating and purifying, and preparing bacterial suspension with physiological saline.
2. Ultraviolet mutagenesis and screening of strains
And (3) pouring the sterilized MRS culture medium into culture dishes, after solidification, coating the bacterial suspension obtained in the step (1) on a flat plate, controlling bacterial colonies to be about 50-80 in each culture dish, culturing for 12 hours, and then, carrying out mutagenesis for 30s at a distance of 20cm from an ultraviolet lamp.
Selecting the mutagenized strain, inoculating the strain in an MRS liquid culture medium, culturing for 24 hours, measuring the OD value, selecting the strain with higher growth speed, coating a proper amount of bacterial suspension on an MRS plate, culturing for 24 hours in a constant-temperature incubator at 37 ℃, and carrying out the next step of gram staining.
3. Gram staining of the Strain
Dropping a drop of sterilized distilled water on a glass slide, selecting a single bacterium which grows faster after mutagenesis, dissolving the single bacterium in the water, scraping the single bacterium by a scraper, and drying and fixing the single bacterium on an alcohol lamp. Dripping crystal violet staining solution, staining for 2min, washing with water, and naturally drying; dripping iodine solution for 2min, washing with water, and naturally drying; dropwise adding 50S of alkaline fuchsin ethanol solution, washing with water, and naturally drying; when the bacteria are observed on a common optical microscope, the bacteria are positive if purple, and negative if red.
II, identification of the strain MA.WTPQJ01
Screening to obtain a strain MA.WTPQJ01, and performing morphological, physiological, biochemical and genetic identification on the strain MA.WTPQJ01, wherein the specific steps are as follows:
1. morphological identification
Single colonies of strain ma.wtpqj01 in log growth phase and stable colony size are described below: the size of a single colony is less than or equal to 1mm, the colony is circular, milky, smooth, convex, neat in edge and opaque, and the colony morphology is shown in figure 1.
Gram staining was performed on the strain ma.wtpqj01 in the logarithmic growth phase, and the morphology of the cells was observed by an optical microscope. The isolated and screened strain MA.WTPQJ01 was gram-positive and spherical in cell morphology, and the results are shown in FIG. 2.
2. Physiological and biochemical identification
The results of the tests using the bacterial micro biochemical identification tube of Qingdao Haibobo Biometrics Ltd are shown in Table 1.
TABLE 1 physiological and biochemical identification results
3. 16S rRNA Gene sequence homology analysis
The total DNA of bacteria is extracted by adopting a bacterial genome DNA extraction kit of Tiangen Biochemical technology Co. The extracted sample is sent to Shanghai Megi biological medicine science and technology Limited for sequencing. BLAST homology comparison is carried out on the determination result in a GenBank database, and the strain type of the strain ma.wtpqj01 is determined to be Pediococcus pentosaceus (Pediococcus pentosaceus). The sequencing result is shown in SEQ ID NO. 1.
And (3) identifying the strain as Pediococcus pentosaceus by 16S rRNA gene sequencing according to a sequencing result and the microbiological characteristics and physicochemical characteristics.
Pediococcus pentosaceus MA.WTPQJ01 has been deposited in China general microbiological culture Collection center (CGMCC for short, address: No. 3, institute of microbiology, Japan academy of sciences, Japan) of China Committee for culture Collection of microorganisms, 2.25.2021, and named as Pediococcus pentosaceus with the deposition number of CGMCC No. 21825.
Example 2 stress resistance assay of Pediococcus pentosaceus MA.WTPQJ01
1. Heat resistance test
The bacterial liquid of the pediococcus pentosaceus MA.WTPQJ01 is put into a water bath kettle for 20 minutes, and is respectively treated at 60 ℃, 80 ℃ and 100 ℃, wherein the treatment is repeated for 3 times, and the viable count is measured by adopting a pouring method after the treatment is finished.
After 8 lg (cfu/ml) of pediococcus pentosaceus MA.WTPQJ01 is treated at 60 ℃ for 20 minutes, the viable count is 4.97lg (cfu/ml), and after 20 minutes of treatment at 80 ℃, the viable count is close to 0, which indicates that the pediococcus pentosaceus is not resistant to high temperature and needs to be coated in industrial production.
2. Acid resistance detection
To a concentration of 108CFU/ml Pediococcus pentosaceus MA.WTPQJ01 was inoculated into MRS medium with pH values of 2.0, 3.0, and 4.0, respectively, and viable cell counts were measured by plate decantation at 1h, 2h, 3h, and 4h, respectively.
The Pediococcus pentosaceus can grow normally in an acidic environment, i.e. at pH of 4.0 and 3.0, and has an inhibitory effect on the growth of Pediococcus pentosaceus at pH of 2.0, but the viable count of the Pediococcus pentosaceus can still be maintained at 10 after 2h6CFU/ml above (FIG. 3). The result shows that the pediococcus pentosaceus MA.WTPQJ01 has strong acid tolerance and can resist the influence of gastric acid.
3. Detection of bile salt resistance
Performing multiple dilution on activated Pediococcus pentosaceus MA.WTPQJ01 by using sterile normal saline, selecting a proper dilution gradient, sucking 200 mu L of diluent, placing the diluent in a sterile culture dish, setting 6 times of dilution, pouring a flat plate by using MRS culture medium containing sodium taurocholate (0.1%, 0.2%, 0.3% and 0.4%) with different concentrations, culturing for 4 hours at 37 ℃, counting colonies every 1 hour, pouring the flat plate by using the MRS culture medium without the sodium taurocholate, culturing for 48 hours at 37 ℃, counting the colonies, and taking the counted colonies as a control group.
As a result, as shown in FIG. 4, the number of viable bacteria at different concentrations of bile salts did not significantly decrease with time. The bile salt effects of 0.1% and 0.2% have weak influence on the pediococcus pentosaceus MA.WTPQJ01, and hardly affect the normal growth thereof. After 0.3% of bile salt acts for 4 hours, the number of viable bacteria is slightly reduced. After 0.4% of bile salt acts for 2 hours, the viable count can still be maintained at about 104CFU/ml indicates that the pediococcus pentosaceus MA.WTPQJ01 has stronger bile salt resistance.
Example 3 antibiotic susceptibility testing of Pediococcus pentosaceus MA. WTPQJ01
The pediococcus pentosaceus MA.WTPQJ01 with a suitable concentration was spread on MRS medium, 1 drug sensitive paper sheet was uniformly attached to each dish, cultured for 36 hours, and the size of the zone of inhibition was observed, with the results shown in Table 2.
TABLE 2 sensitivity results of Pediococcus pentosaceus MA. WTPQJ01 to different antibiotics
The experimental results show that the pediococcus pentosaceus MA.WTPQJ01 does not have good drug resistance, so the pediococcus pentosaceus is safe and reliable to use as a feeding probiotic.
Example 4 bacteriostatic test of Pediococcus pentosaceus MA.WTPQJ01 against gram-negative bacteria
Pouring 10ml of agar culture medium into a culture dish, placing an oxford cup after the culture medium is solidified, pouring LB culture medium mixed with 1% of pathogenic bacteria into the upper layer, and pulling out the oxford cup after solidification. 200 mu L of the bacterial liquid of the pediococcus pentosaceus MA.WTPQJ01 is added into the sample adding hole, the sample is carefully placed into a constant temperature incubator at 37 ℃ for upright culture for 24 hours, and the size of the inhibition zone is observed.
The results show that the pediococcus pentosaceus MA.WTPQJ01 has strong inhibition capacity on aeromonas hydrophila ATCC7966, Citrobacter freundii ATCC43864 and Salmonella pullorum CVCC1791, and the diameters of inhibition zones respectively reach 2.20cm, 2.10cm and 3.10cm (figure 5).
Example 5 degradation experiment of Pediococcus pentosaceus MA. WTPQJ01 on mycotoxins
Pulverizing and mixing several mildewed feeds, mixing with mildewless feed to adjust vomitoxin concentration to 1000 μ g/mg (detecting vomitoxin by enzyme-linked immunosorbent assay), centrifuging activated M.WTPQJ01 bacterial liquid, discarding supernatant, collecting viable bacteria, and adjusting concentration to 10 with sterile distilled water8CFU/ml. Taking 100g of fodder with vomitoxin concentration of 1000 μ g/mg, and mixing 5ml of fodder with concentration of 10% according to 5%8The CFU/ml Pediococcus pentosaceus bacterial solution is uniformly sprayed on the feed, the feed is placed in a 37 ℃ incubator, the concentration of the vomitoxin is 467 mu g/mg after 24 hours, 282 mu g/mg after 48 hours and 113 mu g/mg after 72 hours. The calculation formula of the degradation rate is as follows: (mycotoxin addition amount-mycotoxin residual amount)/mycotoxin addition amount x 100%. The degradation rate of the pentacoccus pentosaceus MA.WTPQJ01 on vomitoxin is calculated to be 88.70%.
Example 6 growth Curve assay of Pediococcus pentosaceus MA.WTPQJ01
The growth curve represents the dynamic change of the overall process of bacteria growth in a new and suitable environment until senescence and death. Inoculating Pediococcus pentosaceus MA.WTPQJ01 into MRS culture medium at an inoculation amount of 10% (v/v), culturing at 37 deg.C for 24 hr, and measuring OD every 2 hr with MRS culture medium without adding bacteria liquid as blank control600And calculating the viable count. The experiment was repeated three times, the results were averaged, the data were recorded andand drawing a growth curve. As shown in FIG. 6, the growth curve shows that Pediococcus pentosaceus MA.WTPQJ01 is in logarithmic growth phase at 2-16 hours, and the propagation rate is high. The number of Pediococcus pentosaceus MA.WTPQJ01 tends to be stable at 16-24 hours and is in a plateau stage.
Example 7 preparation of a preparation of Pediococcus pentosaceus MA.WTPQJ01
The present example provides a method for culturing pediococcus pentosaceus ma.wtpqj01 and preparing a preparation thereof, which comprises the following steps:
1. a fermentation medium was inoculated with a 20-hour old strain of Pediococcus pentosaceus MA.WTPQJ01 at an inoculum size of 5% (v/v).
The formula of the fermentation medium is as follows: 30g/L of whey powder, 30g/L of soybean meal, 5g/L of glucose, 4g/L of sodium chloride, 0.1g/L of zinc sulfate, 0.6g/L of manganese sulfate, 0.8g/L of magnesium sulfate, 1g/L of diammonium hydrogen citrate and 0.01% (v/v) of defoaming agent. Dissolving the above materials in water to obtain fermentation culture medium, and sterilizing with 115 deg.C high temperature steam for 30 min. And (3) inoculating the pediococcus pentosaceus MA.WTPQJ01 when the temperature of the fermentation culture medium is reduced to 37 ℃.
2. After inoculation, stirring at 37 deg.C and rotation speed of 220rpm, introducing mixed gas of nitrogen and hydrogen at a volume ratio of 9:1, fermenting for 24 hr, and discharging to obtain Pediococcus pentosaceus with viable count of 1 × 109cfu/ml fermentation broth.
3. And centrifuging the fermentation liquor at 4 ℃ to obtain precipitate to obtain bacterial sludge, adding 100mL of freeze-drying protective agent into 300g of bacterial sludge, and uniformly mixing by using an oscillator to prepare bacterial suspension. Precooling at minus 80 ℃ for 1.5 hours, and quickly transferring the frozen sample to a freeze dryer for freeze-drying for 24 hours to ensure that the water content of the fungus powder reaches about 3 percent. The formula of the freeze-drying protective agent is as follows: 10% of skimmed milk powder and 6% of lactose.
Example 8 evaluation of safety of the preparation of Pediococcus pentosaceus MA.WTPQJ01
In this example, a mouse is used as an experimental animal, and the safety of pediococcus pentosaceus ma.wtpqj01 is evaluated by a gavage test method, which specifically includes:
1. taking the freeze-dried powder of the pediococcus pentosaceus MA.WTPQJ01 prepared by the method of example 7, and measuring by plate counting, wherein the lyophilized powder of the pediococcus pentosaceus MA.WTPQJ01The number of viable bacteria is 2 × 109cfu/g。
2. Selecting about 8 weeks old mice 72, randomly dividing into 4 groups (group A is control group, and is administered with sterile normal saline, group B is high dose group, and is 2 × 109Filling the bacterial liquid at the dose of cfu/cfu; group C is medium dose group, according to 2X 108Filling the bacterial liquid at the dose of cfu/cfu; group D is low dose group, according to 2X 107cfu/dose of bacteria) 3 replicates per group, 6 mice per replicate.
3. The administration is carried out once every nine morning hours for 21 days.
The laboratory mouse room controls the constant temperature and humidity, the natural illumination, the mouse freely takes food and drinks water, and the mouse cage is cleaned once every 7 days. In the experimental process, the state, survival condition, presence or absence of clinical abnormal symptoms and the like of the mice were observed and recorded every day.
Detection indexes are as follows:
(1) on the day of experiment, blood samples of experimental mice are obtained by adopting a heart blood taking mode, and serum is obtained after static centrifugation and is used for detecting blood biochemical indexes such as albumin, total protein, high-density lipoprotein, low-density lipoprotein, triglyceride, cholesterol, urea, tumor cell necrosis factor and the like in the serum.
(2) The whole heart, liver, spleen and kidney were weighed (bilaterally) and wet-weighed, and the heart index ═ heart wet weight/body weight × 100%, liver index ═ liver wet weight/body weight × 100%, spleen index ═ spleen wet weight/body weight × 100%, and kidney index ═ kidney wet weight/body weight × 100% were calculated, respectively.
Survival of mice from different treatment groups is shown in table 3.
TABLE 3 survival of mice in different treatment groups
| Group A | Group B | Group | Group D | ||
| 7 days | Survival | | Survival | Survival | |
| 14 days | Survival | Survival | Survival | Survival | |
| 21 days | Survival | Survival | Survival | Survival |
As can be seen from Table 3, after the mice were gavaged with Pediococcus pentosaceus MA.WTPQJ01 for 21 days, the mice of each treatment group survived, indicating that Pediococcus pentosaceus MA.WTPQJ01 is safe for animals.
The statistical results of the organ index of the mice of the different treatment groups are shown in table 4.
TABLE 4 organ index of mice of different treatment groups
| Group A | Group B | Group C | Group D | |
| Heart and heart | 0.61 | 0.63 | 0.67 | 0.65 |
| Liver disease | 5.59 | 5.61 | 5.55 | 5.56 |
| Spleen | 0.42 | 0.41 | 0.47 | 0.42 |
| Kidney (A) | 1.36 | 1.31 | 1.34 | 1.35 |
As can be seen from table 4, the organ index of the treated mice was not significantly changed compared to the control group, indicating that pediococcus pentosaceus ma.wtpqj01 did not cause abnormality of the organs of the mice.
Example 9 application of the preparation of Pediococcus pentosaceus MA.WTPQJ01
Selecting 72 piglets of 28-day old Dudu grown-up ternary hybrid weaned piglets, dividing the piglets into 2 groups according to a random block group design in an experimental period of 45 days, wherein each group has 6 repetitions, and each repetition has 6 pigs. Group A was a control group (basal diet group), group B was a treatment group (basal diet supplemented with 260g/t of the Pediococcus pentosaceus preparation prepared in example 7, and the effective viable count was 2X 109cfu/g)。
During the test period, piglets are raised in a totally enclosed nursing pigsty, the temperature is controlled to be 25-27 ℃, and the piglets are fed with free food and water. The basal diet does not contain any antibiotics, and the immunization of the piglets is carried out according to a conventional immunization program.
Measurement indexes are as follows: the production performance of the weaned piglets of each treatment group specifically comprises the following indexes:
1. the piglet feed intake was recorded every day and the average daily feed intake was calculated after the experiment was completed.
2. Piglet body weights were recorded on the day the experiment began and ended and the average daily gain was calculated.
3. And (3) calculating the feed-meat ratio according to the test results of the 1 and the 2, wherein the calculation mode is average daily feed intake/average daily gain.
4. During the test period, the fecal condition of the piglets is observed and recorded at 10:00 a day in the morning, and the diarrhea rate of the weaned piglets is calculated, wherein the diarrhea rate (%) is (number of diarrhea heads multiplied by the number of diarrhea days)/(number of pigs multiplied by the number of test days) multiplied by 100%.
The detection results of the above detection indexes are shown in table 5.
TABLE 5 Effect of Pediococcus pentosaceus formulation addition to basal diets on weaned piglet production Performance
| Average daily food intake (kg) | Average daily gain (kg) | Meat ratio of materials | Rate of diarrhea | |
| Group A | 0.494 | 0.342 | 1.44 | 5.66％ |
| Group B | 0.528 | 0.372 | 1.42 | 4.23％ |
As can be seen from table 5, the average daily feed intake and average daily gain of piglets in the treated group were significantly higher than those in the control group (P <0.05), and the feed-meat ratio was lower than that in the control group, indicating that the feed benefit was better with the addition of the formulation of pediococcus pentosaceus ma.wtpqj01. The diarrhea rate of the treated group is obviously reduced compared with that of the control group, which shows that the pediococcus pentosaceus MA.WTPQJ01 has the function of improving piglet diarrhea.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> university of agriculture in China
<120> Pediococcus pentosaceus MA.WTPQJ01 and application thereof
<130> KHP211123025.7
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1471
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gtggcgggct gctataatgc agtcgaacga acttccgtta attgattatg acgtacttgt 60
actgattgag attttaacac gaagtgagtg gcgaacgggt gagtaacacg tgggtaacct 120
gcccagaagt aggggataac acctggaaac agatgctaat accgtataac agagaaaacc 180
gcatggtttt cttttaaaag atggctctgc tatcacttct ggatggaccc gcggcgtatt 240
agctagttgg tgaggtaaag gctcaccaag gcagtgatac gtagccgacc tgagagggta 300
atcggccaca ttgggactga gacacggccc agactcctac gggaggcagc agtagggaat 360
cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa gggtttcggc 420
tcgtaaagct ctgttgttaa agaagaacgt gggtaagagt aactgtttac ccagtgacgg 480
tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540
aagcgttatc cggatttatt gggcgtaaag cgagcgcagg cggtctttta agtctaatgt 600
gaaagccttc ggctcaaccg aagaagtgca ttggaaactg ggagacttga gtgcagaaga 660
ggacagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg 720
cgaaggcggc tgtctggtct gcaactgacg ctgaggctcg aaagcatggg tagcgaacag 780
gattagatac cctggtagtc catgccgtaa acgatgatta ctaagtgttg gagggtttcc 840
gcccttcagt gctgcagcta acgcattaag taatccgcct ggggagtacg accgcaaggt 900
tgaaactcaa aagaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 960
agctacgcga agaaccttac caggtcttga catcttctga cagtctaaga gattagaggt 1020
tcccttcggg gacagaatga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1080
ttgggttaag tcccgcaacg agcgcaaccc ttattactag ttgccagcat taagttgggc 1140
actctagtga gactgccggt gacaaaccgg aggaaggtgg ggacgacgtc aaatcatcat 1200
gccccttatg acctgggcta cacacgtgct acaatggatg gtacaacgag tcgcgagacc 1260
gcgaggttaa gctaatctct taaaaccatt ctcagttcgg actgtaggct gcaactcgcc 1320
tacacgaagt cggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg 1380
ggccttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag ccggtggggt 1440
aaccttttag gagctagccg tctaaggtat c 1471
Claims (10)
1. Pediococcus pentosaceus (Pediococcus pentosaceus) MA.WTPQJ01 is characterized in that the Pediococcus pentosaceus is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 21825.
2. A microbial preparation comprising the Pediococcus pentosaceus (Pediococcus pentosaceus) MA. WTPQJ01 according to claim 1.
3. A feed or feed additive characterized by containing Pediococcus pentosaceus (Pediococcus pentosaceus) MA. WTPQJ01 according to claim 1.
4. A mycotoxin degrading agent comprising Pediococcus pentosaceus (Pediococcus pentosaceus) MA. WTPQJ01 according to claim 1.
5. A preservative comprising Pediococcus pentosaceus (Pediococcus pentosaceus) MA. WTPQJ01 according to claim 1.
6. A pharmaceutical agent comprising Pediococcus pentosaceus (Pediococcus pentosaceus) MA. WTPQJ01 according to claim 1.
7. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) ma.wtpqj01 according to claim 1 or the microbial agent according to claim 2 or the mycotoxin degrading agent according to claim 4 for the degradation of mycotoxins.
8. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) ma.wtpqj01 according to claim 1 or the microbial inoculum according to claim 2 or the feed or feed additive according to claim 3 for increasing the feed intake of animals, for promoting the growth of animals, for promoting the weight gain of animals or for increasing the feed utilization.
9. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) ma.wtpqj01 according to claim 1 or the microbial preparation according to claim 2 for the preparation of a feed, a feed additive or a medicament for improving disease resistance of animals.
10. Use of Pediococcus pentosaceus (Pediococcus pentosaceus) ma.wtpqj01 according to claim 1 or the microbial preparation according to claim 2 or the preservative according to claim 5 or the medicament according to claim 6 for bacteriostasis and preservation.
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| CN114698767A (en) * | 2022-06-07 | 2022-07-05 | 山东健源生物科技有限公司 | Biological agent for removing mycotoxin in feed as well as preparation method and application of biological agent |
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