CN115992066A - Lactobacillus paracasei NHB-LpD2 for liquid fermented feed and application - Google Patents
Lactobacillus paracasei NHB-LpD2 for liquid fermented feed and application Download PDFInfo
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- CN115992066A CN115992066A CN202211078842.XA CN202211078842A CN115992066A CN 115992066 A CN115992066 A CN 115992066A CN 202211078842 A CN202211078842 A CN 202211078842A CN 115992066 A CN115992066 A CN 115992066A
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Abstract
Lactobacillus paracasei NHB-LpD2 for liquid fermented feed and application thereof, belonging to the technology of microbial fermented feed and liquid feedIn the art, the lactobacillus paracasei（Lactobacillus paracasei）NHB-LpD2 is separated from piglet intestinal canal and preserved in China general microbiological culture Collection center (CGMCC) with preservation number of 24436 and preservation date of 2022, 02 month and 28 days. The lactobacillus paracasei NHB-LpD2 does not use amino acid in the fermentation process, has high propagation speed and strong stress resistance, and has remarkable probiotics. Moreover, the lactobacillus paracasei NHB-LpD2 can be used for preparing liquid fermented feed and wet-based fermented feed, and has remarkable benefit for cultured animals.
Description
Technical Field
The invention belongs to the field of microbial fermented feed and liquid feed, and particularly relates to lactobacillus paracasei NHB-LpD2 for liquid fermented feed and application thereof.
Background
With the development of modern society, intensive cultivation has become a mainstream trend, and the lack of antibiotics often causes great damage to the breeders. However, problems of bacterial resistance and drug residues caused by abuse of long-term antibiotics are increasingly serious. At present, the feed industry has entered an era of complete 'forbidden', so development of a tibody product and research on tibody technology have become important subjects in the field of current intensive animal husbandry production.
The probiotics and the metabolites thereof have the effects of promoting digestion and absorption of nutrient substances, improving matrix immunity, inhibiting intestinal inflammation, improving intestinal flora structure and the like. Therefore, probiotics made of probiotics become the main way of replacing antibodies.
Lactobacillus paracasei is a gram-positive homofermentative lactic acid bacterium which is an important member of the genus lactobacillus and which widely exists in the intestinal tract, in the traditional fermented dairy products, in kimchi and other environments. The existing research shows that metabolites such as organic acid, bacteriocin and the like generated by lactobacillus paracasei can inhibit the growth of pathogenic bacteria. Therefore, the method can be used for preparing microecological preparation and fermented feed.
The invention is as follows: lactobacillus paracasei and application thereof, application number 201911341915.8 provides the Lactobacillus paracasei, and the preservation number of the Lactobacillus paracasei is CCTCC NO: M2016667. The lactobacillus paracasei has antibacterial activity on pathogenic bacteria salmonella typhimurium CMCC50015, listeria monocytogenes LM1, micrococcus luteus 10209, staphylococcus myelitis, salmonella typhimurium CMCC50015 or bacillus subtilis BAS2, but has a general inhibition effect, so that the invention provides a novel lactobacillus paracasei NHB-LpD2 with better antibacterial effect and excellent performance and provides application of the novel lactobacillus paracasei NHB-LpD in liquid fermented feed.
Disclosure of Invention
The invention aims to provide lactobacillus paracasei NHB-LpD2 for liquid fermented feed and application thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a lactobacillus paracasei NHB-LpD2, which is classified and named as lactobacillus paracasei (Lactobacillus paracasei) NHB-LpD2 and is preserved in China general microbiological culture collection center (CGMCC) No.24436 at a preservation address of Beijing, wherein the China general microbiological culture Collection center is the 28 th month of 2022.
Preferably, the lactobacillus paracasei NHB-LpD2 is a lactobacillus paracasei strain isolated from the intestinal tracts of piglets, and is determined to be lactobacillus paracasei by colony morphology observation, molecular biological identification and the like, and the 16SrDNA sequence of the lactobacillus paracasei strain is shown as SEQ ID NO. 1.
Preferably, lactobacillus paracasei NHB-LpD has the following microbiological characteristics: lactobacillus paracasei NHB-LpD is a gram-positive short bacillus which grows well on MRS agar culture medium, is cultured for 48 hours to form circular colonies, has the diameter of 2-3mm and is milky white, which shows that the bacteria are not smooth, opaque, convex in the middle and irregular in edge, the colony is in a shape as shown in figure 1, the micro-morphology is in a shape as shown in figure 2, the bacteria are gram-positive and are rod-shaped, free of spores, facultative anaerobic, the growth proper temperature is 10-40 ℃, the optimal growth temperature is 20-35 ℃, the growth pH is 3.0-7.0, and the optimal pH is 4.0-7.0.
Preferably, the method for culturing the lactobacillus paracasei NHB-LpD2 comprises the following steps: collecting seed solution (viable bacteria concentration 10) of Lactobacillus paracasei NHB-LpD2 (preservation number is CGMCC No. 24436) 9 CFU/mL) 17.5mL, inoculated in 350mL shake flask fermentation medium for shake flask fermentation culture; after the shake flask fermentation is finished, 10L of fermentation tank seed culture is carried out, 350mL of shake flask seed liquid is inoculated into the 10L of seed fermentation tank for culture, the liquid amount of the 10L seed tank is 7L, 100L of fermentation tank culture is carried out after the culture is finished, 7L of seed liquid is inoculated into the 100L of fermentation tank for fermentation culture, and the liquid amount of the 100L of fermentation tank is 70L of fermentation culture medium.
Preferably, the shake flask fermentation medium consists of the following components: 0.5 to 5 percent of sucrose, 0.5 to 5 percent of glucose, 0.1 to 2 percent of sodium acetate, 0.5 to 5 percent of yeast powder, 0.5 to 4 percent of soybean peptone, 0.1 to 2 percent of ammonium citrate, 0.01 to 0.5 percent of magnesium sulfate, 0.01 to 0.5 percent of dipotassium hydrogen phosphate, 0.01 to 1.0 percent of calcium carbonate, 0.01 to 0.1 percent of manganese sulfate, 0.05 to 0.5 percent of tween-80 and the balance of water.
Preferably, it is: 1% of sucrose, 0.8% of glucose, 0.4% of sodium acetate, 0.8% of yeast powder, 1% of soybean peptone, 0.5% of ammonium citrate, 0.1% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.04% of manganese sulfate, 0.1% of tween-80 and the balance of water.
The shaking flask fermentation conditions are as follows: the inoculation amount is 1-10% (volume ratio), the fermentation temperature is 20-40 ℃, the initial pH is 4.0-7.0, the fermentation time is 80-300r/min, and the fermentation time is 8-48h.
Preferably, it is: the inoculation amount is 5% (volume ratio), the fermentation temperature is 28 ℃, the initial pH is 6.5, the initial pH is 200r/min, and the fermentation time is 8h.
The culture mediums of the 10L fermentation tank and the 100L fermentation tank are the same, and the components are as follows: 0.5-5% of soft white sugar, 0.5-4% of edible glucose, 0.5-2% of soybean peptone, 0.5-4% of beef extract powder, 0.1-2% of sodium citrate, 0.01-0.5% of magnesium sulfate, 0.01-1.0% of calcium carbonate, 0.01-0.5% of manganese sulfate and the balance of water.
Preferably, it is: 1.5% of soft sugar, 0.5% of edible glucose, 1% of soybean peptone, 1% of beef extract powder, 0.5% of sodium citrate, 0.1% of magnesium sulfate, 0.2% of calcium carbonate, 0.05% of manganese sulfate and the balance of water.
The culture medium of the 100L fermentation tank comprises the following components: the initial pH is 4.0-7.0, the liquid loading amount is 50-70L of culture medium, the tank pressure is controlled to be 0.03-0.06MPa, the inoculation amount is 700mL-7L, the fermentation temperature is 25-40 ℃, the fermentation time is 8-48h, and the stirring rotating speed is 80-300r/min.
Preferably, it is: the initial pH is 6.5, the pH is 4.8, the liquid loading amount is 70L of culture medium, the tank pressure is controlled to be 0.05MPa, the inoculation amount is 7L, the fermentation temperature is 30 ℃, the fermentation time is 14h, and the stirring rotating speed is 300r/min.
The invention provides application of lactobacillus paracasei NHB-LpD2 in preparing liquid fermented feed.
Preferably, the liquid fermented feed improves the daily gain of piglets, reduces the feed-meat ratio of the piglets and reduces the diarrhea rate;
the liquid fermented feed improves the growth performance of the fattening pigs and improves the pork flavor of the fattening pigs.
The invention provides application of lactobacillus paracasei NHB-LpD2 in preparing wet-based fermented feed.
Preferably, the wet-based fermented feed provides the productivity and survival rate of white-feather broilers.
The invention provides application of lactobacillus paracasei NHB-LpD2 in preparation of antibacterial agents.
Preferably, the antimicrobial spectrum of the antimicrobial agent comprises the following species: erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus peng, aeromonas hydrophila and vibrio parahaemolyticus.
The invention provides application of lactobacillus paracasei NHB-LpD2 in preparing a feed additive.
The invention provides application of lactobacillus paracasei NHB-LpD2 in preparing a medicament for preventing and treating animal diarrhea.
The invention provides a liquid fermented feed, which is characterized in that a fermentation strain of the liquid fermented feed is lactobacillus paracasei NHB-LpD2.
The invention provides a feed additive for animal drinking water, which contains lactobacillus paracasei NHB-LpD2.
The invention provides a preparation method of fermented liquid feed containing lactobacillus paracasei NHB-LpD2.
The invention provides application of lactobacillus paracasei NHB-LpD or a feed containing the same in improving feed conversion rate and improving animal production performance.
The beneficial effects of the invention are as follows:
(1) The lactobacillus paracasei NHB-LpD2 has strong antibacterial performance, strong acid production capacity, strong stress resistance, high propagation speed, strong fermentation capacity and no free amino acid in the fermentation process; is suitable for the production of fermentation raw materials and liquid fermentation feeds in biological feeds, and lays a solid strain foundation for the later development.
(2) The lactobacillus paracasei NHB-LpD has remarkable probiotics, can remarkably inhibit the growth of erysipelas on pigs, pseudomonas aeruginosa, klebsiella pneumoniae, listeria immitis, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, clostridium perfringens, proteus penmansis, aeromonas hydrophila and vibrio parahaemolyticus, has broad-spectrum antibacterial property, can inhibit the growth of mixed bacteria in liquid fermented feed, and ensures the stable quality of the liquid fermented feed.
(3) The lactobacillus paracasei NHB-LpD can be used for preparing liquid fermented feed, the prepared liquid fermented feed is rich in lactic acid bacteria, the pH value is obviously reduced, free amino acid is not lost before and after fermentation and in the storage process of the liquid fermented feed, harmful components are not generated, and the prepared liquid fermented feed is stable in property and rich in beneficial microorganisms.
(4) The liquid fermented feed prepared by the lactobacillus paracasei NHB-LpD is applied to weaned pig production, the bodies of the piglets are strong and handsome, the fur is bright, the static time is relatively long, and the odor of the excrement is small. Compared with a control group, the liquid fermentation feed group can obviously improve the daily gain of piglets in the test group, obviously reduce the feed conversion ratio by 0.26 and obviously reduce the diarrhea rate. Promote the growth and feed conversion rate of weaned pigs, thereby improving the pig raising benefit.
(5) The liquid fermented feed of the lactobacillus paracasei NHB-LpD2 is applied to the production of growing-finishing pigs, the feed conversion rate of the test group is obviously lower than that of a control group, the feed conversion rate is improved, and the growth performance is improved by reducing the feed conversion rate by 0.21. The pork flavor of the liquid feed group is obviously improved, and the pork flavor of the liquid feed group is obviously superior to that of a control group, so that the pork quality of the fattening pigs is improved, and the healthy delicious pork is produced, so that the quality pork is provided for people.
(6) The wet-based fermented feed of lactobacillus paracasei NHB-LpD2 is applied to white feather broiler breeding, the feed conversion rate of the test group is obviously lower than that of a control group, the feed conversion rate is reduced by 10.43%, the growth performance is improved, and the feed conversion rate is improved.
Drawings
FIG. 1 is a colony morphology of Lactobacillus paracasei NHB-LpD2 on MRS medium.
FIG. 2 is a gram stain of Lactobacillus paracasei NHB-LpD strain.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.
The percentage "%" referred to in the present invention refers to mass percent unless otherwise specified; however, the percentage of the solution, unless otherwise specified, refers to the grams of solute contained in 100mL of solution.
Example 1
Isolation, screening and identification of Lactobacillus paracasei NHB-LpD2
1. Separation and purification of lactic acid bacteria
(1) Taking the collected fresh piglet intestinal contents back to a laboratory, weighing 10g of a sampling sample under the aseptic condition, and placing the sampling sample into a triangular flask containing 90mL of sterilized normal saline;
(2) After shaking for 1h at 37 ℃, sequentially diluting to 100 ten thousand times by adopting a 10-fold ratio dilution method, and selecting three dilutions of 1 ten thousand times, 10 ten thousand times and 100 ten thousand times;
(3) Sucking 0.1mL, coating on an improved MRS agar plate, inversely culturing for 48 hours at 37 ℃ after the plate coating, picking out a colony of suspected lactobacillus with obvious calcium dissolving ring and larger than 5mm by using an inoculating loop, and carrying out streak separation culture on the MRS agar plate;
(4) After 48h of culture, picking out bacterial colony with better separation effect, transferring the bacterial colony onto MRS agar inclined plane by using an inoculating loop for pure culture, repeating the pure culture for 3 times, suspending bacterial cells in 20% glycerol solution, and preserving in a refrigerator at-80 ℃ for later use.
2. Observation of colony morphology
According to the size of the calcium dissolving ring in the step 1, the strains with strong acid production capacity are preliminarily screened out, and the strains can be preliminarily determined to be lactobacillus, and the total strains is 87 strains. Activating the glycerol tube strain stored in the step 1 for 2-3 times by using an MRS agar plate, inoculating the glycerol tube strain into an MRS broth culture medium, performing shake culture for 18-20 hours at the constant temperature of 37 ℃ for 180r/min, taking a clean glass slide for gram staining, performing microscopic examination, observing the microscopic morphology of the strain, and selecting non-spore-producing bacteria with positive gram staining as standby.
3. Preparation of lactic acid bacteria suspension and fermentation broth
Culturing the lactobacillus obtained in the step 1 on an MRS agar plate at 35 ℃ for 48 hours, picking single colony from the plate, and shake culturing for 24 hours at 35 ℃ and 180r/min in 100mL MRS liquid culture medium to obtain lactobacillus suspension for later use; and continuing to shake-culture at 35 ℃ and 180r/min for 96 hours to obtain lactobacillus fermentation liquor for later use.
4. Screening of lactic acid bacteria for acid producing ability
Inoculating the lactobacillus suspension obtained in the step 3 into MRS liquid culture medium according to an inoculum size of 1% (volume ratio), culturing for 12 hours at 35 ℃, screening out lactobacillus with the pH of fermentation liquor being less than 4.0 for 12 hours, and obtaining 27 strains of lactobacillus in total.
5. Screening of lysine degradation by lactic acid bacteria
Inoculating the lactobacillus suspension obtained in the step 3 into MRS liquid culture medium added with 1% lysine according to an inoculum size of 1% (volume ratio), culturing for 24 hours at 35 ℃, measuring free lysine content every 4 hours, and selecting strains D2, D35, D42, D63 and D71 which do not degrade lysine in the fermentation process.
6. Screening of bacteriostatic lactic acid bacteria
(1) Collecting pathogenic bacteria (such as erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic Escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus pensonii, aeromonas hydrophila, and Vibrio parahaemolyticus) with a concentration of 10 9 2mL of CFU/mL bacterial suspension is added into a pathogenic bacteria culture medium which is filled with 200mL of bacterial suspension and cooled to about 45 ℃ after sterilization, then 10mL of non-solidified bacteria-carrying culture medium is sucked, and the bacteria-carrying culture medium is transferred onto a nutrient agar plate poured with 10mL of bottom plate, so that a plurality of pathogenic bacteria plates are prepared.
(2) Each pathogen nutrient agar plate on the ultra-clean workbench is clamped with 1 sterilized oxford cup (a round small tube with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10mm, 200 mu L of liquid can be added into the tube, and two ends of the tube are smooth) by using sterile forceps, and the sterilized oxford cup is placed on a plate so as to be contacted with a culture medium without gaps;
(3) After several minutes, 200. Mu.L of the fermentation broth of the suspected lactic acid bacteria strain (obtained in step 3) was respectively aspirated into oxford cups and incubated at 37℃for 24h. At least 3 repetitions of each strain are carried out, and the size of a bacteriostasis zone is observed and measured, wherein 2 strains with large bacteriostasis zones are provided, and are respectively marked as D2 and D71, and D2 is selected as a next study object.
7. Identification of strain species
(1) The strain D2 is subjected to morphological and physiological biochemical identification, the strain D2 can well grow on an MRS flat plate, is cultured for 48 hours to form circular colonies, the diameters of the colonies are 2-3mm, the colonies are milky white, the colonies are not smooth and opaque, the middle of the colonies are convex, the edges of the colonies are irregular, the colony morphology is shown in figure 1, the micro morphology is shown in figure 2, the micro morphology is gram-positive, the bacterial strain is rod-shaped and has no spores, the facultative anaerobism is realized, the growth temperature is in a proper range of 10-40 ℃, the optimal growth temperature is 20-35 ℃, the growth pH is 3.0-7.0, and the optimal pH is 4.0-7.0.
(2) Extracting the genome DNA of the D2 strain by using a kit for extracting the bacterial DNA. Sequencing the 16S rDNA gene fragment of the A4 strain by using the primers F and R, wherein the obtained sequence is shown as SEQ ID NO.1, and comparing the measured sequence with the 16S rDNA sequence in GenBank by BLAST analysis, so that the homology of the strain D2 and the lactobacillus paracasei can be seen to reach 100%. Strain D2 was identified as Lactobacillus paracasei (Lactobacillus paracasei) by morphological features of strain D2, 16S rDNA features, and formally designated NHB-LpD2.
8. Preservation of strains
The separated, purified and screened lactobacillus paracasei (Lactobacillus paracasei) NHB-LpD2 is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (address: national institute of sciences of China, including national institute of sciences, including No. 3, beijing area, charpy, no.1, and Fu, having a preservation number of CGMCC No.24436, and classified and named Pediococcus acidilactici (Lactobacillus paracasei).
Wherein, the composition of the modified MRS agar culture medium is as follows: 1.0% of peptone, 0.5% of sodium acetate, 1.0% of beef extract, 2% of glucose, 0.5% of yeast extract, 0.1% of Tween 80, 0.2% of K2HPO4, 0.058% of MgSO4, 0.2% of diammonium citrate, 0.025% of MnSO4, 1.8% of agar, 1% of calcium carbonate, the balance of water and pH 7.0+/-0.2.
MRS agar medium consists of: 1.0% peptone, 0.5% sodium acetate, 1.0% beef extract, 2% glucose, 0.5% yeast extract, 0.1% Tween 80, K 2 HPO 4 0.2%, mgSO4 0.058%, diammonium citrate 0.2%, mnSO 4 0.025%, agar 1.8%, balance water, pH 7.0.+ -. 0.2.
MRS broth medium composition was: 1.0% peptone, 0.5% sodium acetate, 1.0% beef extract, 2% glucose, 0.5% yeast extract, 0.1% Tween 80, K 2 HPO 4 0.2%, mgSO4 0.058%, diammonium citrate 0.2%, mnSO 4 0.025%, the balance being water, pH 7.0.+ -. 0.2.
The pathogen culture mediums are respectively as follows: the erysipelas pig bacillus, the listeria monocytogenes and the streptococcus pneumoniae are TSA+5% defibrinated sheep blood agar;
pseudomonas aeruginosa, klebsiella pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, proteus penmansoni and aeromonas hydrophila are used as nutrient agar culture mediums;
vibrio parahaemolyticus is TCBS culture medium, and Bacillus perfringens is tryptone-sulfite-cycloserine agar culture medium.
The composition of TSA+5% defibrinated sheep blood agar is: 1.5% of tryptone, 0.5% of soyase, 0.5% of sodium chloride, 1.5% of agar, the balance of water, and pH 7.2+/-0.2, and 5% of defibrinated sheep blood is added when the mixture is cooled to 50 ℃ during use.
The nutrient agar medium consists of: 1% of peptone, 0.3% of beef extract, 2% of agar, 0.5% of NaCl and the balance of water, and the pH value is 7.2+/-0.2.
The composition of the TCBS agar medium was: yeast powder 0.5%, peptone 1%, sodium thiosulfate 1%, sodium citrate 1%, ox gall powder 0.5%, sodium taurocholate 0.3%, sucrose 2%, sodium chloride 1%, ferric citrate 0.1%, thymol blue 0.0004%, agar 1.5% and pH 8.6+ -0.1.
The composition of the tryptone-sulfite-cycloserine agar medium was: 1.5% of tryptone, 0.5% of soyase, 0.5% of yeast powder, 0.1% of sodium metabisulfite, 0.1% of ferric ammonium citrate, 2% of agar and the balance of water, wherein pH 7.6+/-0.2 is adopted, and 20mL/250mL of 0.5% D-cycloserine solution which is sterilized by filtration is added when the solution is cooled to 50 ℃ during use.
Example 2
Preparation of Lactobacillus paracasei NHB-LpD2 fermentation broth
(1) Collecting seed solution (viable bacteria concentration 10) of Lactobacillus paracasei NHB-LpD2 (preservation number is CGMCC No. 24436) 9 CFU/mL) 17.5mL, inoculated in 350mL shake flask fermentation medium for shake flask fermentation culture;
(2) After the shake flask fermentation is finished, 10L of fermentation tank seed culture is carried out, 350mL of shake flask seed liquid is inoculated into the 10L of seed fermentation tank for culture, the liquid amount of the 10L seed tank is 7L, 100L of fermentation tank culture is carried out after the culture is finished, 7L of seed liquid is inoculated into the 100L of fermentation tank for fermentation culture, and the liquid amount of the 100L of fermentation tank is 70L of fermentation culture medium.
(3) After fermentation, detecting the viable count of the fermentation broth to be 8.1X10 10 CFU/mL, the fermentation broth was stored in a refrigerator at 4deg.C for use.
The shake flask fermentation medium consists of the following components: 1% of sucrose, 0.8% of glucose, 0.4% of sodium acetate, 0.8% of yeast powder, 1% of soybean peptone, 0.5% of ammonium citrate, 0.1% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate, 0.04% of manganese sulfate, 0.1% of tween-80 and the balance of water.
The shaking flask fermentation conditions are as follows: the inoculation amount is 5% (volume ratio), the fermentation temperature is 28 ℃, the initial pH is 6.5, the initial pH is 200r/min, and the fermentation time is 8h.
The components of the fermentation tank culture medium are as follows: 1.5% of soft sugar, 0.5% of edible glucose, 1% of soybean peptone, 1% of beef extract powder, 0.5% of sodium citrate, 0.1% of magnesium sulfate, 0.2% of calcium carbonate, 0.05% of manganese sulfate and the balance of water.
The fermentation conditions of the fermentation tank are as follows: the initial pH is 6.5, the pH is 4.8, the liquid loading amount is 70L of culture medium, the tank pressure is controlled to be 0.05MPa, the inoculation amount is 7L, the fermentation temperature is 30 ℃, the fermentation time is 14h, and the stirring rotating speed is 300r/min.
Example 3
Probiotic verification of Lactobacillus paracasei NHB-LpD2
(1) Collecting pathogenic bacteria (such as erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic Escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus pensonii, aeromonas hydrophila, and Vibrio parahaemolyticus) with a concentration of 10 9 2mL of CFU/mL bacterial suspension is added into a pathogenic bacteria culture medium which is filled with 200mL of bacterial suspension and cooled to about 45 ℃ after sterilization, then 10mL of non-solidified bacteria-carrying culture medium is sucked, and the bacteria-carrying culture medium is transferred onto a nutrient agar plate poured with 10mL of bottom plate, so that a plurality of pathogenic bacteria plates are prepared.
(2) On an ultra-clean bench, each pathogen nutrient agar plate was clamped with 1 sterilized oxford cup (circular small tube with inner diameter of 6mm, outer diameter of 8mm and height of 10mm, 200. Mu.L of liquid was added to the tube, both ends of the tube were smooth) and placed on the plate, and after 10 minutes, 200. Mu.L of the fermentation broth prepared in example 2 was added dropwise to each small tube without overflowing, and incubated at 37℃for 12 hours, and then the diameter of the inhibition zone was measured. Three replicates per experiment were averaged and the results are shown in table 1.
The pathogen culture mediums are respectively as follows: the erysipelas pig bacillus, the listeria monocytogenes and the streptococcus pneumoniae are TSA+5% defibrinated sheep blood agar;
pseudomonas aeruginosa, klebsiella pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, proteus penmansoni and aeromonas hydrophila are used as nutrient agar culture mediums;
vibrio parahaemolyticus is TCBS culture medium, and Bacillus perfringens is tryptone-sulfite-cycloserine agar culture medium.
The composition of TSA+5% defibrinated sheep blood agar is: 1.5% of tryptone, 0.5% of soyase, 0.5% of sodium chloride, 1.5% of agar, the balance of water, and pH 7.2+/-0.2, and 5% of defibrinated sheep blood is added when the mixture is cooled to 50 ℃ during use.
The nutrient agar medium consists of: 1% of peptone, 0.3% of beef extract, 2% of agar, 0.5% of NaCl and the balance of water, and the pH value is 7.2+/-0.2.
The composition of the TCBS agar medium was: yeast powder 0.5%, peptone 1%, sodium thiosulfate 1%, sodium citrate 1%, ox gall powder 0.5%, sodium taurocholate 0.3%, sucrose 2%, sodium chloride 1%, ferric citrate 0.1%, thymol blue 0.0004%, agar 1.5% and pH 8.6+ -0.1.
The composition of the tryptone-sulfite-cycloserine agar medium was: 1.5% of tryptone, 0.5% of soyase, 0.5% of yeast powder, 0.1% of sodium metabisulfite, 0.1% of ferric ammonium citrate, 2% of agar and the balance of water, wherein pH 7.6+/-0.2 is adopted, and 20mL/250mL of 0.5% D-cycloserine solution which is sterilized by filtration is added when the solution is cooled to 50 ℃ during use.
TABLE 1 antibacterial Effect of Lactobacillus paracasei NHB-LpD2 on pathogenic bacteria
| Pathogenic bacteria | Antibacterial diameter (mm) |
| Erysipelas suis of erysipelothrix rhusiopathiae | 21.32 |
| Pseudomonas aeruginosa Pseudomonas aeruginosa | 24.67 |
| Klebsiella pneumoniae Klebsiella pneumoniae | 18.19 |
| Listeria monocytogenes Listeria ivanovii | 22.31 |
| Streptococcus pneumoniae Streptococcus pneumoniae | 20.45 |
| Intestinal pathogenic Escherichia coli | 31.24 |
| Staphylococcus aureus StBphylococcus Bureus | 30.23 |
| Salmonella typhi Salmonella typhimurium | 23.41 |
| Salmonella | 22.34 |
| Shigella Shigella Castellani | 22.36 |
| Clostridium perfringens Clostridium perfringen | 28.91 |
| Proteus penneri | 18.21 |
| Aeromonas hydrophila Aeromonas hydrophila | 18.23 |
| Vibrio parahaemolyticus Vibrio parahaemolyticus | 17.61 |
From the detection results, the lactobacillus paracasei NHB-LpD2 has an inhibiting effect on various pathogenic bacteria.
EXAMPLE 4 stress resistance verification of Lactobacillus paracasei NHB-LpD2
1. Determination of artificial gastric juice resistance:
10mL of Lactobacillus paracasei NHB-LpD bacterial suspension (prepared according to the method described in step 3 of example 1) is placed in 90mL (250 mL triangular flask) of prepared artificial gastric juice, and is subjected to constant-temperature shaking at 37 ℃ for 180min at 200 r/min; and after the shaking is finished, 10mL of sample solution is taken to adjust the pH value to 7.0, 90mL of physiological saline is added, the shaking is performed for 30min at a constant temperature of 200r/min at 37 ℃, and then the colony culture count of the dilution plate is performed. The results are shown in Table 2.
As can be seen from Table 2, the survival rate of Lactobacillus paracasei NHB-LpD2 treated in artificial gastric juice (containing enzyme) with pH of 1.5, pH of 2.0 and pH of 2.5 respectively for 3 hours is over 95%, which shows that the strain NHB-LpD has higher acid resistance, can resist gastric acid and can smoothly reach intestinal tracts to play a role.
The preparation method of the artificial gastric juice comprises the following steps: preparation of artificial gastric juice referring to preparation method in the "pharmacopoeia of the people's republic of China" 2010 edition, 16.4mL of diluted hydrochloric acid is taken, about 800mL of water and 10g of pepsin are added, shaking is carried out, water is added to be weighed and released into 1000mL, pH values are respectively adjusted to 1.5, 2.0 and 2.5, and microporous filter membrane is sterilized (0.22 μm) for standby.
TABLE 2 survival of Lactobacillus paracasei NHB-LpD2 after 3h of treatment in artificial gastric juice
| Treatment of | pH1.5 | pH2.0 | pH2.5 |
| Initial Activity CFU/mL | 2.13×10 10 | 2.13×10 10 | 2.13×10 10 |
| Post-treatment Activity CFU/mL | 2.05×10 10 | 2.09×10 10 | 2.12×10 10 |
| Survival after treatment% | 96.24 | 98.12 | 99.53 |
2. Determination of Artificial intestinal juice resistance
1mL of Lactobacillus paracasei NHB-LpD bacterial suspension (prepared according to the method described in the 3 rd step of example 1) is placed in 99mL (250 mL triangular flask) of artificial intestinal juice, the temperature is kept constant at 37 ℃ and the shaking is carried out for 5 hours at 200r/min, after the shaking is finished, 1mL of sample liquid is taken, 99mL of physiological saline is added, the temperature is kept constant at 37 ℃ and the shaking is carried out for 30 minutes at 200r/min, and then the colony culture and the count of the diluted flat plate are carried out.
The result shows that the activity of the lactobacillus paracasei NHB-LpD in the artificial intestinal juice is not reduced, and the survival rate is 100%, which shows that the strain can well survive in the intestinal juice and preserve the activity, thereby playing the probiotic effect.
The artificial intestinal juice is prepared by the following steps: preparation of artificial intestinal juice referring to preparation method in the "pharmacopoeia of the people's republic of China" 2010 edition, phosphate buffer solution (containing pancreatin) (pH 6.8), taking 6.8g of monopotassium phosphate, adding 500mL of water to dissolve, and adjusting pH value to 6.8 with 0.1mol/L sodium hydroxide solution; and dissolving 10g of pancreatin in a proper amount of water, mixing the two solutions, diluting the mixture to 1000mL by adding water, and filtering and sterilizing the mixture by using a microfiltration membrane with the thickness of 0.22 mu m.
3. Determination of bile salt resistance
1mL of Lactobacillus paracasei NHB-LpD bacterial suspension (prepared according to the method described in the step 3 of the example 1) is taken and placed in 99mL (250 mL triangular flask) of solutions with different bile salt concentrations, wherein the bile salt concentrations are 0.03%, 0.15% and 0.3% respectively, then the solution is subjected to constant-temperature shaking for 120min at 37 ℃ and 200r/min, after the shaking is completed, 1mL of sample solution is taken, 99mL of physiological saline is added, the solution is subjected to constant-temperature shaking for 30min at 37 ℃ and 200r/min, and then the colony culture count of a dilution plate is carried out.
The results are shown in Table 3. The lactobacillus paracasei NHB-LpD2 is treated in a solution with 0.3 percent of bile salt solubility for 2 hours, and the survival rate is 96.51 percent, which shows that the strain has higher bile salt resistance and can resist bile salt in duodenal juice, so that the strain can reach intestinal tracts to play a role.
The preparation method of the bile salt solution with different concentrations comprises the following steps: 1.8mL, 9mL and 18mL of 5% bile salt solution are respectively added into PBS solution with pH of 7.4, the volume is fixed to 300mL, and the PBS solution containing 0.03%, 0.15% and 0.30% of bile salt is uniformly mixed.
The preparation method of the 5% bile salt solution comprises the following steps: accurately weighing 5.0g of bile salt, dissolving with 100mLPBS solution to constant volume, and sterilizing at 121deg.C for 20min.
The preparation method of the PBS solution comprises the following steps: sodium chloride 0.8%, potassium chloride 0.02%, disodium hydrogen phosphate 0.363%, potassium dihydrogen phosphate 0.024%, the balance being water. Adjusting pH to 7.4 with 6mol/L HCl, sterilizing at 121deg.C for 20min, and keeping.
TABLE 3 survival of Lactobacillus plantarum NHE-LpB11 after 2h treatment in bile salts solutions of different concentrations
| Treatment of | 0.03％ | 0.15％ | 0.3％ |
| Initial Activity CFU/mL | 2.13×10 10 | 2.13×10 10 | 2.13×10 10 |
| Post-treatment Activity CFU/mL | 2.13×10 10 | 2.11×10 10 | 2.06×10 10 |
| Survival after treatment% | 100 | 99.06 | 95.23 |
From the detection results, the lactobacillus paracasei NHB-LpD2 has the properties of artificial gastric juice resistance, artificial intestinal juice resistance and bile salt resistance.
Example 5
Fermentation performance verification of lactobacillus paracasei NHB-LpD2
1. Acid production capacity determination
Collecting seed solution (viable bacteria concentration 10) of Lactobacillus paracasei NHB-LpD2 (preservation number is CGMCC No. 24436) 9 CFU/mL), 15mL, is inoculated into 300mL shaking flask fermentation medium for shaking flask fermentation culture, the change condition of the pH value is measured in the fermentation process, and the measurement result shows that the pH value is not basically reduced in 2 hours before fermentation, the pH value starts to be rapidly reduced in 3 hours until 9 hours, the pH value starts to be slowly reduced after being reduced to 4.1, the pH value is kept stable when being reduced to 15 hours, and the pH value is finally reduced to 3.8.
2. Reproductive performance determination
Collecting seed solution (viable bacteria concentration 10) of Lactobacillus paracasei NHB-LpD2 (preservation number is CGMCC No. 24436) 9 CFU/mL) was inoculated into a sterile MRS broth medium at an inoculum size of 1% (v/v), the liquid loading amount was 300mL/1000mL, shaking culture was performed at 30℃for 200r/min, the number of viable Lactobacillus paracasei NHB-LpD2 in the MRS broth medium was measured every half hour, a growth curve was drawn, the change in viable count in the logarithmic growth phase of lactic acid bacteria was obtained, and then the breeding generation was calculated according to the formula.
The test result shows that the breeding time of the lactobacillus paracasei NHB-LpD2 is 27 minutes.
The generation time is calculated according to the following formula:
wherein X1 is the number of (T1) bacteria at the beginning of the log phase; x2 is the bacterial count in the late logarithmic phase (T2); g is the generation time, namely the breeding generation time.
3. Determination of free amino acid degradation Capacity
Collecting seed solution (viable bacteria concentration 10) of Lactobacillus paracasei NHB-LpD2 (preservation number is CGMCC No. 24436) 9 CFU/mL) was inoculated in an inoculum size of 1% (volume ratio) into MRS liquid medium supplemented with 4% (lysine, methionine, threonine and tryptophan ratio 1:1:1) amino acids, incubated at 30℃for 24h, and the free amino acid content in the broth was determined after fermentation, indicating that free amino acids in the fermentation medium were not degraded during fermentation of strain NHB-LpD2.
Example 6
Acute toxicity test of Lactobacillus paracasei NHB-LpD2
The safety evaluation of lactobacillus paracasei was performed by an acute toxicity test, with reference to the national standard GB15193.3-2003 maximum tolerated dose method. 60 common Kunming mice, namely male and female mice, are half, 18-20g, are subjected to conventional feeding for 1 week, and are subjected to stomach irrigation three times a day, and 0.25g/mL of lactobacillus paracasei NHB-LpD bacterial liquid (equivalent to 15000mg/kg body weight) is continuously irrigated for 2 weeks, so that whether the mice are poisoned or die is observed.
During the test, the mice have good mental state and no poisoning and death phenomena, so that the maximum tolerance dose MTD of the strain of the invention in the acute toxicity test is more than 15000mg/kg, and the strain can be determined to be of a non-toxic grade according to the grading standard, and has higher safety.
Example 7
Preparation of lactobacillus paracasei NHB-LpD2 liquid fermented feed
Activating the preserved 5 strains of lactobacillus strains for 2-3 times by using an MRS agar plate, then inoculating the strains into an MRS broth culture medium, and carrying out shake culture for 24 hours at the constant temperature of 37 ℃ for 180r/min to obtain 4 strains of lactobacillus seed liquid for later use. Uniformly mixing 5 strains of lactobacillus seed liquid to obtain strain mixed liquid. Then adding the mixed materials into liquid feed according to the inoculation amount of 5%, mixing at the mixing speed of 200r/min for 30min, then filling the mixed materials into a fermentation barrel, and sealing and fermenting for 14h to obtain the lactobacillus paracasei NHB-LpD2 liquid fermented feed. The detection results of the related indexes before and after fermentation of the liquid fermented feed are shown in table 4, the pH value of the liquid feed is obviously reduced before and after fermentation, the oxidation-reduction potential is reduced, the microorganism content is obviously improved, and the nutrient substances are not lost.
TABLE 4 detection of relevant indicators before and after fermentation of liquid fermented feed
| Project | Before fermentation | After fermentation |
| pH | 6.82±0.12 a | 3.95±0.23 b |
| Oxidation-reduction potential (mV) | 179±10.21 a | -181±8.34 b |
| Effective viable count of lactic acid bacteria (CFU/mL) | 2.1 a ×10 6 | 4.9 b ×10 9 |
| Free lysine content (mg/mL) | 1.29±0.03 | 1.29±0.09 |
| Total free amino acid content (%) | 1.34±0.08 | 1.62±0.13 |
| Amino acid sum (%) | 18.53±0.34 | 18.57±0.21 |
| Cadaverine content (μg/mL) | 4.12±0.23 | 4.21±0.12 |
The strain mixed solution consists of the following components: pediococcus acidilactici NHB-PaA4 seed solution 30%, lactobacillus paracasei NHB-LpD seed solution 20%, lactobacillus plantarum NHE-LpE seed solution 20%, lactobacillus plantarum NHE-LpE seed solution 20%, lactobacillus plantarum NHE-LpE seed solution 10%.
The liquid feed consists of the following components: 30% of solid feed and the balance of water
The solid feed consists of the following components: 51.43 parts of corn (for medium and large pigs), 15 parts of wheat, 5 parts of wheat embryo (second grade), 11.4 parts of soybean meal (with protein content of 46% or more), 8.1 parts of wheat bran (with crude ash content of 5.5% or less), 5 parts of wheat middling (second grade), 1.03 parts of stone dust, 0.89 part of L-lysine sulfate and fermentation byproducts (70%) thereof, 0.64 part of calcium hydrophosphate (III), 0.41 part of sodium chloride (98.5%), 0.4 part of montmorillonite (95), 0.23 part of L-threonine (98.5%), 0.2 part of Jiaerfu (M-S220D), 0.18 part of DL-methionine (98.5% inlet), 0.04 part of L-tryptophan (98%) 0.03 part of Jiaerfu (V-S203) and 0.02 part of phytase (high temperature resistant 20000).
The preservation number of the pediococcus acidilactici NHB-PaA4 is CGMCC NO.24435, and the pediococcus acidilactici is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 2 and 28 days of 2022.
The preservation number of the lactobacillus plantarum NHE-LpE5 is CGMCC NO.24432, and the lactobacillus plantarum is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms at 2 and 28 days of 2022.
The preservation number of the lactobacillus plantarum NHE-LpE9 is CGMCC NO.24433, and the lactobacillus plantarum is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 2 months and 28 days of 2022.
The preservation number of the lactobacillus plantarum NHE-LpE15 is CGMCC NO.24434, and the lactobacillus plantarum is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) for 2 months and 28 days of 2022.
Example 8
Preparation of lactobacillus paracasei NHB-LpD2 wet-based fermented feed
1. Lactobacillus plantarum NHE-LpB6401 strain activation
Activating the preserved lactobacillus plantarum NHE-LpB6401 strain by operating an MRS agar plate for 2-3 times, then inoculating the strain into an MRS broth culture medium, and performing shake culture at a constant temperature of 37 ℃ for 180 revolutions per minute for 10 hours to obtain lactobacillus plantarum NHE-LpB6401 seed liquid for later use.
2. Pediococcus acidilactici NHE-Pa11403 strain activation
Activating the preserved Pediococcus acidilactici NHE-Pa11403 strain with MRS agar plates for 2-3 times, inoculating into MRS broth culture medium, and shake culturing at 37deg.C for 180r/min for 10 hr to obtain Pediococcus acidilactici NHE-Pa11403 seed solution.
3. Preparation of wet-based fermented feed
Uniformly mixing the seed liquid of the lactobacillus plantarum NHE-LpB6401 and the seed liquid of the pediococcus acidilactici NHE-Pa11403 with the seed liquid of the lactobacillus paracasei NHB-LpD to obtain a strain mixed liquid. And then evenly mixing the lactobacillus paracasei with the composite fermentation raw materials, filling the mixture into a breathing bag for sealing after evenly mixing, and fermenting for 5-7 days to obtain the lactobacillus paracasei NHB-LpD2 wet-based fermented feed.
The strain mixed solution consists of the following components: lactobacillus plantarum NHE-LpB6401 seed solution 30%, pediococcus acidilactici NHE-Pa11403 seed solution 30% and Lactobacillus paracasei NHB-LpD2 seed solution 40% (fermentation broth prepared in example 2).
The composite fermentation raw material consists of the following components: 60% of wheat flour, 30% of bean pulp and 10% of bran.
The preservation number of the lactobacillus plantarum NHE-LpB6401 is CGMCC NO.22645, which is disclosed in Chinese patent CN 113430140A; the strain has been deposited in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms at 5 and 31 of 2021.
The preservation number of the pediococcus acidilactici NHE-Pa11403 is CGMCC NO.21192, which is disclosed in Chinese patent CN 113564072A; the strain has been deposited in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms at 11 and 16 months in 2020.
Example 9
Influence of liquid fermented feed on growth performance of weaned pigs
Three-way hybridization weaned pigs (half of the male and female) with similar weight and weight (Duroc X long white X large white) are selected for the test, 200 weaned pigs are randomly divided into two groups (a control group and a test group), 10 bars are treated each time, 10 heads are treated each time, and the test is carried out for 43 days, so that the feeding test is carried out under the same environment. The diet of the control group is a basic diet (refer to the Chinese pig feeding standard), and the diet of the test group is the liquid fermented feed prepared in example 6. Before the start of the test, the pig house was thoroughly disinfected. The control group pig group adopts a dry feeding mode, and the test pig group adopts a liquid porridge feeder, and can eat and drink water freely. The feeding amount and the health condition are recorded every day, the brightness and the fecal condition of the fur of the pig are observed regularly, and other feeding management are the same. In the test process, the piglets of the test group are strong and handsome in shape, bright in fur, relatively longer in static time and small in fecal odor. Compared with a control group, the liquid fermentation feed group can obviously improve the daily gain of piglets in the test group, obviously reduce the feed conversion ratio by 0.26 and obviously reduce the diarrhea rate. (Table 5)
TABLE 5 influence of liquid fermented feed on growth performance of weaned piglets
| Project | Control group | Test group |
| Initial weight (kg) | 6.41±0.17 | 6.41±0.10 |
| Last weight (kg) | 20.20±1.02 b | 23.85±1.03 a |
| Average daily gain (g/d) | 306±23.56 b | 405±42.18 a |
| Average daily feed intake (kg/d) | 563±56.43 b | 638±43.21 a |
| Feed to meat ratio | 1.83±0.14 a | 1.57±0.21 b |
| Diarrhea rate | 7.14±0.14 A | 0.95±0.01 B |
Note that: the same row of data shoulder marks with different lowercase letters indicate that the difference is significant (p < 0.05), and the same lowercase letters of the shoulder marks or no letters indicate that the difference is not significant (p > 0.05).
Example 10
Effect of liquid fermented feed on growing-finishing pigs
Healthy, about 20kg of body weight, consistent male-female proportion and similar day-old (Duroc X Changbai X Dabai) ternary commercial fattening pigs 80 are selected and completely randomly divided into 2 groups (control group and test group), 4 columns are arranged in each group, and 10 pigs are arranged in each column. Control group: test diet is taken as basic diet (refer to Chinese pig raising standard); the test group diet was the liquid fermented feed prepared in example 6. The test pigs adopt a closed colony house, are fed 3 times a day, eat and drink water freely, clean the colony house every day, and feed for 80 days. The feeding management and the immunization program are the same as the daily management of a pig farm, and the pigs are regularly disinfected and found to be ill and treated in time. The test results showed that the feed meat ratio of the test group was significantly lower than that of the control group, reduced by 0.21, and improved the growth performance (table 6). The pork flavor results showed (table 7) that the liquid feed group had a significantly better pork flavor than the control group.
TABLE 6 Effect of liquid fermented feeds on growing Property of fattening pigs
| Index (I) | Control group | Test group |
| Initial weight (kg) | 22.05±1.21 | 22.26±1.02 |
| Last weight (kg) | 101.34±6.90 b | 117.23±5.74 a |
| Average daily gain (kg/d) | 0.99±0.05 b | 1.19±0.03 a |
| Average daily feed intake (kg/d) | 2.38±0.31 b | 2.61±0.29 a |
| Feed to meat ratio | 2.40±0.11 b | 2.19±0.39 a |
TABLE 7 influence of liquid fermented feeds on the flavor of fattening pork
| Index (I) | Control group | Test group |
| Aldehydes | 226.88±15.54 b | 308.56±14.91 a |
| Alcohols | 28.71±2.89 b | 47.84±4.97 a |
| Esters of | 1.31±0.98 b | 2.42±1.42 a |
| Acids | 61.48±5.70 b | 106.86±10.03 a |
| Ketones (I) | 50.55±3.21 b | 92.60±3.49 a |
| Others | 9.53±1.99 | 9.39±4.14 |
| Totals to | 378.47±30.31 b | 567.66±38.97 a |
Example 11
Application of wet-based fermented feed in production of broiler chickens
1200 white feather broilers with good body condition and 1 day old are randomly divided into 2 groups, and each group is repeated by 6, and 100 chickens are repeated. The test treatments were respectively: (1) a control group basal ration (CON); (2) Test group basal ration +10% wet base fermented feed (prepared in example 8). Basic ration feed formulation (shown in table 8), the henhouse was thoroughly sterilized before the start of the experiment. The experiment adopts a cage culture mode, and each repetition of each treatment is uniformly distributed in the whole henhouse. The method of taking artificial light as a main part and taking natural light as an auxiliary part is adopted, the mode of combining natural ventilation and mechanical ventilation is adopted, the chickens can eat and drink water freely, the chickens are fed twice at eight and fifteen points every day, and the rest management and immunization procedures refer to broiler raising management manuals. The results of the growth performance of the test are shown in Table 9, and the results show that the production performance and the survival rate of white feather broilers can be remarkably improved by adding the wet-based fermented feed prepared from lactobacillus paracasei NHB-LpD2, and compared with a control group, the feed conversion ratio is reduced by 10.43%, and the survival rate is improved by 1.74%.
TABLE 8 basic ration composition and nutrient level (air-dried basis%)
TABLE 9 Effect of wet-based fermented feeds on broiler growth Performance
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (10)
1. Lactobacillus paracasei NHB-LpD2 classified and named as Lactobacillus paracasei（Lactobacillus paracasei）NHB-LpD2 was deposited on the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 2022, 02 and 28The preservation number is CGMCC NO.24436, and the preservation address is Beijing in China.
2. Use of lactobacillus paracasei NHB-LpD2 as claimed in claim 1 in the preparation of liquid fermented feed.
3. The use according to claim 2, wherein the liquid fermented feed increases daily gain of piglets, decreases feed to meat ratio of piglets and decreases diarrhea rate;
the liquid fermented feed improves the growth performance of the fattening pigs and improves the pork flavor of the fattening pigs.
4. Use of lactobacillus paracasei NHB-LpD2 as claimed in claim 1 in the preparation of a wet-based fermented feed.
5. The use according to claim 4, wherein the wet-based fermented feed improves the productivity and survival rate of white-feather broilers.
6. Use of lactobacillus paracasei NHB-LpD2 as claimed in claim 1 in the manufacture of an antibacterial agent.
7. The use according to claim 6, wherein the antimicrobial spectrum of the antimicrobial agent comprises the following species: erysipelas, pseudomonas aeruginosa, klebsiella pneumoniae, listeria monocytogenes, streptococcus pneumoniae, enteropathogenic escherichia coli, staphylococcus aureus, salmonella typhi, salmonella, bacillus perfringens, proteus peng, aeromonas hydrophila and vibrio parahaemolyticus.
8. Use of lactobacillus paracasei NHB-LpD2 as claimed in claim 1 in the preparation of a feed additive.
9. Use of lactobacillus paracasei NHB-LpD2 as claimed in claim 1 in the manufacture of a medicament for the prevention and treatment of diarrhea in animals.
10. A liquid fermented feed, which is characterized in that a fermentation strain of the liquid fermented feed is lactobacillus paracasei NHB-LpD.
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| KR20160006377A (en) * | 2014-07-09 | 2016-01-19 | 주식회사 유담 | yogurt |
| 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 |
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| KR20160006377A (en) * | 2014-07-09 | 2016-01-19 | 주식회사 유담 | yogurt |
| 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 |
| CN111996147A (en) * | 2020-09-01 | 2020-11-27 | 汉臣氏（沈阳）儿童制品有限公司 | Lactobacillus paracasei with triglyceride reducing function and application thereof |
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Effective date of registration: 20240411 Address after: 621000 Mianyang national hi tech Industrial Development Zone, Sichuan Applicant after: NEW HOPE LIUHE Co.,Ltd. Country or region after: China Applicant after: CHENGDU FENGLAN SCIENCE AND TECHNOLOGY Co.,Ltd. Applicant after: SICHUAN NEW HOPE ANIMAL HUSBANDRY TECHNOLOGY Co.,Ltd. Applicant after: CHENGDU CENTURY INVESTMENT Co.,Ltd. Address before: 621000 Mianyang national hi tech Industrial Development Zone, Sichuan Applicant before: NEW HOPE LIUHE Co.,Ltd. Country or region before: China Applicant before: CHENGDU FENGLAN SCIENCE AND TECHNOLOGY Co.,Ltd. Applicant before: SICHUAN NEW HOPE ANIMAL HUSBANDRY TECHNOLOGY Co.,Ltd. |