Lactobacillus rhamnosus MS27 and application thereof in livestock and poultry breeding

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus rhamnosus MS27 and application thereof in livestock and poultry breeding. According to the invention, a strain of lactobacillus rhamnosus MS27 is found through research, and the preservation number of the lactobacillus rhamnosus MS27 is CGMCC (China General Microbiological Culture Collection Center) NO.27412. The lactobacillus rhamnosus MS27 has the advantages that the lactobacillus rhamnosus MS27 can be used for preparing the lactobacillus rhamnosus; the strain resists common pathogenic bacteria of livestock, a fermentation product of the strain has antioxidant activity, the average daily gain and the average feed intake can be remarkably improved and the feed-gain ratio can be reduced when the strain is used for feeding livestock and poultry, the growth performance of the livestock and poultry can be remarkably improved, and the strain has wide application and market prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and more specifically, to Lactobacillus rhamnosus MS27 and its application in livestock and poultry breeding. Background Art

[0002] The scope and depth of the application of probiotics in the field of livestock and poultry breeding are constantly expanding. Especially since the use of antibiotics has been increasingly discouraged in the livestock industry, the demand for feed additives in the livestock industry is continuously expanding. In the field of microecological preparations, lactic acid bacteria products have a continuously expanding actual application demand due to their unique functions of producing lactic acid and regulating the balance of intestinal flora.

[0003] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) is a Gram-positive probiotic, facultatively anaerobic, acid-tolerant, and is one of the normal human flora. It has a high rate of intestinal adhesion and strong colonization ability. As a kind of Lactobacillus, it is widely used as an additive in various foods, health foods, microecological products, or soy products, etc., and is known as the third-generation probiotic. At present, the great potential of Lactobacillus rhamnosus as a new type of probiotic applied to livestock and poultry breeding has been initially verified. However, compared with Bacillus, lactic acid bacteria still face huge challenges in actual livestock and poultry breeding applications due to their high production costs.

[0004] Solid-state fermentation is a fermentation method on a solid matrix without free water. Compared with liquid fermentation, solid-state fermentation has lower costs and is not easily contaminated with bacteria. Especially for some important secondary metabolites, solid-state fermentation has higher productivity. Solid-state fermentation feed of lactic acid bacteria can not only use low-cost crops and their by-products as culture media, such as corn, soybean meal, wheat bran, corncob, etc., but also effectively increase their economic value while fermenting a large number of live bacteria, including secreting high-value secondary metabolites, enhancing soluble dietary fiber, enhancing antioxidant activity, etc. It is an economical and efficient lactic acid bacteria production process technology. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a new Lactobacillus rhamnosus with antioxidant activity, which can promote animal feeding, growth, and improve feed conversion rate.

[0006] The present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27, whose preservation number is CGMCC NO. 27412.

[0007] The Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus)A strain with a relatively high viscosity, named MS27, was selected after ultraviolet mutagenesis from a saliva sample of Bama miniature pigs. It was deposited at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101) on May 23, 2023, and was taxonomically named Lactobacillus rhamnosus Lacticaseibacillus rhamnosus , and the deposit number is CGMCC No. 27412.

[0008] The Lactobacillus rhamnosus Lacticaseibacillus rhamnosus (MS27) of the present invention can significantly inhibit common pathogenic bacteria of livestock and poultry, such as Staphylococcus aureus, Escherichia coli, and Salmonella, in vitro. And its fermentation product has antioxidant activity, can promote animal feeding, and improve growth performance and feed conversion rate.

[0009] The present invention also provides a bacterial agent containing the above-mentioned Lactobacillus rhamnosus Lacticaseibacillus rhamnosus (MS27).

[0010] The bacterial agent of the present invention can be a liquid bacterial agent or a solid bacterial agent. In addition to containing Lactobacillus rhamnosus Lacticaseibacillus rhamnosus (MS27), it may also contain other strains with the same or different functions.

[0011] The present invention also provides a fermentation product of the above-mentioned Lactobacillus rhamnosus Lacticaseibacillus rhamnosus (MS27).

[0012] The fermentation product of the present invention is obtained by solid-state fermentation of the above-mentioned Lactobacillus rhamnosus Lacticaseibacillus rhamnosus (MS27).

[0013] The substrate for the solid-state fermentation described in the present invention includes, by weight: 35-45 parts of wheat bran, 40-55 parts of soybean meal, 5-10 parts of cane molasses, 5-8 parts of calcium carbonate, 8-12 parts of maltodextrin, and 60-80 parts of water.

[0014] Preferably, the substrate for the solid-state fermentation described in the present invention includes, by weight: 35-37 parts of wheat bran, 40-42 parts of soybean meal, 8-10 parts of cane molasses, 5-6 parts of calcium carbonate, 9-11 parts of maltodextrin, and 60-70 parts of water.

[0015] More preferably, the substrate for the solid-state fermentation described in the present invention includes, by weight: 35 parts of wheat bran, 40 parts of soybean meal, 10 parts of cane molasses, 5 parts of calcium carbonate, 10 parts of maltodextrin, and 70 parts of water.

[0016] The conditions for the solid-state fermentation in the present invention are anaerobic fermentation, the fermentation temperature is 35°C to 45°C, and the fermentation time is 36 h to 60 h; and / or, the inoculation amount is 20% to 50% L / kg; Preferably, after the fermentation is completed, it further includes drying and pulverizing steps.

[0017] In the present invention, the viable count of the solid-state fermentation product of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 is 1.0 to 9.0×10 9 CFU / g, and it has antioxidant activity. The total antioxidant activity T-AOC of the solid-state fermentation preparation of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 is ≥150 U / g.

[0018] The present invention also provides a method for solid-state fermenting the above-mentioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27. The substrate for the solid-state fermentation is as described above, and the conditions for the solid-state fermentation are as described above.

[0019] As a specific embodiment, the solid-state fermentation method of the present invention may further include two steps of liquid seed preparation and solid-state anaerobic fermentation. Among them, during liquid seed preparation, the fermentation temperature is 35°C to 39°C, the fermentation inoculation amount is 1% to 3%, the fermentation cycle is 18 h to 36 h, the fermentation rotation speed is 50 to 100 r / min, and the conditions for solid-state anaerobic fermentation are anaerobic culture bags, the fermentation temperature is controlled at 35°C to 39°C, and the fermentation cycle is 36 h to 60 h.

[0020] The present invention also provides the following applications of the above-mentioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 or its bacterial agent or fermentation product in any of the following aspects: (1) Improving the antioxidant activity of feed or feed additives; (2) Increasing the body weight and / or daily weight gain of livestock and poultry; (3) Increasing the feed intake of livestock and poultry; (4) Reducing the feed-to-meat ratio of livestock and poultry; (5) Increasing the survival rate of livestock and poultry; (6) Preparing feed additives; (7) Livestock and poultry farming; Preferably, the livestock and poultry are broiler chickens.

[0021] The present invention also provides a feed additive, which includes the above-mentioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 or its bacterial agent or fermentation product.

[0022] The present invention also provides a livestock and poultry farming method, which includes the step of feeding the above-mentioned fermentation product; preferably, the livestock and poultry are broiler chickens.

[0023] The beneficial effects of the present invention are at least as follows: The Lactobacillus rhamnosus MS27 of the present invention has antioxidant activity, and can significantly improve the growth performance, feed intake of chickens and improve feed conversion efficiency. Description of the Drawings

[0024] Figure 1 It is an agarose gel electrophoresis map of the 16S rDNA fragment of Lactobacillus rhamnosus MS27 CGMCC No.27412.

[0025] Figure 2 It is a phylogenetic tree map of Lactobacillus rhamnosus MS27 CGMCC No.27412.

[0026] Figure 3 It is a colony morphology map of Lactobacillus rhamnosus MS27 CGMCC No.27412.

[0027] Figure 4 It is a Gram staining microscopic examination map of Lactobacillus rhamnosus MS27 CGMCC No.27412.

[0028] Figure 5 It is a growth curve map of Lactobacillus rhamnosus MS27 CGMCC No.27412.

[0029] Figure 6 It is a sensitivity result map of Lactobacillus rhamnosus MS27 CGMCC No.27412 to different antibiotics.

[0030] Figure 7 It is an antibacterial effect map of Lactobacillus rhamnosus MS27 CGMCC No.27412 against different pathogenic bacteria.

[0031] Figure 8 It is a finished product map of the final product of solid-state fermentation of Lactobacillus rhamnosus MS27 CGMCC No.27412. Detailed Description of the Invention

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with the examples. It should be understood that the following examples are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0033] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources or prepared by conventional methods in the art unless otherwise specified.

[0034] Example 1 Isolation and Identification of Lactobacillus rhamnosus Lacticaseibacillus rhamnosus MS27 (1) Isolation of Lactobacillus rhamnosus Lacticaseibacillus rhamnosus MS27 Lactobacillus selective medium: 10 g of tryptone, 5 g of yeast extract powder, 6 g of potassium dihydrogen phosphate, 2 g of ammonium citrate, 25 g of sodium acetate, 0.575 g of magnesium sulfate, 0.12 g of manganese sulfate, 0.034 g of ferrous sulfite, 1 g of Tween 80, 20 g of glucose. Add to 1000 mL of distilled water, stir well, adjust the pH value to 5.5, pipette 1.32 mL of glacial acetic acid, mix well, heat to dissolve, and cool to 45 °C for standby.

[0035] MRS-calcium carbonate solid medium: 10 g of peptone, 8 g of beef extract powder, 4 g of yeast extract powder, 20 g of glucose, 2 g of dipotassium hydrogen phosphate, 2 g of ammonium hydrogen citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 20 g of agar, 1 g of Tween-80. Dissolve in 1000 mL of distilled water, adjust the pH to 6.3 - 6.7, autoclave at 121 °C for 15 min, cool to 55 - 60 °C, add 1% calcium carbonate (calcium carbonate is sterilized separately after adding to distilled water), mix well and pour into sterile petri dishes to prepare solid plates.

[0036] Oral sample collection and strain isolation: Use a sterile swab to dip into the oral mucus of Bama miniature pigs, place it in a sterile centrifuge tube, collect the mucus, filter it through sterile gauze, inoculate the mucus at 1% into 10 mL of Lactobacillus selective medium, anaerobically culture at 45 °C for 24 h - 48 h to enrich Lactobacillus, after gradient dilution with normal saline, then spread it on the MRS-calcium carbonate solid medium plate, anaerobically culture at 37 °C - 45 °C for 24 h - 36 h, observe the colony morphology, pick the oval-shaped strains with obvious calcium dissolution reaction, perform three streaking purifications, and perform sequencing identification after purification.

[0037] Species identification and screening: The selected strains are inoculated into 15 mL of MRS medium and cultured at 37 °C for 24 h - 36 h. Extract the total DNA of the strains according to the method of the bacterial genomic DNA kit. Use the universal bacterial 16S rRNA primers (27F: AGAGTTTGATCMTGGCTCAG, SEQ ID No.1 / 1492R: GGTTACCTTGTTACGACTT, SEQ ID No.2) to amplify the 16S rDNA fragment of the strain. Send the PCR product to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Use the NBCI database to perform BLAST alignment on the sequencing results, and screen out the strains identified as Lactobacillus rhamnosusLacticaseibacillus rhamnosus The strain of ( Lacticaseibacillus rhamnosus ) was inoculated into 15 mL of MRS medium as a single colony. After culturing at 37 °C for 24 h, it was serially diluted with normal saline and spread on an MRS solid plate. After irradiating for 90 s at a distance of 20 cm from a 20 W ultraviolet lamp, it was cultured in the dark at 37 °C for 48 h. A strain of Lactobacillus rhamnosus with high viscosity was screened out by the colony drawing method (

[0038] (2) 16S rRNA sequence homology analysis and phylogenetic tree construction of strain MS27 The 16S rRNA of strain MS27 was carried out according to the above-mentioned strain identification method. As shown in Figure 1, the size of the amplified 16S rDNA product of strain MS27 was about 1500 bp. The sequencing result is shown in SEQ ID No.3. The sequence was aligned with the NBCI database, and homologous and similar 16S rRNA gene sequences were selected from GenBank. MEGA11.0 was used to construct a phylogenetic tree. The sequence alignment was constructed by the Neighbor-joining method, and the Bootstrap method was used for testing. The number of repetitions was 2000 times. The phylogenetic tree of MS27 is as Figure 2 shown. Homology comparison showed that the category of strain MS27 was Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) with a similarity of 99.80%.

[0039] (3) Colony morphology observation After Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 was cultured to the logarithmic growth phase, it was spread on an MRS solid medium using the method of serial dilution with sterile normal saline and cultured at 37 °C for 24 h to 48 h. Plates with colony counts between 30 and 300 were selected for photographing and observation. As Figure 3 shown, Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 presented milky white circular colonies on the MRS solid medium, with a diameter of 1 - 2 mm, a smooth and moist surface, slightly convex, and a drawing feeling when streaked.

[0040] (4) Bacterial Gram staining Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 was observed during the logarithmic growth phase. 1 - 3 drops of the bacterial liquid were spread on a clean glass slide, clamped with forceps and fixed in the flame in front of an alcohol lamp. After cooling, the staining operation was carried out according to the steps of the Gram staining kit. After staining, it was observed and recorded under the oil immersion lens of an optical microscope. The results are as Figure 4 shown. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 was rod-shaped, blue-violet, and was a Gram-positive bacterium.

[0041] (5)Biochemical identification of lactic acid bacteria Prepare Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 that grows single colonies after purification. Use an inoculation needle to pick single colonies from the plate and inoculate them into the biochemical identification tubes of lactic acid bacteria. Culture according to the instructions of the manual, and measure the reaction of strain MS27 to aesculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose respectively. The test results are shown in Table 1. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 shows positive reactions to aesculin, cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, inulin, and lactose, and a negative reaction to raffinose, which is consistent with the biochemical reactions of Lactobacillus rhamnosus, further confirming that MS27 is Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ). After biological preservation, the preservation number of the strain is: CGMCC No. 27412.

[0042] Table 1 Biochemical identification results of lactic acid bacteria Example 2 Growth and physiological and biochemical characteristics of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 (1)Bacterial growth curve Pick single colonies from the solid plate and inoculate them into a 100 mL conical flask containing 50 mL of liquid MRS medium. Culture in a shaker at 37 °C at 100 r / min for 24 h. After the bacterial solution is stable, inoculate it into 500 mL of medium containing 250 mL of liquid MRS medium at a concentration of 1%, set three replicates, culture at 37 °C at 100 r / min, and take out 5 mL every 2 h to detect the changes in its pH, OD 600 and viable cell count. The viable cell count is calculated by the plate counting method. The specific method is to dilute the bacterial solution in a 10-fold gradient, take 100 μL of the bacterial solution and spread it on the MRS solid medium, culture in an incubator at 37 °C for 36 h, and select the plates with colonies between 30 and 300 for colony counting. The test results are as Figure 5 shown. For Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27, as the growth time continues to extend, the viable cell count first increases and then levels off, reaching the highest viable cell count at 22 h, about 1.47×10 9 CFU / mL, OD 600 shows an S-shaped curve. It is inferred that the logarithmic growth phase range of MS27 is about 4 h to 10 h. The pH results show that MS27 grows and produces acid, and the pH continuously decreases with the extension of time. After 24 h of growth, the pH drops to 3.23.

[0043] (2)Drug sensitivity analysis The drug resistance of the strains was evaluated by performing drug sensitivity analysis on representative categories of antibiotics, mainly as follows: penicillins (penicillin, carbenicillin), macrolides (erythromycin), tetracyclines (tetracycline), chloramphenicols (chloramphenicol), and lincosamides (clindamycin). Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 in the logarithmic growth phase was inoculated into MRS solid medium at 45°C - 55°C at a ratio of 1%, and left standing for 20 min. After the medium solidified, the drug sensitivity test paper was picked up with forceps and placed in the center of the solid plate, gently pressed, and placed in an incubator at 37°C for 48 h. The growth of the strains was observed. Among them, a diameter of the inhibition zone less than 15 mm was considered resistant (R), a diameter between 16 mm - 20 mm was considered moderately sensitive (I), and a diameter greater than 20 mm was considered sensitive (S). The results were as Figure 6 , shown in Table 2. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 was sensitive to carbenicillin, erythromycin, and tetracycline, and moderately sensitive to chloramphenicol, clindamycin, and penicillin. These indicated that MS27 did not have obvious drug resistance and was a good probiotic for livestock and poultry.

[0044] Table 2 Antibiotic sensitivity of Lactobacillus rhamnosus MS27 (3) Ability to inhibit pathogenic bacteria in vitro Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 picked from the solid plate was inoculated into MRS liquid medium and fermented at 37°C for 36 h at 100 r / min. The effects of the bacterial liquid (labeled as 1), fermentation supernatant (labeled as 2), and bacterial cells (labeled as 3) on common pathogenic bacteria of livestock and poultry, Staphylococcus aureus, Escherichia coli, and Salmonella, were detected. The bacterial liquid was centrifuged at 4°C and 5000 rpm for 10 min, and the supernatant was obtained after filtering through a membrane. The bacterial cell precipitate was resuspended by adding an equal volume of sterile normal saline.

[0045] Staphylococcus aureus ATCC 25923, Escherichia coli K88, and Salmonella typhimurium SL1344 growing on the solid plate were picked and inoculated into 30 mL of sterilized liquid medium. After culturing at 37°C for 12 h, the concentration was adjusted with sterile normal saline, and the pathogenic bacteria were diluted to 1.0×10 8 CFU / mL for standby. Oxford cups were placed on the plain agar plate. The sterilized solid LB medium was cooled to 45 - 55°C, and 20 mL of solid LB medium + 200 μL of the diluted pathogenic bacteria liquid were mixed evenly and poured onto the plate. After cooling and solidifying, the Oxford cups were removed, and 150 μL of the sample was added to each well. After standing at 4°C for 2 h - 4 h, it was cultured at 37°C for 24 h. The obtained results were asFigure 7 As shown in Figure 7 , the results showed that Lactobacillus rhamnosus had obvious inhibitory effects on common pathogenic bacteria of livestock and poultry, such as Staphylococcus aureus, Escherichia coli and Salmonella. Further, the antibacterial active substance was produced by the bacteria and secreted into the fermentation supernatant, and the bacteria itself had no obvious antibacterial effect.

[0046] Example 3 Solid-state fermentation of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 The solid-state fermentation of Lactobacillus rhamnosus MS27 includes three parts: liquid seed culture, solid-state fermentation and drying. The main contents are as follows: (1) Liquid seed culture: Pick a single colony of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 and inoculate it into 50 mL of liquid MRS medium, culture at 37 °C for 12 h - 24 h, then inoculate at a concentration of 1%, inoculate into a 5 L liquid fermenter, the liquid filling volume is 3.5 L - 4 L, the medium is MRS liquid medium, the tank pressure is controlled at 0.04 mPa, the air flow rate is controlled at 3 L / min, the culture temperature is 35 °C - 45 °C, the rotation speed is 50 - 100 r / min, and the fermentation cycle is 18 h - 36 h.

[0047] (2) Solid-state anaerobic fermentation: The solid-state fermentation medium is composed of the following components by weight: 35 - 45 parts of wheat bran, 40 - 55 parts of soybean meal, 5 - 10 parts of cane molasses, 5 - 8 parts of calcium carbonate, 8 - 12 parts of maltodextrin, 60 - 80 parts of water. The solid medium is sterilized at 121 °C for 10 min - 20 min, and among them, cane molasses and maltodextrin are mixed and sterilized separately, and then formulated into a 10% - 50% aqueous solution and sterilized at 121 °C for 10 min - 20 min. Then, inoculate the fermentation broth prepared in a 5 L fermenter into the above solid-state fermentation medium at an inoculation amount of 20% - 50% L / kg, culture at 35 °C - 45 °C in an anaerobic fermentation bag for 36 h - 60 h, dry the fermentation product at room temperature to prepare the final product, crush it, and pass through a 60-mesh sieve to prepare the final product. The viable count of the final product is about 1.0 - 9.0×10 9 CFU / g.

[0048] Example 4 Antioxidant activity of the solid-state fermentation substrate of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 (1) Liquid seed culture: Pick a single colony of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus)Inoculate a single colony of MS27 into 50 mL of liquid MRS medium and culture it at 37°C for 24 h. Then, inoculate it at a concentration of 1% into a 5 L liquid fermenter with a liquid volume of 4 L. The medium is MRS liquid medium. Control the tank pressure at 0.04 mPa, the air flow rate at 3 L / min, the culture temperature at 37°C, the rotation speed at 100 r / min, and the fermentation cycle at 36 h.

[0049] (2)Solid-state anaerobic fermentation: The solid-state fermentation medium is composed of the following components by weight: 35 parts of wheat bran, 40 parts of soybean meal, 10 parts of cane molasses, 5 parts of calcium carbonate, 10 parts of maltodextrin, and 70 parts of water. Sterilize the solid medium at 121°C for 20 min. Among them, cane molasses and maltodextrin are mixed and sterilized separately, and then added to 30 parts of water to prepare an aqueous solution and sterilized at 121°C for 20 min. Then, inoculate the fermentation broth prepared in a 5 L fermenter into the above solid-state fermentation medium at an inoculation amount of 30% L / kg, culture it at 37°C in an anaerobic fermentation bag for 60 h, dry the fermentation product at room temperature to prepare the final product, pulverize it, and pass it through a 60-mesh sieve to prepare the final product, as Figure 8 shown. The viable count of the final product is approximately 5.7×10 9 CFU / g.

[0050] (3)Preparation of crude body fluid samples Prepare a suspension (dilution factor N = 10) of the dried Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )MS27 solid-state fermentation sample after the above solid-state fermentation according to 2 g + 20 mL of distilled water, put it into an ultrasonic cleaner and ultrasonically extract for 30 min. Transfer the supernatant to a centrifuge tube and centrifuge at 10000 rpm for 10 min. Take the supernatant and filter it through a 0.45 μm filter membrane to obtain the crude extract.

[0051] (4)Determination of total antioxidant capacity (T-AOC) The antioxidant substance Fe 3＋ is reduced to Fe 2＋ , and Fe 2＋ can form a stable complex with phenanthroline substances and has the maximum absorption at a wavelength of 520 nm. Therefore, the total antioxidant activity can be determined by colorimetry. According to the method of the T-AOC determination kit, it is stipulated that at 37°C, when the absorbance (A) of the reaction system per minute per milliliter of antioxidant substance increases by 0.01, it is defined as one unit of total antioxidant activity (U). Set three replicates for the experiment, take the average value, and calculate according to the following formula: ; Where: A1 is the absorbance of the sample; A0 is the absorbance of the control; V1 is the total volume of the reaction solution, 4.1 mL; V0 is the sampling volume, 0.2 mL; N is the dilution factor of the sample, which is 10 in this experiment.

[0052] The test results are shown in Table 3. The total antioxidant activity of the solid fermentation substrate before fermentation was 92.25 U / g. After fermentation by Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), the total antioxidant activity increased to 153.75 U / g, showing good antioxidant activity.

[0053] Table 3 Effects of Lactobacillus rhamnosus MS27 fermentation on the total antioxidant activity of solids Example 5 Application of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 in broiler feeding.

[0054] The final product after solid-state fermentation of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 in Example 4 was added to the basal diet of AA broilers at an addition dose of 0.5%. A total of 216 healthy 1-day-old AA broilers with uniform body weight were purchased from Beijing Dafeng Poultry Breeding Co., Ltd. and randomly divided into 2 treatments (control group and MS27 group), with 6 replicates in each treatment and 18 broilers in each replicate (3 cages, 6 broilers in each cage, male chicks). The experimental period was 42 days, divided into two stages: 1-21 days old and 22-42 days old.

[0055] The test used a corn-soybean meal-based basal diet. The required basal diet for the test was mixed in the same batch and then weighed according to the number of treatment groups and divided into 5 equal parts. The experimental group diet was added with a certain proportion of feed additives to the basal diet. The specific formula is shown in Table 4 below.

[0056] Table 4 Composition and nutrient levels of broiler basal diet (% dry matter basis) The premix in Table 4 is a vitamin and mineral premix, which provides the following nutrients per kilogram: vitamin A, 9,000 IU; vitamin D3, 3,000 IU; vitamin E, 24 mg; vitamin K3, 1.8 mg; vitamin B1, 2.0 mg; riboflavin, 5.0 mg; vitamin B6, 3.0 mg; vitamin B12, 0.1 mg; niacin, 40 mg; pantothenic acid, 15 mg; folic acid, 1.0 mg; biotin, 0.05 mg; choline chloride, 500 mg, iron, 80 mg; copper, 20 mg; zinc, 90 mg; manganese, 80 mg; iodine, 0.35 mg; selenium, 0.30 mg.

[0057] The experiment was conducted in the metabolism room of the Feed Center on the West Campus of China Agricultural University. The chicken house selected was a three - layer cage - type house with three cage racks in total. Each cage rack had 30 cages, and each cage had 6 chickens. Artificial lighting was used in the chicken house, and the chickens were allowed to feed freely and drink water from nipple drinkers. The temperature control and vaccine immunization procedures in the experimental chicken house were carried out according to the feeding management regulations.

[0058] (1)Production performance During the experiment, the health status of the broilers was observed daily. For dead chickens, the death time needed to be recorded in time, the initial weight was weighed, the feed intake and the number of deaths of each replicate were accurately recorded, the final weight was weighed, and the mortality rate, average daily feed intake, average daily weight gain and feed - to - meat ratio of the broilers from 1 to 42 days old were calculated. Record daily abnormal situations, medication and immunization procedures.

[0059] The test results are shown in Table 5. The final product after solid - state fermentation of 0.5% Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )MS27 can significantly improve the average daily weight gain and average daily feed intake of AA broilers ( P <0.01). Among them, the average daily weight gain increased by 10.49%, and the average daily feed intake increased by 4.95%. At the same time, it can significantly improve the feed utilization rate, and the feed - to - meat ratio decreased by 4.97% ( P <0.01). After 42 days of test feeding, all the broilers in the Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )MS27 group survived, and the survival rate reached 100%, while that of the control group was 92.59%.

[0060] Table 5 Effects of Lactobacillus rhamnosus MS27 feeding on the growth performance of AA broilers Represents P <0.01.

[0061] (2)Slaughter performance Taking each replicate as a unit, 1 broiler was randomly selected from each replicate for slaughter. Before slaughter, it was weighed and the live weight was recorded. At 42 days old, 1 rooster was randomly selected from each replicate, and the slaughter rate, eviscerated yield, wing rate, leg muscle rate, breast muscle rate and abdominal fat rate were measured according to the "Nomenclature and Measurement Statistics Methods for Poultry Production Performance" (NY / T 823 - 2020). The calculation formulas are as follows: Slaughter rate (%) = (dressed weight / live weight before slaughter) × 100; Eviscerated yield (%) = (eviscerated weight / dressed weight) × 100; Wing rate (%) = (weight of bilateral wings / eviscerated weight) × 100; Ratio of leg muscle (%) = (leg muscle weight / dressed weight) × 100; Ratio of breast muscle (%) = (breast muscle weight / dressed weight) × 100; Ratio of abdominal fat (%) = (abdominal fat weight / dressed weight) × 100; The test results are shown in Table 6. The feeding of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27 had no significant effect on the slaughter performance of AA broilers. From an economic perspective, the ratio of breast muscle in the MS27 group increased by 4.12% compared with the control group, and the ratio of wings increased by 0.74% compared with the control group.

[0062] Table 6 Effects of feeding Lactobacillus rhamnosus MS27 on the slaughter performance of AA broilers Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) MS27, characterized in that The deposit number is CGMCC NO. 27412.

2. A bacterial agent, characterized in that Containing the Lactobacillus rhamnosus according to claim 1 ( Lacticaseibacillus rhamnosus )MS27.

3. A Lactobacillus rhamnosus according to claim 1 ( Lacticaseibacillus rhamnosus ) Fermentation product of MS27.

4. The fermentation product according to claim 3, characterized in that By treating the Lactobacillus rhamnosus described in claim 1 ( Lacticaseibacillus rhamnosus )MS27 was obtained by solid-state fermentation.

5. The fermentation product according to claim 4, characterized in that The substrate for the solid-state fermentation comprises, by weight, 35-45 parts of wheat bran, 40-55 parts of soybean meal, 5-10 parts of sugarcane molasses, 5-8 parts of calcium carbonate, 8-12 parts of maltodextrin, and 60-80 parts of water.

6. The fermentation product according to claim 4 or 5, characterized in that The solid-state fermentation conditions are anaerobic fermentation, the fermentation temperature is 35°C to 45°C, and the fermentation time is 36 h to 60 h; and / or, the inoculum size is 20% to 50% L / kg; Preferably, after the fermentation is completed, it also includes drying and crushing steps.

7. A solid fermentation method of the Lactobacillus rhamnosus according to claim 1 ( Lacticaseibacillus rhamnosus ) The method of MS27, characterized in that The substrate for the solid-state fermentation is as described in claim 5, and the conditions for the solid-state fermentation are as described in claim 6.

8. The Lactobacillus rhamnosus of claim 1 ( Lacticaseibacillus rhamnosus ) Use of MS27 or the bacterial agent according to claim 2 or the fermentation product according to any one of claims 3 to 6 in any of the following aspects: (1) Enhance the antioxidant activity of feed or feed additives; (2) Increase the body weight and / or daily weight gain of livestock and poultry; (3) Increase feed intake of livestock and poultry; (4) Reduce the feed-to-meat ratio of livestock and poultry; (5) Improve the survival rate of livestock and poultry; (6) Preparation of feed additives; (7) Livestock and poultry farming; Preferably, the livestock and poultry are broiler chickens.

9. A feed additive, characterized in that: The Lactobacillus rhamnosus according to claim 1 ( Lacticaseibacillus rhamnosus ) MS27 or the bacterial agent described in claim 2 or the fermentation product described in any one of claims 3-6.

10. A livestock and poultry breeding method, characterized in that: The method comprises the step of feeding the fermentation product according to any one of claims 3 to 6; preferably, the livestock and poultry are broilers.

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