Lactobacillus crispatus LSC1 and application thereof

Abstract

The invention relates to the technical field of microorganisms and fermentation, in particular to a lactobacillus crispatus LSC1 (Lactobacillus crispatus LSC1) and application of the lactobacillus crispatus LSC1. The preservation number of the lactobacillus crispatus LSC1 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) NO.33007, and the lactobacillus crispatus LSC1 can be used for efficiently inhibiting the growth and reproduction of escherichia coli and has good tolerance to high-temperature, high-cholate and low-pH environments. The fermentation process of the lactobacillus crispatus LSC1 is optimized, the optimized fermentation method can utilize appropriate and cheap carbon sources and nitrogen sources to perform culture fermentation on the strain, probiotic products formed by fermentation can be put into production application, and further research shows that the fermented products can improve the egg laying performance and egg quality of old laying hens, so that the yield of the old laying hens is improved. Good popularization and application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms and fermentation, and particularly relates to a Lactobacillus crispatus Lactobacillus crispatus ) LSC1 and its applications. Background Art

[0002] Avian colibacillosis refers to a focal and systemic infectious disease caused by the infection of avian pathogenic Escherichia coli (APEC) of some specific serotypes such as O1, O2, O35, O78, etc. Some common symptoms include: Escherichia coli septicemia, omphalitis, yolk peritonitis, upper respiratory tract inflammation, enteritis, panophthalmitis, fibrinous airsacculitis, perihepatitis and pericarditis, swollen head syndrome, cellulitis, arthritis, synovitis, encephalitis, Escherichia coli granuloma, yolk peritonitis and salpingitis, etc. Early infection of APEC in chicks is mostly characterized by a high infection rate and mortality; broilers infected with APEC grow slowly, have diarrhea, and gradually dehydrate and emaciate to death; laying hens infected with APEC mostly show sporadic deaths, a decrease in egg production rate and a reduction in egg quality. Avian colibacillosis occurs globally, and chickens of different ages and breeds are likely to be infected throughout the year. Air, water, feed and related equipment can all become the transmission media of the disease. It is roughly estimated that avian colibacillosis causes economic losses of about hundreds of millions of US dollars to the global poultry industry every year.

[0003] For a long time, antibiotics have been the main means of preventing and treating avian colibacillosis. Long-term and high-dose use of antibiotics to treat colibacillosis is likely to make Escherichia coli develop drug resistance, and it also reduces the body's immunity, making it extremely easy to have mixed infections such as mycoplasma and Salmonella, increasing the treatment difficulty, and the drug residue components in livestock and poultry products are likely to exceed the national standards. With the ban on antibiotic products, seeking natural alternative products with the advantages of being green, highly efficient, and having a relatively low cost has become an urgent matter in the current livestock and poultry breeding industry. Probiotics, as a kind of microorganism that can have a series of beneficial effects on the body itself by improving and regulating the body's intestinal microflora, have gradually become antibiotic substitutes for veterinary drugs and feed additives. Therefore, it is urgent to develop a lactic acid bacteria preparation with a good inhibitory effect on Escherichia coli to treat avian colibacillosis.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a Lactobacillus crispatus LSC1 and its applications.

[0006] Specifically, the technical solution of the present invention is as follows: In the first aspect, the present invention provides a Lactobacillus crispatus Lactobacillus crispatus)LSC1, with the preservation number of CGMCC NO. 33007. This strain can effectively inhibit the growth and reproduction of Escherichia coli and has good tolerance to high temperature, high bile salts and low pH environment.

[0007] In the second aspect, the present invention provides a Lactobacillus crispatus preparation, which contains at least one of the cells, fermentation broth, fermentation supernatant, fermentation broth precipitate, and freeze-dried powder of the Lactobacillus crispatus LSC1.

[0008] Preferably, it is an Escherichia coli inhibitor. Based on the stress resistance characteristics of the Lactobacillus crispatus LSC1, it has obvious advantages in inhibiting Escherichia coli in a specific environment.

[0009] In the third aspect, the present invention provides the application of the Lactobacillus crispatus LSC1 or the Lactobacillus crispatus preparation in inhibiting Escherichia coli for non-disease diagnosis purposes.

[0010] In the fourth aspect, the present invention provides a fermentation method of Lactobacillus crispatus LSC1, including: inoculating Lactobacillus crispatus LSC1 into a fermentation medium for fermentation culture.

[0011] Preferably, the composition of the fermentation medium includes: glucose 7.5±1 g / L, beef extract 15.0±2 g / L, yeast powder 7.5±1 g / L, peptone 5.0±1 g / L, trisodium citrate 2±0.5 g / L, anhydrous sodium acetate 5±1 g / L, dipotassium hydrogen phosphate 2±0.5 g / L, magnesium sulfate heptahydrate 0.58±0.1 g / L, manganese sulfate tetrahydrate 0.25±0.05 g / L, Tween-80 1±0.2 mL / L.

[0012] Preferably, the inoculation amount of Lactobacillus crispatus LSC1 is 2%±0.4%.

[0013] Preferably, the fermentation temperature of Lactobacillus crispatus LSC1 is 37±2 °C and the fermentation time is 20±5 h.

[0014] Preferably, the initial pH value of the fermentation of Lactobacillus crispatus LSC1 is 6.0±0.5.

[0015] In the fifth aspect, the present invention provides a fermentation product obtained by the aforementioned fermentation method.

[0016] In the sixth aspect, the present invention provides the application of the Lactobacillus crispatus LSC1 and the fermentation product in improving the intestinal flora or intestinal health of old laying hens. The old laying hens can be over 70 weeks old. Through research, the present invention finds that feeding old laying hens with chicken feed containing the Lactobacillus crispatus LSC1 as an additive can improve the egg production performance of old laying hens and improve the egg quality.

[0017] Preferably, in every 1 kg of chicken feed, the addition amount of Lactobacillus crispatus LSC1 is 1×10 8 ~1×10 10 CFU.

[0018] Beneficial effects: The present invention provides a Lactobacillus crispatus LSC1 and its application, and the preservation number of the Lactobacillus crispatus LSC1 is CGMCC NO. 33007. The Lactobacillus crispatus LSC1 strain provided by the present invention can efficiently inhibit the growth and reproduction of Escherichia coli, and has good tolerance to high temperature, high bile salt and low pH environments. The present invention also optimizes the fermentation process of the Lactobacillus crispatus LSC1, and the optimized fermentation method can use suitable and inexpensive carbon sources and nitrogen sources to culture and ferment the strain. The probiotic product formed by fermentation can be put into production and application, and has good prospects for popularization and application. Description of the drawings

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will describe the drawings required to be used in the embodiments or the description of the prior art.

[0020] Figure 1 It is the colony morphology of the Lactobacillus crispatus LSC1 strain.

[0021] Figure 2 It is the antibacterial effect of the Lactobacillus crispatus LSC1 strain on Escherichia coli.

[0022] Figure 3 It is the result of the acid tolerance test of the Lactobacillus crispatus LSC1 strain.

[0023] Figure 4 It is the result of the bile salt tolerance test of the Lactobacillus crispatus LSC1 strain.

[0024] Figure 5 It is the result of the temperature tolerance test of the Lactobacillus crispatus LSC1 strain.

[0025] Figure 6 It is the phylogenetic tree diagram of the Lactobacillus crispatus LSC1 strain.

[0026] Figure 7 It is the growth curve of the Lactobacillus crispatus LSC1 strain.

[0027] Figure 8 It is the selection of the fermentation carbon source of the Lactobacillus crispatus LSC1 strain.

[0028] Figure 9 It is the optimization of the fermentation carbon source concentration of the Lactobacillus crispatus LSC1 strain.

[0029] Figure 10 It is the selection of the fermentation nitrogen source of the Lactobacillus crispatus LSC1 strain.

[0030] Figure 11 Optimization of the inoculum amount for the fermentation of Lactobacillus crispatus strain LSC1.

[0031] Figure 12 Optimization of the initial pH for the fermentation of Lactobacillus crispatus strain LSC1.

[0032] Figure 13 Optimization of the fermentation temperature for Lactobacillus crispatus strain LSC1. Specific implementation manner

[0033] In the present invention, poultry-derived fecal samples are selected, and the samples are diluted and spread on plates; the cultured strains are isolated, purified and preserved; the isolated and preserved strains are preliminarily screened to obtain avian-derived lactic acid bacteria that inhibit Escherichia coli, and then the lactic acid bacteria are further screened, that is, the abilities of the strains to tolerate acid, bile salts, temperature and artificial gastrointestinal fluid are identified; the genus and species of the screened lactic acid bacteria are identified; the fermentation process of the screened lactic acid bacteria is optimized, including medium optimization: screening of carbon sources and nitrogen sources; fermentation condition optimization: optimization of inoculum amount, initial pH and temperature. Finally, a new probiotic product and its fermentation method for treating avian Escherichia coli disease are provided.

[0034] Specifically, the present invention provides a Lactobacillus crispatus LSC1 that can inhibit Escherichia coli. It is isolated and screened from poultry-derived fecal samples, has an effective inhibitory effect on the growth and reproduction of Escherichia coli, and has good tolerance to high temperature, high bile salts and low pH environments. After identification, strain LSC1 is Lactobacillus crispatus ( Lactobacillus crispatus ). This strain was deposited on December 9, 2024 at the China General Microbiological Culture Collection Center (abbreviated as CGMCC, address: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101), and was classified and named as Lactobacillus crispatus ( Lactobacillus crispatus ), with the deposit number CGMCC NO. 33007.

[0035] The Lactobacillus crispatus LSC1 described in the present invention is isolated and screened by the following method.

[0036] (1) Select a donor chicken and collect the contents of the duodenum, jejunum, ileum and cecum; (2) Weigh 0.5 g of the intestinal contents and add 4.5 mL of sterile physiological saline to prepare a suspension; (3) Gradient dilute the suspension, and then spread 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 suspensions on plates containing 1.5% CaCO 3In the MRS solid medium, anaerobic culture was carried out at 37 °C for 24 h; (4)After the colonies grew out, single colonies with obvious dissolution zones were selected and cultured in MRS broth, and then streaked and purified on the MRS solid medium, repeating three times; (5)The strain obtained in step (4) was cultured, centrifuged at 12,000 r for 10 min, and the supernatant was taken and filtered through a 0.22 μm sterile filter; (6)The Oxford cup method was used to detect the inhibitory effect of the supernatant on Escherichia coli, and lactic acid bacteria with a large diameter of the inhibition zone were selected; (7)The acid tolerance, bile salt tolerance and temperature tolerance of the lactic acid bacteria screened in step (6) were identified, and species identification was carried out; (8)The fermentation process of the lactic acid bacteria screened in step (7) was optimized, including the selection and concentration ratio of the carbon source and nitrogen source of the fermentation medium and the fermentation conditions, including the fermentation temperature, the initial fermentation pH and the inoculum size.

[0037] Finally, a strain of Lactobacillus crispatus LSC1 from poultry source was screened and obtained in the present invention, which can efficiently inhibit the growth and reproduction of Escherichia coli, and its fermentation process was optimized. It can be cultured and fermented using a suitable and inexpensive carbon source and nitrogen source, and the probiotic product formed by fermentation can be put into production and application.

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0039] At the endpoints and any value in the ranges disclosed in this specification, these ranges or values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and a single point value, and between single point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0040] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "specific implementation manners", or "some specific implementation manners" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the embodiments of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0041] In the embodiments provided in this specification, where specific technologies or conditions are not indicated, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. Where the manufacturers of the reagents or instruments used are not indicated, they are all conventional products that can be obtained through regular channels.

[0042] Example 1 This example describes the isolation and purification process of Lactobacillus crispatus LSC1 strain and the research process and results of its biological characteristics.

[0043] 1 Experimental steps 1.1 Isolation and purification of the strain First, items required for isolation such as scissors and forceps were sterilized by high pressure and set aside for use. The contents of the duodenum, jejunum, ileum, and cecum of healthy adult Hy-Line Brown laying hens were taken, and aseptic operation was adopted throughout the experimental process. 0.5 g (±0.001 g) of the sample was taken, and the contents of each intestinal segment were diluted 10 -2 times, 10 -3 times, 10 -4 times, 10 -5 times, 10 -6 times with normal saline. 100 μL of the diluted solutions of different multiples were respectively added to the surface of the prepared MRS agar medium containing 1.5% CaCO 3 , and a disposable spreading rod was used to spread evenly. It was cultured in a constant temperature incubator at 37 °C for 24 h. According to the different colony sizes, morphologies, and surface smoothness, a disposable inoculation loop was used to purify and subculture the colonies on the medium. After multiple streak purifications by the "three-line method", single colonies were picked and placed in MRS liquid medium for enrichment.

[0044] 0.7 ml of the well-enriched MRS liquid medium was taken, 0.7 ml of 50% glycerol normal saline was added, and it was pipetted repeatedly and stored at -20 °C for one day, and then transferred to -80 °C for storage to preserve the strain.

[0045] 1.2 Antibacterial test Preparation of pathogen indicator bacteria solution: Inoculate pathogenic Escherichia coli into sterilized ordinary broth medium, incubate at a constant temperature of 37 °C for 24 h, and then store in a 4 °C refrigerator for standby. Preparation of centrifuged supernatant of isolated strain: Inoculate the isolated strain into sterilized MRS liquid medium, incubate anaerobically at 37 °C for 24 h, centrifuge the cultured bacterial solution at 10000 r for 5 min, and take the supernatant. Oxford cup antibacterial test: Take 100 μL of Escherichia coli and spread it evenly on an EMB agar plate. After the pathogenic bacteria solution has completely penetrated, place Oxford cups horizontally on it. Add 200 μL of centrifuged supernatant to each Oxford cup, place it horizontally in a 4 °C refrigerator for 12 - 16 h, take it out, incubate at 37 °C for 24 h, and then observe and measure the diameter of the antibacterial circle. The test is set with 3 parallel replicates.

[0046] 1.3 Acid tolerance test Use 1 mol / L HCl to adjust the pH values of MRS liquid medium to 3.5, 4.0, 4.5, 5.0, and 5.5 respectively, sterilize at 115 °C under high pressure steam for 30 min, inoculate the activated lactic acid bacteria at an inoculation amount of 2% (v / v) into MRS liquid medium with different pH values respectively, incubate at 37 °C for 24 h, and then take samples to measure the OD of the bacterial solution 600 。

[0047] 1.4 Bile salt tolerance test Add porcine bile salt to MRS liquid medium to make its concentration 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% (w / v) respectively, shake well to completely dissolve it, sterilize at 115 °C under high pressure steam for 30 min, inoculate the activated lactic acid bacteria at an inoculation amount of 2% (v / v) into MRS liquid medium with different bile salt concentrations respectively, incubate at 37 °C for 24 h, and then take samples to measure the OD of the bacterial solution 600 。

[0048] 1.5 Temperature tolerance test Inoculate the activated lactic acid bacteria at an inoculation amount of 2% (v / v) into MRS liquid medium, incubate at 30 °C, 37 °C, 42 °C, 45 °C, and 50 °C for 24 h, and then take samples to measure the OD of the bacterial solution 600 。

[0049] 1.6 Simulated gastric juice and intestinal juice tolerance test Preparation of artificial gastric juice: Hydrochloric acid with a volume fraction of 9.5% - 10.5% was added to a sterile aqueous solution, and the pH was adjusted to 3.0. Then, 2.5 g / L pepsin was added. After complete dissolution, it was filtered through a 0.22 μm sterile filter membrane for sterilization. Preparation of artificial intestinal juice: 3.4 g of potassium dihydrogen phosphate was dissolved in 250 mL of sterile water, and the pH was adjusted to 6.8 with a 0.4% sodium hydroxide solution. 1 g of trypsin was added to every 100 mL of the liquid. After thorough mixing, it was filtered through a 0.22 μm sterile filter membrane for sterilization.

[0050] Take 500 μL of the activated lactic acid bacteria fermentation broth and mix it with 4.5 mL of artificial gastric juice in a 5 mL shaking tube. Incubate at 37 °C for 4 h, and perform viable cell counting. Subsequently, take 500 μL of the cultured lactic acid bacteria and inoculate it into 4.5 mL of artificial intestinal juice, and incubate at 37 °C for 4 h, and perform viable cell counting; the experiment was set with 3 replicates. Calculate the survival rate using the following formula: Survival rate (%) = (number of viable cells after 4 h of culture in gastric juice or intestinal juice / number of viable cells at 0 h) × 100%.

[0051] 1.7 16S rRNA sequence analysis Refer to the 16S rDNA gene sequences of various lactic acid bacteria in GenBank, and use DNAMAN software to compare the 16S rDNA gene sequences of lactic acid bacteria. One pair of primers (Table 1) was designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0052]

[0053] Extraction of genomic DNA of the strain Extract the DNA of the isolated strain using a DNA extraction kit, and strictly follow the steps in the kit instruction manual.

[0054] PCR amplification Using the extracted strain DNA as a template, perform PCR amplification with the designed 16S rDNA primers. The PCR reaction system is shown in Table 2, and the PCR reaction program and its cycling parameters are shown in Table 3.

[0055]

[0056]

[0057] PCR gel electrophoresis verification Add 10 mL of TAE and 0.9 g of agarose powder to 80 mL of double-distilled water. After it is fully dissolved, put it into a conical flask and melt it in the microwave for 3 min. After the agarose powder and TAE are completely fused, add 10 μl of GVⅡ and pour it into the mold. Wait for the liquid to cool and solidify. Then add 3 μl of PCR product, Marker 2000, and blank control in sequence. Subsequently, start the electrophoresis apparatus and set the program to 120 V / 20 min.

[0058] 16S rRNA sequencing The above PCR amplification products were sent to Biomarker Technologies Corporation for second-generation sequencing of the 16S rRNA of the strains. The full-length 16S rRNA sequences of the sequenced strains were input into the GenBank database and used to compare the homology of the sequences with the known standard strain sequences by BLAST.

[0059] 1.8 Determination of the growth curve of lactic acid bacteria After the strain was activated and expanded, it was inoculated into a 100 ml Erlenmeyer flask containing 25 ml of MRS liquid medium and cultured statically for 24 h. Samples were taken every 4 h to measure the absorbance value. The growth curve was plotted with the culture time as the abscissa and the corresponding absorbance value as the ordinate, with three replicates in each group.

[0060] 1.9 Optimization of carbon source types Based on MRS, only the carbon source was changed, and other components in the MRS liquid medium remained unchanged. The original carbon source (glucose) in the MRS medium was replaced with 2% (w / v) maltose, sucrose, glucose, molasses, and fructooligosaccharide, respectively, and the medium without any carbon source was used as the control group. Inoculate at an inoculation amount of 2% into a 100 ml conical flask containing 25 ml of liquid medium, adjust the initial medium pH to 6.0, and ferment statically in a 37 °C constant temperature incubator for 20 h. Then measure the bacterial density and viable count of the fermentation broth to screen out the optimal carbon source.

[0061] 1.10 Optimization of carbon source concentration The concentrations of the selected optimal carbon source were set to 7.5 g / L, 12.5 g / L, 17.5 g / L, 22.5 g / L, and 27.5 g / L, respectively. Other components in the MRS liquid medium remained unchanged. Inoculate at an inoculation amount of 2% into 25 ml of the medium, adjust the initial medium pH to 6.0, and ferment statically in a 37 °C constant temperature incubator for 20 h. Then measure the bacterial density and viable count of the bacterial liquid to obtain the optimal addition amount of the carbon source.

[0062] 1.11 Optimization of nitrogen source types On the basis of optimizing the optimal carbon source and its concentration, while keeping other components of the MRS medium unchanged, the main nitrogen sources in the MRS medium: peptone, beef extract, yeast powder, were respectively replaced with 1.5% (w / v) beef extract, yeast powder, NH 4 Cl, urea, soybean meal powder, and the MRS medium without any nitrogen source was used as a control. Inoculate at an inoculation amount of 2% into 25 ml of the medium, with the initial medium pH being 6.0, and statically ferment in a constant temperature incubator at 37 °C for 20 h, then measure the bacterial density of the bacterial liquid and the viable bacteria count of the fermentation broth to screen out the optimal nitrogen source.

[0063] 1.12 Orthogonal experiment for nitrogen source optimization

[0064] 1.13 Selection of inoculation amount According to the obtained optimized medium, adjust the initial pH of the medium to 6.0, and select inoculation amounts of 1%, 2%, 3%, 4% and 5% respectively, inoculate into the optimized medium, and after statically fermenting in a constant temperature incubator at 37 °C for 20 h, measure the bacterial density and viable bacteria count of the fermentation broth to obtain the optimal inoculation amount.

[0065] 1.14 Selection of temperature Adjust the initial pH of the medium to 6.0, inoculate according to the optimized inoculation amount into the optimized fermentation medium, set the fermentation temperatures to 28 °C, 31 °C, 34 °C, 37 °C, 40 °C respectively, and after statically fermenting for 20 h, measure the bacterial density and viable bacteria count of the fermentation broth to obtain the optimal temperature.

[0066] 1.15 Selection of initial pH Adjust the initial pH of the medium to 5.0, 5.5, 6.0, 6.5, 7.0, inoculate according to the optimized inoculation amount into the optimized fermentation medium, and on the basis of the optimized temperature, after statically fermenting for 20 h, measure the bacterial density and viable bacteria count of the fermentation broth to obtain the optimal initial pH.

[0067] 2 Experimental results 2.1 Isolation of the strain and its antibacterial effect The strain was isolated from chicken manure, Figure 1 is the colony morphology of the strain, Figure 2 is the inhibitory effect of the strain on Escherichia coli.

[0068] 2.2 Acid tolerance test The strain has good tolerance to low pH environments, Figure 3 is the OD 600 value of the strain in different pH environments.

[0069] 2.3 Bile salt tolerance test The strain can survive in bile salt concentrations of 0.3%, 0.4%, and 0.5%, and has good tolerance to high concentrations of bile salts. Figure 4 OD of the strain at different bile salt concentrations 600 value.

[0070] 2.4 Temperature tolerance test As the culture temperature of the strain increases, the cell density decreases, and it has certain tolerance to 45°C and 50°C. Figure 5 OD of the strain at different temperatures 600 value.

[0071] 2.5 Simulated gastric and intestinal fluid tolerance test.

[0072] The strain has good tolerance to artificial simulated gastric and intestinal fluids, and has a high survival rate at 4 h. Table 5 shows the survival rates of the strain in artificial simulated gastric and intestinal fluids.

[0073]

[0074] 2.6 Results of 16S rRNA sequencing experiment The strain was identified as Lactobacillus crispatus by 16S rRNA sequencing and BLAST alignment. Figure 6 This is the phylogenetic tree diagram.

[0075] The sequencing results are as follows:

[0076] 2.7 Growth curve of Lactobacillus crispatus Lactobacillus crispatus LSC1 was in the lag phase within 0 - 2 h, during which the growth of the bacteria was slow. After 2 h, it entered the logarithmic phase, and the number of viable bacteria increased rapidly. It entered the stationary phase from 12 h to 20 h and the decline phase after 20 h. Figure 7 This is the growth curve of...

[0077] 2.8 Selection of fermentation carbon source The fermentation effects of different carbon sources for Lactobacillus crispatus LSC1 were in the order of glucose > sucrose > molasses. The fermentation effects of maltose and fructooligosaccharide were relatively poor. Finally, glucose was determined to be the optimal carbon source for LSC1.

[0078] 2.9 Selection of fermentation carbon source concentration When the glucose concentration for Lactobacillus crispatus LSC1 was 7.5 g / L, the number of viable bacteria in the fermentation broth was the highest. Figure 9 This is the number of viable bacteria of Lactobacillus crispatus LSC1 at different carbon source concentrations.

[0079] 2.10 Selection of fermentation nitrogen source The fermentation effects of different nitrogen sources for Lactobacillus crispatus LSC1 were in the order of yeast extract > beef extract > peptone. Since the mixed nitrogen source provides more abundant nutrients than the single nitrogen source, yeast powder, beef extract, and peptone were selected as the mixed nitrogen source. Figure 10 This is the number of viable bacteria of Lactobacillus crispatus LSC1 under each fermentation nitrogen source.

[0080] 2.11 Optimization of nitrogen source ratio and concentration The results of the orthogonal experiment showed that: under these conditions, beef extract 15.0 g / L, yeast powder 7.5 g / L, peptone 5.0 g / L, and the number of viable bacteria was 2.34×10 9 CFU / mL.

[0081]

[0082] 2.12 Optimization of fermentation inoculum amount The optimal inoculum amount of Lactobacillus crispatus LSC1 was 2%, Figure 11 This is the number of viable bacteria of Lactobacillus crispatus LSC1 at different inoculum amounts.

[0083] 2.13 Optimization of initial fermentation pH The optimal initial pH value of Lactobacillus crispatus LSC1 was 6.0, Figure 12 This is the number of viable bacteria of Lactobacillus crispatus LSC1 at different initial pH values.

[0084] 2.14 Optimization of fermentation temperature The optimal fermentation temperature of Lactobacillus crispatus LSC1 was 37℃,Figure 13 The viable count of Lactobacillus crispatus LSC1 at different fermentation temperatures.

[0085] Thus, the optimal culture conditions for Lactobacillus crispatus LSC1 are as follows: glucose 7.5 g / L, beef extract 15.0 g / L, yeast powder 7.5 g / L, peptone 5.0 g / L, trisodium citrate 2 g, anhydrous sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate heptahydrate 0.58 g, manganese sulfate monohydrate 0.25 g, Tween-80 1 mL / L, initial pH 6.0, inoculum size 2%, and culture temperature 37 °C.

[0086] 3 Study on the improvement of laying performance and egg quality of aged laying hens by Lactobacillus crispatus LSC1 3.1 Experimental design and feeding plan The experiment adopted a completely randomized design. Hy-Line Brown laying hens over 67 weeks old, with the same breed, similar body weight, healthy, and from the same source, were randomly divided into 4 treatment groups, with 6 replicates in each treatment group (1 replicate per chicken cage). Each replicate had 6 chickens, totaling about 144 laying hens. The experimental period was 8 weeks. The experimental treatments included 1) control group; 2) J1 group (adding 1×10 8 CFU bacterial liquid per 1 kg of feed); 3) J2 group (adding 1×10 9 CFU bacterial liquid per 1 kg of feed); 4) J3 group (adding 1×10 10 CFU bacterial liquid per 1 kg of feed). During the entire experimental period, the room temperature was maintained at 25 °C, the daily light time was 16 hours of light and 8 hours of darkness, and the laying hens could eat and drink freely for a total of 8 weeks. Eggs were collected daily, and egg quality was measured at the end of the fourth and eighth weeks.

[0087] 3.2 Data analysis Data were presented as mean ± standard error of the mean (SEM). One-way analysis of variance was performed using SPSS 20.0 (Chicago, USA), followed by Duncan's multiple comparison test. The significance level was indicated by asterisks, * indicating 0.01 < p value < 0.05, ** indicating 0.001 < p value < 0.01, *** indicating p value < 0.001, or significance was marked with letters. If two groups had the same letter label, it indicated that there was no significant difference between them, while if two groups had different letter labels, it indicated a significant difference between the two groups.

[0088] 3.3 Experimental results 1) Effects of Lactobacillus crispatus LSC1 on the production performance of laying hens in the late laying period Compared with the control group, adding Lactobacillus crispatus LSC1 to the feed can increase the qualified egg rate of laying hens in the late laying period and the average daily feed intake of laying hens from 0 to 4 weeks ( P <0.05), and reduce the cracked egg rate and the average egg weight from 4 to 8 weeks and from 0 to 8 weeks ( P <0.05).

[0089] 2) Effects of Lactobacillus crispatus LSC1 on egg quality of laying hens in the late laying period Compared with the control group, adding Lactobacillus crispatus LSC1 from 0 to 4 weeks and from 0 to 8 weeks will reduce the yolk color ( P <0.05). From 0 to 8 weeks, as the dose of Lactobacillus crispatus increases, the eggshell thickness and egg shape index decrease. Adding low-dose and medium-dose Lactobacillus crispatus from 0 to 8 weeks will reduce the egg weight ( P <0.05).

[0090]

[0091] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Lactobacillus crispatus ( Lactobacillus crispatus ) LSC1, characterized in that Its deposit number is CGMCCNO. 33007.

2. A Lactobacillus crispatus preparation, characterized in that: Contains at least one of the bacterial cells, fermentation liquid, fermentation supernatant, fermentation liquid precipitation and freeze-dried powder of Lactobacillus crispatus LSC1 according to claim 1.

3. The Lactobacillus crispatus preparation according to claim 2, characterized in that: It is an inhibitor of E. coli.

4. Use of the Lactobacillus crispatus LSC1 according to claim 1 or the Lactobacillus crispatus preparation according to claim 2 or 3 in inhibiting Escherichia coli for non-disease diagnosis purposes.

5. A fermentation method of Lactobacillus crispatus LSC1, characterized in that: Lactobacillus crispatus LSC1 was inoculated into the fermentation medium for fermentation culture.

6. The fermentation method according to claim 5, characterized in that: The fermentation medium comprises: 7.5±1 g / L glucose, 15.0±2 g / L beef extract, 7.5±1 g / L yeast powder, 5.0±1 g / L peptone, 2±0.5 g / L triammonium citrate, 5±1 g / L anhydrous sodium acetate, 2±0.5 g / L dipotassium hydrogen phosphate, 0.58±0.1 g / L magnesium sulfate dodecahydrate, 0.25±0.05 g / L manganese sulfate tetrahydrate, and 1±0.2 mL / L Tween-80.

7. The fermentation method according to claim 5 or 6, characterized in that: The inoculum size of Lactobacillus crispatus LSC1 was 2% ± 0.4%; Preferably, the fermentation temperature of Lactobacillus crispatus LSC1 is 37±2°C and the fermentation time is 20±5h; Preferably, the initial pH value of the fermentation of Lactobacillus crispatus LSC1 is 6.0±0.

5.

8. A fermentation product obtained by the fermentation method according to any one of claims 5 to 7.

9. Use of the Lactobacillus crispatus LSC1 according to claim 1 or the fermented product according to claim 8 in improving the egg-laying performance of aged laying hens and improving the egg quality.

10. The use according to claim 9, characterized in that: The amount of Lactobacillus crispatus LSC1 added to each 1kg chicken feed was 1×10 8 ~1×10 10 CFU.

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