Lactobacillus crispatus with gastric acid resistance and high phenyllactic acid yield and application of lactobacillus crispatus in poultry breeding

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus crispatus resistant to gastric acid and high in phenyllactic acid yield and application of the lactobacillus crispatus in poultry breeding, and the preservation number of the lactobacillus crispatus is CCTCC NO: M20231841. An applicant screens out a strain of lactobacillus crispatus from intestinal tracts of Jianghan free-range chickens, and experimental detection shows that the lactobacillus crispatus has good broad-spectrum antibacterial ability and acid resistance. Laying hen breeding experiments show that the daily total laying weight of laying hens in an experimental group added with lactobacillus crispatus is increased by 12.8% compared with that of a control group, the laying rate is increased to 92% from 83%, and the feed-egg ratio is decreased to 2.01 from 2.27; the protein content in the eggs is increased by 3.8%, the sterol content is reduced by 26%, the fat content is reduced by 24%, the yolk color and relative density of the eggs are improved, and the eggshell thickness is increased.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a Lactobacillus crispatus strain and application thereof in laying hen breeding. Background Art

[0002] In today's large-scale breeding environment, the breeding environment, breeding density, and epidemic prevention level will have a layered impact on the immune system of laying hens. The increase in breeding density allows some pathogens that are harmful to laying hens to have the opportunity to reproduce, causing harm to the body of laying hens, leading to an increase in the incidence of various diseases, which in turn affects the economic interests of farmers. The use of antibiotics reduces the incidence of diseases, promotes the growth of laying hens, and has promoted the laying hen breeding industry. However, in order to prevent the breeding costs from being too high and increase economic benefits, some farmers add antibiotics without restraint during the breeding process, resulting in drug residues in eggs, affecting the safety of egg quality, and posing a threat to food safety and human health.

[0003] Microbial feed additives, Chinese herbal medicine preparations, enzyme preparations, antimicrobial peptides and fermented feed can not only replace the function of antibiotics in fighting diseases and promoting growth, but also improve the quality of livestock and poultry meat, eggs, milk, etc. On the one hand, it reduces the negative impact of the use of antibiotics on animals, humans and the environment, and on the other hand, it promotes the development of the breeding industry. Therefore, these antibiotic substitutes must be indispensable in future breeding.

[0004] Lactobacillus crispatus is a facultative anaerobic bacterium, Gram-positive, and is a slender, curved, and slender bacillus, belonging to the Firmicutes, Bacillus, Lactobacilales, Lactobacillaceae, and Lactobacillus. CN114891657A discloses a method for preparing a Lactobacillus crispatus and a microencapsulated composite bacterial preparation thereof for inhibiting and promoting growth, and the application thereof in livestock and poultry breeding. The Lactobacillus crispatus has an inhibitory ability against Salmonella, and the Lactobacillus crispatus is prepared in combination with two other bacteria into a microencapsulated composite bacterial preparation, which can improve the body's resistance to Salmonella, improve the intestinal mucosal immune level, and stimulate the body's immune response. Summary of the invention

[0005] The invention aims to provide a strain of Lactobacillus crispatus with a broad antibacterial spectrum, and the preservation number is CCTCC NO: M20231841.

[0006] Another object of the present invention is to provide the use of Lactobacillus crispatus M1027 in the preparation of poultry feed additives.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A strain of Lactobacillus crispatus that is resistant to gastric acid and produces high amounts of phenyllactic acid was isolated by the applicant from the cecum of native chickens in Hubei Province. It was finally identified as Lactobacillus crispatus through physiological and biochemical analysis and 16S rRNA gene sequence identification. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on October 7, 2023, with the classification name: Lactobacillus crispatus M1027, the deposit number is CCTCC NO: M20231841, and the address is: Wuhan University, Wuhan, China.

[0009] The protection scope of the present invention also includes:

[0010] The fermentation liquid of Lactobacillus crispatus M1027 contains live bacteria of Lactobacillus crispatus M1027.

[0011] Fermentation supernatant of Lactobacillus crispatus M1027.

[0012] A composition containing Lactobacillus crispatus M1027, fermentation broth and / or fermentation supernatant thereof.

[0013] Application of Lactobacillus crispatus M1027, fermentation liquid, fermentation supernatant or the above combination in the preparation of poultry breeding feed additives.

[0014] In the above application, preferably, the poultry is chicken.

[0015] Application of Lactobacillus crispatus M1027, fermentation broth, fermentation supernatant or the above composition in the preparation of an antibacterial agent, wherein the antibacterial agent inhibits Gram-positive bacteria, Gram-negative bacteria and / or fungi.

[0016] In the above application, preferably, the Gram-negative bacteria include: Escherichia coli, Salmonella, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus;

[0017] Gram-positive bacteria include: Staphylococcus aureus, Streptococcus, Micrococcus luteus, Nocardia;

[0018] Fungi include: Fusarium, Rhizopus, and Penicillium.

[0019] The above-mentioned Lactobacillus crispatus M1027 high-density solid fermentation method comprises the following steps:

[0020] The cultured Lactobacillus crispatus M1027 seed solution is inoculated into a solid fermentation medium at a volume mass ratio of 5%-15%, cultured at 25-35° C. for 48-72 hours, and dried and crushed at low temperature to obtain the product;

[0021] Solid fermentation medium formula:

[0022] 1000 parts of bran, 1000-2000 parts of water, 20-30 parts of glucose, 5-15 parts of yeast extract powder, 3-7 parts of potassium dihydrogen phosphate, by weight.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The Lactobacillus crispatus provided by the invention is resistant to gastric acid, and has a survival rate greater than 98% after being treated with artificial gastric juice at pH 2.6 for 3 hours.

[0025] The invention provides a strain of Lactobacillus crispatus M1027. The strain has a fast growth rate, a broad-spectrum antibacterial effect, can inhibit most Gram-positive and Gram-negative bacteria and filamentous fungi, and has broad application prospects and potential for popularization and application.

[0026] The strain can use bran as a matrix for high-density solid-state fermentation. Compared with liquid fermentation, the method for high-density solid-state fermentation of Lactobacillus crispatus M1027 provided by the invention has simple equipment, low technical difficulty and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The growth curve of L. crispatus M1027 and the pH value change of the fermentation broth.

[0028] Figure 2 The coaggregation of L. crispatus M1027. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with specific examples. The examples are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operating processes are given, but the protection scope of the present invention is not limited to the following examples. The methods used in the following examples are conventional methods unless otherwise specified.

[0030] Embodiment 1:

[0031] Isolation and identification of Lactobacillus crispatus M1027:

[0032] Free-range Jianghan native chickens were collected from various parts of Hubei Province. After the experimental chickens were killed by bleeding, their body surfaces were disinfected with 75% ethanol. Then, the connecting end of the cecum and the rectum was tied tightly with sterile cotton thread, and the cecum was quickly dissected and separated and placed in a 50mL centrifuge tube. The samples were stored briefly at 4°C for microbial isolation. 1g of cecal content samples were taken in 50mL centrifuge tubes containing 9mL sterile PBS, 3-5 sterilized glass beads were added, and the samples were vortexed to mix them thoroughly, and then sterile gauze was used to filter to remove solid residues. 100μL of the dilution was spread on an MRS plate containing 0.01% bromocresol purple and cultured anaerobically at 37°C for 24h; single colonies of different morphology and color that changed the color of bromocresol purple were picked and streaked on the MRS plate for 1-2 times, and then PCR amplification and sequencing of the 16S rRNA gene sequence were performed. After the sequencing results were correctly compared, the strains were preserved for later use.

[0033] A lactic acid bacterium was isolated from it, and its 16S rRNA gene sequence was aligned with 100% homology with Lactobacillus crispatus by BLAST on NCBI. The strain was deposited in China Center for Type Culture Collection (CCTCC) on October 7, 2023, with classification name: Lactobacillus crispatus M1027, and the deposit number is CCTCC NO: M20231841. Address: Wuhan University, Wuhan, China

[0034] Embodiment 2:

[0035] Growth curve and pH value determination of Lactobacillus crispatus M1027:

[0036] After L. crispatus M1027 was cultured to the logarithmic phase, it was inoculated into MRS liquid medium at a 2% inoculum and cultured at 37°C. Samples were taken at 0h, 6h, 9h, 12h, 24h, 36h, and 48h after inoculation to measure OD 600 and pH values, with 3 replicates for each treatment.

[0037] like Figure 1 As shown in the figure, the hysteresis period of L. crispatus M1027 is not obvious during its growth. It is in the logarithmic phase within 9 hours after inoculation and enters the stable phase after 12 hours of cultivation. 600The pH value of the fermentation broth decreased from about 6.5 to about 4 after 12 hours of cultivation, and then there was no obvious pH change. This shows that the synthesis and secretion of acidic substances that lower the pH of the fermentation broth are coupled with growth.

[0038] Embodiment 3:

[0039] Determination of acid resistance of Lactobacillus crispatus M1027:

[0040] After L. crispatus M1027 was cultured to the logarithmic phase, it was inoculated into 9 mL of artificial gastric fluid (pH 2.6) at a 10% inoculum. After mixing evenly, the mixture was diluted 10 6 After doubling, take 100 μL and spread it on MRS solid medium to detect the number of live bacteria. Incubate at 37°C, repeat the dilution and spreading process at 30min and 180min to detect the number of live bacteria. Strains 62 and 64 in Table 1 are common Lactobacillus crispatus collected by the applicant.

[0041] Table 1 Tolerance of Lactobacillus crispatus to artificial gastric juice

[0042]

[0043] Note: The unit of viable bacteria count is: 10 7 CFU / mL, compared with 0min in this group, * Indicates significant difference (P<0.05); ** Indicates extremely significant difference (P<0.01)

[0044] The survival rate of L. crispatus M1027 after being treated with artificial gastric juice at pH 2.6 for 3 hours was greater than 98%, indicating that the strain can tolerate low pH gastric juice well and has excellent probiotic properties.

[0045] Embodiment 4:

[0046] Antibacterial performance test of Lactobacillus crispatus M1027:

[0047] After culturing L. crispatus M1027 seed liquid for 12 hours, it was inoculated into MRS liquid culture medium at a 2% inoculation rate and cultured at 37°C for 12 hours. The fermentation liquid was sampled for use and then centrifuged at 4000r / min for 15 minutes to separate the fermentation liquid supernatant (corresponding to the supernatant group in Table 2) and the bacteria. The fermentation liquid itself corresponds to the bacterial liquid group in Table 2; the bacteria were washed 2-3 times with sterile PBS buffer and resuspended to the original concentration of the fermentation liquid for use (corresponding to the precipitation group in Table 2).

[0048] The supernatant was treated differently:

[0049] 1) Boiling water bath treatment for 40 min (corresponding to the heating groups in Table 2);

[0050] 2) Proteinase K treatment in a 37°C constant temperature water bath for 1 h (corresponding to the proteinase K groups in Table 2);

[0051] 3) 37℃ constant temperature water bath 2 O 2 Enzyme treatment for 1 h (corresponding to H in Table 2) 2 O 2 enzyme group);

[0052] 4) Adjust the pH to 6.5 (corresponding to the pH 6.5 group in Table 2).

[0053] After the LB solid medium is dissolved, wait for it to cool down to about 45°C, inoculate the activated indicator strain into the medium at a 2% inoculum, pour about 25 mL of the bacterial culture medium into a culture dish with 5 sterile Oxford cups evenly placed, and use sterile tweezers to pull out the Oxford cups after the culture medium solidifies. The indicator strains used in the experiment are:

[0054] Gram-negative bacteria: Escherichia coli, Salmonella, Aeropsymonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus;

[0055] Gram-positive bacteria: Staphylococcus aureus, Streptococcus, Micrococcus luteus, Nocardia;

[0056] Fungi: Pichia pastoris, Fusarium, Rhizopus, Penicillium.

[0057] 250 μL of sample was added to each well, and the plate was placed in a 4°C refrigerator for 1 hour to allow the sample to diffuse in the agar, and then placed in a 37°C constant temperature incubator for 24 hours, and the diameter of the inhibition zone was measured and the data was recorded. Each treatment was repeated 3 times.

[0058] The results are shown in Table 2. The bacterial precipitate of L. crispatus M1027 fermentation broth did not show an inhibitory effect on all indicator strains, indicating that the antibacterial substances exist in the supernatant of the fermentation broth. The antibacterial effect was lost after the pH of the supernatant was adjusted to 6.5. Heating, proteinase K treatment and H 2 O 2 Enzyme treatment did not affect the intensity of antibacterial activity, indicating that the antibacterial substance was not H 2 O 2, works under acidic conditions, is heat stable, and is not a protein in nature. Among the indicator strains, L. crispatus M1027 is more effective against Aeromonas hydrophila among Gram-negative bacteria, Streptococcus among Gram-positive bacteria, and Penicillium among fungi than other strains.

[0059] Table 2 Antibacterial activity of Lactobacillus crispatus M1027

[0060]

[0061] Embodiment 5:

[0062] Coaggregation assay

[0063] The experiment investigated the coaggregation ability of L. crispatus M1027 with Escherichia coli, Salmonella, Streptococcus and Staphylococcus aureus. After activation, the bacterial suspension of Vibrio parahaemolyticus L. crispatus M1027 was centrifuged at 4000r / min for 15min, the supernatant was discarded, and the suspension was washed 2-3 times with sterile PBS buffer and the OD was adjusted. 600 Escherichia coli, Salmonella, Streptococcus and Staphylococcus aureus were inoculated into LB liquid medium and cultured at 37°C for 24 hours. The bacterial solution was centrifuged at 6000r / min for 6 minutes. The cells were washed three times with sterile PBS buffer and then sterile PBS buffer was added to adjust the OD 600 To 0.4 ± 0.05. Take 5 mL of the test bacterial solution and indicator bacterial solution of the adjusted concentration and add them to the sterile PA bottle, vortex and mix. Take samples and measure OD 600 Then place it in a 37°C constant temperature incubator for static culture, and measure the supernatant OD after 24 hours. 600 Each treatment was repeated 3 times. The copolymerization rate was calculated as follows:

[0064] Coagulation rate (%) = (1-A t / A 0 )×100%

[0065] Among them, A t OD of the supernatant of the mixed bacterial solution at 24 h 600 , A 0 is the initial OD of the mixed bacterial solution 600 .

[0066] Probiotics can prevent the formation of pathogenic biofilms in the digestive or reproductive tract by co-aggregating with pathogenic bacteria. Figure 2 As shown, L. crispatus M1027 has a co-aggregation rate of more than 50% for the four indicator strains used. Based on the results of self-aggregation, it can be considered that the S layer protein of L. crispatus M1027 has good properties.

[0067] Embodiment 6:

[0068] Mass spectrometry analysis

[0069] After culturing L. crispatus M1027 for 24 hours, centrifuge at 4000 r / min for 15 minutes to collect the supernatant as the test sample. The mass spectrometry analysis was commissioned to Wuhan Metrowell Biotechnology Co., Ltd.

[0070] The lactic acid derivatives detected in the supernatant of L. crispatus M1027 fermentation broth include 3-indole-lactic acid (CAS#1821-52-9), hydroxyphenyllactic acid (CAS#306-26-0), L-3-phenyllactic acid (CAS#20312-36-1), and 2-methyllactic acid (CAS#594-61-6), and their relative abundances are shown in Table 3. Among them, 3-indole-lactic acid is produced by tryptophan metabolism and has anti-tumor ability; L-3-phenyllactic acid is phenyllactic acid, which is a new type of antibacterial agent. It can be seen that the fermentation products of L. crispatus M1027 have high application value.

[0071] Table 3 Lactic acid derivatives detected in the supernatant of L. crispatus M1027 fermentation broth and their relative abundance

[0072]

[0073] Embodiment 7:

[0074] The method for producing a Lactobacillus crispatus inoculum by high-density solid fermentation comprises the following steps:

[0075] The slant seeds of Lactobacillus crispatus M10272 were picked up with an inoculation loop, inoculated into YPD liquid culture medium, and cultured at 30°C and 100 rpm for 24 h to obtain seed liquid.

[0076] The cultured seed liquid is inoculated into the solid fermentation medium at an inoculation rate of 10% (v / m), the fermentation temperature is 30° C., the fermentation cycle is 60 hours, and the number of viable bacteria can reach 21 billion CFU / g dry material.

[0077] The formula of the solid fermentation medium described above is: 1000g bran, 1000mL water, 25g glucose, 10g yeast extract powder, and 5g potassium dihydrogen phosphate.

[0078] A shallow dish (320 mm long, 240 mm wide, 45 mm deep) was used to enlarge the fermentation and culture of Trichosporon by 10 times, and the bran loading amount was 500 g. After fermentation using the above process, the live bacterial count of Lactobacillus crispatus could reach 18 billion CFU / g dry material.

[0079] 1.0% trehalose and 5% skim milk powder were added as protective agents, and the mixture was dried and crushed at 50°C, and the survival rate was above 75%.

[0080] Embodiment 8:

[0081] Application of Lactobacillus crispatus M1027 in laying hen farming:

[0082] Choose 256 Nongda No. 5 laying hens of 300 days age with similar growth conditions, and randomly divide into 2 groups, each group of 4 repetitions, each repetition 32, every 8 are placed in a cage and fed. After 7d of feeding without any added basal feed, formal test is started, and the experimental period is 28d. The A group feed is not processed, and the B group adds the solid fermentation product of lactobacillus crispatus (lactobacillus crispatus viable count is 12,000,000,000 / g, i.e., the preparation after the protective agent drying and pulverizing is added in embodiment 7) by 400g / t in the basal feed.

[0083] After the experiment officially started, the total number and weight of eggs laid by each group of laying hens were recorded at the same time every day, the feed consumption was counted at a fixed time every week, a series of production performance indicators were calculated, and egg quality indicators were measured.

[0084] Compared with the control group, the total egg weight of the Lactobacillus crispatus group increased by 12.8%, the egg laying rate increased from 83% to 92%, and the feed-egg ratio decreased from 2.27 to 2.01; the protein content in eggs increased by 3.8%, the sterol content decreased by 26%, and the fat content decreased by 24% (Table 4).

[0085] Table 4 Basic index detection of eggs

[0086]

[0087]

Claims

1. An isolated strain of Lactobacillus crispatus ( Lactobacillus crispatus ) M1027, the preservation number of the crispatus Lactobacillus is CCTCC NO: M20231841.

2. The fermentation liquid of Lactobacillus crispatus M1027 according to claim 1, wherein the fermentation liquid contains live bacteria of Lactobacillus crispatus M1027.

3. The fermentation supernatant of Lactobacillus crispatus M1027 according to claim 1.

4. A composition comprising the Lactobacillus crispatus M1027 according to claim 1, the fermentation broth according to claim 2 and / or the fermentation supernatant according to claim 3.

5. Use of the Lactobacillus crispatus M1027 according to claim 1, the fermentation liquid according to claim 2, the fermentation supernatant according to claim 3 or the composition according to claim 4 in the preparation of a poultry feed additive.

6. The use according to claim 5, wherein the poultry is chicken.

7. Use of the Lactobacillus crispatus M1027 according to claim 1, the fermentation broth according to claim 2, the fermentation supernatant according to claim 3 or the composition according to claim 4 in the preparation of an antibacterial agent, wherein the antibacterial agent inhibits Gram-positive bacteria, Gram-negative bacteria and / or fungi.

8. The use according to claim 7, wherein the Gram-negative bacteria comprises: Escherichia coli, Salmonella Salmonella ), Aeropsymonas hydrophila ( Aeromonas hydrophila ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ); Gram-positive bacteria include: Staphylococcus aureus ( Staphylococcus aureus ), Streptococcus ( Streptococcus ), Micrococcus luteus ( Micrococcus luteus )、Nocardia( Nocardia ); Fungi include: Fusarium ( Fusarium )、Rhizopus( Rhizopus )、Penicillium( Penicillium ).

9. The high-density solid fermentation method of Lactobacillus crispatus M1027 according to claim 1, comprising the following steps: The cultured Lactobacillus crispatus M1027 seed solution is inoculated into a solid fermentation medium at a volume-to-mass ratio of 5%-15%, cultured at 25-35° C. for 48-72 h, and dried and crushed at low temperature to obtain the product; Solid fermentation medium formula: 1000 parts of bran, 1000-2000 parts of water, 20-30 parts of glucose, 5-15 parts of yeast extract powder, 3-7 parts of potassium dihydrogen phosphate, by weight.

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