Lactobacillus reuteri RD003, microbial inoculum containing lactobacillus reuteri RD003 and application of lactobacillus reuteri RD003

Abstract

The invention discloses lactobacillus reuteri RD003, a microbial agent containing the lactobacillus reuteri RD003 and application of the lactobacillus reuteri RD003 and belongs to the technical field of microorganisms. The invention aims to provide lactobacillus reuteri which can be added into animal feed to improve the immunity of sika deer and the flora abundance in rumen fluid. The lactobacillus reuteri RD003 is preserved in the China Center for Type Culture Collection, the preservation date is June 9, 2022, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 2022847. The invention provides a theoretical basis for the use of sika deer feed or feed additives.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus reuteri RD003, a bacterial agent containing the bacteria and applications thereof. Background Art

[0002] Since my country banned the addition of antibiotics to feed, the research and development of probiotics and other antibiotic substitutes has become a hot topic. Probiotic preparations are a natural feed additive. It is currently recorded that lactic acid bacteria can be added to feed to replace antibiotics, and can regulate the structure of the flora, increase the body's immunity or prevent diarrhea. However, screening lactic acid bacteria that can be added to feed is a technical problem that needs to be solved at present, and screening sika deer homologous probiotics as a feed strain for Cervidae animals is a key technical problem that needs to be solved at present. Summary of the invention

[0003] The purpose of the invention is to provide a Lactobacillus reuteri which can be added into animal feed to improve the immunity of sika deer and the abundance of flora in rumen fluid.

[0004] The present invention provides a Lactobacillus reuteri RD003, which is deposited in China Center for Type Culture Collection on June 9, 2022, and has a deposit number of CCTCC NO: M 2022847.

[0005] The present invention provides a microbial preparation containing the Lactobacillus reuteri RD003.

[0006] The present invention provides an animal feed additive or animal feed containing the Lactobacillus reuteri RD003.

[0007] The present invention provides a medicine containing the Lactobacillus reuteri RD003.

[0008] The present invention provides an application of the Lactobacillus reuteri RD003, the microbial preparation, the animal feed additive or the animal feed or the medicine in improving the immunity of sika deer.

[0009] It is further defined that improving the immunity of sika deer means increasing the content of immunoglobulin in serum.

[0010] It is further defined that the immunoglobulin is immunoglobulin A.

[0011] The present invention provides an application of the Lactobacillus reuteri RD003, the microbial preparation, the animal feed additive or the animal feed or the medicine in improving the microbial diversity in the rumen fluid of sika deer.

[0012] Further defined, microbial diversity refers to increased relative abundance of Bacteroidota, Euryarchaeota, Rikenellaceae RC9 gut group, and Christensenellaceae R-7 group.

[0013] The present invention provides an application of the Lactobacillus reuteri RD003, the microbial preparation, the animal feed additive or the animal feed or the medicine in improving the digestibility of nutrients or in resistance to artificial gastrointestinal juice, intestinal juice and pig bile salt.

[0014] Beneficial effects: The Lactobacillus reuteri RD003 involved in the present invention has strong adaptability and colonization ability because it is separated from the feces of homologous healthy sika deer.

[0015] Tests on growth performance, acid production capacity and tolerance in a simulated gastrointestinal environment showed that Lactobacillus reuteri RD003 entered the logarithmic growth phase after 4 hours of culture in MRS medium, and the pH value during the stable period was maintained at around 4.10; the survival rate was 93.65% after 3 hours in a simulated gastric fluid environment, and 89.49% after 3 hours in intestinal fluid; the survival rate was 92.56% after 6 hours in a bile salt concentration of 0.1%, further proving its strong growth performance and adaptability.

[0016] Relevant experiments on adding Lactobacillus reuteri RD003 to the diet have proved that it can increase the content of serum immunoglobulin A of sika deer in the antler-growing period, thereby improving the body's immune level; increase the relative abundance of Bacteroidota, Euryarchaeota, Rikenellaceae RC9 gut group and Christensenellaceae R-7 group in rumen fluid, thereby optimizing the flora structure and improving the digestion and absorption of nutrients in feed.

[0017] [Biological preservation information]: A Lactobacillus reuteri RD003, deposited in the China Center for Type Culture Collection, the preservation date is June 9, 2022, the preservation number is CCTCC NO: M 2022847, and the preservation address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The Neighbor-Joining phylogenetic tree of Lactobacillus reuteri RD003 was constructed based on the 16S rRNA gene sequence alignment results with Leuconostocmesenteroides NBRC 100496 (AB681194) as the outer branch;

[0019] Figure 2 This is the result of the microscope (1000X) of Lactobacillus reuteri RD003 strain;

[0020] Figure 3 This is a photo result of the plate of Lactobacillus reuteri RD003 strain;

[0021] Figure 4 It is the growth curve and acid production curve result diagram of Lactobacillus reuteri RD003;

[0022] Figure 5 This is the result of principal coordinate analysis based on Bray-Curtis distance;

[0023] Figure 6 This is the bacterial phylum composition result of rumen fluid of sika deer;

[0024] Figure 7 This is the composition result of bacteria at genus level in rumen fluid of sika deer. DETAILED DESCRIPTION

[0025] Example 1: Isolation and identification of Lactobacillus reuteri RD003

[0026] Fresh feces of a three-year-old male sika deer was collected and added to sterile phosphate buffered saline (PBS) at a ratio of 1:5 (w:v). The mixture was shaken for 30 min and then allowed to stand for 10 minutes. 1 ml of the supernatant was taken for gradient dilution. -3 , 10 -4 and 10 -5 The diluted bacterial solution was spread on MRS solid medium and cultured anaerobically at 37°C for 24 to 48 hours. A round, milky white, shiny single colony was selected for Gram staining microscopy. A single Gram-positive bacillus was streaked and purified on MRS medium for more than 3 times until a purified strain and its biologically pure culture were obtained. The bacterial 16S rRNA sequence was further determined, and a phylogenetic tree was constructed using the NeighborJoining method ( Figure 1 ), and Lactobacillus reuteri RD003 was identified based on parentage relationship analysis. Morphological observation revealed that the colonies of Lactobacillus reuteri RD003 were round, milky white, with neat edges, no wrinkles on the surface, and moist and shiny ( Figure 2 The bacteria are short rod-shaped, arranged singly or in pairs, and are Gram-positive ( Figure 3 ).

[0027] Example 2: Growth curve, acid production capacity and stress resistance test of Lactobacillus reuteri RD003

[0028] 1. Growth curve and acid production capacity

[0029] Take the bacterial liquid of Lactobacillus reuteri RD003 in the logarithmic growth phase, inoculate it into liquid MRS at an inoculum size of 2% (v:v), take the uninoculated liquid MRS as blank, and culture it at 37°C for 24h. Take the culture liquid once every 2h to measure the absorbance (OD)600 value at 600nm and pH, respectively. Draw the growth curve and acid production curve with time as the horizontal axis and the OD600 value and pH of the culture liquid as the vertical axis.

[0030] The results are as follows Figure 4 As shown in the figure, after inoculation, the strain of Lactobacillus reuteri RD003 continued to grow, and after 4 hours, it grew rapidly and entered the logarithmic growth phase. After 14 hours, the strain grew slowly, and the plateau phase lasted until 24 hours without any decline. The pH value of the bacterial solution gradually decreased after inoculation, and the pH value dropped from 6.82 to 5.95 in 0-6 hours, and the pH value of the bacterial solution dropped rapidly from 5.95 to 4.10 in 8-14 hours. After 14 hours, the pH value of the bacterial solution changed slowly. The results show that Lactobacillus reuteri RD003 has good growth performance and acid production ability.

[0031] 2. Resistance to artificial gastrointestinal fluid, intestinal fluid and pig bile salt test

[0032] According to the 2013 edition of the Chinese Pharmacopoeia, artificial gastric juice with a pH value of 3.0 and artificial intestinal juice with a pH value of 6.8 were prepared and filtered with a 0.22 μm microporous sterile filter membrane. Take the bacterial solution of Lactobacillus reuteri RD003 in the logarithmic growth period, centrifuge, resuspend the bacterial solution, mix evenly in a volume ratio of resuspension: artificial gastric juice or artificial intestinal juice = 1:9, and culture at 37°C. Take the 0 and 3h culture solutions, dilute them in multiple ratios, spread them on plates, count the viable bacteria, repeat 3 times and take the average value, and calculate the bacterial survival rate.

[0033] Survival rate (%) = 100 × lgNt / lgN0. Where: N0 is the number of viable bacteria at 0h; Nt is the number of viable bacteria at t (t = 3)h.

[0034] The statistical results are shown in Table 1. After Lactobacillus reuteri RD003 was incubated in artificial gastric juice for 3 hours, its survival rate was 93.65%, and after entering intestinal juice for 3 hours, its survival rate was 89.49%.

[0035] Table 1 Statistics of survival rate of Lactobacillus reuteri RD003 in artificial gastric juice and artificial intestinal juice

[0036]

[0037] MRS liquid culture medium with pig bile salt concentrations of 0.1%, 0.2% and 0.3% was prepared, the pH of the culture medium was adjusted to 8.0 with 1.0 M sodium hydroxide solution, sterilized, and inoculated with the Lactobacillus reuteri RD003 to be tested at a 2% (v:v) inoculation amount after cooling, with 3 replicates per group, and cultured at 37°C for 0 h and 6 h. 1 ml of the culture solution cultured in each culture medium was taken out, diluted in multiple ratios and spread on plates, and live bacteria were counted. The average value of the three replicates was taken to calculate the bacterial survival rate.

[0038] Survival rate (%) = 100 × lgNt / lgN0. Where: N0 is the number of viable bacteria at 0h; Nt is the number of viable bacteria at t (t = 6)h.

[0039] The statistical results are shown in Table 2. When the bile salt concentration in the culture medium was 0.1%, the survival rate of Lactobacillus reuteri RD003 was 92.56%, and when the bile salt concentration in the culture medium was 0.3%, the survival rate was 47.78%.

[0040] The above results show that Lactobacillus reuteri RD003 has good tolerance to the simulated gastrointestinal environment.

[0041] Table 2 Statistics of survival rate of Lactobacillus reuteri RD003 in different concentrations of pig bile salt

[0042]

[0043] Example 3: Preparation of Lactobacillus reuteri RD003 bacterial agent

[0044] Lactobacillus reuteri RD003 fermentation broth: 2% (v:v) Lactobacillus reuteri RD003 seed liquid was inoculated into MRS liquid medium and cultured at 37°C for 24-36 hours. The fermentation broth was taken and the number of viable cells was 10 9-10 cfu / mL.

[0045] Lactobacillus reuteri RD003 powder: 2% (v:v) Lactobacillus reuteri RD003 seed liquid was inoculated into MRS liquid medium, fermented and cultured at 37°C for 24-48h, centrifuged at 4000rpm / min for 15min, and bacterial sludge was taken. A protective agent (a mixture of 10% skim milk and trehalose) was added according to 5 times the weight of the bacterial sludge. After being fully mixed, it was placed in a -80°C refrigerator overnight and vacuum freeze-dried to prepare bacterial powder. The number of viable cells was 10 11-12 cfu / g.

[0046] Example 4: Application of Lactobacillus reuteri RD003 strain on sika deer in the antler-growing stage

[0047] Experimental Materials and Methods

[0048] Ten 3-year-old sika deer in the antler-growing stage were randomly divided into two groups, with 5 deer in each group. They were fed the following diets: basal diet (control group), basal diet + bacterial agent (experimental group), Lactobacillus reuteri RD003 at 10 9 cfu / kg was added to the basal diet for 30 days.

[0049] The basic diet is made of corn flour, soybean meal, corn germ meal, wheat bran, and silage corn straw as the main raw materials, and nutritional additives consisting of minerals, vitamins, etc. are added. The composition of the basic diet is shown in Table 3, and the nutritional level is shown in Table 4.

[0050] Table 3 Basic diet composition

[0051]

[0052]

[0053] a The premix provides per kilogram of diet: MgO 0.083g; MnSO4·H2O 0.047g; FeSO4·H2O 0.058g; ZnSO4·H2O 0.039g; FeSO4·H2O 0.034g; Vitamin A 532.4IU; Vitamin D3 546.48IU; Vitamin E 0.911IU; Vitamin K3 0.253mg; Vitamin B1 0.101mg; Vitamin B2 0.759mg; Vitamin B 12 0.015mg; folic acid 0.025mg; niacin 1.78mg; calcium pantothenate 1.26mg; CaHPO4 5.68g; CaHPO4 5.02g.

[0054] Table 4 Nutritional levels of basic diets

[0055] Nutritional Information content% Dry matter 88.01 Crude Protein 19.89 Crude fat 3.92 Neutral detergent fiber 45.53 Acid detergent fiber 25.53 Total energy (MJ / kg) 18.15

[0056] Sample collection method:

[0057] On the 30th day of the experiment, each sika deer was anesthetized by injection of 0.6-1.3 mg / kg body weight of xylazine hydrochloride before morning feeding, 10 mL of blood was collected from the jugular vein in a coagulation tube, centrifuged at 4°C and 4000 r / min for 12 min, and serum was collected in a 2 mL centrifuge tube and stored in a -80°C refrigerator. Rumen fluid was collected in a 5 mL cryotube using a rumen fluid collector, quickly frozen in liquid nitrogen, and then stored in a -80°C refrigerator.

[0058] Sample determination method:

[0059] The levels of serum immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM) were determined using enzyme-linked immunosorbent assay kits produced by Shanghai ELISA Biotechnology Co., Ltd.

[0060] Rumen fluid genomic DNA extraction and PCR amplification: The genomic DNA of rumen fluid samples was extracted using the CTAB method, and then the concentration and purity of the DNA were detected by electrophoresis. The sample DNA was diluted to 1 ng / μL with sterile pure water. All PCR mixtures were added with 15μL Phussion⑥ High-Fidelity PCR Master Mix, 0.2μM primers and 10ng DNA template for amplification according to the set program. Mixing and purification of PCR products: Qualified PCR products were purified and quantified by magnetic beads, and the PCR products were tested by electrophoresis after mixing according to the quality of the PCR product concentration. The products of the target bands were recovered using a universal DNA purification and recovery kit. Library construction and sequencing: The library construction kit uses NEB Next⑥Ultra TM The library was constructed using ⅡFSDNA PCR-free LibraryPrep Kit, quantified by Qubit and Q-PCR, and finally sequenced by NovaSeq6000 using PE 250.

[0061] Data analysis methods:

[0062] The experimental data were statistically analyzed using the GLM program in SAS software, and multiple comparisons were performed using Ducan's method, where P < 0.01 was considered extremely significant, P < 0.05 was considered significant, and P > 0.05 was considered insignificant. The results were expressed as mean ± standard deviation.

[0063] Results and Conclusions:

[0064] As shown in Table 5, the serum IgA content in the experimental group was significantly higher than that in the control group (P < 0.05). IgA is the main antibody of mucosal immunity, and its main function is to eliminate pathogens with the assistance of nonspecific immune protection mechanisms. This shows that the addition of Lactobacillus reuteri RD003 can improve the immune level of sika deer in the antler-growing period.

[0065] Table 5 Effect of adding Lactobacillus reuteri RD003 on serum immunoglobulin levels in sika deer during antler growth period

[0066]

[0067] The results of principal coordinates analysis (PCoA) based on Bray-Curtis distance are as follows Figure 5 As shown in the figure, the addition of the two coordinate axes can explain 40.82% of the variable composition. The bacterial community composition of some samples in the two groups is similar, but there are differences. Alpha diversity can analyze the richness and diversity of the sample microbial community. Among them, the Chao1 index is used to estimate species richness, and the Shannon index is used to evaluate species diversity. It can be seen from Table 6 that the Chao1 and Shannon indices of the experimental group are significantly higher than those of the control group (P < 0.01), indicating that the addition of Lactobacillus reuteri RD003 can improve the species richness and diversity of rumen bacteria in sika deer during the antler growth period.

[0068] Table 6 Effect of adding Lactobacillus reuteri RD003 on the Alpha diversity index of rumen flora of sika deer during antler growth period

[0069]

[0070]

[0071] The results of the phylum abundance analysis of rumen fluid bacteria of sika deer are as follows Figure 6 As shown in the figure, compared with the control group, the relative abundance of Bacteroidota and Euryarchaeota in the experimental group was significantly increased (P < 0.05). Studies have shown that Bacteroidetes is the dominant flora in the rumen microorganisms of ruminants and an important flora for the utilization of feed carbohydrates and proteins. Euryarchaeota plays an important role in ruminant feed utilization and methane emissions. It can be seen that the addition of Lactobacillus reuteri RD003 has a positive effect on improving the rumen flora structure of sika deer.

[0072] The results of genus-level abundance analysis of rumen fluid bacteria in sika deer are as follows: Figure 7 As shown in the figure, compared with the control group, the relative abundance of Rikenellaceae RC9 gut group and Christensenellaceae R-7 group in the experimental group was significantly increased (P < 0.05). Studies have shown that Rikenellaceae RC9 gut group can decompose structural carbohydrates, and Christensenellaceae R-7 group is involved in amino acid and lipid metabolism. It can be seen that the addition of Lactobacillus reuteri RD003 can promote the digestion and absorption of nutrients in feed by increasing the relative abundance of beneficial bacteria in the rumen.

Claims

1. A Lactobacillus reuteri RD003, characterized in that It is deposited in the China Center for Type Culture Collection, the deposit date is June 9, 2022, and the deposit number is CCTCC NO: M 2022847.

2. A microbial preparation containing the Lactobacillus reuteri RD003 according to claim 1.

3. An animal feed additive or animal feed containing the Lactobacillus reuteri RD003 according to claim 1.

4. A medicine containing the Lactobacillus reuteri RD003 according to claim 1.

5. Use of the Lactobacillus reuteri RD003 according to claim 1, the microbial preparation according to claim 2, the animal feed additive or animal feed according to claim 3 or the drug according to claim 4 in improving the immunity of sika deer.

6. The use according to claim 5, characterized in that: To improve the immunity of sika deer, the content of immunoglobulin in serum is increased.

7. The use according to claim 6, characterized in that: The immunoglobulin is immunoglobulin A.

8. Use of the Lactobacillus reuteri RD003 of claim 1, the microbial preparation of claim 2, the animal feed additive or animal feed of claim 3, or the drug of claim 4 in increasing the microbial diversity in rumen fluid of sika deer.

9. The use according to claim 8, characterized in that: Microbial diversity refers to the increase in the relative abundance of Bacteroidota, Euryarchaeota, Rikenellaceae RC9gut group, and Christensenellaceae R-7group.

10. Use of the Lactobacillus reuteri RD003 of claim 1, the microbial preparation of claim 2, the animal feed additive or animal feed of claim 3, or the drug of claim 4 in improving the digestibility of nutrients or in resistance to artificial gastrointestinal fluid, intestinal fluid and pig bile salts.

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