Lactobacillus IMAU80584, fermentation agent and application thereof

Abstract

The invention provides lactobacillus IMAU80584, a fermentation agent and an application thereof, which belong to the field of microbial technology and are deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC1.2566. The lactobacillus IMAU80584 of the invention has excellent acid production characteristics and good acid tolerance, strong fermentation ability, and has a strong inhibitory effect on Escherichia coli. The lactobacillus IMAU80584 screened by the invention has a wide range of application prospects and huge economic value in the fields of preventing and treating pathogenic bacteria and fermented food as a probiotic.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a lactobacillus strain IMAU80584, a fermentation agent and applications thereof. Background Art

[0002] Probiotics, also known as microecological regulators and live bacteria preparations, refer to live microbial preparations that can promote the ecological balance of the host's intestinal flora and have a beneficial effect on the host. There are a large number of microorganisms in the human intestine, which are divided into beneficial bacteria, harmful bacteria and neutral bacteria. Under normal circumstances, the intestinal flora is in a balanced state, interdependent and mutually restricted, and coexists with the human body in a mutually beneficial way. Intestinal flora imbalance can lead to a series of biological dysfunctions such as diarrhea and infection.

[0003] Escherichia coli is a Gram-negative short rod. Under normal conditions, it is a normal flora of the intestines of humans and animals. It has flagella around its body, can move, and has no spores. However, a small number of special types of Escherichia coli have quite strong toxicity, such as enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), enteropathogenic Escherichia coli (EPEC), etc. Once infected, especially for infants and young livestock (poultry), it will cause severe diarrhea and sepsis, and even death. For example, when humans are infected with EHEC, they will have severe spasmodic abdominal pain and recurrent hemorrhagic diarrhea, accompanied by fever, vomiting, etc. In some seriously infected people, the kidneys are damaged and they are prone to acute renal failure or even death.

[0004] The acid-producing property of lactobacillus is an important property for screening starter cultures for fermented dairy products. Starters used for sour cream, fermented cream, fermented dairy products and similar products are required to have good acid-producing properties. However, most acidic products currently use thermophilic Streptococcus and Bulgarian Lactobacillus, but this composite strain is intolerant to gastric juice, intestinal juice and bile, and is difficult to colonize in the intestines. The survival rate is extremely low, and it is difficult to exert its probiotic effect. Therefore, screening lactobacilli with high bactericidal and acid-producing properties has very important research significance and value. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a Lactobacillus IMAU80584, which has excellent acid production characteristics and effectively inhibits pathogenic bacteria.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a lactobacillus IMAU80584, which is preserved in Guangdong Province Microbiological Culture Collection Center with a preservation number of GDMCC 1.2566.

[0008] Preferably, the growth characteristics of the Lactobacillus IMAU80584 are: growth temperature of 10-45° C., pH value of 3.5-9.5, and mass volume fraction of NaCl solution of 0-6%.

[0009] Preferably, the fermentation substrate of Lactobacillus IMAU80584 is one or more of L-arabinose, D-ribose, D-xylose, esculin ferric citrate, D-maltose, D-sucrose and D-melezitose.

[0010] Preferably, the enzymes expressed in the Lactobacillus IMAU80584 are leucine arylaminease, valine arylaminease, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-uronidase and α-glucosidase.

[0011] The invention provides a fermentation bacterial agent, comprising the above-mentioned Lactobacillus IMAU80584.

[0012] Preferably, the effective live bacterial concentration of Lactobacillus IMAU80584 is 1×10 6 ~×10 8 CFU / mL.

[0013] The present invention also provides a use of any one of the above-mentioned Lactobacillus IMAU80584 or any one of the above-mentioned fermentation bacteria agents in the preparation of fermented milk.

[0014] Preferably, the fermented milk includes fermented soy milk or cow's milk.

[0015] The present invention also provides the use of any one of the above-mentioned Lactobacillus IMAU80584 in the preparation of products for preventing and treating pathogenic bacteria.

[0016] Preferably, the pathogenic bacteria include Escherichia coli.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The lactobacillus IMAU80584 of the present invention is isolated from the traditional naturally fermented dairy product yogurt yak milk in Saichi Village, Sangke Township, Xiahe County, Gansu Province. Experiments have proved that the lactobacillus IMAU80584 of the present invention has excellent acid production characteristics and good acid tolerance, and has strong fermentation ability. The lactobacillus IMAU80584 has a strong inhibitory effect on Escherichia coli. The lactobacillus IMAU80584 screened by the present invention is a probiotic and has broad application prospects and huge economic value in the fields of preventing and treating pathogenic bacteria and fermented foods.

[0019] Collection information:

[0020] The Lactobacillus sp. IMAU80584 provided by the present invention is deposited in the Guangdong Provincial Microbiological Culture Collection Center (abbreviated as GDMCC), the storage address is: 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China, Postal Code 510070, Storage Number: GDMCC 1.2566, and the storage time is April 10, 2021. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The colony morphology of Lactobacillus sp. IMAU80584 in Example 1 is shown;

[0022] Figure 2 This is a microscopic examination image of Lactobacillus sp. IMAU80584 after Gram staining in Example 1;

[0023] Figure 3 The TEM electron microscope morphology of the Lactobacillus sp. IMAU80584 in Example 1;

[0024] Figure 4 The results of carbohydrate metabolism test of Lactobacillus sp. IMAU80584 API 50CHL in Example 1;

[0025] Figure 5 The results of the enzyme activity test of Lactobacillus sp. IMAU80584API-ZYM in Example 1;

[0026] Figure 6 The phylogenetic tree of Lactobacillus sp. IMAU80584 and all model strains of closely related species based on the sequences of two housekeeping genes (rpoA and pheS) in Example 1;

[0027] Figure 7 The phylogenetic tree of Lactobacillus sp. IMAU80584 and all model strains of closely related species in Example 1 based on the whole genome core gene;

[0028] Figure 8 The Oxford cup antibacterial effect of the mixed bacterial solution of Lactobacillus sp. IMAU80584 on Escherichia coli in Example 4;

[0029] Fig. 9This is the Oxford cup antibacterial effect of the sterile supernatant of Lactobacillus sp. IMAU80584 in Example 4 on Escherichia coli. DETAILED DESCRIPTION

[0030] The invention provides a lactobacillus IMAU80584, which is preserved in Guangdong Province Microbiological Culture Collection Center with a preservation number of GDMCC 1.2566.

[0031] In the present invention, the lactobacillus is classified and named as Lactobacillus sp. IMAU80584, and is preserved in the Guangdong Provincial Microbiological Culture Collection Center (abbreviated as GDMCC), the preservation address is: 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China, Postal Code 510070, Collection Number: GDMCC 1.2566, and the preservation time is April 10, 2021.

[0032] The Lactobacillus IMAU80584 provided by the present invention is derived from the traditional naturally fermented dairy product yogurt yak milk in Saichi Village, Sangke Township, Xiahe County, Gansu Province. The Lactobacillus IMAU80584 has the following microbiological characteristics:

[0033] (1) Colony characteristics: The diameter of a single colony is 1 to 2 mm. The color is beige and opaque. The colony surface is smooth and flat with slightly rough edges.

[0034] (2) Morphological characteristics: The bacteria are short rod-shaped, 1.67-1.91 μm long, 0.67-0.83 μm wide, arranged in a single rod, without flagella, non-motile, and Gram-positive;

[0035] (3) Growth characteristics: The minimum growth temperature of Lactobacillus IMAU80584 is 10°C, the maximum growth temperature is 45°C, and the optimum growth temperature is 30-37°C; the maximum growth pH is 9.5, and the minimum growth pH is 3.5; growth can be observed in 0-6% NaCl solution by mass volume fraction, but the best growth is in 0-5% NaCl solution by mass volume fraction.

[0036] In the present invention, the fermentation substrate of Lactobacillus IMAU80584 is preferably one or more of L-arabinose, D-ribose, D-xylose, esculin ferric citrate, D-maltose, D-sucrose and D-melezitose.

[0037] In the present invention, the enzymes expressed in the Lactobacillus IMAU80584 are preferably leucine arylaminease, valine arylaminease, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-uronidase and α-glucosidase.

[0038] The invention provides a fermentation bacterial agent, comprising the above-mentioned Lactobacillus IMAU80584.

[0039] In the present invention, the effective live bacterial concentration of Lactobacillus IMAU80584 is preferably 1×10 6 ~1×10 8 CFU / mL, more preferably 1×10 7 The fermentation agent of the present invention also includes auxiliary materials, the mass percentage of the auxiliary materials in the fermentation agent is 4-8%, and the auxiliary materials include one or more of European corn starch, sucrose, lactose, sodium glutamate, skimmed milk powder, water, glycerol, maltodextrin, soy protein isolate, and sodium ascorbate.

[0040] The present invention also provides a use of any one of the above-mentioned Lactobacillus IMAU80584 or any one of the above-mentioned fermentation bacteria agents in the preparation of fermented milk.

[0041] In the present invention, the fermented milk preferably includes fermented soy milk or cow's milk, and more preferably fermented soy milk. The Lactobacillus IMAU80584 of the present invention has efficient acid production ability and acid resistance, and has broad application prospects in the dairy industry.

[0042] The present invention also provides the use of any one of the above-mentioned Lactobacillus IMAU80584 in the preparation of products for preventing and treating pathogenic bacteria.

[0043] In the present invention, the pathogenic bacteria preferably include Escherichia coli. The Lactobacillus IMAU80584 of the present invention has a strong inhibitory effect on Escherichia coli, can improve the microecological environment in the body, and enhance immunity.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] Isolation and Identification of Lactobacillus sp. IMAU80584

[0047] 1. Isolation of Lactobacillus IMAU80584

[0048] Lactobacillus sp. IMAU80584 was isolated from the traditional naturally fermented dairy product yogurt yak milk in Saichi Village, Sangke Township, Xiahe County, Gansu Province. The specific isolation method is as follows:

[0049] The yak milk sample was diluted 10 times and 10 -5 , 10 -6 and 10 -7After anaerobic culture at 37℃ for 48h, colonies with different morphologies were selected for streaking purification, and single colonies were picked and inoculated into MRS broth medium, cultured at 37℃ for 24h, and all Gram-positive and catalase-negative strains were cultured for three generations, frozen with skim milk, and stored at -80℃, and finally Lactobacillus IMAU80584 was screened out.

[0050] 2. Identification of Lactobacillus IMAU80584 by colony characteristics, morphological characteristics, physiological and biochemical characteristics, and enzyme activity detection

[0051] Lactobacillus sp. IMAU80584 was inoculated into MRS broth liquid medium and cultured at 37°C for 24 h. After three generations of subculture, the strain activity was restored and the bacterial solution was diluted 10-fold to obtain 10 -1 ~10 -8 Dilution gradient, pipette 200 μL 10 -6 , 10 -7 , 10 -8 The gradient dilutions were evenly spread on MRS agar plates and cultured anaerobically at 37°C for 48 h.

[0052] The MRS culture medium is composed of the following weight components: peptone (10g), beef extract (10g), yeast powder (5g), glucose (20g), Tween-80 (1g), dipotassium hydrogen phosphate (2g), sodium acetate (5g), sodium citrate (2g), magnesium sulfate heptahydrate (200mg), and manganese sulfate pentahydrate (54mg); the above weight components are sterilized at 121°C for 15min to obtain the MRS culture medium.

[0053] 1) The colony morphology of Lactobacillus IMAU80584 on the culture dish, the size, color, transparency, colony surface state and colony edge state of the formed colonies were observed and recorded.

[0054] The results showed that the single colony obtained by Lactobacillus IMUA80584 had a diameter of 1-2 mm, was beige and opaque, had a smooth and flat surface, and had slightly rough edges (see Figure 1 ).

[0055] 2) Gram staining of Lactobacillus IMUA80584 was performed, the bacterial morphology was observed using a BX53 optical microscope, the negatively stained cell morphology of the strain was observed using a transmission electron microscope, the catalase activity was examined by observing the bubble production using a 3% (v / v) H2O2 solution, and the cell motility was tested in a semi-solid medium (0.3%), wherein the semi-solid medium was an MRS agar semi-solid medium.

[0056] The results showed that Lactobacillus IMUA80584 was Gram-positive, short rod-shaped, single-rod arranged, without flagella, and non-motile (see Figure 2 ); individual bacterial cells are rod-shaped, 1.67 to 1.91 μm long and 0.67 to 0.83 μm wide, arranged in a single rod (see Figure 3 ); Lactobacillus IMUA80584 did not produce bubbles, was hydrogen peroxide negative, and had no motility.

[0057] 3) Identification of physiological and biochemical characteristics:

[0058] ① Using the three-line method, the seed culture liquid of Lactobacillus IMAU80584 is streaked on MRS agar medium, cultured at 37° C. for 48 h, and prepared according to the instructions a cell inoculum in the suspension having the appropriate optical density required in the process; wherein the MRS agar medium (peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate [(NH4)2HC6H5O7] 2.0 g, glucose (C6H 12 O6·H2O) 20.0g, Tween 801.0mL, sodium acetate (CH3COONa·3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·7H2O) 0.50g, manganese sulfate (MnSO4·H2O) 0.25g, agar 15.0g, distilled water 1000mL, pH 6.2~6.6).

[0059] ② Inoculate the prepared cell inoculum into the biochemical identification tube. After inoculation, culture it aerobically at 37℃ for 24h and 48h, and record the reaction results respectively. Positive and negative tests are evaluated according to the color code indicated by the API system.

[0060] Table 1 Results of CHL carbohydrate metabolism test of Lactobacillus IMAU80584 and related Lactobacillus API50

[0061]

[0062]

[0063]

[0064] Note: 1. “-” means negative, “+” means positive; 2. A: Lactobacillus IMAU80584, B: Lentilactobacillusrapi DSM 19907T, C: Lentilactobacillus kisonensis DSM 19906T, D: Lentilactobacillus diolivorans DSM 14421T.

[0065] From Table 1 and Figure 4 It can be seen that Lactobacillus IMAU80584 can utilize L-arabinose, D-ribose, D-xylose, esculin ferric citrate, D-maltose, D-sucrose and D-melezitose as fermentation substrates.

[0066] 4) Lactobacillus IMAU80584 API-ZYM enzyme activity detection

[0067] A. Using the three-line method, the seed culture of Lactobacillus IMAU80584 was streaked on MRS agar medium and cultured at 37°C for 48h. According to the instructions, a cell inoculum in the suspension of the raw material with the appropriate optical density required in the process was prepared. The MRS agar medium (peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, diammonium hydrogen citrate [(NH4)2HC6H5O7] 2.0g, glucose (C6H 12 O6·H2O) 20.0g, Tween 801.0mL, sodium acetate (CH3COONa·3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·7H2O) 0.50g, manganese sulfate (MnSO4·H2O) 0.25g, agar 15.0g, distilled water 1000mL, pH 6.2~6.6).

[0068] B. Inoculate the prepared cell inoculum into the biochemical identification tube. After inoculation, culture the test strip aerobically at 37°C for 4 hours (the test strip should not be placed under strong light during the culture process). Add one drop of ZYMA and one drop of ZYMB. Wait for 5 minutes and observe the color change of the test strip. Positive and negative tests are evaluated according to the color code indicated by the API system.

[0069] The results are shown in Table 2 and Figure 5 It can be seen that Lactobacillus IMAU80584 can express leucine arylaminase, valine arylaminase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-uronidase and α-glucosidase.

[0070] Table 2 Biochemical reaction results of Lactobacillus IMAU80584 and related Lactobacillus API20 ZYM

[0071]

[0072] Note: 1. "-" means negative, "+" means positive. 2. A: Lactobacillus IMAU80584, B: Lentilactobacillus rapi DSM 19907T, C: Lentilactobacillus kisonensis DSM 19906T, D: Lentilactobacillus diolivorans DSM 14421T.

[0073] 3. Identification of 16S rRNA of Lactobacillus IMAU80584

[0074] First, a single clone of Lactobacillus IMAU80584 was picked and inoculated into MRS broth medium and cultured in a constant temperature anaerobic incubator at 37°C for 24 h. The bacterial sludge was collected when the strain grew to the late exponential phase. The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit according to the manufacturer's instructions. The universal bacterial 16S rRNA primers 27F: 5′-AGAGTTTGACCTGGCTAG-3′ (SEQ ID NO.1) and 1495R: 5′-CTACGGCTCCTTGTTCGA-3′ (SEQ ID NO.2) were used to perform colony PCR amplification, and the DNA quality was detected by gel electrophoresis (0.1% w / v agarose gel). The concentration was determined by spectrophotometry, and the qualified PCR products were sent to Shanghai Sunny Biotechnology Co., Ltd. for 16S rRNA sequencing.

[0075] The 16S rRNA sequence of Lactobacillus IMAU80584 was compared with the 16S rRNA sequences of all determined bacteria in the database at NCBI. The 16S rRNA sequence of the IMAU80584 strain was most homologous to that of Lentilactobacillusrapi DSM 19907T of the genus Lentilactobacillus, and less homologous to other strains, and the strain was determined to be of the genus Lentilactobacillus.

[0076] Then, the sequences of two housekeeping genes, pheS and rpoA, were obtained from the genome sequence (encoding phenylalanyl-tRNA synthase α subunit and RNA polymerase α subunit, respectively), and the phylogenetic tree of the two housekeeping genes pheS and rpoA of Lactobacillus IMAU80584 and all model strains of closely related species was constructed using the neighbor-joining method and the maximum likelihood method (see Figure 6 ), and the analysis was performed using MEGA 7.0 software based on 1000 replicates.

[0077] In summary, based on the 16S rRNA and housekeeping gene sequence analysis and the construction of the phylogenetic tree, strain IMAU80584 was a suspected new species in the genus Lentilactobacillus.

[0078] 4. Whole genome sequencing and identification of Lactobacillus IMAU80584

[0079] Firstly, Lactobacillus IMAU80584 was inoculated into MRS broth medium and cultured at 37°C for 24 h. After three generations of subculture to obtain a pure culture of the strain, it was sent to Beijing Novogene Technology Co., Ltd. for whole genome sequencing. The genome sequencing platform was IlluminaNovaseq6000, and the Illumina PE library was constructed. The genome sequencing data of the obtained strain was quality evaluated. After filtering, the software package SOAPdenovo (v2.04) was used to splice and assemble high-quality reads, and appropriate kmer values ​​were selected for splicing and assembly. The filtered data were single-base corrected, and the assembly results were evaluated using genome size, number of scaffolds, N50 length, N90 length and GC content as indicators.

[0080] Then, a phylogenetic tree was constructed by combining the 88 core genes of all model strains of the genus Lentilactobacillus published by NCBI, and it was found that Lactobacillus sp. IMAU80584 and Lentilactobacillusrapi DSM 19907 T Clustered together, but independently formed a sub-branch ( Figure 7 ), indicating that strain IMAU80584 is a new species in the genus Lentilactobacillus.

[0081] The results showed that Lactobacillus IMAU80584 and Lentilactobacillus rapi DSM 19907 T The ANI (average nucleotide identity) and GGDC (genomic distance) values ​​of the genus were 93.14 and 52.8, respectively, both below the threshold for defining a species; Figure 7 It is known that Lactobacillus IMAU80584 and Lentilactobacillus rapi DSM 19907 T The strains clustered together but formed a subclade independently, indicating that strain IMAU80584 was a new species within the genus Lentilactobacillus.

[0082] Example 2

[0083] Detection of Growth Characteristics of Lactobacillus IMAU80584

[0084] Lactobacillus IMAU80584 was inoculated into sterile MRS broth medium and cultured anaerobically at 37°C for 24 hours before being taken out for use.

[0085] 1. pH tolerance test

[0086] The seed culture of Lactobacillus IMAU80584 was inoculated into MRS broth medium with pH values ​​of 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5 and 9.5 at an inoculation rate of 2%, and cultured anaerobically at 37°C for 24 h. The absorbance values ​​(OD600) at 0 h and 24 h were measured, and the growth of the strain was judged based on the increase in the absorbance value.

[0087] Table 3 pH tolerance of Lactobacillus IMAU80584

[0088]

[0089] Note: -: not growing; + growing; w- weak

[0090] The results in Table 3 show that Lactobacillus IMAU80584 grew better when cultured at pH 4.5, 5.5, 6.5, 7.5, and 8.5, grew slowly when cultured at pH 3.5 and 9.5, and did not grow at pH 2.5.

[0091] 2. NaCl tolerance test

[0092] The seed culture of Lactobacillus IMAU80584 was inoculated into MRS broth medium with NaCl concentrations of 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% at an inoculation rate of 2%, and cultured anaerobically at 37°C for 24h. The absorbance (OD600) at 0h and 24h was measured, and the growth of the strain was judged based on the increase in the absorbance.

[0093] Table 4 NaCl tolerance of Lactobacillus IMAU80584

[0094]

[0095] Note: -: not growing; + growing; w- weak

[0096] The results in Table 4 show that Lactobacillus IMAU80584 grows weakly in MRS broth medium containing 6% NaCl, and hardly grows in MRS broth medium containing more than 6% NaCl.

[0097] 3. Temperature tolerance test

[0098] The seed culture solution of Lactobacillus IMAU80584 was inoculated into MRS broth medium at an inoculum volume of 2%, and cultured anaerobically for 24 h at 4°C, 10°C, 20°C, 30°C, 37°C and 45°C. The absorbance values ​​(OD600) at 0 h and 24 h were measured, and the growth of the strain was judged according to the change in the absorbance value.

[0099] Table 5 Temperature tolerance of Lactobacillus IMAU80584

[0100]

[0101] Note: -: not growing; + growing; w- weak

[0102] The results in Table 5 show that Lactobacillus IMAU80584 does not grow when cultured at 4°C, but grows at 10-45°C.

[0103] Example 3

[0104] Determination of fermentation capacity of Lactobacillus IMAU80584

[0105] 1. Soymilk fermentation

[0106] 1) Preparation method of soybean milk fermentation liquid

[0107] Use soybean powder with a protein content of 42% to prepare a soymilk fermentation liquid with a protein content of 5%, add 6% sucrose, and choose 100mL conical flasks to evenly package the soymilk fermentation liquid, with 40mL in each conical flask. After sealing, sterilize at 121℃ for 15min, rapidly cool to 40℃, and set aside.

[0108] 2) Method for fermenting soy milk using Lactobacillus IMAU80584

[0109] The seed culture of Lactobacillus IMAU80584 was cultured overnight in MRS broth medium, inoculated into the prepared soymilk fermentation broth at an inoculum amount of 8% of the volume of the soymilk, and fermented at 37° C. for 24 hours.

[0110] 3) Evaluation of the acid production capacity of soymilk fermented by Lactobacillus IMAU80584

[0111] After inoculation, the pH values ​​of soy milk at different fermentation times were measured at 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h.

[0112] The results showed that the pH value of fermented soy milk was 6.4 at the beginning of fermentation. As the fermentation time increased, the pH value of fermented soy milk gradually decreased. When the fermentation time was 15 hours, the pH value of fermented soy milk reached the lowest pH of 4.7 and then tended to stabilize.

[0113] 2. Milk fermentation

[0114] 1) Preparation method of milk fermented liquid

[0115] Use skim milk powder to prepare 89% milk fermentation liquid, add 6% sucrose, choose 100mL conical flasks to evenly package the milk fermentation liquid, 40mL in each conical flask, seal it, sterilize it at 121℃ for 15min, quickly cool it to 40℃, and set it aside.

[0116] 2) Method for fermenting milk with Lactobacillus IMAU80584

[0117] The seed culture of Lactobacillus IMAU80584 was cultured overnight in MRS broth medium, inoculated into the prepared milk fermentation liquid at an inoculum amount of 8% of the volume of milk, and fermented at 37° C. for 24 hours.

[0118] 3) Evaluation of the acid production capacity of Lactobacillus IMAU80584 fermented milk

[0119] After inoculation, the pH value of milk at different fermentation times was measured at 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h.

[0120] The results showed that the pH value of the fermented milk was 6.1 at the beginning of fermentation. As the fermentation time increased, the pH value of the fermented milk gradually decreased. When the fermentation time was 15 hours, the pH value of the fermented milk reached the lowest pH of 4.6 and then tended to stabilize.

[0121] Example 4

[0122] Antibacterial ability test of Lactobacillus IMAU80584

[0123] Escherichia coli was selected as the pathogen indicator bacteria.

[0124] The experimental method is carried out in accordance with the national standard GB / T 39101-2020. The specific operations are as follows:

[0125] 1) Take the seed liquid of Lactobacillus IMAU80584 and inoculate it into liquid culture medium at 2% inoculation volume. After culturing at 37°C for 24 hours, take 500 μl of bacterial liquid, centrifuge and take the supernatant. Filter the supernatant with a 0.22 μm microporous filter membrane, and mix the remaining bacterial precipitate with the same volume of sterile physiological saline for later use.

[0126] 2) Determination of the antibacterial activity of Lactobacillus IMAU80584 by Oxford cup antibacterial method

[0127] Inoculate the indicator bacteria into a test tube containing 5.0 mL of NB medium and subculture for three generations at 37°C as the culture medium. Heat and dissolve the prepared NA (nutrient agar) medium, cool it to 45°C to 50°C, and dilute the prepared indicator bacteria suspension to 10 -4 Gradient, add 7% addition to NA culture medium, mix thoroughly, pour into a sterile plate, gently shake the plate to make it evenly flat, wait for it to solidify and set aside, place 3 Oxford cups at an equal distance on the test plate containing indicator bacteria, measure 100 μL of Lactobacillus IMAU80584 mixed bacterial solution and sterile supernatant, slowly add it to the Oxford cup, repeat each treatment 3 times, and one plate has 3 parallels.

[0128] 4. Place the plate with the mixed bacterial solution in a 37°C constant temperature incubator and incubate upright until the inhibition zone is clear. Use an inhibition zone measuring instrument to measure the diameter of the inhibition zone. Measure each inhibition zone three times in different directions and record the average value.

[0129] 5. The plate with sterile supernatant added was moved to a 4°C refrigerator for pre-diffusion for 8 hours. After taking out the plate, it was placed in a 37°C constant temperature incubator and cultured upright until the inhibition zone was clear. The diameter of the inhibition zone was measured using an inhibition zone measuring instrument. Each inhibition zone was measured 3 times in different directions and the average value was recorded. The results are shown in Table 6.

[0130] Table 6 Antibacterial results of Lactobacillus IMAU80584

[0131] sample Inhibition zone size Original solution 11~12mm Supernatant 14~15mm

[0132] from Figure 8 , Fig. 9 It can be seen from the antibacterial results in Table 6 that the Lactobacillus IMAU80584 strain of the present invention has a relatively obvious antibacterial effect on Escherichia coli. Therefore, the Lactobacillus IMAU80584 strain of the present invention has a good development potential in the preparation of products for preventing and treating pathogenic bacteria.

[0133] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. Sequence Listing <110> Inner Mongolia Agricultural University <120> Lactobacillus IMAU80584, fermentation agent and application thereof <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 agagtttgac ctggctag 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 ctacggctcc ttgttcga 18

Claims

1. A Lactobacillus ( Lactobacillus sp. ) Use of IMAU80584 in the preparation of a product for preventing and treating Escherichia coli, characterized in that: The Lactobacillus ( Lactobacillus sp. )IMAU80584 is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC 1.2566.

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