Lactobacillus mucosus MX-2 and application thereof

Abstract

This invention discloses a strain of fermenting *Lactobacillus mucinus* (… Limosilactobacillus fermentum MX-2, with its accession number GDMCC No:64164 at the Guangdong Provincial Center for Microbial Culture Collection, effectively inhibits the growth of bacteria and fungi. It is sensitive to multiple antibiotics and has high safety. It exhibits high acid and bile salt resistance and high β-galactosidase production capacity, which can solve the problems of poor gastrointestinal tolerance, narrow antibacterial spectrum, and low lactase hydrolysis activity of existing strains. It improves the flavor of dairy products and helps eliminate lactose intolerance symptoms in humans. It provides high-quality strain resources and industrial application solutions for food biopreservation and the development of low-lactose functional foods.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology technology, specifically relating to a strain of fermenting *Lactobacillus mucilaginosus* (… Limosilactobacillus fermentum MX-2 and its applications. Background Technology

[0002] With the increasing nutritional needs of people, the intake of milk and dairy products has also increased, making lactose intolerance a relatively common health problem. Lactose intolerance can cause gastrointestinal discomfort such as bloating, abdominal pain, and diarrhea in the short term; long-term deliberate avoidance of dairy products can also lead to insufficient intake of calcium and high-quality protein, increasing the risk of osteoporosis and continuously disrupting the balance of the gut microbiota. Currently, the mainstream methods for alleviating lactose intolerance fall into three categories: first, lactose removal processing of dairy products, including the mature but costly and time-consuming β-galactosidase hydrolysis method, and ultrafiltration, which relies on the physical separation of lactose through double-layer membranes; second, oral lactase supplements, which need to be taken before meals, have limited duration of action, and vary in effectiveness from person to person; and third, lactase-producing lactic acid bacteria, which, when added to fermented foods, can hydrolyze lactose and alleviate intolerance symptoms. Therefore, developing foods and food supplements containing lactic acid bacteria can help alleviate the symptoms of lactose intolerance, which is one of the most promising application areas for lactic acid bacteria.

[0003] Lactic acid bacteria (LAB) is a collective term for a group of Gram-positive bacteria that utilize carbohydrates for fermentation while simultaneously producing large amounts of lactic acid. *Lactobacillus fermentum*, as an important category of lactobacilli, is one of the dominant strains in traditional fermented foods. It is widely found in various foods such as fermented foods, meat products, and dairy products, and is also commonly found in human saliva and the intestines of humans and animals.

[0004] β-galactosidase, also known as lactase, hydrolyzes lactose into glucose and galactose and is widely found in animals, plants, and microorganisms. Due to their rapid growth, high metabolic rate, and strong adaptability, microorganisms are the primary source of industrially produced enzyme preparations on a commercial and industrial scale. Summary of the Invention

[0005] This invention provides a strain of fermenting *Lactobacillus mucinus* (… Limosilactobacillus fermentum MX-2 was deposited on December 14, 2023, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 64146; deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] Another objective of this invention is to utilize the aforementioned fermented *Lactobacillus mucilaginosus* (… Limosilactobacillus fermentumMX-2 is used in the preparation of preparations to regulate human lactose intolerance. Fermented Lactobacillus MX-2 has acid and bile salt resistance, strong antibacterial activity, and high β-galactosidase production capacity. It is used to prepare fermented foods and food supplements containing fermented Lactobacillus MX-2 to help alleviate the symptoms of lactose intolerance.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: 1. Strains Isolation and Purification After crushing the collected Yunnan traditional sour rice noodles, they were inoculated into sterilized MRS liquid medium and cultured in a 37°C constant temperature shaker. Then, the cultured bacterial solution was serially diluted, and different concentrations of the diluted solution were evenly spread on MRS solid medium. Each gradient was performed in triplicate. The spread solid medium was placed in a 37°C constant temperature incubator for culture. Single colonies were picked and repeatedly spread on MRS agar plates for bacterial isolation and purification, resulting in 105 purified strains. Lactic acid bacteria producing protease were initially screened using the skim milk plate diffusion method, and then lactic acid bacteria producing β-galactosidase were screened again using the 5-bromo-4-chloro-3-indole-D-galactopyranoside (X-Gal) plate colorimetric method. Finally, the β-galactosidase activity was quantitatively detected using the o-nitro-β-D-galactopyranoside (ONPG) spectrophotometric method. Among them, strain MX-2 had the highest β-galactosidase activity. 2. Identification of strain MX-2 (1) Strain MX-2 colonies on MRS agar medium are round, raised, with neat edges, milky white in color, and Gram-positive. (2) Genomic DNA of strain MX-2 was extracted using the QIAamp genomic DNA and RNA kit. Using the extracted genomic DNA as a template, PCR amplification was performed using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3') for the 16S rRNA gene. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were compared with the GeMRSank results in the NCBI database using BLAST. Based on morphological characteristics and molecular identification results, strain MX-2 was finally identified as *Lactobacillus fermentatus* (a type of *Lactobacillus*). Limosilactobacillus fermentum ); 3. The relevant activities of strain MX-2 were detected by acid resistance test, bile salt resistance test, drug sensitivity test and antibacterial test. The experimental results showed that strain MX-2 has antibacterial activity and is resistant to acid and bile salts.

[0008] Advantages and technical effects of the present invention: This invention provides *Lactobacillus mucinus* MX-2 obtained from fermented foods, which can effectively inhibit the growth of bacteria and fungi, is sensitive to multiple antibiotics, and has high safety. It has high acid and bile salt resistance and high β-galactosidase production capacity, which can solve the problems of poor gastrointestinal tolerance, narrow antibacterial spectrum, and low lactose hydrolysis activity of existing strains. It can improve the flavor of dairy products and help eliminate lactose intolerance symptoms in humans. It provides high-quality strain resources and industrial application solutions for food biopreservation and the development of low-lactose functional foods. Attached Figure Description

[0009] Figure 1 This is a colony morphology diagram of *Lactobacillus mucinus* MX-2. Figure 2 Image showing Gram staining results; Figure 3 This is a phylogenetic tree. Detailed Implementation

[0010] The technical solution of the present invention will be further described in detail below through embodiments, but the content of the present invention is not limited thereto. Unless otherwise specified, the methods in this embodiment are conventional methods, and the materials and reagents used are obtained from commercial sources or prepared according to conventional methods unless otherwise specified. Example 1: Isolation, purification, and identification of Lactobacillus fermentum MX-2 1. Lactobacillus fermentum MX-2 was isolated from traditional Yunnan fermented rice noodles. The specific steps were as follows: 5g of the collected fermented rice noodles were weighed, crushed, and quickly inoculated into a sterilized 5mL MRS liquid culture medium test tube. The test tube was placed in a 37℃ constant temperature shaker for 24h. Then, the cultured bacterial solution was serially diluted, and 100μL of 10 mL of each solution was taken. 5 10 6 10 7 The diluted solution was evenly spread on MRS solid medium, and each gradient was performed in triplicate. The spread solid medium was placed in a 37°C incubator and incubated for 24 hours. Single colonies were picked and repeatedly spread on MRS agar plates for bacterial isolation and purification, resulting in 105 purified strains. 2. β-Galactosidase Activity Assay Lactic acid bacteria producing protease were initially screened using the skim milk plate diffusion method, followed by secondary screening using the 5-bromo-4-chloro-3-indole-D-galactopyranoside (X-Gal) plate colorimetric method for producing β-galactosidase. Finally, β-galactosidase activity was quantitatively detected using the o-nitro-β-D-galactopyranoside (ONPG) spectrophotometric method. The specific implementation is as follows: (1) Screening protease-producing lactic acid bacteria using the skim milk plate diffusion method One hundred and five frozen lactic acid bacteria strains were rejuvenated. 20 μL of the preserved bacterial culture was inoculated into 5 mL of sterile MRS liquid medium and incubated at 37°C for 24 h. After mixing, 20 μL of the culture was transferred to 5 mL of gelatin induction medium and induced at 37°C for 72 h. 1 mL of the induced bacterial culture was centrifuged at 8000 rpm for 5 min, and 100 μL of the supernatant was dropped onto 6.0 mm diameter skim milk agar plates and incubated at 37°C for 48 h. Lactic acid bacteria with large clear zones were screened and retained, with three replicates for each strain. Fifty-one protease-producing lactic acid bacteria strains were obtained through screening. (2) Screening of lactic acid bacteria producing β-galactosidase using the 5-bromo-4-chloro-3-indole-D-galactoside (X-Gal) plate colorimetric method. Prepare a 20 mg / mL X-Gal dimethylamide solution and store it at -20℃ in the dark. Inoculate 5 mL of the protease-producing lactic acid bacteria from step (1) into sterile MRS liquid medium and incubate at 37℃ for 48 h; take 50 μL of bacterial solution and spread it on an MRS agar plate, incubate at 37℃ for 24 h until a single colony grows, add 2 mL of X-Gal solution, and observe the colony color development after standing at 37℃ for 24 h. Pick the blue colonies and retain them. 26 strains of lactic acid bacteria with β-galactosidase activity were screened out. (3) β-galactosidase activity analysis Rejuvenate the X-Gal selected strains by centrifuging 500 μL of bacterial culture at 8000 r / min for 10 min at low temperature; wash the bacterial pellet 2-3 times with phosphate buffer, add 100 μL of 0.2% lysozyme and incubate at 37℃ for 1 h to break the cell walls; add 100 μL of 4 mol / L NaCl, mix well, and centrifuge again for 10 min; take 500 μL of supernatant, preheat at 37℃ for 5 min, add an equal volume of 20 mmol / L ONPG at 37℃, and react at 37℃ for 30 min; stop the reaction by adding 1.5 mL of 0.5 M Na2CO3, and measure the absorbance at 420 nm to calculate the enzyme activity; ONP standard curve: y = 0.0051x - 0.0429, R 2 =0.9968.

[0011] Enzyme activity unit (U / mL): At 37℃ and pH 6.8, 1 mL of enzyme solution hydrolyzing ONPG to generate 1 μmol of ONP per minute is defined as 1 enzyme activity unit; the results are shown in Table 1; the results show... L. fermentum The β-galactosidase activity of MX-2 was the highest, reaching 6.47 U / mL; Table 1 ; 3. Morphological identification Strain MX-2 was identified by catalase test, Gram staining, and microscopic examination. The results of Gram staining and microscopic examination are as follows: Figure 1 ,2 As shown, MX-2 is a Gram-positive bacterium; on MRS agar plates, the colonies are round, raised, with neat edges, and are milky white in color. The catalase test result is negative.

[0012] 4. Molecular biological identification Genomic DNA was extracted from the strain (DNA extraction was performed using the QIAamp genomic DNA and RNA kit). Using the extracted genomic DNA as a template, the 16S rRNA gene of strain MX-2 was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'). The reaction system consisted of 10 μL of 2×TaqPCR Master Mix, 0.5 μL each of forward and reverse primers, 1 μL of DNA template, and 8 μL of ddH2O. The reaction program was as follows: 95℃ for 5 min; 94℃ for 1 min, 55-58℃ for 1 min, 72℃ for 90 s, 30 cycles; 72℃ for 10 min. The PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for gene sequencing. The sequence is shown in SEQ ID NO: 1. The 16S rRNA gene was then... The rRNA gene sequence was BLAST-aligned using GeMRSank in the NCBI database. The bacterial standard strain with the highest similarity to the isolated strain was selected to determine the strain species. A phylogenetic tree was then constructed using MEGA 7.0 software. The phylogenetic tree for strain MX-2 is shown below. Figure 3 As shown; combining morphological characteristics and molecular identification results, strain MX-2 was ultimately identified as *Lactobacillus fermentans* (…). Limosilactobacillus fermentum ) Example 2: Acid Resistance Test Strain MX-2 was inoculated into MRS broth medium at an inoculum rate of 4‰. After two generations of activation at 37℃, the activated bacterial suspension (1.58 × 10⁻⁶) was inoculated into acidic MRS liquid medium at pH 6.2 ± 0.2 (blank control group) and pH 3, respectively. 9 (cfu / mL), incubated at 37℃ for 3 h, viable cell counts were performed at 0 and 3 h, and the survival rate of the strains was calculated according to the following formula: Strain survival rate / % = N1 / N0 × 100, where N1: number of viable bacteria after treatment; N0: initial number of viable bacteria.

[0013] The final concentration of strain MX-2 after 3 hours of acid treatment was 1.17 × 10⁻⁶. 9 The cfu / mL concentration yielded a survival rate of 74%.

[0014] Example 3: Bile Salt Tolerance Test After activating strain MX-2, the strain was inoculated into fresh MRS medium containing 0.3% and 0% (blank control group) bile salts at an inoculation rate of 4‰. After treatment at 37℃ for 0 and 3 h, viable cell counts were performed on plates at 0 and 3 h. The survival rate was calculated as follows: strain survival rate / % = N1 / N0 × 100, where N1: viable cell count after treatment; N0: initial viable cell count.

[0015] The results show L. fermentum The initial concentration of MX-2 was 1.58 × 10⁻⁶. 9 The final concentration of fermented *Lactobacillus mucinus* MX-2 after 3 hours of bile salt treatment was 1.16 × 10⁻⁶ cfu / mL. 9 The cfu / mL concentration yielded a survival rate of 73%.

[0016] Example 4: Drug Sensitivity Test The susceptibility of bacterial strains to drugs was determined using the disk diffusion method. The specific steps are as follows: Filter paper was cut into 5mm diameter circular pieces, sterilized at 121℃, and then dried in an oven. The filter paper pieces were then immersed in drug solutions of specific concentrations to prepare drug susceptibility test discs with standardized drug content, which were then dried for later use. The bacterial strain was activated twice with MRS liquid medium. When the viable count reached 1×10⁻⁶, the bacterial strain was activated. 8 When CFU / mL, take 1 mL of bacterial suspension, centrifuge to remove the supernatant, wash the bacterial suspension twice with 0.9% sterile physiological saline, resuspend the bacterial suspension, and take an appropriate amount of bacterial suspension to spread evenly on MRS solid medium. After the medium solidifies, place the self-made drug sensitivity test strips on it, with a spacing of not less than 24 mm and a distance of more than 15 mm from the inner edge of the plate. After placing them on the plate, quickly place it in a constant temperature incubator and incubate at 37℃ for 24 h. Then measure the diameter of the inhibition zone. Use the standard sensitive bacteria Staphylococcus aureus ATCC25923 (… Staphylococcus golden ATCC25923 was used as a control strain.

[0017] The results are shown in Table 2. L. fermentum MX-2 showed varying sensitivities to different antibiotics, being sensitive to ampicillin, penicillin, and kanamycin, but resistant to chloramphenicol, norfloxacin, and ofloxacin. Based on the results... L. fermentum MX-2 has a high safety profile and can be used in subsequent food development. Table 2 ;

[0018] Note: R: resistance; I: intermediate resistance; S: susceptibility.

[0019] Example 5: Antibacterial Test The antibacterial activity of strain MX-2 was determined using the Oxford cup method: Approximately 10 mL of MRS solid medium was poured evenly onto the bottom of an agar plate, allowed to dry, and then an Oxford cup was placed in the center of the plate. 10 mL of MRS solid medium was added to the inside of the Oxford cup. 8 CFU / mL of fermented Lactobacillus mucin MX-2 was added to the culture medium after it had cooled to 40°C, bringing the final concentration of the pathogen to 10. 6 CFU / mL. The pathogens were Staphylococcus aureus (on BHI solid medium, purchased from Guangdong Huankai Microbial Technology Co., Ltd.), Escherichia coli (on LB solid medium, purchased from Guangdong Huankai Microbial Technology Co., Ltd.), and Candida albicans (on Sabouraud dextrose agar, purchased from Guangdong Huankai Microbial Technology Co., Ltd.). After complete cooling and solidification, the cultures were incubated at 37°C for 24 hours. The diameter of the inhibition zone was measured. All experiments were repeated in triplicate.

[0020] For ease of comparison, in addition to the experimental strain L. fermentum In addition to MX-2, the following were also selected L. fermentum MX-5 L. fermentum MX-8 L. fermentum MX-9 L. plantarum YM5-3 and L. plantarum YM4-1 was used as a control experiment; antibacterial tests were conducted on Escherichia coli, Staphylococcus aureus, and Candida albicans, respectively; the results are shown in Table 3. Table 3 ; The results showed that strain MX-2 of this invention had the strongest antibacterial effect against Escherichia coli, and also had a high antibacterial effect against Staphylococcus aureus and Candida albicans.

[0021] The strain MX-2 of this invention was isolated from traditional Yunnan fermented rice noodles and identified through molecular biology as *Lactobacillus fermentans*. This strain is naturally safe and has clear traceability. It efficiently secretes β-galactosidase, which not only breaks down lactose and produces lactic acid, optimizing the flavor and texture of food, but also inhibits the growth of other bacteria through its metabolites, extending shelf life and enhancing nutritional value. Furthermore, it hydrolyzes large lactose molecules into glucose and galactose, significantly improving lactose intolerance in humans. Compared to existing commercially available lactobacilli, MX-2 combines high lactase production with broad-spectrum antibacterial properties, aligning with current trends in clean label and functional fermented foods, and possesses broad application prospects in the dairy processing and fermented food industries.

Claims

1. A strain of fermenting *Lactobacillus mucilaginosus* ( Limosilactobacillus fermentum MX-2, whose accession number at the Guangdong Provincial Center for Microbial Culture Collection is GDMCC No: 64164.

2. The use of the fermented Lactobacillus MX-2 of claim 1 in the preparation of a formulation for regulating human lactose intolerance.

3. The application according to claim 2, characterized in that: Fermented Lactobacillus mucinus MX-2 exhibits acid and bile salt resistance, strong antibacterial activity, and high β-galactosidase production capacity.

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