Lactobacillus rhamnosus that does not utilize lactulose but can utilize galactose and its application

Abstract

The invention discloses a rhamnosus lactobacillus that does not utilize lactulose but can utilize galactose and its application. The strain is rhamnosus lactobacillus grx21, and its preservation number is CGMCC No.26009. The rhamnosus lactobacillus grx21 of the invention can utilize galactose but cannot utilize lactulose, has a fast metabolic rate and a high survival rate, can be used to remove the byproduct galactose in the hot alkali synthesis lactulose system, is used to prepare high-purity lactulose suitable for use by people with galactosemia, and is used as a probiotic in food or medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a Lactobacillus rhamnosus which does not utilize lactulose but can utilize galactose and an application thereof. Background Art

[0002] Lactulose is also known as lactulose, isomerized lactose, and has a molecular formula of C 12 H 22 O 11 Lactulose cannot be hydrolyzed by human digestive enzymes and is not absorbed in the small intestine. After entering the colon, it is used as a carbon source for intestinal microorganisms, promoting the proliferation of bifidobacteria and lactobacilli. At the same time, it is metabolized to produce lactic acid and short-chain fatty acids, acidifying the intestinal environment, lowering the intestinal reduction potential, and inhibiting the growth of harmful bacteria such as Salmonella and Clostridium. The acidified colon environment will diffuse freely NH 3 Converted to NH 4+ , effectively treat hepatic encephalopathy caused by high blood ammonia. Therefore, lactulose is often used as a prebiotic in food and medicine.

[0003] However, lactulose does not exist in nature and is generally produced by isomerization of lactose using hot alkali catalysis. The traditional hot alkali method of converting lactose into lactulose is accompanied by the presence of a large amount of byproduct galactose, which cannot meet the needs of patients with galactosemia. Galactosemia patients have inherited carbohydrate metabolism disorders, and the intake of galactose can cause jaundice, kidney problems and even death. In industry, high-purity lactulose is generally obtained by using an activated carbon column with continuous ethanol elution to remove galactose or by continuously removing the byproduct galactose through an anion and cation exchange column, which is cumbersome and expensive.

[0004] Lactic acid bacteria are a type of non-spore, Gram-positive bacteria that ferment sugars to produce lactic acid. Some lactic acid bacteria, such as Streptococcus thermophilus, Lactobacillus casei, and Lactobacillus rhamnosus, can phosphorylate galactose to galactose-6-phosphate due to the presence of a galactose-specific phosphotransferase system, and transport it into the cell, where it metabolizes galactose through the EMP pathway and the tagatose-6-phosphate pathway. Literature 1 screened 7 strains with the ability to utilize galactose from 44 strains of Streptococcus thermophilus, but Streptococcus thermophilus has more stringent environmental requirements than Lactobacillus rhamnosus, grows slowly at low pH, and consumes incomplete galactose (Lactose / Galactose Metabolism of Lactic Acid Bacteria and Its Application, Zhang Susu, Shandong University, May 2020 Thesis). Literature 2 explored the utilization of galactose by Lactobacillus casei and found that the acid production of Lactobacillus casei was relatively simple (Towards galactose accumulation in dairy foods fermented by conventional starter cultures: Challenges and strategies Qinglong Wu Trends in Food Science & Technology Volume 41, Issue 1, January 2015, Pages 24-36). At present, there are few literature reports comparing galactose and lactulose in strain metabolism. By utilizing the metabolic specificity of lactic acid bacteria, the galactose metabolism in the hot alkaline catalyzed lactose system is completely used to prepare high-purity lactulose, and lactic acid bacteria that can metabolize galactose and do not utilize lactulose are screened. This is a green, low-cost, and high-purity lactulose preparation method suitable for galactosemia people.

[0005] At the same time, lactic acid bacteria have a good immunomodulatory effect. They stimulate the intestinal mucosa to promote the differentiation of lymphocytes into plasma cells to produce antibodies, thereby enhancing the body's immunity. Lactic acid bacteria have the function of improving intestinal function, mainly through metabolites such as lactic acid and organic acids, directly inhibiting or killing intestinal pathogens to regulate the balance of intestinal microecological flora. The lactic acid bacteria metabolism hot alkaline method is used to catalyze the byproduct galactose in the lactose system to produce organic acids and other beneficial biomass. Lactic acid bacteria and galactose work together to produce synbiotics rich in lactulose, lactic acid bacteria, and organic acids. Summary of the invention

[0006] The invention provides a Lactobacillus rhamnosus which does not utilize lactulose but can utilize galactose and application thereof.

[0007] The Lactose-independent Lactobacillus rhamnosus described in the present invention is grx21, which was deposited in the General Microbiology Center of the China Culture Collection Administration on November 2, 2022, with a deposit number of CGMCC No. 26009 and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The Lactobacillus rhamnosus grx21 of the present invention cannot utilize lactulose, but can utilize galactose. There is no significant difference between Lactobacillus rhamnosus grx21 in MRS medium with lactulose as the only carbon source and in sugar-free medium, and it hardly grows. After 24 hours of fermentation in MRS medium, OD 600 was 0.987, and was metabolized to produce lactic acid, acetic acid, and butyric acid; after fermentation for 24 h in MRS medium with galactose as the only carbon source, OD 600 It is 0.907, and is metabolized to produce lactic acid, acetic acid, propionic acid, and citric acid.

[0009] The present invention also provides the use of the above-mentioned rhamnosus lactobacillus grx21 in preparing high-purity lactulose. The specific method is: inoculating rhamnosus lactobacillus grx21 into a system for preparing lactulose by hot alkali catalysis of lactose, and separating lactulose after anaerobic fermentation for a period of time.

[0010] Furthermore, the present invention provides an application of Lactobacillus rhamnosus grx21 in the preparation of lactulose synbiotics. The specific method is: inoculating Lactobacillus rhamnosus grx21 into a system for preparing lactulose by hot alkali catalysis of lactose, and after anaerobic fermentation for a period of time, collecting the fermentation product to obtain the lactulose synbiotics.

[0011] The lactulose synbiotic of the present invention is a lactulose synbiotic containing Lactobacillus rhamnosus grx21, lactulose and short-chain fatty acids.

[0012] The short-chain fatty acids described in the present invention are lactic acid, acetic acid, propionic acid and gamma-aminobutyric acid.

[0013] The system for preparing lactulose from lactose by hot alkali catalysis of the present invention is a commercial lactulose preparation system, which is a chemical synthesis system. Reducing lactose is heat-treated under alkaline conditions so that the glucose on the lactose unit is directly isomerized into fructose, thereby producing lactulose.

[0014] Furthermore, the present invention provides the use of the Lactobacillus rhamnosus grx21 as a probiotic in food or medicine.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] (1) The Lactobacillus rhamnosus grx21 of the present invention metabolizes galactose quickly and can completely metabolize galactose within 6 hours after being inoculated into MRS galactose culture medium.

[0017] (2) The survival rate of the Lactobacillus rhamnosus grx21 of the present invention in artificial gastric juice with a pH of 3.0 and a 0.2% bile salt medium is greater than 100%. At the same time, in a system of lactose catalyzed by hot alkali to produce lactulose, under the condition of a carbon source of up to 200 g / L, the Lactobacillus rhamnosus grx21 can still metabolize galactose therein and has a high survival rate.

[0018] (3) The rhamnosus Lactobacillus grx21 of the present invention can utilize galactose but cannot utilize lactulose, and can be used to remove the byproduct galactose in the hot alkali synthesis lactulose system, and to prepare high-purity lactulose suitable for people with galactosemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The figure shows the utilization of lactulose (a) and galactose (b) by the 81 lactic acid bacteria strains in Example 1.

[0020] Figure 2 The metabolism of lactulose and galactose by strain grx21 in Example 1 is shown.

[0021] Figure 3 This is a colony morphology diagram of strain grx21 in Example 1 on MRS solid culture medium.

[0022] Figure 4 This is an optical microscope morphological diagram of the strain grx21 in Example 1.

[0023] Figure 5 The acid production of strain grx21 in Example 1 after fermentation in MRS medium and MRS-Gal medium for 24 hours respectively.

[0024] Figure 6 The fermentation results of galactose and lactulose in the hot alkali lactulose synthesis system by strain grx21 in Example 2 are shown.

[0025] Figure 7 This is the metabolic acid production of strain grx21 in Example 2 in the hot alkaline lactulose synthesis system. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with embodiments, drawings and tables.

[0027] Example 1

[0028] Screening of lactic acid bacteria that do not utilize lactulose but utilize galactose

[0029] The inventors isolated and purified Gram-positive strains from samples collected in the longevity area of ​​Bama, Guangxi, screened out the bacilli through microscopic examination, and used cell turbidity test to measure the absorbance values ​​(OD) at 600 nm in MRS medium with lactulose as the sole carbon source. 600-Lu ) and the absorbance at 600 nm (OD 600-Gal ) to screen strains that do not utilize lactulose but utilize galactose.

[0030] 1. Growth of lactic acid bacteria in MRS medium, lactulose and galactose MRS medium

[0031] MRS liquid culture medium: glucose 20.00 g, sodium acetate 5.00 g, potassium hydrogen phosphate 2.00 g, peptone 10.00 g, Tween-80 1.00 mL, beef extract 10.00 g, manganese sulfate tetrahydrate 0.05 g, yeast extract 5.00 g, diammonium citrate 2.00 g, magnesium sulfate heptahydrate 0.20 g, add distilled water to make up to 1000 mL, and sterilize at 121°C for 15 min.

[0032] MRS medium with lactulose as the sole carbon source (MRS-Lu): Replace the glucose in the MRS medium with lactulose, keep the rest unchanged, sterilize at 121°C for 15 min, and set aside.

[0033] MRS medium with galactose as the sole carbon source (MRS-Gal): Replace the glucose in the MRS medium with galactose, keep the rest unchanged, sterilize at 121°C for 15 min, and set aside.

[0034] Sugar-free culture medium: Remove the glucose from the MRS culture medium, keep the rest unchanged, sterilize at 121℃ for 15 min, and set aside.

[0035] The strain was inoculated into MRS-Lu medium and MRS-Gal medium at a 3% addition rate, respectively, and fermented in a 37°C incubator. Samples were taken at 0h and 24h, and the absorbance value (OD) of the fermentation broth at 600nm was measured by an enzyme marker. 600 ), the growth of lactic acid bacteria in MRS-Lu and MRS-Gal medium was recorded, and MRS medium was used as a positive control and sugar-free medium as a negative control.

[0036] like Figure 1 As shown in the figure, one of the 81 lactic acid bacteria strains had an OD of 600 Below 0.1, 8 strains had an OD value of 0.1 in MRS-Gal medium. 600 Above 0.8, screened in MRS-Lu medium OD 600 Below 0.1, in MRS-Gal medium OD 600The strain with an OD higher than 0.8 was numbered 1. The growth of strain 1 in glucose, lactulose, galactose, and sugar-free MRS medium was further studied. The results showed that strain 1 hardly grew in sugar-free MRS medium and MRS-Lu medium. 600-Lu The OD value was only 0.011, and the growth was better in MRS-Gal culture. 600 -Gal is 0.907.

[0037] 2. Evaluation of lactic acid bacteria's ability to ferment lactulose and galactose

[0038] After strain No. 1 was activated for two generations in MRS liquid culture medium, it was inoculated into MRS-Lu culture medium and MRS-Gal culture medium respectively and fermented in an incubator at 37°C. 1 ml of fermentation supernatant was taken at 0 h and 24 h, centrifuged at 8000 r / min for 2 min, and the supernatant was filtered through a 0.22 μm aqueous phase filter membrane for HPLC detection to analyze the content of lactulose and galactose in the supernatant.

[0039] like Figure 2 As shown, after strain 1 was fermented in MRS-Lu MRS medium for 24 hours, the lactulose content decreased from 20 g / L to 19.262 g / L; after fermented in MRS-Gal MRS medium for 24 hours, the galactose content decreased from 20 g / L to 0 g / L.

[0040] 3. Identification of lactic acid bacteria

[0041] 3.1 Morphological observation and Gram staining

[0042] The characteristics of the colonies on the MRS solid plate were observed with the naked eye, and a single colony was picked and placed on a glass slide. After Gram staining, the morphology of the strain was observed under a biological microscope.

[0043] like Figure 3 As shown in the figure, the colony is milky white, nearly round and smooth with neat edges. Figure 4 As shown, after Gram staining, the bacteria were Gram-positive, long rod-shaped, and had no spores.

[0044] 3.2 Identification of strain 16S rDNA

[0045] (1) Extracting the genome: After the strain is activated, take 1 ml of the bacterial solution and centrifuge it, and use a bacterial genomic DNA extraction kit to extract DNA;

[0046] (2) PCR amplification: The genomic DNA of the strain was used as a template for amplification. The PCR amplification program was as follows: 95°C, 5 min; 95°C, 30 s; 55°C, 15 s; 72°C, 2 min. After 30 cycles, the cells were extended at 72°C for 5 min.

[0047] The PCR product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing after electrophoresis detection. Sequencing results: the nucleotide length was about 1500 bp, and the sequence obtained by sequencing was compared with the GenBank database of NCBI for homology analysis. The 16S rDNA sequence had a homology of 97.39% with Lacticaseibacilus rhamnosus strain TMPC43372, so the strain of the present invention was named Lactobacillus rhamnosus grx21.

[0048] Table 1 Homology comparison results of lactic acid bacteria 16S rDNA gene sequences

[0049]

[0050] 4. Determination of galactose metabolites fermented by Lactobacillus rhamnosus grx21

[0051] After grx21 was activated for 2 generations in MRS medium, it was inoculated into MRS medium and MRS-Gal medium respectively and fermented in an incubator at 37°C. 1 ml of fermentation supernatant was taken at 0 h and 24 h, centrifuged at 8000 r / min for 2 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane and used for HPLC detection to analyze the organic acid content in the supernatant.

[0052] like Figure 5 As shown, Lactobacillus rhamnosus grx21 produced 0.14 g / L lactic acid, 2.10 g / L acetic acid, and 4.26 g / L butyric acid in MRS medium after fermentation for 24 hours; and metabolized to produce 0.12 g / L lactic acid, 2.14 g / L acetic acid, 4.96 g / L propionic acid, and 1.11 g / L citric acid in MRS-Gal medium after fermentation for 24 hours.

[0053] 5. Evaluation of the acid and bile resistance of Lactobacillus rhamnosus grx21

[0054] After Lactobacillus rhamnosus grx21 was activated for 2 generations in MRS liquid medium, it was inoculated into artificial gastric juice with a pH of 3.0 at a 3% inoculum. Mix well, culture at 37°C, take samples at 0 and 3h, dilute them serially with sterile water at a ratio of 1:10, culture them with MRS solid medium, count the live bacteria, and calculate the strain's tolerance to pH 3.0 gastric acid.

[0055]

[0056] After grx21 was activated for 2 generations in MRS liquid medium, it was inoculated into 0.2% bile salt medium at a 3% inoculum size. After culturing at 37°C for 3 h, it was cultured in MRS solid medium and the live bacteria were counted to calculate the strain's tolerance to 0.2% bile salt.

[0057]

[0058] The results showed that the survival rate of Lactobacillus rhamnosus grx21 in artificial gastric juice with a pH of 3.0 and cultured at 37°C for 3 hours was 152.19%±2, which was greater than 100%, and it had strong low pH tolerance; in 0.2% bile salt medium, cultured at 37°C for 3 hours, the survival rate of the strain was 108.34%±1, which was greater than 100%, and it had strong bile salt tolerance. In summary, the strain has high tolerance to the digestive tract, especially low pH and bile salt tolerance.

[0059] Example 2

[0060] 1. Lactobacillus rhamnosus grx21 can be used to prepare lactulose

[0061] In the early stage of the experiment, the inventor prepared lactulose by hot alkali catalysis of lactose at 70°C, pH 12, and reaction time of 50 minutes. 67g / L lactulose and 64g / L byproduct galactose were generated in the system. After inoculation with Lactobacillus rhamnosus grx21 for 24 hours of fermentation, the galactose content was 0.0g / L, while the lactulose content remained unchanged. In industry, high-purity lactulose is generally obtained by using an activated carbon column with ethanol continuous elution to remove the byproduct galactose or by continuous anion and cation exchange columns to remove galactose, which is expensive and cumbersome to operate. grx21 can be used for the preparation of lactulose and is suitable for use by people with galactosemia.

[0062] 2. Lactobacillus rhamnosus grx21 can be used for the preparation of lactulose synbiotics

[0063] Activate grx21 in MRS medium for 2 generations, transfer to 400mL MRS medium with 10% inoculation, ferment at 37℃ for 24h, centrifuge at 6000rpm for 15min, collect bacterial cells, wash three times with saline, transfer to hot alkaline catalysis lactose to prepare lactulose system, make the final concentration of bacteria to 10g / L, introduce nitrogen to maintain anaerobic environment, the dissolved oxygen rate in the tank is 2%, and start fermentation. During the fermentation process, the pH dropped, and 10mol / LNaOH was added by peristaltic pump to keep the pH constant at 7.5±0.1, and ferment at 37℃ and 100rpm for 24h. Figure 6 , 7 As shown, HPLC determined that galactose was completely fermented, lactulose had no significant change, and lactic acid, acetic acid, propionic acid, and γ-aminobutyric acid were metabolized to form lactic acid, acetic acid, propionic acid, and γ-aminobutyric acid, with contents of 4.08 (g / L), 0.69 (g / L), 14.21 (g / L), and 3.10 (g / L), respectively. This embodiment can obtain a lactulose synbiotic rich in rhamnosus Lactobacillus grx21, lactulose, and short-chain fatty acids.

[0064] 3. The strain of the present invention can be prepared into probiotic powder and applied to food and medicine

[0065] The lactic acid bacteria are frozen at -40°C to -60°C and freeze-dried to form a powder. The powder can be added to various beverages or foods as a supplement and can also be used in medicines.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )grx21, the deposit number is CGMCCNo.26009, the deposit date is November 2, 2022, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The use of Lactobacillus rhamnosus in the preparation of high-purity lactulose according to claim 1, characterized in that: The specific method is: inoculating Lactobacillus rhamnosus grx21 into a system for preparing lactulose by hot alkali catalysis of lactose, and obtaining lactulose after anaerobic fermentation for a period of time.

3. The use of Lactobacillus rhamnosus grx21 in the preparation of lactulose synbiotics according to claim 1, characterized in that: The specific method is as follows: Lactobacillus rhamnosus grx21 is inoculated into a system for preparing lactulose by hot alkali catalysis of lactose, and after anaerobic fermentation for a period of time, the fermentation product is collected to obtain a lactulose synbiotic.

4. The use according to claim 3, characterized in that: The lactulose synbiotic is a lactulose synbiotic containing rhamnosus lactobacillus grx21, lactulose and short-chain fatty acids.

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