WO2023240936A1 - 一种延长寿命、抗衰老、减脂的罗伊氏乳杆菌及其产品与应用 - Google Patents

一种延长寿命、抗衰老、减脂的罗伊氏乳杆菌及其产品与应用 Download PDF

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WO2023240936A1
WO2023240936A1 PCT/CN2022/135531 CN2022135531W WO2023240936A1 WO 2023240936 A1 WO2023240936 A1 WO 2023240936A1 CN 2022135531 W CN2022135531 W CN 2022135531W WO 2023240936 A1 WO2023240936 A1 WO 2023240936A1
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lactobacillus reuteri
product
food
present
nematodes
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PCT/CN2022/135531
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English (en)
French (fr)
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罗卫飞
蒙丽丽
柴智慧
黄连飞
卢恩秋
肖桂龙
庞世福
甘彩玉
韦新利
刘力源
林艳金
余秀奇
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广西爱生生命科技有限公司
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Priority to PCT/CN2022/135531 priority Critical patent/WO2023240936A1/zh
Priority to CN202280067122.5A priority patent/CN118019840A/zh
Publication of WO2023240936A1 publication Critical patent/WO2023240936A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3571—Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the invention belongs to the technical field of screening and application of microorganisms, and specifically relates to Lactobacillus reuteri that prolongs lifespan, resists aging, and reduces fat, and its products and applications.
  • Lactobacillus reuteri is a lactic acid bacterium that has been reported to exist naturally in the intestines of almost all vertebrates and mammals and has certain broad-spectrum antibacterial properties.
  • the Ministry of Health of China approved Lactobacillus reuteri as a microbial strain for human health care products, and the bacterium has been recognized internationally as a new type of probiotic lactic acid bacteria and has high theoretical research and production application value.
  • the present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes Lactobacillus reuteri that prolongs lifespan, resists aging, and reduces fat, as well as its products and applications.
  • the present invention successfully isolated a strain of Lactobacillus reuteri A21041, and found that this strain has a better life-extending effect than other known Lactobacillus reuteri, and , it can also have both gastrointestinal colonization effect and fat reduction effect, and has extremely high development and practical value, providing new directions and ideas for the development and application of life-extending, anti-aging, and fat-reducing products.
  • the first aspect of the present invention provides a strain of Lactobacillus reuteri, which is Lactobacillus reuteri A21041 and is taxonomically named Lactobacillus reuteri. It was deposited in Guangdong City on September 23, 2022.
  • Microbial Culture Collection Center (Guangdong Microbial Culture Collection Center, GDMCC) (the collection address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong province, China), the collection number is: GDMCC No: 62832.
  • the Lactobacillus reuteri A21041 is isolated from fecal samples of centenarians in Hepu County, Beihai City, Guangxi Zhuang Autonomous Region, China.
  • the colony shape of Lactobacillus reuteri A21041 is round, convex, smooth, white or milky white colonies with neat edges.
  • the 16S sequence of Lactobacillus reuteri A21041 is shown in SEQ ID NO: 5.
  • the complete gene information of Lactobacillus reuteri A21041 is shown in Table 2.
  • the screening and isolation method of Lactobacillus reuteri A21041 is: taking fecal samples from centenarians in Hepu County, Beihai City, Guangxi Zhuang Autonomous Region, China, into MRS solid culture medium, and under anaerobic conditions After culturing for 0.2 to 3 days, pick out the colonies in the medium that are round, convex, smooth, and white or milky white with neat edges. After PCR amplification and identification, Lactobacillus reuteri A21041 can be obtained.
  • the culture time is 15h to 24h.
  • the culture temperature of Lactobacillus reuteri A21041 is 30°C to 37°C, and the rotation speed is 150rpm to 220rpm.
  • the PCR amplification identification uses Lactobacillus reuteri-specific amplification primers.
  • the Lactobacillus reuteri-specific amplification primers are as shown in SEQ ID NO: 1 and 2.
  • the amplification system for PCR amplification identification is:
  • the amplification program of the PCR amplification identification is: 95°C for 3 minutes; 95°C for 40 seconds, 56°C for 30 seconds, 72°C for 1 minute, 30 cycles; and 72°C for 8 minutes.
  • a second aspect of the present invention provides products containing Lactobacillus reuteri according to the first aspect of the present invention, and the products include food, food additives, feed, feed additives, medicines and cosmetics.
  • the Lactobacillus reuteri is Lactobacillus reuteri A21041.
  • the mass fraction of Lactobacillus reuteri in the product is greater than or equal to 5%.
  • the mass fraction of Lactobacillus reuteri in the product is greater than or equal to 8%.
  • the mass fraction of Lactobacillus reuteri in the product is greater than or equal to 10%.
  • the mass fraction of Lactobacillus reuteri in the product is 5% to 15%.
  • the product also contains other auxiliary materials.
  • the auxiliary materials include pharmaceutically acceptable auxiliary materials, food additives, and cosmetic auxiliary materials.
  • the pharmaceutically acceptable auxiliary agents include but are not limited to diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbing agents (such as calcium sulfate, calcium hydrogen phosphate etc.), wetting agents (such as ethanol), binders (such as hypromellose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, crospovidone, etc.), lubricants ( Such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), etc. Osmotic regulators (such as glucose, sodium chloride, etc.), chelating agents (such as disodium EDTA).
  • diluents such as starch, dextrin, sucrose, lactose,
  • the food additives include, but are not limited to, colorants, enzyme preparations, thickeners, leavening agents, and sweeteners.
  • the cosmetic auxiliary materials include but are not limited to flavors, pigments, preservatives, and antioxidants.
  • the form of Lactobacillus reuteri in the product includes freeze-dried powder, bacterial liquid, and granular inoculant.
  • freeze-dried powder bacterial liquid
  • granular inoculant inoculation.
  • the term "lyophilized powder” refers to using the vacuum freeze-drying method of a freeze dryer to freeze the moisture in the medicinal solution in advance, and then sublimating the frozen moisture in the medicinal solution in a vacuum sterile environment, thereby A freeze-dried product was obtained.
  • the term "granular inoculant” refers to the inoculant used in order to prevent the powdered inoculant from directly contacting the bactericide or chemical fertilizer during use, resulting in a reduction in the effect. It usually refers to the combination of bacterial liquid and granular inoculant.
  • the type of inoculant obtained after mixing carriers such as biochar, vermiculite, etc.).
  • the effective bacterial activity of Lactobacillus reuteri in the product is 10 7 cfu/mL to 10 9 cfu/mL.
  • the third aspect of the present invention provides a method for preparing the product described in the second aspect of the present invention, which includes the following steps: after activating and propagating Lactobacillus reuteri, it is processed into a bacterial agent, and then mixed with other auxiliary materials to obtain .
  • the Lactobacillus reuteri is Lactobacillus reuteri A21041.
  • the activation and propagation conditions of Lactobacillus reuteri are: culture time is 15h-24h, culture temperature is 30°C-37°C, and rotation speed is 150rpm-220rpm.
  • the bacterial agent includes but is not limited to freeze-dried powder, bacterial liquid, and granular inoculant agent.
  • the processing technology of the bacterial agent is carried out according to conventional methods in the art.
  • the freeze-dried powder is prepared by freeze-drying Lactobacillus reuteri A21041 bacterial liquid.
  • the auxiliary materials include pharmaceutically acceptable auxiliary materials, food additives, and cosmetic auxiliary materials.
  • the fourth aspect of the present invention provides a food additive containing Lactobacillus reuteri according to the first aspect of the present invention.
  • the food additive includes Lactobacillus reuteri A21041 freeze-dried powder, plant extract, and starch hydrolyzate. Polysaccharides and dietary fiber.
  • the plant extracts include but are not limited to plant essential oils, saponins, alkaloids, polysaccharides, polyphenols, and flavonoids.
  • the plant extract is tea polyphenols and bayberry anthocyanin extract.
  • the starch hydrolyzable polysaccharide includes but is not limited to glycogen, inulin, cellulose, hemicellulose, pectic substances, peptidoglycan, and maltodextrin.
  • the starch hydrolyzable polysaccharide is maltodextrin.
  • the dietary fiber includes soluble dietary fiber and insoluble dietary fiber.
  • the dietary fiber is soluble dietary fiber.
  • the food additive in terms of mass percentage, includes 5-15% Lactobacillus reuteri A21041 freeze-dried powder, 15-45% plant extract, 40-60% starch hydrolyzed polysaccharide and 5 ⁇ 15% dietary fiber.
  • the food additives include 5 to 15% Lactobacillus reuteri A21041 freeze-dried powder, 5 to 15% tea polyphenols, and 10 to 30% bayberry anthocyanin extract. Material, 40-60% maltodextrin and 5-15% soluble dietary fiber.
  • the food additives include 8 to 12% Lactobacillus reuteri A21041 freeze-dried powder, 10 to 15% tea polyphenols, and 10 to 15% bayberry anthocyanin extract. material, 45-55% maltodextrin and 8-12% soluble dietary fiber.
  • the food additives include 10% Lactobacillus reuteri A21041 freeze-dried powder, 15% tea polyphenols, 15% bayberry anthocyanin extract, 50% malt paste Essence and 10% soluble dietary fiber.
  • the food additive is prepared by thoroughly mixing Lactobacillus reuteri A21041 freeze-dried powder, plant extracts, starch hydrolyzable polysaccharides and dietary fiber.
  • the fifth aspect of the present invention provides the use of the Lactobacillus reuteri described in the first aspect of the present invention in the preparation of food, food additives, feed, feed additives, medicines and cosmetics.
  • the food, food additives, feed, feed additives, medicines and cosmetics have at least one of the following functions (1) to (4):
  • a nematode (Caenorhabditis elegans) screening platform is used to verify the relevant effects.
  • Caenorhabditis elegans has a simple structure, a transparent body, is easy to observe, has a short development cycle, is easy to cultivate artificially, and its signaling pathways are highly conserved. This makes the Caenorhabditis elegans model effect verification more accurate and the experimental cost low.
  • Lactobacillus reuteri A21041 has excellent life-extending, anti-aging, weight loss and lipid-lowering effects at the same time, and based on Lactobacillus reuteri A21041 can survive in the simulated gastrointestinal tract environment. Therefore, it can be shown that it has the potential to colonize the gastrointestinal tract of the body and exert its life-extending, anti-aging, weight loss and lipid-lowering effects stably and long-term.
  • a sixth aspect of the present invention provides a method for extending lifespan and anti-aging, which includes the following steps: administering an effective amount of Lactobacillus reuteri A21041 to a desired subject.
  • the effective amount (for humans) is 0.02-0.04g/kg body weight.
  • the administration is oral.
  • the administration frequency is once every 1 to 24 hours.
  • the administration frequency is: every 1 hour, every 2 hours, every 4 hours, every 6 hours, every 8 hours, every 12 hours or every 24 hours.
  • a seventh aspect of the present invention provides a method for reducing fat, comprising the following steps: administering an effective amount of Lactobacillus reuteri A21041 to a desired subject.
  • the effective amount (for humans) is 0.02-0.04g/kg body weight.
  • the administration is oral.
  • the administration frequency is once every 1 to 24 hours.
  • the administration frequency is: every 1 hour, every 2 hours, every 4 hours, every 6 hours, every 8 hours, every 12 hours or every 24 hours.
  • the present invention discovered for the first time a strain of Lactobacillus reuteri that has excellent resistance to artificial gastrointestinal fluids and bile salts, and has excellent life-extending, anti-aging, weight loss and lipid-lowering effects, so that it can be based on this strain Effective development of related products has extremely high practical value.
  • the product of the present invention contains Lactobacillus reuteri A21041, which can effectively colonize in the gastrointestinal tract of the body and exert life-extending, anti-aging, weight loss and lipid-lowering effects stably and for a long time.
  • Figure 1 is a specific PCR electrophoresis diagram of Lactobacillus reuteri A21041 in Example 1;
  • Figure 2 is a comparative diagram of the effects of Lactobacillus reuteri A21041 and the control group OP50 on the life span of nematodes in Example 4;
  • Figure 3 is a comparative diagram of the effects of Lactobacillus reuteri A21041 and the control group OP50 on nematode aging in Example 5;
  • Figure 4 is a comparison diagram of nematode fat staining on the 7th day when Lactobacillus reuteri A21041 and the control group OP50 were stained in Example 6;
  • Figure 5 is a comparison diagram of nematode fat staining on the 5th day when Lactobacillus reuteri A21041 and the control group OP50 were stained in Example 7;
  • Figure 6 is a diagram showing the effect of Lactobacillus reuteri A21041 on nematode life span and fat metabolism gene expression levels in Example 7, and a diagram of the related mechanism pathways;
  • Figure 7 is a comparison chart of the effects of commercial Lactobacillus reuteri 17938 and the control group OP50 on the life span of nematodes in Comparative Example 1;
  • Figure 8 is a comparison chart of nematode fat staining between the commercial Lactobacillus reuteri 17938 and the control group OP50 in Comparative Example 2;
  • Figure 9 is a comparison chart of the effects of OP50+Metformin (10mM) and the control group OP50 on the life span of nematodes in Comparative Example 3;
  • Figure 10 is a comparison diagram of the colony morphology of Lactobacillus reuteri A21041-B and Lactobacillus reuteri A21041 in Comparative Example 4;
  • Figure 11 is a comparison chart of nematode fat staining between Lactobacillus reuteri A21041-B in Comparative Example 4 and the control group OP50.
  • the experimental materials and reagents used are all conventional consumables and reagents available from commercial sources.
  • the MRS solid medium formula used is: peptone 10g/L, beef extract powder 5g/L, yeast extract powder 4g/L, glucose 20g/L, dipotassium hydrogen phosphate 2g/L, triscitrate Ammonium 2g/L, sodium acetate 5g/L, magnesium sulfate 0.2g/L, manganese sulfate 0.05g/L, agar 15g/L, Tween-80 1g/L.
  • add 1L deionized water adjust the pH value to 6.2 ⁇ 0.2, and sterilize at 121°C for 15 minutes to obtain MRS solid culture medium.
  • Lactobacillus reuteri A21041 isolation and purification steps of Lactobacillus reuteri A21041 in the embodiments of the present invention are specifically as follows:
  • PCR amplification solution (2 ⁇ Taq PCR StarMix with Loading Dye, purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.) to the cultured PCR tube, and add Lactobacillus reuteri-specific amplification primers to perform PCR amplification (The amplification system is shown in Table 1).
  • Lactobacillus reuteri-specific amplification primers are:
  • Upstream primer F 5’-GCTTCACTCGCTGCAGTTAA-3’ (SEQ ID NO: 1);
  • Downstream primer R 5’-CCAACCAAACCTCGGTCAGA-3’ (SEQ ID NO: 2).
  • the amplification program was: 95°C for 3 min; 95°C for 40 s, 56°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 8 min.
  • nucleotide sequence of the 16S primer used is:
  • Upstream primer 16S-F 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO: 3);
  • Downstream primer 16S-R 5’-TACGGCTACCTTGTTACGACTT-3’ (SEQ ID NO: 4).
  • the confirmed culture solution was purified by plate streaking twice, and the purified single colony was picked and cultured repeatedly to obtain a bacterial liquid.
  • the bacterial liquid and 30% glycerol preservation solution were mixed at a volume ratio of 1:1 to freeze the strain. tube and stored in -80°C refrigerator.
  • the Lactobacillus reuteri (named Lactobacillus reuteri A21041) obtained in the above example was sent to the Guangdong Provincial Microbial Culture Collection Center (GDMCC) for preservation.
  • GDMCC Guangdong Provincial Microbial Culture Collection Center
  • Its taxonomic name is: Lactobacillus reuteri
  • the preservation date is: On September 23, 2022, the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong province, China, and the deposit number is: GDMCC No: 62832.
  • the survival rate of Lactobacillus reuteri A21041 in artificial gastrointestinal fluid and bovine bile salt solution was calculated based on the number of single colonies in the above experiments.
  • the survival rates of Lactobacillus reuteri A21041 after treatment with 0.3g/L and 0.5g/L bovine bile salt solutions for 24 hours were 260% and 80% respectively. It can be seen that Lactobacillus reuteri A21041 in the embodiment of the present invention has the potential to colonize the human gastrointestinal tract for a long time.
  • Lactobacillus reuteri A21041 In order to ensure the safety of Lactobacillus reuteri A21041 in various uses, the safety evaluation of the strain was carried out in this example, and the testing institution was the Microbial Analysis and Testing Center of Guangdongzhou, China. Referring to the determination criteria in Appendix A of China's Technical Guidelines for Safety Inspection and Evaluation of Bacteria for Raw Materials in Health Foods (2020 Edition), Lactobacillus reuteri A21041 has no adverse effects on the general health of mice; body weight and other indicators are compared with those of the control group The difference is not statistically significant; therefore, under the conditions of the examples of the present invention, the sample is not pathogenic.
  • Lactobacillus reuteri A21041 was cultured in MRS liquid medium, and Escherichia coli OP50 was cultured in LB liquid medium.
  • the culture conditions were: temperature 37°C, time 20h, rotation speed 200rpm.
  • Lactobacillus reuteri A21041 and Escherichia coli OP50 bacterial liquid both bacterial liquid concentrations are 10 7 CFU/mL were plated on nematode growth medium (NGM) and cultured overnight.
  • the NGM culture medium formula is: 3g sodium chloride, 17g agar powder, 2.5g peptone, 975mL deionized water. After the above components are fully mixed, seal it with tin foil. After high-pressure steam sterilization for 20 minutes, the water bath was cooled to 55°C. Add the following sterile solutions under sterile conditions: 1 mL of 1 M CaCl 2 , 1 mL of 1 M MgSO 4 , 25 mL of 1 M KPO 4 buffer, 1 mL of 5 mg/mL cholesterol (dissolved in 95% ethanol).
  • the thawed nematodes (Caenorhabditis elegans) were centrifuged and added to the NGM medium containing OP50, and placed in a 20°C incubator for 3 days.
  • M9 buffer to suspend the cultured nematodes in step (1), suck them into the culture tube, add lysis solution (NaOH with a mass ratio of 5N and a sodium hypochlorite solution with a volume ratio of 5%) to each tube, lyse for 6 minutes, and centrifuge at 3500r/min. 1 min, discard the supernatant, and wash 4 times with M9 buffer. Centrifuge again and discard the supernatant. Transfer the precipitate to NGM medium and culture it overnight at a constant temperature of 20°C to obtain L1-stage nematode larvae.
  • lysis solution NaOH with a mass ratio of 5N and a sodium hypochlorite solution with a volume ratio of 5%
  • the obtained synchronized nematodes were randomly divided into two groups (control group (OP50) and experimental group (A21041)), with 150 worms in each group.
  • the day when L4 stage nematodes were picked was recorded as day 0.
  • the NGM medium containing OP50 and A21041 was changed every 2 days and the life span of the nematodes was counted until all nematodes died. Abnormally dead nematodes are not included in the statistics.
  • GraphPad Prism 5 was used for statistical analysis, and one-way analysis of variance was used to compare the significance of differences between multiple groups; the t test method was used to analyze the differences between two groups. P ⁇ 0.05 means there is a significant difference, P ⁇ 0.01 means there is a very significant difference.
  • Lactobacillus reuteri A21041, Escherichia coli OP50 and Caenorhabditis elegans were cultured according to the method in the above example, and the nematodes were synchronized.
  • the synchronized nematodes were randomly divided into two groups (control group (OP50) and experimental group (A21041)), with 150 worms in each group.
  • the day when L4 stage nematodes were picked was recorded as day 0.
  • the nematodes were replaced with NGM medium containing OP50 and A21041 every 2 days.
  • the frequency of head shaking of the nematodes within 30 s is counted one by one on the NGM plate containing M9 buffer.
  • the swing of the nematode's head from one side to the other and back to the original position was counted as one swing.
  • Lactobacillus reuteri A21041 can significantly increase the frequency of head swinging of nematodes and increase the vitality of nematodes. Therefore, Lactobacillus reuteri Lactobacillus A21041 can significantly delay nematode aging.
  • Lactobacillus reuteri A21041, Escherichia coli OP50 and Caenorhabditis elegans were cultured according to the method in the above example, and the nematodes were synchronized.
  • 100 synchronized nematodes were transferred to plates coated with OP50 and A21041 for culture. They were cultured for 7 days and the plates were changed every other day. After washing three times with chilled M9 buffer on day 7, the nematodes were resuspended in 4% paraformaldehyde. Shake slightly at room temperature for 1 hour, centrifuge at 3000-4000 rpm for 1 minute, remove the supernatant and wash twice with M9 buffer. Then resuspend the nematodes in a mixed PBS solution containing 60% isopropyl alcohol and 0.01% Triton agent and incubate on a shaker at 25°C for 1 to 2 hours. Remove the dye and wash twice with M9 buffer.
  • Example 7 Fluorescence quantitative PCR determination of the effects of Lactobacillus reuteri A21041 on life span and fat metabolism gene loci
  • Lactobacillus reuteri A21041, Escherichia coli OP50 and Caenorhabditis elegans were cultured according to the method in the above example, and the nematodes were synchronized.
  • RNA samples from each group were taken for reverse transcription PCR amplification according to the instructions of the reverse transcription kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.). Store the PCR amplification products at -20°C until use. Use the primer pairs of each gene shown in Table 3 to perform conventional fluorescence quantitative PCR, and use 2- ⁇ Ct for data statistics.
  • Lactobacillus reuteri A21041 strain is fermented and multiplied, and then the bacterial liquid after fermentation and multiplication is centrifuged and freeze-dried to obtain Lactobacillus reuteri A21041 freeze-dried powder. Then compound according to the following formula to obtain a food additive or dietary supplement containing Lactobacillus reuteri A21041.
  • the formula of food additives or dietary supplements containing Lactobacillus reuteri A21041 is: 10% Lactobacillus reuteri A21041 freeze-dried powder, 15% tea polyphenols (purchased from Shanenkang Biotechnology Co., Ltd.), 15% bayberry anthocyanin extract (purchased from Shanenkang Biotechnology Co., Ltd.), 50% maltodextrin and 10% soluble dietary fiber (purchased from Shanenkang Biotechnology Co., Ltd.) Technology Co., Ltd).
  • the preparation method is as follows: thoroughly mixing the above raw materials.
  • the source and culture method of the commercial Lactobacillus reuteri 17938 strain are the same as the above-mentioned comparative example 1, and the nematode fat particle test method is the same as the above-mentioned example.
  • Metformin a drug for treating type 2 diabetes, has the effect of prolonging the life span of nematodes, and therefore, was used as a positive drug in this comparative example.
  • Lactobacillus reuteri A21041, Escherichia coli OP50 and Caenorhabditis elegans were cultured according to the method in the above example, and the nematodes were synchronized.
  • the synchronized nematodes were randomly divided into two groups (control group (OP50) and positive group (OP50+Metformin (final concentration: 10mM))), with 150 nematodes in each group. The day when L4 stage nematodes were picked was recorded as day 0. During the experiment, The nematodes were replaced with NGM medium containing OP50 every 2 days and the lifespan was counted until all nematodes died. Abnormally dead nematodes were not included in the statistics. Statistical analysis was performed using GraphPad Prism 5, and one-way analysis of variance was used to compare the differences between multiple groups. Significance of difference; use t test method to analyze the difference between the two groups. P ⁇ 0.05 means there is a significant difference, and P ⁇ 0.01 means there is an extremely significant difference.
  • Example 1 From the sample in Example 1, a strain of Lactobacillus reuteri A21041-B was also isolated. Observed under a microscope, the colony morphology of Lactobacillus reuteri A21041-B and Lactobacillus reuteri A21041 were different. A21041 The edge of the colony of -B is thinner and the overall colony is more transparent; while the edge of the colony of A21041 is thicker, as shown in Figure 9. In addition, the 16S sequenced nucleotide sequence of A21041-B is the same as SEQ ID NO:5, indicating that it is also a strain of Lactobacillus reuteri.
  • A21041-B was subjected to the same separation and purification, artificial gastrointestinal fluid resistance, artificial bile salt resistance and fat particle tests.
  • Lactobacillus reuteri A21041 has excellent life-extending, anti-aging, weight loss and lipid-lowering effects at the same time, and is based on Lactobacillus reuteri A21041 can survive in the simulated gastrointestinal tract environment. Therefore, it can be shown that it has the potential to colonize the gastrointestinal tract of the body and exert its life-extending, anti-aging, weight loss, and lipid-lowering effects stably and long-term.

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Abstract

本发明公开了一种延长寿命、抗衰老、减脂的罗伊氏乳杆菌及其产品与应用，该罗伊氏乳杆菌为罗伊氏乳杆菌A21041，分类学命名为Lactobacillus reuteri，于2022年9月23日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC No:62832。该罗伊氏乳杆菌具有优异的耐人工胃肠液、耐胆盐效果，且兼具优异的延长寿命、抗衰老、减肥降脂功效的罗伊氏乳杆菌，从而能够基于该菌株有效开发相关产品，具有极高的实用价值。

Description

一种延长寿命、抗衰老、减脂的罗伊氏乳杆菌及其产品与应用 技术领域
本发明属于微生物的筛选及应用技术领域，具体涉及一种延长寿命、抗衰老、减脂的罗伊氏乳杆菌及其产品与应用。
背景技术
根据相关研究调查数据显示，截止至2017年底，中国60岁及以上老年人口有2.4亿人，占总人口的17.3％，2017年新增老年人口超1000万人，中国社会正在逐渐步入老龄化阶段，给国家经济、医疗系统带来了一定的压力与挑战。而随着人体衰老的发生，机体内会伴随许多的疾病产生，如高血压等。高血压的主要原因是机体肥胖。世界卫生组织己经宣布肥胖是首要的健康问题之一，中国《2014年国民体质监测公报》显示，在2014年，老年人的超重率已经超过了40％。而且随着经济的增长和生活水平的提升，肥胖症的患病率也在不断上升，使其逐渐成为发生率持续升高的一类严重危害人体健康的疾病。目前，肥胖症的治疗仍是一项重大的难题，世界范围内急需一种更高效、安全的肥胖症治疗手段。
补充益生菌是目前众多科研工作者青睐的肥胖治疗思路。相关技术表明，益生菌可有效促进人体营养物质的消化吸收及代谢、提高机体抗氧化水平、提高机体免疫力等。罗伊氏乳杆菌(Lactobacillus reuteri)是目前已报道的几乎天然存在于所有脊椎动物和哺乳动物肠道内的乳酸菌，具有一定的广谱抗菌性。中国卫生部于2003年批准了罗伊氏乳杆菌可作为人类保健品的微生物菌种，且该菌已是国际上公认的新型益生乳酸菌，具有很高的理论研究和生产应用价值。但对于利用罗伊氏乳杆菌开发延长寿命、抗衰老、减脂产品或在相关方面的应用却鲜有报道。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种延长寿命、抗衰老、减脂的罗伊氏乳杆菌及其产品与应用。本发明基于中国广西壮族自治区百岁老人粪便样本，成功分离得到了一株罗伊氏乳杆菌A21041，并发现该菌株相对于其他已知的罗伊氏乳杆菌具有更好的寿命延长效果，而且，其还能够兼具胃肠道定殖效果和减脂效果，具有极高的开发价值和实用价值，为延长寿命、抗衰老、减脂产品的开发与应用提供了新的方向和思路。
本发明的第一个方面，提供一株罗伊氏乳杆菌，所述罗伊氏乳杆菌为罗伊氏乳杆菌A21041，分类学命名为Lactobacillus reuteri，于2022年9月23日保藏于广东省微生物菌种保藏中心(Guangdong Microbial Culture Collection Center，GDMCC)(保藏地址为：中国广东省广州市先烈中路100号大院59号楼5楼)，保藏编号为：GDMCC No:62832。
在本发明的一些实施方式中，所述罗伊氏乳杆菌A21041分离自中国广西壮族自治区北海市合浦县百岁老人的粪便样本。
在本发明的一些实施方式中，所述罗伊氏乳杆菌A21041的菌落形态为圆形、凸起、光滑、边缘整齐的白色或乳白色的菌落。
在本发明的一些实施方式中，所述罗伊氏乳杆菌A21041的16S序列如SEQ ID NO:5所示。
在本发明的一些实施方式中，所述罗伊氏乳杆菌A21041的全基因信息如表2所示。
在本发明的一些实施方式中，所述罗伊氏乳杆菌A21041的筛选分离方法为：取中国广西壮族自治区北海市合浦县百岁老人的粪便样本至MRS固体培养基中，在厌氧条件下培养0.2～3天，挑取培养基中菌落形态为圆形、凸起、光滑、边缘整齐的白色或乳白色的菌落，经PCR扩增鉴定后，即得罗伊氏乳杆菌A21041。
在本发明的一些实施方式中，所述培养时间为15h～24h。
在本发明的一些实施方式中，所述罗伊氏乳杆菌A21041的培养温度为30℃～37℃，转速150rpm～220rpm。
在本发明的一些实施方式中，所述PCR扩增鉴定使用罗伊氏乳杆菌特异性扩增引物。
在本发明的一些实施方式中，所述罗伊氏乳杆菌特异性扩增引物如SEQ ID NO:1和2所示。
在本发明的一些实施方式中，所述PCR扩增鉴定的扩增体系为：
组分 含量
10μM上游引物F 1μL
10μM下游引物R 1μL
PCR扩增液 12.5μL
DNA模板 1μL
ddH 2O 补至25μL
在本发明的一些实施方式中，所述PCR扩增鉴定的扩增程序为：95℃3min；95℃40s，56℃30s，72℃1min，循环30次；72℃8min。
当然，本领域技术人员也可以根据实际使用需求，合理调整PCR扩增体系中的含量与浓度，以及扩增程序，从而实现鉴定。
本发明的第二个方面，提供含有本发明第一个方面所述罗伊氏乳杆菌的产品，所述产品包括食品、食品添加剂、饲料、饲料添加剂、药品和化妆品。
在本发明的一些实施方式中，所述罗伊氏乳杆菌为罗伊氏乳杆菌A21041。
在本发明的一些实施方式中，所述产品中的罗伊氏乳杆菌的质量分数大于等于5％。
在本发明的一些实施方式中，所述产品中的罗伊氏乳杆菌的质量分数大于等于8％。
在本发明的一些实施方式中，所述产品中的罗伊氏乳杆菌的质量分数大于等于10％。
在本发明的一些实施方式中，所述产品中的罗伊氏乳杆菌的质量分数为5％～15％。
在本发明的一些实施方式中，所述产品中还含有其他辅料。
在本发明的一些实施方式中，所述辅料包括药学上可接受的辅剂、食品添加剂、化妆品辅料。
在本发明的一些实施方式中，所述药学上可接受的辅剂包括但不限于稀释剂(如淀粉、糊精、蔗糖、乳糖、甘露醇等)、吸收剂(如硫酸钙、磷酸氢钙等)、润湿剂(如乙醇)、粘合剂(如羟丙甲纤维素、聚维酮等)、崩解剂(如羟甲基淀粉钠、交联聚维酮等)、润滑剂(如滑石粉、氢化植物油、聚乙二醇等)、着色剂(如二氧化钛、亚甲蓝等)、包衣材料、溶剂、pH调节剂、抗菌剂(如亚硫酸钠、硫代硫酸钠等)、等渗调节剂(如葡萄糖、氯化钠 等)、螯合剂(如EDTA二钠)。
在本发明的一些实施方式中，所述食品添加剂包括但不限于着色剂、酶制剂、增稠剂、膨松剂、甜味剂。
在本发明的一些实施方式中，所述化妆品辅料包括但不限于香精、色素、防腐剂、抗氧化剂。
在本发明的一些实施方式中，所述产品中的罗伊氏乳杆菌的形式包括冻干粉、菌液、颗粒接种菌剂。当然，需要理解的是，本领域技术人员也可以根据实际使用需求和现有的处理工艺，合理选择适当的罗伊氏乳杆菌形式用于使用，包括但不限于上述的冻干粉、菌液、颗粒接种菌剂。
在本发明中，术语“冻干粉”是指采用冷冻干燥机的真空冷冻干燥法预先将药液里面的水分冻结，然后在真空无菌的环境下将药液里面被冻结的水分升华，从而得到冷冻干燥而成的制品。
在本发明中，术语“颗粒接种菌剂”是指为了防止粉状菌剂在使用时直接与杀菌剂或化学化肥接触，导致效果降低而采用的菌剂，通常是指将菌液与颗粒状载体(如生物炭、蛭石等)混合后得到的菌剂类型。
在本发明的一些实施方式中，所述产品中的罗伊氏乳杆菌的有效菌活为10 7cfu/mL～10 9cfu/mL。
当然，本领域技术人员可以根据所述产品中的罗伊氏乳杆菌的实际形式合理调整其活性，从而实现稳定的技术效果。
本发明的第三个方面，提供本发明第二个方面所述产品的制备方法，包括如下步骤：将罗伊氏乳杆菌活化扩繁后，加工为菌剂，然后与其他辅料混合，即得。
在本发明的一些实施方式中，所述罗伊氏乳杆菌为罗伊氏乳杆菌A21041。
在本发明的一些实施方式中，所述罗伊氏乳杆菌的活化扩繁条件为：培养时间为15h～24h，培养温度为30℃～37℃，转速150rpm～220rpm。
在本发明的一些实施方式中，所述菌剂包括但不限于冻干粉、菌液、颗粒接种菌剂。
在本发明的一些实施方式中，所述菌剂的加工工艺按照本领域中的常规方法进行。示例性地，冻干粉的制备为：将罗伊氏乳杆菌A21041菌液进行冷冻干燥，即得。
在本发明的一些实施方式中，所述辅料包括药学上可接受的辅剂、食品添加剂、化妆品辅料。
本发明的第四个方面，提供一种含有本发明第一个方面所述罗伊氏乳杆菌的食品添加剂，所述食品添加剂包括罗伊氏乳杆菌A21041冻干粉、植物提取物、淀粉水解多糖和膳食纤维。
在本发明的一些实施方式中，所述植物提取物包括但不限于植物精油、皂苷、生物碱、多糖、多酚、黄酮类化合物。
在本发明的一些实施方式中，所述植物提取物为茶多酚和杨梅花青素提取物。
在本发明的一些实施方式中，所述淀粉水解多糖包括但不限于糖原、菊糖、纤维素、半纤维素、果胶物质、肽聚糖、麦芽糊精。
在本发明的一些实施方式中，所述淀粉水解多糖为麦芽糊精。
在本发明的一些实施方式中，所述膳食纤维包括可溶性膳食纤维和不可溶性膳食纤维。
在本发明的一些实施方式中，所述膳食纤维为可溶性膳食纤维。
在本发明的一些实施方式中，按照质量百分比计，所述食品添加剂包括5～15％罗伊氏乳杆菌A21041冻干粉、15～45％植物提取物、40～60％淀粉水解多糖和5～15％膳食纤维。
在本发明的一些实施方式中，按照质量百分比计，所述食品添加剂包括5～15％罗伊氏乳杆菌A21041冻干粉、5～15％茶多酚、10～30％杨梅花青素提取物、40～60％麦芽糊精和5～15％可溶性膳食纤维。
在本发明的一些实施方式中，按照质量百分比计，所述食品添加剂包括8～12％罗伊氏乳杆菌A21041冻干粉、10～15％茶多酚、10～15％杨梅花青素提取物、45～55％麦芽糊精和8～12％可溶性膳食纤维。
在本发明的一些实施方式中，按照质量百分比计，所述食品添加剂包括10％罗伊氏乳杆菌A21041冻干粉、15％茶多酚、15％杨梅花青素提取物、50％麦芽糊精和10％可溶性膳食纤维。
在本发明的一些实施方式中，所述食品添加剂的制备方法为：将罗伊氏乳杆菌A21041冻干粉、植物提取物、淀粉水解多糖和膳食纤维充分混合后即得。
本发明的第五个方面，提供本发明第一个方面所述罗伊氏乳杆菌在制备食品、食品添加剂、饲料、饲料添加剂、药品和化妆品中的用途。
在本发明的一些实施方式中，所述食品、食品添加剂、饲料、饲料添加剂、药品和化妆品具有如下(1)～(4)中至少一种功能：
(1)延长寿命；
(2)促进胃肠道菌群定殖；
(3)抗衰老；
(4)减脂。
在本发明中，使用线虫(秀丽隐杆线虫)筛选平台来进行相关效果的验证。秀丽隐杆线虫(Caenorhabditis elegans)构造简单、身体透明、便于观察、发育周期短、易于人工培养且信号通路高度保守，从而使得秀丽隐杆线虫模型效果验证准确率较高，实验成本低。而本发明中，通过基于秀丽隐杆线虫的寿命试验、抗衰试验和减脂试验，有效证明了罗伊氏乳杆菌A21041同时兼具优异的延长寿命、抗衰老、减肥降脂功效，并且基于罗伊氏乳杆菌A21041在拟胃肠道环境中可以存活，因此，能够说明其有潜力定殖在机体胃肠道中，稳定并长久地发挥延长寿命、抗衰老、减肥降脂效果。
本发明的第六个方面，提供一种延长寿命、抗衰老的方法，包括如下步骤：向所需受试者给药有效量的罗伊氏乳杆菌A21041。
在本发明的一些实施方式中，所述有效量(人用)为0.02～0.04g/kg体重。
当然，本领域技术人员可以根据上述人用有效量根据转换公式得到其他动物用有效量，包括但不限于鼠、兔、狗、猪和猴子。
在本发明的一些实施方式中，所述给药方式为口服。
在本发明的一些实施方式中，所述给药频率为每1～24小时使用1次。
在本发明的一些实施方式中，所述给药频率为：每1小时、每2小时、每4小时、每6小时、每8小时、每12小时或每24小时使用一次。
本发明的第七个方面，提供一种减脂的方法，包括如下步骤：向所需受试者给药有效量的罗伊氏乳杆菌A21041。
在本发明的一些实施方式中，所述有效量(人用)为0.02～0.04g/kg体重。
当然，本领域技术人员可以根据上述人用有效量根据转换公式得到其他动物用有效量，包括但不限于鼠、兔、狗、猪和猴子。
在本发明的一些实施方式中，所述给药方式为口服。
在本发明的一些实施方式中，所述给药频率为每1～24小时使用1次。
在本发明的一些实施方式中，所述给药频率为：每1小时、每2小时、每4小时、每6小时、每8小时、每12小时或每24小时使用一次。
本发明的有益效果是：
1.本发明首次发现了一株具有优异的耐人工胃肠液、耐胆盐效果，且兼具优异的延长寿命、抗衰老、减肥降脂功效的罗伊氏乳杆菌，从而能够基于该菌株有效开发相关产品，具有极高的实用价值。
2.本发明中的产品中含有罗伊氏乳杆菌A21041，能够有效定殖在机体胃肠道中，稳定并长久地发挥延长寿命、抗衰老、减肥降脂效果。
附图说明
图1为实施例1中罗伊氏乳杆菌A21041的特异性PCR电泳图；
图2为实施例4中罗伊氏乳杆菌A21041与对照组OP50对线虫寿命影响的对比图；
图3为实施例5中罗伊氏乳杆菌A21041与对照组OP50对线虫衰老影响的对比图；
图4为实施例6中罗伊氏乳杆菌A21041与对照组OP50在第7天染色时的线虫脂肪染色对比图；
图5为实施例7中罗伊氏乳杆菌A21041与对照组OP50在第5天染色时的线虫脂肪染色对比图；
图6为实施例7中罗伊氏乳杆菌A21041对线虫寿命、脂肪代谢基因表达水平影响图及相关机制通路图；
图7为对比例1中商业罗伊氏乳杆菌17938与对照组OP50对线虫寿命影响的对比图；
图8为对比例2中商业罗伊氏乳杆菌17938与对照组OP50的线虫脂肪染色对比图；
图9为对比例3中OP50+Metformin(10mM)与对照组OP50对线虫寿命影响的对比图；
图10为对比例4中罗伊氏乳杆菌A21041-B与罗伊氏乳杆菌A21041的菌落形态对比图；
图11为对比例4中罗伊氏乳杆菌A21041-B与对照组OP50的线虫脂肪染色对比图。
具体实施方式
为了使本发明的发明目的、技术方案及其技术效果更加清晰，以下结合具体实施方式，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的具体实施方式仅仅是为了解释本发明，并非为了限定本发明。
所使用的实验材料和试剂，若无特别说明，均为常规可从商业途径所获得的耗材和试剂。
在下述实施例中，所用的MRS固体培养基配方为：蛋白胨10g/L，牛肉浸粉5g/L，酵母浸粉4g/L，葡萄糖20g/L，磷酸氢二钾2g/L，柠檬酸三铵2g/L，醋酸钠5g/L，硫酸镁0.2g/L，硫酸锰0.05g/L，琼脂15g/L，吐温-80 1g/L。将上述组分混合后，加入1L去离子水，调节pH值至6.2±0.2，121℃灭菌15min，即得MRS固体培养基。
实施例1罗伊氏乳杆菌A21041的分离与纯化
本发明实施例中的罗伊氏乳杆菌A21041的分离纯化步骤具体如下：
取中国广西壮族自治区北海市合浦县百岁老人的粪便样本，取适量粪便样本至MRS固体培养基中，涂抹均匀后，放置在厌氧工作站中，在37℃、90％氮气+10％二氧化碳条件下培养两天。培养结束后，挑取平板中菌落形态为圆形、凸起、光滑、边缘整齐的白色或乳白色的菌落至少量MRS液体培养基中，混合均匀后转移至PCR管中，置于37℃培养箱中继续培养24h。
向培养后的PCR管中加入PCR扩增液(2×Taq PCR StarMix with Loading Dye，购自北京康润诚业生物科技有限公司)，并加入罗伊氏乳杆菌特异性扩增引物，进行PCR扩增(扩增体系如表1所示)。
其中，罗伊氏乳杆菌特异性扩增引物为：
上游引物F：5’-GCTTCACTCGCTGCAGTTAA-3’(SEQ ID NO:1)；
下游引物R：5’-CCAACCAAACCTCGGTCAGA-3’(SEQ ID NO:2)。
表1罗伊氏乳杆菌PCR扩增体系
组分 含量
10μM上游引物F 1μL
10μM下游引物F 1μL
PCR扩增液 12.5μL
DNA模板 1μL
ddH 2O 补至25μL
扩增程序为：95℃3min；95℃40s，56℃30s，72℃1min，循环30次；72℃8min。
取扩增产物进行凝胶电泳。
经电泳跑胶确定为罗伊氏乳杆菌，胶图如图1所示。
同时，取部分培养液使用16S引物按照表1所示的PCR扩增体系进行PCR扩增后(扩增程序同上述实施例)，将扩增产物送至生工生物工程(上海)股份有限公司进行测序鉴定。
其中，所用的16S引物的核苷酸序列为：
上游引物16S-F：5’-AGAGTTTGATCCTGGCTCAG-3’(SEQ ID NO:3)；
下游引物16S-R：5’-TACGGCTACCTTGTTACGACTT-3’(SEQ ID NO:4)。
经测序鉴定，确认为罗伊氏乳杆菌，其16S序列为：5’-GAGGGACGGGCGGGTGCTATACATGCAAGTCGTACGCACTGGCCCAACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCGCATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAA AGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTGCGCTAACCTTAGAGATAAGGCGTTCCCTTCGGGGACGCAATGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTACTAGTTGCCAGCATTGAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGTCGCAAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCGTTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAACGCCCAAAGTCGGTGGCCTAACCTTTATGGAGGGGACCGCGCCCCCCCCTATAACGC-3’(SEQ ID NO:5)。对确认后的培养液进行两次平板划线纯化，挑取纯化后的单菌落进行重复培养，得到菌液，将菌液与30％甘油保存液以1：1的体积比混合于菌株冻存管中，放置于-80℃冰箱保藏。
进一步对上述实施例中得到的罗伊氏乳杆菌进行全基因测序(交由安诺优达基因科技(北京)有限公司进行测序)。结果如表2所示。
表2罗伊氏乳杆菌A21041的全基因测序结果
样品ID 重叠群(contigs) 总长度/bp GC比(％) CDS/个 Gene/个
A21041 128 2011591 38.77 1977 2048
将上述实施例中得到的罗伊氏乳杆菌(命名为罗伊氏乳杆菌A21041)送至广东省微生物菌种保藏中心(GDMCC)进行保藏，其分类学命名为：Lactobacillus reuteri，保藏日期为：2022年9月23日，保藏地址为：中国广东省广州市先烈中路100号大院59号楼5楼，保藏编号为：GDMCC No:62832。
实施例2罗伊氏乳杆菌A21041的耐人工肠胃液、耐胆盐试验
具体实验步骤如下：
取上述罗伊氏乳杆菌A21041进行过夜培养活化，稀释菌液浓度至OD600＝1。按体积比10％的接种量，将菌液接种于pH＝2.5的人工胃液(1mol/mL的稀盐酸加水稀释得到)中，分别于0h、1h、3h后各取100μL溶液涂布于MRS平板中，在生化培养箱37℃培养48h后进行单菌落数量计数。
按照上述同样的方法，按体积比10％的接种量，将菌液接种于pH＝8的人工肠液(pH6.8，取磷酸二氢钾6.8g，加水500mL溶解，用0.1mol/L氢氧化钠溶液调节pH值至6.8，加入胰酶水溶液(胰酶10g加水溶解)，加水稀释至1000mL即得)中，分别于2h、4h、6h、8h后各取100μL溶液涂布于MRS平板中，在生化培养箱37℃培养48h后进行单菌落数量计数。
同时，按体积比10％的接种量，将菌液分别接种于0.3g/L和0.5g/L牛胆盐的MRS液体培养基中，分别于0h、2h、24h后各取100μL溶液涂布于MRS平板中，在生化培养箱37℃ 培养48h后进行单菌落数量计数。
根据上述实验的单菌落数量计算罗伊氏乳杆菌A21041在人工胃肠液及牛胆盐溶液的存活率。
结果发现，罗伊氏乳杆菌A21041在pH＝2.5的人工胃液处理3h后存活率为90％，在pH＝8.0的人工肠液处理8h后存活率为80％左右。罗伊氏乳杆菌A21041在0.3g/L和0.5g/L牛胆盐溶液处理24h后存活率分别为260％和80％。由此可见，本发明实施例中的罗伊氏乳杆菌A21041有潜力能在人体胃肠道中长期定殖。
实施例3罗伊氏乳杆菌A21041的安全性评价
为保证罗伊氏乳杆菌A21041在各类用途中的安全性，在本实施例中进行了菌株的安全性评价，检测机构为中国广东省微生物分析检测中心。参照中国保健食品原料用菌种安全性检验与评价技术指导原则(2020年版)附录A判定标准，罗伊氏乳杆菌A21041对小鼠的一般健康情况无不良影响；体重等指标与对照组相比差异无统计学意义；故在本发明实施例条件下，该样品不具有致病性。同时，对A21041进行了全基因组测序，并通过Diamond(version 2.1.0.4)和RGI(version 5.2.0)软件进行全基因组分析，根据毒力因子数据库标准(匹配度≥85％和e值<10 -5为无毒性基因或致病因子)，A21041的匹配度≥95％、e值<10 -5，因此从基因水平上评估，A21041无毒性。因此，罗伊氏乳杆菌A21041是安全的益生菌。
实施例4罗伊氏乳杆菌A21041的寿命试验
(1)罗伊氏乳杆菌A21041、大肠杆菌OP50和秀丽隐杆线虫的培养：
将罗伊氏乳杆菌A21041培养于MRS液体培养基中，将大肠杆菌OP50培养于LB液体培养基中，培养条件均为：温度37℃，时间20h，转速200rpm。取罗伊氏乳杆菌A21041和大肠杆菌OP50菌液(菌液浓度均为10 7CFU/mL)分别涂板于线虫培养基(nematode growth medium，NGM)上过夜培养。
其中，NGM培养基配方为：氯化钠3g，琼脂粉17g，蛋白胨2.5g，去离子水975mL，将上述组分充分混合均匀后，用锡箔纸封口。高压蒸汽灭菌20min后，水浴锅冷却至55℃。在无菌条件下加入下列灭菌的溶液：1mL的1M CaCl 2，1mL的1M MgSO 4，25mL的1M KPO 4buffer，1mL的5mg/mL胆固醇(溶于95％乙醇中)。
将解冻的线虫(秀丽隐杆线虫)离心后分别加至有OP50的NGM培养基上，置于20℃培养箱培养3天。
(2)线虫同期化处理：
使用M9缓冲液悬浮步骤(1)中培养后的线虫，吸至培养管中，向每管中加入裂解液(质量比5N的NaOH和体积比5％次氯酸钠溶液)，裂解6min，3500r/min离心1min，弃上清液，用M9缓冲液反复清洗4次。再次离心弃上清液。将沉淀转移至NGM培养基上，20℃恒温过夜培养，即得L1期线虫幼虫。用M9缓冲液冲洗离心一次后，将线虫幼虫转移至含有OP50的NGM平板上，20℃培养28-30h即可得到同期化线虫，即L4期线虫。
(3)寿命试验：
将得到的同期化线虫随机分成两组(对照组(OP50)和实验组(A21041))，每组150条。挑取L4期线虫当天记为第0天，实验过程中，线虫每2天更换一次含有OP50和A21041的NGM培养基并统计寿命，直到所有线虫死亡为止。非正常死亡线虫不计入统计中。采用GraphPad Prism 5进行统计分析，采用单因素方差分析比较多组间的差异显著性；用t检验 法分析两两组间差异。P＜0.05为具有显著差异，P＜0.01为具有极显著差异。
结果如图1所示，可以发现，与OP50对照组相比，线虫在喂养A21041后显著地延长了寿命(OP50对照组、A21041实验组的平均寿命分别为18天(d)和21.5天(d))。相对于OP50对照组，A21041延长了线虫19.66％的寿命(P＜0.05)。
实施例5抗衰试验
按照上述实施例中的方法进行罗伊氏乳杆菌A21041、大肠杆菌OP50和秀丽隐杆线虫的培养，并进行线虫同期化处理。
然后，将同期化线虫随机分成两组(对照组(OP50)和实验组(A21041))，每组150条。挑取L4期线虫当天记为第0天，实验过程中，线虫每2天更换一次含有OP50和A21041的NGM培养基。将线虫养至第5天、10天和15天时，在含M9缓冲液的NGM平板中逐一统计线虫在30s内头部摆动的频率。以线虫头部从一侧摆至另一侧再回到原位记为摆动一次。
结果如图2所示，可以发现，在第10天和第15天(线虫的中老年期)，罗伊氏乳杆菌A21041可以显著提高线虫的头部摆动频率，提升线虫活力，因此罗伊氏乳杆菌A21041可显著延缓线虫衰老。
实施例6减脂试验
按照上述实施例中的方法进行罗伊氏乳杆菌A21041、大肠杆菌OP50和秀丽隐杆线虫的培养，并进行线虫同期化处理。
分别将100条同期化线虫转移至涂有OP50和A21041的平板中进行培养，培养7天，隔天换板一次。在第7天用冷却的M9缓冲液洗三次后，将线虫重悬在4％的多聚甲醛中。室温下轻微摇动1h，3000～4000rpm离心1min，去除上清后用M9缓冲液洗两次。随后将线虫重悬在含有体积比60％异丙醇和体积比0.01％Triton X-100的混合PBS溶液中，孵育15min；待线虫沉降后，去除异丙醇，加入1mL的40％油红O染色剂，在25℃摇床孵育1～2h。去除染料，用M9缓冲液清洗两次。加入200μL M9缓冲液，在倒置荧光显微镜下逐条对线虫拍照。采用Image J统计图像数据，用GraphPad Prism 5统计脂肪粒降解率数据，P＜0.05为具有显著差异，P＜0.01为具有极显著差异。
结果如图3所示，可以发现，与OP50对照组相比，喂食罗伊氏乳杆菌A21041的实验组中的线虫脂肪粒明显颜色较淡，脂肪粒较小，表明罗伊氏乳杆菌A21041对线虫脂肪粒有明显的抑制作用，抑制率高达68.77％(P＜0.01)。
重复上述实验，将培养时间调整为5天。
结果如图4所示，在喂养A21041的第5天，对线虫进行染色后，发现与OP50对照组相比，喂食罗伊氏乳杆菌A21041的实验组中的线虫脂肪粒明显颜色较淡，脂肪粒较小，表明仅投喂5天的罗伊氏乳杆菌A21041即可对线虫脂肪粒有明显的抑制作用，抑制率为54.47％。
实施例7荧光定量PCR测定罗伊氏乳杆菌A21041影响寿命、脂肪代谢基因位点情况
按照上述实施例中的方法进行罗伊氏乳杆菌A21041、大肠杆菌OP50和秀丽隐杆线虫的培养，并进行线虫同期化处理。
分别将800条同期化线虫转移至涂有OP50和A21041的平板中进行培养，培养5天，隔天换板一次。在第5天用M9缓冲液清洗线虫1-2次。用液氮冻干后研磨线虫，按照总RNA提取试剂盒(购自洛麦格(北京)生物技术有限公司)操作说明进行线虫总RNA提取。然后 各组取等量的RNA样本按照反转录试剂盒(购自天根生化科技(北京)有限公司)说明书进行反转录PCR扩增。将PCR扩增产物置于-20℃保存待用。使用表3中所示的各基因的引物对进行常规荧光定量PCR，采用2-ΔΔCt进行数据统计。
表3荧光定量PCR检测位点及对应的引物序列信息
Figure PCTCN2022135531-appb-000001
结果如图5所示，可以看出，nhr-80、daf-12基因的表达较为显著上调，尤其是nhr-80，与对照组相比，上调了近2倍。因此，上述结果可以说明A21041延长线虫寿命可能与nhr-80、daf-12两个基因调控的信号通路相关，即与核激素受体信号通路相关。
实施例8基于罗伊氏乳杆菌A21041制备延长寿命、抗衰老、减脂的产品
取罗伊氏乳杆菌A21041菌株进行发酵扩繁，然后对发酵扩繁后的菌液进行离心、冻干，即得罗伊氏乳杆菌A21041冻干粉。然后按照下述配方进行复配，即可得到含有罗伊氏乳杆菌A21041的食品添加剂或膳食补充剂。
以人体的使用量为标准，按照重量百分含量计算，含有罗伊氏乳杆菌A21041的食品添加剂或膳食补充剂的配方为：10％罗伊氏乳杆菌A21041冻干粉，15％茶多酚(购自善恩康生 物科技有限公司)，15％杨梅花青素提取物(购自善恩康生物科技有限公司)，50％麦芽糊精和10％可溶性膳食纤维(购自善恩康生物科技有限公司)。制备方法为：将上述原料充分混合后即得。
对比例1
在本对比例中，商业罗伊氏乳杆菌17938菌株分离自森宝益生菌滴露，培养方式以及线虫寿命测试方法同上述实施例。
效果对比结果如图6所示，可以发现，与OP50对照组相比，线虫在喂养罗伊氏乳杆菌17938后可延长线虫寿命(OP50对照组、罗伊氏乳杆菌17938实验组的平均寿命分别为18d和20d)。相对于OP50对照组，17938延长了线虫11.11％的寿命。但对比罗伊氏乳杆菌A21041，罗伊氏乳杆菌A21041的寿命延长效果更加显著，说明罗伊氏乳杆菌A21041相比现有的罗伊氏乳杆菌具有更强的寿命延长效果。
对比例2
在本对比例中，商业罗伊氏乳杆菌17938菌株来源、培养方式同上述对比例1，线虫脂肪粒测试方法同上述实施例。
效果对比结果如图7所示，可以发现，喂食商业罗伊氏乳杆菌17938菌株的线虫脂肪粒颜色与对照组无明显差别，抑制率仅有33.85％，不具有减脂效果，而罗伊氏乳杆菌A21041减脂效果显著。
对比例3
治疗2型糖尿病药物二甲双胍(Metformin)具有延长线虫寿命的作用，因此，在本对比例中作为阳性药物使用。
在本对比例中，按照上述实施例中的方法进行罗伊氏乳杆菌A21041、大肠杆菌OP50和秀丽隐杆线虫的培养，并进行线虫同期化处理。
将同期化线虫随机分成两组(对照组(OP50)和阳性组(OP50+Metformin(终浓度为10mM))，每组150条。挑取L4期线虫当天记为第0天，实验过程中，线虫每2天更换一次含有OP50的NGM培养基并统计寿命，直到所有线虫死亡为止，非正常死亡线虫不计入统计中。采用GraphPad Prism 5进行统计分析，采用单因素方差分析比较多组间的差异显著性；用t检验法分析两两组间差异。P＜0.05为具有显著差异，P＜0.01为具有极显著差异。
效果对比结果如图8所示，可以发现，与对照组相比，线虫在喂养加了Metformin(10mM)的OP50后可延长线虫寿命(OP50对照组、OP50+Metformin(10mM)阳性组的平均寿命分别为18d和19d)，相对于对照组，Metformin(10mM)延长了线虫5.56％的寿命，但对比罗伊氏乳杆菌A21041的喂养效果，喂养罗伊氏乳杆菌A21041后的线虫寿命延长效果要显著优于加入了Metformin的阳性组。
对比例4
从实施例1中的样本中，还分离得到了一株罗伊氏乳杆菌A21041-B，从显微镜下观察，罗伊氏乳杆菌A21041-B与罗伊氏乳杆菌A21041菌落形态不相同，A21041-B的菌落边缘较薄，菌落整体较透明；而A21041的菌落边缘则较厚，具体如图9所示。此外，A21041-B的16S测序后的核苷酸序列同SEQ ID NO:5，说明其也是一株罗伊氏乳杆菌。
按照上述实施例中的操作，对A21041-B进行相同的分离纯化、耐人工肠胃液、耐人工胆盐和脂肪粒测试。
结果如图10所示，可以发现，A21041-B虽然也具有一定的耐人工胃肠液、耐胆盐效果，但抑制线虫脂肪粒的效果不如罗伊氏乳杆菌A21041，抑制率仅为18.24％。
综上所述，上述结果说明，在现有的罗伊氏乳杆菌中，仅罗伊氏乳杆菌A21041同时兼具优异的延长寿命、抗衰老、减肥降脂功效，并且基于罗伊氏乳杆菌A21041在拟胃肠道环境中可以存活，因此，能够说明其有潜力定殖在机体胃肠道中，稳定并长久地发挥延长寿命、抗衰老、减肥降脂效果。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一株罗伊氏乳杆菌，其特征在于，所述罗伊氏乳杆菌为罗伊氏乳杆菌A21041，分类学命名为Lactobacillus reuteri，于2022年9月23日保藏于广东省微生物菌种保藏中心，保藏编号为：GDMCC No:62832。
  2. 含有权利要求1所述罗伊氏乳杆菌的产品，其特征在于，所述产品包括食品、食品添加剂、饲料、饲料添加剂、药品和化妆品。
  3. 根据权利要求2所述的产品，其特征在于，所述产品中的罗伊氏乳杆菌的质量分数大于等于5％，优选大于等于8％，更优选大于等于10％。
  4. 根据权利要求2所述的产品，其特征在于，所述产品中还含有其他辅料，所述辅料包括药学上可接受的辅剂、食品添加剂、化妆品辅料。
  5. 根据权利要求1所述的产品，其特征在于，所述产品中的罗伊氏乳杆菌的形式包括冻干粉、菌液、颗粒接种菌剂。
  6. 权利要求2～5任一项所述产品的制备方法，包括如下步骤：将罗伊氏乳杆菌活化扩繁后，加工为菌剂，然后与其他辅料混合，即得。
  7. 一种含有权利要求1所述罗伊氏乳杆菌的食品添加剂，其特征在于，所述食品添加剂包括罗伊氏乳杆菌A21041冻干粉、植物提取物、淀粉水解多糖和膳食纤维。
  8. 根据权利要求7所述的食品添加剂，其特征在于，按照质量百分比计，所述食品添加剂包括5～15％罗伊氏乳杆菌A21041冻干粉、15～45％植物提取物、40～60％淀粉水解多糖和5～15％膳食纤维。
  9. 权利要求1所述罗伊氏乳杆菌在制备食品、食品添加剂、饲料、饲料添加剂、药品和化妆品中的用途。
  10. 根据权利要求9所述的用途，其特征在于，所述食品、食品添加剂、饲料、饲料添加剂、药品和化妆品具有如下(1)～(4)中至少一种功能：
    (1)延长寿命；
    (2)促进胃肠道菌群定殖；
    (3)抗衰老；
    (4)减脂。
PCT/CN2022/135531 2022-11-30 2022-11-30 一种延长寿命、抗衰老、减脂的罗伊氏乳杆菌及其产品与应用 WO2023240936A1 (zh)

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CN110452828A (zh) * 2019-03-24 2019-11-15 广西大学 罗伊氏乳杆菌菌株及其应用
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