CN115181710A - Lactobacillus salivarius B12WU and application thereof - Google Patents
Lactobacillus salivarius B12WU and application thereof Download PDFInfo
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- CN115181710A CN115181710A CN202211106671.7A CN202211106671A CN115181710A CN 115181710 A CN115181710 A CN 115181710A CN 202211106671 A CN202211106671 A CN 202211106671A CN 115181710 A CN115181710 A CN 115181710A
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- b12wu
- lactobacillus salivarius
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- bacterial agent
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Abstract
The invention provides a lactobacillus salivarius B12WU with high acid-base tolerance and application thereof, and relates to the technical field of microorganisms and application thereof. The lactobacillus salivarius is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.24787. The lactobacillus salivarius B12WU provided by the invention can synthesize beta-nicotinamide mononucleotide NMN, and can be subjected to the test of a gastrointestinal strong-acid-base environment. In addition, the B12WU strain has high alpha amylase or alpha glucosidase inhibition activity, so the B12WU strain has wide application prospects in the aspect of preparing products for supplementing NMN, reducing blood sugar level and improving diabetes.
Description
Technical Field
The invention relates to the technical field of microorganisms and application thereof, in particular to lactobacillus salivarius (lactobacillus: (a)Lactobacillus salivarius) Strain B12WU and uses thereof.
Background
beta-Nicotinamide Mononucleotide (NMN) as Nicotinamide adenine dinucleotide NAD + The precursor of (Nicotinamide adenine dinuclleotide coenzyme I) can be converted into NAD with special activity through related biochemical processes + Can promote the metabolism of sugar, fat and protein in human body, improve the blood sugar level of diabetes patients, control weight, repair DNA damage, activate longevity protein and delay aging; especially in aging, NAD + Is the only substrate of the longevity protein Sirtuins, but with age, NAD in vivo + The content is continuously reduced, so how to increase the in vivo NAD + The level, further enhancing the physical constitution and delaying the aging process, has become the hot spot of the current scientific research and market demand.
In recent years, more and more studies have shown that old mice are given additional NAD supplementation + Can also prolong the service life and maintain health; can supplement NMN level in vivo and also can increase NAD in vivo + Horizontal effect, and further delaying aging. Because the NMN content in food is very low, the effect of supplementing NMN by only depending on normal diet intake is poor, therefore, exogenous NMN supplementation is an effective way for improving NMN in vivo. NMN is now approved by developed countries in Europe, america, japan and the like as a food raw material of health products to develop various NMN-related health products, and at present, NMN is mainly synthesizedThree methods are as follows: chemical synthesis, biological enzyme synthesis and fermentation. The NMN in many health products sold on the market is not high-purity active natural beta-form NMN, and the contained NMN may be produced by a traditional chemical method. Chemical synthesis techniques are simple, but require multiple reactions, and the subsequent separation and purification of chiral substances is quite costly, so that many chemically synthesized NMNs are not high in active purity. More importantly, the impurities such as reagents and the like participating in the chemical synthesis reaction are accumulated continuously after entering the human body, and the serious harm to the human health is possibly caused. Compared with the prior art, the chiral purity of NMN obtained by a biosynthesis method (an enzymatic method and a fermentation method) is higher and can reach more than 99 percent, and the method is an effective method for producing high-activity NMN at present, but the method has higher technical barrier, relates to the modification of engineering bacteria and a large amount of biological enzymes, and has much lower yield compared with a chemical method. Therefore, the method for safely and effectively synthesizing NMN has wide market significance.
Intestinal microorganisms, particularly probiotics have a non-negligible regulating effect on the health of a human body, such as regulating blood sugar and blood fat, regulating emotion and improving gastrointestinal diseases, and a probiotic-based developed food Linlanganymu relates to yoghourt, beverages, candies and the like. Not only intestinal microorganisms can be used as probiotics, but also metabolites thereof can be developed as metazoan. Because the intestinal flora, especially probiotics, has higher safety and low separation and purification cost, and NMN synthesized by the intestinal flora is active beta-NMN, the probiotics capable of generating the NMN are screened based on a proper detection method, so that a safer and more reliable raw material source is provided for NMN related products, and the development of probiotics food or medicine with various health care effects based on the probiotics food or medicine has very wide market prospect.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a lactobacillus salivarius with high acid-base tolerance (Lactobacillus salivarius) The strain B12WU can synthesize beta-nicotinamide mononucleotide NMN and can withstand the test of the strong acid-base environment of the stomach and intestine. In addition, the B12WU strain hasHigher alpha amylase or alpha glucosidase inhibitory activity, and thus the B12WU strain also has the potential to lower the body's blood glucose level.
The technical scheme provided by the invention is as follows:
in one aspect, the invention provides a high acid-base tolerance lactobacillus salivarius (Lactobacillus salivarius) The strain B12WU is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.24787.
The lactobacillus salivarius strain B12WU is separated and screened from feces of centenarian old people living in Chengmei in Hainan China for a long time, and in-vitro experiments prove that the strain can synthesize NMN and can withstand the examination of the strong acid-base environment of the stomach and intestine. In addition, the B12WU strain has a higher amylase inhibitory activity, particularly inhibiting α -amylase inhibitory activity. The substance with amylase inhibiting activity can inhibit the activities of salivary amylase and pancreatic amylase, so that the B12WU strain has the potential of reducing the blood sugar level of the body, and is used for preparing a product for reducing the blood sugar level and improving diabetes.
In one aspect, the invention provides a microbial inoculum comprising said lactobacillus salivarius strain B12WU.
In specific embodiments, the microbial inoculum is a solid formulation or a liquid formulation; the solid microbial inoculum is powder prepared by adopting a freeze-drying method.
In one aspect, the invention provides the use of said lactobacillus salivarius strain B12WU or a bacterial agent in the synthesis of β -nicotinamide mononucleotide.
In one aspect, the invention provides the use of the high acid-base tolerance lactobacillus salivarius strain B12WU or a microbial inoculum for inhibiting alpha amylase or alpha glucosidase activity. Further provides application of the high-acid-base-tolerance lactobacillus salivarius strain B12WU or microbial inoculum in preparation of products for treating or preventing diabetes or in preparation of diabetes adjuvant therapy medicines. The bacterial strain and the microbial inoculum can be used for treating and preventing diabetes, for example, preparing medicaments for preventing or relieving type 1 diabetes and type 2 diabetes, or preparing medicaments for preventing or relieving intestinal flora disorder caused by diabetes. And further can be used alone or in combination with other products having hypoglycemic or antidiabetic activity. The products can be prepared into microbial preparation, functional food, health product or medicine.
In one embodiment, the high acid-base tolerant lactobacillus salivarius is one or more of an inactivated or non-inactivated fermentation supernatant, bacterial suspension, and cell disruption supernatant.
In one embodiment, the mode of inactivation is pasteurization.
In another aspect, the invention provides the application of the lactobacillus salivarius strain B12WU or the microbial inoculum as probiotics in food or health care product components, such as yoghurt, beverage, candy and the like.
In another aspect, the invention provides a medicament containing the high acid-base tolerant lactobacillus salivarius strain B12WU and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers include, but are not limited to: one or more of a filler, a wetting agent, a disintegrant, a binder, or a lubricant.
In one embodiment, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch, or dextrin; the wetting agent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, cross-linked povidone or low-substituted hydroxypropyl cellulose; the adhesive is one or more of starch paste, syrup, maltose or liquid glucose; the lubricant is one or more of magnesium stearate, talcum powder or silicon dioxide.
In one embodiment, the medicament is in a dosage form for oral administration; preferably, the medicament is in the form of a solution, a suspension, an emulsion, a powder, a lozenge, a pill, a syrup, a buccal lozenge, a tablet and a capsule.
In another aspect, the present invention provides a method for improving blood glucose metabolism by adding the high acid-base tolerant lactobacillus salivarius strain B12WU or a microbial inoculum to a system containing blood glucose. The method is for non-disease diagnostic or therapeutic purposes.
Biological sample preservation information: lactobacillus salivarius (Lactobacillus salivarius) The strain B12WU is preserved in the China general microbiological culture Collection center on 27 th 4 th 2022 with the preservation number: CGMCC No.24787; and (4) storage address: western road No. 1 institute of microbiology, china institute of academy of sciences, no. 3, north chen yang district, beijing, china, zip code 100101. The strains were detected as viable by the depository at 2022, 4 months and 27 days.
Has the advantages that:
1. the lactobacillus salivarius can synthesize NMN and can withstand the test of the gastrointestinal strong acid-base environment.
2. The lactobacillus salivarius has high amylase inhibition activity, particularly alpha amylase and alpha glucosidase inhibition activity. The substance with amylase inhibition activity can inhibit the activities of salivary amylase and pancreatic amylase, so that the B12WU strain has the potential of reducing the blood sugar level of an organism and has wide application prospect in the aspect of preparing products for reducing the blood sugar level and improving diabetes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a mass spectrometric identification of Lactobacillus salivarius B12WU of the present invention;
FIG. 2 is a standard curve drawn for a standard used in the detection of NMN by chemiluminescence in accordance with the present invention.
FIG. 3 is a high performance liquid chromatography assay of NMN in Lactobacillus salivarius B12WU of the invention.
FIG. 4 shows the inhibitory effect of Lactobacillus salivarius B12WU suspensions and fermentation supernatants on glucosidase activity (A) and amylase activity (B), p <0.05, p <0.01, p <0.001.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 identification and preparation of Lactobacillus salivarius B12WU Strain
Feces of centenarian living in the long-life countryside of the Chengmei world in China are collected as a screening sample, and the collection target population does not take antibiotic medicines before collection, and has no probiotic taking history and no gastrointestinal disease history.
The collected fecal samples are diluted and spread on MRS solid culture medium (peptone 10 g, beef extract 10 g, yeast extract 5 g, diammonium hydrogen citrate 2 g, glucose 20 g, tween 80 mL, sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar 18 g, distilled water 1000 mL), anaerobic culture 24-48h at 37 ℃, separate to obtain different single colonies on the plate, then each single colony is streaked on a new MRS solid culture medium plate respectively to obtain purified colonies, each purified single colony is spread on a mass spectrum plate, auxft substrate and a lysate are added on a new MRS solid culture medium plate respectively to be identified by mass spectrometer after drying, and the strains are identified on a mass spectrometer (MALDI-TOF 80, and mass spectrometer after the identification.
The identification result of the Lactobacillus salivarius B12WU strain related to the invention is shown in figure 1, and the B12WU strain has high similarity with the Lactobacillus salivarius, so that the strain is named as the Lactobacillus salivarius B12WU strain.
Example 2 preparation of B12WU viable bacteria solution, cell lysate and metabolite
(1) Coating the frozen bacterium liquid at minus 80 ℃ on an MRS solid plate (peptone 10 g, beef extract 10 g, yeast extract 5 g, diammonium hydrogen citrate 2 g, glucose 20 g, tween 80 mL, sodium acetate 5 g, dipotassium hydrogen phosphate 2 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar 18 g and distilled water 1000 mL), inversely culturing for 24-48h at 37 ℃, inoculating a single colony in an MRS liquid culture medium, and inversely culturing for 18-24h at 37 ℃ to obtain a first-generation bacterium liquid; taking 10% (v/v) of a first-generation bacterium liquid, inoculating the first-generation bacterium liquid to a fresh MRS liquid culture medium, and culturing at 37 ℃ for 18-24h to obtain a second-generation bacterium liquid; inoculating 10% (v/v) of the second-generation bacterial liquid into a fresh MRS liquid culture medium, and culturing at 37 ℃ for 18-24h to obtain the working bacterial liquid.
For functional studies on lactobacillus salivarius B12WU, the present invention will prepare samples as follows:
(1) Viable bacteria liquid: and (3) centrifuging the working bacterial liquid of the lactobacillus salivarius B12WU for 15 min at 13000 rpm and 4 ℃, removing the supernatant, collecting the precipitate, and resuspending the precipitate by using sterile PBS to obtain the live bacterial liquid with live bacteria.
(2) Cell lysate: and (2) centrifuging the working bacterium solution of the lactobacillus salivarius B12WU at 13000 rpm at 4 ℃ for 15 min, removing the supernatant, collecting the precipitate, adding 1mL of ultrapure water, placing the precipitate in an ice water mixture, crushing the precipitate by using an ultrasonic crusher to obtain a thallus lysate, and placing the lysate on an ice box for later use after the lysate is crushed.
(3) Fermentation supernatant liquid: the working bacterium liquid of the lactobacillus salivarius B12WU is placed at 13000 rpm and 4 ℃ for centrifugation for 15 min, and then the supernatant is collected and stored at 4 ℃ for later use, wherein the supernatant is a strain fermentation supernatant sample and can be used for in-vitro enzyme activity experiments in the following embodiments.
Viable cell liquid can also be obtained by other means in the technical field as long as the cells can be enriched from the culture solution. This can be achieved, for example, by means of centrifugation and/or filtration.
Example 3 detection of NMN content in Lactobacillus salivarius B12WU Strain
3.1 Detection by chemiluminescence method
Taking 2 mL of B12WU viable bacteria liquid, centrifuging at 12000 rpm for 5 min, collecting thallus precipitate, adding 1mL ultrapure water into the thallus precipitate for re-suspension, and carrying out ultrasonic crushing in an ice water mixture to obtain a thallus lysate. A clean 1.5 mL centrifuge tube is taken on a centrifuge tube rack, 104 mu L of different concentrations of standard (0.21 mg/L, 1.67 mg/L, 3.34 mg/L, 5.01 mg/L, 8.36 mg/L) or thallus lysate, 42 mu L of 2M KOH,42 mu L of 20% acetophenone are added, after vortex mixing, the mixture is placed on an ice box for standing for 2 min, 188 mu L of 88% formic acid is added, the mixture is placed on a shaker at 37 ℃ for mixing (the temperature is required to be higher than 37 ℃), 100 mu L of reaction solution is transferred into black Kong Yingguang wells after 10 min, 3 multiple wells are set up experimentally, excitation light 382 zxft 382 is read by a Tecan microplate reader, light emission is emitted to nm, then the concentration of the N standard and the concentration of the NMN standard and the concentration of the NM standard are plotted in a standard curve (the NMxFT 3525N strain), and the average value of the concentration of the N of the strain can be calculated as an NMxN strain 3525 WU + 12 strain B, and the average value of the strain can be calculated as a strain WU < 12 strain.
3.2 High performance liquid chromatography detection
To further confirm the ability of lactobacillus salivarius B12WU to produce NMN, we centrifuged the live lactobacillus salivarius B12WU solution, collected the pellet, disrupted and lysed, and then subjected to HPLC detection. The instrument parameter conditions were as follows: instrument waters 2695+2489; a chromatographic column: symmetryC18 5um 4.6mm 250mm; detection wavelength: 210 nm; mobile phase: 0.02M potassium dihydrogen phosphate: methanol =90, flow rate: 1.0 mL/min; the sample size is 10 mul; column temperature: at 30 ℃. FIG. 3 is a high performance liquid chromatography assay of NMN in Lactobacillus salivarius B12WU of the invention. The detection result shows that the Lactobacillus salivarius B12WU strain has the capacity of producing nicotinamide mononucleotide, and the content of NMN in the Lactobacillus salivarius B12WU is 430.8461 mu g/g through calculation.
Example 4 acid-base tolerance of Lactobacillus salivarius B12WU
4.1 preparation of Artificial stomach and intestine solution
The preparation of the artificial gastrointestinal fluid used in the invention refers to Chinese pharmacopoeia.
Artificial gastric juice: 234 ml concentrated hydrochloric acid is measured and added with water to be diluted to 1000 ml to obtain 9.5% -10.5% diluted hydrochloric acid, then the prepared diluted hydrochloric acid 16.4 ml is taken, 800 ml and pepsin 10 g are added with water, after shaking up, the diluted hydrochloric acid is added with water to be diluted to 1000 ml to obtain the artificial gastric juice.
Artificial small intestine liquid: taking monopotassium phosphate 6.8 g, adding water 500 ml to fully dissolve the monopotassium phosphate, adjusting the pH value to 6.8 by using 0.1 mol/L sodium hydroxide solution, weighing pancreatin 10 g, adding water to dissolve the pancreatin 10.1 mol/L sodium hydroxide solution, mixing the two solutions, adding water to dilute the two solutions to 1000 ml to obtain the artificial intestinal juice.
4.2 Tolerance testing of strains
Collecting cultured viable bacteria, centrifuging at 13000 rpm at 4 deg.C for 15 min, discarding supernatant, collecting thallus precipitate, re-suspending thallus with physiological saline, respectively inoculating to artificial stomach/intestine solution according to viable count of 109 CFU/ml, culturing at 37 deg.C for 3 hr, sampling, detecting viable count, and calculating survival rate with viable count of 0h as control, wherein survival rate (%) = (viable count of 3 hr/viable count of 0 hr) = 100%. The results show that the survival rates of the lactobacillus salivarius B12WU strain in the invention are 74.3 percent and 78.9 percent respectively after the lactobacillus pentosus strain is incubated and cultured in artificial gastric juice and intestinal juice for 4 hours; the B12WU strain is shown to have better acid-base tolerance, can be subjected to the test of the gastrointestinal strong acid-base environment after being ingested, can retain more bacterial quantity in the intestinal tract, and is beneficial to fully exerting the effect of probiotics.
Example 5 in vitro enzyme Activity assay
5.1 Lactobacillus salivarius B12WU inhibition of alpha amylase activity assay
Taking 2 mg/ml alpha amylase solution 2 ml (prepared by 50mM PBS buffer solution with pH 7.0), respectively adding 2 ml sample liquid (B12 WU viable bacteria suspension and fermentation supernatant), reacting at 37 ℃ for 30 min, adding 2 ml 1% soluble starch, reacting at 37 ℃ for 15 min, adding iodine solution for color development, and measuring the absorbance OD660 at 660 nm by using an enzyme labeling instrument. Among them, MRS medium was used as a negative control, and acarbose was used as a positive control. Through calculation, the average inhibition rates of the live B12WU suspension and the fermentation supernatant on the amylase activity are 23.1% and 22.8% respectively, and the inhibition rate of acarbose can reach 27.6% (part A on the left side in fig. 4); the B12WU strain and the metabolite thereof have high amylase inhibition activity. The substance with amylase inhibiting activity can inhibit the activity of salivary amylase and pancreatic amylase, so that the B12WU has the potential of reducing the blood sugar level of the body.
5.2 Lactobacillus salivarius B12WU inhibition of alpha glucosidase activity assay
Mixing 30 mul of Lactobacillus salivarius B12WU sample (including viable bacteria suspension and fermentation supernatant) with 30 mul of alpha glucosidase enzyme solution (0.1U/ml), incubating for 10 min at 37 ℃, adding 60 mul of substrate pNPG (0.5 mM), reacting for 20 min at 37 ℃, and adding 100 mul of 2M sodium carbonate solution to terminate the reaction. The absorbance at 405 nm is then measured using a microplate reader. Among them, MRS medium was used as a negative control, and acarbose was used as a positive control. The inhibition rates of the B12WU viable bacteria suspension and the fermentation supernatant on alpha glucosidase are close, the average values are 24.7 percent and 25.1 percent respectively, and the inhibition rate of acarbose can reach 28.8 percent (part B on the right side in figure 4); the B12WU strain and the metabolite thereof have stronger alpha glucosidase inhibition activity. At present, acarbose, which is a known alpha glucosidase inhibitor, is a widely used hypoglycemic drug, so the B12WU strain has the potential to prevent and treat postprandial hyperglycemia and improve diabetes mellitus by inhibiting the activity of alpha glucosidase.
According to the experimental results, the B12WU strain provided by the invention can be used for synthesizing NMN and can be tested in the gastrointestinal strong acid-base environment. In addition, the B12WU strain has higher activity of inhibiting alpha amylase or alpha glucosidase. The substance with amylase inhibition activity can inhibit the activities of salivary amylase and pancreatic amylase, so that the B12WU strain has the potential of reducing the blood sugar level of an organism and has wide application prospect in the aspect of preparing products for reducing the blood sugar level and improving diabetes.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. Lactobacillus salivarius with high acid-base tolerance (Lactobacillus salivarius) The strain B12WU is characterized in that the Lactobacillus salivarius is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.24787.
2. A bacterial agent comprising lactobacillus salivarius strain B12WU as claimed in claim 1.
3. Use of lactobacillus salivarius strain B12WU as claimed in claim 1 or a bacterial agent as claimed in claim 2 in the synthesis of β -nicotinamide mononucleotide.
4. Use of a lactobacillus salivarius strain B12WU as claimed in claim 1 or a bacterial agent as claimed in claim 2 for lowering blood glucose by inhibiting alpha amylase or alpha glucosidase activity.
5. Use of lactobacillus salivarius strain B12WU as claimed in claim 1 or a bacterial formulation as claimed in claim 2 in the manufacture of a product for the treatment or prophylaxis of diabetes.
6. Use of the lactobacillus salivarius strain B12WU as claimed in claim 1 or a bacterial agent as claimed in claim 2 as an ingredient in food or health care products.
7. The use according to any one of claims 3 to 6, wherein the Lactobacillus salivarius is one or more of an inactivated or non-inactivated fermentation supernatant, a bacterial suspension and a cell disruption supernatant.
8. Use according to claim 7, wherein the inactivation is pasteurization.
9. A medicament comprising the lactobacillus salivarius strain B12WU of claim 1 or the bacterial agent of claim 2, and a pharmaceutically acceptable carrier.
10. The medicament of claim 9, wherein the medicament is in a dosage form for oral administration.
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