CN113430153B - Lactobacillus reuteri ZJuuds 09 for reducing blood pressure and application thereof - Google Patents
Lactobacillus reuteri ZJuuds 09 for reducing blood pressure and application thereof Download PDFInfo
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- CN113430153B CN113430153B CN202110951511.1A CN202110951511A CN113430153B CN 113430153 B CN113430153 B CN 113430153B CN 202110951511 A CN202110951511 A CN 202110951511A CN 113430153 B CN113430153 B CN 113430153B
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- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/16—Agglomerating or granulating milk powder; Making instant milk powder; Products obtained thereby
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- A—HUMAN NECESSITIES
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- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- 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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/70—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor
- A23L13/72—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor using additives, e.g. by injection of solutions
- A23L13/74—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor using additives, e.g. by injection of solutions using microorganisms or enzymes
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- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
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- 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
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- A—HUMAN NECESSITIES
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- A61K35/66—Microorganisms or materials therefrom
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- A—HUMAN NECESSITIES
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- A61P9/12—Antihypertensives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23V2400/173—Reuteri
Abstract
The invention relates to the technical field of food microorganisms, and particularly discloses Lactobacillus reuteri ZJUIDS09 with the collection number of CGMCC NO. 22843. The invention also provides the application of the Lactobacillus reuteri ZJUIDS09 in preparing products with the function of reducing blood pressure.
Description
Technical Field
The invention relates to the technical field of food microorganisms, in particular to lactobacillus reuteri ZJUIDS09 with a blood pressure reducing function and application thereof.
Background
Hypertension has now become a metabolic disease in a large number of people in china and the world. Hypertension has serious influence on human health, and is accompanied by clinical syndromes of functional or organic damage of organs such as heart, brain, kidney, etc. Hypertension is the most common chronic disease and also the most major risk factor for cardiovascular and cerebrovascular diseases. Angiotensin Converting Enzyme (ACE) plays an important role in blood pressure, it converts Angiotensin I into Angiotensin II, which has a vasoconstrictive function, and it also hydrolyzes bradykinin, which has a vasodilatory action. Antihypertensive peptides in fermented milk can induce vasodilatory reaction by inhibiting ACE activity, thereby lowering blood pressure. Fermented milk is recommended as a non-drug treatment for hypertension, mainly because it has substantially no adverse side effects. The hypotensive effect of fermented milk is mainly due to the hypotensive peptides in the milk proteins, which peptides can be released during fermentation or gastrointestinal digestion by specific lactic acid bacteria.
Lactobacillus fermentation is an important way for producing antihypertensive peptides, and lactobacillus is closely related to human life and is one of beneficial microorganisms widely applied to the fields of food fermentation, industrial lactic acid fermentation and medical care. Lactobacillus reuteri, a kind of Lactobacillus, is added to food to improve the taste, texture and flavor of the food. The lactobacillus reuteri can also be planted in a human body to play a probiotic role, such as regulating intestinal flora, inhibiting the growth of intestinal pathogenic bacteria, reducing blood pressure, enhancing the immunity of the organism, improving lactose digestion, resisting tumors, resisting oxidation and the like.
CN110384719A invention, "Lactobacillus reuteri strain GMNL-263 for improving hypertension" and its composition "discloses Lactobacillus reuteri GMNL 263, which has the effect of lowering blood pressure by inhibiting IL 1 β, an inflammatory-promoting cytokine, and promoting the growth of Bifidobacterium for killing bacteria by heat. The scheme embodies the application effect of the composition of the lactobacillus reuteri strain GMNL-263 and the bifidobacterium in the crowd, and the inactivated lactobacillus reuteri strain GMNL-263 adopted in the scheme plays a role in inhibiting an inflammation-promoting cell hormone IL 1 beta and promoting bifidobacterium, but the GMNL-263 viable bacteria have a good blood pressure reducing effect.
Disclosure of Invention
The invention aims to solve the technical problem of providing lactobacillus reuteri ZJUIDS09 for reducing blood pressure and application thereof.
In order to solve the technical problem, the invention provides Lactobacillus reuteri ZJUIDS09 with the preservation number of CGMCC NO. 22843.
As an improvement of Lactobacillus reuteri (Lactobacillus reuteri) ZJUIDS09 of the present invention: the 16S rDNA full sequence is SEQ ID No: 1, the preparation method is as follows.
The invention also provides application of the Lactobacillus reuteri ZJUIDS09 in preparation of products with the function of reducing blood pressure.
The product with the function of reducing blood pressure is, for example, food, medicine, health care product and pet food with the function of reducing blood pressure.
The food with blood pressure lowering function comprises fermented fruit and vegetable juice (such as fermented pumpkin and dragon fruit juice) with blood pressure lowering function, yogurt with blood pressure lowering function, and cheese with blood pressure lowering function;
the feed having the function of lowering blood pressure is a pet food (for example, pet milk powder) having the function of lowering blood pressure.
As an improvement of the application of the invention:
the medicine with blood pressure lowering function comprises viable bacteria preparation with blood pressure lowering function, wherein viable count of Lactobacillus reuteri ZJuIDS09 is 1.0 × 1010～1×1011CFU/g。
The lactobacillus reuteri ZJUID 09 can be used for preparing silage with the function of reducing blood pressure by single fermentation or mixed fermentation with other strains.
The strain ZJUIDS09 of the invention has the preservation name: lactobacillus reuteri; the preservation unit: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.22843, preservation time 2021 year 07 month 07 day.
The Lactobacillus reuteri ZJUIDS09 strain is screened out, and the strain is identified by combining the morphological, physiological and culture characteristics of bacteria, 16S rDNA sequencing and the like.
The colony morphology characteristics of the Lactobacillus reuteri ZJUIDS09(Lactobacillus reuteri ZJUIDS09) provided by the invention are as follows: obvious colonies are formed on an MRS agar culture medium, and the size of the colonies is 0.3-1.5 mm. The colony is round, the edge is neat, white, and the surface is moist and smooth. The morphological characteristics of the thallus are as follows: gram staining is positive, no spore is produced, and the bacillus circulans is single, paired or short chain.
The ACE inhibition rate capability of the Lactobacillus reuteri ZJUIDS09(Lactobacillus reuteri ZJUIDS09) provided by the invention reaches 90.41%. It has the following capabilities:
1. has stronger ACE inhibition rate and IC50Namely, has stronger capability of reducing blood pressure.
2. The bacterium also has excellent oxidation resistance, can resist acid and bile salt, and has certain proliferation capacity; sensitivity to common antibiotics; has antibacterial activity;
namely, it can resist the gastrointestinal environment, has no antibiotic resistance, and can inhibit pathogenic bacteria harmful in the intestine.
In conclusion, the lactobacillus reuteri ZJUIDS09 with strong blood pressure lowering function is screened from probiotics separated from traditional fermented food. The strain has strong ACE inhibition rate and IC50The inhibition rate. The strain has obvious advantages in oxidation resistance, acid resistance and bile salt resistance compared with other lactic acid bacteria, is suitable for gastrointestinal environment and has proliferation capacity, and the ACE inhibition rate of products after in vivo digestion is also high. Has no antibiotic resistance and has antibacterial activity. The lactobacillus provided by the invention can be widely used for developing blood pressure reduction related probiotic functional products.
Drawings
FIG. 1 is a colony morphology chart of Lactobacillus reuteri ZJUIDS 09.
FIG. 2 is a gram-stained bacterial morphology of Lactobacillus reuteri ZJUIDS 09.
FIG. 3 is an electrophoretic identification chart of 16S rDNA of Lactobacillus reuteri ZJUIDS 09;
note: m is 5000, 3000, 2000, 1000, 750, 500, 250 and 100bp from top to bottom in sequence; lanes 1 and 2 are both PCR images of Lactobacillus reuteri ZJUIDS 09;
FIG. 4 is an example of a plate of Lactobacillus reuteri ZJUIDS09 sensitive to antibiotics;
in FIG. 4, 1 to 10 represent penicillin G, chloramphenicol, ampicillin, gentamicin, erythromycin, ciprofloxacin, lincomycin, trimethoprim, clarithromycin, and tetracycline, respectively.
FIG. 5 is an example of a plate of antibacterial activity of Lactobacillus reuteri ZJUIDS 09;
in fig. 5, the inhibition zones of salmonella, large intestine, staphylococcus aureus and listeria monocytogenes are shown from left to right.
FIG. 6 is a graph showing ACE inhibition efficiency of different yoghurts fermented by Lactobacillus reuteri ZJUIDS 09;
buffalo milk, buffalo whey, common cow milk and common whey are sequentially arranged from left to right.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 screening and characterization of lactobacillus reuteri ZJUIDS 09:
1. screening of lactic acid bacteria
1.1 sample sources
The strain used by the invention is derived from a dairy fan self-made by farmers in Yunnan. 15 parts of sample are collected, and the sample is collected, refrigerated immediately and mailed to a laboratory for separating and purifying the strain.
1.2 isolation and purification of the Strain
Approximately 5g of fresh sample was collected in a sterile tube and immediately sent to the laboratory for strain isolation. Putting 1g of sample into 9mL of MRS liquid culture medium, carrying out vortex mixing, and then carrying out enrichment culture at 37 ℃ for 48 h; then sucking 1mL of enrichment liquid in a super clean bench, performing tenfold gradient dilution by using sterile physiological saline, and selecting 10-6、10-7、10-8And (3) three dilution gradients, wherein 100 mu L of bacterial liquid of each gradient is taken and smeared on an MRS agar culture medium, and the bacterial liquid is cultured for 48h at 37 ℃. After the culture is finished, selecting a plate with 50-150 single colonies growing in an agar culture medium, picking typical colonies, carrying out streaking purification on an MRS agar plate for many times until the colony forms on the whole plate are consistent, and picking single colonies to an MRS liquid culture medium for enrichment culture. The obtained strains were all stored in MRS liquid medium containing 40% glycerol at-80 deg.C by freezing.
The obtained strain with good genetic stability was named ZJUIDS 09.
2. Identification of Lactobacillus reuteri ZJUIDS09
2.1 characteristics of the colonies
After lactobacillus reuteri ZJUIDS09 is cultured in MRS agar medium for 48 hours, the diameter is 0.3-1.5 mm, the colony is circular, the edge is neat, the color is white, and the surface is moist and smooth, as shown in figure 1.
2.2 microscopic morphology:
lactobacillus reuteri ZJUIDS09 colony smear: gram-positive, non-sporulating, rectus rotundus, single, paired, or short chain, see fig. 2.
2.316S rDNA identification
Extracting the target strain genome DNA by using an Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out 16S rDNA PCR experiment by using bacterial universal primers 27F and 1492R, and after the PCR amplification reaction is finished, taking a PCR product to carry out agarose gel detection and photographing, wherein the length of an amplified fragment is about 1.5kb, and the figure is 3. The PCR product was sent to Huada Gene (Wuhan) for sequencing, and the results are shown in SEQ ID NO: 1, BLAST sequence alignment on NCBI website, shows that the sequence has over 99% homology with the 16S rDNA sequence of Lactobacillus reuteri.
Combining the sequence alignment result of the strain ZJUIDS09 with the physiological and biochemical results to determine that the screened Lactobacillus ZJUIDS09 is Lactobacillus reuteri.
The deposit information of strain ZJUIDS09 is as follows: the preservation name is: lactobacillus reuteri; the preservation unit: china general microbiological culture Collection center, preservation Address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.22843, preservation time 2021 year 07 month 07 day.
Example 2 confirmation of the ability of Lactobacillus reuteri ZJuuds 09 to lower blood pressure
ACE inhibition ratio
Preparation of Strain fermentation broth
The strain ZJUIDS09 preserved in a glycerol tube is firstly streaked and activated for 2-3 times on an MRS agar plate, and then a single colony is picked up and is subjected to amplification culture for 18h at 37 ℃ in an MRS liquid culture medium to ensure that the concentration of the bacterial liquid is increasedTo about 108CFU/mL or so was used as a culture medium. The culture broth was inoculated into sterilized reconstituted skim milk (11%, w/v) at an inoculum size of 2% (vol%), incubated at 37 ℃ for 48 hours, then removed, and placed in a refrigerator at 4 ℃ overnight. Taking out the fermented milk from the refrigerator, adjusting the pH to 4.6, centrifuging 8000g for 10min to obtain supernatant, adjusting the pH to 8.2, centrifuging 8000g for 10min, and collecting the supernatant as the fermented supernatant of the strain ZJUIDS 09.
The sterilized reconstituted skim milk (11%, w/v) is: the skim reconstituted milk was prepared at a concentration of 11g/100ml and then sterilized at 121 ℃ for 15 minutes.
FAGG is used as a simulated substrate of angiotensin I, and the ACE inhibitory activity of the strain fermentation supernatant is determined by means of an enzyme-labeling instrument. The determination steps are as follows: measuring the absorbance A1 (before reaction) at 340nm on a 96-well enzyme label plate by using an enzyme label instrument according to the table 1, continuously reacting the reactant on the enzyme label plate at the constant temperature of 37 ℃ for 40min, measuring the absorbance A2 (after reaction) again, carrying out parallel experiments for 3 times, and taking an average value of the results. Δ a (Δ a ═ a1-a2) was calculated, and ACE enzyme activity was expressed as a change in absorbance per unit time, while PBS buffer was used as a negative control and other strains were used as control strains.
The change of the absorbance value at 340nm is measured by a microplate reader to represent the ACE enzyme activity, and the inhibition rate can be represented by the following formula:
in the formula: delta A is the change in absorbance within 40min when buffer is added; Δ B is the change in absorbance over 40min upon addition of inhibitor (fermentation supernatant). The experiments were performed in parallel 3 times, and the results were averaged.
TABLE 1 ACE inhibitory ratio measuring reagent and addition amount
IC50Measurement of (2)
The concentration of inhibitor achieved by the inhibitory activity isThe half inhibitory concentration was designated IC 50. Freeze-drying fermented milk strain (i.e. freeze-drying fermented supernatant of strain ZJUIDS09 at-40 deg.C for 24 hr), adding HEPES buffer solution (pH 8.3, 0.08M) to obtain 5 concentration gradients of 0.3, 0.5, 6, 15, and 21mg/mL, respectively, using the 5 concentration gradients as ACE inhibitor, determining ACE inhibition rate of each concentration gradient by the above method, drawing standard curve, and calculating ACE inhibition IC of sample by Graph Pad Prism 8 software50The value is obtained.
The inhibitor concentration at which the ACE inhibition rate reached 50% was half the inhibitory concentration and was recorded as IC50. Fermentation liquor preparation and ACE inhibition ratio (%) and IC of other strains50The measurement of (2) was as above.
TABLE 2 ACE inhibition (%) and IC of the supernatant from the fermentation of the strains50
Marked by significant difference, P <0.05
As shown in Table 2, the lactobacillus reuteri ZJUIDS09 screened by the invention is remarkably superior to other strains in the aspect of fermentation supernatant inhibition rate. IC of Lactobacillus reuteri ZJUIDS0950The content is remarkably low, which indicates that the lactobacillus reuteri ZJUIDS09 fermentation liquor can reach 50 percent of ACE inhibition rate under low concentration (0.33 g/L). ACE inhibition and IC50The results show that the lactobacillus reuteri ZJUIDS09 fermentation product has obvious blood pressure lowering effect.
The ACE inhibition ratio of the fermentation supernatant of Lactobacillus reuteri GMNL 263 was significantly lower than that of zjuid 09.
2. Simulation of ACE inhibition after gastrointestinal digestion
Mixing the fermented supernatant (50mL) with artificial gastric juice at a ratio of 2:1(v/v), digesting in a constant temperature water bath oscillator at 37 ℃ for 2h, heating in a boiling water bath for 10min to terminate the reaction, and sampling to determine the ACE inhibition rate; mixing the residual sample (50mL) digested by artificial gastric juice with artificial intestinal juice at a ratio of 1:1(v/v), then digesting for 2h in a thermostatic waterbath oscillator at 37 ℃, and heating in boiling water bath for 10min to terminate the reaction. Sampling and determining ACE inhibitory activity, and determining ACE inhibitory rate.
Preparing artificial gastric juice: 1.6mL of dilute hydrochloric acid (hydrochloric acid having a concentration of 2M) was added with 80mL of water and 1g of pepsin, and after mixing them well, the mixture was diluted with water to 100 mL.
Preparing artificial intestinal juice: 0.68g of monopotassium phosphate is taken and dissolved in 100mL of water, 0.1mol/L of sodium hydroxide solution is taken to adjust the pH value to 6.8, 10g of trypsin is added, and after uniform mixing, the mixture is diluted to 1000 mL.
TABLE 3 simulated post-digestion ACE inhibition (%)
abc represents significant differences, P <0.05
The results in table 3 show that the fermentation supernatant of lactobacillus reuteri zjuid 09 has 76.39% ACE inhibition rate after passing through simulated gastric juice and intestinal juice, which is significantly higher than the fermentation broth of other strains. The lactobacillus reuteri ZJUIDS09 fermentation liquid can play a good blood pressure reducing effect in the gastrointestinal tract of a human body.
Example 3 confirmation of antioxidant Capacity of Lactobacillus reuteri ZJuuds 09
The culture solution prepared in example 2 was centrifuged at 8000r/min at 4 ℃ for 5min to collect mycelia, washed 2 times with phosphate buffer (pH 6.8), and suspended in MRS liquid medium to adjust viable cell count to about 108CFU/mL to obtain a bacterial suspension;
the bacterial suspension and the fermentation supernatant prepared in example 2 were used as samples. Meanwhile, the antioxidant activity is measured by taking lactobacillus rhamnosus ATCC53103 as a positive control strain.
1. Total antioxidant capacity (FRAP method)
The method for measuring the total antioxidant capacity was slightly modified according to the method of Giuberti et al. To the microplate, 150. mu.L of TPTZ working solution (0.3M acetic acid-sodium acetate buffer, 20mM ferric chloride solution, 10mM TPTZ buffer, mixed at V: V: V: 10:1:1, ready-to-use) and 20. mu.L of the sample were added, mixed by shaking, reacted at 37 ℃ for 10min, and the absorbance of the solution at 593nm was measured. The absorbance measured by the sample is substituted into a ferrous sulfate standard curve, and the antioxidant capacity of the sample is expressed by ferrous sulfate equivalent (mu mol FeSO4/mL sample). Each sample was repeated 3 times and averaged.
Ferrous sulfate standard curve: ferrous sulfate solutions with different mass concentrations (0. mu.M, 50. mu.M, 100. mu.M, 200. mu.M, 400. mu.M, 600. mu.M, 800. mu.M) were prepared, ferrous sulfate solutions with different molar concentrations, 10mM TPTZ buffer solution, and 0.3M acetate buffer solution were mixed at V: V: V ═ 1:1:10, 170. mu.L of the mixed solution was added to an ELISA plate, the reaction was carried out at 37 ℃ for 10min, and the absorbance of the solution at 593nm was measured. And drawing a standard curve by taking the absorbance as a vertical coordinate and the ferrous sulfate mass concentration as a horizontal coordinate.
2. Reducing power
The reduction ability was measured by the method of Lin et al with some modifications. 1mL of sample was put into a centrifuge tube, and 1mL of each of 0.2M PBS buffer solution (pH6.6) and 1% (w/v) potassium ferricyanide solution was added and mixed. Water bath at 50 deg.c for 20min and ice bath cooling. Then adding 1mL of 10% trichloroacetic acid, centrifuging at 6000r/min for 5min, taking 1mL of supernatant, adding 1mL of 0.1% (w/v) ferric trichloride and 1mL of distilled water, mixing uniformly, standing for reaction for 10min, and measuring the absorbance at 700 nm. The samples were replaced with PBS buffer or MRS broth medium as a blank. Each sample was repeated 3 times and averaged.
Reducing power (%) [ (As-Ab)/Ab ]. multidot.100
In the formula: as-absorbance of the sample set;
ab-blank absorbance.
3. DPPH radical scavenging ability
The DPPH radical scavenging ability was determined by reference to Shimada et al with some modifications. Preparing 1000mg/ml VC standard solution, and diluting to different concentration gradients (0-30 mug/ml). Adding 100 μ L of sample to be tested (or VC standard solution) and 100 μ L of 0.2mM DPPH ethanol solution (prepared with absolute ethanol, stored at 4 deg.C in dark place, and used as prepared) into an ELISA plate, shaking, keeping dark for 30min at room temperature, and measuring the absorbance of the solution at 517 nm; replacing 100 mu L of ethanol solution of LDPPH with 100 mu L of absolute ethanol to obtain a blank group; the control group was prepared by replacing 100. mu.L of the test sample with 100. mu.L of PBS buffer (or MRS broth), and the blank was zeroed with 100. mu.L of a mixture of PBS buffer (or MRS broth) and absolute ethanol. Each sample was repeated 3 times and averaged.
DPPH radical scavenging capacity (%) [1- (As-Ab)/Ac ]. 100
In the formula: as-absorbance of the sample set;
ab-blank absorbance;
ac-control absorbance;
the resulting structure is shown in table 4.
TABLE 4 antioxidant Activity of Lactobacillus reuteri ZJUIDS09
Indicates significant difference, P < 0.05; marked differences, P < 0.01.
The total antioxidant capacity, the reducing capacity and the DPPH free radical of the fermentation supernatant fluid of the lactobacillus reuteri ZJUIDS09 screened by the invention are all obviously higher than those of the standard strain lactobacillus rhamnosus ATCC 53103. Thus, the lactobacillus reuteri ZJUIDS09 fermentation supernatant and bacterial suspension have high antioxidant capacity.
The total antioxidant capacity of both the Lactobacillus reuteri GMNL 263 suspension and the fermentation supernatant was far inferior to that of Lactobacillus rhamnosus ATCC 53103.
Example 4 confirmation of acid resistance and cholate resistance of Lactobacillus reuteri ZJUIDS09
1. Acid resistance test
The culture solution prepared in example 2 was centrifuged at 8000r/min at 4 ℃ for 5min to collect cells, which were washed 2 times with phosphate buffer (pH 6.8), and the cells were suspended in MRS liquid medium adjusted to pH 3.0 to adjust viable cell count to about 108CFU/mL to obtain bacterial suspension for acid-resistant experiments;
directly (culturing at 37 ℃ for 0h) carrying out bacterial colony counting on the bacterial suspension for the acid resistance experiment by adopting a pouring plate method (culturing at 37 ℃ for 48h), and recording the obtained number as N0；
Culturing the bacterial suspension for acid resistance experiments at 37 ℃ for 3h, then counting colonies by adopting a pour plate method (culturing at 37 ℃ for 48h), and recording the number of the obtained colonies as Nt；
The acid-resistant survival rate calculation formula is as follows:
in the above formula, N0The number of viable bacteria (CFU/mL) of the test strain is 0 h; n is a radical oftTo test the viable count of the strain for 3h (CFU/mL).
2. Bile salt resistance test
The bacterial suspension (obtained in example 3) was inoculated in an amount of 10% to MRS liquid medium containing 0.3% (m/v) of bovine bile salt (control is MRS liquid medium containing no bovine bile salt), and cultured at 37 ℃ for 3 hours. The number of viable bacteria in the sample was then counted using the pour plate method. The poured plates were incubated at 37 ℃ for 48 h. The bile salt tolerance of the strain is expressed as the logarithm of the difference between the viable count per mL of medium containing bile salts and the viable count per mL of medium without bile salts at 3h (log CFU/mL).
3. And (3) adopting different strains to carry out the acid resistance and cholate resistance performance measurement.
4. As shown in Table 4, the acid and bile salt resistance of Lactobacillus reuteri ZJUIDS09 was significantly better than that of the control strain. Its survival rate in MRS medium with pH 3.0 is as high as 98%. The viable count in the environment containing 0.3% of ox bile salt still reaches 106CFU/mL or above indicates that the bile salt tolerance is better. Experiments prove that the lactobacillus reuteri ZJUIDS09 has higher gastrointestinal tract viability.
TABLE 5 results of the strains' tolerance to acids and bile salts
5. Probiotics must be able to survive a range of adverse environments such as gastric acid and bile in the gastrointestinal tract to exert their probiotic effects. The lactobacillus reuteri ZJUIDS09 provided by the invention can grow and proliferate under the condition of pH 3.0, and can smoothly pass through the acidic environment in the stomach to reach the small intestine. Meanwhile, the lactobacillus reuteri ZJUIDS09 can resist bile salt, can survive in intestinal tracts and further can effectively play a role in reducing blood pressure.
Lactobacillus reuteri (Lactobacillus reuteri) GMNL 263 bile salt tolerance is far less than that of Lactobacillus reuteri ZJUIDS09 of the present invention.
Example 5 confirmation of the hydrophobic Capacity of Lactobacillus reuteri ZJUIDS09
1. Measurement of hydrophobicity
Lactobacillus reuteri ZJUIDS09 cells (cells prepared in step 1 of example 3) were washed twice with clean PBS buffer (0.2M, pH6.8) and resuspended to OD610The absorbance of the strain is about 0.5, and a suspension of the lactobacillus reuteri ZJUIDS09 bacteria is obtained; suspensions of other bacteria were prepared as above.
Thoroughly mixing 2ml of Lactobacillus reuteri ZJUIDS09 suspension and 2ml of xylene, shaking in water bath at 37 deg.C for 5min, and measuring OD of water phase after 0h and 2h respectively610And (4) light absorption value.
A0Absorbance of 0h, AtTh absorbance.
The results obtained are shown in the following table.
TABLE 6 surface hydrophobicity of different strains (%)
2. Analysis of results
The hydrophobicity of lactobacillus reuteri ZJUIDS09 was measured to be 51.43% significantly higher than other strains. The result shows that the strain has stronger adhesive capacity, can be adhered to the intestinal tract of a human and improves the health of intestinal flora.
Example 6 confirmation of antibiotic susceptibility of Lactobacillus reuteri ZJUIDS09
To a concentration of about 108The suspension of the CFU/mL Lactobacillus reuteri ZJUIDS09 strain is added into sterilized MRS agar culture medium cooled to about 45 ℃ according to the amount of 1%, mixed fully, and added into 15 mL/dish quantitatively. After coagulation, the drug sensitive paper is taken out with tweezers and placed on the culture medium. The plate was placed right side up in a 37 ℃ incubator for 24 h. Paper without antibiotic was used as a blank control. And measuring the diameter of the inhibition zone. Each was repeated three times.
The diameters of the antibiotic-sensitive zone of inhibition of Lactobacillus reuteri ZJUIDS09 are shown in Table 7. With reference to CLSI (2017) drug susceptibility test standards, Lactobacillus reuteri ZJUIDS09 exhibits sensitivity to penicillin, clarithromycin, tetracycline, erythromycin, lincomycin, and chloramphenicol. Intermediaries are presented for gentamicin. The experimental results show that lactobacillus reuteri zjuid 09 is sensitive to common antibiotics.
TABLE 7 results of susceptibility of Lactobacillus reuteri ZJUIDS09 to antibiotics
Note: s, sensitivity; i, an intermediary; r, drug resistance
With the wide application of antibiotics in clinical treatment, the drug resistance of lactic acid bacteria is more and more serious, and the intake of drug-resistant lactic acid bacteria for a long time brings great difficulty to clinical treatment. The lactobacillus reuteri ZJUIDS09 provided by the invention is sensitive to common antibiotics and cannot cause harm to human health.
Example 7 confirmation of the inhibitory Activity against pathogenic bacteria of Lactobacillus reuteri ZJUIDS09
The antibacterial activity of the lactic acid bacteria is determined by adopting an international universal agar diffusion method. 10mL of LB agar medium was poured into a sterile petri dish, and cooled to prepare a lower layer medium. The concentration of the bacteria is about 107Adding the CFU/mL indicator bacterium suspension in an amount of 1% and cooling toSterilized LB agar medium at about 45 ℃ was mixed well and added quantitatively to a 10 ml/dish. Placing the sterilized oxford cup on the upper surface. After the upper medium was condensed, the Oxford cup was gently pulled out. A sample of fermentation supernatant of Lactobacillus reuteri ZJUIDS09 was added at 100. mu.L/well and phosphate buffer (0.2M, pH6.8) was used as a control. The strains with obvious inhibition zones around the small holes are selected, the diameters of the inhibition zones are measured, and each is repeated three times.
As shown in Table 8, the metabolite of Lactobacillus reuteri ZJUIDS09 has certain inhibitory effect on Staphylococcus aureus, Escherichia coli and Salmonella enteritidis, and is superior to the bacteriostatic effect of the control lincomycin. The metabolite of the strain can be seen to have bacteriostatic properties.
TABLE 8 results of the inhibitory potency of the strains on pathogenic bacteria
Staphylococcus aureus is the most common pathogenic bacterium in human pyogenic infections, Escherichia coli can cause severe diarrhea and septicemia, and Salmonella species can also cause food poisoning in humans. The bacteriocin, organic acid, hydrogen peroxide and other bacteriostatic substances generated by the metabolism of the lactobacillus reuteri ZJUIDS09 can individually or jointly inhibit the growth of the pathogenic bacteria. The metabolite of the lactobacillus reuteri ZJUIDS09 provided by the invention has better antibacterial effects on staphylococcus aureus, escherichia coli and listeria monocytogenes than the control, and shows that the metabolite has obvious antibacterial effects on the three bacteria, plays an important role in maintaining intestinal microecological balance and has a health promotion effect.
Example 8 preparation of functional fermented yogurts and whey Using Lactobacillus reuteri ZJUIDS09
(1) Raw materials: 2L of whole UHT sterilized milk or fresh cow's milk, whey, buffalo milk, buffalo whey;
(2) preheating: putting into a container and heating to 63 ℃;
(3) homogenizing and sterilizing: homogenizing in homogenizer under pressure of 15-25MPa, pouring the mixed solution into iron tank, adding 100g white sugar, and sterilizing in 90 deg.C water bath environment for 10 min;
(4) and (3) cooling: cooling the sterilized milk to 40-50 ℃ for later use;
(5) preparing a leaven: lactobacillus reuteri ZJUIDS09 strain was inoculated into a test tube containing sterilized skim milk (12%, w/v) under aseptic conditions and cultured at 37 ℃ for 20 hours. The inoculation amount of each passage is 2-4% (v/v), the vitality is restored after 2-3 passages, and the mixture is placed in a refrigerator at 4 ℃ for preservation;
(6) inoculation and fermentation: inoculating activated Lactobacillus reuteri ZJUIDS09 into cow milk or whey under aseptic condition, wherein the inoculation amount is 2-4% (v/v); fermenting at 42 deg.C for 6-10 h;
(7) after-ripening: after fermentation, putting the mixture into a refrigerator with the temperature of 4 ℃ for after-ripening for 12-24 h;
(8) filling and refrigerating: after the after-ripening was completed, the mixture was filled into 250mL sterilized glass bottles and sent to a freezer for refrigeration.
The prepared yoghurt or yoghurt whey was tested for ACE inhibition as described in example 2 and the results are shown in figure 6. The results in fig. 6 show that the ordinary cow's milk and buffalo milk prepared by lactobacillus reuteri zjuid 09 both have an ACE inhibition rate of nearly 90%, and whey also has an ACE inhibition rate of more than 35%, indicating that lactobacillus reuteri zjuid 09 is suitable for preparing antihypertensive yogurt.
Example 9 preparation of functional fermented fruit and vegetable juice Using Lactobacillus reuteri ZJUIDS09
(1) Raw materials: selecting fresh pumpkins and dragon fruits;
(2) cleaning and cutting: cleaning, peeling (removing pulp from fructus Cucurbitae Moschatae), and cutting into small pieces;
(3) flash evaporation: inactivating enzyme by a flash evaporation method, treating for 0.5-1 min at 121 ℃, and rapidly exhausting;
(4) pulping: according to the weight ratio of 1:1, the pumpkin and water are gradually put into a colloid mill to be ground, and coarse grinding and fine grinding are carried out once respectively; pulping the dragon fruit by a pulping machine until the pulp is uniform and has no blocks;
(5) blending and homogenizing: according to the weight percentage, 15 percent of pumpkin juice, 30 percent of dragon fruit juice, 3 percent of cane sugar and the balance of water; adding 0.2% of CMC (carboxymethyl cellulose) as a stabilizer, uniformly mixing, and adopting a two-stage homogenization method, wherein the low pressure (15MPa) is firstly adopted, and the high pressure (25MPa) is secondly adopted, so that the diameter and the grain diameter of the melon pulp particles are 2-3 mu m.
(6) And (3) sterilization and cooling: keeping the temperature of the blended composite fruit and vegetable juice at 100 ℃ for 10min, and cooling to about 40 ℃.
(7) Inoculation and fermentation: inoculating activated Lactobacillus reuteri ZJuuds 09 under aseptic condition, and controlling initial bacterial count at 107CFU/mL. Fermenting at 37 deg.C for 24 h.
(8) After-ripening: after the fermentation is finished, putting the mixture into a refrigerator with the temperature of 4 ℃ for 3 hours.
(9) Filling and refrigerating: after the after-ripening was completed, the mixture was filled into 250mL sterilized glass bottles and sent to a freezer for refrigeration.
Example 10 preparation of functional fermented sour meat Using Lactobacillus reuteri ZJUIDS09
(1) Slicing: commercially available fresh streaky pork was cut into 3cm square slices.
(2) Mixing glutinous rice flour: 1000g of raw meat is mixed with 250g of glutinous rice flour, and 1% of glucose is added.
(3) Inoculation and fermentation: inoculating activated Lactobacillus reuteri ZJuuds 09 under aseptic condition, and controlling initial bacterial count at 107CFU/mL. Fermenting at 37 deg.C for 18 h.
(4) Pickling: adding 2% salt, and pickling at 25 deg.C for 20 d.
Example 11 preparation of antihypertensive bacteria powder using Lactobacillus reuteri ZJUIDS09
1. Preparation of lactobacillus reuteri ZJUIDS09 bacterial paste
A single colony of Lactobacillus reuteri ZJUIDS09 is selected and inoculated in 50mL of MRS liquid culture medium, and the mixture is placed in an incubator at 37 ℃ for culturing for 18 h. Then activated in 250mL MRS liquid culture medium according to the inoculum size of 5 percent, and cultured for 24h in an incubator at 37 ℃. Finally, the activated Lactobacillus reuteri ZJUIDS09 was cultured at 5% inoculum size in 10L fermentor at high density and at 37 deg.C and pH6.8 for 18 h. Then centrifuging at 8000r/min and 4 deg.C for 15min, discarding supernatant, collecting thallus precipitate, and rinsing thallus with sterile phosphate buffer (pH 6.8) for 2 times. Thus obtaining the lactobacillus reuteri ZJUIDS09 bacterial mud.
2. Preparation of the protective agent
The freeze-drying protective agent contains 15% of skim milk powder, 5% of trehalose, 3% of sodium glutamate, 1% of glycerol and 0.5% of cysteine hydrochloride. Water is used as the solvent. Sterilizing at 110 deg.C for use.
3. Preparation of lactobacillus reuteri ZJUIDS09 bacterial powder
The prepared lactobacillus reuteri ZJUIDS09 thallus precipitate is fully mixed with a protective agent solution according to the proportion of 1: 5. Pre-freezing for 5h at-40 ℃ to uniformly freeze the lactobacillus reuteri powder on the inner wall of the container, then carrying out vacuum freeze drying, and drying for 18-20 h to obtain the lactobacillus reuteri ZJUIDS09 powder. Rehydrating with normal saline, washing twice, and determining viable count of Lactobacillus reuteri ZJUIDS09 powder to be 1.0 × 1010～1×1011CFU/g。
Example 12 preparation of probiotic milk powder for pets Using Lactobacillus reuteri ZJUIDS09
1. Preparation of lactobacillus reuteri ZJUIDS09 bacterial powder
Lyophilized powder of Lactobacillus reuteri ZJUIDS09 strain prepared in reference example 11, the viable count of the strain powder was 1.0X 1010-1×1011CFU/g。
2. Preparation of pet formula powder
Primary selection of raw materials: milk powder, fish meal, bone meal, grains, vegetable oil and additives: vitamins, trace elements, functional factors, and others;
automatic batching: putting the obtained material raw materials into a material bin according to a formula;
crushing: crushing the weighed materials by a crusher;
mixing: adding vegetable oil and trace elements into the crushed materials, and adding the materials into a mixer for uniformly mixing;
puffing: the mixed materials are made into granular materials through a bulking machine;
drying: drying the mixed materials by a dryer, and controlling the temperature to be 65-70 ℃;
grading and screening: passing the material flow through a grading sieve, and controlling the particle size to be 2.5-5 mm;
3. preparation of probiotic formula powder for pets
Mixing the bacterial powder prepared in the step 1 and the pet feed prepared in the step 2 according to the ratio of 1: 1000, and the live bacteria leaving the factory in the final product are 108CFU/g is higher than the standard. And (5) after filling, storing and selling the product.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Zhejiang university
<120> Lactobacillus reuteri ZJUIDS09 for reducing blood pressure and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1402
<212> DNA
<213> Lactobacillus reuteri (Lactobacillus reuteri)
<400> 1
tggctcagga tgaacgccgg cggtgtgcct aatacatgca agtcgtacgc actggcccaa 60
ctgattgatg gtgcttgcac ctgattgacg atggatcacc agtgagtggc ggacgggtga 120
gtaacacgta ggtaacctgc cccggagcgg gggataacat ttggaaacag atgctaatac 180
cgcataacaa caaaagccac atggcttttg cttgaaagat ggctttggct atcactctgg 240
gatggacctg cggtgcatta gctagttggt aaggtaacgg cttaccaagg cgatgatgca 300
tagccgagtt gagagactga tcggccacaa tggaactgag acacggtcca tactcctacg 360
ggaggcagca gtagggaatc ttccacaatg ggcgcaagcc tgatggagca acaccgcgtg 420
agtgaagaag ggtttcggct cgtaaagctc tgttgttgga gaagaacgtg cgtgagagta 480
actgttcacg cagtgacggt atccaaccag aaagtcacgg ctaactacgt gccagcagcc 540
gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaagc gagcgcaggc 600
ggttgcttag gtctgatgtg aaagccttcg gcttaaccga agaagtgcat cggaaaccgg 660
gcgacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtggaa tgcgtagata 720
tatggaagaa caccagtggc gaaggcggct gtctggtctg caactgacgc tgaggctcga 780
aagcatgggt agcgaacagg attagatacc ctggtagtcc atgccgtaaa cgatgagtgc 840
taggtgttgg agggtttccg cccttcagtg ccggagctaa cgcattaagc actccgcctg 900
gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 960
agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atcttgcgct 1020
aaccttagag ataaggcgtt cccttcgggg acgcaatgac aggtggtgca tggtcgtcgt 1080
cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgttactagt 1140
tgccagcatt aagttgggca ctctagtgag actgccggtg acaaaccgga ggaaggtggg 1200
gacgacgtca gatcatcatg ccccttatga cctgggctac acacgtgcta caatggacgg 1260
tacaacgagt cgcaagctcg cgagagtaag ctaatctctt aagccgttct cagttcggac 1320
tgtaggctgc aactcgccta cacgaagtcg gaatcgctag taatcgcgga tcagcatgcc 1380
gcggtgaata cgttcccggg cc 1402
Claims (4)
1. Lactobacillus reuteri: (Lactobacillus reuteri）ZJuIDS09, characterized in that: the preservation number is CGMCC NO. 22843.
2. Lactobacillus reuteri (L) according to claim 1 (L., (L.))Lactobacillus reuteri）ZJuIDS09, characterized in that: the 16S rDNA full sequence is SEQ ID No: 1, the preparation method is as follows.
3. Lactobacillus reuteri (L) according to claim 1 or 2 (L.), (L.) according to claim 2Lactobacillus reuteri）The application of ZJuIDS09 in preparing medicine with blood pressure lowering effect is provided.
4. Use according to claim 3, characterized in that:
the medicine with the function of reducing blood pressure comprises a live bacteria preparation with the function of reducing blood pressure, and lactobacillus reuteri (L.) (Lactobacillus reuteri）The viable count of ZJuIDS09 was 1.0X 1010~1.0×1011 CFU/g。
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