CN113350383B - Bifidobacterium lactis BL-99 capable of resisting oxidation and regulating blood pressure and application thereof - Google Patents
Bifidobacterium lactis BL-99 capable of resisting oxidation and regulating blood pressure and application thereof Download PDFInfo
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Abstract
The invention provides bifidobacterium lactis BL-99 for resisting oxidation and regulating blood pressure and application thereof. The invention firstly provides application of Bifidobacterium lactis (Bifidobacterium lactis) BL-99 in preparing a composition with the effects of resisting oxidation, regulating blood pressure and/or regulating sleep, wherein the preservation number of the Bifidobacterium lactis is CGMCC No.15650. The study of the invention finds that the bifidobacterium lactis strain fermented emulsion has the application in the directions of resisting oxidation, regulating blood pressure, regulating sleep and the like, and can be widely applied to the fields of food, medicine, feed, chemical industry and the like.
Description
Technical Field
The invention relates to the technical field of microbial fermentation, in particular to related application of Bifidobacterium lactis BL-99 (with the preservation number of CGMCC No. 15650) in fermentation preparation of a composition with effects of resisting oxidation, regulating blood pressure and/or regulating sleep.
Background
Aging is the biggest killer of human organs, and free radicals (free radials) generated by the body are the most important cause of organ aging. When the body's mechanisms regulating free radicals are lost or unbalanced, excessive free radical production can damage cellular DNA, alter intracellular protein structures, attack cell membranes, and ultimately lead to somatic cell death. Skin aging is also caused by a rapid increase in the amount of active oxygen induced by long-term exposure to ultraviolet (UV exposure) or heat (heat exposure), resulting in diseases such as skin darkness and skin cancer.
Hypertension, hyperlipemia and hyperglycemia are collectively called as "three highs", and is a cardiovascular and cerebrovascular disease which is harmful to human health. According to the speculation of experts, the number of hypertension patients worldwide will exceed 15 hundred million in 2025. Effective treatment or prevention of hypertensive disorders has become a worldwide health problem. At present, the related research and products of the fermented dairy products with the effect of reducing blood pressure mainly focus on simple addition and application of functional components.
From the last 90 century, the research on the antihypertensive of the relevant microorganisms, especially lactobacillus, is highly regarded, and related documents report that the lactobacillus can reduce the concentration of serum cholesterol, improve the SOD activity in serum and reduce the level of lipid peroxide, and has the functions of assisting in reducing blood pressure, blood fat and resisting oxidation, but the functions of assisting in reducing blood pressure, blood fat and resisting oxidation of the lactobacillus have the difference of strains and even strains, and the effect of the lactobacillus applied to the specific fermented dairy product needs to be further examined.
Disclosure of Invention
An object of the present invention is to provide a novel use of bifidobacterium lactis BL-99.
Bifidobacterium lactis BL-99 strain has been deposited in 26.04.2018 in China general microbiological culture Collection center CGMCC (address: no. 3, institute of microbiology, national academy of sciences, north Chen Xilu No.1, beijing, inward region), and is named after classification: bifidobacterium lactis (Bifidobacterium lactis); the preservation number is CGMCC No.15650.
The invention discovers that Bifidobacterium lactis (Bifidobacterium lactis) BL-99 takes feed liquid (milk substrate) mainly containing milk components as a fermentation substrate, and the fermentation product contains substances such as superoxide dismutase, angiotensin converting enzyme inhibitor, gamma-aminobutyric acid, B vitamins and the like, has excellent total antioxidant (T-AOC) capability, and has the effects of resisting oxidation, regulating blood pressure and/or regulating sleep.
Therefore, the invention provides the application of Bifidobacterium lactis (Bifidobacterium lactis) in preparing a composition with the effects of resisting oxidation, regulating blood pressure and/or regulating sleep, wherein the preservation number of the Bifidobacterium lactis is CGMCC No.15650.
According to a specific embodiment of the present invention, bifidobacterium lactis is fermented to produce superoxide dismutase, angiotensin converting enzyme inhibitor, gamma-aminobutyric acid and/or B vitamins to have antioxidant, blood pressure regulating and/or sleep regulating effects.
According to a specific embodiment of the present invention, the bifidobacterium lactis of the present invention is used in the preparation of a composition having antioxidant, blood pressure regulating and/or sleep regulating effects, which may be a food composition, a feed composition, a cosmetic composition or a pharmaceutical composition.
According to a specific embodiment of the invention, in the application of the invention, bifidobacterium lactis CGMCC No.15650 is used for fermentation, and a fermentation product is prepared and used as or further for preparing the composition with the effects of resisting oxidation, regulating blood pressure and/or regulating sleep.
According to a particular embodiment of the invention, the fermentation substrate used in the fermentation may be an emulsion. Preferably, the milk may be raw milk or reconstituted milk, and may be whole milk, low fat milk or skim milk. The emulsion may optionally contain 5% to 10% sucrose.
According to a specific embodiment of the present invention, the fermentation conditions are: fermenting for 1-7 days at 35-45 ℃.
According to a particular embodiment of the invention, the bifidobacterium lactis is inoculated in the fermentation substrate in the form of a seed liquid, in an amount of between 2% and 5%. The preparation of the seed liquid may be carried out according to conventional techniques in the probiotic field.
According to a specific embodiment of the present invention, the milk substrate used as the fermentation substrate of bifidobacterium lactis BL-99 in the present invention may be milk (whole milk, low-fat milk, or skim milk) containing milk of 80% or more, and if necessary, auxiliary materials such as sweeteners and stabilizers may be optionally added, for example, fermentation substrates commonly used in the preparation of fermented milk or milk beverages, and the production of angiotensin converting enzyme inhibitors, superoxide dismutase, γ -aminobutyric acid, B-group vitamins, and the like can be achieved by the fermentation of bifidobacterium lactis BL-99 in the present invention.
Angiotensin Converting Enzyme (ACE), also known as kininase II or peptidyl-carboxypeptidase, belongs to the vascular endothelial cell membrane binding enzyme. The main functions of ACE are: catalyzes the conversion of angiotensin I to angiotensin II; inactivating bradykinin. Because of the two functions, angiotensin converting enzyme becomes an ideal target for treating diseases such as hypertension.
Superoxide Dismutase (SOD) is an antioxidant metalloenzyme existing in organisms, can catalyze Superoxide anion free radical disproportionation to generate oxygen and hydrogen peroxide, and plays a vital role in the balance of oxidation and antioxidation of the organisms.
Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid, is used as an important inhibitory neurotransmitter, and has various physiological functions of soothing the nerves, reducing blood pressure, improving sleep and the like for mammals. It has been reported that GABA plays an antioxidant role as a regulator of oxidative metabolites, arabidopsis thaliana SSADH mutants grow under high temperature, and active oxygen intermediates (ROIs) of the arabidopsis thaliana SSADH mutants accumulate to cause plant death, while GABA can reduce the accumulation of the ROIs to prevent organisms from oxidative damage and peroxidation death caused by high temperature.
The antioxidant efficacy of the B vitamins is well known and can also help to remove liver fat, lower cholesterol, prevent arteriosclerosis. In addition, vitamin B can prevent sunburn and skin cancer, keep skin smooth and moist, delay the appearance of wrinkles and promote healthy hair growth. The research reports that the vitamin B in the B vitamins 2 Vitamin B 3 Vitamin B 6 The same degree participates in the oxidation-reduction process of organisms, and plays an important role in an antioxidant defense system in vivo.
According to a specific embodiment of the invention, superoxide dismutase, angiotensin converting enzyme inhibitor, gamma-aminobutyric acid and/or B vitamins in the supernatant of BL-99 fermented emulsion are measured, and the supernatant of the fermented product is used as or further used for preparing the composition with the effects of resisting oxidation, regulating blood pressure and/or regulating sleep.
According to some embodiments of the present invention, the Angiotensin Converting Enzyme (ACE) inhibition ratio of the bifidobacterium lactis BL-99 fermented emulsion of the present invention can reach 10 to 55%.
According to some embodiments of the invention, the total antioxidant capacity (T-AOC) of the bifidobacterium lactis BL-99 fermented emulsion reaches 1.0-2.5U/mL, and the total antioxidant capacity decreases slowly with the progress of the fermentation process.
According to some embodiments of the invention, the bifidobacterium lactis BL-99 fermented emulsion has a superoxide anion radical scavenging capacity of 20 to 40%.
According to some specific embodiments of the invention, the total superoxide dismutase (T-SOD) activity of the Bifidobacterium lactis BL-99 fermented emulsion can reach 20-30U/mL.
According to some embodiments of the present invention, the content of gamma-aminobutyric acid (GABA) in the Bifidobacterium lactis BL-99 fermented emulsion of the present invention may be 10 to 20 μ g/mL.
According to some embodiments of the invention, vitamin B is present in the Bifidobacterium lactis BL-99 fermented emulsion of the invention 1 The content can reach 60-100 mug/mL; vitamin B 2 The content can reach 2.8-3.1 mug/mL; vitamin B 3 40-60 mug/mL; vitamin B 6 The content is 9-14 mu g/mL; vitamin B 9 The content of (b) is 1.5-1.6 mu g/mL.
According to a specific embodiment of the present invention, the fermentation product obtained from the fermented milk substrate of bifidobacterium lactis BL-99 of the present invention can be directly used as food (e.g. fermented milk, milk drink, milk powder, solid beverage, etc.), daily chemical (cosmetics, etc.), feed (including pet food or probiotic product for animal husbandry), or medicine, etc., or be further used for preparing such food, daily chemical, feed, or medicine product. The process for preparing food, daily chemical, feed or pharmaceutical products from the fermentation product can be carried out with reference to the prior art in the field.
In a particular embodiment of the present invention, the BL-99 fermentation product of the present invention is used as or further for the preparation of a food product, preferably a fermented milk or a fermented milk beverage. The emulsion used as a fermentation substrate may further comprise other materials commonly used in fermented milk or fermented milk drinks, such as appropriate amounts of sweeteners, stabilizers, nutritional additives, and the like. The fermented milk or fermented milk beverage may be fermented milk or fermented milk beverage containing active bacteria preserved at low temperatureThe fermented milk may be sterilized fermented milk or fermented milk beverage stored at room temperature. In some more specific embodiments of the present invention, the fermented milk or fermented milk beverage has bifidobacterium lactis CGMCC No.15650 in an amount of 1 × 10 7 cfu/mL～1×10 11 cfu/mL。
In conclusion, the invention provides a new application of bifidobacterium lactis BL-99, and the invention discovers that the strain single-bacterium fermented milk substrate can generate substances such as superoxide dismutase, angiotensin converting enzyme inhibitor, gamma-aminobutyric acid, B vitamins and the like, and has the potential of resisting oxidation, regulating blood pressure and regulating sleep. The bifidobacterium lactis BL-99 strain can be used for producing fermented milk, milk drinks, milk powder, solid beverages, daily chemicals and the like with the effects of resisting oxidation, regulating blood pressure and regulating sleep.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description taken in conjunction with the accompanying specific embodiments, and the technical solutions of the present invention are described, it being understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
Example 1: determination of growth dynamics characteristics of fermented milk containing bifidobacterium lactis BL-99
1. Experimental strains
The bifidobacterium lactis BL-99 (CGMCC No. 15650) used in the technical scheme of the invention;
control strain 1: commercial Bifidobacterium lactis strain BB-12.
2. Activation of strains and liquid culture of seeds
(1) Strain activation: activating the bifidobacterium lactis by using a BSM culture medium, taking a strain growing to a logarithmic phase, and storing a glycerol tube for later use. Wherein, BSM culture medium (g/L): 5.0g of peptone, 5.0g of beef extract, 10.0g of tryptone, 5.0g of yeast extract powder, 0.5% fructo-oligosaccharide, 10.0g of glucose, 1.0mL of Tween-80, K 2 HPO 4 2.0g, sodium acetate 5.0g, 2.0g of diammonium hydrogen citrate 4 ·7H 2 O 0.25g，MgSO 4 ·7H 2 O 0.1g，1.0％CaCO 3 (endogenous adjustment of pH) distilled water was added to 1000mL.
(2) Preparation of strain seed liquid: dissolving 12% whole milk powder and 6% white sugar in water at normal temperature, sterilizing at 95 deg.C for 5min, cooling to 36 + -1 deg.C, inoculating strain, fermenting at 36 + -1 deg.C for 48 + -2 h, and culturing to logarithmic phase for 72 + -2 h to obtain seed liquid.
3. Detection of antioxidant capacity of lactobacillus bifidus fermented milk
Preparing fermented milk: adding sucrose 6.5% of the total weight of raw milk into raw milk, stirring, heating to 95 deg.C, sterilizing for 5min, cooling the sterilized emulsion to 37 deg.C, adding strain seed solutions (inoculum size 3%), fermenting at 37 deg.C for 24, 48, 72, 96, and 120h, centrifuging the fermented emulsion at 8000r/min for 15min, collecting the fermented supernatant, filtering with 0.22 μm filter membrane, and measuring antioxidant ability of the filtrate.
(1) Total antioxidant (T-AOC) Capacity determination
In the invention, the antioxidation capacity of the bifidobacterium lactis is evaluated mainly by measuring the inhibition rate of Angiotensin Converting Enzyme (ACE), the total antioxidation (T-AOC) capacity and the superoxide anion free radical scavenging capacity.
The Angiotensin Converting Enzyme (ACE) determination method mainly comprises a colorimetric method, an enzyme coupling method and the like. In the present invention, the Angiotensin Converting Enzyme (ACE) inhibitory activity is measured by ultraviolet spectrophotometry: taking a 2mL centrifuge tube, adding 50 mu L of 50mmol/L sodium borate buffer solution with 5mmol/L Hippuryl-His-Leu (HHL) as a substrate, adding 20 mu L of liquid to be detected, mixing, preheating for 3min in a water bath at 37 ℃, adding 10 mu L ACE enzyme solution, reacting for 30min at 37 ℃, and adding 100 mu L1 mol/L hydrochloric acid to stop the reaction. 1.7mL of ethyl acetate was added, followed by shaking for 15s, centrifugation at 3000 Xg for 10min, and separation of the upper 1mL of ethyl acetate layer (layer on which the sample was present). Heating to volatilize and remove the organic solvent, adding 1mL of deionized water, shaking to completely dissolve hippuric acid, and measuring the light absorption value of the sample at 228nm by using a micro ultraviolet spectrophotometry. The ACE inhibition ratio (ACEI) is calculated as follows:
wherein A represents the absorbance of ACE with HHL to completely produce hippuric acid (control); b represents the light absorption value (sample) of hippuric acid generated after the liquid to be detected reacts with ACE and HHL; c represents the light absorption value of hippuric acid generated after the reaction of adding ACE hydrochloride and HHL.
The total antioxidant capacity (T-AOC) is measured by adopting a total antioxidant capacity (T-AOC) detection kit purchased from Nanjing, and the operation is carried out according to the kit instruction. The antioxidant can reduce ferric ions into ferrous ions which can form a stable complex with phenanthroline substances, and the oxidation resistance can be measured by a colorimetric method. In the determination process, when a sample to be detected is mixed with a reagent provided by the kit, the mixture is fully mixed by a vortex mixer, placed in water bath at 37 ℃ for 30 minutes for 10 minutes, the wavelength is 520nm, the optical path is 1cm, double distilled water is adjusted to zero, and the absorbance value of each tube is determined.
The superoxide anion free radical scavenging ability is measured by a pyrogallol-spectrophotometer method. The pyrogallol-spectrophotometry is widely applied to the primary screening of antioxidants in the food and medicine industries and the evaluation of the antioxidant function of various active substances for eliminating superoxide anion free radicals. The pyrogallol autoxidation process is a chain reaction and can generate superoxide anion free radicals, the content of the oxidation products of the pyrogallol autoxidation process can be detected by a spectrophotometer, and the oxidation resistance of the antioxidant substances can be indirectly evaluated by the method. The method comprises the following specific steps: 0.88mL of 0.1mol/L Tris-HCl buffer (pH 8.2) was put in a test tube, and 0.2mL of 1.0mmol/L ethylenediaminetetraacetic acid (EDTA), 0.2mL of the sample, and 0.3mL of distilled water were added in this order. Reacting in 25 ℃ water bath for 10min, adding 0.4mL 9.0mmol/L pyrogallol, reacting for 60min, and adding 100 uL 12.0mol/L HCl to terminate the reaction. All above were averaged 3 times and the experiment was repeated 3 times. Calculating the formula: clearance = (Ac-As)/Ac × 100%, where As: measuring the absorbance at a wavelength of 325 nm; ac: the blank tube was used with 0.2mL of distilled water instead of the sample, and the absorbance was measured by the same tube as the operation method.
In the present invention, the results of angiotensin converting enzyme inhibition assay of probiotic fermentation supernatants at different fermentation times are shown in Table 1, the results of T-AOC potency assay are shown in Table 2, and the results of superoxide anion radical scavenging potency assay are shown in Table 3.
TABLE 1 angiotensin-converting enzyme inhibition
TABLE 2 Total antioxidant Capacity
TABLE 3 superoxide anion radical scavenging ability
(2) Detection of other antioxidant-related abilities in BL-99 fermentation samples
In the invention, other oxidation resistance related capacities in BL-99 fermentation samples are also detected, including total superoxide dismutase (T-SOD) activity, gamma-aminobutyric acid (GABA) content and B vitamin content of the supernatant at different fermentation times.
In the invention, the activity of the total superoxide dismutase (T-SOD) is determined by adopting a total superoxide dismutase (T-SOD) kit which is purchased from Nanjing and operated according to the kit instruction. The reaction system of xanthine and xanthine oxidase produces superoxide anion radical which oxidizes hydroxylamine to form nitrite, which is purple red under the action of color developing agent, and the absorbance of the product is measured with visible spectrophotometer. When the tested sample contains SOD, it has specific inhibition action on superoxide anion free radical, so that the formed nitrite is reduced, and the absorbance value of the measuring tube is lower than that of the control tube in the colorimetric process, and the activity of SOD in the sample can be obtained by means of calculation.
In the invention, the method for measuring the content of gamma-aminobutyric acid (GABA) is carried out according to QB/T4587-2013 gamma-aminobutyric acid.
In the invention, the content of B vitamins is determined by a high performance liquid chromatograph, and the specific operation conditions refer to HPLC detection of water-soluble vitamins (Liyan, food research and development, 2018) in black skin termitomyces albuminosus.
In the present invention, the results of measuring total superoxide dismutase (T-SOD) activity, gamma-aminobutyric acid (GABA) content and B vitamins of BL-99 probiotic fermentation supernatants at different fermentation times are shown in Table 4.
TABLE 4 BL-99 measurement of the antioxidant capacity of fermentation supernatants
The measurement results show that the BL-99 fermented emulsion has excellent antioxidant capacity and can be used for producing fermented milk, milk drinks, milk powder, solid beverages, daily chemicals and the like with the effects of resisting oxidation, regulating blood pressure and regulating sleep.
Example 2 fermentation preparation of milk beverage with antioxidant, blood pressure regulating and sleep regulating potential by Bifidobacterium lactis BL-99
The embodiment provides the milk beverage which is prepared by fermenting the bifidobacterium lactis BL-99 and has the potential of resisting oxidation, regulating blood pressure and regulating sleep. The preparation method of the beverage comprises the following steps:
(a) Inoculating bifidobacterium lactis BL-99 into skim milk for fermentation to obtain fermented milk;
(b) Blending with a sweetening agent and/or a sour agent to form a blending liquid, uniformly mixing the blending liquid with the fermented milk prepared in the step (a), and homogenizing to prepare the fermented milk beverage.
Further, the temperature of the fermentation in the step (a) is 37 ℃, and the fermentation time is 72h.
Further, in the step (b), the mixing volume ratio of the fermented milk to the blending liquid is 1.
Further, the pressure for homogenization in step (b) is 30MPa.
Further, a sterilization process may be used in step (b), preferably at 120 ℃ for 15min.
The beverage of the embodiment has good potential of resisting oxidation, regulating blood pressure and regulating sleep in two product categories of low temperature and normal temperature.
Example 3 fermentation of Bifidobacterium lactis BL-99 fermented milk with antioxidant, blood pressure regulating and sleep regulating potential
Heating skim milk to more than 50 ℃, adding 6% of white granulated sugar, stirring until the white granulated sugar is completely dissolved, preheating to 60-65 ℃, homogenizing under 20Mpa pressure, carrying out heat treatment at about 90 ℃ for 5-8 minutes, cooling to 37 ℃, and inoculating bifidobacterium lactis BL-99 seed liquid, wherein the inoculation amount is 2-3%. Fermenting at 37 deg.C for 6-8h to obtain functional fermented milk product for direct drinking. The product has sweet and sour taste and good flavor, and also has effects of resisting oxidation, regulating blood pressure, and regulating sleep.
Claims (12)
1. Application of Bifidobacterium lactis (CGMCC) in preparing composition with effects of resisting oxidation and/or regulating blood pressure is disclosed, wherein the accession number of Bifidobacterium lactis is CGMCC No.15650.
2. The use of claim 1, wherein the composition further has sleep regulating efficacy.
3. Use according to claim 1, wherein the bifidobacterium lactis produces superoxide dismutase, angiotensin converting enzyme inhibitors, gamma-aminobutyric acid and/or B vitamins by fermentation.
4. Use according to claim 1 or 2, wherein the composition is a food composition, a feed composition, a cosmetic composition or a pharmaceutical composition.
5. Use according to claim 1 or 2, wherein the emulsion is fermented with bifidobacterium lactis CGMCC No.15650 to produce a fermented product, which is used as or further for producing the composition.
6. Use according to claim 5, wherein the emulsion is fresh or reconstituted milk, with or without 5-10% sucrose.
7. Use according to claim 6, wherein the fermentation conditions are: fermenting for 1-7 days at 35-45 ℃.
8. Use according to claim 6, wherein the Bifidobacterium lactis is inoculated in the emulsion in the form of a seed liquid, in an amount of between 2% and 5%.
9. Use according to claim 6, wherein the supernatant of the fermentation product is used as or further in the preparation of the composition.
10. Use according to claim 5, wherein the composition is fermented milk, fermented milk beverage or milk powder.
11. Use according to claim 5, wherein the composition is a solid beverage.
12. The use according to claim 10, wherein the fermented milk or fermented milk beverage contains bifidobacterium lactis CGMCC No.15650 in an amount of1×10 7 cfu/mL～1×10 11 cfu/mL。
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| CN115386513A (en) * | 2022-08-02 | 2022-11-25 | 诺佰克（武汉）生物科技有限公司 | Probiotics capable of producing hyaluronic acid, helping skin cells to produce hyaluronic acid and preventing photoaging and application of probiotics |
| CN117736911A (en) * | 2023-11-28 | 2024-03-22 | 金华银河生物科技有限公司 | Bifidobacterium breve B-169 and application thereof in reducing blood pressure |
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