CN102093964A - Tomato-derived lactic acid bacteria with functions of resisting oxidation and lowering blood fat - Google Patents
Tomato-derived lactic acid bacteria with functions of resisting oxidation and lowering blood fat Download PDFInfo
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- CN102093964A CN102093964A CN2010105669911A CN201010566991A CN102093964A CN 102093964 A CN102093964 A CN 102093964A CN 2010105669911 A CN2010105669911 A CN 2010105669911A CN 201010566991 A CN201010566991 A CN 201010566991A CN 102093964 A CN102093964 A CN 102093964A
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Abstract
The invention relates to lactic acid bacteria with functions of resisting oxidation and lowering blood fat, which are derived from fresh tomato juice. The pharmacological experiment proves that the tomato-derived lactic acid bacteria have the effects of enhancing activities of antioxidases of hepatic and nephridial tissues of organisms and lowering the blood fat, have the advantages of long-time action and no toxic or side effect, and have wide practical value.
Description
Technical field
The present invention relates to a kind of milk-acid bacteria of improving body activities of antioxidant enzymes and blood fat reducing level, it obtains from tomato juice.
Background technology
Some metabolic process of environmental pollution, ultraviolet radiation and vital movement and the generation of activity in vivo oxyradical have substantial connection.Find that at present remains of pesticide, chemical reagent and the influence of various radiating thereof all can cause the body free-radical contents to raise, and modern medicine is thought, human aging is relevant with intravital free radical isoreactivity molecule.System of defense is being kept the physiological equilibrium of free-radical generating and removing in the normal circumstances lower body, but when system of defense can not effectively remove or have ectogenic free radical to invade in a large number, active oxygen was such as superoxide anion (O
2 -), hydrogen peroxide (H
2O
2) and hydroxy radical qiao (OH) thus etc. level will exceed the appearance that normal range causes oxidative stress, the hereditary material DNA of infringement cell, act on physiologically active substances such as intravital enzyme, protein, thereby cause aging and cause multiple disease, as cancer, diabetes, cardiovascular disorder, hypertension etc., so free radical is one of human body diseases, aging and dead direct inducement.
Up to now, existing hundreds of antioxidants are found, and most of antioxidant derives from chemosynthesis and natural product extraction.But along with the progress of science, the human harm of recognizing the chemosynthesis antioxidant gradually; The extraction of crude substance is again because the restriction of problems such as isolation identification, composite and modification hinders its development.Found in recent years to contain a lot of antioxidant in the meta-bolitess of a lot of bacteriums, and Microbial resources are abundant, with short production cycle, raw material is cheap, so microbial fermentation is expected to become an effective way of extensive acquisition edible antioxidant.
But milk-acid bacteria is meant a group fermentable carbohydrates and produces the anaerobism of a large amount of lactic acid and bacteriocin or the general designation of amphimicrobian gram-positive cocci or bacillus, extensively exists at nature.Studies show that in a large number, overwhelming majority milk-acid bacterias are important physical floras very in normal people and animals' enteron aisle, undertaking the multiple important physical function of body, can regulate the body gastrointestinal tract normal microflora, keep microecological balance, reduce serum cholesterol, antitumor and immunity effect effect, can suppress corrupt bacteria growing breeding and spoilage product in the enteron aisle generation, have additional nutrients, promote immunological competence, thereby to the generation effects such as physiological function, immune response, tumour generation, aging course and stress reaction of body.The present invention separates it, and the effect of and reducing blood-fat anti-oxidant to it studies, and finds that this milk-acid bacteria has tangible raising activities of antioxidant enzymes and reducing blood lipid.
Summary of the invention
The objective of the invention is to from tomato, be separated to a kind of milk-acid bacteria, have anti-oxidant and effect reducing blood-fat by experimental results show that it in external and the body.
Embodiment
Below come further to set forth the beneficial effect of milk-acid bacteria of the present invention by experiment, these experiments have comprised the pharmacodynamic experiment and the clinical observation on the therapeutic effect experiment of medicine of the present invention.
Milk-acid bacteria supernatant preparation of fermentation liquid
Bacterial classification is inserted 30 ℃ of cultivation 48h in the sterilized MRS liquid nutrient medium, continuous passage 3 times.Nutrient solution is through the centrifugal 10min of 4000 * g, collects supernatant liquor, and it is stand-by to be put in 4 ℃ of refrigerators.
The measuring method of the outer resistance of oxidation of milk-acid bacteria supernatant fermented liq
Remove the mensuration of superoxide radical ability
Get the Tris-Hcl (0.05mol/L of 4.5mL, pH 8.2) place 25 ℃ of water bath with thermostatic control preheating 20min, add sample solution 0.1mL, add the 0.4mL pyrogallol solution (2.5mol/L) that preheating is good in 25 ℃ of waters bath with thermostatic control again, behind the mixing in 25 ℃ of waters bath with thermostatic control accurate response 4min, add 0.1mL concentrated hydrochloric acid (8mol/L) termination reaction then.Reactant is measured absorbancy at 320nm wavelength place.
Clearance rate (%)=[1-(A
b-A
c)/A
a] * 100%
A
a-do not add sample, add pyrogallol
A
b-adding sample and pyrogallol
A
c-add sample, do not add pyrogallol
Remove the mensuration of hydroxy radical qiao ability
The neighbour of 1mL-F beautiful jade (2.5mmol/L), phosphate buffered saline buffer (the PBS of 1mL, 0.02mol/L, pH 7.4) mix with the distilled water of 1mL, (FeSO4 2.5mmol/L), fully adds 1mL superoxol (H2O2 behind the mixing once more to add the copperas solution of 1mL then, 20mmol/L), measure absorbancy behind 37 ℃ of water-bath 1.5h in the 536nm place.Blank effective distilled water replaces.
Clearance rate (%)=(A
s-A
p)/(A
b-A
p) * 100%
A
b-add sample, do not add H
2O
2
A
p-do not add sample, add H
2O
2
A
s-adding sample and H
2O
2
The mensuration of anti peroxidation of lipid ability
Fresh egg yellow liquor and phosphate buffer soln (pH 7.4 for PBS, 0.1mol/L) equal-volume mixes and magnetic agitation 10min.Add this PBS solution dilution according to the ratio of 1: 25 (V/V) then and become the yolk suspension.(0.1mol/L pH7.4) and behind the copperas solution mixing is incubated vibration 1.5h in 37 ℃ with 1mL yolk suspension, 0.5mL sample, 1mLPBS.Mixed solution adds 1mL trichoroacetic acid(TCA) (TCA, massfraction are 2.5%), behind the mixing in the centrifugal 20min of 4000 * g.Get the thiobarbituricacid (TBA, massfraction are 0.8%) that supernatant liquor 3mL adds 2mL, in 100 ℃ of heating 10min, measure absorbancy at the 532nm place behind the mixing.
Inhibiting rate (%)=(A
0-A
1)/A
0* 100%
A
0-blank replaces with the equivalent sample solvent
A
1-sample
The antioxidation in vitro test-results shows that the superoxide radical clearance rate of this milk-acid bacteria supernatant fermented liquid is 23.81%, and the clearance rate of hydroxy radical qiao is 80.47%, and the inhibiting rate of lipid peroxide is 52.92%.
Continuation is carried out pharmaceutical research by animal experiment to milk-acid bacteria of the present invention, observes its influence to Mouse Liver and nephridial tissue activities of antioxidant enzymes and blood lipid level.
1. experiment material:
1.1 animal
Health, maturation, body weight (22 ± 3) g, ICR kind cleaning level mouse, male and female half and half are provided by Chinese Military Medical Science Institute Experimental Animal Center.Mouse raising condition is 22 ± 2 ℃ of room temperatures, and 12h illumination and dark cycle are freely drunk water and ingested 7 days, begin after the adaptation to experimentize.24 mouse are divided into 4 groups at random, promptly normal group, high fat control group, the normal feed group of milk-acid bacteria and milk-acid bacteria high lipid food group, 6 every group.The conventional raising 2 days gives 2 * 10 respectively before the experiment
9The milk-acid bacteria of CFU/mL is hanged 0.2mL, and high fat control group and normal control group are given and equal-volume physiological saline, successive administration 28 days.
1.2 reagent
The milk-acid bacteria suspension; Catalase (CAT) mensuration test kit (visible light method), superoxide-dismutase (SOD) testing cassete, Selenoperoxidase (GSH-px) test kit and mda (MDA) mensuration test kit build up bio-engineering research by Nanjing to be provided; Total cholesterol test kit-TC (enzyme process), triglyceride level test kit-TG (enzyme process), high density lipoprotein cholesterol test kit HDL-c (selective precipitation method) and low density lipoprotein cholesterol test kit LDL-c (selective precipitation method) are provided by the safe clinical reagent of Beijing northization company limited.Anti-mouse CD4
+, CD8
+Provide by U.S. santa cruzebiotechnology company with the Nrf2 monoclonal antibody.
1.3 the hyperlipemia model mouse makes up
Lard 12%, cholesterol 1%, egg yolk 0.5% and basal feed 86.5%, the mouse 28 days fed continuously.
1.4 the mensuration of liver and nephridial tissue activities of antioxidant enzymes
After the off-test, each is organized mouse and be can't help water 12h by fasting, is under etherization dissected, and takes out liver,kidney,spleen and thymic tissue fast, and with ice-cold physiological saline rinsing and weigh, the tissue homogenate of preparation 10% detects with corresponding mensuration test kit.
1.5 the mensuration of blood lipids index
After the off-test, mouse is used etherization, be fixed on and dissect on the plate, syringe is moistening with heparin, get blood 2mL from eye socket, note not haemolysis, 4000rpm immediately after the taking-up, 4 ℃, centrifugal 10min, obtain serum, utilize test kit to measure serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-c) and high-density lipoprotein (HDL) (HDL-c) index.
2. method and result
2.1 milk-acid bacteria is to the influence of Mouse Liver and nephridial tissue antioxidase
Measured each group and tried Mouse Liver and these 4 kinds of activities of antioxidant enzymes indexs of nephridial tissue superoxide-dismutase (SOD), mda (MDA), Selenoperoxidase (GSH-px) and catalase (CAT), the result is as shown in table 1.By the result as can be seen, compare with the normal control group, the hepatic tissue SOD vigor of milk-acid bacteria normal group mouse significantly raises (p＜0.05), and MDA content significantly reduces (p＜0.01), and the GSH-px activity of nephridial tissue significantly increases (p＜0.05); Compare with high fat control group, the hepatic tissue SOD vigor of the high fat group of milk-acid bacteria mouse significantly raises (p＜0.05), and the MDA content of liver and nephridial tissue significantly reduces (p＜0.01).
Table 1 milk-acid bacteria is to the influence of mouse tissue activities of antioxidant enzymes
Annotate: this value is mean+SD.Utilize SPSS13.0 software to carry out significance analysis.
* represent to compare p＜0.05 with the normal control group, * * represents to compare p＜0.01 with the normal control group;
+ expression is compared p＜0.05 with high fat control group, ++ expression is compared p＜0.01 with high fat control group.
2.2 milk-acid bacteria is to the influence of blood lipids index
After the off-test, water 12h is can't help in the animal fasting, wins eyeball then and gets blood, measures TC, TG, LDL-c and HDL-c index.Experimental result sees Table 2.By the result as can be seen, compare with high fat control group, TC, the TG of the high fat group of milk-acid bacteria and LDL-c index reduce obviously (p＜0.01).
Table 2 milk-acid bacteria is to the influence of mouse blood lipids index
Annotate: this value is mean+SD.Utilize SPSS13.0 software to carry out significance analysis.
+ expression is compared p＜0.05 with high fat control group, ++ expression is compared p＜0.01 with high fat control group.
2.3 milk-acid bacteria is to the influence of mouse lymphocyte subgroup
Each group is tried mouse and got blood at the state inferior orbit of etherization after being irritated stomach milk-acid bacteria 28h, behind the anticoagulant heparin to its peripheral blood CD4
+And CD8
+T lymphocyte subset group's quantity is measured.Measurement result such as table 3 expression.Each group is tried its corresponding control group of mouse and is compared mouse peripheral blood CD4
+The lymphocytic per-cent of T all has rising, and difference reaches significance level (p＜0.01).The milk-acid bacteria normal group is compared with the normal control group, and the high fat group of milk-acid bacteria is compared its CD4 with high fat control group
+/ CD8
+Value raise obviously significant difference (p＜0.01).This shows that this milk-acid bacteria can be improved the immunizing power of mouse.
Table 3 milk-acid bacteria is to the influence of mouse peripheral blood mononuclear lymphocyte subgroup
Annotate: this value is mean+SD.Utilize SPSS13.0 software to carry out significance analysis.
* represent to compare p＜0.05 with the normal control group, * * represents to compare p＜0.01 with the normal control group;
+ expression is compared p＜0.05 with high fat control group, ++ expression is compared p＜0.01 with high fat control group.
2.4 the influence that milk-acid bacteria is expressed the anti-oxidant specific proteins of murine liver tissue cell
Each group was tried the mouse stomach milk-acid bacteria after 28 days, adopted Flow Cytometry to detect the situation of murine liver tissue cell Nrf2 protein expression, and the result is as shown in table 4.Test-results shows, irritates the mouse that feeds milk-acid bacteria mouse liver cell Nrf2 expressing quantity is increased, thereby illustrate that the Nrf2 great expression that is activated resists oxidative damage under high smectic attitude.
Table 4 milk-acid bacteria is to the influence of mouse liver cell Nrf2 protein expression
Annotate: this value is mean+SD.Utilize SPSS13.0 software to carry out significance analysis.
* represent to compare p＜0.05 with the normal control group, * * represents to compare p＜0.01 with the normal control group;
+ expression is compared p＜0.05 with high fat control group, ++ expression is compared p＜0.01 with high fat control group.
2.5 acute toxinology experiment of the present invention
20 mouse are divided into 2 groups at random, 10 every group, male and female half and half.The test group animal is irritated stomach milk-acid bacteria suspension, and control group is irritated the physiological saline of stomach equivalent, and 1 time on the one, the mental status of animal in the week and death condition etc. are observed in drug withdrawal then, and the result finds no animal dead, shows that tentatively this milk-acid bacteria is safer, sees table 5 for details.
Table 5 acute toxicity test in mice result
Annotate: "-" expression is normal.
3. discuss
This experimentation on animals is reliable through the repeated experiments proving effect.Hyperlipidaemia group mouse anti oxidase activity and blood lipid level are on the low side in this research, and activities of antioxidant enzymes of milk-acid bacteria group mouse and blood lipids index have rising in various degree, illustrate that this milk-acid bacteria can play the effect of removing interior free yl and blood fat reducing, can be used for the treatment of hyperlipidaemia.
In addition, the present invention can be prepared into active bacteria formulation in clinical application according to practical situation.
Claims (3)
1. the milk-acid bacteria of a strain and reducing blood lipid anti-oxidant from having of tomato, it is characterized in that: described milk-acid bacteria is taken in the fresh tomato juice, has the body of raising oxidase activity, the medical use of blood fat reducing level.
2. milk-acid bacteria according to claim 1 is characterized in that: above-mentioned milk-acid bacteria can be prepared into oral liquid, capsule, powder, granule or tablet in clinical application.
3. according to claim 1,2 described milk-acid bacterias, it is characterized in that: can add appropriate amount of starch or other auxiliary material as additive.
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|---|---|---|---|
| CN2010105669911A CN102093964A (en) | 2010-11-26 | 2010-11-26 | Tomato-derived lactic acid bacteria with functions of resisting oxidation and lowering blood fat |
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|---|---|---|---|
| CN2010105669911A CN102093964A (en) | 2010-11-26 | 2010-11-26 | Tomato-derived lactic acid bacteria with functions of resisting oxidation and lowering blood fat |
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2010
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Application publication date: 20110615 |