CN1464046A - Bacillus adhaerens and its use in preparing health food having thrombolysis property - Google Patents
Bacillus adhaerens and its use in preparing health food having thrombolysis property Download PDFInfo
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Abstract
本发明涉及的纳豆芽孢杆菌为Bacilliis nattoNLSSe，2002年3月8日保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏号为CGMCCNO.0724；其革兰氏染色阳性，不抗酸杆菌，直或近直形，0.6－0.8×1.5－2.0um；好氧，有芽孢，芽孢中生、不膨大，在一个孢子囊细胞中，芽孢不多于一个，细胞壁化学组分中含有内消旋二氨基庚二酸和甘氨酸；无特征性糖；能运动，鞭毛周生；在蛋自胨查氏琼脂、桑塔斯琼脂、牛肉浸汁琼脂、黄豆粉琼脂或葡萄糖酵母精琼脂五种天然有机培养基上生长良好，利用黄豆饼粉培养基形成较厚菌膜，菌落乳脂色，边缘光滑；可在含有氮源的培养剂中培养得到含有血栓溶解性物质纳豆激酶的培养物，并可对培养物提取粉未状物质，可用来在制备具有血栓溶解性保健食品或保健食品添加剂。The Bacillus natto involved in the present invention is Bacilliis nattoNLSSe, which was preserved on March 8, 2002 in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and its preservation number is CGMCCNO.0724; its Gram staining is positive, and it is not an acid-fast bacillus , straight or nearly straight, 0.6-0.8×1.5-2.0um; aerobic, with spores, middle-growing spores, not enlarged, no more than one spore in a sporangia cell, and the chemical components of the cell wall contain endolysis Spin diaminopimelic acid and glycine; non-characteristic sugar; able to move, flagellum perinatal; on peptone Chase agar, Santas agar, beef infusion agar, soybean flour agar or glucose yeast agar five natural It grows well on organic medium, using soybean cake powder medium to form a thicker bacterial film, the colony is creamy in color, and the edge is smooth; it can be cultured in a culture medium containing nitrogen source to obtain a culture containing nattokinase, a thrombus-dissolving substance, and The powdery substance can be extracted from the culture, and can be used to prepare a thrombus-dissolving health food or health food additive.
Description
Invention field
The invention belongs to biological technical field, particularly a kind of bacillus natto and have thrombolysis Sexual health promotion Application in Food in preparation.
Background technology
At present, the modern times " ciril disease " such as hyperlipidemia, hypertension, diabetes, cancer have replaced communicable disease becomes human No.1 killer.The report of the World Health Organization 1998 is pointed out, 1,500 ten thousand people that have an appointment every year in the world die from the blood circulation disease, and wherein 7,200,000 people die from coronary heart disease, and 4,600,000 people die from apoplexy, 3,500,000 people die from other heart troubles, and whole world per minute has 23 people to die from vascular disease.According to Ministry of Health's statistics, China's cardiovascular and cerebrovascular diseases number of patients has reached 200,000,000, and wherein, patient over half does not know that oneself is ill.To the end of the year 1997, China hyperpietic has reached 1.1 hundred million, and increases with annual 3500000 people's speed.
Exploitation has the important means that the regulating blood fat health care foods is prevention and diseases such as treatment hyperlipidemia, hypertension.In recent years, health functional food has been subjected to more and more liking according to statistics, have 93% children, 98% old man and a considerable amount of the young and the middle aged all to use or using various health functional foods in the domestic big city.The annual health-product market sales volume of China is up to 45,000,000,000 yuans.In the international market, health functional food also has same development trend.At present, the healthcare products that have the regulating blood fat function on the China market are of a great variety, and dragons and fishes jumbled together, and many kinds all are that there is a big difference with natto health-care product on the function without the imitative healthcare products of Ministry of Health's approval.
Natto comes from China, import Japan into after, be developed to plurality of health care functions food, and accepted by countries in the world, produced huge economic benefit.Modern study finds that natto has blood pressure regulation, reduces the function of blood cholesterol levels, and hyperlipidemia, hypertension, thrombus disease etc. are had the assisting therapy effect.Therefore, in Japan, natto has been used as the representative of health functional food.Its nourishing function comprises: 1) prevention of arterial sclerosis prevents vessel aging; 2) make blood salubrious, blood flow is unimpeded; 3) thrombus; 4) generation of prevention oxyradical; 5) enhance metabolism; 6) blood pressure regulation; 7) prevention of brain infraction; 8) prevent and improve dementia symptom; 9) prevention constrictive cardiomyopathy and myocardial infarction; 10) reduce low density lipoprotein cholesterol content; 11) reduce neutral fat content.The effect of " preventing heart disease " of natto has also obtained the approval of U.S. FDA.
Though natto is generally acknowledged to have above-mentioned nourishing function, the thrombolysis activity of natto is very low, has only a large amount of long-term edible competence exertion thrombolysis effectiveness.
Summary of the invention
The objective of the invention is to overcome directly natto and goods thereof limitation as health-care product, and provide a kind of bacillus natto that can in multiple nitrogenous substratum, efficiently synthesize Nattokinase (Nattokinase), the Nattokinase that is produced is the protein kinase of a kind of can reaching " per os fibrinolytic therapy " purpose, active quite stable in enteron aisle, compare with other thrombolysis medicine, except that having stronger thrombolytic effect, also have long action time in the body, advantage such as have no side effect; This Nattokinase directly enters blood circulation through intravenous drip, or orally, intestinal absorption directly acts on thrombus after entering blood circulation, can also induce liver or blood vessel endothelium to produce TPA (the no TPA antigenicity of Nattokinase itself), make the activation of organism self plasmin system, thereby it is gentle, improve the fibrinolytic of blood constantly, compare with other plasmin, its security is the highest;
Another object of the present invention is the application of bacillus natto in producing protective foods.
Embodiment of the present invention are as follows:
Bacillus natto provided by the invention, it is characterized in that: this bacillus natto is Bacilliis nattoNLSSe, be preserved on March 8th, 2002 " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number are CGMCC NO.0724;
Bacterial strain NLSSE is Gram-positive, not acid-fast bacilli; Aerobic, gemma is arranged, give birth in the gemma, do not expand, in a sporocyst cell, no more than one of gemma contains meso-DAP (meso diaminopimelic acid), atypism sugar in the cell walls chemical composition; Bacterial strain NLSSe is shaft-like, straight or proximal straight type, and 0.6-0.8 * 1.5-2.0um can move flagellum Zhousheng; Well-grown on five kinds of natural organic substratum utilizes the soybean cake powder substratum to form thicker mycoderm, the bacterium colony creamy, and the edge is smooth.
The screening method of bacillus natto NLSSe provided by the invention is:
After the granule soybean drip washing of selected northeast, add the long-pending water logging bubble of N diploid and spend the night, get the swollen soybean 121 ℃ of temperature, steam sterilizing 30 minutes is got straw and is wrapped, and 35 ℃ of temperature were fermented 24 hours; Get fermented soybean and carry out strain separating; Separate the single bacterium colony that obtains and in 10% soya-bean milk, cultivate, detect its thrombolysis activity, the higher bacterial strain of screening thrombolysis activity with fibrin plate; Be further purified, obtain a purebred bacterial strain, send Institute of Microorganism, Academia Sinica culture presevation evaluating center to identify; By being named NLSSe, be preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number are CGMCC NO.0724 on March 8th, 2002 into bacillus natto Bacillusnatto;
Bacillus natto of the present invention is from natural fermented soybean, with natto kinase activity as index, separation and purification and obtaining, its biological property is as follows:
A. morphological feature
Cultivated 2-4 days in 30 ℃, bacterial strain NLSSe is shaft-like, chaining not, no sporangiocyst; Straight or proximal straight type, 0.6-0.8 * 1.5-2.0um, peritrichous can move, and gemma is arranged, life in the gemma, do not expand, in a sporocyst cell, no more than one of gemma (seeing Fig. 1,2), Gram-positive, not antiacid;
B. cultural characteristic
This bacterial strain NLSSe 30 ℃ of cultural characteristics of cultivating 2-4 days in following five kinds of substratum see Table 1,
The cultural characteristic of table 1 bacterial strain NLSSe on 5 kinds of substratum
Substratum colony colour and shape
The dirty white of peptone Cha Shi agar bacterium colony, mycoderm is thick, and the edge is smooth
Sang Tasi agar bacterium colony almond white, mycoderm is thick, and the edge is smooth
Ox meat extract agar bacterium colony oyster white, the mycoderm moderate, the edge is smooth
The little yellow of analysis for soybean powder agar bacterium colony, mycoderm is thick, the high and steep shape in edge
The smart agar bacterium colony of glucose yeast yellowish white, mycoderm is thin, edge roughness
C. chemical composition
Press instant thin-layer chromatography method (the Thin Layer Chromography of Hasegawa T. etc., TLC) this bacterial strain NLSSe is carried out full cell hydrolyzed solution sugar type and amino acid analysis, confirm that this bacterium contains meso-DAP (meso diaminopimelic acid, Diaminopimelic acid) and glycine, atypism sugar (sugared type C);
D. physiological and biochemical property
With reference to the method for " Bergey ' s Manual of Systematic Bacteriology ", the physiological and biochemical property of bacterial strain NLSSe is as shown in table 2.
The physiological and biochemical property of table 2 bacterial strain NLSSe
| Feature result | Feature result |
| On gelatin liquefaction+Starch Hydrolysis+indole reaction-nitrate reduction+catalase+cellulose growth-VP reaction-lecithinase-tyrosine hydrolysis-Lao ?decomposition+gas generate 5%+growth 7%+10% among-2%+NaCl- | D-galactolipin+maltose+melibiose-L-rhamnose-sucrose+D-wood sugar-Arabinose-D-glycosides dew alcohol+L-inositol-D-Fructose+D-Glucose+10+30+growth 45+temperature 50+(℃) 55-60- |
Bacillus natto NLSSe bacterial strain of the present invention all can be grown well at pH4-9, and is growth temperature 25-45 ℃, aerobic; Virulence experiment proof bacterial strain NLSSe does not have overt toxicity to mouse.
Bacillus natto provided by the invention has thrombolysis Sexual health promotion Application in Food in preparation: bacillus natto provided by the invention is containing cultivates the culture that obtains containing the thrombolysis material in the substratum of nitrogenous source, and can have thrombolysis Sexual health promotion food as preparation to culture extracting liq shape or powdered material; Described nitrogenous source is beans such as soybean, black soya bean, and protein extract.
The Nattokinase that bacillus natto provided by the invention produced (Nattokinase) is the protein kinase of a kind of can reaching " per os fibrinolytic therapy " purpose.Nattokinase is active quite stable in enteron aisle, compares with other thrombolysis medicine, except that having stronger thrombolytic effect, also has long action time in the body, and advantage such as have no side effect.Nattokinase directly enters blood circulation through intravenous drip, or orally, intestinal absorption directly acts on thrombus after entering blood circulation, can also induce liver or blood vessel endothelium to produce TPA (the no TPA antigenicity of Nattokinase itself), make the activation of organism self plasmin system, thereby it is gentle, improve the fibrinolytic of blood constantly, compare with other plasmin, its security is the highest; Bacillus natto bacterial strain of the present invention is to separate the bacterial classification that obtains from natural fermented soybean, can be in liquid culture such as solid culture such as soybean, black soya bean and soya-bean milk, accent pH is 6-8, temperature 20-45 ℃, this bacterium can produce a kind of highly active thrombus dissolving enzyme, can directly utilize, and also can make bacterium liquid or powdered and make an addition in the food, manufacture the protective foods with thrombus dissolving functions, it has a extensive future.
Description of drawings
The electromicroscopic photograph (750X) of accompanying drawing 1 bacterial strain NLSSe thalline and gemma form
The spore surface feature electromicroscopic photograph (20000X) of accompanying drawing 2 bacterial strain LSSe J-1
Embodiment
Embodiment 1
Produce the health-care natto functional food with bacillus natto bacterial strain NLSSe fermented soybean provided by the invention:
The preparation of bacillus natto strain inclined plane:
Medium component: soyflour 2%, sodium-chlor 1%, soy peptone 0.2%, agar 1.5%, PH7.0, sterilized 30 minutes by 121 ℃; Each test tube adds 10 milliliters of substratum, makes the test tube slant; Insert bacillus natto bacterial strain NLSSe, 30 ℃, cultivated 24 hours, obtain slant strains; Slant strains is washed with stroke-physiological saline solution, and pour into and shake in bottle liquid nutrient medium, 30 ℃, cultivated 24 hours on 150 rev/mins of shaking tables, obtain liquid spawn;
Get northeast and produce 1 kilogram of soybean, 17 ℃ were soaked 15 hours, and behind the draining 121 ℃, High Temperature High Pressure boiling 20 minutes.Add 30 milliliters of bacterial strain NLSSe liquid spawns, 40 ℃ of insulations, relative temperature 90% was fermented 24 hours.Be cooled to 20 ℃, continue fermentation 24 hours;
Fermented soybean is 40 ℃ of vacuum-dryings, and then, aseptic condition is pulverized down, adds 1.5 times of pure salad oils of volume, vitamin-E, and colloidal mill grinds, is mixed, and incapsulates, and obtains the capsule health-care food of present embodiment.
The major function composition and the content of the capsule health-care food of present embodiment are:
Embodiment 2
| Linolic acid (g/100g) | ????30 |
| Vitamin-E (mg/100g) | ????100 |
| Soybean isoflavones (mg/100g) | ????50 |
| Crude polysaccharides (mg/g) | ????50 |
Produce health-care natto food with bacillus natto bacterial strain NLSSe fermented soybean provided by the invention:
Bacillus natto strain inclined plane preparation: medium component: soyflour 2%, sodium-chlor 1%, soy peptone 0.2%, agar 1.5%, PH7.0, sterilized 30 minutes by 121 ℃; Each test tube adds 10 milliliters of substratum, makes the test tube slant.Insert bacillus natto bacterial strain NLSSe, 30 ℃, cultivated 24 hours, obtain slant strains; Slant strains is washed with stroke-physiological saline solution, and pour into and shake in bottle liquid nutrient medium, 30 ℃, cultivated 24 hours on 150 rev/mins of shaking tables, obtain liquid spawn;
Get northeast and produce 1 kilogram of soybean, 17 ℃ were soaked 20 hours, and behind the draining 121 ℃, High Temperature High Pressure boiling 30 minutes.Add 20 milliliters of bacterial strain NLSSe liquid spawns, 40 ℃ of insulations, relative temperature 90% was fermented 48 hours.Be cooled to 20 ℃, continue fermentation 24 hours; Fermented soybean is 40 ℃ of vacuum-dryings, and then, aseptic condition is pulverized down, adds 1.5 times of pure salad oils of volume, vitamin-E, and colloidal mill grinds, is mixed, and incapsulates, and obtains the capsule health-care food of present embodiment;
The major function composition and the content of the capsule health-care food of present embodiment are:
Embodiment 3
| Linolic acid (g/100g) | ????35 |
| Vitamin-E (mg/100g) | ????150 |
| Soybean isoflavones (mg/100g) | ????75 |
| Crude polysaccharides (mg/g) | ????75 |
Produce health-care natto food with bacillus natto bacterial strain NLSSe fermented soybean provided by the invention:
Bacillus natto strain inclined plane preparation: medium component: soyflour 2%, sodium-chlor 1%, soy peptone 0.2%, agar 1.5%, PH7.0, sterilized 30 minutes by 121 ℃; Each test tube adds 10 milliliters of substratum, makes the test tube slant.Insert bacillus natto bacterial strain NLSSe, 30 ℃, cultivated 24 hours, obtain slant strains; Slant strains is washed with stroke-physiological saline solution, and pour into and shake in bottle liquid nutrient medium, 30 ℃, cultivated 24 hours on 150 rev/mins of shaking tables, obtain liquid spawn;
Get northeast and produce 1 kilogram of soybean, 20 ℃ were soaked 8 hours, and behind the draining 121 ℃, High Temperature High Pressure boiling 20 minutes.Add 50 milliliters of bacterial strain NLSSe liquid spawns, 40 ℃ of insulations, relative temperature 90% was fermented 24 hours.Be cooled to 10 ℃, continue fermentation 48 hours; Fermented soybean is 40 ℃ of vacuum-dryings, and then, aseptic condition is pulverized down, adds 1.5 times of pure salad oils of volume, vitamin-E, and colloidal mill grinds, is mixed, and incapsulates, and obtains the capsule health-care food of present embodiment;
The major function composition and the content of the capsule health-care food of present embodiment are:
Embodiment 4, bacterial strain NLSSe fermented soybean are produced the health-care natto functional food
| Linolic acid (g/100g) | ????40 |
| Vitamin-E (mg/100g) | ????200 |
| Soybean isoflavones (mg/100g) | ????5100 |
| Crude polysaccharides (mg/g) | ????100 |
Bacillus natto strain inclined plane preparation: medium component: soyflour 2%, sodium-chlor 1%, soy peptone 0.2%, agar 1.5%, pH7.0, sterilized 30 minutes by 121 ℃; Each test tube adds 10 milliliters of substratum, makes the test tube slant; Insert bacterial strain NLSSe, 30 ℃, cultivated 24 hours, obtain slant strains; Slant strains is washed with stroke-physiological saline solution, and pour into and shake in bottle liquid nutrient medium, 30 ℃, cultivated 24 hours on 150 rev/mins of shaking tables, obtain liquid spawn;
Get northeast and produce 1 kilogram of soybean, 20 ℃ were soaked 8 hours, and behind the draining 121 ℃, High Temperature High Pressure boiling 20 minutes; Add 50 milliliters of bacterial strain NLSSe liquid spawns, 40 ℃ of insulations, relative temperature 90% was fermented 24 hours; Be cooled to 10 ℃, continue fermentation 48 hours; Fermented soybean is 40 ℃ of vacuum-dryings, and then, aseptic condition is pulverized down, adds 1.5 times of pure salad oils of volume, vitamin-E, and colloidal mill grinds, is mixed, and incapsulates, and obtains capsule health-care food.
The major function composition and the content of this health functional food are:
Embodiment 5: the clinical trial of health-care natto functional food
| Linolic acid (g/100g) | 40 |
| Vitamin-E (mg/100g) | 200 |
| Soybean isoflavones (mg/100g) | 5100 |
| Crude polysaccharides (mg/g) | 100 |
Take 6 health-care natto functional food capsules for twice for every day the treatment component, take 6 capsulae vacuuses to control group.Regularly euglobulin lysis time (ELT), scleroproein (former) degraded product (FDP) and histiotype plasminogen incitant (TPA) are measured in blood sampling respectively, characterize the fibrinolytic of blood plasma with this.Experimental result shows test group behind the clothes capsule, and ELT obviously prolongs in the blood, and FDP and TPA obviously increase, and illustrate that this health-care natto functional food can improve the fibrinolytic of blood plasma.Nattokinase in the health-care natto functional food and thrombus have special avidity, can single-minded thrombus, Fibrinogen there is not degrading activity, a large amount of uses can not cause bleeding, can prevent and the following illness of assisting therapy: cerebral thrombosis, cerebral infarction, myocardial infarction, thrombus sequela, thrombophlebitis, vasculitis etc.
| Project | Group (routine number) | Before the medication | After the medication | The P value |
| ?ELT(min) | Treatment group control group | 215.25±70.33 188.62±81.21 | ?176.41±71.22 ?183.25±69.38 | ＜0.002 ＞0.2 |
| ?FDP(μg/ml) | Treatment group control group | 1.02±0.19 0.93±0.18 | ?3.28±0.46 ?0.95±0.21 | ＜0.001 ＞0.2 |
| ?TPA(μg/ml) | Treatment group control group | 7.22±1.09 6.89±1.22 | ?12.41±1.78 ?6.86±1.39 | ＜0.002 ＞0.2 |
Claims (5)
1. a strain bacillus natto, it is characterized in that: this bacillus natto is Bacilliis natto NLSSe, is preserved in that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", its preserving number are CGMCC NO.0724 on March 8th, 2002.
2. by the described bacillus natto of claim 1, it is characterized in that: the Gram-positive of this bacillus natto, acid-fast bacilli not, straight or proximal straight type, 0.6-0.8 * 1.5-2.0um; Aerobic, gemma is arranged, give birth in the gemma, do not expand, in a sporocyst cell, no more than one of gemma; Flagellum Zhousheng can move.
3. by the described bacillus natto of claim 1, it is characterized in that: contain meso diaminopimelic acid and glycine, atypism sugar in this bacillus natto cell walls chemical composition.
4. by the described bacillus natto of claim 1, it is characterized in that: this bacillus natto is at egg well-grown on five kinds of natural organic substratum of the smart agar of peptone Cha Shi agar, Sang Tasi agar, ox meat extract agar, analysis for soybean powder agar or glucose yeast, utilize the soybean cake powder substratum to form thicker mycoderm, the bacterium colony creamy, the edge is smooth.
5. the described bacillus natto of claim 1 has thrombolysis Sexual health promotion Application in Food in preparation.
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| CN100436576C (en) * | 2005-01-31 | 2008-11-26 | 中国科学院微生物研究所 | Nattokinase and its coding gene and clone and expressing method |
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| CN100436576C (en) * | 2005-01-31 | 2008-11-26 | 中国科学院微生物研究所 | Nattokinase and its coding gene and clone and expressing method |
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| CN102008110A (en) * | 2010-11-11 | 2011-04-13 | 唐清池 | Multi-strain microbial composite beverage preparation and preparation method thereof |
| CN102154112A (en) * | 2010-12-03 | 2011-08-17 | 天津科技大学 | Plasmin high-yield strain and method for preparing plasmin |
| CN102154112B (en) * | 2010-12-03 | 2012-11-14 | 天津科技大学 | Plasmin high-yield strain and method for preparing plasmin |
| CN103409355A (en) * | 2013-08-28 | 2013-11-27 | 湖南农业大学 | Bacillus natto for producing nattokinase by virtue of rapeseed dregs and application of bacillus natto |
| CN103409355B (en) * | 2013-08-28 | 2014-04-30 | 湖南农业大学 | Bacillus natto for producing nattokinase by virtue of rapeseed dregs and application of bacillus natto |
| CN103859404A (en) * | 2014-03-18 | 2014-06-18 | 蔡之宣 | Micro-ecological fruit-vegetable enzyme preparation method |
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| CN109652355A (en) * | 2019-02-12 | 2019-04-19 | 西安培华学院 | A kind of hypoglycemic bafillus natto and preparation method and application |
| CN111088196A (en) * | 2020-01-18 | 2020-05-01 | 河北省科学院生物研究所 | Strain SWS-001 for preparing highly active nattokinase and its application |
| CN111088196B (en) * | 2020-01-18 | 2022-04-22 | 河北省科学院生物研究所 | Strain SWS-001 for preparing high-activity nattokinase and application thereof |
| CN111840525A (en) * | 2020-08-01 | 2020-10-30 | 武汉真福医药股份有限公司 | A composition for treating thromboangiitis and preparation method thereof |
| CN111840525B (en) * | 2020-08-01 | 2023-10-31 | 武汉真福医药股份有限公司 | Composition for treating thromboangiitis and preparation method thereof |
| CN113881608A (en) * | 2021-11-17 | 2022-01-04 | 内蒙古普泽生物制品有限责任公司 | A kind of Bacillus natto and its application in natto fermentation to produce nattokinase |
| CN118813504A (en) * | 2024-09-19 | 2024-10-22 | 青岛诺和诺康生物科技有限公司 | Lactobacillus crispatus for improving thrombosis and vascular health and its products and applications |
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