CN104357355A - Bacillus natto capable of producing MK-7 and application of bacillus natto - Google Patents
Bacillus natto capable of producing MK-7 and application of bacillus natto Download PDFInfo
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Abstract
The invention discloses bacillus natto capable of producing MK-7 and application of the bacillus natto. The bacillus natto capable of producing MK-7 is named as bacillus natto BLCC1-0053 and preserved in the China Center for Type Culture Collection on January 16th, 2014 with the preservation number of CCTCC M 2014028. The bacillus natto BLCC10053 is capable of producing MK-7, and MK-7 is one active ingredient of frequently-used vitamin K2, can be easily absorbed by a human body, can be used for enhancing bone health, preventing osteoporosis, inhibiting angiosteosis, maintaining arterial elasticity, preventing arteriosclerosis and protecting cardiovascular health. The bacillus natto is capable of producing MK-7, the yield reaches up to 3.68733mg/g, and VK2 required by metabolism of the human body is supplemented, so that the effect of preventing and treating multiple diseases is achieved.
Description
Technical field
The invention belongs to technical field of bioengineering, particularly relate to a kind of bacillus natto and the application thereof with product MK-7 ability.
Background technology
Osteoporosis (osteoporosis, OP) be with bone amount reduce and osseous tissue micro-architectural deterioration (Grafting Cancellous Bone Bolt girder attenuates, number of breaks minimizing, cortex bone porous, thinning) for feature, so that a kind of systemic skeletal disease that the fragility of bone increases and risk of fractures increases.Pathogenic factor it is generally acknowledged with calcium and vitamin deficiency, endocrine disturbance, nutritional trouble and motion relevant with illumination deficiency.Any crowd and any age can be betided, be more common in the elderly, especially menopausal women.Osteoporotic serious consequence is fracture, and osteoporotic fracture can hinder the rehabilitation process of patient, increases medical expense, even strengthens mortality ratio.Along with the prolongation of human longevity, osteoporosis and concurrent osteoporotic fracture thereof have become serious major global public health problem, and the women of nearly 30% ~ 50% and the male sex of 15% ~ 30% can run into the fracture of osteoporosis initiation in some stage of life.
Research finds, the concentration of the multiprenylmenaquinone in osteoporosis patient blood is obviously on the low side, and the deficiency of multiprenylmenaquinone is a reason of induced osteoporosis disease, and Japan is using the medicine of multiprenylmenaquinone as osteoporosis.Conventional multiprenylmenaquinone active body has MK-7, MK-4, MK-9, and its biological activity is all significantly higher than vitamin K1.In human homergy, when functions of intestines and stomach standard state, under normal intestinal flora participates in by vitamin K1 and or vitamin K3, methylnaphthohydroquinone be just absorbed and used after being converted into multiprenylmenaquinone active body.
At present, most domestic be merely by supplementary calcium, increase the absorption of calcium from enteron aisle to blood and carry out preventing osteoporosis disease, but take in calcium agent and can cause calcium a large amount of deposition and increase the generation of the diseases such as atherosclerosis, elevation of blood pressure, apoplexy, sacroiliitis, urinary stone disease in blood and cartilage etc. in a large number for a long time.Vitamin K particularly supplementing of multiprenylmenaquinone can evade above risk, promotes calcium further conversion in blood and effectively utilizes.The object of the present invention is to provide can the bacterial strain of activeconstituents MK7 of high yield multiprenylmenaquinone, provides the fermentation culture method of this bacterial strain and the application in preventing osteoporosis.
Summary of the invention
The object of the embodiment of the present invention is that providing a kind of has the bacillus natto and application thereof that produce MK-7 ability, be intended to solve at present, most domestic be merely by supplementary calcium, increase the absorption of calcium from enteron aisle to blood and carry out preventing osteoporosis disease, take in calcium agent and can cause calcium a large amount of deposition and increase the pathogenetic problems of disease such as atherosclerosis, elevation of blood pressure, apoplexy, sacroiliitis, urinary stone disease in blood and cartilage etc. in a large number for a long time.
The embodiment of the present invention realizes like this, a kind of have the bacillus natto producing MK-7 ability, this has the bacillus natto called after bacillus natto BLCC1-0053 producing MK-7 ability, be preserved in China typical culture collection center on January 16th, 2014, its deposit number is CCTCC M2014028.
Further, this bacillus natto biological characteristics with product MK-7 ability is as follows: thalline is shaft-like, little chaining, even dyeing; Gemma ovalize, circular or irregular shape on substratum; Surface colour is dark, can be wrinkling, becomes faint yellow or light brown; Gram-positive; Usually can grow at 35 ~ 50 DEG C, seed liquor optimum temperuture 42 DEG C, upper tank fermentation optimum temperuture 45 DEG C.
Another object of the embodiment of the present invention is to provide a kind of application with the bacillus natto producing MK-7 ability, and this application with the bacillus natto producing MK-7 ability comprises the following steps:
Step one, slant culture: be inoculated in by freeze-dried vaccine powder on solid slant culture base, cultivates 20 ~ 28h at 35 ~ 45 DEG C;
Step 2, first order seed is cultivated: get cultured inclined-plane, be aseptically inoculated in 50mL ~ 100mL seed liquid nutrient medium, under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h, obtained primary seed solution;
Step 3, enlarged culturing: with the inoculum size of 1 ~ 5%, is connected in 500mL ~ 1000mL seed liquid nutrient medium by primary seed solution, under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h, obtained secondary seed solution;
Step 4, fermentor cultivation: with the inoculum size of 1 ~ 5%, is connected to secondary seed solution in liquid fermentation medium, 37 DEG C, and 120rpm cultivates 12 ~ 24h, under going to 40 ~ 50 DEG C of conditions, quiescent culture 5 ~ 7d;
Step 5, after fermentation ends, immediately that fermented liquid is centrifugal and with clean water, so repeatedly 2 ~ 3 times postlyophilizations, pulverize, be bacterium powder finished product; Or after fermentation ends, spraying dry immediately, obtains bacterium powder finished product;
Step 6, after fermented liquid and the pre-treatment of freeze-dried vaccine powder, loading, the content of high effective liquid chromatography for measuring MK-7.
Further, in step 2 and step 3, seed liquid medium component is: glucose 0.2%, peptone 1.0%, sodium-chlor 0.5%, yeast extract paste 0.5%, pH value 7.0; Fermentation medium components is: peptone 10%, glycerol 3%, NaCl 0.5%, K
2hPO
40.02%, pH 7.0-7.2.
Further, in step 4, seed liquor culture condition is under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h; Fermentation culture conditions is: 37 DEG C, and 12 ~ 24h cultivated by 120rpm shaking table, under going to 40 ~ 50 DEG C of conditions, and quiescent culture 5 ~ 7d.
Further, in step 4, fermented liquid pre-treating process is: fermented liquid supernatant concentration ratio 10-100: 1, through concentrated solution same volume chloroform-normal hexane mixing solutions, and volume ratio chloroform: normal hexane=4: 1 ~ 1: 1, lixiviate, magnetic agitation 30min ~ 2h after ultrasonic 20 ~ 40min in frozen water, separating funnel liquid-liquid separation, collects organic solvent nitrogen and blows, normal hexane redissolves, and 0.22um membrane filtration twice rear loading is carried out HPLC and detected analysis.
Further, in step 5, freeze-drying thalline pre-treating process is: freeze-drying thalline is weighed, through chloroform-normal hexane mixing solutions, and volume ratio chloroform: normal hexane ratio is 4: 1 ~ 1: 1,30min ~ 2h is left standstill after ultrasonic dissolution 20 ~ 40min, 25 DEG C, the centrifugal 5 ~ 15min of 5000 ~ 10000rpm, nitrogen dries up, chloroform-normal hexane mixing solutions redissolves, and 0.22um membrane filtration twice rear loading is carried out HPLC and detected analysis.
Further, in step 6, HPLC detects MK-7 content, and detector is UV-detector; Moving phase is methyl alcohol: acetonitrile=60%-80%: 40%-20%; Column temperature is 20-40 DEG C; Determined wavelength is 248nm; Flow velocity is 1.0mL/min; Mark product solvent is normal hexane; Mark product concentration is 0.00176-0.088mg/ml.
The technique effect that the present invention realizes: successfully screen the bacillus natto bacterial strain having and produce MK7 ability, and determine the fermentation culture method of practicability and effectiveness, experimental result display is taken this bacterium powder and effectively can be improved osteoporotic conditions, is expected to the new type of health product being developed as safety, nontoxic preventing osteoporosis.Bacillus natto of the present invention can produce MK-7, and output, up to 3.68733mg/g, supplements the VK2 required for body metabolism, thus reaches prevention and the effect for the treatment of various diseases.
Accompanying drawing explanation
Fig. 1 is the applicating flow chart with the bacillus natto producing MK-7 ability that the embodiment of the present invention provides;
Fig. 2 is that the bacillus natto BLCC1-0053 that provides of the embodiment of the present invention ferments after 6d, and thalline MK-7 HPLC analyzes schematic diagram;
Fig. 3 is the microscopic morphology figure of bacillus natto BLCC1-0053 provided by the invention.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
The bacillus natto with product MK-7 ability of the embodiment of the present invention: this bacterial classification called after bacillus natto BLCC1-0053 (Bacillus natto BLCC1-0053), be preserved in China typical culture collection center, Wuhan University on January 16th, 2014; Its deposit number is CCTCC M 2014028; The culture title of request preservation and dated diagnostic characteristics: bacillus natto BLCC1-0053 (Bacillus natto BLCC1-0053); This culture is preserved in China typical culture collection center on January 16th, 2014 and receives, and register on the books, as requested, preservation 30 years by 16 days January in 2014, receive before expiring after the request of culture samples is provided and continue preservation again 5 years, this preservation of viability center of this culture is detected complete on January 22nd, 2014, and result is survival.
Its biological characteristics is as follows: thalline is shaft-like, little chaining, even dyeing; Gemma ovalize, circular or irregular shape on substratum; Surface colour is dark, can be wrinkling, becomes faint yellow or light brown; Gram-positive; Usually can grow at 35 ~ 50 DEG C, seed liquor optimum temperuture 42 DEG C, upper tank fermentation optimum temperuture 45 DEG C.
There is the bacillus natto producing MK-7 ability, be applied to the diseases such as protect against osteoporosis with the form of lyophilized powder.
As shown in Figure 1, the application with the bacillus natto producing MK-7 ability of the embodiment of the present invention comprises the following steps:
S101: slant culture: be inoculated in by freeze-dried vaccine powder on solid slant culture base, cultivates 20 ~ 28h at 35 ~ 45 DEG C;
S102: first order seed is cultivated: get cultured inclined-plane, be aseptically inoculated in 50mL ~ 100mL seed liquid nutrient medium, under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h, obtained primary seed solution;
S103: enlarged culturing: with the inoculum size of 1 ~ 5%, is connected in 500mL ~ 1000mL seed liquid nutrient medium by primary seed solution, under 35 ~ 45 DEG C of conditions, and quiescent culture 12 ~ 24h, obtained secondary seed solution;
S104: fermentor cultivation: with the inoculum size of 1 ~ 5%, is connected to secondary seed solution in liquid fermentation medium, 37 DEG C, and 120rpm cultivates 12 ~ 24h, under going to 40 ~ 50 DEG C of conditions, and quiescent culture 5 ~ 7d;
S105: after fermentation ends is centrifugal by fermented liquid immediately and with clean water, so repeatedly 2 ~ 3 times postlyophilizations, pulverize, be bacterium powder finished product; Or: after fermentation ends, spraying dry immediately, obtains bacterium powder finished product;
S106: after fermented liquid and the pre-treatment of freeze-dried vaccine powder, loading, high performance liquid chromatography (HPLC) measures the content of MK-7.
In step S102 and S103, seed liquid medium component is: glucose 0.2%, peptone 1.0%, sodium-chlor 0.5%, yeast extract paste 0.5%, pH value 7.0; Fermentation medium components is: peptone 10%, glycerol 3%, NaCl 0.5%, K2HPO4 0.02%, pH 7.0-7.2.
In step S104, seed liquor culture condition is under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h; Fermentation culture conditions is: 37 DEG C, and 12 ~ 24h cultivated by 120rpm shaking table, under going to 40 ~ 50 DEG C of conditions, and quiescent culture 5 ~ 7d;
In step S104, fermented liquid pre-treating process is: fermented liquid supernatant concentrates (ratio 10-100: 1), through concentrated solution same volume chloroform-normal hexane mixing solutions (volume ratio chloroform: normal hexane=4: 1 ~ 1: 1) lixiviate, magnetic agitation 30min ~ 2h after ultrasonic 20 ~ 40min in frozen water, separating funnel liquid-liquid separation, collect organic solvent nitrogen to blow, normal hexane redissolves, and 0.22um membrane filtration twice rear loading is carried out HPLC and detected analysis.
In step S105, freeze-drying thalline pre-treating process is: freeze-drying thalline is weighed, 30min ~ 2h is left standstill after chloroform-normal hexane mixing solutions (volume ratio chloroform: normal hexane ratio is 4: 1 ~ 1: 1) ultrasonic dissolution 20 ~ 40min, 25 DEG C, centrifugal 5 ~ the 15min of 5000 ~ 10000rpm, nitrogen dries up, and chloroform-normal hexane mixing solutions redissolves, and 0.22um membrane filtration twice rear loading is carried out HPLC and detected analysis.
In step s 106, HPLC detects MK-7 content, it is characterized in that: detector is UV-detector; Moving phase is methyl alcohol: acetonitrile=60%-80%: 40%-20%; Column temperature is 20-40 DEG C; Determined wavelength is 248nm; Flow velocity is 1.0mL/min; Mark product solvent is normal hexane; Mark product concentration is 0.00176-0.088mg/ml.
Specific embodiments of the invention:
Embodiment 1:
Bacillus natto primary dcreening operation
1 materials and methods:
1.1 experimental strains, bacillus natto BLCC1-0048, bacillus natto BLCC1-0053, bacillus natto BLCC1-0054;
1.2 fermention mediums and culture condition:
Two kinds of culture medium prescriptions:
Seed culture medium: glucose 0.2%, peptone 1%, extractum carnis 0.5%, NaCl 0.5%, pH 7.2-7.4;
Fermention medium: yeast extract paste 5%, glycerol 5%, peptone 10%, K
2hPO
40.06%;
Culture condition: shaking table cultivates 37 DEG C, 24h, rotating speed 120rpm, then 45 DEG C of quiescent culture 5d.
After 1.3 fermentation ends, centrifugation fermented liquid and thalline, thalline lyophilize.
MK-7 assay in 1.4 fermentation thalli and fermented liquid:
Take appropriate freeze-drying thalline (about 25mg), through chloroform: normal hexane=2: the lixiviate of 1 (volume ratio) mixing solutions, 2h is left standstill after the ultrasonic 30min of frozen water, 25 DEG C, the centrifugal 10min of 8000rpm, collect organic solvent nitrogen to dry up, chloroform: normal hexane=2: 1 (volume ratio) mixing solutions redissolves, after the membrane filtration twice of 0.22um, loading is carried out HPLC and is detected and analyze.
Fermented liquid supernatant concentrates (ratio 10-100: 1), and (chloroform: normal hexane=2: 1) mix lixiviate, the ultrasonic 30min of frozen water, magnetic agitation 2h, separating funnel Liquid liquid Separation, collects concentrated solution same volume organic solvent
Organic solvent nitrogen blows, and normal hexane redissolves, and membrane filtration twice rear loading is carried out HPLC and detected analysis.
Detector is UV-detector; Moving phase is methyl alcohol: acetonitrile=60%: 40%; Column temperature is 40 DEG C; Determined wavelength is 248nm; Flow velocity is 1.0mL/min; Mark product solvent is normal hexane; Mark product concentration is 0.088mg/ml.
2 results and analysis:
After three strain bacillus nattos cultivate 6d in two kinds of substratum, centrifugation thalline and fermented liquid, after process in early stage, loading carries out efficient liquid phase chromatographic analysis (HPLC), and experimental data statistics is as shown in table 1:
Table 1. three strain bacillus natto MK-7 content detection result
Learnt by table 1, after Bacillus strain BLCC 10053 ferments in two kinds of substratum, the content of MK-7 is the highest, and therefore subsequent experimental selects Bacillus strain BLCC 10053 as experimental subjects.
Embodiment 2:
The mensuration of bacillus natto to ferment 6d thalline MK-7 content under different culture media condition:
1 materials and methods:
1.1 experimental strain bacillus natto bacterial strain BLCC 10053;
1.2 fermention mediums and culture condition:
Three kinds of basal medium formulation:
N1: glycerol 3%, peptone 10%, NaCl 0.5%, K
2hPO
40.02%, pH value 7.0-7.2
N2: glucose 0.2%, yeast extract paste 0.5%, peptone 1.0%, sodium-chlor 0.5%, pH value 7.0-7.2
N3: yeast extract paste 2.5%, glycerol 2.5%, peptone 9.5%, K
2hPO
40.03%, pH value 7.0-7.2
Culture condition:
Specific experiment scheme is as shown in table 2, and in the bacillus natto access seed culture medium of activation, 16h cultivated by 42 DEG C of shaking tables, and the inoculum size with 5% is transferred in fermention medium, proceeds to quiescent culture 5d in 45 DEG C of incubators after 1d cultivated by 37 DEG C of shaking tables.
After 1.3 fermentation ends, 10000rpm centrifugation tunning and thalline, thalline lyophilize.
MK-7 assay in 1.4 fermentation thalli:
Take appropriate freeze-drying thalline (about 25mg), through chloroform: normal hexane=2: the lixiviate of 1 (volume ratio) mixing solutions, 2h is left standstill after the ultrasonic 30min of frozen water, 25 DEG C, the centrifugal 10min of 8000rpm, collect organic solvent nitrogen to dry up, chloroform: normal hexane=2: 1 (volume ratio) mixing solutions redissolves, after the membrane filtration twice of 0.22um, loading is carried out HPLC and is detected and analyze.
Detector is UV-detector; Moving phase is methyl alcohol: acetonitrile=60%: 40%; Column temperature is 40 DEG C; Determined wavelength is 248nm; Flow velocity is 1.0mL/min; Mark product solvent is normal hexane; Mark product concentration is 0.088mg/ml.
2 results and analysis:
Bacterial strain bacillus natto BLCC 10053 is after experience seed culture medium as shown in table 2 and fermention medium intersect and ferment, centrifugation thalline and fermented liquid, thalline is after process in early stage, and loading carries out efficient liquid phase chromatographic analysis (HPLC), and experimental data statistics is as shown in table 2.Result display selects No. 2 substratum (N2) as seed culture medium, and when No. 1 substratum (N1) is as fermention medium, bacillus natto BLCC 10053 thalline MK-7 output is maximum.
Table 2. bacillus natto to ferment 6d thalline MK-7 content (mg/L)
Embodiment 3:
Bacillus natto thalline MK-7 content under different fermentations time conditions:
1 materials and methods:
1.1 experimental strain bacillus natto bacterial strain BLCC 10053;
1.2 fermention mediums and culture condition;
Seed culture medium N2: glucose 0.2%, peptone 1.0%, sodium-chlor 0.5%, yeast extract paste 0.5%, pH value 7.0-7.2; Fermention medium N1: peptone 10%, glycerol 3%, NaCl 0.5%, K
2hPO
40.02%, pH 7.0-7.2.
In the bacillus natto access seed culture medium of activation, 16h cultivated by 42 DEG C of shaking tables, and the inoculum size with 5% is transferred in fermention medium, proceeds to quiescent culture 3d in 45 DEG C of incubators, 4d, 5d, 6d, 7d after 1d cultivated by 37 DEG C of shaking tables.
After 1.3 fermentation ends, centrifugation tunning and thalline, thalline lyophilize.
MK-7 assay in 1.4 fermentation thalli and fermented liquid;
Take appropriate freeze-drying thalline (about 25mg), through chloroform: normal hexane=2: the lixiviate of 1 (volume ratio) mixing solutions, 2h is left standstill after the ultrasonic 30min of frozen water, 25 DEG C, the centrifugal 10min of 8000rpm, collect organic solvent nitrogen to dry up, chloroform: normal hexane=2: 1 (volume ratio) mixing solutions redissolves, after the membrane filtration twice of 0.22um, loading is carried out HPLC and is detected and analyze.
Fermented liquid supernatant concentrates (ratio 10-100: 1), concentrated solution same volume organic solvent (chloroform: normal hexane=2: 1) mix lixiviate, the ultrasonic 30min of frozen water, magnetic agitation 2h, separating funnel Liquid liquid Separation, collect organic solvent nitrogen to blow, normal hexane redissolves, and membrane filtration twice rear loading is carried out HPLC and detected analysis.
Detector is UV-detector; Moving phase is methyl alcohol: acetonitrile=60%: 40%; Column temperature is 40 DEG C; Determined wavelength is 248nm; Flow velocity is 1.0mL/min; Mark product solvent is normal hexane; Mark product concentration is 0.088mg/ml.
2 results and analysis:
Along with the prolongation of bacillus natto to ferment time, the content of MK-7 increases, and when 8d, the content of MK-7 is uprushed, in often liter of fermented liquid, the MK-7 content (on average) of thalline is 20.3680mg, and in fermented liquid, content is 0.8004mg, and experimental result is specifically in table 3.
Table 3. bacillus natto different fermentations time MK-7 total content (mg/L)
Embodiment 4:
Bacillus natto pilot scale fermentation is tested:
The bacillus natto BLCC10053 of activation accesses in seed culture medium, and 16h cultivated by 42 DEG C of shaking tables, and the inoculum size with 5% is transferred in fermention medium, proceeds to quiescent culture 6d in 45 DEG C of incubators after 1d cultivated by 37 DEG C of shaking tables.After fermentation ends, 8000rpm centrifugation fermented liquid and thalline, thalline is weighed after vacuum lyophilization, measures MK-7 output in thalline.Pilot scale fermentation liquid amounts to 16.7L, and drying obtains thalline 165.62g, and MK-7 content in display thalline analyzed by high performance liquid chromatography (HPLC) is that 3.68733mg/g experimental result is specifically in table 4.
MK-7 content in table 4. pilot scale fermentation thalline
| Duplicate Samples | Example weight (g) | Sample concentration (mg/L) | MK-7(mg/g) |
| 1 | 0.0259 | 50.87802 | 3.92880 |
| 2 | 0.0280 | 54.53029 | 3.89502 |
| 3 | 0.0268 | 51.05754 | 3.81026 |
| 4 | 0.0266 | 41.83139 | 3.14522 |
| On average | 3.68733 |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. one kind has the bacillus natto producing MK-7 ability, it is characterized in that, this has the bacillus natto producing MK-7 ability: this bacillus natto kind called after bacillus natto BLCC1-0053, be preserved in China typical culture collection center on January 16th, 2014, its deposit number is CCTCC M 2014028.
2. have the bacillus natto producing MK-7 ability as claimed in claim 1, it is characterized in that, it is as follows that this has the bacillus natto biological characteristics producing MK-7 ability: thalline is shaft-like, little chaining, even dyeing; Gemma ovalize, circular or irregular shape on substratum; Surface colour is dark, can be wrinkling, becomes faint yellow or light brown; Gram-positive; Usually can grow at 35 ~ 50 DEG C, seed liquor optimum temperuture 42 DEG C, upper tank fermentation optimum temperuture 45 DEG C.
3. have an application for the bacillus natto producing MK-7 ability, it is characterized in that, this application with the bacillus natto producing MK-7 ability comprises the following steps:
Step one, slant culture: be inoculated in by freeze-dried vaccine powder on solid slant culture base, cultivates 20 ~ 28h at 35 ~ 45 DEG C;
Step 2, first order seed is cultivated: get cultured inclined-plane, be aseptically inoculated in 50mL ~ 100mL seed liquid nutrient medium, under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h, obtained primary seed solution;
Step 3, enlarged culturing: with the inoculum size of 1 ~ 5%, is connected in 500mL ~ 1000mL seed liquid nutrient medium by primary seed solution, under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h, obtained secondary seed solution;
Step 4, fermentor cultivation: with the inoculum size of 1 ~ 5%, is connected to secondary seed solution in liquid fermentation medium, 37 DEG C, and 120rpm cultivates 12 ~ 24h, under going to 40 ~ 50 DEG C of conditions, quiescent culture 5 ~ 7d;
Step 5, after fermentation ends, immediately that fermented liquid is centrifugal and with clean water, so repeatedly 2 ~ 3 times postlyophilizations, pulverize, be bacterium powder finished product; Or after fermentation ends, spraying dry immediately, obtains bacterium powder finished product;
Step 6, after fermented liquid and the pre-treatment of freeze-dried vaccine powder, loading, the content of high effective liquid chromatography for measuring MK-7.
4. there is the application of the bacillus natto producing MK-7 ability as claimed in claim 3, it is characterized in that, in step 2 and step 3, seed liquid medium component is: glucose 0.2%, peptone 1.0%, sodium-chlor 0.5%, yeast extract paste 0.5%, pH value 7.0; Fermentation medium components is: peptone 10%, glycerol 3%, NaCl0.5%, K
2hPO
40.02%, pH7.0-7.2.
5. have the application of the bacillus natto producing MK-7 ability as claimed in claim 3, it is characterized in that, in step 4, seed liquor culture condition is under 35 ~ 45 DEG C of conditions, quiescent culture 12 ~ 24h; Fermentation culture conditions is: 37 DEG C, and 12 ~ 24h cultivated by 120rpm shaking table, under going to 40 ~ 50 DEG C of conditions, and quiescent culture 5 ~ 7d.
6. there is the application of the bacillus natto producing MK-7 ability as claimed in claim 3, it is characterized in that, in step 4, fermented liquid pre-treating process is: fermented liquid supernatant concentration ratio 10-100: 1, through concentrated solution same volume chloroform-normal hexane mixing solutions, volume ratio chloroform: normal hexane=4: 1 ~ 1: 1, lixiviate, magnetic agitation 30min ~ 2h after ultrasonic 20 ~ 40min in frozen water, separating funnel liquid-liquid separation, collect organic solvent nitrogen to blow, normal hexane redissolves, and 0.22um membrane filtration twice rear loading is carried out HPLC and detected analysis.
7. there is the application of the bacillus natto producing MK-7 ability as claimed in claim 3, it is characterized in that, in step 5, freeze-drying thalline pre-treating process is: freeze-drying thalline is weighed, through chloroform-normal hexane mixing solutions, volume ratio chloroform: normal hexane ratio is 4: 1 ~ 1: 1,30min ~ 2h is left standstill after ultrasonic dissolution 20 ~ 40min, 25 DEG C, centrifugal 5 ~ the 15min of 5000 ~ 10000rpm, nitrogen dries up, and chloroform-normal hexane mixing solutions redissolves, and 0.22um membrane filtration twice rear loading is carried out HPLC and detected analysis.
8. have the application of the bacillus natto producing MK-7 ability as claimed in claim 3, it is characterized in that, in step 6, HPLC detects MK-7 content, and detector is UV-detector; Moving phase is methyl alcohol: acetonitrile=60%-80%: 40%-20%; Column temperature is 20-40 DEG C; Determined wavelength is 248nm; Flow velocity is 1.0mL/min; Mark product solvent is normal hexane; Mark product concentration is 0.00176-0.088mg/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410621082.1A CN104357355A (en) | 2014-11-06 | 2014-11-06 | Bacillus natto capable of producing MK-7 and application of bacillus natto |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711237A (en) * | 2015-03-20 | 2015-06-17 | 安徽工程大学 | Method for regulating and controlling activity of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase |
| CN104738623A (en) * | 2015-03-17 | 2015-07-01 | 徐州医学院 | Lentinus edodes powder capsules rich in vitamin K2 and calcium and preparation method thereof |
| CN105055458A (en) * | 2015-06-15 | 2015-11-18 | 山东凤凰生物有限公司 | Probiotic preparation for preventing and treating osteoporosis, and preparation method thereof |
| CN105130685A (en) * | 2015-09-30 | 2015-12-09 | 富朗（中国）生物科技有限公司 | Preparation method for organic fertilizer containing Nu-polyglutamic acid |
| CN106701719A (en) * | 2017-01-16 | 2017-05-24 | 中国科学院合肥物质科学研究院 | Method for simultaneously producing vitamin K2 and nattokinase through Bacillus natto fermentation |
| CN107118991A (en) * | 2017-05-25 | 2017-09-01 | 山东凤凰生物有限公司 | A kind of bafillus natto with the production abilities of MK 7 and its application |
| CN107475312A (en) * | 2017-09-14 | 2017-12-15 | 中国科学院合肥物质科学研究院 | A kind of method for effectively producing farnoquinone by improving permeability of cell membrane |
| CN110129234A (en) * | 2019-05-27 | 2019-08-16 | 沈阳农业大学 | The bacillus subtilis strain of high yield Agua-Mephyton 2 through mutagenesis and its application |
| CN111394405A (en) * | 2019-05-14 | 2020-07-10 | 江苏臻大天园健康科技有限公司 | Extraction process of vitamin K2 in natto fermentation liquor |
| WO2023284853A1 (en) * | 2021-07-16 | 2023-01-19 | 湖北美琪健康科技有限公司 | Bacillus natto for producing menaquinone-7 and use thereof |
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2014
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| AYDIN BERENJIAN, ET AL.: "ADVANCES IN MENAQUINONE-7 PRODUCTION BY BACILLUS SUBTILIS NATTO: FED-BATCH GLYCEROL ADDITION", 《AMERICAN JOURNAL OF BIOCHEMISTRY AND BIOTECHNOLOGY》 * |
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| CN104738623A (en) * | 2015-03-17 | 2015-07-01 | 徐州医学院 | Lentinus edodes powder capsules rich in vitamin K2 and calcium and preparation method thereof |
| CN104711237A (en) * | 2015-03-20 | 2015-06-17 | 安徽工程大学 | Method for regulating and controlling activity of 1,4-dihydroxy-2-naphthoic acid-polyisoprene transferase |
| CN104711237B (en) * | 2015-03-20 | 2018-06-12 | 安徽工程大学 | A kind of method of regulation and control 1,4- dihydroxy-2-naphthoic acids-polyisoprene transfer enzyme activity |
| CN105055458A (en) * | 2015-06-15 | 2015-11-18 | 山东凤凰生物有限公司 | Probiotic preparation for preventing and treating osteoporosis, and preparation method thereof |
| CN105055458B (en) * | 2015-06-15 | 2018-10-23 | 山东凤凰生物有限公司 | Prevent the probiotics preparation and preparation method thereof of osteoporosis |
| CN105130685A (en) * | 2015-09-30 | 2015-12-09 | 富朗（中国）生物科技有限公司 | Preparation method for organic fertilizer containing Nu-polyglutamic acid |
| CN106701719B (en) * | 2017-01-16 | 2020-04-21 | 中国科学院合肥物质科学研究院 | Fermentation and co-production of vitamin K by utilizing bacillus natto2Method for producing nattokinase |
| CN106701719A (en) * | 2017-01-16 | 2017-05-24 | 中国科学院合肥物质科学研究院 | Method for simultaneously producing vitamin K2 and nattokinase through Bacillus natto fermentation |
| CN107118991A (en) * | 2017-05-25 | 2017-09-01 | 山东凤凰生物有限公司 | A kind of bafillus natto with the production abilities of MK 7 and its application |
| CN107475312A (en) * | 2017-09-14 | 2017-12-15 | 中国科学院合肥物质科学研究院 | A kind of method for effectively producing farnoquinone by improving permeability of cell membrane |
| CN111394405A (en) * | 2019-05-14 | 2020-07-10 | 江苏臻大天园健康科技有限公司 | Extraction process of vitamin K2 in natto fermentation liquor |
| CN110129234A (en) * | 2019-05-27 | 2019-08-16 | 沈阳农业大学 | The bacillus subtilis strain of high yield Agua-Mephyton 2 through mutagenesis and its application |
| CN110129234B (en) * | 2019-05-27 | 2021-03-02 | 沈阳农业大学 | Mutagenized bacillus subtilis strain with high natural vitamin K2 yield and application thereof |
| WO2023284853A1 (en) * | 2021-07-16 | 2023-01-19 | 湖北美琪健康科技有限公司 | Bacillus natto for producing menaquinone-7 and use thereof |
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