CN110432324A - A kind of bacillus subtilis and its application with anti-thrombus function - Google Patents
A kind of bacillus subtilis and its application with anti-thrombus function Download PDFInfo
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- CN110432324A CN110432324A CN201910712777.3A CN201910712777A CN110432324A CN 110432324 A CN110432324 A CN 110432324A CN 201910712777 A CN201910712777 A CN 201910712777A CN 110432324 A CN110432324 A CN 110432324A
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- bacillus subtilis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
- A23C9/1216—Other enzymes
Abstract
The invention discloses a kind of fermentation of bacillus subtilis cream and preparation method thereof with anti-thrombus function, belongs to technical field of health care food.The bacterial strain that the present invention uses is for bacillus subtilisB.subtilisSp.nk1(CGMCC18057), there is high plasmin activity and high milk-curdling activity.Acidified milk of the present invention is added using fresh cow milk/reconstituted milk or is added without soy meal as raw material, ferment using the bacillus subtilis with high activity fibrinolysin and renin.The finished product of the acidified milk is fine and smooth uniformly to have gloss, and smooth in taste has special sour-sweet taste and milk fragrance.Preparation method is divided into two key links, first is that inoculation bacillus subtilis is fermented, second is that the enzyme activity protective agent such as refrigerating after-ripening and mTG enzyme being added.The present invention solves the problems such as Nattokinase beverage mouthfeel is poor, and validity period is short, and absorption rate is low, and fermentation condition is mild, and method is easy, is suitable for industrialized production.
Description
Technical field
The present invention relates to a kind of bacillus subtilis with anti-thrombus function and its applications, belong to microorganism and its culture
And application field.
Background technique
In modern society's life, the interior thrombosis of human vas will lead to the serious heart such as cerebral infarction and myocardial infarction
Vascular diseases can especially cause very big harm to the health of the middle-aged and the old, and there are thrombosis patients 15,000,000 in the whole world, potential
Thrombolytics market have 2,000,000,000 dollars, but current thrombolytics, as urokinase, streptokinase, recombinant tissue-type plasminogen activation
Agent etc., there are half-life short, side effect it is big, expensive and the various shortcomings such as cannot be administered orally, it is difficult to as applicable
Public drug.And Nattokinase has many advantages, such as that safety is good, effect is rapid, at low cost, Orally-administrable.The enzyme can be by withered grass
Fermentation of bacillus production.So there are extremely tempting bright prospects for developing thrombolytics or health food of new generation.
A kind of proteolytic enzyme that Nattokinase is generated by microorganism has and promotes local blood circulation, increases tissue
Permeability and antiinflammation are generally used for various thrombotic diseases.If Nattokinase is directly oral, can gradually lose under one's belt
Living, efficient lower, therefore, oral agents need to carry out certain protection to its activity in design, production, can be only achieved ideal
Effectiveness.
There are two types of current Nattokinase health drinks, and one is high protein beverage stoste is carried out microbial fermentation generation
Nattokinase and be made；Another kind is will to ferment the Nattokinase for extracting and obtaining directly as in ingredient addition reconciliation beverage.This
A little Nattokinase health drinks are there are two main problems, and first, the flavor of Nattokinase fermenting beverage is not much to consume
Person is received；Second, Nattokinase can be inactivated gradually in gastric environment after taking in human body, and the effectiveness time is short, and absorption rate is not
It is high.There are also products to use cyclodextrin, and gelatin etc. embeds Nattokinase, however these embedding mediums can drink protein
The mouthfeel of material impacts, using being restricted.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of bacillus subtilises with anti-thrombus function
(B.subtilis sp), nk1 is it is characterized in that, the bacterial strain is preserved in China General Microbiological culture presevation administrative center, preservation
Number is CGMCC18057.The present invention also provides a kind of acidified milks with anti-thrombus function.The present invention passes through withered grass gemma
Bacillus fermentation cow's milk generates Nattokinase, makes a kind of working up curd drink with anti-thrombus function；Manufacturing process uses two
Step fermentation, 37-45 DEG C of working up curd after first step inoculation refrigerate after-ripening and protector of enzyme activity are added after second step demulsification.
Acidified milk with anti-thrombus function of the invention is using cow's milk or reconstituted milk as primary raw material, using withered grass
Made from fermentation of bacillus；In the raw material, added with or not added with soy meal.
This acidified milk is fermented using bacillus subtilis strain B.subtilis.sp.nk1, wherein seed culture based formulas
It is as follows:
Casein 10g/L
Sucrose 50g/L
Pure water surplus
High-yield nattokinase bacillus subtilis strain B.subtilis sp.nk1, activating used culture medium is ox
Milk culture medium is formulated as follows:
Fresh milk 500g/L
Sucrose 50g/L
Pure water surplus
Activation condition is 37 DEG C of shaking table culture 12h, and bacterium amount is 1 × 10 after activation8-5×108CFU/mL。
In one embodiment, the soy meal accounts for the 0%-10% (w/v) of raw material.
In one embodiment, the raw material further includes 2-10% (w/v) table sugar.
In one embodiment, the preparation of the acidified milk, comprising the following steps:
1) milk homogeneous；
2) raw material mixes；
3) it sterilizes, is cooling；
4) inoculation produces the bacillus subtilis of Nattokinase and renin；
5) it ferments；
6) it stirs or does not stir, after-ripening；
7) protector of enzyme activity and thickener is added；
8) filling, refrigeration.
In one embodiment, the raw material includes: fresh milk/reconstituted milk, and soybean protein 1-5% (w/v) eats sugarcane
Sugared 1-10% (w/v)；
In one embodiment, the sterilization process in step 3), sterilising conditions are 90-95 degrees Centigrade, sterilizing
Time 5-15 minute；
In one embodiment, the bacillus subtilis of the step 4) is that the high fibrinolytic screened from natto is lived
Power, high condenser water level bacillus subtilis strain B.subtilis sp.nk1 (CGMCC 18057).
In one embodiment, the bacterium solution being inoculated in step 4), inoculum concentration is with the viable bacteria density in lotion after being inoculated with
Meter is 1 × 106-1×107CFU/ml。
In one embodiment, the bacillus subtilis being inoculated in step 4) has before each inoculation at 37 DEG C
It is expanded and is activated under the conditions of oxygen, used medium formula is fresh milk 500g/L, sucrose 50g/L, pure with milk equivalent
Water purification.
In one embodiment, the fermentation of the step 5) is condensed in 35 DEG C of -45 DEG C of fermentations 4-12 hours to milk
For one block of complete gel.
In one embodiment, fermentation process described in the step 5), fermentation temperature are 35-45 DEG C.
In one embodiment, the fermentation time of the step 5) is 4-12 hours.
In one embodiment, it can be carried out in the step 6) or without stirring, stirred type or coagulating type hair is made
Kefir milk.
In one embodiment, the After-ripening being previously mentioned in the step 6) is that 2-10 DEG C of refrigeration after-ripening 6-24 is small
When.
In one embodiment, the after-ripening of the step 6) be 4 DEG C refrigeration 6-24 hours；
In one embodiment, the enzyme activity protective agent being added in the step 7) includes but is not limited to TG enzyme, and TG enzyme adds
Dosage is 0.1%-1%.
In one embodiment, the enzyme activity protective agent added in the step 7) is TG enzyme, additive amount 0.3-3g/L.
In one embodiment, the thickener being added in the step 7) includes but is not limited to agar, and hydroxypropyl two forms sediment
Powder phosphate, trehalose, gelatin.
The invention has the following beneficial effects:
Bacterial strain B.subtilis sp.nk1 (CGMCC 18057) acidified milk of the present invention that the present invention uses, with fresh cow milk/
It is raw material that reconstituted milk, which is added or is added without soy meal, is carried out using the bacillus subtilis with high activity fibrinolysin and renin
It ferments.The finished product of the acidified milk is fine and smooth uniformly to have gloss, and smooth in taste has special sour-sweet taste and milk fragrance.Its
Preparation method is divided into two key links, first is that inoculation bacillus subtilis is fermented, second is that refrigerating after-ripening and mTG enzyme being added
Etc. enzyme activity protective agent.The present invention solves the problems such as Nattokinase beverage mouthfeel is poor, and validity period is short, and absorption rate is low, and sends out
Ferment mild condition, method is easy, is suitable for industrialized production.
Detailed description of the invention
Fig. 1: the bacillus subtilis B.subtilis with high fibrinolytic, milk-clotting activity of monoclonal screening purifying
sp.nk1。
The producing enzyme situation of Fig. 2: B.subtilis sp.nk1 shake flask fermentation in producing enzyme fermentation medium.
Fig. 3: the appearance of the solidified-type fermented milk of embodiment 1.
Fig. 4: the bacillus subtilis in the solidified-type fermented milk smear of embodiment 1；
Fig. 5: the enzymatic activity of the storage different time solidified-type fermented milk of embodiment 1: 1. 0 day, 2. 1 day, 3. 7 days, 4. 14
It, 5. 21 days, 6. core sample be storage 60 days.
Fig. 6: the stirred type acidified milk appearance of embodiment 2.
Fig. 7: the enzymatic activity of the storage different time stirred type acidified milk of embodiment 2；1. fermentation ends, 2. after after-ripening, 3.
It stores 7 days, 4. store 21 days.
Specific embodiment
Embodiment 1:
(1), Spawn incubation and activation
This acidified milk is fermented using bacillus subtilis strain B.subtilis.sp.nk1, wherein seed culture based formulas
It is as follows:
Casein 10g/L
Sucrose 50g/L
Pure water surplus
High-yield nattokinase bacillus subtilis strain B.subtilis sp.nk1, activating used culture medium is ox
Milk culture medium is formulated as follows:
Fresh milk 500g/L
Sucrose 50g/L
Pure water surplus
Activation condition is 37 DEG C of shaking table culture 12h, and bacterium amount is 1 × 10 after activation8-5×108CFU/mL。
(2), bacterial strain produces fibrinolysin and milk-curdling activity identification in shaking flask
Use the enzymatic productivity of shake flask fermentation identification bacillus subtilis strain B.subtilis sp.nk1, producing enzyme fermentation
Producing culture medium used in Nattokinase and renin simultaneously is kidney bean culture medium, and producing enzyme fermentative medium formula is as follows:
Milk-curdling activity measurement uses Arima method.With 0.01mol/L CaCl2Liquid prepares 10% separated milk.This is molten
Liquid uses after being placed at room temperature for 40min after preparing.It takes 10% skimmed milk power liquid of 5mL in 35 DEG C of heat preservation 10min, adds 0.5mL appropriate
Diluted enzyme solution (35 DEG C of enzyme solution heat preservations), shakes up immediately, starts timing.The time (s) that accurate recording is solidified from starting lotion,
The enzyme amount of 40min condensation 10% skimmed milk of 1mL is defined as a Soxhlet unit Soxhlet unit (SU)
It ferments 24 hours under this condition, milk-curdling activity reaches highest, is 812.8SU/ml (such as Fig. 2).
Compared to producing lab ferment bacillus subtilis reported in the literature, such as: Gao Yan 2012, bacillus subtilis JAASB,
440SU/ml；Chwen-Jen Shieh 2009, bacillus subtilis B.subtilis Takahashi, 685.7SU/ml, this
Bacterial strain B.subtilis sp.nk1 used in inventing has higher curdled milk enzyme activity.
Meanwhile the fermentation liquid curdled milk enzyme activity of B.subtilis sp.nk1 is significantly higher than the production Nattokinase in other sources
Bacillus subtilis strain (such as table 1).
| Bacillus subtilis natto bacterial strain | Fermentation liquid condenser water level SU/ml |
| B.subtilis sp.nk1 | 812.8SU/ml |
| Bacillus subtilis ATCC 7058 | 30.2 |
| Bacillus subtilis ATCC 7059 | 11.5 |
| Bacillus subtilis IFO 13169 | 40.1 |
| Bacillus subtilis IFO 3335 | 71.1 |
Thrombolysis activity detection is control with urokinase, uses agarose-fibrinogen plate assay measurement Nattokinase
Activity, operating method are as follows:
1) by fibrin ferment, plasminogen is dissolved in phosphate buffer (pH 7.4), concentration of thrombin 0.1mg/mL, plasminogen
Concentration 0.5mg/mL.Agarose is mixed and heated to 60 DEG C with distilled water to be completely dissolved, with above-mentioned fibrin ferment/plasminogen solution
1:1 mixing, mixing are poured into culture dish, and detection plate is made after cooling.It is spaced 3cm or more on plate to punch, aperture
About 2mm.
2) urokinase is configured to the standard solution of 100IU/mL in phosphate buffer (pH7.4), in each hole of plate
2,4,8,16,32 μ L are separately added into, with the diameter of transparent solusphere around each hole of vernier caliper measurement after 37 DEG C of heat preservation 18h.As a result
The relationship for showing total enzyme activity and solusphere diameter is ln U=0.6318D (mm) -0.6314
Fermentation 42 hours, natto kinase activity reaches highest, is 14520U/ml.
(3), the small lot production of solidified-type fermented milk
1) fresh milk is carried out high-pressure homogeneous；
2) fermentation raw material is mixed.Fermentation raw material includes: 1000mL fresh milk, 40g sucrose, 20g soy meal；
3) 95 DEG C of heating 10min sterilizings；
4) 45 DEG C are cooled to, inoculation produces the bacillus subtilis of Nattokinase；
5) it ferments 8 hours for 40 DEG C, until milk is integrally condensed into one piece of complete ziega；
6) 4 DEG C of refrigeration after-ripening 12h are placed into after naturally cooling to 25 DEG C；
7) storage condition is 4 DEG C of refrigerations.
The solidified-type fermented milk appearance that above method fermentation obtains is delicate, delicate mouthfeel, and just melt in the mouth has Cow Milk Fermentation
Intrinsic flavour and special sour-sweet taste, pleasant impression afterwards is long.
(3), thrombolysis activity detects
It is control with urokinase, uses agarose-fibrinogen plate assay measurement Nattokinase activity, operating method
It is as follows:
1) by fibrin ferment, plasminogen is dissolved in phosphate buffer (pH 7.4), concentration of thrombin 0.1mg/mL, plasminogen
Concentration 0.5mg/mL.Agarose is mixed and heated to 60 DEG C with distilled water to be completely dissolved, with above-mentioned fibrin ferment/plasminogen solution
1:1 mixing, mixing are poured into culture dish, and detection plate is made after cooling.It is spaced 3cm or more on plate to punch, aperture
About 2mm.
2) urokinase is configured to the standard solution of 100IU/mL in phosphate buffer (pH7.4), in each hole of plate
2,4,8,16,32 μ L are separately added into, with the diameter of transparent solusphere around each hole of vernier caliper measurement after 37 DEG C of heat preservation 18h.As a result
The relationship for showing total enzyme activity and solusphere diameter is ln U=0.6318D (mm) -0.6314.
3) sample to be tested phosphate buffer is diluted and is adjusted to pH7.4, taken in 7 μ L adding holes respectively, in another hole
It is control that urokinase standard solution, which is added, measures aperture after a certain period of time, calculates to obtain sample total enzyme activity power, such as Fig. 4.Testing result table
Fermentation of bacillus subtilis cream that is bright, making according to the above method, initial natto kinase activity reach 519.4U/ml, refrigerate 21 days,
Its Nattokinase enzymatic activity remains in 246.1U/mL.
Embodiment 2
(1), actication of culture
High-yield nattokinase bacillus subtilis strain B.subtilis sp.nk1, activating used culture medium is ox
Milk culture medium is formulated as follows:
Fresh milk 500g/L
Sucrose 50g/L
Pure water surplus
Activation condition is 37 DEG C of shaking table culture 12h, and bacterium amount is 2 × 10 after activation8CFU/mL。
(2), the small lot production of stirred type acidified milk
1) fresh milk is carried out high-pressure homogeneous；
2) fermentation raw material is mixed.Fermentation raw material includes: 1000mL fresh milk, 40g sucrose, 20g soy meal；
3) 95 DEG C of heating 10min sterilizings；
4) 45 DEG C are cooled to, inoculation produces the bacillus subtilis of Nattokinase, and inoculum concentration is that 30mL activates bacterium solution；
5) it ferments 8 hours for 40 DEG C, until milk is integrally condensed into one piece of complete ziega；
6) 25 DEG C are cooled to, stirring.
7) 4 DEG C of refrigeration after-ripening 12h；
8) TG enzyme 1g is added, hydroxypropyl PASELLI EASYGEL 3g, agar 3g are stirred evenly；
9) storage condition is 4 DEG C of refrigerations.
The stirred type acidified milk of above-mentioned steps production has certain viscosity, and lotion is fine and smooth, uniformly has gloss.Mouthfeel is refreshing
It is sliding, there is special sour-sweet taste and milk fragrance.
(3), thrombolysis activity detects
Use above-mentioned agarose-fibrinogen plate assay measurement stirred type acidified milk natto kinase activity and storage
Time, the influence to enzymatic activity.
Testing result shows the fermentation of bacillus subtilis made according to the above method cream, its natto kinase activity after after-ripening
It up to 617.4U/mL, refrigerates 21 days, Nattokinase enzymatic activity is 284.6U/mL, such as Fig. 7.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (12)
1. it is a kind of with anti-thrombus function bacillus subtilis (B. subtilis sp), which is characterized in that the bacterial strain preservation
In China General Microbiological culture presevation administrative center, deposit number is CGMCC 18057.
2. the application that bacillus subtilis described in claim 1 is used to prepare antithrombotic food.
3. application according to claim 2, which is characterized in that the food is acidified milk.
4. application according to claim 3, which is characterized in that be using cow's milk or reconstituted milk as primary raw material, use is withered
Made from careless fermentation of bacillus；In the raw material, added with or not added with soy meal.
5. the white 1-5%(w/v of bacillus subtilis described in claim 1), edible sucrose 1-10%(w/v).
The obtained acidified milk 6. CGMCC 18057 according to claim 1 ferments.
7. application according to claim 4, which is characterized in that the inoculum concentration of the bacillus subtilis, with cream after inoculation
Viable bacteria densitometer in liquid is 1 × 106-1×107 CFU/ml。
8. acidified milk according to claim 6, which is characterized in that the preparation of the acidified milk, comprising the following steps:
1) milk homogeneous；
2) raw material mixes；
3) it sterilizes, is cooling；
4) inoculation produces the bacillus subtilis of Nattokinase and renin；
5) it ferments；
6) it stirs or does not stir, after-ripening；
7) protector of enzyme activity and thickener is added；
8) filling, refrigeration.
9. acidified milk according to claim 8, which is characterized in that the enzyme activity protective agent additive amount being added in the step 7)
For 0.1%-1%.
10. acidified milk according to claim 9, which is characterized in that the enzyme activity protective agent being added in the step 7) is TG
Enzyme.
11. acidified milk according to claim 8, which is characterized in that the fermentation of the step 5) is in 5 DEG C of -45 DEG C of fermentations
One block of complete gel is condensed into milk within 4-12 hours.
12. acidified milk according to claim 8, which is characterized in that the fermentation time of the step 5) is 4-10 hours.
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Cited By (3)
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| CN111544371A (en) * | 2020-06-12 | 2020-08-18 | 洋浦吉商生物科技有限公司 | Whitening and repairing mask containing probiotic active extracting solution and nicotinamide |
| CN113151074A (en) * | 2021-04-09 | 2021-07-23 | 江南大学 | Bacillus subtilis mutant strain for high yield of nattokinase and application thereof |
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| CN111544371B (en) * | 2020-06-12 | 2020-11-20 | 洋浦吉商生物科技有限公司 | Whitening and repairing mask containing probiotic active extracting solution and nicotinamide |
| CN113151074A (en) * | 2021-04-09 | 2021-07-23 | 江南大学 | Bacillus subtilis mutant strain for high yield of nattokinase and application thereof |
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