CN106085991B - A kind of method that solid state fermentation prepares Nattokinase - Google Patents
A kind of method that solid state fermentation prepares Nattokinase Download PDFInfo
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Abstract
The invention belongs to microbial fermentation engineering technical fields, it is related to a kind of method that solid state fermentation prepares Nattokinase, slant medium is first prepared respectively, seed liquid culture medium and fermentation medium, after bafillus natto is inoculated on slant medium the bafillus natto after culture is activated again 1~2 ring of picking into seed liquid culture medium shaken cultivation as fermentation seed liquid, then naturally cold after fermentation medium being sterilized, fermentation seed liquid is sprayed in fermentation medium, and water is added and stirs evenly time in beaker middle berth straticulation, it successively ferments after being sealed with gauze, physiological saline is added in after-ripening afterwards for 24 hours, extraction, the supernatant for being rich in Nattokinase is obtained after centrifugation；Its preparation process is simple, easy to operate, and raw material is easy to get, at low cost, and the natto kinase activity of preparation is high, and economic benefit is obvious.
Description
Technical field:
The invention belongs to microbial fermentation engineering technical field, it is related to one kind using peanut meal as raw material, using solid state fermentation
Technique prepares the technology of Nattokinase, the method that especially a kind of solid state fermentation prepares Nattokinase.
Background technique:
Thrombus is formed by fibrinogen and platelet aggregation, and a series of diseases are often caused, and is a kind of serious harm
The cardiovascular disease of human health.(nattokinase abbreviation NK is by bafillus natto (Bacillus to Nattokinase
Subtilisl natto) a kind of serine protease for generating, Nattokinase has been found to be a kind of to treat cardiovascular and cerebrovascular disease
Ideal candidates drug, compared with other traditional Thrombolytic Drugs, have safety it is good, it is at low cost, be easily absorbed by the body, effect directly
Rapidly, many advantages, such as acting duration is long.Therefore, active drug of the Nattokinase as a new generation's treatment thrombus, has
Broader development and application prospect.Currently, published Nattokinase preparation method has very much, such as
CM201510495062.9 discloses a kind of rapidly and efficiently natto kinase purifying preparation method that can amplify,
CN201510731704.0 discloses a kind of method for extracting Nattokinase using deep fermentation, and the fermentation material of this method is
Soya bean, red bean, fruit or vegetables；N201210512373.8 discloses a kind of manufacturing method of Nattokinase, utilizes classical deep layer
Fermentation technique prepares Nattokinase by introducing olecranon bean powder as raw material composition mixed culture medium, and 201310154402.2 is public
A kind of method that high-activity nattokinase is prepared as raw material using semen sojae atricolor has been opened, has been that raw material preparation high activity natto swashs using semen sojae atricolor
Enzyme, but the problems such as to be substantially all that there are enzyme activities low for these methods, and productivity is low, high production cost.Therefore, there is an urgent need to seek
A kind of productivity is high, Nattokinase preparation method at low cost.Peanut meal (PNM) is shelled peanut gained after oil plant is refined in squeezing
Byproduct, gross protein value is 38.0%~47.0%, and crude fiber content is 4.0%~7.0%, and crude fat content is
0.5%~2.0%, it now mostly uses hot squeezing process to extract peanut oil, peanut protein is caused excessively to be denaturalized, added value reduces, only
Can be used for poultry aquatic feeds, this strongly limits peanut meal crude protein food and non food area effective use.At present
The research for preparing anti-oxidation peptide using microbial fermentation peanut meal is more, and Ming Qiangqiang et al. utilizes saccharomycete, lactic acid bacteria, aspergillus niger
Deng five kinds of microorganism solid fermentation peanut meals, exploration prepares the process conditions of anti-oxidation peptide.Liu Hongmei et al. is embraced with withered grass bud
Four kinds of microorganism fluid preparing peanut antioxidant peptide by fermentation such as bacillus AS1.398, Bacillus licheniformis2709 and lactobacillus plantarum,
But it is had not been reported using the research that bacillus natto to ferment peanut meal prepares Nattokinase.
Summary of the invention:
It is an object of the invention to overcome disadvantage of the existing technology, seeks design and a kind of peanut meal solid state fermentation is provided
The method for preparing Nattokinase is prepared high living by adjusting fermentation time, temperature, inoculum concentration, solid-liquid ratio and nutritive salt additional amount
The Nattokinase of power provides theoretical foundation and technical support to reduce the production cost of Nattokinase, is the high-valued of peanut meal
Offer new way is provided.
To achieve the goals above, the present invention prepares the detailed process of Nattokinase are as follows:
(1) preparation of culture medium: agar 15g/L, peptone 10g/L, powdered beef 3g/L and sodium chloride 5g/L are mixed equal
The slant medium that pH value is 7.1~7.5 is obtained in 121 DEG C of sterilizing 20min after even；By peptone 10g/L, beef extract 5g/L,
Sterilizing 20min obtains seed liquor culture at 121 DEG C after mixing by sodium chloride 3g/L, glucose 10g/L and yeast extract 5g/L
Base；By peanut meal, wheat bran, dregs of beans, oat, soybean respectively with clear water clean and add up 3 times of weight water impregnate 15h after by 6:1:
The weight ratio of 1:1:1 mixes, and the % sucrose of peanut meal, wheat bran, dregs of beans, oat and soybean total weight 1.5,0.21% is added
Magnesium sulfate, 0.27% calcium chloride mixing composition nutritive salt, be uniformly mixed at 121 DEG C sterilize 20min obtain fermented and cultured
Base；
(2) preparation of bafillus natto seed liquor: in an aseptic environment, bafillus natto oese is chosen 1
~2 rings are inoculated on the fresh slant medium after sterilizing, and the natto gemma after being activated for 24 hours is cultivated in 37 DEG C of incubators
Bacillus, by the bafillus natto after activation aseptically 1~2 ring of picking into seed liquid culture medium, 37 DEG C,
Shaken cultivation 18h is spare as fermentation seed liquid under the conditions of 160r/min；
(3) fermentation medium is naturally cooled to after steam sterilizing 20min under 0.22MPa pressure through 121 DEG C 55 DEG C with
Under, aseptically, 3~5mL fermentation seed liquid is sprayed in 30g fermentation medium, and fermentation medium weight is added
40%~60% water stirs evenly, and the thin layer of 2-3cm is paved into the beaker of 200mL, at 33~41 DEG C after being sealed with gauze
Under the conditions of successively ferment after 18~30h, 4 DEG C of after-ripening for 24 hours, 5 times of total weight of physiological saline is added, after 4 DEG C of extraction 2h
10000r/min is centrifuged 10min, obtains the supernatant for being rich in Nattokinase.
Nattokinase vigor prepared by the present invention can reach 3162U/mL, and soluble nitrogen content can reach 5.6mg/mL, hydroxyl
Free radical scavenging activity can reach 74%, and iron reducing power OD value can reach 0.906.
There is higher natto compared with prior art, the present invention carrying out solid state fermentation using peanut meal as primary raw material and obtaining
The product of kinase activity reduces the production cost of thrombolysis kinases, brings glad tidings for vast Patients with Cardiovascular/Cerebrovascular Diseases, Er Qiezeng
The added value of peanut meal is added, economic advantages are obvious, and the increase wealth of development and peasant for China's peanut industry has emphatically
Big meaning, preparation process is simple, easy to operate, and raw material is easy to get, at low cost, and the natto kinase activity of preparation is high, economy effect
It is beneficial obvious.
Detailed description of the invention:
Fig. 1 is the growth curve chart of bafillus natto described in the embodiment of the present invention.
Fig. 2 is urokinase vigor canonical plotting described in the embodiment of the present invention.
Fig. 3 is influence curve figure of the fermentation temperature described in the embodiment of the present invention to ferment effect.
Fig. 4 is influence curve figure of the inoculum concentration described in the embodiment of the present invention to ferment effect.
Fig. 5 is influence curve figure of the fermentation time described in the embodiment of the present invention to ferment effect.
Fig. 6 is influence curve figure of the solid-liquid ratio described in the embodiment of the present invention to ferment effect.
Specific embodiment:
The invention will be further described by way of example and in conjunction with the accompanying drawings.
Embodiment 1:
The present embodiment prepares the detailed process of Nattokinase are as follows:
(1) preparation of culture medium: agar 15g/L, peptone 10g/L, powdered beef 3g/L and sodium chloride 5g/L are mixed equal
The slant medium that pH is 7.1~7.5 is obtained in 121 DEG C of sterilizing 20min after even；By peptone 10g/L, beef extract 5g/L, chlorine
Changing sodium 3g/L, glucose 10g/L and yeast extract 5g/L, sterilizing 20min obtains seed liquid culture medium at 121 DEG C after mixing；
By peanut meal, wheat bran, dregs of beans, oat, soybean respectively with clear water clean and add up 3 times of weight water impregnate after by 6:1:1:1:1
Weight ratio mixing, and the sulfuric acid of the % sucrose of peanut meal, wheat bran, dregs of beans, oat and soybean total weight 1.5,0.21% is added
Magnesium, 0.27% calcium chloride mixing composition nutritive salt, be uniformly mixed at 121 DEG C sterilize 20min obtain fermentation medium；
(2) preparation of bafillus natto seed liquor: in an aseptic environment, bafillus natto oese is chosen 1
~2 rings are inoculated on the fresh slant medium after sterilizing, and the natto gemma after being activated for 24 hours is cultivated in 37 DEG C of incubators
Bacillus, by the bafillus natto after activation aseptically 1~2 ring of picking into seed liquid culture medium, 37 DEG C,
Shaken cultivation 18h is spare as fermentation seed liquid under the conditions of 160r/min；
(3) fermentation medium is naturally cooled to after steam sterilizing 20min under 0.22MPa pressure through 121 DEG C 55 DEG C with
Under, aseptically, 3~5mL fermentation seed liquid is sprayed in 30g fermentation medium, and fermentation medium weight is added
40%~60% water stirs evenly, and 2~3cm thin layer is paved into the beaker of 200mL, in 33~41 DEG C of items after being sealed with gauze
It is successively fermented after 18~30h, 4 DEG C of after-ripening for 24 hours under part, 5 times of total weight of physiological saline, the 10000r/ after 4 DEG C of extraction 2h is added
Min is centrifuged 10min, obtains the supernatant for being rich in Nattokinase.
Embodiment 2:
The fermentation seed liquid that the present embodiment prepares 1 step of embodiment (2) is sampled every 3h, measures the OD value at 660nm,
Using the seed culture fluid for not being inoculated with bafillus natto as blank control, the growth curve of bafillus natto is drawn (such as Fig. 1 institute
Show), determine the kind age of access fermentation medium；And successively change the step the fermentation temperature in (3), inoculum concentration (fermentation seed liquid
Fountain height), fermentation time, the nutritive salt in solid-liquid ratio (fermentation medium and the weight ratio of water being added thereto) and step (1),
Influence of the measurement single factor test to Nattokinase vigor and soluble nitrogen content respectively, fermentation temperature is respectively 29,33,37,41,45
℃；Inoculum concentration is respectively 1,2,3,4,5mL；Fermentation time is respectively 12,18,24,30,36h；Solid-liquid ratio is respectively 1:0.2,1:
0.3,1:0.4,1:0.5,1:0.6；Its measurement result is respectively as shown in Fig. 3,4,5,6；Nutritive salt: group is not added in E1 addition group, E2,
Then according to experiment of single factor as a result, selection temperature, time, inoculum concentration and solid-liquid ratio four influence more significant factor progress
Orthogonal experiment, experimental design are shown in Table 1:
1 factor of table with it is horizontally arranged
Orthogonal experiment results are shown in Table 2:
Table 2: Orthogonal Experiment and Design and range analysis result
In the influence of fermentation temperature, K2 > K1 > K3, K2 are maximum, illustrate 37 DEG C of influences to Nattokinase vigor most
Greatly；K2 > K3 > K1, K2 is maximum in the influence of fermentation time, illustrates that the influence to Nattokinase vigor is maximum for 24 hours；It is being inoculated with
In the influence of amount, K3 > K2 > K1, K3 are maximum, illustrate that influence of the 5mL to Nattokinase vigor is maximum；In the influence of solid-liquid ratio
K1 > K2 > K3, K1 are maximum, illustrate that influence of the 1:0.4 to Nattokinase vigor is maximum.
The continuous mode of Nattokinase vigor described in the present embodiment are as follows: urokinase standard curve is first made, according to WS1 mono-
(X-052) 2001Z-2010, reference literature " Astrup T, Muller S.The fibrin plate method for
Estimating fibrinolytie activity [J] .Arch Biochemical Biopys, 1995,40:346-351 "
Disclosed method, using the logarithm of urokinase standard items units as ordinate, the logarithm that vertical two diameters product is enclosed in dissolution is cross
Coordinate is drawn standard curve (as shown in Figure 2), and regression equation y=4.9864x+0.5141, R is calculated2=0.9947,
Take supernatant liquid spotting on fibrin plate again, vertical two diameter of circle, two diameter products are dissolved in 37 DEG C of incubation 18h, measurement
Logarithm substitute into regression equation, calculate sample enzyme activity, every group of data are measured in parallel 3 times；The measurement of soluble nitrogen content is joined
According to document, " Ming Qiangqiang, Yu Lina, Yang Qingli wait aspergillus niger solid state fermentation to prepare peanut protein peptide and antioxidant activity research
[J] food science and technology, 2014,39 (2): method measurement 17-22 "；When measuring antioxidant activity, Scavenging action to hydroxyl free radical, iron
The measurement of reducing power is according to document " Yu L N, Sun J, Liu S F, et a1.Ultrasonic-assisted
enzymolysis to improve the antioxidant activities of peanut(Arachin
conarachin L.)antioxidant hydrolysate[J].International Journal of Molecular
The method of Sciences.2012,13:9051-9068 " measure.
For the growth curve of the present embodiment bafillus natto as shown in Figure 1, in 0~12h, bafillus natto growth is fast
Degree is slower, is in lag phase；Bafillus natto growth is rapid after 12h, into logarithmic growth phase；21h starts natto gemma bar
Bacterium growth slows down, and bacterium number tends towards stability, into stationary phase；30h starts bafillus natto quantity and starts to reduce, into decline
Phase thereby determines that most suitable kind of age of bafillus natto is 18h.
As shown in figure 3, within the scope of 29~45 DEG C, Nattokinase is living for influence of the present embodiment fermentation temperature to ferment effect
Power presentation first increases the trend reduced afterwards, when fermentation temperature is 37 DEG C, bafillus natto producing enzyme vigor highest；Soluble nitrogen
The trend of reduction after first increase is also presented in content, and when temperature is 41 DEG C, temperature is wanted in the growth of soluble nitrogen content highest, strain
Ask higher, in preference temperature scope, bafillus natto ability fast-growth breeding generates a large amount of protease hydrolytic substrate
Albumen.When temperature is higher than the growth scope that bacillus natto is suitable for, the growth and breeding of bacillus natto is suppressed, and leads to thallus number
Amount is reduced, influence of the overall merit fermentation temperature to this 2 indexs, determines that 33~41 DEG C are used as orthogonal experiment fermentation temperature model
It encloses.
Influence of the present embodiment inoculum concentration to ferment effect as shown in figure 4, inoculum concentration within the scope of l~5mL, in fermentation liquid
Nattokinase vigor first increases to be reduced afterwards, Nattokinase vigor highest when inoculum concentration 4mL, the increase trend of soluble nitrogen content with
Nattokinase vigour changes are almost the same, and with the increase of inoculum concentration, the albumen enzyme amount generated in growth reproductive process increases
More, the peptide that proteolysis generates increases, and soluble nitrogen content increases in tunning, as inoculum concentration 5mL, Nattokinase vigor
It is reduced with soluble nitrogen content.This may it is big with the bafillus natto quantity of inoculation, the nutriment of consumption is excessive, cause
The growth of bafillus natto is suppressed, and influence of the overall merit bacterium solution amount to fermentation selects 3~5mL inoculum concentration as just
Hand over experimental level range.
Influence of the present embodiment fermentation time to ferment effect as shown in figure 5, within the scope of 12~36h, Nattokinase
Vigor is first to increase to reduce afterwards, for 24 hours when Nattokinase vigor it is maximum；Soluble nitrogen content, which shows, first increases becoming of reducing afterwards
Gesture.At solid state fermentation initial stage, growth breeding is vigorous, generates various enzyme amount and increases, by the larger molecular organics of fermentation substrate
It is hydrolyzed to small-molecule substance, is conducive to the generation of peptide, then soluble nitrogen content is high in tunning.With prolonging for fermentation time
Long, growth enters stationary phase, and the enzyme amount of generation is reduced, in addition, bafillus natto will consume nutrition during the growth process
Substance causes soluble nitrogen content to reduce, the influence of overall merit fermentation time, determines that 18~30h ferments as orthogonal experiment
Time range.
Influence of the present embodiment solid-liquid ratio to ferment effect is as shown in fig. 6, most suitable solid-liquid ratio is 1 as can be seen from Figure:
0.5, solid state fermentation moisture content is too low, influences the conditions such as the dissolution of nutrient matrix and the swollen of transmitting and particle, is unfavorable for
Growth, moisture content is excessively high to be unfavorable for ventilating, and keeps matrix agglomerating, influences the transmitting and fermentation heat abstraction of oxygen, and natto
The protease that bacillus generates is diluted, and is declined protease and substrate protein contact probability, is influenced hydrolysis rate, fermentation liquid
Middle soluble nitrogen content is few, and the vigor of Nattokinase is lower, and influence of the overall merit solid-liquid ratio to fermentation determines 1:0.4~1:
0.6 is used as orthogonal experiment horizontal extent.
The present embodiment addition nutritive salt E1 group Nattokinase vigor and soluble nitrogen content be respectively 2680U/mL,
5.1mg/mL；The E2 group that do not add is respectively 2260U/mL, 4.6mg/mL, and E1 group is apparently higher than E2 group
Influence of the present embodiment by fermentation time it can be seen from Orthogonal experiment results to Nattokinase vigor in tunning
Maximum, remaining is followed successively by inoculum concentration, solid-liquid ratio, temperature, theoretical optimum combination be fermentation time for 24 hours, 37 DEG C of fermentation temperature, inoculation
5mL, solid-liquid ratio 1:0.4 are measured, the Nattokinase vigor that fermentation liquid is measured under this fermentation condition reaches 3162U/mL, compares single factor test
In highest enzyme activity improve 18% (2680U/mL), soluble nitrogen content 5.6mg/mL, Scavenging action to hydroxyl free radical 74%,
Iron reducing power OD value is 0.906.
Claims (1)
1. a kind of method that solid state fermentation prepares Nattokinase, it is characterised in that prepare the detailed process of Nattokinase are as follows:
(1) preparation of culture medium: after mixing by agar 15g/L, peptone 10g/L, powdered beef 3g/L and sodium chloride 5g/L
The 20min that sterilizes at 121 DEG C obtains the slant medium that pH value is 7.1~7.5；By peptone 10g/L, beef extract 5g/L, chlorination
Sterilizing 20min obtains seed liquid culture medium at 121 DEG C after mixing by sodium 3g/L, glucose 10g/L and yeast extract 5g/L；It will
Peanut meal, wheat bran, dregs of beans, oat, soybean respectively with clear water clean and add up 3 times of weight water impregnate 15h after by 6:1:1:1:1
Weight ratio mixing, and the sulfuric acid of the % sucrose of peanut meal, wheat bran, dregs of beans, oat and soybean total weight 1.5,0.21% is added
Magnesium, 0.27% calcium chloride mixing composition nutritive salt, be uniformly mixed at 121 DEG C sterilize 20min obtain fermentation medium；
(2) preparation of bafillus natto seed liquor: in an aseptic environment, bafillus natto oese is chosen into 1~2 ring
It is inoculated on slant medium, cultivates the bafillus natto after being activated for 24 hours in 37 DEG C of incubators, by receiving after activation
Aseptically 1~2 ring of picking vibrates training into seed liquid culture medium to beans bacillus under the conditions of 37 DEG C, 160r/min
Feeding 18h is spare as fermentation seed liquid；
(3) fermentation medium is naturally cooled to 55 DEG C hereinafter, In under 0.22MPa pressure through 121 DEG C after steam sterilizing 20min
Under aseptic condition, 5mL fermentation seed liquid is sprayed in 30g fermentation medium, and the water of fermentation medium weight 40% is added
It stirs evenly, the thin layer of 2-3cm is paved into the beaker of 200mL, ferment for 24 hours, after 4 DEG C under the conditions of 37 DEG C after being sealed with gauze
It is ripe for 24 hours after, 5 times of total weight of physiological saline is added, 10000r/min is centrifuged 10min after 4 DEG C of extraction 2h, obtains rich in natto
The supernatant of kinases；The Nattokinase vigor of preparation can reach 3162 U/mL, and soluble nitrogen content can reach 5.6mg/mL, hydroxyl
Free radical scavenging activity can reach 74%, and iron reducing power OD value can reach 0.906.
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| CN107254423B (en) * | 2017-07-03 | 2020-04-07 | 成都市农林科学院 | Preparation and application method of nattokinase microbial inoculum |
| CN107254422A (en) * | 2017-07-03 | 2017-10-17 | 成都市农林科学院 | A kind of culture medium for improving Nattokinase producing strains stability and natto kinase activity |
| CN107236774A (en) * | 2017-07-17 | 2017-10-10 | 青岛大学 | The peanut meal fermentation composition that a kind of anti-aging hypermnesia is recalled |
| CN108410847B (en) * | 2018-03-13 | 2021-01-29 | 西安交通大学 | Method for preparing nattokinase dry powder and natto bacillus feed based on liquid fermentation method and application |
| CN110093334A (en) * | 2019-05-08 | 2019-08-06 | 江苏博晟康生物科技有限公司 | A method of Nattokinase is prepared using soybean and semen sojae atricolor fermentation |
| CN113208057B (en) * | 2021-06-08 | 2022-05-10 | 北京燕京中发生物技术有限公司 | Natto containing high-activity nattokinase and preparation method thereof |
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