CN106473023A - A kind of preparation method of the Stichopus japonicus ovum tunning with blood pressure lowering anticoagulant functions - Google Patents
A kind of preparation method of the Stichopus japonicus ovum tunning with blood pressure lowering anticoagulant functions Download PDFInfo
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- stichopus japonicus
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of preparation method of the Stichopus japonicus ovum tunning with blood pressure lowering anticoagulant functions, comprise the steps：Take Stichopus japonicus ovum cleaning, lyophilizing, obtain Stichopus japonicus ovum lyophilized powder, standby；Stichopus japonicus ovum lyophilized powder is mixed with water, adds glucose, adjust initial pH, prepared Stichopus japonicus ovum basal medium；After basal medium is sterilized；Add Bacillus natto bacteria suspension, shaking table culture, obtain Stichopus japonicus ovum fermentation liquid；Stichopus japonicus ovum fermentation liquid is centrifuged, collects supernatant；Obtain supernatant concentration, lyophilizing with blood pressure lowering, anticoagulant functions Stichopus japonicus ovum tunning lyophilized powder.Stichopus japonicus ovum through fermenting bacillus natto, not only can be produced the fibrinolytic enzyme with thrombus effect by the present invention, also produce the polypeptide beneficial to absorption with anticoagulation and buck functionality.Stichopus japonicus ovum tunning, not only directly can use as food, also can be developed into health care product further across concentrating, separating etc..
Description
Technical field
The present invention relates to food of sea cucumber processing technique field, more particularly, to a kind of Stichopus japonicus ovum with blood pressure lowering anticoagulant functions
The preparation method of tunning.
Background technology
With the continuous improvement of people's living standard, average life constantly extends, and society gradually steps into aging, heart and brain blood
Pipe disease illness trend presents to be increased year by year.《Chinese cardiovascular report 2015》Point out, 2014, Chinese cardiovascular death rate
Still occupy the first place of disease death composition, higher than tumor and other diseases.Wherein, cardiovascular death rate in rural area is super from 2009
Cross and persistently higher than city level.Cardiovascular diseasess occupy people's disease death and are formed in rural area is 44.60%, in city is
42.51%.In the every 5 dead people in the whole nation, just there are 2 to be to die from cardiovascular and cerebrovascular disease, and cerebrovascular then becomes the first in China
The cause of the death.Hypertension be a kind of with systolic arterial pressure or the diastolic pressure clinical syndrome that is characterized of rising, be cause the heart, brain, kidney and
The various complication such as blood vessel and a significant risk factor leading to apoplexy, arteriosclerosis, coronary heart disease.Countries in the world are averagely ill
Rate is 10～12%, and Crowds Distribute is wide.Therefore, Research on Cardiovascular medicine, treatment and prevention and cure of cardiovascular disease are today's society
One heat subject of concern.Antihypertensive drugs have many kinds at present, and wherein angiotensin-convertion enzyme inhibitor (ACEI) is should
With most common, be also be currently considered to the most safely, be best suited for one of old people's antihypertensive drug.But most ACEI are to change
Learn synthesis class medicine, in clinical practice, there is different degrees of side effect.Then, find curative effect high, Small side effects natural
The ACEI in source, especially Angiotensin-Converting (ACE) peptide for inhibiting, is whole world focus of attention.
Stichopus japonicus ovum is the gonadal one kind of Stichopus japonicuss, is the by-product during Holothurian machining.Stichopus japonicus ovum rich in nutrition content,
Direct-edible, have and improve the physiological functions such as vision, defying age, health invigorating, prophylaxis of cancer.In recent years, domestic scholars pair
Stichopus japonicus ovum carries out systematic Study, obtains the active substance with special physiological function by modes such as enzymolysis, fermentations, such as class
Carotene and anti-oxidation peptide etc..
Bacillus natto (Bacillus natto) belongs to antibacterial section, bacillus, is isolatable from traditional zymotic soybean system earliest
Product natto, is to prepare a kind of probioticss that tunning is commonly used, can produce multiple enzyme systems.1987, Sumi et al. was from receiving
Extract the nattokinase (nattokinase, NK) with notable thrombolytic effect in bean, promote Chinese scholars to natto spore
Bacillus and its research of tunning.Research display, natto fermentation product has fibrinolytic, cholesterol reducing, blood fat reducing, antibacterial and resists
Multiple alimentary health-care functions such as oxidation.
Content of the invention
It is an object of the invention to widening the application of Stichopus japonicuss raw material, make full use of Holothurian machining by-product Stichopus japonicus ovum money
Source, exploitation is a kind of to be had the feature of Stichopus japonicus ovum fermented product lifting Stichopus japonicus ovum food of blood pressure lowering and anticoagulant functions concurrently and adds
Value.
For reaching above-mentioned purpose, the invention provides a kind of preparation with blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning
Method, comprises the steps：
S1, pretreatment of raw material：Take Stichopus japonicus ovum cleaning, decontamination, -80 DEG C of pre-freeze 4～5h, -50 DEG C of lyophilizing 40～50h, powder
Broken, obtain Stichopus japonicus ovum lyophilized powder, standby；
S2, making basal medium：The Stichopus japonicus ovum lyophilized powder that step S1 is obtained presses mass volume ratio 1 with water:10～50
(g/mL) mix, prepared mixed liquor；It is proportionally added into glucose, described glucose is 1 with the mass volume ratio of described mixed liquor
～7:100 (g/mL), add hydrochloric acid or sodium hydroxide to adjust initial pH to 6～10, prepared Stichopus japonicus ovum basal medium；
S3, sterilizing：The Stichopus japonicus ovum basal medium that step S2 is obtained, 121 DEG C of sterilizing 20min, obtain sterilising medium；
S4, inoculation fermentation：When the sterilising medium temperature that step S3 is obtained is reduced to 40～50 DEG C, add described sterilizing
The Bacillus natto bacteria suspension of culture volume 2～9%, final concentration of the 10 of Bacillus natto in gained sterilising medium7CFU/mL, fully
After mixing, in 31～43 DEG C, ventilation, under shaking speed 140～300r/min, cultivate 32～40h, obtain Stichopus japonicus ovum fermentation liquid；
S5, separation product：The Stichopus japonicus ovum fermentation liquid that step S4 is obtained is centrifuged 20 minutes under 7000r/min, in collection
Clear liquid；Obtain described supernatant concentration, lyophilizing with blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning lyophilized powder.
Under optimal way, Stichopus japonicus ovum lyophilized powder standby period described in step S1 frozen in -20 DEG C；With anti-oxidation and rotten.
Under optimal way, Stichopus japonicus ovum lyophilized powder described in step S2 is 1 with the mass volume ratio of water:30(g/mL).
Under optimal way, the addition of glucose described in step S2 is 2:100(g/mL).
Under optimal way, step S2 adopts NaOH to adjust pH to 7.2.
Under optimal way, hydrochloric acid or sodium hydroxide described in step S2, is added to adjust original ph for design load+0.2.In step
In rapid S3 sterilization process, the pH value of culture medium can decline 0.2, so when adjusting initial pH, being adjusted to design load+0.2.
Under optimal way, step S4 is：After the sterilization fermentation mixture that step S3 is obtained is cooled to 40～50 DEG C, add
The Bacillus natto bacteria suspension of described sterilising medium volume 5%, final concentration of the 10 of Bacillus natto in gained sterilising medium7CFU/
mL；Under 37 DEG C of ventilation conditions, in rotating speed is for the shaking table of 180r/min, cultivate 36h, obtain Stichopus japonicus ovum fermentation liquid.
Under optimal way, step S5 concentration is specially：By described supernatant concentration to original volume 1/5.At concentration
Reason can effectively shorten freeze-drying time.
Under optimal way, the concrete operations of lyophilizing described in step S5 are：- 20 DEG C of pre-freeze 6～8h, -50 DEG C of lyophilizing 24～
40h.
After such scheme, compared with the prior art the present invention has advantages below：
1st, the culture medium containing Stichopus japonicus ovum raw material is first processed for (121 DEG C, 20min) by the present invention through high pressure, improves Stichopus japonicuss
The sensitivity to protease secreted during fermenting bacillus natto for the ovum raw material, makes Stichopus japonicus ovum be more easy to be hydrolyzed, and improves sample after fermentation
Content of peptides in product, and kill pathogenic bacterium and putrefaction bacteria it is ensured that Product safety.The method that Folin- phenol is combined by TCA
In determination sample, content of peptides finds, in the sample of fermentation 0h, content of peptides is 0.25mg/mL, polypeptide in sample after fermentation 36h
Content is 4.3mg/mL；By Folin- phenol mensure soluble protein it was found that 0h Stichopus japonicus ovum sample is due to High Temperature High Pressure,
Dissolution Partial Protein, protein content is 3.5mg/mL, and the protein content in sample after fermentation 36h is 11mg/mL；From Tricine-
SDS-PAGE small molecule electrophoresis result understands, fermented after, in Stichopus japonicus ovum, high molecular weight protein is fully degraded, rich in tunning
Containing small molecular protein and polypeptide, its molecular weight is below 26Kd, and most of tunning is close to 1Kd, and tunning albumen returns
Yield is about 50%.
2nd, the thrombin time (TT) of the non-fermented sample of Stichopus japonicus ovum raw material is 21.95s, and prothrombin time (PT) is
13.6s, activated partial thromboplastin time (APTT) is 56.65s；The APTT of the Stichopus japonicus ovum tunning that the inventive method is obtained
For 69.5s, PT is 15.95s, and TT is 22.4s；The clotting time of the Stichopus japonicus ovum tunning that the inventive method is obtained substantially prolongs
Long, the Stichopus japonicus ovum tunning that the inventive method is obtained has stronger anticoagulant active.
3rd, when Stichopus japonicus ovum sample is to start fermentation, almost without ACE inhibitory activity, ACE suppression ratio is 7.8%, side of the present invention
The ACE inhibitory activity of Stichopus japonicus ovum tunning that method is obtained significantly improves, and ACE suppression ratio is up to for 90%.
4th, rich in the polypeptide with blood pressure lowering anticoagulant functions in the Stichopus japonicus ovum tunning that the inventive method is obtained, to high blood
Pressure disease, high blood viscosity disease and thrombotic disease have potential auxiliary therapeutic action；Meanwhile, contain in the tunning that the present invention is obtained
There is multiple protein enzyme, the people's group energy for digestive enzyme hyposecretion promotes fully digesting and assimilating of its gastrointestinal tract food；The present invention
The Stichopus japonicus ovum tunning that method is obtained can be applicable to the aspects such as beverage, health product, nourishing additive agent and pharmaceutical developmentses.
To sum up, compared with direct beche-de-mer ovum, the present invention by Stichopus japonicus ovum through fermenting bacillus natto, not only can produce have molten
The fibrinolytic enzyme of solution thrombosis effect, also produces beneficial to the polypeptide digesting and assimilating, having anticoagulation and buck functionality.Stichopus japonicus ovum fermentation is produced
Thing, not only directly can use as food, also can be developed into health care product further across concentrating, separating etc..Cause
This, the present invention enriches the application mode of Stichopus japonicus ovum and enhances its biological activity and Development volue.
Brief description
Fig. 1 is the change of Stichopus japonicus ovum sweat TCA solubility oligopeptide content；
Fig. 2 is the change of Stichopus japonicus ovum sweat soluble protein content；
Fig. 3 is the gelatin zymogram spectrogram of Stichopus japonicus ovum tunning；
Fig. 4 is the Tricine-SDS-PAGE spectrogram of Semen Glycines powder and Stichopus japonicus ovum tunning；
Fig. 5 is Semen Glycines powder and the anticoagulant active of Stichopus japonicus ovum tunning.
Specific embodiment
The invention provides a kind of preparation method of the Stichopus japonicus ovum tunning with blood pressure lowering anticoagulant functions, with Stichopus japonicus ovum
For raw material, by fermenting bacillus natto, the ACE inhibitory activity according to tunning and anticoagulant active optimization of fermentation conditions, obtain
Optimum tunning.Terminate fermentation, tunning carried out be centrifuged, filter, concentrate, lyophilizing, prepare blood pressure lowering anticoagulation Stichopus japonicus ovum
Tunning.
The Stichopus japonicus ovum lyophilized powder adopting in following examples is obtained by following methods：Stichopus japonicus ovum is cleaned, removes surface
The impurity of adhesion, drains, and carries out pre-freeze at -80 DEG C, and the quality of the Stichopus japonicus ovum according to pre-freeze and volume settings pre-freeze time 4～
5h, then carries out freeze concentration using vacuum freezing concentrator (freeze dryer), and first freeze dryer is carried out with pre-cooling, about 30 minutes
Afterwards, freeze dryer temperature stabilization, after -50 DEG C, is put into the Stichopus japonicus ovum sample handled well, is opened vacuum pump, rapid pressure drop arrives
Under 10pa, freeze dryer stable operation 40～50h, by whole for Stichopus japonicus ovum sample lyophilizing.Stichopus japonicus ovum after lyophilizing is beaten powder, puts
Enter -20 DEG C of refrigerators standby.
The embodiment of the present invention and comparative example are characterized using following methods：
Test 1：The assay method of TCA solubility oligopeptide content is as follows：
It is 20% trichloroacetic acid (TCA) that fermented sample 100 μ L adds 100 μ L concentration, and concussion mixes, and stands 20min,
It is centrifuged 15min under 16500g/min, take supernatant to be diluted, take the testing sample after dilution 100 μ L, using the side of Folin- phenol
Method measures TCA solubility oligopeptide content, adds Folin- phenol solution A 500 μ L, room temperature reaction 10min, adds Folin- phenol second liquid
100 μ L, 30 DEG C of reaction 30min, 500nm survey absorbance, determine solubility oligopeptide content according to standard curve.Specification Curve of Increasing
As follows：With crystallizing bovine serum albumin, be configured to 0 according to its purity, 0.05,0.10,0.20,0.30,0.40,0.50mg/mL, according to
Secondary addition 500 μ l Folin- phenol solution A, mix.Place 10min under room temperature, add 50 μ l Folin- phenol second liquid, shake immediately
Swing uniformly, under room temperature, place 30min.Then, under 500nm wavelength, measure optical density value with microplate reader.Dense with bovine serum albumin
Spend for abscissa, light absorption value (500nm) is vertical coordinate, draws standard curve.
Test 2：The assay method of soluble protein content is as follows：
Take the testing sample after dilution 100 μ L, add Folin- phenol solution A 500 μ L, room temperature reaction 10min, add
Folin- phenol second liquid 100 μ L, 30 DEG C of reaction 30min, 500nm survey absorbance, determine soluble protein content according to standard curve.
Specification Curve of Increasing is with test 1.
Test 3：The assay method of ACE inhibitory activity is as follows：
ACE inhibitory activity blank group：In 1.5mL centrifuge tube, take 25 μ L ultra-pure waters, (enzyme activity is to add the ACE of 25 μ L
0.047U/mL), whirlpool concussion 2min, 37 DEG C of incubation 5min.Add 50 μ L, 5mmol/L substrate hippuroyl-histidyl--leucine
(HHL), 37 DEG C, react 1h.It is subsequently adding 20 μ L, 0.2MHCl terminating reaction.After reactant liquor crosses the film that aperture is 0.45 μm, use
HPLC (Waters) detects hippuric acid (HA) growing amount.
ACE inhibitory activity sample sets：In 1.5mL centrifuge tube, take the above-mentioned fermentation liquid of 25 μ L (protein content 20mg/mL),
Add the ACE (enzyme activity is 0.047U/mL) of 25 μ L, whirlpool shakes 2min, 37 DEG C of 5min.Add 50 μ L, 5mmol/L substrate
HHL, 37 DEG C of reaction 1h.It is subsequently adding 20 μ L, 0.2MHCl terminating reaction.After reactant liquor crosses the film that aperture is 0.45 μm, use HPLC
(Waters) detect HA growing amount.
ACE suppression ratio=(A-B)/A*100%；(wherein A:Add the peak area of HA in fermented sample group；B:In blank group
The peak area of HA).
Test 4：Anticoagulant active assay method is as follows：
Anticoagulant active measures：By 30 parts of blood from Healthy People health check-up, add 3.8% sodium citrate as anticoagulant
Agent, mix homogeneously, 15min is centrifuged with 3000r/min, collects blood plasma.Test plasma is added test tube, often pipe 0.9mL, add and send out
Ferment product 0.1mL.By kit specification operation, measure TT, PT and APTT.
TT, PT, APTT are the common indexs of detection coagulation function：TT is reaction thrombin activity and fibrinogen content
Index, normal range 14.0～21.0s；The function of PT dominant response exogenous cruor pathway, normal range 9.5～15.5s；
APTT dominant response intrinsic coagulation pathway about the activity of the factor, normal range 23.0～43.0s.
Embodiment 1
Take Stichopus japonicus ovum lyophilized powder 1.49g, add 30mL deionized water, add 0.6g glucose, adjust culture medium with NaOH
PH to 7.2 (design load is 7), culture medium is sealed, is placed in 121 DEG C and sterilizes 20 minutes, after end subject to sterilization, takes out sterilizing culture
Base.Culture medium temperature subject to sterilization, when 40～50 DEG C, adds 0.9mL Bacillus natto bacteria suspension, so that culture medium bacteria concentration is finally reached
107CFU/mL, after Bacillus natto bacteria suspension and sterilising medium are sufficiently mixed, is placed in 37 DEG C, in the shaking table of 180r/min, ventilation
Culture 0～72h.
Take fermentation liquid once every 12h, the present embodiment is measured using the assay method of TCA solubility oligopeptide content and fermented
Cheng Zhong, the content of the solvable oligopeptide of TCA in tunning, result is as shown in Figure 1.During 0h, TCA solubility oligopeptide content is very low, says
The large protein of the Stichopus japonicus ovum high molecular weight protein of the bright High Temperature High Pressure dissolution in 0h and the non-dissolution of Stichopus japonicus ovum is not decomposed by Bacillus natto；
With the prolongation of fermentation time, TCA solubility oligopeptide content increases, and illustrates that Bacillus natto is fermented in Stichopus japonicus ovum culture medium,
High molecular weight protein in culture medium is degraded to small molecular protein or peptide, so that TCA solubility oligopeptide content in tunning is shown
Write and increase.
In 0～72h, take one time fermentation liquid every 12h, with protein content in Folin- phenol method determination sample, result is such as
Fig. 2.In 0h Stichopus japonicus ovum sample due to High Temperature High Pressure, dissolution part soluble protein, its content is 3.5mg/mL, after fermentation 36h
Soluble protein content in sample reaches 11mg/mL, changes less afterwards.
Take the Product samples of above-mentioned fermentation 0h and 36h respectively, after 7000r/min centrifugation 20min, take supernatant 100 μ
L, carries out anticoagulant active mensure, result such as table 1.After fermentation 36h, TT, PT, APTT have significant prolongation, illustrate to ferment
Journey can promote the formation of some anticoagulins so that cruor time extending, plays certain blood coagulation resisting function.
Table 1 Stichopus japonicus ovum fermentation 0h and 36h anticoagulant active
| Stichopus japonicus ovum fermentation 0h sample | Stichopus japonicus ovum fermentation 36h sample | |
| TT/s | 21.95 | 22.4 |
| PT/s | 13.6 | 15.95 |
| APTT/s | 56.65 | 69.5 |
Embodiment 2
Take Stichopus japonicus ovum lyophilized powder 1.1g, add water 30mL, add glucose 0.4g, adjustment pH to be 7.2 (design load is 7),
121 DEG C of high pressure steam sterilization 20min, after being cooled to 40～50 DEG C, access 1.5mL Bacillus natto bacteria suspension, final concentration of 107CFU/
ML, at 37 DEG C, under the conditions of 180r/min, aerlbic culture 36h.Fermentation liquid is centrifuged 20min, supernatant volume through 7000r/min
It is about 30mL, measures Supernatant protein content through forint phenol method and be about 20mg/mL, supernatant is through concentrating, lyophilizing is obtained sea
Ginseng ovum fermentation lyophilized powder is about 0.6g.
The present embodiment product albumen response rate about 54.5%, is measured by TCA solubility oligopeptide content assaying method
Oligopeptide content 18mg/mL arriving.
Gelatin zymogram (active electrophoresis) shows, as shown in figure 3, in tunning, also there is multiple protein enzyme, these eggs
White enzyme not only can promote Stichopus japonicus ovum albuminolysis to form active polypeptide, supplemented with some consumers' digestion enzyme secretion not
Foot, promotes fully digesting and assimilating of gastrointestinal tract food.
Embodiment 3
Take Stichopus japonicus ovum lyophilized powder 3.8g, add water 40mL, add glucose 1.2g, adjustment pH to be 7.2 (design load is 7),
121 DEG C of high pressure steam sterilization 20min, after cooling, access 2mL Bacillus natto bacteria suspension, final concentration of 107CFU/mL, at 37 DEG C,
Under the conditions of 180r/min, aerlbic culture 36h.It is centrifuged 20min through 7000r/min after fermentation ends, take supernatant, supernatant volume
It is about 32mL, protein content is about 23mg/mL after measured, supernatant is through concentrating, lyophilizing is obtained Stichopus japonicus ovum fermentation lyophilized powder about
For 0.736g.
The present embodiment product albumen response rate is 31%, is obtained by TCA solubility oligopeptide content assaying method measurement
Oligopeptide content 20mg/mL.
Protein content/total protein * 100% after protein recovery=fermentation
By the use of Semen Glycines powder as Stichopus japonicus ovum tunning anticoagulant active survey comparative example.Measure in Semen Glycines powder through kjeldahl determination
Total protein content is about 39% (butt), and in Stichopus japonicus ovum, total protein content is about 62% (butt), and result shows, Stichopus japonicus ovum belongs to
High protein, low-fat protein sources, are the good raw material preparing biological activity protein and polypeptide.
Comparative example 1
Take the Semen Glycines powder 1.8g of lyophilizing under the same terms it is ensured that the total protein content that is added to embodiment 2 in culture medium
(0.68g) consistent, ferment 36h under the same conditions as in practical example 2, is centrifuged 20min through 7000r/min after fermentation ends,
Take supernatant, supernatant volume is about 20mL, protein content is about 33mg/mL after measured, supernatant is through concentrating, lyophilizing is obtained greatly
Bean fermentation lyophilized powder is about 0.66g.
This comparative example product albumen response rate is 28%.
The fermented supernatant fluid of Example 2 and comparative example 1 carries out the mensure of ACE inhibitory activity respectively, and result shows, Semen Glycines powder
The ACE suppression ratio of tunning is 86.07%, and the suppression ratio of Stichopus japonicus ovum tunning is 90.06%, illustrates that Stichopus japonicus ovum is suitable
Produce the raw material that ACE suppresses polypeptide, ACE inhibitory activity is slightly above Semen Glycines powder.
In Example 2 and comparative example 1, the Stichopus japonicus ovum and Semen Glycines powder supernatant after 36h that ferments carries out Tricine-SDS- respectively
PAGE small molecule electrophoresis, result shows such as Fig. 4, wherein M represents Ultra-low molecular weight marker, the 1st band represents Semen Glycines powder fermentation 0h,
2nd band represents Semen Glycines powder fermentation 36h, the 3rd band represents Stichopus japonicus ovum fermentation 0h, the 4th band represents Stichopus japonicus ovum fermentation 36h.Find
In 0h, Semen Glycines powder and Stichopus japonicus ovum are all existed with high molecular weight protein, and after fermentation 36h, the high molecular weight protein of Stichopus japonicus ovum is all degraded,
No high molecular weight protein band above 26.6KD, has protein band between 1～26KD, high molecular weight protein quilt in sweat is described
It is degraded into small molecular protein and polypeptide, and Stichopus japonicus ovum creates the small molecule egg of different molecular weight distribution with Semen Glycines powder fermented sample
In vain, this species diversity may lead to the different main cause of the two tunning biological activity.
Comparative example 2
Using the method that the total protein content being added in culture medium is consistent, take the Semen Glycines powder 6g of lyophilizing under the same terms (total
Protein content is 2.36g) add water 40mL, and add glucose 1.2g, adjustment pH to be 7.2 (design load is 7), steam in 121 DEG C of high pressure
Vapour sterilizing 20min, after cooling, access 2mL Bacillus natto bacteria suspension, final concentration of 107CFU/mL, at 37 DEG C, 180r/min condition
Under, aerlbic culture 36h.It is centrifuged 20min through 7000r/min after fermentation ends, take supernatant, supernatant volume is about 27mL, warp
Measure protein content and be about 31mg/mL, supernatant is through concentrating, lyophilizing is obtained fermented soybean lyophilized powder and is about 0.837g.
This comparative example product albumen response rate is 35%.
The fermented supernatant fluid 100 μ L of Example 3 and comparative example 2, measures its anticoagulant active respectively.
Result, can be to the heart as shown in figure 5, Stichopus japonicus ovum tunning has higher anticoagulant active than Semen Glycines powder tunning
Cerebrovascular disease has potential auxiliary therapeutic action.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art in the technical scope of present disclosure, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, all should be included within the scope of the present invention.
Claims (9)
1. a kind of preparation method of the Stichopus japonicus ovum tunning with blood pressure lowering anticoagulant functions is it is characterised in that include following walking
Suddenly：
S1, pretreatment of raw material：Take Stichopus japonicus ovum cleaning, decontamination, -80 DEG C of pre-freeze 4～5h, -50 DEG C of lyophilizing 40～50h, pulverize, obtain
To Stichopus japonicus ovum lyophilized powder, standby；
S2, making basal medium：The Stichopus japonicus ovum lyophilized powder that step S1 is obtained presses mass volume ratio 1 with water:10～50 (g/
ML) mix, prepared mixed liquor；It is proportionally added into glucose, the mass volume ratio of described glucose and described mixed liquor is 1～
7% (g/mL), adds hydrochloric acid or sodium hydroxide to adjust initial pH to 6～10, prepared Stichopus japonicus ovum basal medium；
S3, sterilizing：The Stichopus japonicus ovum basal medium sealing that step S2 is obtained, 121 DEG C of sterilizing 20min, obtain sterilising medium；
S4, inoculation fermentation：When the sterilising medium temperature that step S3 is obtained is reduced to 40～50 DEG C, add described sterilizing culture
Matrix amasss 2～9% Bacillus natto bacteria suspension, final concentration of the 10 of Bacillus natto in gained sterilising medium7CFU/mL, fully mixes
Afterwards, in 31～43 DEG C, ventilation, under shaking speed 140～300r/min, cultivate 32～40h, obtain Stichopus japonicus ovum fermentation liquid；
S5, separation product：The Stichopus japonicus ovum fermentation liquid that step S4 is obtained is centrifuged 20 minutes under 7000r/min, collects supernatant；
Obtain described supernatant concentration, lyophilizing with blood pressure lowering, anticoagulant functions Stichopus japonicus ovum tunning lyophilized powder.
2. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
Stichopus japonicus ovum lyophilized powder standby period described in step S1 frozen in -20 DEG C.
3. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
Stichopus japonicus ovum lyophilized powder described in step S2 is 1 with the mass volume ratio of water:30(g/mL).
4. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
The mass volume ratio of the addition of glucose described in step S2 is 2% (g/mL).
5. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
Step S2 adopts NaOH to adjust pH to 7.2.
6. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
Hydrochloric acid or sodium hydroxide is added to adjust original ph for design load+0.2 described in step S2.
7. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
Step S4 is：After the sterilization fermentation mixture that step S3 is obtained is cooled to 40～50 DEG C, add described sterilising medium volume
5% Bacillus natto bacteria suspension, final concentration of the 10 of Bacillus natto in gained sterilising medium7CFU/mL；In 37 DEG C, ventilation condition
Under, cultivate 36h in rotating speed is for the shaking table of 180r/min, obtain Stichopus japonicus ovum fermentation liquid.
8. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
Step S5 concentration is specially：By described supernatant concentration to original volume 1/5.
9. there is the preparation method of blood pressure lowering anticoagulant functions Stichopus japonicus ovum tunning according to claim 1 it is characterised in that
The concrete operations of lyophilizing described in step S5 are：- 20 DEG C of pre-freeze 6～8h, -50 DEG C of lyophilizing 24～40h.
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| CN110250451A (en) * | 2019-05-15 | 2019-09-20 | 池州市月亮湾生物科技有限公司 | The preparation method and gained sea cucumber intestine ovum freeze-dried powder of sea cucumber intestine ovum freeze-dried powder |
| CN114292765A (en) * | 2021-09-24 | 2022-04-08 | 江西康之康中药科技有限公司 | Bacillus subtilis subspecies natto R3 and application thereof |
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| CN103725739A (en) * | 2013-11-29 | 2014-04-16 | 天基神元生物科技（大连）有限公司 | Method for preparing low-molecule sea cucumber peptide by using bacillus natto fermentation method |
| CN105767954A (en) * | 2016-02-29 | 2016-07-20 | 青岛大学 | Method for preparing blood pressure reducing functional food by utilizing bacillus natto-fermented fresh shellfish meat |
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| CN103725739A (en) * | 2013-11-29 | 2014-04-16 | 天基神元生物科技（大连）有限公司 | Method for preparing low-molecule sea cucumber peptide by using bacillus natto fermentation method |
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| CN107668570A (en) * | 2017-11-06 | 2018-02-09 | 刘海涛 | A kind of preparation method of sea cucumber pulvis, sea cucumber tablet and sea cucumber capsule |
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Application publication date: 20170308 |