CN110195086A - A method of gamma-polyglutamic acid is produced using soy sauce residues solid state fermentation - Google Patents
A method of gamma-polyglutamic acid is produced using soy sauce residues solid state fermentation Download PDFInfo
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Abstract
The invention belongs to bioengineering fermentation technical field more particularly to a kind of methods using soy sauce residues solid state fermentation production gamma-polyglutamic acid, include the following steps: the preparation of soy sauce residues solid-state fermentation culture medium and the activation culture of Bacillus licheniformis strain；Inoculation and solid ferment process: being inoculated with bacillus licheniformis in the prepared soy sauce residues solid-state fermentation culture medium of step 1, carries out solid state fermentation, generates gamma-polyglutamic acid；The extraction of gamma-polyglutamic acid: the culture medium by step 2 containing gamma-polyglutamic acid carries out separating-purifying and obtains purification gamma-polyglutamic acid.The beneficial effects of the present invention are: the agricultures waste auxiliary material such as soy sauce residues and wheat bran, cavings is used to produce gamma-polyglutamic acid as solid state fermentation substrate, gamma-polyglutamic acid can not only be made, it also can solve soy sauce residues processing not in time or problem of environmental pollution caused by dealing with improperly, soy sauce residues turned waste into wealth.
Description
Technical field
The invention belongs to bioengineering fermentation technical fields more particularly to a kind of utilization soy sauce residues solid state fermentation to produce γ-
The method of polyglutamic acid.
Background technique
Gamma-polyglutamic acid, molecular weight have application in 10-1000kDa various fields, can be used in terms of food
Antifreeze, thickener, except astringent etc., can be used for moisture saver mask etc. in cosmetic industry, in pharmaceuticals industry, can be used as drug
Carrier, adhesive etc. have preferable water-retaining property to edaphophyte, also can be used as fertilizer synergist etc., industrially in agricultural
It can be used for high-molecular biologic flocculant, heavy metal absorbent etc., it can thus be seen that gamma-polyglutamic acid all has in many fields
There is good application prospect.The production method for the gamma-polyglutamic acid studied at present is mostly liquid fermentation, solution fermentation cost
Height, fermentation process are limited by dissolved oxygen and concentration of substrate, pass oxygen transfer low efficiency, therefore solid state fermentation has very high answer
Use prospect.
Need to prepare solid state fermentation base-material when solid state fermentation, general solid state fermentation base-material uses dregs of beans, wheat bran, stalk, paddy
Class etc., it is auxiliary material solid state fermentation that not yet discovery, which uses traditional mode to mix the agricultures wastes such as wheat bran, cavings by matrix of soy sauce residues,
Produce γ-polyglutamic acid method.
Soy sauce residues are the residues made after soy sauce, mainly make the raw soybeans soya-bean cake or dregs of beans, semen viciae fabae, pea of soy sauce
Solid substance is generated after beans, wheat and salt etc. are fermented.Due to without dry soy sauce residues moisture content height and containing rich
Rich protein and carbohydrate, the easy growth and breeding of microorganism is easily putrid and deteriorated, generates foul odour, if without and
When processing will cause environmental pollution.Currently, due to unmature soy sauce residues reutilization technology, low salt solid sauce slag frequently as
Fertilizer or feed are undersold, and the squeezing soy sauce residues that high-salt dilute technique generates are often excessively high because of salt content, cannot be direct
It is used for animal as feed, part of the manufacturer to be handled by the way of directly filling, but will lead to fill soil salinization.As soy sauce
The maximum China of slag yield, other than having the research report for being used as feed, fertilizer, otherwise research is carried out few, is
It reduces it to endanger caused by ecological environment and animal, recycles its resource section, China copes with the research of soy sauce residues
Cause enough attention.Therefore, the utilizability for studying soy sauce residues is just particularly important.
109182405 A of Chinese patent CN is related to a kind of utilization lichens bud pole bacterium solid state fermentation production gamma-polyglutamic acid
Method, which is done using dregs of beans and natural minerals as solid state fermentation base-material, and a certain proportion of nutrient solution, inoculation ground are mixed into
Clothing bud pole bacterium, the method for producing gamma-polyglutamic acid by solid state fermentation, promotes comprehensive exploitation, the higher value application of dregs of beans
And reduce production cost.
Chinese patent CN105316370A, which is provided, a kind of utilizes bafillus natto solid state fermentation production gamma-polyglutamic acid
Method, the method for the invention include: to prepare bean pulp solid-state fermentation base-material, are mixed into nutritional supplement, solid-state fermentation culture medium is made,
It is inoculated with bafillus natto, constant-temperature moisture-keeping 4-10 days, blunges and extracts gamma-polyglutamic acid, is filtered, is added after filtrate concentrated by rotary evaporation
Enter ethyl alcohol, precipitate gamma-polyglutamic acid, centrifugation recycling obtains gamma-polyglutamic acid.
Above two patents are using dregs of beans as solid state fermentation base-material, and dregs of beans is one obtained after soybean extracting bean oil
Kind byproduct, as a kind of high protein, dregs of beans is the primary raw material for making livestock and poultry feed, can be also used for production cake
Food, healthy food and cosmetics and antibiotic material are selected, is easily absorbed and utilized, diseases prevention, efficiency of feed utilization can be improved.Although grinding
Study carefully and dregs of beans as solid state fermentation base-material and can generate gamma-polyglutamic acid, but dregs of beans can be to ecological ring unlike soy sauce residues
Border and animal cause damages, and because of the problem of urgent processing may be not present, this two inventions are merely provided and produced using dregs of beans
The method of gamma-polyglutamic acid unlike the present invention in this way, having made research aiming at the problem that soy sauce residues pollute environment, and can incite somebody to action
Soy sauce residues produce gamma-polyglutamic acid as solid state fermentation substrate, so research of the invention has important meaning to environmental protection
Justice.
Summary of the invention
In order to overcome the above-mentioned deficiencies of the prior art, the present invention provides a kind of poly- using soy sauce residues solid state fermentation production γ-
The method of glutamic acid uses the agricultures waste auxiliary material such as soy sauce residues and wheat bran, cavings to produce γ-polyglutamic as solid state fermentation substrate
Gamma-polyglutamic acid can not only be made in acid, moreover it is possible to solve soy sauce Slag treatment not in time or environmental pollution caused by dealing with improperly is asked
Topic, soy sauce residues are turned waste into wealth.
To achieve the goals above, the present invention adopts the following technical scheme:
A method of gamma-polyglutamic acid is produced using soy sauce residues solid state fermentation, it is characterised in that is included the following steps:
Step 1: the preparation of soy sauce residues solid-state fermentation culture medium and the activation culture of Bacillus licheniformis strain；
Step 2: inoculation and solid ferment process: being inoculated with ground in the prepared soy sauce residues solid-state fermentation culture medium of step 1
Clothing bacillus carries out solid state fermentation, generates gamma-polyglutamic acid；
Step 3: the extraction of gamma-polyglutamic acid: the culture medium by step 2 containing gamma-polyglutamic acid carries out separating-purifying and obtains
To purification gamma-polyglutamic acid.
In the step 1, the process for preparation of soy sauce residues solid-state fermentation culture medium includes: the pretreatment of raw material, soy sauce residues
The preparation of solid state fermentation base-material, the preparation of soy sauce residues solid-state fermentation culture medium.
The pretreatment of the raw material includes: the desalting processing of soy sauce residues；The drying of soy sauce residues；The crushing of soy sauce residues and auxiliary
The crushing of material.
The desalting processing process of the soy sauce residues are as follows: it is uniformly mixed, is sufficiently stirred with the mass ratio 1:2 of soy sauce residues and water,
With 4 layers of filtered through gauze, the filter residue that will be obtained is laid in tray；
Preferably, the drying course of the soy sauce residues are as follows: the soy sauce residues after above-mentioned desalination are placed in 60 DEG C of forced air drying
In case, drying is for 24 hours；
Preferably, the crushing process of the crushing of the soy sauce residues and auxiliary material are as follows: put the soy sauce residues of desalination drying and auxiliary material
Enter in pulverizer and crush respectively, it is spare to cross 20 meshes.
The process for preparation of the soy sauce residues solid state fermentation base-material are as follows: by smashed soy sauce residues and auxiliary material be by weight 1:
1 is uniformly mixed.
The preparation process of the soy sauce residues solid-state fermentation culture medium are as follows: nutrition benefit is filled into soy sauce residues solid state fermentation base-material
Agent is filled, the weight ratio of the two is 1:0.5-1.
The nutritional supplement includes: glucose, sodium glutamate, K2SO4、(NH4)2SO4And water, glucose, glutamic acid
Sodium, K2SO4、(NH4)2SO4Thousand points of concentration of weight respectively be: 60-80 ‰, 20-60 ‰, 10-20 ‰, 5-10 ‰, remaining
For water, pH 7.0-7.2.
The activation culture process of Bacillus licheniformis strain in the step 1 are as follows: by Bacillus licheniformis strain in level-one
On inclined-plane for 24 hours in 37 DEG C of cultures, it is inoculated into seed culture medium, 37 DEG C of shaking flask cultures extremely growth logarithmic phase conduct in 12-18 hours
Solid fermentation strain is spare；
Preferably, the primary inclined plane is the inclined-plane LB, and the medium component on the inclined-plane LB is peptone 1%, and yeast is taken out
Extract 0.5%, sodium chloride 1%, natural PH.
Inoculation and solid ferment process in the step 2 are as follows: bacillus licheniformis is pressed into envelope-bulk to weight ratio 5-10% (V/
W it) is inoculated in soy sauce residues solid-state fermentation culture medium, is sufficiently stirred, strain is made to be uniformly distributed in media surface, the training after inoculation
It supports case and keeps 35-37 DEG C of constant temperature, humidity is strictly maintained at 50% or more in incubator, ferments 3-5 days.
The extraction process of gamma-polyglutamic acid in the step 3 are as follows: added water and stirred in solid-state fermentation content, be centrifuged and collect
Ethyl alcohol is added in clear liquid concentrated by rotary evaporation, then stillness of night after concentration to staticly settle, after centrifugation in obtained sediment plus ethyl alcohol
Washing, vacuum freeze drying, the gamma-polyglutamic acid refined；
Preferably, in the step 3 gamma-polyglutamic acid extraction process are as follows: be added 5 times of volumes of solid-state fermentation content water stir
Extraction 1-2 hours is mixed, after 5000r/min is centrifuged, clear liquid is collected by clear liquid concentrated by rotary evaporation to 50ml and adds 4 times of volumes
Ethyl alcohol, stand 6-24 hour, precipitating be collected by centrifugation, then with the ethanol washing of 2 times of volumes, liquid nitrogen, which is handled, to be ground, and vacuum is cold
Dry, to be refined gamma-polyglutamic acid is lyophilized.
The beneficial effects of the present invention are:
It 1, is fermentation base-material with soy sauce residues, with agriculture waste wheat bran, cavings etc. for auxiliary material, addition-certainty ratio nutrient solution,
Solid state fermentation gamma-polyglutamic acid provides the-method of kind efficient mass production gamma-polyglutamic acid, solves soy sauce factory soy sauce
The reluctant problem of residue after squeezing, avoids environmental pollution caused by soy sauce residues are dealt with improperly, while promoting soy sauce
The comprehensive exploitation of slag, higher value application, and reduce production cost.
2, biography oxygen effect in fermentation process can preferably be improved using the method for solid state fermentation, standing for fermentation reduces shearing
The unfavorable factor that power reduces polyglutamic acid molecular weight, production cost is lower, compares compared with liquid state fermentation and improves gamma-polyglutamic acid
Yield, be a kind of economical and eco-friendly sustainable technical method.
3, it is left to ferment using traditional solid state fermentation mode, has saved mass energy, there is natural free of contamination effect,
Solid residue after fermentation may be directly applied to farmland production, do not pollute the environment not only, also to soil by rational allocation
There is the effect of moisturizing synergy, soy sauce residues are turned waste into wealth, and provide a kind of new research direction for the processing of soy sauce residues, gives
The application of soy sauce residues is had laid a good foundation.
4, the gamma-polyglutamic acid that production obtains has application in various fields, can be used for antifreeze in terms of food, increases
Thick dose, except astringent etc., can be used for moisture saver mask etc. in cosmetic industry, can be used as pharmaceutical carrier, bonding in pharmaceuticals industry
Agent etc., has preferable water-retaining property to edaphophyte in agricultural, also can be used as fertilizer synergist etc., industrially can be used for high score
Sub- biological flocculant, heavy metal absorbent etc., therefore, gamma-polyglutamic acid all have a good application prospect in many fields.
Specific embodiment
Explanation is further explained to the present invention below with reference to embodiment.
Example 1:
The activation culture of bacillus licheniformis.The technology is the prior art, detailed process are as follows: primary inclined plane is oblique using LB
The strain of preservation is cultivated for 24 hours on the inclined-plane LB in 37 DEG C, is inoculated into seed culture medium, 37 DEG C of shaking flask culture 12-18 are small by face
It is spare as solid fermentation strain up to growth logarithmic phase.Wherein the strain of preservation is by the hundred limited public affairs of river biotechnology of the Tianjin northeast part of China
Department provides.The ingredient of LB culture medium is peptone 1%, yeast extract 0.5%, sodium chloride 1%, natural PH.
Using 20g soy sauce residues and 20g wheat bran as solid fermentation base-material；By glucose 2.4g, sodium glutamate 2.4g, K2SO4
0.4g、(NH4)2SO40.2g is sufficiently dissolved with 40ml water, is 7.2 with HCl or NaOH aqueous solution tune pH value, as nutritional supplementation
Agent is stand-by；Soy sauce residues are smashed it through into 20 meshes and wheat bran is mixed as solid fermentation base-material；Above-mentioned solid fermentation base-material is mixed
Entering nutritional supplement, the two is mixed into solid fermentation culture medium according to the weight ratio of 1:1, is placed in shaking flask, and 121 DEG C
20min steam sterilizing after being cooled to room temperature, is seeded in above-mentioned solid fermentation culture medium according to the ratio of 10%v/w, stirring
After uniformly, in solid fermentation 3 days in 37 DEG C of incubators, ambient humidity turned daily 50% or more, kept culture mixing equal
It is even.
After fermentation 3 days, with fermentation material in 200ml distillation water retting shaking flask, continue 1h in constant-temperature table, constant-temperature table
Revolving speed is 150r/h, takes out 50ml leachate, is centrifuged 10min in centrifuge with the speed of 5000r/min, collects clear liquid, clearly
Liquid carry out rotary evaporation be concentrated into 50ml, into liquid be added 200ml ethyl alcohol, be sufficiently stirred, in centrifuge with 15000 turns/
The speed of minute is centrifuged 10 minutes, and what is obtained is precipitated as the semifinished product of gamma-polyglutamic acid.
The ethanol washing of 2 times of volumes of the semifinished product of obtained gamma-polyglutamic acid is precipitated, recycling, liquid nitrogen processing are filtered
Grinding, vacuum freeze drying obtain gamma-polyglutamic acid highly finished product, and the gamma-polyglutamic acid yield of per kilogram raw material dry matter is
24.12g。
Example 2:
The activation and culture of bacillus licheniformis.Primary inclined plane uses the inclined-plane LB, for 24 hours in 37 DEG C of cultures, is inoculated into seed
In culture medium, 12-18 hours extremely growth logarithmic phases of 37 DEG C of shaking flask cultures are spare as solid fermentation strain.The wherein strain of preservation
It is provided by Tianjin Peiyang Biotrans Biotech Co., Ltd.The ingredient of LB culture medium be peptone 1%, yeast extract 0.5%,
Sodium chloride 1%, natural PH.
Using 20g soy sauce residues and 20g wheat bran as solid fermentation base-material；By glucose 3.2g, sodium glutamate 2.4g,
K2SO40.8g、(NH4)2SO40.4g is sufficiently dissolved with 40ml water, is 7.0 with HCl or NaOH aqueous solution tune pH value, as nutrition
Replenishers are stand-by；Soy sauce residues grinding and sieving and wheat bran are mixed and are used as solid fermentation base-material；Above-mentioned solid fermentation base-material is mixed
Entering nutritional supplement, the two is mixed into solid fermentation culture medium according to the weight ratio of 1:1, is placed in shaking flask, and 121 DEG C
20min steam sterilizing after being cooled to room temperature, is seeded in above-mentioned solid fermentation culture medium according to the ratio of 10%v/w, stirring
After uniformly, in solid fermentation 5 days in 37 DEG C of incubators, ambient humidity turned daily 50% or more, kept culture mixing equal
It is even.
After fermentation 5 days, with fermentation material in 200ml distillation water retting shaking flask, continue 1h in constant-temperature table, constant-temperature table
Revolving speed is 150r/h, takes out 50ml leachate, is centrifuged 10min in centrifuge with the speed of 5000r/min, collects clear liquid, clearly
Liquid carry out rotary evaporation be concentrated into 50ml, into clear liquid be added 200ml ethyl alcohol, be sufficiently stirred, in centrifuge with 15000 turns/
The speed of minute is centrifuged 10 minutes, and what is obtained is precipitated as the semifinished product of gamma-polyglutamic acid.
The ethanol washing of 2 times of volumes of the semifinished product of obtained gamma-polyglutamic acid is precipitated, recycling, liquid nitrogen processing are filtered
Grinding, vacuum freeze drying obtain gamma-polyglutamic acid highly finished product, and the gamma-polyglutamic acid yield of per kilogram raw material dry matter is
52.14g。
Example 3:
The activation and culture of bacillus licheniformis.Primary inclined plane uses the inclined-plane LB, for 24 hours in 37 DEG C of cultures, is inoculated into seed
In culture medium, 12-18 hours extremely growth logarithmic phases of 37 DEG C of shaking flask cultures are spare as solid fermentation strain.The wherein strain of preservation
It is provided by Tianjin Peiyang Biotrans Biotech Co., Ltd.The ingredient of LB culture medium be peptone 1%, yeast extract 0.5%,
Sodium chloride 1%, natural PH.
Using 20g soy sauce residues and 20g wheat bran as solid fermentation base-material；By glucose 3.2g, sodium glutamate 2g,
K2SO40.4g、(NH4)2SO40.4g is sufficiently dissolved with 40ml water, is 7.0 with HCl or NaOH aqueous solution tune pH value, as nutrition
Replenishers are stand-by；Soy sauce residues are smashed it through into 20 meshes and wheat bran is mixed as solid fermentation base-material；By above-mentioned solid fermentation substrate
Material is mixed into nutritional supplement, and the two is mixed into solid fermentation culture medium according to the weight ratio of 1:1, is placed in shaking flask, and 121
DEG C 20min steam sterilizing, after being cooled to room temperature, is seeded in above-mentioned solid fermentation culture medium according to the ratio of 8%v/w, stirs
After mixing uniformly, in solid fermentation 5 days in 35 DEG C of incubators, ambient humidity turned daily 50% or more, kept culture mixing equal
It is even.
After fermentation 5 days, with fermentation material in 200ml distillation water retting shaking flask, continue 1h in constant-temperature table, constant-temperature table
Revolving speed is 150r/h, takes out 50ml leachate, is centrifuged 10min on centrifuge with the speed of 5000r/min, collects clear liquid, clearly
Liquid carries out rotary evaporation and is concentrated into 50ml, and 200ml ethyl alcohol is added into liquid, is sufficiently stirred, and 15000 revs/min are centrifuged 10 points
Clock, what is obtained is precipitated as the semifinished product of gamma-polyglutamic acid.
The ethanol washing of 2 times of volumes of the semifinished product of obtained gamma-polyglutamic acid is precipitated, recycling, liquid nitrogen processing are filtered
Grinding, vacuum freeze drying obtain gamma-polyglutamic acid highly finished product, and the gamma-polyglutamic acid yield of per kilogram raw material dry matter is
21.26g。
Example 4:
The activation and culture of bacillus licheniformis.Primary inclined plane uses the inclined-plane LB, for 24 hours in 37 DEG C of cultures, is inoculated into seed
In culture medium, 12-18 hours extremely growth logarithmic phases of 37 DEG C of shaking flask cultures are spare as solid fermentation strain.The wherein strain of preservation
It is provided by Tianjin Peiyang Biotrans Biotech Co., Ltd.The ingredient of LB culture medium be peptone 1%, yeast extract 0.5%,
Sodium chloride 1%, natural PH.
Using 20g soy sauce residues and 20g wheat bran as solid fermentation base-material；By glucose 1.2g, sodium glutamate 1g,
K2SO40.2g、(NH4)2SO40.2g is sufficiently dissolved with 20ml water, and using HCl or NaOH aqueous solution tune pH value is 7.1 as nutrition
Replenishers are stand-by；Soy sauce residues are smashed it through into 20 meshes and wheat bran is mixed as solid fermentation base-material；By above-mentioned solid fermentation substrate
Material is mixed into nutritional supplement, and the two is mixed into solid fermentation culture medium according to the weight ratio of 1:1, is placed in shaking flask, and 121
DEG C 20min steam sterilizing, after being cooled to room temperature, is seeded in above-mentioned solid fermentation culture medium according to the ratio of 10%v/w, stirs
After mixing uniformly, in solid fermentation 4 days in 35 DEG C of incubators, ambient humidity turned daily 50% or more, kept culture mixing equal
It is even.
After fermentation 4 days, with fermentation material in 200ml distillation water retting shaking flask, continue 1h in constant-temperature table, constant-temperature table
Revolving speed is 150r/h, takes out 50ml leachate, is centrifuged 10min in centrifuge with the speed of 5000r/min, collects clear liquid, clearly
Liquid carry out rotary evaporation be concentrated into 50ml, into liquid be added 200ml ethyl alcohol, be sufficiently stirred, in centrifuge with 15000 turns/
The speed of minute is centrifuged 10 minutes, and what is obtained is precipitated as the semifinished product of gamma-polyglutamic acid.
The ethanol washing of 2 times of volumes of the semifinished product of obtained gamma-polyglutamic acid is precipitated, recycling, liquid nitrogen processing are filtered
Grinding, vacuum freeze drying obtain gamma-polyglutamic acid highly finished product, and the gamma-polyglutamic acid yield of per kilogram raw material dry matter is
20.10g。
Example 5:
The activation and culture of bacillus licheniformis.Primary inclined plane uses the inclined-plane LB, for 24 hours in 37 DEG C of cultures, is inoculated into seed
In culture medium, 12-18 hours extremely growth logarithmic phases of 37 DEG C of shaking flask cultures are spare as solid fermentation strain.The wherein strain of preservation
It is provided by Tianjin Peiyang Biotrans Biotech Co., Ltd.The ingredient of LB culture medium be peptone 1%, yeast extract 0.5%,
Sodium chloride 1%, natural PH.
Using 20g soy sauce residues and 20g wheat bran as solid fermentation base-material；By glucose 3g, sodium glutamate 1.6g,
K2SO40.4g、(NH4)2SO40.4g is sufficiently dissolved with 40ml water, is 7.0 with HCl or NaOH aqueous solution tune pH value, as nutrition
Replenishers are stand-by；Soy sauce residues are smashed it through into 20 meshes and wheat bran is mixed as solid fermentation base-material；By above-mentioned solid fermentation substrate
Material is mixed into nutritional supplement, and the two is mixed into solid fermentation culture medium according to the weight ratio of 1:1, is placed in shaking flask, and 121
DEG C 20min steam sterilizing, after being cooled to room temperature, is seeded in above-mentioned solid fermentation culture medium according to the ratio of 5%v/w, stirs
After mixing uniformly, in solid fermentation 5 days in 37 DEG C of incubators, ambient humidity turned daily 50% or more, kept culture mixing equal
It is even.
After fermentation 5 days, with fermentation material in 200ml distillation water retting shaking flask, continue 1h in constant-temperature table, constant-temperature table
Revolving speed is 150r/h, takes out 50ml leachate, is centrifuged 10min in centrifuge with the speed of 5000r/min, collects clear liquid, clearly
Liquid carry out rotary evaporation be concentrated into 50ml, into liquid be added 200ml ethyl alcohol, be sufficiently stirred, in centrifuge with 15000 turns/
Minute speed is centrifuged 10 minutes, and what is obtained is precipitated as the semifinished product of gamma-polyglutamic acid.
The ethanol washing of 2 times of volumes of the semifinished product of obtained gamma-polyglutamic acid is precipitated, recycling, liquid nitrogen processing are filtered
Grinding, vacuum freeze drying obtain gamma-polyglutamic acid highly finished product, and the gamma-polyglutamic acid yield of per kilogram raw material dry matter is
33.48g。
Several examples of the invention are described in detail above, but the content is only preferable implementation of the invention
Example, should not be considered as limiting the scope of the invention.It is all according to all the changes and improvements made by the present patent application range
Deng should still be within the scope of the patent of the present invention.
Claims (10)
1. a kind of method using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that include the following steps:
Step 1: the preparation of soy sauce residues solid-state fermentation culture medium and the activation culture of Bacillus licheniformis strain；
Step 2: lichens bud inoculation and solid ferment process: is inoculated in the prepared soy sauce residues solid-state fermentation culture medium of step 1
Spore bacillus carries out solid state fermentation, generates gamma-polyglutamic acid；
Step 3: the extraction of gamma-polyglutamic acid: the culture medium by step 2 containing gamma-polyglutamic acid carries out separating-purifying and obtains essence
Gamma-polyglutamic acid processed.
2. the method according to claim 1 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that institute
It states in step 1, the process for preparation of soy sauce residues solid-state fermentation culture medium includes: the pretreatment of raw material, soy sauce residues solid state fermentation base
The preparation of material, the preparation of soy sauce residues solid-state fermentation culture medium.
3. the method according to claim 2 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that institute
The pretreatment for stating raw material includes: the desalting processing of soy sauce residues；The drying of soy sauce residues；The crushing of soy sauce residues and the crushing of auxiliary material.
4. the method according to claim 3 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that:
The desalting processing process of soy sauce residues are as follows: be uniformly mixed, be sufficiently stirred, with 4 layers of gauze with the mass ratio 1:2 of soy sauce residues and water
Filtering, the filter residue that will be obtained are laid in tray；
Preferably, the drying course of soy sauce residues are as follows: the soy sauce residues after above-mentioned desalination are placed in 60 DEG C of air dry oven, are dried
24h；
Preferably, the crushing process of the crushing of soy sauce residues and auxiliary material are as follows: the soy sauce residues of desalination drying and auxiliary material are put into pulverizer
It is middle to crush respectively, it is spare to cross 20 meshes.
5. the method according to claim 2 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that sauce
The process for preparation of dregs of fat solid state fermentation base-material are as follows: be uniformly mixed smashed soy sauce residues and auxiliary material for 1:1 by weight.
6. the method according to claim 2 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that sauce
The preparation process of dregs of fat solid-state fermentation culture medium are as follows: nutritional supplement, the weight of the two are mixed into soy sauce residues solid state fermentation base-material
Amount is than being 1:0.5-1.
7. the method according to claim 6 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that institute
The nutritional supplement component stated includes: glucose, sodium glutamate, K2SO4、(NH4)2SO4And water, by glucose, sodium glutamate,
K2SO4、(NH4)2SO4It is dissolved in water, nutritional supplement is made, wherein glucose, sodium glutamate, K in nutritional supplement2SO4、
(NH4)2SO4Thousand points of concentration of weight be respectively as follows: 60-80 ‰, 20-60 ‰, 10-20 ‰, 5-10 ‰, pH 7.0-7.2.
8. the method according to claim 1 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that institute
State the activation culture process of Bacillus licheniformis strain in step 1 are as follows: by Bacillus licheniformis strain in 37 in primary inclined plane
DEG C culture for 24 hours, is inoculated into seed culture medium, and 37 DEG C of shaking flask cultures 12-18 hour are to growing logarithmic phase as solid fermentation bacterium
Kind is spare；
Preferably, the primary inclined plane is the inclined-plane LB, and the ingredient of culture medium is peptone 1%, extraction from yeast on the inclined-plane LB
Object 0.5%, sodium chloride 1%, natural PH.
9. the method according to claim 1 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that institute
State the inoculation in step 2 and solid ferment process are as follows: bacillus licheniformis is inoculated in soy sauce residues by envelope-bulk to weight ratio 5-10%
It in solid-state fermentation culture medium, is sufficiently stirred, strain is made to be uniformly distributed in media surface, the incubator after inoculation keeps 35-37
DEG C constant temperature, humidity is strictly maintained at 50% or more in incubator, ferments 3-5 days.
10. the method according to claim 1 using soy sauce residues solid state fermentation production gamma-polyglutamic acid, it is characterised in that
The extraction process of gamma-polyglutamic acid in the step 3 are as follows: added water and stirred in solid-state fermentation content, be centrifuged and collect clear liquid revolving
Ethyl alcohol is added in concentration, then stillness of night after concentration to staticly settle, after centrifugation in obtained sediment plus ethanol washing, vacuum
Freeze-drying, the gamma-polyglutamic acid refined；
Preferably, in the step 3 gamma-polyglutamic acid extraction process are as follows: be added 5 times of volumes of solid-state fermentation content water stirring mentions
It takes 1-2 hours, after 5000r/min is centrifuged, collects clear liquid for clear liquid concentrated by rotary evaporation to 50ml and add the second of 4 times of volumes
Alcohol stands 6-24 hours, precipitating is collected by centrifugation, and then with the ethanol washing of 2 times of volumes, liquid nitrogen, which is handled, to be ground, and vacuum refrigeration is dry
Gamma-polyglutamic acid that is dry, being refined.
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