CN109182407A - A kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member - Google Patents
A kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member Download PDFInfo
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- CN109182407A CN109182407A CN201811104690.XA CN201811104690A CN109182407A CN 109182407 A CN109182407 A CN 109182407A CN 201811104690 A CN201811104690 A CN 201811104690A CN 109182407 A CN109182407 A CN 109182407A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
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Abstract
A kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member, by degreasing pupa albumen, mixture is obtained after yeast powder and peptone mixing, then purified water is added into the mixture and is configured to the feed liquid that mass fraction is 5 ~ 20%, it is heated to 80 ~ 100 DEG C after mixing evenly, the protease that the mixture weight 1 ~ 10% is added after being subsequently cooled to 40 ~ 60 DEG C carries out 2 ~ 15h of enzymatic hydrolysis, then enzymolysis liquid is heated to 90 ~ 120 DEG C of enzyme deactivations, enzymolysis solution after enzyme deactivation obtains filtrate after filtering, filtrate be concentrated and dried up to tryptophan fermentation special nutritional member.The present invention produces tryptophan fermentation special nutritional member by enzyme digestion reaction, first using tryptophan fermentation special nutritional in tryptophan fermentation, can greatly improve the production acid and conversion ratio of tryptophan, improve the level of fermentation technology of tryptophan.
Description
Technical field
The present invention relates to a kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritionals
Member belongs to Fermentation Engineering and enzyme engineering field of biotechnology.
Background technique
Tryptophan is one of eight kinds of essential amino acids in human body and animal life activity, is existed with free state or reference state
In organism.Very important effect, the referred to as second required amino are played to growth and development, the metabolism of humans and animals
Acid is widely used in all various aspects such as medicine, food and feed.The production of tryptophan relies primarily on chemical synthesis and egg earliest
White matter Hydrolyze method, but with deepening continuously to Production by Microorganism Fermentation tryptophan research, this method has been in main
Lead status.With the continuous increase of the tryptophan market demand, fermentation method production tryptophan technology has also obtained global extensive
Concern.Currently, China's fermenting and producing tryptophan totality acid yield is lower, there are also larger gaps for acid yield and world level.
It is horizontal to improve tryptophan fermentation and acid, on the one hand sets about from strain improvement, the tryptophan-producing Strain of breeding high-yield, on the one hand optimizes
The culture medium condition of tryptophan, the result of experiment proves the apparent production acid for influencing tryptophan of the composition of the culture medium of fermentation, excellent
Changing fermentation medium is particularly important link to improving tryptophan to produce acid.In tryptophan fermentation medium, nitrogen source is not only
The source of the nitrogen substances such as bacterium protein, nucleic acid is constituted, and is the source for synthesizing amino in tryptophan.General select does color
The substance of propylhomoserin fermentation organic nitrogen source has yeast extract, corn pulp, brewer's wort, soya-bean cake hydrolyzate etc., these organic nitrogen sources are in color ammonia
Using slowly in acid fermentation incubation, utilization rate is not high, and generates many impurity, influences the metabolism of tryptophan-producing Strain
Journey, the residue not being fully utilized influence the rear abstraction process of tryptophan.
Summary of the invention
The purpose of the present invention is to provide a kind of flavored type mushroom sauce and its processing methods.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that a kind of fermentation of tryptophan is special
With nutrition member, mixture is obtained after degreasing pupa albumen, yeast powder and peptone are mixed, is then added into the mixture
Purified water is configured to the feed liquid that mass fraction is 5 ~ 20%, is heated to 80 ~ 100 DEG C after mixing evenly, is subsequently cooled to 40 ~ 60 DEG C
The protease that the mixture weight 1 ~ 10% is added afterwards carries out 2 ~ 15h of enzymatic hydrolysis, and enzymolysis liquid is then heated to 90 ~ 120 DEG C and is gone out
Enzyme, enzymolysis solution after enzyme deactivation obtain filtrate after filtering, be concentrated and dried up to tryptophan fermentation special nutritional to filtrate
Member.
Preferred technical solution are as follows: material of the mixing package containing following percent mass ratio: degreasing pupa albumen 40 ~
80%, yeast powder 10 ~ 40%, peptone 5 ~ 20%.
Preferred technical solution are as follows: the protease be compound protease, alkali protease and flavor protease in extremely
Few one kind.
Preferred technical solution are as follows: the filtering, which refers to, first carries out micro-filtration filtering for enzymolysis solution after enzyme deactivation, obtained filter
Liquid is filtered with ultrafiltration apparatus again, then is concentrated and dried to the filtrate that ultrafiltration apparatus obtains；The retention of the ultrafiltration apparatus
Molecular weight is 1000 ~ 5000Da.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that a kind of fermentation medium, often
Rising the fermentation medium includes: the glucose of 5.0 ~ 100.0g, the tryptophan fermentation special nutritional member of 0.5 ~ 20.0g, 0.1 ~
(the NH of 10.0g4)2SO4, 0.1 ~ 10.0g K2HPO4·3H2O, the MgSO of 0.1 ~ 10g4·7H2O, the fermentation medium
PH value is 7.0 ~ 7.2.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that a kind of tryptophan preparation side
Method is cultivated in the starting of tryptophan fermentation or pilot process using above-mentioned fermentation medium.
Since above-mentioned technical proposal is used, the present invention has the advantage, that compared with prior art
The present invention produces tryptophan fermentation special nutritional member by enzyme digestion reaction, is sent out in tryptophan fermentation using the tryptophan
Ferment special nutritional member can greatly improve the production acid and conversion ratio of tryptophan, improve the level of fermentation technology of tryptophan.The present invention
In the case where not needing to increase significantly equipment investment and human input, the production of tryptophan fermenting and producing can be increased substantially
Can, production cost is reduced, this method is simple and easy, is suitable for tryptophan industrialized production and promotes, can greatly improve China's color ammonia
Acid fermentation industrial level.
Specific embodiment
Embodiments of the present invention are illustrated by particular specific embodiment below, those skilled in the art can be by this implementation
Content disclosed by example is understood other advantages and efficacy of the present invention easily.
The purpose of term used herein, which is only that, illustrates particular embodiment, it is not intended to be limited to the present invention.It removes
Non- context is explicitly shown, otherwise singular " one " used herein, "one" also include plural form.
When illustrating preferred embodiment, it is potentially based on clear purpose and uses special term；However, this specification institute
Revealer is not intended to be limited in the selected special term；And it is to be understood that each particular element includes having
Identical function, all equivalence techniques for operating in a similar manner and reaching similar effects.
The condition of culture of tryptophan fermenting microbe used in following embodiment and comparative example is as follows:
Seed culture medium: glucose 30.0g/L, yeast powder 20.0g/L, (NH4)2S0420.0g/L KH2PO41.1g/L
MgSO4•7H2O 0.5g/L, 1.5 g/L of urea.
Tryptophan-producing Strain kind is accessed in seed culture medium, in shaking under the conditions of 36 DEG C, pH are 7.0 and dissolved oxygen is 30%
Bottle in culture 10 ~ for 24 hours to logarithmic phase.Tryptophan-producing Strain kind is specifically as follows: Corynebacterium glutamicum (Corynebacterium glutamicum) it is strain.
Comparative example
Fermentation medium: glucose 52.5g/L, yeast powder 10.25g/L, (NH4)2S045.05g/L K2HPO4•3H2O5.05 g/
L, MgSO4•7H2O 5.05/L, initial pH7.1.
The tryptophan-producing Strain bacterium solution of logarithmic phase is controlled automatically by 10% 10L of the inoculum concentration access containing fermentation medium
In fermentor processed, initial constant volume 5L；Condition of culture: 35 DEG C of temperature, pH value 7, dissolved oxygen >=30%, tank press 0.06 MPa, fermentation process
Middle residual sugar content control passes through stream plus proper quantity of defoaming agent defoaming 0.11%.It ferments to 40h and stops, when putting tank, L-Trp
Producing acid and saccharic acid conversion ratio see the table below.
| Test number | L-Trp produces acid | Saccharic acid conversion ratio |
| One | 34.85g/L | 15.32% |
| Two | 35.14g/L | 15.59% |
| Three | 35.02g/L | 15.10% |
| It is average | 35.00g/L | 15.34% |
Embodiment 1: a kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member
Purified water 500mL is added into beaker, accurately weighs degreasing pupa albumen 20g, yeast powder 20g, peptone 10g, puts into
In beaker, stir evenly, by indirect heating to 90 DEG C maintain 15 minutes, fast cooling is to 55 DEG C.Compound protease is added
1.6g, alkali protease 1.2g, flavor protease 0.8g keep 55 ± 1 DEG C of temperature enzymatic hydrolysis.After digesting 6hr, it is heated to 90 DEG C of dimensions
It holds 10min and carries out enzyme deactivation.Reaction solution is separated by solid-liquid separation by micro-filtration filter.Micro-filtration clear liquid is tested by 2000Da and is surpassed
Filter membrane is filtered.Ultrafiltration clear liquid is concentrated with Rotary Evaporators, after being concentrated into one third volume, stops concentration.Make
Concentrate is dried with spray drying lab scale equipment, obtains powdered tryptophan fermentation special nutritional member 41.6g.
Prepare fermentation medium: glucose 52.5g/L, tryptophan fermentation special nutritional member 8g/L, (NH4)2SO45.05g/
L, K2HPO4·3H2O5.05g/L, MgSO4·7H205.05g/L, initial pH7.1；The tryptophan-producing Strain bacterium solution of logarithmic phase is pressed
10% inoculum concentration accesses the 10L containing fermentation medium and automatically controls in fermentor, initial constant volume 5L；Condition of culture: temperature 35
DEG C, pH value 7, dissolved oxygen >=30%, tank press that 0.06 MPa, the control of residual sugar content disappears by stream plus in right amount 0.11% in fermentation process
Infusion defoaming.Fermentation stops to 40h, and when putting tank, the production acid of L-Trp is 40.54g/L, saccharic acid conversion ratio 16.87%, phase
For comparative example, L-Trp produces acid and improves 15.83%, and saccharic acid conversion ratio improves 9.97%, has and significantly improves.
Embodiment 2: a kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member
Purified water 200L is added into 2000L enzymatic vessel, accurately weighs degreasing pupa albumen 8kg, yeast powder 5kg, peptone 2kg
Put into enzymatic vessel in, stir evenly, by indirect heating to 90 DEG C maintain 15 minutes, fast cooling is to 55 DEG C.Compound protease
0.6kg, alkali protease 0.6kg, flavor protease 0.3g keep 55 ± 1 DEG C of temperature enzymatic hydrolysis.After digesting 8hr, it is heated to 100
DEG C maintain 10min carry out enzyme deactivation.Reaction solution is separated by solid-liquid separation by micro-filtration filter.Micro-filtration clear liquid is surpassed by 1000Da
Filter is filtered.Ultrafiltration clear liquid is concentrated with vacuum evaporation equipment, after being concentrated into one third volume, is stopped dense
Contracting.Concentrate is dried using spray dryer, obtains powdered tryptophan fermentation special nutritional member about 14.2kg.
Prepare fermentation medium: glucose 52.5g/L, tryptophan fermentation special nutritional member 5g/L, (NH4)2SO45.05g/
L, K2HPO4·3H2O5.05g/L, MgSO4·7H205.05g/L, initial pH7.1；The tryptophan-producing Strain bacterium solution of logarithmic phase is pressed
10% inoculum concentration accesses the 10L containing fermentation medium and automatically controls in fermentor, initial constant volume 5L；Condition of culture: temperature 35
DEG C, pH value 7, dissolved oxygen >=30%, tank press that 0.06 MPa, the control of residual sugar content disappears by stream plus in right amount 0.11% in fermentation process
Infusion defoaming, the tryptophan for starting stream plus 50m% content when cultivating to 12h ferment special nutritional member sterile liquid, and flow acceleration is
4g/h adds until 36h stops stream.Fermentation stops to 40h, and when putting tank, the production acid of L-Trp is 48.60 g/L, saccharic acid conversion ratio
It is 17.46%, relative to comparative example, L-Trp produces acid and improves 38.86%, and saccharic acid conversion ratio improves 13.82%, has aobvious
It writes and improves.
Prepare fermentation medium: glucose 52.5g/L, tryptophan fermentation special nutritional member 5g/L, (NH4)2SO45.05g/
L, K2HPO4·3H2O5.05g/L, MgSO4·7H205.05g/L, initial pH7.1；The tryptophan-producing Strain bacterium solution of logarithmic phase is pressed
10% inoculum concentration accesses the 50m containing fermentation medium3It automatically controls in fermentor, initial constant volume 25m3；Condition of culture: temperature
35 DEG C, pH value 7, dissolved oxygen >=30%, tank press 0.06 MPa, the control of residual sugar content is 0.11% in fermentation process, by stream plus appropriate
Defoaming agent defoaming, the tryptophan for starting stream plus 50% content when cultivating to 12h ferment special nutritional member sterile liquid, flow acceleration
For 20kg/h, add until 36h stops stream.Fermentation stops to 40h, and when putting tank, the production acid of L-Trp is 48.28 g/L, and saccharic acid turns
Rate is 18.17%, and relative to comparative example, L-Trp produces acid and improves 37.94%, and saccharic acid conversion ratio improves 18.45%, tool
It improves a lot, more conducively tryptophan fermentation industry production improves production capacity, reduces cost.
Embodiment 3: a kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member
A kind of tryptophan fermentation special nutritional member, obtains mixture after degreasing pupa albumen, yeast powder and peptone are mixed, so
Purified water is added in the backward mixture and is configured to the feed liquid that mass fraction is 5%, is heated to 80 DEG C after mixing evenly, then
The protease that the mixture weight 1% is added after being cooled to 40 DEG C carries out enzymatic hydrolysis 2h, and enzymolysis liquid is then heated to 90 DEG C and is gone out
Enzyme, enzymolysis solution after enzyme deactivation obtain filtrate after filtering, be concentrated and dried up to tryptophan fermentation special nutritional to filtrate
Member.
Preferred embodiment are as follows: material of the mixing package containing following percent mass ratio: degreasing pupa albumen
40%, yeast powder 40%, peptone 20%.
Preferred embodiment are as follows: the protease be compound protease,.
Preferred embodiment are as follows: the filtering, which refers to, first carries out micro-filtration filtering for enzymolysis solution after enzyme deactivation, obtained filter
Liquid is filtered with ultrafiltration apparatus again, then is concentrated and dried to the filtrate that ultrafiltration apparatus obtains；The retention of the ultrafiltration apparatus
Molecular weight is 1000Da.
A kind of fermentation medium, every liter of fermentation medium includes: the tryptophan fermentation of the glucose, 20.0g of 5.0g
(the NH of special nutritional member, 10.0g4)2SO4, 10.0g K2HPO4·3H2O, the MgSO of 10g4·7H2O, the fermentation medium
PH value be 7.2.Condition of culture are as follows: 40 DEG C of temperature, pH value 7.5, dissolved oxygen >=30%, tank press 0.10 MPa, residual sugar in fermentation process
Content is controlled 0.2%.
A kind of tryptophan preparation method uses preceding claim fermentation medium in the initial phase of tryptophan fermentation
It is cultivated.
Embodiment 4: a kind of tryptophan preparation method and its fermentation medium and tryptophan that use fermentation special nutritional member
A kind of tryptophan fermentation special nutritional member, obtains mixture after degreasing pupa albumen, yeast powder and peptone are mixed, so
Purified water is added in the backward mixture and is configured to the feed liquid that mass fraction is 20%, is heated to 100 DEG C after mixing evenly, so
The protease that the mixture weight 10% is added after being cooled to 60 DEG C afterwards carries out enzymatic hydrolysis 15h, and enzymolysis liquid is then heated to 120
DEG C enzyme deactivation, enzymolysis solution after enzyme deactivation obtain filtrate after filtering, filtrate be concentrated and dried ferment up to tryptophan it is dedicated
Nutrition member.
Preferred embodiment are as follows: material of the mixing package containing following percent mass ratio: degreasing pupa albumen
80%, yeast powder 10%, peptone 10%.
Preferred embodiment are as follows: the protease is alkali protease.
Preferred embodiment are as follows: the filtering, which refers to, first carries out micro-filtration filtering for enzymolysis solution after enzyme deactivation, obtained filter
Liquid is filtered with ultrafiltration apparatus again, then is concentrated and dried to the filtrate that ultrafiltration apparatus obtains；The retention of the ultrafiltration apparatus
Molecular weight is 1000 ~ 5000Da.
In order to achieve the above objects and other related objects, present invention provide the technical scheme that a kind of fermentation medium, often
Rising the fermentation medium includes: the glucose of 100.0g, the tryptophan fermentation special nutritional member of 20.0g, 10.0g
(NH4)2SO4, 10.0g K2HPO4·3H2O, the MgSO of 10g4·7H2O, the pH value of the fermentation medium are 7.0.Cultivate item
Part are as follows: 30 DEG C of temperature, pH value 6.5, dissolved oxygen >=30%, tank press 0.02 MPa, in fermentation process the control of residual sugar content 0.01%.
A kind of tryptophan preparation method is trained in the pilot process of tryptophan fermentation using above-mentioned fermentation medium
It supports.
As described above is only to be not intended to tool to explain the preferred embodiments of the invention to do any shape to the present invention
Limitation in formula should all wrap therefore all have any modification or change for making the related present invention under identical spirit
It includes in the scope that the invention is intended to protect.
Claims (6)
1. a kind of tryptophan fermentation special nutritional member, it is characterised in that: after mixing degreasing pupa albumen, yeast powder and peptone
Mixture is obtained, purified water is then added into the mixture and is configured to the feed liquid that mass fraction is 5 ~ 20%, after mixing evenly
Be heated to 80 ~ 100 DEG C, be added after being subsequently cooled to 40 ~ 60 DEG C the mixture weight 1 ~ 10% protease carry out enzymatic hydrolysis 2 ~
Enzymolysis liquid is then heated to 90 ~ 120 DEG C of enzyme deactivations by 15h, and enzymolysis solution after enzyme deactivation obtains filtrate after filtering, to filtrate into
Row is concentrated and dried up to tryptophan fermentation special nutritional member.
2. tryptophan fermentation special nutritional member according to claim 1, it is characterised in that: the mixing package contains following matter
Measure the material of percentage: degreasing pupa albumen 40 ~ 80%, yeast powder 10 ~ 40%, peptone 5 ~ 20%.
3. tryptophan fermentation special nutritional member according to claim 1, it is characterised in that: the protease is compound protein
At least one of enzyme, alkali protease and flavor protease.
4. tryptophan fermentation special nutritional member according to claim 1, it is characterised in that: the filtering refers to will be after enzyme deactivation
Enzymolysis liquid first carry out micro-filtration filtering, obtained filtrate is filtered with ultrafiltration apparatus again, then to the filtrate that ultrafiltration apparatus obtains
It is concentrated and dried；The molecular cut off of the ultrafiltration apparatus is 1000 ~ 5000Da.
5. a kind of fermentation medium, it is characterised in that: every liter of fermentation medium includes: the glucose of 5.0 ~ 100.0g, 0.5
(the NH of the fermentation special nutritional of tryptophan described in claim 1 ~ 4 any claim of ~ 20.0g member, 0.1 ~ 10.0g4)2SO4, 0.1 ~ 10.0g K2HPO4·3H2O, the MgSO of 0.1 ~ 10g4·7H2O, the pH value of the fermentation medium is 7.0 ~
7.2。
6. a kind of tryptophan preparation method, it is characterised in that: in the starting of tryptophan fermentation or pilot process, wanted using right
Fermentation medium described in asking 5 is cultivated.
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Cited By (1)
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