CN101550437A - Method for preparing fermented liquid rich in L-tryptophan by utilizing rice - Google Patents

Abstract

The invention relates to a method for preparing fermented liquid rich in L-tryptophan by utilizing rice, which is applied in the preparation of fermented liquid containing L-tryptophan. The method comprises the following steps of: (1) preparation of double enzyme glucose solution, which includes zymohydrolysis and saccharification of rice slurry by using high-temperature amylase and efficient saccharifying enzyme; (2) preparation of seed culture, which includes the steps of (2.1) dividing the double enzyme glucose solution obtained in (1) into three parts of X, Y and Z by weight according to the proportion that X : Y : Z is equal to 1 : 4 : 95, wherein X is used as sugar liquid needed by seed culture medium, Y is used as sugar liquid needed by fermentation medium and Z is used as sugar liquid needed by fed-batch sugar; and (2.2) preparing seed culture medium containing double enzyme glucose solution with the amount of X by using a conventional method and equally dividing the seed culture medium into two parts: one part is used as seed culture medium for recombinant Escherichia coli; and the other part is used as seed culture medium for corynebacterium glutamicum; and (3) fed-batch sugar fermentation by using the seed culture. The method has the advantages of improving the acid yield and the yield coefficient, shortening the fermentation period and saving the energy consumption.

Description

Utilize the rice preparation to be rich in the method for L-tryptophane fermented liquid

Technical field

The present invention relates to a kind of method of utilizing the rice preparation to be rich in L-tryptophane fermented liquid, be applied to prepare L-tryptophane fermented liquid.

Background technology

L-tryptophane formal name used at school tryptophane, chemical name L-2-amino-3-indyl propionic acid, another name L-pancreas Argine Monohydrochloride, L-amino indole propionic acid, molecular formula C11H12O2N2, relative molecular mass 204.21.L-tryptophane (L-Tryptophan) is the neutral die aromatischen Aminosaeuren that contains indyl, is white in color or slightly yellowy leaflet crystal or powder, and odorless or little smelly is dissolved in hot pyridine, is slightly soluble in ethanol, is insoluble to chloroform, ether.Dissolve in diluted acid or diluted alkaline, long-time illumination is then painted.Be total to a small amount of indoles of thermogenesis with water,, then produce the volume indoles as heating in the presence of NaOH or CuSO4.In the dark add thermally labile with acid.More stable in alkali lye, but then easily decompose when having other amino acid or carbohydrate.Rapidly during heating in 210 ℃ of jaundice, 290 ℃ of fusions (decomposition).The L-tryptophane has three kinds of optical isomers.Solubleness is 1.14% (25 ℃), 2.8% (75 ℃) in the water.Specific rotatory power ${\left[\mathrm{\α}\right]}_D^{20}=+2.8(1\mathrm{NHCl},C=1).$ PKa (25 ℃) is 2.38 and 9.39, iso-electric point pI=5.89.Its structural formula is:

The L-tryptophane is present in the natural protein widely, is one of people and the indispensable indispensable amino acid of various animal growths, and the L-tryptophane also is widely used in aspects such as medicine, food, feed except playing an important role in Nutrition and Metabolism.

The method of the existing L-of preparation tryptophane fermented liquid is to be raw material with the oral glucose, produces at single tryptophane under the effect of bacterium, produces the tryptophane fermented liquid.The purity of oral glucose is higher, only can provide L-tryptophane strain fermentation required carbon source, nitrogenous source, adding inorganic salt provides growth required trace element, add inorganic salt outside adding the required activator of bacterial classification in, introduce other foreign ions, brought difficulty for the purification of product in the follow-up fermented liquid.Too much or the very few nutrition-allocated proportion that all will change fermented liquid of the adding of inorganic salt influences fermentation and acid, makes the fermentation and acid level not high, and the index of fermentative production is 22g/L at present.

The fermentation of L-tryptophane is a microbial metabolism, and the metabolism of the L-tryptophane bacterial strain that occurs stream is very faint now, and is subjected to the inhibition or the interference of big quantity of material, thereby changes the pathways metabolism of bacterial strain, causes the purpose product to change.Therefore at present it is unstable that fermenting process produces acid, or suppress or disturb to cause producing metabolism and change because of producing halfway, causes producing the acid purpose product that even do not produce on the low side.

Summary of the invention

The purpose of this invention is to provide the high rice preparation that utilizes of a kind of L-tryptophane yield and be rich in the method for L-tryptophane fermented liquid, it is raw material with rice.

Starting point of the present invention is: rice under the effect of alpha-amylase, high-effective glucoamylase, under conditions such as certain temperature, time, is made two enzymatic glucose liquid by fermentation.

The required liquid glucose of seed culture medium in the fermenting process, fermention medium and the stream sugaring of adding during the fermentation provides by above-mentioned pair of enzymatic glucose liquid, produces in the presence of the bacterium at the L-tryptophane and ferments, and obtains being rich in the fermented liquid of L-tryptophane.

Described stream sugaring is to explain during the fermentation, because of the fermentation of bacterial classification makes the sugared content in the fermented liquid reduce, need constantly add for fermentation is normally carried out, to reach the required best sugar degree of normal fermentation.

Very faint because of the metabolism of L-tryptophane bacterial strain stream, and be subjected to the inhibition or the interference of big quantity of material, thereby change the pathways metabolism of bacterial strain, cause purpose product content to reduce even change, thus the L-tryptophane ferment in bacterial classification play crucial effects.The mode that adopts bacterial classification to add is fermented, and the bacterial classification that the vigor of adding timely is stronger impels fermentation to carry out along the approach that produces the purpose product, shortens fermentation period when improving spawn activity and acid producing ability.

Purpose of the present invention can reach like this: a kind of method of utilizing the rice preparation to be rich in L-tryptophane fermented liquid is characterized in that comprising following (1), (2), (3) three operations:

(1), the two enzymatic glucose liquid operations of preparation, this operation comprises in regular turn:

(1.1) will under the grinding machine effect that has grinding abrasive disk, make slurry behind the rice warm water soaking;

(1.2) utilize alpha-amylase, high-effective glucoamylase the slurry enzymatic saccharification in succession by the condition of executing enzyme;

(1.3) begin the enzyme that goes out when producing sugared climax when the slurry saccharification;

(1.4) and then add powder activity carbon decoloring and perlite powder flocculation, static 30min is after conventional filtration obtains two enzymatic glucose liquid;

(2), preparation inoculum operation, this operation comprises:

(2.1) the two enzymatic glucose liquid of (1) operation gained are divided into X, Y, Z three parts by weight, the each several part ratio is X: Y: Z=1: 4: 95; X is as the needed liquid glucose of seed culture medium, and Y is as the needed liquid glucose of fermention medium, and Z is as the needed liquid glucose of stream sugaring;

(2.2) the conventional making contained the seed culture medium that above-mentioned X measures two enzymatic glucose liquid, and it is divided equally two portions: a part is as the recombination bacillus coli seed culture medium; Another part is as the Corynebacterium glutamicum seed culture medium;

(2.3) 1% inclined-plane recombination bacillus coli original seed is inserted the recombination bacillus coli seed culture medium, 36 ℃ of holding temperatures adopt the optical density(OD) of 752 spectrophotometric instrumentation liquid mediums at visible light 660 nano wave lengths, can culture transferring to optical density(OD) 〉=15 o'clock; At interval after 8 hours time differences, the Corynebacterium glutamicum original seed with 1% inserts the Corynebacterium glutamicum seed culture medium, and 36 ℃ of holding temperatures adopt the optical density(OD) of 752 spectrophotometric instrumentation liquid mediums at visible light 660 nano wave lengths, can culture transferring to optical density(OD) 〉=15 o'clock;

(3), utilize inoculum to flow the sugaring fermentation procedure, this operation comprises:

(3.1) will add fermentor tank as two enzymatic glucose liquid that the required liquid glucose of fermention medium is used, and routine is made fermention medium in jar;

(3.2) temperature of keeping fermention medium by the water quench fermentor tank is 35 ℃, keeps substratum pH value 6.0～6.5 by add liquid ammonia to substratum; Press 10% long-pending inoculum size of the two enzymatic glucose liquid of the interior fermention medium of fermentor tank successively the recombination bacillus coli liquid medium and the Corynebacterium glutamicum liquid medium of interval 8h immigration (2) gained;

(3.3) afterwards, add the stream sugaring by fed-batch mode, to keep the glucose concn 2%[M/V of substratum in the fermentor tank all the time], till adding, two enzymatic glucose liquid of using to direct current sugaring amount promptly get the fermented liquid that is rich in the L-tryptophane.

The present invention compares with prior art has following advantage:

1, because two enzymatic glucose is that rice is through amylase, two enzyme effects of saccharifying enzyme produce, compare with oral glucose, it also has the large amount of organic composition except glucose, these are organic to exist for the tryptophane fermentation provides a large amount of nutrition, the microbial transformation approach that exists for the tryptophane fermentation of inorganics such as metal ion provides the thalline activator in simultaneously two enzymatic glucoses, stimulate thalli growth, activate the activeconstituents of thalline, shorten the growth and the adaptive phase of thalline, strengthen the thalline vigor, improve acid production rate.Acid yield is increased to 30g/L by 22g/L, and yield also is increased to 12.5% by 9%.

2, prior art is veryer long to the biosynthetic pathway of L-tryptophane from glucose, its metabolism stream is also more weak, and the multiple precursor of synthetic needs of L-tryptophane, if think further to improve the accumulation volume of L-tryptophane, just must manage to strengthen the metabolism stream of synthetic these precursors.On the other hand, the regulatory mechanism more complicated in the L-tryptophane biosynthetic pathway except feedback inhibition and this coarse adjustment system of feedback repression, also exists the fine tuning system---the attenuator system.Therefore the present invention adopts the mode of adding bacterial classification, has strengthened the metabolism stream of L-tryptophane, makes fermentation carry out along the approach that produces purpose product L-tryptophane, and adds the fermentative activity that bacterial classification has improved bacterial strain greatly, has shortened fermentation period, has saved energy consumption.Fermentation period foreshortens to 29～31h by 33～35h, and steam mono-consumption 20T/T tryptophane is reduced to the 18T/T tryptophane.

Embodiment

Preparing fermented liquid with 5.5T rice below the present invention is that example is further described.

The two enzymatic glucose liquid of preparation.With 5.5T rice defibrination, volume is 20 cubic metres slurry A.Use soda ash or hydrochloric acid to regulate pH to 5.8～6.0 of slurry A, preferred 5.9.In slurry A, add alpha-amylase then and get slurry B for 38 liters.Again slurry B is heated under 120 ℃ of vapor action liquefier; Again the gained liquefier is all pumped into saccharifying tank, the liquefier temperature stirs to 60-62 ℃ of scope in the regulating tank, and pH value transfers to 4.4.At this moment, drop into high-effective glucoamylase and also stir, saccharification begins the enzyme that goes out when producing sugared climax, adds Powdered Activated Carbon and perlite powder, and static 30min is after conventional filtration obtains the two enzymatic glucose liquid of 4295.0Kg.

Above-mentioned " producing sugared maximum " is that statement adopts Fei woods reagent that liquid glucose content is measured, and reaches the maximum value of sugared content; " Powdered Activated Carbon " plays the decolorization of liquid glucose; " perlite powder " plays the effect of flocculation, is the suspended solid in the liquid glucose is flocculated into oarse-grained material, is beneficial to filtration.Alpha-amylase, high-effective glucoamylase are sold, are provided by letter (China) Investment Co., Ltd of Novi.

The preparation inoculum.The two enzymatic glucose liquid of 4295.0Kg are divided into X, Y, Z three parts by weight, and the each several part ratio is X: Y: Z=1: 4: 95; X has 42.95Kg as the needed liquid glucose of seed culture medium; Y has 171.8Kg as the needed liquid glucose of fermention medium; Z has 4080.5Kg as the needed liquid glucose of stream sugaring.The conventional making contained the seed culture medium that above-mentioned X measures two enzymatic glucose liquid, and it is divided equally two portions: a part is used as the recombination bacillus coli seed culture medium; Another part is used as the Corynebacterium glutamicum seed culture medium.Each component of seed culture medium is carried out proportioning: KH in following ratio ₂PO ₄0.8g/L, KCl 2.2g/L, yeast extract powder 0.5g/L, MgSO ₄1.2g/L, (NH4) ₂SO ₄1.8g/L, trisodium citrate 1.4g/L, defoamer 0.3g/L; Two enzymatic glucose liquid 40g/L; PH value 6.5.During the preparation seed culture medium, two enzymatic glucose fluid components will be with the separately sterilization adding of front nutrient media components.

1% inclined-plane recombination bacillus coli original seed is inserted the recombination bacillus coli seed culture medium, and 36 ℃ of holding temperatures adopt the optical density(OD) of 752 spectrophotometric instrumentation liquid mediums at visible light 660 nano wave lengths, o'clock can culture transferring to optical density(OD) 〉=15.At interval after 8 hours time differences, the Corynebacterium glutamicum original seed with 1% inserts the Corynebacterium glutamicum seed culture medium, and 36 ℃ of holding temperatures adopt the optical density(OD) of 752 spectrophotometric instrumentation liquid mediums at visible light 660 nano wave lengths, o'clock can culture transferring to optical density(OD) 〉=15.Above-mentioned recombination bacillus coli kind and Corynebacterium glutamicum kind are provided by life science institute of Fujian Normal University public offering.

Utilize inoculum to carry out stream and add sugar-fermenting.The two enzymatic glucose liquid 171.8Kg sterilization back adding volumes that to use as the required liquid glucose of fermention medium are 20 cubic metres fermentor tank.By following each component preparation fermention medium: KH ₂PO ₄0.8g/L, KCl 1.0g/L, yeast extract powder 0.5g/L, MgSO ₄1.5g/L, (NH4) ₂SO ₄1.3g/L, citric acid 2g/L, defoamer 0.3g/L; Two enzymatic glucose liquid 7.5g/L.Transfer pH6.0～6.5, preferred 6.2 with liquefied ammonia.

The temperature of keeping substratum by the water quench fermentor tank is 35 ℃, by add to substratum liquid ammonia keep substratum pH value 6.0～6.5. by fermentor tank in long-pending 10% inoculum size of the two enzymatic glucose liquid of substratum successively at interval 8h move into the recombination bacillus coli liquid medium and the Corynebacterium glutamicum liquid medium of B gained.Afterwards, add the stream sugaring by fed-batch mode, to keep the glucose concn 2%[M/V of substratum in the fermentor tank all the time], till two enzymatic glucose liquid of using to direct current sugaring amount add.The result adds end at 29h stream sugaring 4080.5Kg, and 30h exhausts because of sugar, Seed Aging, and product acid is fainter, fermentation ends.

Fermentation and acid 32g/L, lower tank amasss 16T, and gained theoretical acid=16 * 32=512kg adds pure sugared 4300kg altogether, and glucose acid invert ratio is 11.9%, and this numerical value is yield.The present invention is because fermentation period shortens, and the heating energy source steam consumption that regulating and controlling temperature is used in the fermenting process also just correspondingly obtains reducing.

Claims (1)

1, a kind of method of utilizing the rice preparation to be rich in L-tryptophane fermented liquid is characterized in that comprising following three operations:

(1), the two enzymatic glucose liquid operations of preparation, this operation comprises in regular turn:

(1.1) will under the grinding machine effect that has grinding abrasive disk, make slurry behind the rice warm water soaking;

(1.2) utilize alpha-amylase, high-effective glucoamylase the slurry enzymatic saccharification in succession by the condition of executing enzyme;

(1.3) begin the enzyme that goes out when producing sugared climax when the slurry saccharification;

(1.4) and then add powder activity carbon decoloring and perlite powder flocculation, static 30min is after conventional filtration obtains two enzymatic glucose liquid;

(2), preparation inoculum operation, this operation comprises:

(2.1) the two enzymatic glucose liquid of (1) operation gained are divided into X, Y, Z three parts by weight, the each several part ratio is X: Y: Z=1: 4: 95; X is as the needed liquid glucose of seed culture medium, and Y is as the needed liquid glucose of fermention medium, and Z is as the needed liquid glucose of stream sugaring;

(2.2) the conventional making contained the seed culture medium that above-mentioned X measures two enzymatic glucose liquid, and it is divided equally two portions: a part is as the recombination bacillus coli seed culture medium; Another part is as the Corynebacterium glutamicum seed culture medium;

(2.3) 1% inclined-plane recombination bacillus coli original seed is inserted the recombination bacillus coli seed culture medium, 36 ℃ of holding temperatures adopt the optical density(OD) of 752 spectrophotometric instrumentation liquid mediums at visible light 660 nano wave lengths, can culture transferring to optical density(OD) 〉=15 o'clock; At interval after 8 hours time differences, the Corynebacterium glutamicum original seed with 1% inserts the Corynebacterium glutamicum seed culture medium, and 36 ℃ of holding temperatures adopt the optical density(OD) of 752 spectrophotometric instrumentation liquid mediums at visible light 660 nano wave lengths, can culture transferring to optical density(OD) 〉=15 o'clock;

(3), utilize inoculum to flow the sugaring fermentation procedure, this operation comprises:

(3.1) will add fermentor tank as two enzymatic glucose liquid that the required liquid glucose of fermention medium is used, and routine is made fermention medium in jar;

(3.2) temperature of keeping fermention medium by the water quench fermentor tank is 35 ℃, keeps substratum pH value 6.0～6.5 by add liquid ammonia to substratum; Press 10% long-pending inoculum size of the two enzymatic glucose liquid of the interior fermention medium of fermentor tank successively the recombination bacillus coli liquid medium and the Corynebacterium glutamicum liquid medium of interval 8h immigration (2) gained;

(3.3) afterwards, add the stream sugaring by fed-batch mode, to keep the glucose concn 2%[M/V of substratum in the fermentor tank all the time], till adding, two enzymatic glucose liquid of using to direct current sugaring amount promptly get the fermented liquid that is rich in the L-tryptophane.