CN104694591A - Fermentation medium for biotin defined amount fermentation production glutamic acid, fermentation method for producing glutamic acid by using fermentation medium and application - Google Patents
Fermentation medium for biotin defined amount fermentation production glutamic acid, fermentation method for producing glutamic acid by using fermentation medium and application Download PDFInfo
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Abstract
The invention relates to a fermentation medium for biotin defined amount fermentation production glutamic acid. Components and the final concentration of the components in a formula comprises: 425-495 g / L of corn starch hydrolysis sugar, 3.5-5 g / L of corn steep liquor, 2.6-3.6 g / L of molasses, 0.6-0.9 g / L of MgSO4.7H2O, 0.0025-0.005 g / L of MnSO4, 0.0025-0.005 g / L of FeSO4, 0.001-0.0025 g / L of VB1, 2-3 g / L of KC1 and 3-8 g / L of H3PO4. Dissolving solvent is water. Phosphoric acid is neutralized by alkali, so that pH of the fermentation medium ranges from 6.8 to 7.2. According to the appropriate ratio of the carbon source, the nitrogen source, the carbonic oxide and the phosphorus content, the acid production rate is improved, the thallus acid production is prompted, finally the acid production level is obviously improved, the improvement is about 4 percent, and the yield of glutamic acid and the conversion rate of sugar acid are improved.
Description
Technical field
The invention belongs to bio-fermentation engineering field, especially a kind of fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid and the fermentation process utilizing it to produce L-glutamic acid and application.
Background technology
Inorganic salt are the indispensable materials of microbial life activity.Need the nutritive element such as phosphorus, potassium in glutamic acid fermentation process, phosphorus is the principal element of oxidative phosphorylation in metabolic process in inorganic salt, and it is also the moiety of nucleic acid, phosphatide, some coenzyme; High-energy phosphate bond plays an important role in energy storage with transmission, phosphoric acid salt or a kind of important buffer reagent.No exception in glutamic acid fermentation, the height of phosphorus content is very large on its impact, and phosphorus concentration is too high, and bacterial metabolism has the tendency turning to synthesis to pick propylhomoserin.Phosphorus concentration is too low, and thalli growth is bad, shows as that consumption sugar is slow, residual sugar is high, and thalli growth is slow.The phosphoric acid salt such as Sodium phosphate dibasic (Na2HPO412H2O), dipotassium hydrogen phosphate (K2HPO43H2O) is often used to provide phosphoric in production; Repone K (KCl) provide potassium element.
L-glutamic acid is first amino acid products in the world, and China is L-glutamic acid first big producing country.According to the statistics of Chinese biological fermentation industry association, the output to China's L-glutamic acid in 2011 and sodium salt (monosodium glutamate) thereof has reached 2,420,000 tons, accounts for more than 70% of Gross World Product.Different from other amino acid products, the topmost purposes of L-glutamic acid produces the seasonings such as monosodium glutamate, and L-glutamic acid is also widely used in medicine, agricultural, daily use chemicals and the raw material as other leavened prods simultaneously.
Through the development in more than 40 years, China's L-glutamic acid and the research of monosodium glutamate industry in strain improvement, process optimization, quality product improvement and energy-saving and emission-reduction, refuse process etc. all achieve great successes.1986, China replaces Japan became monosodium glutamate first big producing country.Between 2000 ~ 2006 years after entering the new millennium, China's monosodium glutamate industry is with the speed rapid growth of every annual 15.3%.Through the violent integration of 2006 ~ 2008 years, industry concentration ratio improved constantly, and had begun to take shape leader group of enterprises at present.Fu Feng group and plum blossom group rely on clear assurance to industry moving law and strong competitive power, show one's talent from the market competition of fierceness, form the gesture of tripartite confrontation with Japanese aginomoto.A few years from now on monosodium glutamate industry will eliminate the manufacturing enterprise that production technology level falls behind and competitive power is weak further, and industry annexs integration dynamics and will aggravate further.
As a ripe fermentation industry, L-glutamic acid and glutamate production profit meagre, the height of production level directly affects the final and decisive juncture of enterprise.Recent year monosodium glutamate enterprise through continuing technological transformation production cost is declined to a great extent, but in acid yield, raw material and plant factor etc. with still have certain gap abroad.Because L-glutamic acid and monosodium glutamate industry are the ripe production that technology barriers are low and fund input is large, because of in the industry often using direct rate war as main competitive method.Thus, manufacturing enterprise must consider from manufacture link itself, seeks the maximization of production efficiency and minimumization of comprehensive cost as far as possible, just likely stands firm in the market competition of fierceness.Facing to the powerful competitive power of domestic and international opponent, COFCO must pay attention to independent research, promotes glutamic acid fermentation production level further, compresses comprehensive production cost, improve rate of gross profit, only in this way just can meet following fiercer industry competition and be expected to degree of depth developing relevant market.
The fermentative production bacterial strain of current L-glutamic acid and monosodium glutamate industry is mainly vitamin H defective type, " suboptimal dose " glutamic acid fermentation technique that domestic glutamate production corporate boss will adopt, namely be utilize this characteristic of producing bacterial classification, by controlling the concentration of vitamin H in substratum, bacterial classification is made to be produce acid type cell in the specified phase of fermenting process by long bacterial type cell transition.
When vitamin H is abundant, the cytolemma synthesis of glutamic acid producer is complete, and L-glutamic acid can not penetrate into film in film, and the glutamic acid accumulation in born of the same parents to a certain extent, carries out feedback inhibition to glutamate dehydrogenase, thus stops the biosynthesizing of L-glutamic acid.When vitamin H is limited the quantity, because cytolemma synthesis is imperfect, L-glutamic acid can penetrate into born of the same parents in born of the same parents, and the content of L-glutamic acid in born of the same parents is reduced, and L-glutamic acid is lacked of proper care to the feedback inhibition of glutamate dehydrogenase, and L-glutamic acid is constantly preferentially synthesized.Utilize fermentative Production L-glutamic acid except selecting excellent glutamate producing bacterium, also according to the characteristic of bacterial strain uses therefor, suitable fermention medium must be selected and control best technological condition for fermentation.Good Medium Proportion can give full play to the biosynthesis ability producing bacterial classification, makes it to reach maximum production effect.But use the L-glutamic acid of existing fermention medium fermentative production, not only lower than rate of producing acid, fermentation period is longer, and, existing fermention medium mostly uses phosphoric acid salt, but the time due to the complete water-soluble needs of phosphoric acid salt in process for preparation is longer, easily causes the mixing of fermention medium uneven, thus acid production rate and transformation efficiency is caused to decline, and extend the work period, reduce production efficiency.As can be seen here, the fermention medium of the L-glutamic acid of existing fermentative production can not meet user demand completely, needs a kind of fermention medium of new fermentative production L-glutamic acid badly.
By retrieval, find following two sections of patent publication us relevant to present patent application:
1, a kind of high-efficiency fermenting produces the method (CN101402979A) of Pidolidone, with vitamin H deficient strain for producing bacterial strain, nutritious and the substratum of equilibrium is mixed with by a certain percentage as fermention medium using some nutrition compositions, in fermenting process, by regulating fermentor tank mixing speed and air quantity, dissolved oxygen is controlled in suitable level, by Feeding ammonia water control pH, and add certain density glucose solution by stream residual sugar is controlled at lower level, fermentation 32h stops.The present invention is not when increasing any extras and human input, shorten whole fermentation period, and significantly improve output (more than 140g/L) and the transformation efficiency (more than 60%) of Pidolidone, whole simple operation of process, production cost is lower, is extremely suitable for suitability for industrialized production.
2, the research (author: Liu Lifeng, Yu Weihong, ferment scientific and technological communication the 36th volume) that affects glutamic acid fermentation of phosphoric acid salt consumption, in order to improve the acid production rate of L-glutamic acid, finds breach from the formula of substratum.First consider from increase sylvite, the consumption of dipotassium hydrogen phosphate is increased to 0.20% by 0.17%.Viewed from test-results, produce acid low, reason is: in whole fermenting process, microcosmic salt number to be out of shape with thalline and the speed of growth has very large relation, microcosmic salt is many, and thalline is short and thick, from 10 ~ 20h, thalline is bred in a large number, and consumption sugar, finally cause consumption urea few, produce acid low, determine with 16% two enzyme sugar substratum in, better with the consumption of dipotassium hydrogen phosphate 0.14%, its acid production rate is steady, average 10.55%, transformation efficiency steadily and higher, average 65.95%.
By contrast, there are the different of essence in patent application of the present invention and above-mentioned patent publication us.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art part, a kind of fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid is provided and utilizes its fermentation process producing L-glutamic acid and application, this fermention medium can improve than rate of producing acid, shortens fermentation period, promote that thalline produces acid, improves acid production rate and glucose acid invert ratio, and the time of this substratum water-soluble needs in process for preparation is shorter, fermention medium mixes, shorten the preparation time of fermention medium, improve production efficiency; Meanwhile, this fermentation process is easy and simple to handle, and production cost is lower, is extremely suitable for suitability for industrialized production.
The present invention realizes the technical scheme that goal of the invention adopts:
For a fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid, its composition and the final concentration of each composition in this formula are:
W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 3.5 ~ 5g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o0.6 ~ 0.9g/L, MnSO
40.0025 ~ 0.005g/L, FeSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 3g/L, H
3pO
43 ~ 8g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with alkali neutralising phosphoric acid.
And the mass concentration of described W-Gum hydrolysis sugar is 30-35%; Or the concentration of described corn steep liquor is 18-25 degree Beaume.
And described alkali is sodium hydroxide, sodium carbonate, ammoniacal liquor or potassium hydroxide.
The application of fermention medium as above in fermentative production L-glutamic acid.
Utilize fermention medium as above to produce the fermentation process of L-glutamic acid, step is as follows:
(1) prepare burden: according to proportioning, various raw material weighing is good, dissolve and be mixed with fermention medium completely;
(2) sterilizing: by the fermention medium sterilizing prepared, temperature 108 ~ 110 DEG C, holds time 8 ~ 10 minutes, the substratum after sterilizing be cooled in fermentation 33 DEG C etc. to be seeded;
(3) by OD value be the vitamin H defective type glutamate producing bacterium seed culture fluid of 0.3 ~ 0.8 in seed culture fluid: the volume ratio of fermention medium is in 10 ~ 15% ratios access fermention mediums; Leavening temperature controls at 32 ~ 40 DEG C; Supplementing nitrogenous source is 6.8 ~ 7.2 to pH, and control air flow is 2 ~ 10L/min; Fermentation period is 24-28h, obtains L-glutamic acid.
And, described step (3) in fermenting process in, when the concentration of glucose in fermention medium is lower than 5-6%, the mode being added W-Gum hydrolysis sugar by stream makes the final concentration of glucose in fermention medium maintain between 0.5-5%, and the mass concentration that stream adds W-Gum hydrolysis sugar is 50-70%.
And described vitamin H defective type glutamate producing bacterium is Tianjin tyrothricin (Brevibacterium tianjinese) S9114 or CM415.
And the preparation process of described temperature vitamin H defective type glutamate producing bacterium seed culture fluid is as follows:
(1) actication of culture: get slant preservation bacterial classification 1 ~ 2 ring and be inoculated in and be equipped with in the slant tube of seed culture medium, 31 ~ 33 DEG C of constant temperature quiescent culture 24h;
(2) seed culture: access activated spawn by the triangular flask that 100 ~ 200mL seed culture medium is housed of own sterilizing, 31 ~ 33 DEG C of shaking culture 12 ~ 18h, obtain the bacterial strain activation solution that OD value is 0.3 ~ 0.8, are placed in 4 DEG C of refrigerator cold-storages for subsequent use.
The advantage that the present invention obtains and positively effect are:
1, carbon source suitable in basal culture medium, nitrogenous source, carbon-nitrogen ratio and phosphorus content improve and compare rate of producing acid, promote that thalline produces acid, final acid yield obtains remarkable lifting, can about 4% be improved, improve L-glutamic acid yield and glucose acid invert ratio, and in this substratum, use phosphoric acid to replace temperature sensitive type strain fermentation to produce phosphoric acid salt in L-glutamic acid substratum, the time of this substratum water-soluble needs in process for preparation is shorter, fermention medium mixes, shorten the preparation time of fermention medium, simultaneously, alkaline matter is utilized to neutralize fermention medium, the PH of fermention medium is made to meet thalli growth requirement, improve production efficiency, there is wide prospects for commercial application.
2, sugared concentration glutamic acid fermentation is had a significant impact in fermention medium, glutamic acid yield increases with sugared concentration and increases within the specific limits, but sugared excessive concentration, because osmotic pressure increases, affect thalli growth, fermentation period is extended, produce acid not easily to stablize, therefore this fermentation process adopts the lower W-Gum hydrolysis sugar of concentration to be used for preparing substratum, adopts the W-Gum hydrolysis sugar of miscarrying and adding higher concentration in fermention medium to maintain the needs of thalli growth and acid in fermentation culture process; Nitrogenous source is the source of the nitrogenous substancess such as synthesising thalli protein matter, nucleic acid and synthesizing amino acid amino, and a part of ammonia, for regulating pH value, forms glutaminate during the fermentation simultaneously, and therefore the nitrogenous source of glutamic acid fermentation needs is higher than general fermentation industry; Simultaneously, phosphorus is some protein and the moiety with nucleic acid, phosphorus amount is very large on the impact of L-glutamic acid, and when phosphorus concentration is too high, the metabolism of thalline turns to synthesis α-amino-isovaleric acid, but phosphorus content is too low, thalli growth is bad, and this fermentation process uses the phosphoric acid salt in phosphoric acid replacement temperature sensitive type strain fermentation production L-glutamic acid substratum, and the time of this substratum water-soluble needs in process for preparation is shorter, fermention medium mixes, and shortens the preparation time of fermention medium; Good Medium Proportion can give full play to the biosynthesis ability producing bacterial classification, makes it to reach maximum production effect.
3, when this fermentation process does not increase any extras and human input compared with existing production technique, significantly improve acid production rate (more than 14%) and the transformation efficiency (more than 67%) of Pidolidone, whole simple operation of process, production cost is lower, is extremely suitable for suitability for industrialized production.
Embodiment
Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The raw material used in the present invention, if no special instructions, is conventional commercially available prod; The method used in the present invention, if no special instructions, is the ordinary method of this area.
Adopt working method and the process control condition of fermention medium fermentative production L-glutamic acid of the present invention, add explanation if not special, all with the known method of operating of phosphatic fermention medium fermentative production L-glutamic acid and process control condition consistent.
The microorganism strains used in the present invention can be the corynebacterium glutamicum S9114 (Corynebacterium glutamicum S9114) of vitamin H defective type; The mass concentration of the W-Gum hydrolysis sugar used in the present invention can be 30-35%, and the concentration of the corn steep liquor used can be 18-25 degree Beaume.
Embodiment 1:
For a fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid, the proportioning raw materials of described fermention medium is as follows:
W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 3.5 ~ 5g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o0.6 ~ 0.9g/L, MnSO
40.0025 ~ 0.005g/L, FeSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 2.5g/L, H
3pO
43.0 ~ 4.0g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with sodium hydroxide neutralising phosphoric acid.
Utilize above-mentioned fermention medium to produce the fermentation process of L-glutamic acid, processing step and the condition of its fermentation culture are as follows:
(1) prepare burden: according to proportioning, various raw material weighing is good, dissolve and be made into 6L fermention medium completely;
The proportioning raw materials of described fermention medium is as follows: W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 3.5 ~ 5g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o 0.6 ~ 0.9g/L, MnSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 2.5g/L, H
3pO
43.0 ~ 4.0g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with sodium hydroxide neutralising phosphoric acid.
(2) sterilizing: the fermention medium prepared to be transferred in 10L fermentor tank and to carry out sterilizing, temperature 108 ~ 110 DEG C, holding time 8 ~ 10 minutes, the substratum after sterilizing be cooled in fermentation 33 DEG C etc. to be seeded;
(3) inoculation fermentation: be that the seed culture fluid of the vitamin H deficient strain of 0.7 ~ 1.0 is by seed culture fluid 600mL access fermention medium by OD value; Leavening temperature controls at 32 ~ 40 DEG C; Supplementing nitrogenous source is 7.0 to pH, and control air flow is 2 ~ 10L/min; Fermentation period is 28h, obtains L-glutamic acid;
During the fermentation, when remaining sugar concentration in fermention medium, (remaining sugar concentration refers to concentration of reduced sugar contained in fermention medium, herein remaining sugar concentration mensuration adopt Fehlings reagent) lower than 5-6% time, the mode being added W-Gum hydrolysis sugar by stream makes remaining sugar concentration in fermention medium maintain between 0.5-5%, and the mass concentration that stream adds W-Gum hydrolysis sugar is 50-70%.
The step of the preparation method of above-mentioned vitamin H defective type glutamate producing bacterium seed liquor is as follows:
(1) the vitamin H defective type glutamate producing bacterium on a ring slant medium is accessed in the seed culture medium of own sterilizing, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the bacterial strain activation solution that OD value is 0.6 ~ 0.8;
(2) by bacterial strain activation solution be by volume 7% ~ 15% ratio access seed culture medium in, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the vitamin H defective type glutamate producing bacterium seed liquor that OD value is 0.7 ~ 1.0.
Measure the content of glutamic acid in fermented liquid after fermentation ends, and calculate acid production rate and transformation efficiency, and carry out cost keeping.
Embodiment 2:
For a fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid, the proportioning raw materials of described fermention medium is as follows:
W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 3.5 ~ 5g/L, molasses 2.6 ~ 3.0g/L, MgSO47H
2o0.6 ~ 0.8g/L, MnSO
40.0025 ~ 0.004g/L, FeSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 3g/L, H
3pO
43 ~ 8g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with sodium carbonate neutralising phosphoric acid.
Utilize above-mentioned fermention medium to produce the fermentation process of L-glutamic acid, processing step and the condition of its fermentation culture are as follows:
(1) prepare burden: according to proportioning, various raw material weighing is good, dissolve and be made into 6L fermention medium completely;
The proportioning raw materials of described fermention medium is as follows: W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 3.5 ~ 5g/L, molasses 2.6 ~ 3.0g/L, MgSO47H
2o 0.6 ~ 0.8g/L, MnSO
40.0025 ~ 0.004g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 3g/L, H
3pO
43.0 ~ 8.0g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with sodium carbonate neutralising phosphoric acid.
(2) sterilizing: the fermention medium prepared to be transferred in 10L fermentor tank and to carry out sterilizing, temperature 108 ~ 110 DEG C, holding time 8 ~ 10 minutes, the substratum after sterilizing be cooled in fermentation 33 DEG C etc. to be seeded;
(3) inoculation fermentation: be that the seed culture fluid of the vitamin H deficient strain of 0.7 ~ 1.0 is by seed culture fluid 600mL access fermention medium by OD value; Leavening temperature controls at 32 ~ 40 DEG C; Supplementing nitrogenous source is 7.0 to pH, and control air flow is 2 ~ 10L/min; Fermentation period is 28h, obtains L-glutamic acid;
During the fermentation, when remaining sugar concentration in fermention medium, (remaining sugar concentration refers to concentration of reduced sugar contained in fermention medium, herein remaining sugar concentration mensuration adopt Fehlings reagent) lower than 5-6% time, the mode being added W-Gum hydrolysis sugar by stream makes remaining sugar concentration in fermention medium maintain between 0.5-5%, and the mass concentration that stream adds W-Gum hydrolysis sugar is 50-70%.
The step of the preparation method of above-mentioned vitamin H defective type glutamate producing bacterium seed liquor is as follows:
(1) the vitamin H defective type glutamate producing bacterium on a ring slant medium is accessed in the seed culture medium of own sterilizing, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the bacterial strain activation solution that OD value is 0.6 ~ 0.8;
(2) by bacterial strain activation solution be by volume 7% ~ 15% ratio access seed culture medium in, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the vitamin H defective type glutamate producing bacterium seed liquor that OD value is 0.7 ~ 1.0.
Measure the content of glutamic acid in fermented liquid after fermentation ends, and calculate acid production rate and transformation efficiency, and carry out cost keeping.
Embodiment 3:
For a fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid, the proportioning raw materials of described fermention medium is as follows:
W-Gum hydrolysis sugar 450 ~ 480g/L, corn steep liquor 3.8 ~ 4g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o0.6 ~ 0.8g/L, MnSO
40.0025 ~ 0.004g/L, FeSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 3g/L, H
3pO
43 ~ 8g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with ammonia neutralization phosphoric acid.
Utilize above-mentioned fermention medium to produce the fermentation process of L-glutamic acid, processing step and the condition of its fermentation culture are as follows:
(1) prepare burden: according to proportioning, various raw material weighing is good, dissolve and be made into 6L fermention medium completely;
The proportioning raw materials of described fermention medium is as follows: W-Gum hydrolysis sugar 450 ~ 480g/L, corn steep liquor 3.8 ~ 4g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o 0.6 ~ 0.8g/L, MnSO
40.0025 ~ 0.004g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 3g/L, H
3pO
43 ~ 8g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with ammonia neutralization phosphoric acid.
(2) sterilizing: the fermention medium prepared to be transferred in 10L fermentor tank and to carry out sterilizing, temperature 108 ~ 110 DEG C, holding time 8 ~ 10 minutes, the substratum after sterilizing be cooled in fermentation 33 DEG C etc. to be seeded;
(3) inoculation fermentation: be that the seed culture fluid of the vitamin H deficient strain of 0.7 ~ 1.0 is by seed culture fluid 600mL access fermention medium by OD value; Leavening temperature controls at 32 ~ 40 DEG C; Supplementing nitrogenous source is 7.0 to pH, and control air flow is 2 ~ 10L/min; Fermentation period is 24h, obtains L-glutamic acid;
During the fermentation, when remaining sugar concentration in fermention medium, (remaining sugar concentration refers to concentration of reduced sugar contained in fermention medium, herein remaining sugar concentration mensuration adopt Fehlings reagent) lower than 5-6% time, the mode being added W-Gum hydrolysis sugar by stream makes remaining sugar concentration in fermention medium maintain between 0.5-5%, and the mass concentration that stream adds W-Gum hydrolysis sugar is 50-70%.
The step of the preparation method of above-mentioned vitamin H defective type glutamate producing bacterium seed liquor is as follows:
(1) the vitamin H defective type glutamate producing bacterium on a ring slant medium is accessed in the seed culture medium of own sterilizing, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the bacterial strain activation solution that OD value is 0.6 ~ 0.8;
(2) by bacterial strain activation solution be by volume 7% ~ 15% ratio access seed culture medium in, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the vitamin H defective type glutamate producing bacterium seed liquor that OD value is 0.7 ~ 1.0.
Measure the content of glutamic acid in fermented liquid after fermentation ends, and calculate acid production rate and transformation efficiency, and carry out cost keeping.
Embodiment 4:
For a fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid, the proportioning raw materials of described fermention medium is as follows:
W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 4.0 ~ 5g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o0.6 ~ 0.9g/L, MnSO
40.0025 ~ 0.005g/L, FeSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2.5 ~ 2.8g/L, H
3pO
43 ~ 8g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with potassium hydroxide neutralising phosphoric acid.
Utilize above-mentioned fermention medium to produce the fermentation process of L-glutamic acid, processing step and the condition of its fermentation culture are as follows:
(1) prepare burden: according to proportioning, various raw material weighing is good, dissolve and be made into 6L fermention medium completely;
The proportioning raw materials of described fermention medium is as follows: W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 4.0 ~ 5g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o 0.6 ~ 0.9g/L, MnSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2.5 ~ 2.8g/L, H
3pO
43 ~ 8g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with potassium hydroxide neutralising phosphoric acid.
(2) sterilizing: the fermention medium prepared to be transferred in 10L fermentor tank and to carry out sterilizing, temperature 108 ~ 110 DEG C, holding time 8 ~ 10 minutes, the substratum after sterilizing be cooled in fermentation 33 DEG C etc. to be seeded;
(3) inoculation fermentation: be that the seed culture fluid of the vitamin H deficient strain of 0.7 ~ 1.0 is by seed culture fluid 600mL access fermention medium by OD value; Leavening temperature controls at 32 ~ 40 DEG C; Supplementing nitrogenous source is 7.0 to pH, and control air flow is 2 ~ 10L/min; Fermentation period is 28h, obtains L-glutamic acid;
During the fermentation, when remaining sugar concentration in fermention medium, (remaining sugar concentration refers to concentration of reduced sugar contained in fermention medium, herein remaining sugar concentration mensuration adopt Fehlings reagent) lower than 5-6% time, the mode being added W-Gum hydrolysis sugar by stream makes remaining sugar concentration in fermention medium maintain between 0.5-5%, and the mass concentration that stream adds W-Gum hydrolysis sugar is 50-70%.
The step of the preparation method of above-mentioned vitamin H defective type glutamate producing bacterium seed liquor is as follows:
(1) the vitamin H defective type glutamate producing bacterium on a ring slant medium is accessed in the seed culture medium of own sterilizing, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the bacterial strain activation solution that OD value is 0.6 ~ 0.8;
(2) by bacterial strain activation solution be by volume 7% ~ 15% ratio access seed culture medium in, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the vitamin H defective type glutamate producing bacterium seed liquor that OD value is 0.7 ~ 1.0.
Measure the content of glutamic acid in fermented liquid after fermentation ends, and calculate acid production rate and transformation efficiency, and carry out cost keeping.
Comparative examples:
For a method for vitamin H suboptimal dose fermentative production L-glutamic acid, processing step and the condition of its fermentation culture are as follows:
(1) prepare burden: according to proportioning, various raw material weighing is good, dissolve and be made into 6L fermention medium completely;
The proportioning raw materials of described fermention medium is as follows:
W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 3.5 ~ 5g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o0.6 ~ 0.9g/L, MnSO
40.0025 ~ 0.005g/L, FeSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 3g/L, Na
2hPO
45 ~ 9g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with ammonia neutralization phosphoric acid.
(2) sterilizing: the fermention medium prepared to be transferred in 10L fermentor tank and to carry out sterilizing, temperature 108 ~ 110 DEG C, holding time 8 ~ 10 minutes, the substratum after sterilizing be cooled in fermentation 33 DEG C etc. to be seeded;
(3) inoculation fermentation: be that the seed culture fluid of the vitamin H deficient strain of 0.7 ~ 1.0 is by seed culture fluid 600mL access fermention medium by OD value; Leavening temperature controls at 32 ~ 40 DEG C; Supplementing nitrogenous source is 7.0 to pH, and control air flow is 2 ~ 10L/min; Fermentation period is 32h, obtains L-glutamic acid;
The step of the preparation method of above-mentioned vitamin H defective type glutamate producing bacterium seed liquor is as follows:
(1) the vitamin H defective type glutamate producing bacterium on a ring slant medium is accessed in the seed culture medium of own sterilizing, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the bacterial strain activation solution that OD value is 0.6 ~ 0.8;
(2) by bacterial strain activation solution be by volume 7% ~ 15% ratio access seed culture medium in, 30 ~ 35 DEG C of shaking culture 6 ~ 10h, obtain the vitamin H defective type glutamate producing bacterium seed liquor that OD value is 0.7 ~ 1.0.
Measure the content of glutamic acid in fermented liquid after fermentation ends, and calculate acid production rate and transformation efficiency, and carry out cost keeping.
Detected result of the present invention:
The contrast of embodiment experimental result is as shown in the table:
Table 1 embodiment experimental result
Table 2 quality product contrast information slip
| Sequence number | Index name | Industry standard | The inventive method |
| 1 | Content % (L-glutamic acid butt meter) | 95 | 98 |
| 2 | Specific rotation [a] D20 DEG C/HCl solution | 31.3 | 30.8 |
| 3 | Transmittance (%) | 30 | 38 |
| 4 | SO4 2-(％) | 0.35 | 0.2 |
Note: for embodiment 1
Table 3 L-glutamic acid production technique index contrast table
| Sequence number | Index name | Known technology | The inventive method |
| 1 | Leavening temperature (DEG C) | 32-38.5 | 32-40 |
| 2 | Fermentation period (h) | 28-32 | 24-28 |
| 3 | Produce acid (g/L) | 13.5 | 14 |
| 4 | Glucose acid invert ratio (%) | 65 | 67 |
As can be seen from above-mentioned experimental result: utilize culture medium prescription provided by the invention, acid production rate and the glucose acid invert ratio of L-glutamic acid are improved; Fermentation period also shortens 2 hours, and quality product also increases.
Claims (8)
1. for a fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid, it is characterized in that: its composition and the final concentration of each composition in this formula are:
W-Gum hydrolysis sugar 425 ~ 495g/L, corn steep liquor 3.5 ~ 5g/L, molasses 2.6 ~ 3.6g/L, MgSO47H
2o0.6 ~ 0.9g/L, MnSO
40.0025 ~ 0.005g/L, FeSO
40.0025 ~ 0.005g/L, V
b10.001 ~ 0.0025g/L, KCl2 ~ 3g/L, H
3pO
43 ~ 8g/L, dissolution solvent is water, makes the pH of fermention medium be 6.8 ~ 7.2 with alkali neutralising phosphoric acid.
2. according to the fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid described in claim 1, it is characterized in that: the mass concentration of described W-Gum hydrolysis sugar is 30-35%; Or the concentration of described corn steep liquor is 18-25 degree Beaume.
3. the fermention medium for vitamin H suboptimal dose fermentative production L-glutamic acid according to claim 1 and 2, is characterized in that: described alkali is sodium hydroxide, sodium carbonate, ammoniacal liquor or potassium hydroxide.
4. the application of the fermention medium as described in any one of claims 1 to 3 in fermentative production L-glutamic acid.
5. utilize the fermention medium as described in any one of claims 1 to 3 to produce the fermentation process of L-glutamic acid, it is characterized in that: step is as follows:
(1) prepare burden: according to proportioning, various raw material weighing is good, dissolve and be mixed with fermention medium completely;
(2) sterilizing: by the fermention medium sterilizing prepared, temperature 108 ~ 110 DEG C, holds time 8 ~ 10 minutes, the substratum after sterilizing be cooled in fermentation 33 DEG C etc. to be seeded;
(3) by OD value be the vitamin H defective type glutamate producing bacterium seed culture fluid of 0.3 ~ 0.8 in seed culture fluid: the volume ratio of fermention medium is in 10 ~ 15% ratios access fermention mediums; Leavening temperature controls at 32 ~ 40 DEG C; Supplementing nitrogenous source is 6.8 ~ 7.2 to pH, and control air flow is 2 ~ 10L/min; Fermentation period is 24-28h, obtains L-glutamic acid.
6. the fermentation process utilizing fermention medium to produce L-glutamic acid according to claim 5, it is characterized in that: described step (3) in fermenting process in, when remaining sugar concentration in fermention medium is lower than 5-6%, the mode being added W-Gum hydrolysis sugar by stream makes remaining sugar concentration in fermention medium maintain between 0.5-5%, and the mass concentration that stream adds W-Gum hydrolysis sugar is 50-70%.
7. the fermentation process utilizing fermention medium to produce L-glutamic acid according to claim 5, is characterized in that: described vitamin H defective type glutamate producing bacterium is Tianjin tyrothricin (Brevibacterium tianjinese) S9114 or CM415.
8. the fermention medium that utilizes according to claim 5 or 6 produces the fermentation process of L-glutamic acid, it is characterized in that: the preparation process of described temperature vitamin H defective type glutamate producing bacterium seed culture fluid is as follows:
(1) actication of culture: get slant preservation bacterial classification 1 ~ 2 ring and be inoculated in and be equipped with in the slant tube of seed culture medium, 31 ~ 33 DEG C of constant temperature quiescent culture 24h;
(2) seed culture: access activated spawn by the triangular flask that 100 ~ 200mL seed culture medium is housed of own sterilizing, 31 ~ 33 DEG C of shaking culture 12 ~ 18h, obtain the bacterial strain activation solution that OD value is 0.3 ~ 0.8, are placed in 4 DEG C of refrigerator cold-storages for subsequent use.
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Cited By (5)
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| CN106701855A (en) * | 2017-03-01 | 2017-05-24 | 中粮生化能源（龙江）有限公司 | Method for fermenting temperature-sensitive strains by phosphoric acid to produce glutamic acid |
| CN106906255A (en) * | 2017-03-01 | 2017-06-30 | 中粮生化能源（龙江）有限公司 | A kind of method that phosphoric acid is used for biotin suboptimal dose fermenting and producing glutamic acid |
| CN110353247A (en) * | 2019-08-19 | 2019-10-22 | 湖北中轻食品有限公司 | A kind of production technology of bran acid fermentation nutrition fortifier |
| CN112708645A (en) * | 2020-11-04 | 2021-04-27 | 呼伦贝尔东北阜丰生物科技有限公司 | Method for efficiently producing monosodium glutamate |
| CN116113333A (en) * | 2020-07-17 | 2023-05-12 | 国际香料和香精公司 | Taste enhancing composition |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701855A (en) * | 2017-03-01 | 2017-05-24 | 中粮生化能源（龙江）有限公司 | Method for fermenting temperature-sensitive strains by phosphoric acid to produce glutamic acid |
| CN106906255A (en) * | 2017-03-01 | 2017-06-30 | 中粮生化能源（龙江）有限公司 | A kind of method that phosphoric acid is used for biotin suboptimal dose fermenting and producing glutamic acid |
| CN110353247A (en) * | 2019-08-19 | 2019-10-22 | 湖北中轻食品有限公司 | A kind of production technology of bran acid fermentation nutrition fortifier |
| CN116113333A (en) * | 2020-07-17 | 2023-05-12 | 国际香料和香精公司 | Taste enhancing composition |
| CN112708645A (en) * | 2020-11-04 | 2021-04-27 | 呼伦贝尔东北阜丰生物科技有限公司 | Method for efficiently producing monosodium glutamate |
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