CN113151380A - Culture process for improving titer of nisin - Google Patents

Culture process for improving titer of nisin Download PDF

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CN113151380A
CN113151380A CN202110307349.XA CN202110307349A CN113151380A CN 113151380 A CN113151380 A CN 113151380A CN 202110307349 A CN202110307349 A CN 202110307349A CN 113151380 A CN113151380 A CN 113151380A
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李珊
林青
王绘砖
陈钊
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HEBEI SHENGXUE DACHENG PHARMACEUTICAL CO Ltd
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Abstract

A culture process for improving the titer of nisin comprises the following steps: adding CaCl with a certain concentration into a fermentation culture medium2From Ca (OH)2Adjusting pH to 7.0-7.2, inoculating seed liquid after disinfection, controlling pH of fermentation liquid to 6.0-6.3 by feeding liquid alkali in the fermentation process, feeding glucose and corn steep liquor at variable speed after synthesis of nisin at initial logarithmic growth stage is started, and finishing fermentation for 20-30 h. By adding small amounts of CaCl2While the inhibition of lactic acid is removed, the proper feed supplement proportion for promoting the growth of the bacteria and producing the elements is added according to the variable speed flow of the growth of the bacteria and the synthesis characteristics of the products, the technical level is obviously improved, and the biological potency of the nisin is measured to be more than 18000 IU/mL. Simultaneously, the introduction amount of Ca ions is reduced, the extraction and the filtration are convenient, the equipment investment is reduced, and the power cost is reducedThe production efficiency is improved, and the method is suitable for industrial production.

Description

Culture process for improving titer of nisin
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a culture process for improving the titer of nisin.
Background
Nisin (Nisin) is a polypeptide substance produced by fermentation of streptococcus lactis (Lactococcus lactis) and contains 34 amino acids. It can effectively inhibit the propagation of gram-positive bacteria and spores thereof, can be quickly digested and absorbed by human body after being eaten, is a natural food preservative with high efficiency, no toxicity, safety and no side effect, and is widely applied to various foods such as dairy products, meat products, canned products, fruit juice, alcoholic beverages and the like.
There are 2 problems with streptococcus lactis in the fermentative production of nisin: firstly, a large amount of lactic acid is produced as a byproduct, which causes the reduction of pH, inhibits the growth of thalli and also inhibits the synthesis of nisin. Secondly, because the fermentation period is short, the nutrient utilization speed is high, the titer fluctuation amplitude is reduced or basically does not increase in the later period of fermentation, nisin is used as a primary metabolite, the fermentation formula is relatively simple, carbon sources and nitrogen sources are main influencing factors, the commonly used carbon sources are glucose and sucrose, and the nitrogen sources are yeast extract powder, peptone, corn steep liquor and the like, and are all used for thallus growth and product synthesis. Glucose can provide energy and carbon skeleton, and yeast extract powder, corn steep liquor, etc. can provide amino acids, vitamins, growth factors, etc.
The mixed fermentation of yeast and streptococcus lactis is reported to remove lactic acid inhibition, but the streptococcus lactis has strong bacteriostatic ability, the requirement on mixed fermentation nutrient components is high, the cost of raw materials is increased, and the method is not suitable for industrial production; addition of CaCO to the culture medium has also been reported3The pH is regulated, but the subsequent extraction, separation and purification are not facilitated.
Chinese patent CN104789624B (application No. CN201510190841.8) discloses a method for co-producing calcium lactate by fermenting nisin, wherein the pH of fermentation liquid is controlled by feeding calcium hydroxide suspension with a certain concentration, and the titer reaches 12300-13030IU/mL after the fermentation is finished.
By adding Ca (OH) in a flowing manner2The method controls the pH, can remove partial lactic acid inhibition, but greatly increases the turbidity of the fermentation liquor, can not completely intercept titer in the extraction and filtration process, and causes the reduction of the extraction yield; at the same time, due to Ca (OH)2The solution is suspension, and is used during applicationAnd a stirring device is required to be added for continuous stirring, the uniform mixing suspension state is kept, the equipment investment and the power cost are increased, and the production efficiency is reduced.
In conclusion, how to remove the inhibition of lactic acid on fermentation has a great influence on the improvement of titer, and how to optimize the thallus to maximize growth and products, improve the activity of the thallus in the later period, and promote the titer increase is also the key.
Disclosure of Invention
The invention aims to solve the problems of the prior art and provides a fermentation production process which can improve the titer, remove the inhibition of lactic acid, provide a nutrition ratio suitable for the growth of thalli and production elements, improve the property of fermentation liquor, facilitate the extraction and separation operation, reduce the equipment investment, reduce the power cost and improve the production efficiency.
The technical scheme adopted by the invention is as follows: a culture process for improving the titer of nisin comprises the following steps: firstly, CaCl with a certain concentration is added into a fermentation medium2Then, Ca (OH)2Adjusting the pH value to 7.0-7.2; then sterilizing the fermentation culture medium, and inoculating a seed solution into the fermentation culture medium after the sterilization for fermentation culture; and controlling the pH value of the fermentation liquor to be 6.0-6.3 by feeding liquid alkali in the fermentation culture process, and simultaneously feeding glucose and corn steep liquor at a variable speed after the synthesis of nisin at the initial logarithmic growth stage is started, wherein the fermentation culture period is finished for 20-30h, so that the nisin fermentation liquor is obtained.
Further, the preparation method of the seed liquid comprises the following steps: inoculating Streptococcus lactis liquid into seed tank containing seed culture medium, performing two-stage amplification culture, diluting the liquid by 5 times of OD600nm, and transferring seed when the absorbance reaches above 0.6 and pH is 6.0-6.5.
Further, the inoculation amount of the seed solution inoculated into the fermentation medium is as follows: the seed liquid accounts for 5-8% of the fermentation culture medium, and the fermentation culture period is 22-26 h.
Further, the seed culture medium comprises the following components in percentage by weight: 0.5-1.0% of glucose, 0.5-1.0% of bovine bone peptone, 0.5-1.0% of yeast extract powder and KH2PO40.3-0.7％，NaCl0.1-0.3％，MgSO4·7H20.01 to 0.03 percent of O, and the balance being filled with deionized water.
Further, the fermentation medium comprises the following components in percentage by weight: 1.0-1.5% of glucose, 0.5-1.0% of yeast extract powder and KH2PO40.1-0.3％，CaCl2Or CaSO40.5-1.0％，MgSO4·7H20.01 to 0.03 percent of O, 0.2 to 0.8 percent of corn steep liquor dry powder, 800.1 to 0.2 percent of Tween-and the balance being filled with deionized water.
Further, the sterilization conditions of the fermentation medium are as follows: the temperature is 95-100 deg.C, and the time is 10 min.
Further, the Ca (OH)2The concentration of (A) is 20-25%.
Further, the conditions of the fermentation culture are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr and 0Mpa in the second 4 hr at 30 + -1 deg.C.
Further, the glucose feeding rate is: 7.0-10.0mL/L/h in 4-10h, 3.5-5.0mL/L/h in 10-16h, and 1.25-2.5mL/L/h after 16-fermentation is finished; the corn slurry flow acceleration rate is as follows: 0.25-0.4mL/L/h for 4-12h, and 0.08-0.24mL/L/h for 12-16 h.
Further, the concentration of the glucose is 40-60%; the concentration of the corn steep liquor is 20-30%, wherein the total amino acid content of the corn steep liquor is more than or equal to 50%.
The beneficial effects obtained by the invention are as follows: the invention determines a culture process for improving the titer of nisin, and CaCl with a certain concentration is added into a fermentation medium2From Ca (OH)2Adjusting pH to 7.0-7.2, inoculating seed liquid after disinfection, controlling pH of fermentation liquid to 6.0-6.3 by feeding liquid alkali in the fermentation process, feeding glucose and corn steep liquor at variable speed after synthesis of nisin at initial logarithmic growth stage is started, and finishing fermentation for 20-30 h. By adding small amounts of CaCl2While the inhibition of lactic acid is removed, the proper feed supplement proportion for promoting the growth of the bacteria and producing the elements is added according to the variable speed flow of the growth of the bacteria and the synthesis characteristics of the products, the technical level is obviously improved, and the biological potency of the nisin is measured to be more than 18000 IU/mL. Simultaneously, the introduction amount of Ca ions is reduced, the extraction and the filtration are convenient, the equipment investment is reduced, and the reduction of the content of Ca ionsThe power cost is reduced, the production efficiency is improved, and the method is suitable for industrial production.
Drawings
FIG. 1 is a graph showing the comparison of the titer of example 1 of the present invention and comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.
Example 1:
the seed culture medium is prepared according to the following proportion (percent) by weight: 0.5 percent of glucose, 0.5 percent of bovine bone peptone, 0.5 percent of yeast extract powder and KH2PO40.3，NaCl 0.1，MgSO4·7H2O0.01, the balance being filled with deionized water, and the pH value being 7.0 after the deionized water is removed.
And (3) sterilization: sterilizing at the temperature of 121-.
Inoculating lactic acid streptococcus strain liquid to carry out two-stage amplification culture. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. And (3) detecting the feed liquid diluted by 5 times of OD600nm, wherein the light absorption value reaches 0.603, the pH value is 6.09, and transferring the seeds.
The fermentation medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 0.5 percent of yeast extract powder and KH2PO40.1％，CaCl20.5％，MgSO4·7H20.01 percent of O, 0.2 percent of corn steep liquor dry powder, 800.1 percent of Tween-and the balance of deionized water.
And (3) sterilization: boiling at 95-100 deg.C for 10min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air. After disinfection, 20% Ca (OH) is used2The pH was adjusted to 7.0.
Inoculating the cultured seed solution into fermentation culture medium, culturing under non-aerated condition under 0.04 + -0.005 Mpa for the first 4 hr, and culturing under 0Mpa for 4 hr at 30 + -1 deg.C under 1.5 hr, and regulating pH to 6.0 by regulating liquid alkali.
And (3) material supplementing process control: after fermentation for 4 hours, 40% of glucose and 20% of corn steep liquor are added, wherein the glucose adding rate is as follows: 7.0mL/L/h after 4-10h, 3.5mL/L/h after 10-16h, and 1.25mL/L/h after 16-fermentation is finished. The corn steep liquor replenishing rate is as follows: 0.25-0.4mL/L/h for 4-12h, and 0.08-0.24mL/L/h for 12-16 h.
After the fermentation culture is finished, the fermentation period is 22h, and the biological titer of the nisin is 18172U/mL.
Example 2
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.75 percent of glucose, 0.75 percent of bovine bone peptone, 0.75 percent of yeast extract powder and KH2PO40.5，NaCl 0.2，MgSO4·7H2O0.02, and the balance of deionized water, and the pH value is 6.99 after the deionized water is removed.
And (3) sterilization: sterilizing at the temperature of 121-.
Inoculating lactic acid streptococcus strain liquid to carry out two-stage amplification culture. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. And (3) detecting the feed liquid diluted by 5 times of OD600nm, wherein the light absorption value reaches 0.612, the pH value is 6.14, and transferring the seeds.
The fermentation medium is prepared according to the following proportion (percent) by weight: 1.25% glucose, 0.75% yeast extract powder, KH2PO40.3，CaCl20.75，MgSO4·7H2O0.03, corn steep liquor dry powder 0.5, Tween-800.15, and deionized water in balance, and the pH value is 6.86 after the deionized water is removed.
And (3) sterilization: boiling at 95-100 deg.C for 10min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air. After the disinfection is finished, 22.5 percent of Ca (OH) is used2The pH was adjusted to 7.1.
Inoculating the cultured seed solution into fermentation culture medium, culturing under non-aerated condition under 0.04 + -0.005 Mpa for the first 4 hr, and culturing under 0Mpa for 4 hr at 30 + -1 deg.C under 1.5 hr, and regulating pH to 6.15 with alkali.
And (3) material supplementing process control: after fermentation for 4 hours, 50% of glucose and 25% of corn steep liquor are supplemented, wherein the glucose supplementing rate is as follows: 8.5mL/L/h after 4-10h, 4.25mL/L/h after 10-16h, and 1.9mL/L/h after 16-fermentation is finished. The corn steep liquor replenishing rate is as follows: 0.325mL/L/h for 4-12h, and 0.16mL/L/h for 12-16 h.
And (5) after the fermentation culture is finished, the fermentation period is 22h, and the biological value of nisin is detected to be 18039U/mL.
Example 3
The seed culture medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 1.0 percent of bovine bone peptone, 1.0 percent of yeast extract powder and KH2PO40.7，NaCl 0.3，MgSO4·7H2O0.03, the balance being filled with deionized water, and the pH value being 6.96 after the deionized water is removed.
And (3) sterilization: sterilizing at the temperature of 121-.
Inoculating lactic acid streptococcus strain liquid to carry out two-stage amplification culture. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. And (3) detecting the feed liquid diluted by 5 times of OD600nm, enabling the light absorption value to reach 0.609, enabling the pH value to be 6.18, and transplanting the seeds.
The fermentation medium is prepared according to the following proportion (percent) by weight: glucose 1.5, yeast extract powder 1.0, KH2PO40.3，CaCl21.0，MgSO4·7H2O0.03, corn steep liquor dry powder 0.8, Tween-800.2, and deionized water in balance, and the pH value is 6.92 after the deionized water is removed.
And (3) sterilization: boiling at 95-100 deg.C for 10min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air. After the disinfection is finished, 25% Ca (OH) is used2The pH was adjusted to 7.2.
Inoculating the cultured seed solution into fermentation culture medium, culturing under non-aerated condition under 0.04 + -0.005 Mpa for the first 4 hr, and culturing under 0Mpa for 4 hr at 30 + -1 deg.C under 1.5 hr, and regulating pH to 6.3 with alkali.
And (3) material supplementing process control: after fermentation for 4 hours, 50% of glucose and 25% of corn steep liquor are supplemented, wherein the glucose supplementing rate is as follows: 10mL/L/h for 4-10h, 5mL/L/h for 10-16h, and 2.5mL/L/h for 16-end of fermentation. The corn steep liquor replenishing rate is as follows: 0.4mL/L/h for 4-12h, and 0.24mL/L/h for 12-16 h.
After the fermentation culture is finished, the fermentation period is 22h, and the biological value of the nisin is detected to be 18281U/mL.
Comparative example 1
The seed culture medium is prepared according to the following proportion (percent) by weight:0.5 percent of glucose, 0.5 percent of bovine bone peptone, 0.5 percent of yeast extract powder and KH2PO40.3，NaCl 0.2，MgSO4·7H2O0.02, and the balance of deionized water, and the pH value is 7.0 after the deionized water is removed.
And (3) sterilization: sterilizing at the temperature of 121-.
Inoculating lactic acid streptococcus strain liquid to carry out two-stage amplification culture. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. The solution is diluted by 5 times of OD600nm, the absorbance reaches 0.617, the pH value is 6.21, and the seeds are transferred.
The fermentation medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 0.75 percent of yeast extract powder and KH2PO40.2，NaCl 0.2，MgSO4·7H2O0.02, corn steep liquor dry powder 0.5, Tween-800.1, and deionized water in balance, and the pH value is 6.89 after the deionized water is removed.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the cultured seed solution into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.2 by adjusting liquid alkali.
And (3) material supplementing process control:
sugar supplement: when the fermentation culture is carried out for 4 hours, the glucose is reduced to 0.55 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3 to 0.5 percent.
And (3) material supplementing process control: and (3) beginning to supplement 50% of glucose after fermenting for 4 hours, wherein the glucose supplement rate is as follows: 4-10h5mL/L/h, and the intermediate control sugar concentration is 0.3-0.5%.
And (5) finishing the fermentation culture, wherein the fermentation period is 22h, and the biological value of the nisin is detected to be 13318U/mL.
Comparative example 2
The seed culture medium is prepared according to the following proportion (percent) by weight: 0.75 percent of glucose, 0.75 percent of bovine bone peptone, 0.75 percent of yeast extract powder and KH2PO40.5，NaCl 0.2，MgSO4·7H2O0.02, and the balance of deionized water, and the pH value is 6.95 after the deionized water is removed.
And (3) sterilization: sterilizing at the temperature of 121-.
Inoculating lactic acid streptococcus strain liquid to carry out two-stage amplification culture. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. The material liquid is diluted by 5 times and detected under OD600nm, the light absorption value reaches 0.615, the pH value is 6.21, and the seeds are transferred.
Fermentation culture: the volume of the culture medium is 40T.
The fermentation medium is prepared according to the following proportion (percent) by weight: 1.25% glucose, 0.75% yeast extract powder, KH2PO40.2，NaCl 0.2，MgSO4·7H2O0.02, corn steep liquor dry powder 0.5, Tween-800.15, and deionized water for supplementing balance, and having pH of 6.9.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the cultured seed solution into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr, and culturing under 0Mpa in the second 4 hr at 30 + -1 deg.C while controlling pH value to 6.2 by adjusting liquid alkali.
And (3) material supplementing process control:
sugar supplement: when the fermentation culture is carried out for 4.5h, the glucose is reduced to 0.51 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3-0.5 percent.
1.5% corn steep liquor: the fermentation was incubated for 8h and fed-through at a rate of 0.3% volume of broth.
After the fermentation culture is finished, the fermentation period is 22h, and the biological value of the nisin is 13274U/mL.
Comparative example 3
The seed culture medium is prepared according to the following proportion (percent) by weight: 1.0 percent of glucose, 1.0 percent of bovine bone peptone, 1.0 percent of yeast extract powder and KH2PO40.7，NaCl 0.3，MgSO4·7H2O0.03, and the balance being filled with deionized water, and the pH value being 7.01 after the deionized water is removed.
And (3) sterilization: sterilizing at the temperature of 121-.
Inoculating lactic acid streptococcus strain liquid to carry out two-stage amplification culture. The aeration is closed, the pressure of the tank is maintained at 0.04 +/-0.05 Mpa, and the culture temperature is maintained at 30 +/-1 ℃. And (3) detecting the feed liquid diluted by 5 times of OD600nm, wherein the light absorption value reaches 0.614, the pH value is 6.13, and transferring the seeds.
The fermentation medium is prepared according to the following proportion (percent) by weight: glucose 1.5, yeast extract powder 1.0, KH2PO40.3，NaCl 0.3，MgSO4·7H2O0.03, corn steep liquor dry powder 0.8, Tween-800.2, and deionized water in balance, and the pH value is 6.92 after the deionized water is removed.
And (3) sterilization: boiling at 100 deg.C for 30min, cooling to 30 + -1 deg.C, adding glucose, and maintaining the pressure with sterile air.
And (4) inoculating the cultured seed solution into a fermentation tank, culturing for 22-26h, and finishing fermentation. The fermentation culture conditions are as follows: culturing under non-aerated condition at 30 + -1 deg.C under 0.04 + -0.005 Mpa for the first 4 hr and 0Mpa for the second 4 hr, and adding Ca (OH) during the whole process2The pH value is controlled to be within 6.3.
And (3) material supplementing process control:
sugar supplement: when the fermentation culture is carried out for 4 hours, the glucose is reduced to 0.5 percent, the sugar is supplemented, and the intermediate sugar concentration is controlled to be 0.3 to 0.5 percent.
And (5) finishing the fermentation culture, wherein the fermentation period is 22h, and the biological value of the nisin is detected to be 14427U/mL.

Claims (10)

1. A culture process for improving the titer of nisin is characterized in that: the method comprises the following steps: firstly, CaCl with a certain concentration is added into a fermentation medium2Then, Ca (OH)2Adjusting the pH value to 7.0-7.2; then sterilizing the fermentation culture medium, and inoculating a seed solution into the fermentation culture medium after the sterilization for fermentation culture; controlling the pH of the fermentation liquid to 6.0-6.3 by feeding liquid alkali in the fermentation culture process, and feeding glucose and corn steep liquor at variable speed after the synthesis of nisin at the initial logarithmic growth stage is started, wherein the fermentation culture period is 20-30h, and the nisin is obtainedA bacteriocin fermentation broth.
2. The fermentation production process of high yield nisin according to claim 1, wherein: the preparation method of the seed liquid comprises the following steps: inoculating Streptococcus lactis liquid into seed tank containing seed culture medium, performing two-stage amplification culture, diluting the liquid by 5 times of OD600nm, and transferring seed when the absorbance reaches above 0.6 and pH is 6.0-6.5.
3. The fermentation production process of high yield nisin according to claim 1, wherein: the inoculation amount of the seed liquid inoculated into the fermentation medium is as follows: the seed liquid accounts for 5-8% of the fermentation culture medium, and the fermentation culture period is 22-26 h.
4. The fermentation production process of high yield nisin according to claim 2, wherein: the seed culture medium comprises the following components in percentage by weight: 0.5-1.0% of glucose, 0.5-1.0% of bovine bone peptone, 0.5-1.0% of yeast extract powder and KH2PO40.3-0.7％，NaCl 0.1-0.3％，MgSO4·7H20.01 to 0.03 percent of O, and the balance being filled with deionized water.
5. The fermentation production process of high yield nisin according to claim 1 or 3, wherein: the fermentation medium comprises the following components in percentage by weight: 1.0-1.5% of glucose, 0.5-1.0% of yeast extract powder and KH2PO40.1-0.3％，CaCl2Or CaSO40.5-1.0％，MgSO4·7H20.01 to 0.03 percent of O, 0.2 to 0.8 percent of corn steep liquor dry powder, 800.1 to 0.2 percent of Tween-and the balance being filled with deionized water.
6. The fermentation production process of high yield nisin according to claim 1, wherein: the sterilization conditions of the fermentation medium are as follows: the temperature is 95-100 deg.C, and the time is 10 min.
7. The fermentation production process of high yield nisin according to claim 1, wherein: the Ca (OH)2The concentration of (A) is 20-25%.
8. The fermentation production process of high yield nisin according to claim 3, wherein: the conditions of the fermentation culture are as follows: culturing under 0.04 + -0.005 Mpa in the first 4 hr and 0Mpa in the second 4 hr at 30 + -1 deg.C.
9. The fermentation production process of high yield nisin according to claim 1, wherein: the glucose feeding rate is as follows: 7.0-10.0mL/L/h in 4-10h, 3.5-5.0mL/L/h in 10-16h, and 1.25-2.5mL/L/h after 16-fermentation is finished; the corn slurry flow acceleration rate is as follows: 0.25-0.4mL/L/h for 4-12h, and 0.08-0.24mL/L/h for 12-16 h.
10. The fermentation production process of high yield nisin according to claim 9, wherein: the concentration of the glucose is 40-60%; the concentration of the corn steep liquor is 20-30%, wherein the total amino acid content of the corn steep liquor is more than or equal to 50%.
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Citations (7)

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