Fermentation medium and method for low-pH fermentation of nisin

Abstract

The present invention relates to a fermentation medium and method for low-pH fermentation of nisin, belonging to the field of microbial fermentation technology, the components of the fermentation medium are by weight percentage: sucrose 1.0%, glucose 1.0%, yeast extract 0.5%, potassium dihydrogen phosphate 0.5%, sodium chloride 0.2%, magnesium sulfate 0.03%, corn steep liquor dry powder 2.0%, Tween-80 0.1% and tetrahydropyrimidine, the fermentation medium is adjusted to pH 7.0 with 15% (m / m) sodium hydroxide solution before inoculation, and the specific method is: seed liquid preparation; shake flask fermentation culture or fermentation tank fermentation culture. The present invention adds tetrahydropyrimidine to the fermentation medium, can control the lower fermentation pH without reducing the fermentation titer, reduce the probability of bacterial infection, and can reduce the amount of sodium hydroxide and hydrochloric acid used in the preparation process of nisin, avoid waste of resources, and achieve the reduction of the production and preparation cost of nisin.

Description

Technical Field

[0001] The invention relates to a fermentation medium and a method for fermenting nisin at a low pH, and belongs to the technical field of microbial fermentation. Background Art

[0002] Nisin is a polypeptide antibiotic secreted by lactococcus, which can inhibit most Gram-positive bacteria and has a strong inhibitory effect on Bacillus spores. After consumption, it is quickly hydrolyzed into amino acids in the human body and is harmless to the human body. It is widely used as a food preservative.

[0003] The stability and solubility of nisin are closely related to pH value. Nisin is relatively active under acidic conditions and has a small amount of adsorption. The increase in environmental pH will lead to the degradation and solubility of nisin. The thermal stability is good when the pH value is below 4, and the thermal stability decreases significantly when the pH value is greater than 5. The solubility is 12% at pH 2.5 and drops to 4% at pH 5.0. It is insoluble in water under neutral and alkaline conditions. However, the nisin-producing nisin strain requires a neutral fermentation process to grow well. In the fermentation production process of nisin, in order to achieve a high cell density, a large amount of alkali solution needs to be added to maintain the pH above 6.0. After the fermentation is completed, a large amount of hydrochloric acid needs to be used to adjust the pH to 2.0-3.0 for extraction. There is a certain waste of resources, which increases the production and preparation cost of nisin. Summary of the invention

[0004] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a fermentation medium and method for fermenting nisin at a low pH, which can reduce the amount of alkali solution used in the fermentation process and the amount of hydrochloric acid used in the extraction process, the low fermentation pH has a low probability of bacterial contamination, and can improve the fermentation titer.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] A fermentation medium for fermenting nisin at a low pH. The components of the fermentation medium are as follows by weight: 1.0% sucrose, 1.0% glucose, 0.5% yeast extract, 0.5% potassium dihydrogen phosphate, 0.2% sodium chloride, 0.03% magnesium sulfate, 2.0% corn steep liquor powder, 0.1% Tween-80 and ectoine. The pH of the fermentation medium is adjusted to 7.0 with a 15% (m / m) sodium hydroxide solution before inoculation.

[0007] Furthermore, the concentration of added ectoine is 50ppm to 200ppm.

[0008] The method for low pH fermentation of nisin based on fermentation medium comprises the following steps:

[0009] Step 1: Seed solution preparation:

[0010] Inoculate 1 ml of lactic acid Streptococcus seed solution stored at -20°C into a shake flask containing 200 ml of seed culture medium, and place it in a 30°C incubator for 8 to 10 hours, with an OD of 1.8 to 2.0;

[0011] Step 2: Shake flask fermentation or fermentation tank fermentation:

[0012] If it is shake flask fermentation culture: transfer the cultured seed liquid to a shake flask filled with 200 ml fermentation medium at a transfer rate of 8%, place it in a 30°C incubator and culture it for 24 hours to obtain the nisin fermentation liquid, and measure the final fermentation pH and fermentation titer;

[0013] If it is fermentation culture in a fermenter: transfer the cultured seed liquid to a fermenter containing 5L fermentation medium at a transfer rate of 8%, culture temperature at 30°C, rotation speed at 50rpm, feed 50% glucose, control sugar concentration at 3-8g / L, control pH at 6.8 by feeding 15% (m / m) sodium hydroxide solution for 0-12h, control pH at 4.5-6.2 by feeding 15% (m / m) sodium hydroxide solution for 12-22h, obtain nisin fermentation broth, measure the fermentation titer after 22h, and calculate the volume of sodium hydroxide solution consumed by 1L fermentation broth and the volume of hydrochloric acid solution used to adjust pH 2.5 of 1L fermentation broth with 30% (m / m) hydrochloric acid solution.

[0014] Furthermore, the components of the seed culture medium are as follows by weight: 1.5% peptone, 1.5% yeast extract, 2.0% potassium dihydrogen phosphate, 2.0% sucrose, 0.15% sodium chloride and 0.015% magnesium sulfate. The pH of the seed culture medium is adjusted to 7.0 with 15% (m / m) sodium hydroxide solution before inoculation.

[0015] Furthermore, during fermentation in a fermenter, the lactic acid streptococcus is fermented for 12 hours and then the fermentation pH is controlled to be relatively low, wherein the range of the relatively low fermentation pH is 4.5 to 5.0.

[0016] The beneficial effects of the present invention are as follows: by adding tetrahydropyrimidine to the fermentation medium, a lower fermentation pH can be controlled without reducing the fermentation titer, thereby reducing the probability of bacterial contamination, and at the same time, the amount of sodium hydroxide and hydrochloric acid used in the preparation process of nisin can be reduced, thereby avoiding waste of resources and reducing the production and preparation cost of nisin. DETAILED DESCRIPTION

[0017] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. At the same time, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials, and instruments are all prior art and can be obtained from commercial channels unless otherwise specified.

[0018] A fermentation medium for fermenting nisin at a low pH. The components of the fermentation medium are as follows by weight: 1.0% sucrose, 1.0% glucose, 0.5% yeast extract, 0.5% potassium dihydrogen phosphate, 0.2% sodium chloride, 0.03% magnesium sulfate, 2.0% corn steep liquor powder, 0.1% Tween-80 and ectoine. The pH of the fermentation medium is adjusted to 7.0 with a 15% (m / m) sodium hydroxide solution before inoculation; wherein the added concentration of ectoine is 50ppm-200ppm.

[0019] The components of the seed culture medium are as follows by weight: 1.5% peptone, 1.5% yeast extract, 2.0% potassium dihydrogen phosphate, 2.0% sucrose, 0.15% sodium chloride and 0.015% magnesium sulfate. The seed culture medium is adjusted to pH 7.0 with 15% (m / m) sodium hydroxide solution before inoculation.

[0020] The method for identifying the potency of nisin refers to GB 1886.231-2016 "National Food Safety Standard Food Additive Nisin".

[0021] Example 1: A method for low pH fermentation of nisin based on a fermentation medium, comprising the following steps:

[0022] Step 1. Seed solution preparation: inoculate 1 ml of lactic acid streptococcus seed solution stored at -20°C into a shake flask containing 200 ml of seed culture medium, and place it in a 30°C incubator for static culture for 9 h, OD 2.0.

[0023] Step 2, shake flask fermentation: transfer the cultured seed liquid into a shake flask containing 200 ml fermentation medium (with 50 ppm of tetrahydropyrimidine) at a transfer rate of 8%, place it in a 30°C incubator for 24 hours, and measure the final fermentation pH and fermentation titer.

[0024] Example 2: The method is the same as that of Example 1, except that the concentration of ectoine added to the fermentation medium is 100 ppm.

[0025] Comparative Example 1:

[0026] The method is the same as that of Example 1, except that ectoine is not added to the fermentation medium.

[0027] The fermentation titer and pH of the fermentation broth of Example 1, Example 2 and Comparative Example 1 were measured according to standard GB 1886.231-2016. The results are shown in Table 1:

[0028] Table 1. Effects of different ectoine additions on shake flask fermentation of nisin

[0029]

[0030] As can be seen from Table 1, the final fermentation pH of Example 1 was 4.32, and the fermentation titer was 7660 IU / ml, which was 0.28 lower than the final fermentation pH of Comparative Example 1 of 4.6, and the fermentation titer was 12.75% higher than the fermentation titer of 6794 IU / ml of Comparative Example 1; the final fermentation pH of Example 2 was 4.16, and the fermentation titer was 8434 IU / ml, which was 0.44 lower than the final fermentation pH of Comparative Example 1 of 4.6, and the fermentation titer was 24.14% higher than the fermentation titer of 6794 IU / ml of Comparative Example 1. It can be seen from the above results that the low pH fermentation nisin culture medium of the present invention can improve the tolerance of nisin to low pH and improve the fermentation titer of nisin.

[0031] Example 3: A method for low pH fermentation of nisin based on a fermentation medium, comprising the following steps:

[0032] Step 1. Seed solution preparation: inoculate 1 ml of lactic acid streptococcus seed solution stored at -20°C into a shake flask containing 200 ml of seed culture medium, and place it in a 30°C incubator for static culture for 8.5 h, OD 2.0.

[0033] Step 2, fermentation culture in a fermenter: transfer the cultured seed liquid to a fermenter containing 5 L of fermentation medium (the added concentration of tetrahydropyrimidine is 100 ppm) at a transfer rate of 8%, the culture temperature is 30°C, the rotation speed is 50 rpm, 50% glucose is added to control the sugar concentration to 5 g / L, 15% (m / m) sodium hydroxide solution is added to control the pH to 6.8 for 0 to 12 h, and sodium hydroxide solution is added to control the pH to 6.2 for 12 to 22 h. The fermentation titer of 22 h is measured, and the volume of sodium hydroxide solution consumed by 1 L of fermentation liquid and the volume of hydrochloric acid solution used to adjust the pH of 1 L of fermentation liquid to 2.5 with 30% (m / m) hydrochloric acid solution are calculated.

[0034] Example 4: The method is the same as that of Example 3, except that the pH is controlled to 5.0 by adding 15% (m / m) sodium hydroxide for 12 to 22 hours.

[0035] Example 5: The method is the same as that of Example 3, except that the pH is controlled at 4.5 by adding 15% (m / m) sodium hydroxide for 12 to 22 hours.

[0036] Comparative Example 2:

[0037] The method is the same as that of Example 3, except that ectoine is not added to the fermentation medium.

[0038] Comparative Example 3:

[0039] The method was the same as that of Example 3, except that ectoine was not added to the fermentation medium, and the pH was controlled at 5.0 by feeding 15% (m / m) sodium hydroxide for 12 to 22 h.

[0040] Comparative Example 4:

[0041] The method is the same as that of Example 3, except that ectoine is not added to the fermentation medium, and the pH is controlled at 4.5 by feeding 15% (m / m) sodium hydroxide for 12 to 22 hours.

[0042] The fermentation broth of Examples (3-5) and Comparative Examples (2-4) fermented for 22 hours was used to determine the fermentation titer according to standard GB 1886.231-2016, and the amount of sodium hydroxide solution (ml / L), the amount of hydrochloric acid solution (ml / L), the titer increase rate (%), the sodium hydroxide reduction rate (%) and the hydrochloric acid reduction rate (%) were calculated. The specific calculation method is:

[0043] ① The amount of sodium hydroxide solution used (ml / L) = the total volume of sodium hydroxide consumed in fermentation / the volume of the tank.

[0044] ②Amount of hydrochloric acid solution used (ml / L) = volume of hydrochloric acid solution consumed to adjust the pH of 1L fermentation liquid to 2.5 / 1L.

[0045] ③Titer improvement rate (%) = (treatment fermentation titer / control fermentation titer-1) × 100%.

[0046] ④Sodium hydroxide reduction rate (%) = (1-amount of treated sodium hydroxide solution / amount of control sodium hydroxide solution) × 100%.

[0047] ⑤ Hydrochloric acid reduction rate (%) = (1-amount of treated hydrochloric acid solution / amount of control hydrochloric acid solution) × 100%.

[0048] The results are shown in Table 2, Table 3 and Table 4:

[0049] Table 2. Effects of different pH and fermentation medium on nisin fermentation

[0050]

[0051] As can be seen from Table 2, when the pH of the fermentation control was reduced from 6.2 to 5.0 during 12h to 22h, the fermentation titer of Example 4 decreased by 2.46% compared with Example 3, which was significantly less than the 14.92% decrease in the fermentation titer of Comparative Example 3 compared with Comparative Example 2; when the pH of the fermentation control was reduced from 6.2 to 4.5 during 12h to 22h, the fermentation titer of Example 5 decreased by 3.83% compared with Example 3, which was significantly less than the 27.50% decrease in the fermentation titer of Comparative Example 4 compared with Comparative Example 2. It can be seen from the above results that with the decrease of the fermentation control pH, the fermentation titer decreased to varying degrees, and the lower the fermentation pH control, the more significant the decrease in the fermentation titer; the decrease in the titer of nisin in the low pH fermentation medium of the present invention with the decrease in the pH of the fermentation liquid was significantly less than that in the comparative fermentation medium.

[0052] Table 3. Effect of low pH nisin fermentation medium on fermentation under the same fermentation pH conditions

[0053]

[0054] As can be seen from Table 3, when the pH of the fermentation was controlled at 6.2 for 12h to 22h, the fermentation titer of Example 3 was 5.36% higher than that of Comparative Example 2, when the pH of the fermentation was controlled at 5.0, the fermentation titer of Example 4 was 20.79% higher than that of Comparative Example 3, and when the pH of the fermentation was controlled at 4.5, the fermentation titer of Example 5 was 39.76% higher than that of Comparative Example 4. From the above results, it can be seen that when the pH of the fermentation was controlled at the same level for 12h to 22h, the fermentation titer of the low pH fermentation medium of the present invention was improved to varying degrees compared with the comparative example, and the lower the pH, the more significant the improvement in fermentation titer, and the preferred fermentation pH range was 4.5 to 5.0.

[0055] Table 4. Effect of low pH fermentation medium on nisin fermentation

[0056]

[0057] As can be seen from Table 4, in Example 4, the pH of the fermentation control during 12h to 22h is 1.2 lower than that of Comparative Example 2, the fermentation titer is 2.76% higher than that of Comparative Example 2, the amount of sodium hydroxide solution used in the fermentation is reduced by 15.53%, and the amount of hydrochloric acid solution used to adjust the pH of the fermented liquid to 2.5 is reduced by 23.91%; in Example 5, the pH of the fermentation control during 12h to 22h is 1.7 lower than that of Comparative Example 2, the fermentation titer is 1.33% higher than that of Comparative Example 2, the amount of sodium hydroxide solution used in the fermentation is reduced by 26.21%, and the amount of hydrochloric acid solution used to adjust the pH of the fermented liquid to 2.5 is reduced by 36.96%. It can be seen from the above results that the fermentation medium and method for low pH fermentation of nisin of the present invention can control a lower fermentation pH without reducing the fermentation titer, reduce the amount of sodium hydroxide and hydrochloric acid, and the probability of bacterial infection is low when the fermentation pH is low.

[0058] In summary, in the method of low-pH fermentation of nisin, adding tetrahydropyrimidine to the fermentation medium can control a lower fermentation pH without reducing the fermentation titer, reduce the probability of contamination, and at the same time reduce the amount of sodium hydroxide and hydrochloric acid used in the preparation process of nisin, avoid waste of resources, and reduce the production and preparation cost of nisin.

Claims

1. A fermentation medium for low pH fermentation of nisin, characterized in that: The components of the fermentation medium are as follows by weight percentage: sucrose 1.0%, glucose 1.0%, yeast extract 0.5%, potassium dihydrogen phosphate 0.5%, sodium chloride 0.2%, magnesium sulfate 0.03%, corn steep liquor dry powder 2.0%, Tween-80 0.1% and ectoine; the pH of the fermentation medium is adjusted to 7.0 with 15% (m / m) sodium hydroxide solution before inoculation; the added concentration of the ectoine is 50ppm-200ppm.

2. A method for fermenting nisin using the low pH of the fermentation medium according to claim 1, characterized in that: The following steps are involved: Step 1: Seed solution preparation: Inoculate 1 ml of lactic acid Streptococcus seed solution stored at -20°C into a shake flask containing 200 ml of seed culture medium, and place it in a 30°C incubator for 8 to 10 hours, with an OD of 1.8 to 2.0; Step 2: Shake flask fermentation or fermentation tank fermentation: If it is shake flask fermentation culture: transfer the cultured seed liquid to a shake flask filled with 200 ml fermentation medium at a transfer rate of 8%, place it in a 30°C incubator and culture it for 24 hours to obtain the nisin fermentation liquid, and measure the final fermentation pH and fermentation titer; In case of fermentation culture in a fermenter: transfer the cultured seed liquid to a fermenter containing 5 L fermentation medium at a transfer rate of 8%, culture temperature at 30°C, rotation speed at 50 rpm, feed 50% glucose, control sugar concentration at 3-8 g / L, control pH at 6.8 by feeding 15% (m / m) sodium hydroxide solution for 0-12 h, control pH at 4.5-6.2 by feeding 15% (m / m) sodium hydroxide solution for 12-22 h, obtain nisin fermentation broth, measure the fermentation titer after 22 h, and calculate the volume of sodium hydroxide solution consumed by 1 L fermentation broth and the volume of hydrochloric acid solution used to adjust pH to 2.5 with 30% (m / m) hydrochloric acid solution for 1 L fermentation broth.

3. The method for low pH fermentation of nisin according to claim 2, characterized in that: The components of the seed culture medium are as follows by weight: 1.5% peptone, 1.5% yeast extract, 2.0% potassium dihydrogen phosphate, 2.0% sucrose, 0.15% sodium chloride and 0.015% magnesium sulfate. The pH of the seed culture medium is adjusted to 7.0 with 15% (m / m) sodium hydroxide solution before inoculation.

4. The method for low pH fermentation of nisin according to claim 3, characterized in that: When the fermentation culture is carried out in a fermenter, the lactic acid streptococcus is fermented for 12 hours and the fermentation pH is controlled to be lower, and the lower fermentation pH range is 4.5-5.0.

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