Method for improving fermentation rate of yeast under stress environment based on redox potential regulation and control

Abstract

The invention belongs to the field of fermentation engineering, and particularly relates to a method for improving the fermentation rate in a yeast stress environment based on redox potential regulation, which comprises the following steps: adding fermentation liquor after a fermentation tank is sterilized, inoculating yeast according to a certain proportion, and fermenting the yeast in stress environments such as low temperature, high temperature, peracid and peracid; and monitoring the oxidation-reduction potential of the fermentation liquid, and regulating and controlling the oxidation-reduction potential in a set range by controlling the gas introduction amount or a chemical substance control method until the fermentation index reaches the standard. By optimizing the oxidation-reduction potential, the fermentation performance is improved, the fermentation time is shortened, the yield of a target product is increased, and meanwhile, the energy consumption of fermentation is reduced. The method is suitable for the yeast fermentation process, and an efficient and energy-saving technical solution is provided for the yeast fermentation process.

Description

Technical Field

[0001] The present invention belongs to the field of fermentation engineering and relates to a method for improving the fermentation rate under yeast stress environment based on redox potential (ORP) regulation, and specifically relates to a method for improving the fermentation rate of yeast under low temperature, high temperature, over-acid and over-alkali stress conditions based on redox potential regulation, aiming to enhance yeast tolerance, improve fermentation efficiency and optimize industrial production. Background Art

[0002] Yeast fermentation is an important process in the industrial production of food, beverages and biofuels. However, in practical applications, yeast is often affected by stress conditions such as low temperature (<20°C), high temperature (>37°C), over-acidity (pH<4.0) or over-alkalinity (pH>8.0). These environmental stresses can lead to a decrease in yeast metabolic rate and cell activity, thereby extending the fermentation cycle and reducing production efficiency. Traditional countermeasures rely on adjusting temperature or pH, but these methods may affect product quality and energy loss, such as increased by-product generation. Traditional methods respond to stress by changing environmental conditions, which may require additional energy consumption to maintain temperature or pH adjustment. This not only increases production costs, but also creates an additional burden on the environment, especially in large-scale industrial production. For example, an article entitled Effect of low temperature on the shaping of yeast-derived metabolite compositions during wine fermentation studied low-temperature and room-temperature wine fermentation. Yeast can produce more flavor substances that improve the taste of wine under low-temperature conditions, but this method has the problem of long fermentation time. This is also the problem that the present invention needs to solve.

[0003] Redox potential (also known as oxidation-reduction potential, ORP) is an important parameter reflecting the redox state of a solution. It is the embodiment of the macroscopic redox properties of all substances in the solution, reflects the ability of the solution to gain or lose electrons, and indirectly reflects the physiological metabolism of cells. It is mainly affected by temperature, ventilation, pH, culture medium components and metabolites.

[0004] By regulating ORP, the metabolic activity and stress resistance of yeast can be effectively affected. Studies have shown that appropriate ORP regulation can promote the adjustment of yeast metabolic pathways, increase fermentation rate and maintain flavor quality. For example, the invention patent with application number CN201110267251.2 discloses a method for producing butanol by anaerobic fermentation regulated by redox potential, which is used to regulate butanol production in industrial applications. In the article Development of redoxpotential-controlled schemes for very-high-gravity ethanol fermentation published by Liu CG, ORP was reported to be used to accelerate ethanol fermentation production. However, ORP regulation strategies under complex stress conditions such as low temperature, high temperature, over-acidity and over-alkali have not been systematically studied. Therefore, it is of great significance to develop a comprehensive method based on ORP regulation to increase the fermentation rate of yeast under stress conditions. Summary of the invention

[0005] The present invention proposes a method for increasing the fermentation rate under yeast stress environment based on redox potential regulation, which improves fermentation performance, shortens fermentation time, increases target product yield, and reduces fermentation energy consumption by optimizing ORP level. The method is applicable to yeast fermentation process and provides an efficient and energy-saving technical solution for yeast fermentation process.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for increasing the fermentation rate under yeast stress environment based on redox potential regulation comprises the following steps:

[0008] After the fermentation tank is sterilized, fermentation liquid is added and yeast is inoculated in a certain proportion to allow the yeast to ferment under a stress environment. During the fermentation process, the redox potential of the fermentation liquid is monitored and controlled in real time within the set range until the fermentation index reaches the standard and the fermentation is terminated.

[0009] Furthermore, the stressful environment is one or more of the following conditions coexisting:

[0010] 1) Low temperature conditions: fermentation temperature is below 20°C;

[0011] 2) High temperature conditions: fermentation temperature is higher than 37°C;

[0012] 3) Overacid conditions: pH value of fermentation broth is less than 4.0;

[0013] 4) Over-alkaline conditions: the pH value of the fermentation liquid is greater than 8.0.

[0014] Furthermore, the yeast includes one of eukaryotic unicellular microorganisms such as Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces pastorianus, and Yarrowia lipolytica.

[0015] Furthermore, the fermentation broth includes but is not limited to one of YPD medium, malt extract medium, grape juice medium, and lignocellulose hydrolyzate, and the content of the medium components is adjusted according to actual conditions.

[0016] Furthermore, the yeast inoculation amount is 5%-10% of the fermentation volume or the number of viable bacteria is 10 6 -10 8 CFU / mL.

[0017] Furthermore, the redox potential setting range is determined according to stress conditions, oxygenation conditions and strain characteristics. The determination process is: determining the working range of ORP through preliminary experiments, and then selecting the optimal ORP range therein.

[0018] Furthermore, a redox electrode is used to monitor the redox potential of the fermentation liquid.

[0019] Furthermore, the redox potential of the fermentation liquid is controlled by controlling the gas flow rate or using a chemical substance control method, specifically:

[0020] The method for controlling the amount of gas introduced is specifically as follows: for anaerobic fermentation, when the redox potential is lower than the lower limit of the set range, sterile air is introduced into the fermentation liquid until the redox potential returns to the set range. Since the anaerobic fermentation process is a process in which the redox potential continues to decrease, if the redox potential is higher than the upper limit of the set range, the reducing substances produced by the yeast will reduce the redox potential, and no other measures are required; for non-anaerobic fermentation, when the redox potential is lower than the lower limit of the set range, the rate of ventilation into the fermentation liquid is increased, and when the redox potential is higher than the upper limit of the set range, the rate of ventilation into the fermentation liquid is reduced or nitrogen is introduced into the fermentation liquid;

[0021] The chemical substance control method is specifically as follows: when the redox potential is higher than the upper limit of the set range, a reducing agent is added; when the redox potential is lower than the lower limit of the set range, an oxidizing agent is added. The reducing agent is selected from one of dithiothreitol (DDT), sodium sulfite, and ascorbic acid, and the oxidizing agent is selected from one of hydrogen peroxide, potassium persulfate, and hematoxylin.

[0022] The innovation of the present invention lies in that the present invention improves the fermentation performance of yeast under stress environment, shortens the fermentation time and increases the ethanol yield by optimizing the ORP level. The mechanism is that regulating ORP can promote the activation of metabolic pathways related to stress resistance in eukaryotic unicellular microorganisms such as yeast. Under redox controlled conditions, intracellular reactive oxygen species (ROS) are reduced, thereby giving yeast tolerance to extreme conditions and increasing the fermentation rate and product (such as ethanol, lactic acid, succinic acid and extracellular protein, etc.) yield.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] 1. Improvement of fermentation efficiency: The present invention achieves significant improvement in sugar consumption rate and target product yield under stress environment through ORP regulation, thereby shortening the fermentation cycle. The fermentation efficiency is significantly improved compared with traditional methods such as adjusting temperature or pH.

[0025] 2. Maintaining flavor quality; ORP control can effectively balance the fermentation rate and accumulation of flavor substances during the fermentation process of food and beverages, and will not cause a significant increase in flavor substances that affect the taste, such as diacetyl and pentanedione, while enhancing the production of ester substances.

[0026] 3. Energy saving and consumption reduction: The present invention adopts micro-ventilation technology to achieve precise control of ORP, which significantly reduces the demand for oxygen supply, thereby reducing the energy consumption of fermentation. In addition, the present invention does not require equipment adjustment, and provides an economical and efficient solution for large-scale industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The graphs are as follows: the biomass, glucose, ethanol, and glycerol contents change over time under low-temperature fermentation in Example 1 of the present invention and Comparative Example 1, wherein (a) is the final value of the biomass, (b) is the graph of the glucose change over time, (c) is the graph of the glycerol change over time, and (d) is the graph of the ethanol change over time.

[0028] Figure 2 The graphs are graphs of higher alcohol and ester contents under low-temperature fermentation in Example 1 of the present invention and Comparative Example 1, wherein (a) is a graph of higher alcohol content, and (b) is a graph of ester content. DETAILED DESCRIPTION

[0029] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0030] Example 1: Comparison of low-temperature beer fermentation results using brewer's yeast.

[0031] This embodiment provides a method for increasing the fermentation rate under yeast stress environment based on redox potential regulation, comprising the following steps:

[0032] (1) Fermentation condition setting: Pasteurella was inoculated into YPD medium fermentation liquid, and the inoculation amount was 10% of the fermentation volume; the low-temperature fermentation temperature was set to 12°C, the pH value of the fermentation liquid was 5.5, and the anaerobic static fermentation was set to -150 mV±5 mV according to the target redox potential.

[0033] (2) ORP control: During the fermentation process, the ORP of the fermentation liquid is monitored in real time every 5 seconds by a redox electrode, and the ORP of the fermentation liquid is maintained at the target value by controlling the amount of gas introduced: when the ORP is lower than the target value, sterile air is introduced into the fermentation liquid through the ventilation system at a ventilation volume of 10 -3 vvm, when ORP returns to the target value, stop ventilation; since the anaerobic fermentation process is a process in which the redox potential continues to decrease, if the redox potential is higher than the upper limit of the set range, the reducing substances produced by the yeast will lower the redox potential, and no other measures are needed.

[0034] Comparative Example 1

[0035] The fermentation conditions in this comparative example were the same as those in Example 1, except that ORP control was not performed during the fermentation process.

[0036] The glucose consumption, ethanol production, cell biomass, changes in redox potential, and accumulation of flavor substances (such as diacetyl, pentanedione, and higher alcohols) in the fermentation broth of Example 1 and Comparative Example 1 were monitored and recorded, and the monitoring results were compared. Figure 1 , Figure 2 .

[0037] As can be seen from the figure:

[0038] As for the fermentation time, when the standard ethanol concentration of 25.00 g / L is reached, the time of Example 1 is 97 hours, which is 11.11% shorter than that of Comparative Example 1. As for the accumulation of flavor substances, in Example 1, the amounts of higher alcohols and total esters are 168.10 ppm and 5.91 ppm, respectively, while in Comparative Example 1, the amounts are 95.65 ppm and 4.14 ppm, respectively. The contents of aromatic substances in Example 1 are significantly higher than those in Comparative Example 1. It can be seen that the method provided in Example 1 can shorten the fermentation time and improve the flavor of beer.

[0039] Example 2: Comparison of fermentation results of fuel ethanol produced by brewer's yeast under high temperature conditions.

[0040] This embodiment provides a method for increasing the fermentation rate under yeast stress environment based on redox potential regulation, comprising the following steps:

[0041] (1) Fermentation condition setting: Saccharomyces cerevisiae was inoculated into YPD medium fermentation liquid, and the inoculation amount was 5% of the fermentation volume; the high temperature fermentation temperature was set to 40°C, the pH value of the fermentation liquid was 5.5, no ventilation was performed, and the stirring rate was 150 rpm; according to the target redox potential, it was set to -100 mV±5 mV.

[0042] (2) ORP control: During the fermentation process, the ORP of the fermentation liquid was monitored in real time every 5 seconds by a redox electrode, and the ORP of the fermentation liquid was maintained at the target value by controlling the amount of gas introduced. When the ORP was lower than the target value, sterile air was introduced into the fermentation liquid through the ventilation system with a ventilation volume of 2×10 -3 vvm, when ORP returns to the target value, stop ventilation.

[0043] (3) Data collection: Monitor and record the glucose consumption, ethanol production, cell biomass, and redox potential changes in the fermentation broth.

[0044] (4) Fermentation results: In this example, the final target product ethanol content was 79.32 g / L, which was 44.61% higher than the final ethanol content of 54.85 g / L in the fermentation without ORP control under the same fermentation conditions.

[0045] Example 3: Comparison of fermentation results of fuel ethanol production by Saccharomyces cerevisiae under acidic conditions.

[0046] This embodiment provides a method for increasing the fermentation rate under yeast stress environment based on redox potential regulation, comprising the following steps:

[0047] (1) Fermentation condition setting: Saccharomyces cerevisiae was inoculated into the lignocellulose hydrolysate fermentation broth with a pH of 5.0, and the inoculation amount was 8% of the fermentation volume; the fermentation temperature was set to 30°C, the ventilation volume was 1 vvm, the stirring rate was 100 rpm, and according to the target redox potential, it was set to -50 mV ± 5 mV; after 12 h of fermentation, the pH dropped to below 4.0, reaching the peracid stress condition.

[0048] (2) ORP control: During the fermentation process, the ORP of the fermentation broth was maintained at the target value by chemical substance control. When the ORP was lower than the set value, hydrogen peroxide was added at a rate of 1 g / L·h until the ORP returned to the set value. When the redox potential was higher than the upper limit of the set range, dithiothreitol was added at a rate of 0.5 g / L·h until the ORP returned to the set value.

[0049] (3) Data collection: Monitor and record the glucose consumption, ethanol production, cell biomass, and redox potential changes in the fermentation broth.

[0050] (4) Fermentation results: In this example, the final target product ethanol content was 43.08 g / L, which was 44.71% higher than the final ethanol content of 29.77 g / L in the fermentation without ORP control under the same fermentation conditions.

[0051] Example 4: Comparison of fermentation results for producing fuel ethanol under alkaline conditions of Pichia pastoris.

[0052] This embodiment provides a method for increasing the fermentation rate under yeast stress environment based on redox potential regulation, comprising the following steps:

[0053] (1) Fermentation conditions: Saccharomyces cerevisiae was inoculated into the lignocellulose hydrolysate fermentation broth with a pH of 8.0, and the inoculation amount was 6% of the fermentation volume; the fermentation temperature was set to 30°C, the ventilation volume was 2 vvm, the stirring rate was 100 rpm, and the target redox potential was set to -50 mV ± 5 mV.

[0054] (2) ORP control: During the fermentation process, the ORP of the fermentation broth was maintained at the target value by chemical substance control. When the ORP was lower than the set value, hydrogen peroxide was added at a rate of 1 g / L·h until the ORP returned to the set value. When the redox potential was higher than the upper limit of the set range, dithiothreitol was added at a rate of 0.5 g / L·h until the ORP returned to the set value.

[0055] (3) Data collection: Monitor and record the glucose consumption, ethanol production, cell biomass, and redox potential changes in the fermentation broth.

[0056] (4) Fermentation results: In this example, the final target product ethanol content was 38.42 g / L, which was 58.89% higher than the final ethanol content of 24.18 g / L in the fermentation without ORP control under the same fermentation conditions.

[0057] The above-mentioned embodiments are only embodiments of the present invention, but they should not be construed as limiting the scope of the present invention. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation, characterized in that: The method comprises: adding fermentation liquid to the sterilized fermentation tank, inoculating yeast in a certain proportion, and allowing the yeast to ferment under a stress environment; during the fermentation process, real-time monitoring and controlling the redox potential of the fermentation liquid within a set range until the fermentation index reaches the standard, and then ending the fermentation.

2. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 1, characterized in that: The stressful environment is one or more of the following conditions coexisting: 1) Low temperature conditions: fermentation temperature is below 20°C; 2) High temperature conditions: fermentation temperature is higher than 37°C; 3) Overacid conditions: pH value of fermentation broth is less than 4.0; 4) Over-alkaline conditions: the pH value of the fermentation liquid is greater than 8.

0.

3. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 1, characterized in that: The yeast comprises one of Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces pastorianus and Yarrowia lipolytica.

4. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 1, characterized in that: The fermentation liquid comprises one of YPD culture medium, malt extract culture medium, grape juice culture medium and lignocellulose hydrolyzate.

5. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 1, characterized in that: The inoculation amount of the yeast is 5%-10% of the fermentation volume or 10 6 -10 8 CFU / mL.

6. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 1, characterized in that: The redox potential setting range is determined according to stress conditions, oxygenation conditions and strain characteristics.

7. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 1, characterized in that: The redox potential of the fermentation broth can be controlled by controlling the amount of gas introduced or by using chemical substances.

8. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 7, characterized in that: The redox potential of the fermentation liquid is controlled by controlling the gas flow rate, specifically: For anaerobic fermentation: when the redox potential is lower than the lower limit of the set range, sterile air is introduced into the fermentation liquid until the redox potential returns to the set range; For non-anaerobic fermentation: when the redox potential is lower than the lower limit of the set range, the rate of aeration into the fermentation liquid is increased; when the redox potential is higher than the upper limit of the set range, the rate of aeration into the fermentation liquid is reduced or nitrogen is introduced into the fermentation liquid.

9. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 7, characterized in that: The redox potential of the fermentation broth is controlled by using a chemical substance control method: when the redox potential is higher than the upper limit of the set range, a reducing agent is added, and when the redox potential is lower than the lower limit of the set range, an oxidizing agent is added.

10. The method for increasing the fermentation rate of yeast under stress environment based on redox potential regulation according to claim 9, characterized in that: The reducing agent is selected from one of dithiothreitol, sodium sulfite and ascorbic acid, and the oxidizing agent is selected from one of hydrogen peroxide, potassium persulfate and hematoxylin.

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