Method for regulating pH abnormity in early fermentation stage of abamectin

Abstract

The invention discloses a method for regulating and controlling abnormal pH in the early fermentation stage of abamectin. Supplemented materials are composed of glucose, yeast peptone, ammonium sulfate and water. The mass ratio of the ammonium sulfate to the glucose is 0.20-0.30%, the mass ratio of the glucose to the yeast peptone is 20.0-30.0%, the mass ratio of the yeast peptone to the water is 2.0-3.0%, and the balance is water; the temperature is adjusted from 27-30 DEG C of normal culture to 25-26 DEG C when the pH is abnormal, and the temperature is adjusted to 27-30 DEG C on the third day of material supplement. Regulation and control of feed supplement are combined with culture temperature, abnormal metabolism PH is stimulated to restore normal metabolism, and the production level can be improved in the preparation process.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of biopesticide raw materials, and in particular to a method for regulating abnormal pH in the early stage of avermectin fermentation. Background Art

[0002] Avermectin, also known as Avermectins in English, is a macrolide disaccharide compound. It is a natural product isolated from soil microorganisms. It has contact and stomach poisoning effects on insects and mites, and has a weak fumigation effect. It has no systemic effect, but has a strong penetration effect on leaves, can kill pests under the epidermis, and has a long residual effect. Avermectin does not have an ovicidal effect. Its mechanism of action is different from that of general insecticides. It interferes with neurophysiological activities and stimulates the release of γ-aminobutyric acid, which has an inhibitory effect on the nerve conduction of arthropods. Mites, nymphs, insects, and larvae will show paralysis symptoms after contact with the agent, and will be inactive and not feed. They will die after 2 to 4 days. Because it does not cause rapid dehydration of insects, the lethal effect of avermectin is slow. Although it has a direct killing effect on predatory and parasitic natural enemies, it has little damage to beneficial insects because there are few residues on the surface of plants.

[0003] Market Prospect Analysis: Abamectin has a large potential market, broad domestic and foreign markets, and promising prospects. The demand for this product in developing countries and regions is gradually increasing. In the domestic market, due to the relatively moderate price of Abamectin, the main users include economic crops such as fruit trees and vegetables, as well as a wider range of common crops, and it can be widely promoted and used throughout the country. China's ban on highly toxic pesticides such as methamidophos, parathion, methyl parathion, and monocrotophos, China's accession to the WTO, and the stringent pesticide residue standards in international agricultural products and food trade have undoubtedly provided huge opportunities for the development of biological pesticides such as Abamectin. If the production of Abamectin can be carried out in accordance with market demand, total quantity control can be implemented from a macro perspective, more development can be carried out from a process perspective, and technology can be continuously improved to control the reduction in yield caused by metabolic deviation during the fermentation process, increase yield and reduce costs, the market prospects of Abamectin are still full of hope and brightness.

[0004] At present, avermectin is fermented in batches, and the required nutrients are added to the culture medium at one time until the fermentation of this batch is completed, and sugar is slightly supplemented during the fermentation process. After the fermentation tank is inoculated, the pH metabolism first decreases, then increases, then decreases, and then increases, and then begins a steady downward trend. The pH in the early stage of fermentation during the process presents a "W" trend. The common pH metabolism phenomenon is that after fermentation for about 50 hours, the pH should be in a state of slow decline, but in this process, the pH will suddenly increase sharply during the slow decline, and the tank batches with this phenomenon will affect the fermentation unit of the batch. The fermentation yield of avermectin is seriously affected. In response to this phenomenon, the following measures are currently taken to restore the fermentation to normal, release part of the fermentation liquid, and press a small part of the normal fermentation liquid into the tank; in actual application, it is found that the above methods are slow to restore pH and fermentation units, and often cannot be restored to normal production levels. Summary of the invention

[0005] The object of the present invention is to provide a method for regulating abnormal pH in the early stage of avermectin fermentation. The process of regulating feed supplementation combined with culture temperature stimulates abnormal metabolic pH to restore normal metabolism, so as to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The method for regulating abnormal pH in the early stage of avermectin fermentation comprises the following steps:

[0008] Step 1: The prepared feed is placed outside the fermentation reactor, through a special sterilization device, and is continuously heated, maintained and cooled in different equipment, and then enters the feed tank;

[0009] Step 2: Determine the specific weight of each feed, put them into the batching tank, stir and dissolve them evenly to form a uniform liquid, and transfer the prepared liquid to the sterilization device. The sterilization temperature is controlled at 130-150℃ for sterilization, and the material flow rate is 20-40m 3 / h, wait for the feeding tank to cool down to 40-50℃, and then get the sterilized feeding for standby use;

[0010] Step 3: When pH metabolism is abnormal in the early stage of fermentation, add the above feed continuously for 3-5 days;

[0011] Step 4: When pH abnormalities occur, reduce the culture temperature to 25-26°C.

[0012] Furthermore, the feed comprises the following components in percentage by weight:

[0013] Ammonium sulfate is 0.20-0.30%, glucose is 20.0-30.0%, yeast peptone is 2.0-3.0%, and the rest is water.

[0014] Furthermore, the metabolic abnormality is the phenomenon that the pH in the fermentation tank rebounds sharply during the downward trend after the fermentation cycle of 50 hours, which is the pH metabolic abnormality.

[0015] Furthermore, the amount of the supplementary feed is 3-4% of the mass of the feed liquid.

[0016] Furthermore, the supplementary feed is added for three consecutive days.

[0017] Furthermore, the temperature is regulated in time when abnormal pH metabolism is found, and the temperature is restored on the third day after feeding.

[0018] Furthermore, the sterilization method of the feed in step 1 is continuous sterilization.

[0019] Furthermore, the glucose is used as a fermentation carbon source, and ammonium sulfate and yeast peptone are used as nitrogen sources.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The regulation of this process can effectively and quickly restore abnormal pH, and continuous digestion has high efficiency in preserving nutrients in the materials, which is beneficial to the synthesis of fermentation products.

[0022] 2. The process equipment is commonly used production equipment, simple and easy to operate, effectively improving the fermentation production level, and does not affect the intrinsic quality of the product itself and downstream products. It has obvious economic benefits and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a curve chart comparing the pH recovery range and titer increase at different glucose concentrations when the ammonium sulfate concentration is 0.25% and the yeast peptone concentration is 2.5%;

[0024] Figure 2 This is a curve chart showing the effect of different yeast peptone concentrations on pH recovery and titer increase when the ammonium sulfate concentration is 0.25% and the glucose concentration is 25%;

[0025] Figure 3 This is a curve chart showing the effect of different ammonium sulfate concentrations on the pH recovery range and titer increase when the yeast peptone concentration is 2.5% and the glucose concentration is 25%;

[0026] Figure 4 The figure is a comparison chart of the PH and potency increase of normal batches and the PH and potency increase of abnormal batches. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The control method for abnormal pH in the early stage of avermectin fermentation and the control method for restoring pH metabolism of avermectin fermentation are feed supplement combined with culture temperature control. The feed supplement consists of glucose, yeast peptone, ammonium sulfate and water; according to the sum of the mass ratios of each component, 0.20-0.30% ammonium sulfate, 20.0-30.0%, yeast peptone 2.0-3.0%, and the rest is water; the temperature control is adjusted from 27-30°C in normal culture to 25-26°C when pH abnormality occurs. The temperature is controlled to 27-30°C on the third day of feed supplement.

[0029] Example 1: The mass percentage of each component is as follows: ammonium sulfate is 0.25%, glucose is 25%, yeast peptone is 2.5%, and the rest is water, specifically: 1000kg of glucose, 10kg of ammonium sulfate, 100kg of yeast peptone, 2890kg of water, and the total amount of avermectin fermentation feed is 4 tons.

[0030] The feeding method comprises the following steps:

[0031] (1) Dissolve the formulated material in water and transfer it to the sterilization device. The sterilization temperature is controlled at 150°C for sterilization and the material flow rate is 20m 3 / h. After replacing the feed tank, the temperature is lowered to 45°C to obtain the sterilized feed for standby use;

[0032] (2) The fermentation cycle is about 50 hours, and the pH should be in a state of slow decline. However, during this process, the pH suddenly rises sharply during the slow decline, which is a phenomenon of abnormal pH metabolism. The culture temperature is promptly reduced to 25°C and the above feed is added at 3% of the mass of the liquid in the fermentation tank for 3 consecutive days. The fermentation weighs 110 tons and 3.3 tons of liquid are added. The culture temperature is restored to 28°C on the third day.

[0033] Example 2: The mass percentage of each component is as follows: ammonium sulfate is 0.2%, glucose is 20%, yeast peptone is 2%, and the rest is water, specifically: 800kg of glucose, 8kg of ammonium sulfate, 80kg of yeast peptone, 3112kg of water, and the total amount of avermectin fermentation feed is 4 tons.

[0034] The feeding method comprises the following steps:

[0035] (1) Dissolve the formulated material in water and transfer it to the sterilization device. The sterilization temperature is controlled at 150°C for sterilization and the material flow rate is 20m 3 / h. After replacing the feed tank, the temperature is lowered to 45°C to obtain the sterilized feed for standby use;

[0036] (2) The fermentation cycle is about 50 hours, and the pH should be in a state of slow decline. However, during this process, the pH suddenly rises sharply during the slow decline, which is a phenomenon of abnormal pH metabolism. The culture temperature is promptly reduced to 25°C and the above feed is added at 3% of the mass of the liquid in the fermentation tank for 3 consecutive days. The fermentation weighs 110 tons and 3.3 tons of liquid are added. The culture temperature is restored to 28°C on the third day.

[0037] Example 3: The mass percentage of each component is as follows: ammonium sulfate is 0.25%, glucose is 25%, yeast peptone is 2.5%, and the rest is water, specifically: 1000kg of glucose, 10kg of ammonium sulfate, 100kg of yeast peptone, 2890kg of water, and the total amount of avermectin fermentation feed is 4 tons.

[0038] The feeding method comprises the following steps:

[0039] (1) Dissolve the formulated material in water and transfer it to the sterilization device. The sterilization temperature is controlled at 150°C for sterilization and the material flow rate is 20m 3 / h. After replacing the feed tank, the temperature is lowered to 45°C to obtain the sterilized feed for standby use;

[0040] (2) The fermentation cycle is about 70 hours, and the pH should be in a state of slow decline. However, during this process, the pH suddenly rises sharply during the slow decline, which is a phenomenon of abnormal pH metabolism. The culture temperature is promptly reduced to 25°C and the above feed is added at 3% of the mass of the liquid in the fermentation tank for 3 consecutive days. The fermentation weighs 110 tons and 3.3 tons of liquid are added. On the third day, the culture temperature is restored to 28°C.

[0041] Example 4: The mass percentage of each component is as follows: ammonium sulfate is 0.2%, glucose is 20%, yeast peptone is 2%, and the rest is water, specifically: 800kg of glucose, 8kg of ammonium sulfate, 80kg of yeast peptone, 3112kg of water, and the total amount of avermectin fermentation feed is 4 tons.

[0042] The feeding method comprises the following steps:

[0043] (1) Dissolve the formulated material in water and transfer it to the sterilization device. The sterilization temperature is controlled at 150°C for sterilization and the material flow rate is 20m 3 / h. After replacing the feed tank, the temperature is lowered to 50°C to obtain the sterilized feed for standby use;

[0044] (2) The fermentation cycle is about 70 hours, and the pH should be in a state of slow decline. However, during this process, the pH suddenly rises sharply during the slow decline, which is an abnormal pH metabolism. The culture temperature is promptly reduced to 26°C and the above feed is added at 3% of the mass of the liquid in the fermenter for 3 consecutive days. The fermentation weighs 110 tons and 3.3 tons of liquid are added. The culture temperature is restored to 30°C on the third day.

[0045] Example 5: The mass percentage of each component is as follows: ammonium sulfate is 0.3%, glucose is 30%, yeast peptone is 3%, and the rest is water, specifically: 1200kg of glucose, 12kg of ammonium sulfate, 120kg of yeast peptone, 2668kg of water, and the total amount of avermectin fermentation feed is 4 tons.

[0046] The feeding method comprises the following steps:

[0047] (1) Dissolve the formulated material in water and transfer it to the sterilization device. The sterilization temperature is controlled at 150°C for sterilization and the material flow rate is 20m 3 / h. After replacing the feed tank, the temperature is lowered to 50°C to obtain the sterilized feed for standby use;

[0048] (2) The fermentation cycle is about 90 hours, and the pH should be slowly decreasing. However, during this process, the pH suddenly rises sharply, which is an abnormal pH metabolism. The culture temperature is promptly reduced to 25°C and the above feed is added at 3% of the mass of the liquid in the fermenter for 3 consecutive days. The fermentation weighs 110 tons and 3.3 tons of liquid are added. On the third day, the culture temperature is restored to 27°C.

[0049] Five batches of fermentation tanks of Examples 1-5 were selected, and different glucose concentrations, different yeast peptone concentrations, and different ammonium sulfate concentrations were used for feeding at 50h, 70h, and 90h of the fermentation cycle of the fermentation tank. The temperature was adjusted to 25-26 degrees at 50 hours, and the temperature was restored to 27-30 degrees at 90 hours. The titer increase and pH recovery were measured respectively.

[0050] Potency test method: High performance liquid chromatography, in accordance with the method specified in national standard GB 29696-2013.

[0051] Figure 1 The curve diagram of the influence of different glucose concentrations on the average titer increase and pH recovery for three consecutive days is shown in the following figure. The ammonium sulfate is 0.25%, the yeast peptone is 2.5%, the horizontal axis is the glucose concentration, and the vertical axis is the titer increase and pH recovery. At the same time, the culture temperature is reduced to 25-26℃ at 50h, and the temperature is restored to 27-30℃ at 90h. Titer increase = current titer - titer of the previous day;

[0052] from Figure 1 It can be seen that when the glucose concentration in the feed is 25%, the feed titer increase and pH recovery range are optimal.

[0053] Figure 2 The influence curve of the average titer increase and pH recovery for three consecutive days is shown in the following figure: the materials with different yeast peptone concentrations are fed at 50h, 70h, and 90h of the fermentation cycle in the fermenter and the corresponding temperature is controlled. The ammonium sulfate is 0.25%, the glucose concentration is 25%, the horizontal axis is the yeast peptone concentration, and the vertical axis is the titer increase and pH recovery. At the same time, the culture temperature is reduced to 25-26℃ at 50h, and the temperature is restored to 27-30℃ at 90h. Titer increase = current titer - titer of the previous day; from Figure 2 It can be seen that when the yeast peptone concentration in the feed is 2.5%, the feed titer increase and pH recovery range are optimal.

[0054] Figure 3 The curve of the influence of different ammonium sulfate concentrations on the average titer increase and pH recovery for three consecutive days was shown in the following figure. The yeast peptone concentration was 0.25%, the glucose concentration was 25%, the horizontal axis was the yeast peptone concentration, and the vertical axis was the titer increase and pH recovery. At the same time, the culture temperature was reduced to 25-26°C at 50h, and the temperature was restored to 27-30°C at 90h. Titer increase = current titer - titer of the previous day; from Figure 3 It can be seen that when the ammonium sulfate concentration in the feed is 0.25%, the feed titer increase and pH recovery range are optimal.

[0055] like Figure 4 , showing the comparison between the pH metabolic trend and potency increase of the normal batch from around 50 hours to the third day and the abnormal batch from around 50 hours to the third day.

[0056] In summary, when the yeast peptone concentration in the feed is 2.5%, the ammonium sulfate concentration is 0.25%, and the glucose concentration is 25%, when pH metabolism abnormalities occur around 50h of the fermentation cycle, feed is added and the culture temperature is reduced to 25-26℃ at 50h, and the temperature is restored to 27-30℃ at 90h. The titer increase and pH recovery range are the best. This is because the fermentation broth uses carbon sources for carbon metabolism in the early stage of fermentation culture, and then uses nitrogen sources for nitrogen metabolism. However, when pH abnormalities occur under certain conditions, according to the phenomenon, it is the result of premature nitrogen metabolism. Abnormal metabolism may cause some intermediate metabolites to accumulate, the growth environment changes, the pH changes, and the enzyme activity is affected, thereby inhibiting microbial growth and product synthesis, resulting in a weak fermentation unit growth after the abnormality occurs. Glucose provides a fast-acting carbon source for fermentation and provides a superior environment for carbon utilization. At the same time, the culture temperature is regulated to temporarily reduce the metabolic rate, correct the abnormal nitrogen metabolism to carbon metabolism, and adjust the pH environment of the fermentation broth.

[0057] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for controlling abnormal pH in the early stage of avermectin fermentation, characterized in that: The steps include: Step 1: The prepared feed is placed outside the fermentation reactor, through a special sterilization device, and is continuously heated, maintained and cooled in different equipment, and then enters the feed tank; Step 2: Determine the specific weight of each feed, put them into the batching tank, stir and dissolve them evenly to form a uniform liquid, and transfer the prepared liquid to the sterilization device. The sterilization temperature is controlled at 130-150℃ for sterilization, and the material flow rate is 20-40m 3 / h, wait for the feeding tank to cool down to 40-50℃, and then get the sterilized feeding for standby use; Step 3: When pH metabolism is abnormal in the early stage of fermentation, add the above feed continuously for 3-5 days; Step 4: When pH abnormalities occur, reduce the culture temperature to 25-26°C.

2. The method for controlling abnormal pH in the early stage of avermectin fermentation according to claim 1, characterized in that: The feed comprises the following components in percentage by weight: Ammonium sulfate is 0.20-0.30%, glucose is 20.0-30.0%, yeast peptone is 2.0-3.0%, and the rest is water.

3. The method for controlling abnormal pH in the early stage of avermectin fermentation according to claim 1, characterized in that: The metabolic abnormality is the phenomenon that the pH in the fermenter rebounds sharply during the downward trend after the fermentation cycle of 50 hours, which is the abnormal pH metabolism.

4. The method for controlling abnormal pH in the early stage of avermectin fermentation according to claim 1, characterized in that: The amount of the supplementary material added is 3-4% of the mass of the feed liquid.

5. The method for controlling abnormal pH in the early stage of avermectin fermentation according to claim 1, characterized in that: The supplementary feeding time is three consecutive days.

6. The method for controlling abnormal pH in the early stage of avermectin fermentation according to claim 1, characterized in that: The temperature control timing is to timely control the temperature when abnormal pH metabolism is found, and the temperature is restored on the third day after feeding.

7. The method for controlling abnormal pH in the early stage of avermectin fermentation according to claim 1, characterized in that: The sterilization method of the feed in step 1 is continuous sterilization.

8. The method for controlling abnormal pH in the early stage of avermectin fermentation according to claim 2, characterized in that: The glucose is the fermentation carbon source, and ammonium sulfate and yeast peptone are the nitrogen sources.

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