Expanded culture technique of microbial strains
The expanded culture of the strain is the first process of fermentation production, which is also called seed preparation. Seed preparation not only increases the number of cells, but more importantly, it produces high-quality seed for seed production through fermentation. Therefore, how to provide a production strain with high fermentation yield, stable production performance, sufficient quantity and no contamination by other bacteria is the key to the seed preparation process. Seed expansion training task The greater the scale of industrial production, the more seeds are required for each fermentation. In order for small microorganisms to complete such a large fermentation conversion task in a short period of several tens of hours, it is necessary to have a large number of microbial cells. The purpose of expanding the culture of the strain is to provide a considerable amount of metabolically rich seeds for each fermenter feed. Because the length of the fermentation time is related to the size of the inoculum, the inoculum is large and the fermentation time is short. Connecting a large number of mature cells into the fermenter will help shorten the fermentation time, increase the utilization rate of the fermenter, and also help reduce the chance of infection. Therefore, the task of expanding the seed culture is not only to obtain pure and strong cells, but also to obtain vigorous and inoculated bacteria. For the fermentation process of different products, it is necessary to determine the number of stages of seed expansion and cultivation according to the growth rate of the growth and reproduction of the strain. In the production of Antibiotics, the cells of the actinomycetes grow slowly and often, and the three-stage seed is often used for expansion. Generally, 50 t fermenters use tertiary fermentation, and some even use four-stage fermentation, such as streptomycin production. Some enzyme preparations also use tertiary fermentation for fermentation production. The strain used in the fermentation of glutamic acid and other amino acids is bacteria, which grows and multiplies rapidly, and generally adopts secondary fermentation. Process of Seed Preparation <br> Seed preparation of bacteria and yeast is a process in which the number of cells is increased. The slant culture medium of bacteria mostly adopts a formula with limited carbon source and rich nitrogen source, and beef extract and peptone are often used as organic nitrogen sources. The bacterial culture temperature is mostly 37 °C, a few is 28 °C, the bacterial culture time is generally 1 to 2 days, and the spore-forming bacteria are cultured for 5 to 10 days. Seed preparation of molds and actinomycetes generally involves two processes, a spore preparation for producing a large number of spores on a solid medium, and a seed preparation process for producing a large amount of hyphae in a liquid medium. Spore preparation (1) Preparation of actinomycete spores Spore culture of actinomycetes is generally carried out using agar slant medium containing some nutrients suitable for sporulation, such as bran, pea dip, peptone and some inorganic salts. The carbon source and nitrogen source should not be too rich (the carbon source is about 1%, the nitrogen source does not exceed 0.5%), and the carbon source is rich, which is easy to cause a physiological acidic nutrient environment, which is not conducive to the formation of actinomycetes spores. Hyphae reproduction is not conducive to sporulation. In general, drying and limiting nutrients can induce sporulation directly or indirectly. The incubation temperature of the actinomycetes was mostly 28 °C, a few were 37 °C, and the culture time was 5-14 days. Which generation of slant spores is used to access the liquid culture, depending on the characteristics of the strain. The use of mother slant spores to access liquid culture medium is beneficial to prevent the variation of strains. The use of slanted spores to access liquid medium can save the amount of bacteria. There are two ways in which the strain enters the seed tank. One is a spore-in method, in which a spore suspension made of a spore is directly connected to a seed tank. The method can reduce the difference between the batch and the batch, has the advantages of convenient operation, simple process, and convenient control of spore quality, and the spore infusion method has become a direction of fermentation production. Another method is the shake flask mycelium method, which is suitable for some slow-growing actinomycetes. The advantage of this method is that the cultivation time of the seeds in the seed tank can be shortened. 2. Seed preparation Shake the bottle into the tank, often using the mother bottle, the sub-bottle two-stage culture, and sometimes the mother bottle seed can also directly enter the tank. The seed culture medium is rich and complete, and it is easy to be decomposed and utilized by the bacteria. The abundant nitrogen source is beneficial to the growth of mycelium. In principle, the various nutrients should not be too rich, and the concentration of the sub-bottle medium is slightly higher than that of the mother bottle, which is closer to the medium formula of the seed tank. (2) Seed tank seed preparation Seed tank seed preparation process varies from strain to strain, and can generally be divided into first-stage seed, second-stage seed and third-grade seed preparation. Spores (or shake flasks) are inserted into small-sized seed tanks, and after cultivation, a large number of hyphae are formed. Such seeds are called first-class seeds, and the first-stage seeds are transferred to the fermenter for fermentation. For secondary fermentation. If the primary seed is inserted into a larger seed tank and more hyphae are formed by cultivation, the seed thus prepared is called a secondary seed, and the secondary seed is transferred to the fermenter for fermentation, which is called tertiary fermentation. . By the same token, fermentation using a three-stage seed is called a four-stage fermentation. The number of seed tanks is mainly determined by the nature of the strain and the growth rate of the bacteria and the rational application of the fermentation equipment. The purpose of seed preparation is to form a certain number and quality of cells. When spore germination and bacterial growth begin, the amount of bacteria is small and can be carried out in a small tank. The purpose of the fermentation is to obtain a large amount of fermentation product. The product is formed after a large number of cells are formed and reaches a certain growth stage, and needs to be carried out in a large fermenter. At the same time, the production of several fermentation products has different requirements for nutrition and culture conditions at different growth stages. Therefore, two biological processes with different purposes and different process requirements are placed in a large tank, which not only affects the yield of the fermentation product, but also causes waste of power and equipment. The reduction of the number of seed tanks facilitates the simplification of the production process and the control of the fermentation process, and can reduce the fluctuation of fermentation caused by abnormal seed growth. Seed culture <br> Seed culture requires a certain amount of seeds, and in a suitable medium, certain culture conditions and culture methods are controlled to ensure the normal growth of the seeds. The industrial microbial culture method is divided into two types: static culture and aeration culture. The static culture method is to hold the culture medium in the fermentation container, and after the inoculation, the air is not cultured. The aerated culture method uses aerobic bacteria and facultative aerobic bacteria. The environment in which they grow must supply air to maintain a certain level of dissolved oxygen, so that the bacteria grow and ferment rapidly, also known as aerobic culture. . Surface culture method 2. Solid culture method 3. Liquid deep culture (1) Deep medium Three control points for this operation 2 After the temperature control medium is sterilized, it is cooled to the culture temperature for seed culture. Since heat is generated along with the growth and reproduction of microorganisms, the heat is generated by the stirring, so to maintain the temperature constant, it is necessary to be in the jacket or in the coil. Cooling water circulation. 3 Before venting and stirring the air into the seed tank, remove the bacteria through the air filter to make sterile air, then enter the bottom of the tank, and then disperse the air into tiny bubbles by stirring. In order to prolong the bubble retention time, a baffle can be placed in the tank to generate eddy currents. In addition to increasing dissolved oxygen, the microorganisms in the culture solution can be uniformly dispersed in the seed tank, promote heat transfer, pH equalization, and uniform dispersion of the added acid and alkali. (2) Several deep culture methods 2 Carrier culture method The carrier culture method is born out of the tune culture, and at the same time absorbs the advantages of liquid culture. It is a newly developed culture method in recent years. The feature is that a natural or synthetic porous material is used instead of a solid substrate such as bran as a carrier for microorganisms, and nutrients can be strictly controlled. At the end of the fermentation, as long as the cells and the medium are squeezed out for extraction, the carrier can be reused. 3 Two-step method In the two-step liquid submerged culture of enzyme preparation, the cells are the same in each step and the culture conditions are different, because the optimum conditions for the growth of microorganisms and enzyme production are quite different. For example, the addition of glucose to the culture medium can greatly increase the growth of the cells or hyphae, but it severely hinders the synthesis of many enzymes. Although the aeration of the culture medium can promote the growth of microorganisms, in many cases, the synthesis of the enzyme is inhibited. In order to obtain the maximum high-environment enzyme, a method of regulation must be established, which requires that the unit enzyme activity of the cell is high and the number of cells is required, that is, the cells are created under different physiological conditions. Two-step liquid submerged culture is one of the specific measures to achieve this adjustment. The two-step process for the production of enzyme preparations is characterized by the distinction between cell growth conditions (growth phase) and enzyme production conditions (production phase). The bacteria first multiply on the rich medium, then collect the bacterial concentrate, wash it and then transfer it to the enzyme-producing medium with the inducer. During this period, the bacteria accumulate a large amount of enzymes, generally no longer multiply, nutrition The ingredients or inducers are fully utilized. Seed quality control (1) Factors affecting spore quality and their control Medium In addition, the impact of water quality cannot be ignored. Differences in the region, seasonal changes and pollution from water sources can all be the cause of fluctuations in water quality. In order to avoid the influence of water quality fluctuation on spore quality, an appropriate amount of inorganic salt may be added to distilled water or no salt water for use in the preparation medium. For example, when preparing a slant culture medium for producing tetracycline, 0.03% (NH4)2HPO4, 0.028% KH2PO4 and 0.01% MgSO4 are sometimes added in the absence of saline to ensure spore quality and increase tetracycline fermentation yield. In order to ensure the quality of the spore culture medium, the main raw materials used in the slant culture medium, the sugar, nitrogen and phosphorus contents must be subjected to chemical analysis and shake flask fermentation test before they can be used. The quality of the medium after sterilization should be strictly controlled when preparing the medium. Before the slant medium is used, it should be placed at a suitable temperature for a certain period of time, so that the slanted surface is free of condensed water, and the moderate moisture is favorable for spore growth. The preparation of spore media should also consider the selection of multiple amino acids by colonies of different metabolic types. The strains can present colonies of many different metabolic types on solid medium, and various amino acids have different expressions on colonies. The more varieties of nitrogen sources, the more types of colonies appear, which is not conducive to the stability of production. The use of a single nitrogen source on the slant medium can inhibit the appearance of some abnormal colonies; while the plate medium for isolation and screening needs to add a more complex nitrogen source to fully display the various colony types. filter. Therefore, there are two experiences in the preparation of solid media: 1 spore culture medium for production or as a medium for preparing sand spores or passages to use a relatively simple nitrogen source in order to maintain the advantages of normal colony type; For plate culture media for seed selection or isolation, a more complex organic nitrogen source is required in order to facilitate the selection of specially metabolized colonies. 2. Cultivate temperature and humidity Microorganisms grow over a wide temperature range. However, to obtain high quality spores, the optimum temperature range is very narrow. In general, increasing the culture temperature can accelerate the metabolic activity of the bacteria and shorten the culture time. However, the enzymes of sugar metabolism and nitrogen metabolism of the bacteria have different sensitivity to temperature. Therefore, the physiological state of the bacteria is different depending on the culture temperature, and if the spores are cultured at the optimum temperature, the productivity will be lowered. The optimum temperature required by different strains is different and needs to be determined through practice. For example, the optimum temperature of the Streptomyces serrata slope is 36.5-37 °C. If it is higher than 37 °C, the spores mature early and are easy to age. After the fermenter is connected, the mycelium will be slowly used for sugar and nitrogen, and the amino nitrogen will be used. The recovery is advanced, and the fermentation yield is reduced. The lower the culture temperature control, it is conducive to the formation of spores. The Streptomyces cerevisiae slant was first cultured at 36.5 °C for 3 days and then at 28.5 °C for 1 day. The number of spores obtained was increased by 3 to 7 times compared with the number of spores cultured at 36.5 °C for 4 days. When the spores are cultured, the relative humidity of the culture chamber has a great influence on the speed, quantity and quality of spore formation. When the relative humidity in the air is high, the water in the medium evaporates less; when the relative humidity is low, the water in the medium evaporates more. For example, in the dry areas of northern China, due to the dry climate in winter, the relative humidity of the air is low, and the water in the slanting medium evaporates quickly, causing the lower part of the slope to contain a certain amount of water, while the upper part is easy to dry, and the spores grow faster and The lower part of the slope is long upwards. In summer, the relative humidity of the air is high, and the water in the slope is evaporated slowly. At this time, the spores grow from the upper part to the lower part, and the lower part often accumulates condensed water, causing the spores to grow slowly or the spores to grow. Tests have shown that when the spores are cultured under certain conditions, the relative humidity in the north is controlled at 40% to 45%, while the relative humidity in the south is controlled at 35% to 42%, and the obtained spores are of good quality. In general, fungi require high humidity, while actinomycetes have low humidity requirements. The optimum culture temperature and humidity are relative, for example, relative humidity, medium composition, and influence on the optimal temperature of the microorganism. The culture temperature and the difference in the composition of the medium also affect the optimum relative humidity of the microorganism culture. 3. Cultivation time and refrigerating time (1) Spore culture time The nuclear matter and cytoplasm inside the mycelium and aerial hyphae are in a flowing state. If the hyphae are broken, the contents of each hyphae are different, and some fragments contain nuclear particles. Some fragments have no nuclear particles, and the number of nuclear particles is not uniform. The hyphae at this stage are not suitable for the preservation and passage of strains. The spore itself is an independent genetic body, and its genetic material is relatively complete, so the spores can be used for passage and preservation to maintain the basic characteristics of the original strain. But spore itself also has the difference between young and aging. In general, senescence spores are not as young as spores, because the senescence spores are gradually entering the germination stage, and the nuclear material tends to differentiate. The spore culture process generally chooses to terminate the culture at the stage of spore maturation. At this time, the spores or free scattered spores can be seen under the microscope. If the culture continues, the senescence mycelium enters the autolysis stage of the slanted surface, which is characterized by the appearance of the chamfer and discoloration. Dark or yellow, the bacteria layer is sunken, sometimes white spots or blackening. The white spot indicates that the spore germination grows a second-generation hyphae, and the black shows that the hyphae are autolyzed. The culture time of spores has an important effect on the quality of spores. Too young spores can not withstand refrigeration. For example, the terrapin culture is slanted for 4.5 days, the spores are not fully mature, and the hyphae begin to autolyze after 7-8 days of refrigeration. While the culture time is extended for half a day (ie, culture for 5 days), the spores are fully mature and can be refrigerated for 20 days without autolysis. Spores that are too old will lead to a decrease in productivity, and the culture time of spores should be controlled at the stage where the amount of spores is large, the spores are mature, and the fermentation yield is normal. (2) Spore refrigerating time The refrigerating time of the spores has an effect on the spore quality. The effect varies with the strain. The general principle is that the refrigerating time should be short and not long. It has been reported that in streptomycin production, the fermentation unit of the slant spores after being chilled at 6 °C for 2 months is 18% lower than that of the one month after refrigerating, and after 3 months of refrigeration, it is reduced by 35%. 4. Inoculum size The amount of spore inoculation into the seed tank also has an effect on the fermentation production. For example, the number of spores of the globular bacteria of one of the penicillin-producing bacteria has a great influence on the yield of penicillin fermentation. If the number of spores is too small, the spheroids grown after the cans are too large, affecting the ventilation effect; if the number of spores is too large, The spheroids are not well maintained after entering the tank. In addition to the above factors to be controlled, in order to obtain high quality spores, the quality of the strains needs to be controlled. The strains preserved by various methods should be naturally isolated once every one year, and a single colony inoculated with spore culture medium having good morphology and production performance is selected. The prepared slant spores are subjected to shake flask fermentation test and can be used for fermentation production after passing the test. (2) Factors affecting seed quality and their control The quality of the seed is one of the important factors for the normal fermentation. Seed preparation is not only to provide a certain number of bacteria, but more importantly to provide fermentation-producing bacteria with a certain physiological state. The control of seed quality will be used as a starting point. Medium 2. Culture conditions For the small can seeds produced by penicillin, the feeding process can be used to improve the seed quality, that is, after the seed tank is cultured for a certain period of time, a certain amount of seed culture medium is added, and as a result, the volume of the seed tank is increased, and the seed quality is also improved. The mycelium group is obviously reduced, the accumulation in the hyphae is increased, the mycelium is thick, and the fermentation unit is increased. 3. Age The optimum age varies greatly depending on the species. The age of bacteria is generally 7 to 24 h, the age of mold is generally 16 to 50 h, and the age of actinomycetes is generally 21 to 64 h. Different batches of the same strain are cultured for the same time, and the quality of the obtained seeds is not completely consistent. Therefore, the optimum age should be tested several times, especially according to the quality of the seeds in this batch. Ivacaftor Cas 873054-44-5,Iva-Int Cas 13721-01-2,Tezacaftor Cas 1152311-62-0,Lumacaftor Cas 936727-05-8 Shijiazhuang Dingmin pharmaceutical Sciences Co.,Ltd , https://www.dingminpharma.com
Spore preparation is the beginning of seed preparation and an important part of fermentation production. The quality and quantity of spores have a significant impact on the growth, reproduction and fermentation yield of mycelium. The spore preparation process of different strains has different characteristics.
(2) Preparation of mold spores Spore culture of molds, generally using natural agricultural products such as rice, millet, corn, bran, and wheat grains as the medium. This is because the nutrients in these agricultural products are more suitable for the spores of mold, and the medium surface area of ​​this type of medium is large, and a large number of spores can be obtained. The culture of the mold is generally 25 to 28 ° C, and the culture time is 4 to 14 days.
Seed preparation is a process in which spores or cells cultured on a solid medium are transferred to a liquid medium to be propagated into a large number of hyphae or cells. The medium and other process conditions used in seed preparation are beneficial for spore germination, hyphal reproduction or bacterial growth.
(1) Shake flask seed preparation Some strains with slow spore germination and hyphal reproduction speed need to be spun the flask into a hyphae and then into the seed tank. This is the shake flask seed. The shake flask is equivalent to a miniature seed tank, and its medium formulation and culture conditions are similar to those of the seed tank.
The surface culture method is an aerobic static culture method. The medium state of the container is further divided into liquid surface culture and solid surface culture. The larger the surface area relative to the volume of the medium in the container, the easier it is to promote the transfer of oxygen from the gas-liquid interface into the medium. The growth rate of the bacteria in this method is related to the depth of the medium, and the larger the surface area per unit volume, the faster the growth rate.
Solid culture is further divided into shallow dish solid culture and deep solid culture, which are collectively referred to as koji culture. It originated from the traditional koji technology unique to China's brewing production. Its greatest feature is the high enzyme activity of the solid koji.
The liquid deep seed tank is vented from the bottom of the tank, and the air that is fed is dispersed into fine bubbles by the stirring blades to promote the dissolution of oxygen. This culture method of agitating and stirring the bottom of the tank is called a submerged culture method with respect to the surface culture method in which oxygen is dissolved by natural diffusion from the gas-liquid interface. It is characterized by easy selection of optimal culture conditions according to the nutrient requirements of the production strain for metabolism and the conditions of aeration, agitation, temperature and hydrogen ion concentration in the medium at different physiological periods.
1 Sterilization The fermentation industry requires pure culture, so the medium must be heat sterilized before seed culture. Therefore, the seed tank has a steam jacket for heat sterilization of the culture medium and the seed tank, or the medium is sterilized by a continuous heating sterilizer and continuously conveyed into the seed tank.
1 Control culture method According to the change of the inside of the tank, the change of the state variable in a short period of time and the influence of the environmental factors that may be measured on the microbial metabolic activity are grasped, and the control culture is carried out based on this, so as to achieve the optimal culture condition of the product. To this end, data is acquired by a sensor that measures state variables, and comprehensive analysis is performed by an electronic computer, and the result is used as a signal for feedback adjustment, and the environment (culture conditions) is controlled within a given reference. This is called electronic computer control training. It has been widely used in the fermentation of large-scale beer in the open air.
Seed quality is an important factor affecting the level of fermentation production. The quality of seed quality depends mainly on the genetic characteristics and culture conditions of the strain itself. This means that both good strains and good culture conditions are required to obtain high quality seeds.
The quality of spores is related to the medium, culture temperature, humidity, culture time, inoculum size, etc. These factors are related to each other and affect each other. Therefore, various factors must be fully considered and carefully controlled.
The raw materials, spores, processing methods and dosages of the spore culture medium have a certain influence on the spore quality. The instability of spore quality during production is often caused by unstable raw material quality. The difference in the origin, variety and processing method of raw materials will lead to changes in the content of trace elements and other nutrients in the medium. For example, due to the different raw materials and production processes used to produce peptone, peptone has different trace element content, phosphorus content and amino acid composition, and these nutrients play an important role in cell growth and sporulation. The agar grades have different effects on spore quality, which is caused by different grades of agar containing different inorganic ions.
When incubating in the incubator, if the relative humidity is low, the water-filled dish can be placed to increase the relative humidity in the incubator. In order to ensure the exchange of fresh air, the incubator should be opened several times a day to facilitate spore growth. The modern incubator is constant temperature, constant humidity, and can be ventilated without manual control.
The growth and development of filamentous fungi on slant medium can be divided into five stages: 1 spore germination and intracellular mycelial growth stage; 2 aerial hyphae growth stage; 3 sporulation stage; 4 spore maturation stage; 5 bevel The aging mycelium autolysis stage.
The inoculum size when preparing spores should be moderate, and the inoculum size is too large or too small to affect the spore quality. Because the size of the inoculum affects the number of individual spores in a certain amount of medium, which affects the physiological state of the cells. After inoculation, the colonies are evenly distributed throughout the slope, and the colony can be subdivided into normal inoculation. If the inoculum is too small, the colonies growing on the slope will be sparse. If the inoculum is too large, the colony will be dense on the slope. Generally, the spores used for passage are required to have a relatively small colony distribution, and are suitable for selecting a single colony for subculture. Inoculation of shake flasks or beveled spores into the tank requires that the colony density is moderate or slightly dense, and the number of spores meets the required standard. Generally, a test tube with a height of 20 cm and a diameter of 3 cm is required, and the number of filamentous spores is required to reach 107 or more.
Seed quality is mainly affected by factors such as spore quality, culture medium, culture conditions, age and inoculation amount. The quality of the shake flask seed is mainly based on the appearance color, potency, mycelial concentration or viscosity, sugar and nitrogen metabolism, pH value, etc., and meets the requirements before entering the tank.
The control of the quality of the raw material of the seed medium is similar to the control of the quality of the raw material of the spore medium. The nutrient composition of the seed culture medium should be suitable for the needs of seed culture. Generally, some mediums suitable for spore germination and mycelial growth are selected, which are easily absorbed and utilized by the bacteria in nutrition, and the nutrient components are appropriately enriched and completely, nitrogen The source and vitamin content are higher, which can make the hyphae thick and have strong vitality. On the other hand, the nutrient content of the medium should be as close as possible to the fermentation medium to suit the needs of fermentation, and such seeds can be easily adapted to the culture conditions of the fermenter once they are transferred to the fermenter. The purpose of the fermentation is to obtain as much fermentation product as possible, the medium is generally relatively concentrated, and the seed medium is preferably slightly thin. The pH of the seed medium should be relatively stable to suit the growth and development of the bacteria. Changes in pH cause changes in the activity of various enzymes, and have a great influence on mycelial morphology and metabolic pathways. For example, pH control of seed media has a significant effect on tetracycline fermentation.
Seed culture should choose the optimum temperature, as described above. The control of aeration and agitation during the cultivation process is very important. The oxygen demand of different stages of seed tanks or the same level seed tanks should be different. It should be controlled differently. Generally, the oxygen demand in the early stage is less, and the oxygen demand in the later stage is more. Increase the amount of oxygen supplied. Adequate aeration can improve seed quality during seed preparation of penicillin production. For example, the resulting lower hypoventilation adequate ventilation and access are two cases seed fermenter, the fermentation thereof may be a difference of 1 unit times. However, in the fermentation production of oxytetracycline, the aeration of the primary seed tank is small but beneficial to the fermentation. Insufficient aeration and agitation can cause abnormal phenomena such as hyphae agglomeration and hyphae sticking. During the production process, sometimes the seed culture will produce a large amount of foam and affect the normal aeration and agitation. At this time, it should be strictly controlled, and even the medium formulation may be considered to reduce foaming.
The seed culture time is called the age of the seed. In the seed tank, as the culture time is prolonged, the amount of the bacteria gradually increases. However, the bacteria multiply to a certain extent. Due to the consumption of nutrients and the accumulation of metabolites, the amount of bacteria does not continue to increase, but gradually ages. Since the physiological activities of the cells in different growth stages vary greatly during the growth and development of the cells, the control of the inoculation age is very important. In industrial fermentation production, it is generally selected in the logarithmic growth phase where vitality is extremely strong, and the amount of bacteria has not yet reached the peak. At this time, the seeds can quickly adapt to the environment, fast growth and reproduction, can greatly shorten the lag phase in the fermentor (adjustment), the non-shortened product synthesis time in the fermentor, and improve the utilization of the fermenter, saving power consumption . If the age control is not appropriate, when the seed with too young age is connected to the fermenter, there will often be a slow growth in the early stage, a lot of foam, a prolonged fermentation cycle, and a mycelial agglomeration due to too little bacteria, causing abnormal fermentation, etc. When the seeds of the old age are connected to the fermenter, the production capacity will decline due to the aging of the bacteria. In the production of oxytetracycline, the age of the first-class seeds differs by 2 to 3 h, and the metabolism of the cells will be significantly different after being transferred to the fermenter.