01 Strain degradation and aging

1.1. Strain degradation
Strain degradation refers to the phenomenon that the genetic variation of the strain occurs in the process of reproduction, which leads to the change of its character and the decline of the quality and yield of the strain. Strain degradation is a very common problem, which not only affects the development of industry, but also brings challenges to microbiology research.

a. Causes of strain degradation:

• Environmental factors: inappropriate culture conditions, such as high temperature, high humidity, hypoxia, etc., will lead to strain degradation.
• Genetic factors: changes in genetic material, such as gene mutations, chromosome aberrations, etc., can lead to strain degradation.
• Unreasonable use: Unreasonable use of strains, such as frequent passage, overuse, etc., will lead to strain degradation.

b. Impact:

• Decreased yield: Due to the degradation of the strain, the number of cultured offspring is reduced, and the yield is decreased.
• Quality decline: Due to the degradation of the strain, the quality of the cultured offspring is decreased, such as abnormal shape and uneven color.
• Decline in stability: Due to the degradation of the strain, the stability of the offspring is reduced, such as inconsistent growth rate and decreased ability to adapt to the environment.

c. Solution:

• Selection of excellent original species: selection of genetically stable and excellent traits of the original species for culture.
• Improve culture conditions: optimize the culture conditions, such as reducing the temperature, controlling humidity, increasing oxygen and so on.
• Rational use of strains: avoid frequent passage and excessive use of strains.

1.2. Strain aging
Strain aging refers to the phenomenon that the growth rate and reproduction ability of the strain decrease gradually in the process of long-term culture or preservation. Strain aging is also a serious problem, which not only affects the quality and yield of the strain, but also brings difficulties to microbiology research.

a. Causes of strains aging:

• Time factor: long-term preservation and culture will lead to strain aging.
• Environmental factors: inappropriate preservation conditions, such as high temperature, high humidity, hypoxia, etc., will lead to strain aging.
• Culture conditions: Inappropriate culture conditions, such as insufficient nutrition, temperature fluctuations, will lead to strain aging.

b. Impact:

• Decreased growth rate: due to the aging of the strain, the growth rate is slowed down and the culture cycle is prolonged.
• Decreased reproductive capacity: due to the aging of the strain, the reproductive capacity is decreased and the number of offspring is reduced.
• Decline in genetic stability: due to the aging of the strain, genetic stability is reduced, such as gene mutations, chromosome aberrations, etc.

c. Solution:

• Optimize storage conditions: select suitable storage conditions, such as low temperature, dry, low oxygen, etc.
• Optimize the culture conditions: optimize the culture conditions, such as increasing nutrition, controlling temperature, etc.
• Regular replacement of strains: regular replacement of strains to avoid strain aging.

02 Measures to control degradation

In order to effectively cope with strain degradation, we need to analyze the causes of degradation in the deep perspective.They include genetic instability of strains, unsuitable culture conditions, changeable culture environment and so on. By analyzing the causes of degradation, countermeasures can be taken to prevent further degradation of the strains.

2.1. Strain selection and improvement: In order to improve the stability and performance of the strain, we need to carry out strain selection and improvement. It includes the selection of strains with good genetic stability, the screening of mutants, and the use of genetic engineering technology for genetic modification. Through breeding and improvement, the adaptability and yield of the strain can be improved, and the risk of strain degradation can be reduced.

2.2. Optimization of culture conditions: Culture conditions have an important impact on the growth and metabolism of strains. To prevent strain degradation, we need to optimize the culture conditions. They include adjusting the composition of the medium, controlling the culture temperature, adjusting the pH value and so on. By optimizing the culture conditions, the growth and metabolism of the strains could be improved, and the risk of strain degradation could be reduced.

2.3. Establishment of scientific strain management system: The scientific strain management system can effectively prevent strain degradation. It includes the establishment of strain storage bank, the regulation of strain use and transmission process, and the regular identification and performance testing of strains. By establishing the scientific culture management system, the stability and reliability of culture can be guaranteed and the risk of culture degradation can be reduced.

2.4. Use of molecular biology technology to assist identification: Molecular biology technology can help us more accurately identify strains and judge their genetic stability and performance. It Includes DNA fingerprinting technology, PCR technology, gene sequencing and so on. With the aid of molecular biology technology, the phenomenon of strain degradation can be more accurately identified, the causes of degradation can be deeply analyzed, and the corresponding countermeasures can be taken.

2.5. Strengthen strain safety assessment: In the process of strain use and transmission, we need to strengthen safety assessment to prevent strain from leakage and pollution of the environment. This includes testing the safety of the strains and assessing the impact of the strains on the environment. By strengthening the culture safety assessment of strain, the potential risks to the environment and human health can be reduced, and the safety and reliability of microbial fermentation process can be ensured.

03 Strain aging and rejuvenation operation

In the process of microbial fermentation, the aging of strains may lead to the decline of production capacity and the instability of product quality. In order to solve this problem, we need to carry out strain aging and rejuvenation operations. The following content will introduce the aging and rejuvenation operation of the strain from seven aspects to help you restore the production capacity and stability of the strain.

3.1. Screening of dominant strains that produce active substances: In the aging and rejuvenation operation of strains, screening of dominant strains that produce active substances is the first step. We can screen out the dominant strains with higher production capacity by means of plate streak method and liquid culture method. Through the screening of dominant strains, the adaptability and yield of strains could be improved, which provided a good basis for the subsequent rejuvenation of strains.

3.2. Reasonable seed aging: Seed aging is an important step in the aging and rejuvenation operation of strains. Reasonable seed aging can promote the growth and metabolism of the strains and improve the ability of the strains to produce active substances. According to the characteristics of the strains and culture conditions, we can determine the appropriate aging time, temperature and types of aging agents. Reasonable seed aging can promote the recovery and optimization of strains.

3.3. Use of appropriate culture conditions to promote the growth of strains: Culture conditions have an important impact on the growth and metabolism of strains. In the operation of aging and rejuvenation of strains, we can promote the growth of strains by optimizing the culture conditions. For example, controlling the culture temperature, adjusting the pH value, adding the appropriate nutrients and so on. Through suitable culture conditions, the growth and metabolism of the strains can be improved, and the ability of producing active substances can be increased.

3.4. Timely separation and purification to obtain high-purity strains: In the aging and rejuvenation operation of strains, timely separation and purification is the necessary step. Through separation and purification, high purity strains can be obtained to ensure the purity and performance of strains. We can use appropriate separation methods and purification agents to separate and purify strains. By means of separation and purification, the quality and production capacity of the strains can be further improved.

3.5. Reasonable strain preservation to ensure the survival and stability of the strain: Reasonable strain preservation can ensure the survival and stability of the strain, and avoid the mutation and pollution of the strain during the process of use. We can use storage methods which is suitable for strain characteristics, such as low temperature storage, vacuum drying storage, etc. The reliability and stability of the strains can be ensured through reasonable strain preservation, which provides the strong guarantee for the subsequent production.

3.6. Modification and optimization through genetic breeding technology to improve the ability of strains to produce active substances: In the aging and rejuvenation operation of strains, modification and optimization through genetic breeding technology is an effective means to improve the performance of strains. For example, gene recombination and transfer technology can be used to change the genetic characteristics of strains and improve the ability of strains to produce active substances. Through the modification and optimization of genetic breeding technology, the adaptability and yield of strains can be further improved, which provides the good basis for the optimization of fermentation conditions.

3.7. Optimization of fermentation conditions is used to improve production efficiency: We can optimize fermentation conditions according to the characteristics of strains and production requirements, such as controlling temperature, adjusting pH value, adding appropriate nutrients, etc. Through the optimization of fermentation conditions, the growth and metabolism of the strains can be improved, and the production efficiency and product quality can be improved.

04 Process and principle of strain expansion

Taking beer yeast as an example, whether beer yeast is pure or not has the great influence on beer fermentation and beer quality. The yeast used in beer factory production is cultivated by the preserved pure yeast, and after reaching the certain amount, it is used on the production site. Every brewery should save the pure yeast suitable for the use of the factory to ensure that the beer produced has the stable style and characteristics.

4.1. Process of culture expansion

• Laboratory expansion culture: inclined test tube (original strain)一Rich flask or tube culture 一Pasteur bottle or triangle flask一 carlsberg flask culture.
• Production site culture: Hanseng tank culture一 yeast expansion culture tank一 yeast propagation tank 一 fermenter.

4.2. Principles of expanded cultivation

• The key to yeast expansion culture is the use of excellent single-cell starter strain. The initial strain was first identified by physiological characteristics and production performance, and then put into use, and ensured that there is no pollution and no variation in the process of expanding culture. The residual liquid after each step of enlargement should be inspected for contamination and no variation.
• In the early stage of culture, in order to improve the yeast proliferation rate and shorten the culture time, the laboratory expanded the culture stage and  the optimal yeast propagation temperature of 25°C is adopted. After each expansion, the temperature is correspondingly reduced, so that the yeast gradually adapt to the requirements of low temperature fermentation. However, the cooling amplitude should not be too large each time to prevent yeast activity from being inhibited.
• In order to shorten the yeast growth stagnation period and shorten the culture time, it is best to expand the yeast culture at each stage and transplant it during the yeast logarithmic growth period, specifically, before the yeast proliferation rate begins to drop back. At this time, the germination rate of yeast is the highest, the death rate is the lowest, and the proliferation is rapid after transplantation.
• Yeast proliferation relies on the biological oxidation of sugars, that is, respiration, to obtain energy. Therefore, ventilation for oxygen is absolutely necessary in yeast expansion culture. From the triangular flask to the Cassia pot culture stage, the container is generally shaken regularly to make the yeast and the air contact the air in the upper space of the container, and also make contact opportunities exist between the yeast and oxygen. After transplantation to the production site for expanded culture, attention should be paid to ventilation for oxygen supply.
• Cultivating yeast should use nutrient-rich high-quality medium. At the stage of laboratory culture, the superior malt with high α-amino nitrogen content should be selected, and the self-made medium should not be added.
• Regarding the expansion factor, in the laboratory stage, due to the high culture temperature, short proliferation time, and better aseptic operating conditions, the expansion factor can be higher, such as 1:10 to 20, or even higher. After the expansion culture of Hanseng pot, due to the gradual decrease of temperature, yeast doubling time is extended, in order to quickly make yeast rise, maintain the growth advantage of yeast, enhance its anti-pollution ability, the expansion ratio of 1:5 is appropriate. According to this principle, the number and volume of the intermediate expansion tank can be easily determined.
• Regarding ventilation, at the early stage of culture, the glucose content in the culture medium is high, and the proliferation effect of continuous ventilation of yeast may not be as good as ideal due to the influence of Crabtree effect, so intermittent ventilation is appropriate. In short, ventilation should be adequate, but not too much. Traditionally, the upper yeast uses continuous ventilation, while the lower yeast does not, which may be due to the difference in fermentation temperature (9°C and 20 °C) and fermentation time (3 days and 10 days).
• Sterility of expanded culture wort. The expanded culture of the production site generally uses the cold wort from the saccharification workshop, often due to the long transport distance, it also needs to be heated and sterilized.