What is fermentation?
Fermentation is the process of inoculating microorganisms into a suitable culture medium and controlling their growth and metabolic environment to enable the microorganisms to perform unique functions. Its main contents include strain selection, seed expansion, culture medium preparation and sterilization, inoculation and fermentation control, etc.
◆Type of fermentation
Fermentation can be divided into different types from different perspectives. According to whether oxygen is needed, it can be divided into anaerobic fermentation (static fermentation) and aerobic fermentation (aeration fermentation); according to the properties of the fermentation medium, it can be divided into solid fermentation and liquid fermentation; according to the different fermentation methods, it can be divided into batch fermentation , fed-batch fermentation and continuous fermentation, etc.
Cosmetic fermentation anaerobic fermentation is suitable for the fermentation of anaerobic or facultative anaerobic microorganisms, such as acetone butanol fermentation, yeast alcohol fermentation, lactic acid fermentation, etc. Clostridium, which produces acetone and butanol, is an obligate anaerobic microorganism, and its cell fermentation and fermentation should be carried out under anaerobic conditions. Yeast is a type of facultative anaerobic microorganism. According to its physiological characteristics, seed preparation should be carried out under stirring and ventilation conditions to promote its growth and reproduction, while alcoholic fermentation is carried out under anoxic conditions. Most lactic acid bacteria are aerobic anaerobic bacteria, and their growth and metabolite synthesis have little to do with the presence or absence of oxygen. However, in order to prevent contamination by miscellaneous bacteria, it is better to ferment in an anoxic environment.
•Liquid fermentation
Refers to the state of the fermentation medium being liquid. The vast majority of purebred fermentations are liquid fermentations, such as penicillin fermentation. Early penicillin fermentations were carried out in liquid shallow trays. Due to the shortcomings of this method, such as low yield, easy contamination, and high labor intensity, it has been replaced by the deep liquid ventilation fermentation method.
Deep liquid ventilation fermentation is carried out in a closed ventilation fermentation tank. The seeds are inserted into a sterilized fermentation medium, and then fermented under suitable temperature and dissolved oxygen. There are snake tubes or jackets in the fermentation tank to control the temperature and dissolved oxygen. The concentration can be controlled by adjusting the mixer speed or the flow of sterile air.
•Batch fermentation
It refers to a fermentation method that does not exchange other materials with the outside world during the fermentation process, except for continuous aeration (aerobic fermentation) and the addition of acid and alkali solutions to adjust the pH of the fermentation liquid. The amount of culture medium is added at one time and the product is harvested at one time.
It is a widely used fermentation method.
Its advantages are:
❶Low temperature requirements and simple process operation;
❷It is easier to solve problems such as bacterial contamination and bacterial strain degradation;
❸The utilization efficiency of nutrients is high, and the product concentration is higher than that of continuous fermentation.
The disadvantages are:
❶It consumes a lot of manpower, material resources and power;
❷The production cycle is long, because the bacteria have a certain growth pattern during batch fermentation, and they all have to go through the lag phase, the logarithmic growth phase, the stable phase and the decline phase, and Each batch of fermentation must go through stages such as strain expansion and fermentation, equipment flushing, and sterilization;
Production efficiency is low, and production efficiency is often calculated by volumetric productivity (expressed in grams of metabolites per liter of fermentation product per hour). , in the batch fermentation process, the productivity of the entire process must be calculated, that is, the time includes not only the fermentation time, but also the time for feeding, tank washing, feeding, sterilization, etc.
◆Purification steps
•Solid-liquid separation
Regardless of whether the required fermentation products are extracellular metabolites, intracellular substances, or even the bacteria themselves, solid-liquid separation must first be performed to separate cells or other solid objects from the fermentation broth. The main methods include pretreatment, filtration, centrifugation and sedimentation.
•Active ingredient extraction
❶Cell disruption To extract enzymes, polysaccharides, nucleic acids, etc. in cells, cells must first be disrupted. Most of the products of genetic engineering accumulate in cells. To obtain these products, cells must first be broken.
❷Concentration and precipitation Concentration is the process of removing a certain amount of solvent (including water) from a low-concentration solution into a high-concentration solution. There are three common types: evaporation concentration, freeze concentration and absorption concentration.
❸Lon exchange and adsorption. Many biological substances are amphoteric substances. Depending on the pH value of the solution, they can exist in three forms: cations, anions and dipole ions. These molecules can reversibly bind to ion exchange resins by electrostatic forces. Adsorption can have two purposes. One is to adsorb and concentrate fermentation products on the adsorbent, and the other is to use the adsorbent to remove impurities or harmful substances in the fermentation broth.
❹Distillation is an effective method for separating liquid mixtures. It is a process of separating required substances from liquid mixtures based on the different boiling points of fermentation products.
❺Extraction is a process in which a specific solvent is added to the fermentation broth mixture and the required substances are separated based on the different solubilities of the fermentation broth components in the aqueous phase and organic phase. This method is often used in the isolation of antibiotics. If the antibiotic fermentation broth is mixed with an organic solvent and adjusted to an appropriate pH, most of the antibiotics will be dissolved in the organic phase. After the organic phase is separated, the pH can be adjusted to transfer the antibiotics to the aqueous phase. By repeating this process, purer antibiotics can be obtained. This method can also be used to extract red pigment from red yeast fermentation broth. The extraction method has the advantages of fast mass transfer speed, short production cycle, easy continuous operation, automatic control, high separation efficiency, and large production capacity, so it is widely used.
•Super extraction technology
Due to the development of technology, extraction does not necessarily take place between the aqueous phase and the organic phase. New technologies such as aqueous two-phase extraction, reverse micelle extraction, and supercritical fluid extraction have been developed. Supercritical fluid extraction technology is widely used in the cosmetics industry. It has the following characteristics:
❶Supercritical extraction can be performed near room temperature (35-40°C) and under a CO2 gas envelope, effectively preventing the oxidation and escape of heat-sensitive substances. Therefore, the active ingredients of medicinal plants are maintained in the extract, and substances with high boiling points, low volatility, and easy pyrolysis can be extracted at temperatures far below their boiling points;
❷Using SFE is the cleanest extraction method. Since no organic solvent is used in the entire process, the extract has no residual solvent substances, thus preventing the presence of harmful substances to the human body and environmental pollution during the extraction process, ensuring 100% purity. naturalness;
❸Extraction and separation are combined into one. When the saturated dissolved CO2 fluid enters the separator, due to the drop in pressure or the change in temperature, the CO2 and the extract quickly become two phases (gas-liquid separation) and separate immediately. Not only does extraction It has high efficiency and low energy consumption, which improves production efficiency and reduces costs;
❹CO2 is an inactive gas. No chemical reaction occurs during the extraction process, and it is a non-flammable gas. It is tasteless, odorless, non-toxic and very safe;
❺CO2 gas is cheap, has high purity, is easy to prepare, and can be recycled repeatedly in production, thus effectively reducing costs;
❻Both pressure and temperature can become parameters for adjusting the extraction process. The purpose of extraction is achieved by changing the temperature and pressure. When the pressure is fixed, substances can also be separated by changing the temperature; conversely, by fixing the temperature, the extract can be separated by lowering the pressure. Therefore, the process is simple and easy to master, and the extraction speed is fast.
Chromatography: When the fermentation broth concentrate flows through a chromatography column equipped with a stationary phase with the mobile phase, each substance in the mixture is separated due to different molecular sizes. It is a fast, simple and efficient separation technology that was only developed in the 1960s.
The technology has a wide range of applications, such as desalination, removal of pyrogenic substances, concentration of polymer solutions, determination of relative molecular weight, purification of antibiotics and other fields. The principle is based on the different molecular sizes of the substances to be separated. It is difficult for macromolecular substances to enter the micropores of gel particles (stationary phase), so they flow downward quickly; in addition to diffusing between gel particles, small molecule substances can also It can enter the micropores of the gel, so the downward flow rate is slow.
New methods such as gas chromatography, medium pressure liquid chromatography, and high performance liquid chromatography have been developed to separate samples.
The biggest feature of chromatography is its high separation efficiency, which can separate various substances with very similar properties. Moreover, it can be used not only for the analysis and identification of small amounts of substances, but also for the separation, purification and preparation of large amounts of substances. Therefore, as an important analysis and separation means and method, it is widely used in scientific research and industrial production. It plays a very important role in petroleum, chemical industry, medicine and health, biological science, environmental science, agricultural science and other fields.
Membrane separation relies on the process of a specific membrane allowing substances to pass through or be retained. Membrane separation is similar to the sieving process, and the purpose of separation is achieved based on the size of the pore size of the filter membrane. According to the size of separated particles or molecules, it is divided into six processes: dialysis, electrodialysis, microfiltration, ultrafiltration, reverse osmosis and nanofiltration. Microfiltration is mainly used in the field of cosmetics. In view of the separation characteristics of microporous filter membranes, the application scope of microporous filter membranes is mainly to intercept particles, bacteria and other pollutants from the gas phase and liquid phase to achieve purification, separation and concentration. the goal of.
•Key steps in purification
The key steps in the extraction process of biological purification include: extraction, chromatography and membrane separation.
Post-processing of fermented products
❶Crystallization is an efficient method of preparing pure substances. Because the crystallization process is highly selective, only similar molecules or ions can form crystals, so the precipitated crystals are very pure. In the fermentation industry, crystallization is widely used in the production of small molecules such as antibiotics, amino acids, organic acids, sugars, nucleotides, vitamins, and coenzymes.
❷The water content in dry biological products can easily cause hydrolytic denaturation and affect the quality, so drying must be carried out. There are many drying equipment in industry, but because most biological products are heat-sensitive substances and are easily deactivated, the drying of biochemical products must be fast and efficient. The heating temperature cannot be too high, and the contact time between the product and the drying medium cannot be too long, and it is To prevent impurities from mixing in and keep it clean and dry, the drying process must be carried out under closed conditions.
Advantages of biological fermentation and Purification technology
The purity and activity of some plant essential oils and plant essences obtained through traditional methods are generally around 20%-30%. The purity of plant active essences and plant essential oils obtained through biological fermentation and purification technology can be as high as 99.99%.