Computational Molecular Biology 2025, Vol.15, No.1, 53-64 http://bioscipublisher.com/index.php/cmb 56 3.3 Advantages and limitations of enzymatic extraction Enzymatic extraction of Cordyceps polysaccharides is indeed becoming increasingly popular, mainly due to the four words "mild and highly efficient". Unlike traditional methods that frequently require high temperature and high pressure, enzymatic methods can be accomplished at normal temperature and normal pressure, and a significant amount of electricity can be saved (Yang et al., 2017). The key point is that under such mild conditions, the polysaccharide structure will not be damaged-those delicate active groups can all be well preserved. The most wonderful thing is the specificity of the enzyme, just like a precise scalpel, specifically targeting components such as cellulose and pectin in the cell wall. However, in actual operation, the price of enzymes is indeed not cheap. Moreover, if the raw materials vary greatly from batch to batch, the proportion of enzyme types needs to be readjusted. Recently, it has been discovered that combining enzymatic methods with short-term ultrasound can not only maintain mildness but also increase extraction efficiency by 20% to 30%, which can be regarded as a new approach of complementing each other's strengths. Indeed, although enzymatic extraction of Cordyceps polysaccharides has obvious advantages, it also has many shortcomings. The most painful thing is the price of enzyme preparations-a bottle of cellulase costs thousands of yuan, and this cost is really unbearable when large-scale production is carried out. What's more troublesome is that the enzyme is too "delicate". If the temperature in the workshop fluctuates slightly or the pH value is not controlled stably, the enzyme activity will disappear without a trace. Last time we conducted a pilot test, we encountered this situation. The temperature sensor of the reaction vessel malfunctioned, deviating by 2 ℃, and the polysaccharide yield of the entire pot of extract was directly halved. So now all of them have to be equipped with high-precision temperature control systems, and pH also needs to be monitored in real time. The investment in these devices has further increased the cost. However, recently there have been studies attempting immobilized enzyme technology, which is said to be able to be reused 5 to 6 times. If it can really be industrialized, it would be a good way to reduce costs. In addition, mixing in some metal ions during the pretreatment of raw materials can also cause the enzymes to "stop working". We have fallen into this trap before, and now we have to be extra cautious in the pretreatment process. 4 Application of Ultrasound-Assisted Extraction inCordyceps Polysaccharides 4.1 Principle of ultrasound-assisted extraction The process of extracting Cordyceps polysaccharides by ultrasonic waves, to put it bluntly, relies on "sound wave bombs" to break the cell walls. When ultrasonic waves are injected into a liquid, countless tiny bubbles are generated-these bubbles form and burst instantly, just like countless miniature bombs exploding on the cell wall. The local temperature during the burst can soar to 5 000 ℃, and the pressure is comparable to that of the deep sea (Cheung et al., 2015). Even the strongest cell wall cannot withstand such a disturbance. Even better, this kind of "explosion" will also stir up microcurrents in the solution, allowing the solvent to drill into the cells as if it had legs. Laboratory data show that with ultrasound assistance, two-thirds of the time can be saved and the solvent usage can also be halved. However, in actual operation, the control of ultrasonic power and time is particularly crucial-too much power can easily shatter the polysaccharide structure, while too long time may cause local overheating. Recently, it has been discovered that pulsed ultrasound is milder than continuous ultrasound and has a better protective effect on the structure of polysaccharides. 4.2 Optimization parameters for ultrasound-assisted extraction Ultrasonic extraction of Cordyceps polysaccharides is like playing a precise sound game-the combination of power and frequency is particularly important. Usually, when the power is adjusted to around 300 W and combined with a 20 kHz sound wave frequency, the effect is the best. At this time, the cavitation bubbles produced are both dense and violent, and can tear the cell wall just right. But time control is the real technical job. The laboratory found that 15-20 minutes is a sweet spot. If it is too short, it cannot be fully extracted. If it exceeds half an hour, the polysaccharide chain begins to break. The temperature should be controlled within the range of 50 ℃-60 ℃(Li et al., 2011), which can maintain the solvent activity without reducing the cavitation effect. Interestingly, recently, someone attempted to introduce inert gas during ultrasound and found that it could produce
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