The preparation of gelatin mainly includes two steps of collagen gelatinization and heat treatment. Gelatinization of collagen is a very important step in the preparation of gelatin. Through this process, the oxygen bonds and covalent cross-links that maintain the stability of the tertiary and quaternary structure of collagen can be effectively destroyed, so that the three-dimensional structure is sufficiently loosened to facilitate subsequent gel extraction. Therefore, the often said gelatin preparation method mainly refers to the collagen gelatinization method.
At present, the most commonly used collagen gelatinization methods mainly include the acid-base method, modified acid-base method, and biological enzymatic hydrolysis. The difference lies in the different media for inducing gelatinization of gelatin. In addition, the application of new processing technologies such as ultra-high pressure in inducing collagen gelatinization has attracted much attention in recent years.
1 Traditional collagen gelation method
1.1 The acid and alkali method
The acid and alkali method uses acid (sulfuric acid, etc.) or alkali (lime water, etc.) to combine with the basic or acidic groups of collagen molecules to break the ionic and oxygen bonds within or between the molecules and release the procollagen molecules, and expand the volume of the raw materials, loose the organization, which is conducive to hot water extraction. The degree of acid-base treatment in this process is very important. Excessive acid or alkali concentration and too long treatment time will lead to excessive hydrolysis of collagen and secondary degradation of macromolecular subunits, resulting in a decrease in gelatin gel strength.
In addition, alkali treatment can also cause hydrolysis in the pyrrolidine-rich area, destroy the structure of the gelatin, reduce the content, and reduce the strength of the gelatin gel. The type, age, and nutritional status of animals all have an impact on the microstructure of collagen, resulting in different batches of raw material quality of gelatin, making it difficult to grasp the degree of acid-base treatment. At present, finding a more controllable gelatinization method has become a research hotspot.
1.2 The acid salt method and the salt-alkali method
The acid salt method and the salt-alkali method use salt (sodium sulfate) and acid or alkali to form a mixed solution instead of a single acid and alkali to pretreat the raw materials. The research and development of this method are mainly aimed at excessive hydrolysis which is easily caused by traditional acid-base treatment. Adding salt can increase the osmotic pressure of the solution and reduce the degree of acid-base penetration, reduce the degradation of collagen by acid or alkali, prevent excessive hydrolysis of collagen molecules, and promote the production of high gel strength gelatin.
Studies have shown that a mixture of hydrochloric acid, lime, sodium hydroxide, and sodium sulfate is used to treat tilapia skin. The results show that the gelatin gel obtained by the latter has higher strength than the former two. This is because sodium hydroxide fully swells fish skin collagen fibers, breaking the non-covalent cross-links in the collagen, and promoting the release of procollagen molecules. At the same time, sodium sulfate has a dehydration and salting-out effect on collagen, shrinks collagen fibers, and prevents them from reaching maximum expansion. This inhibits the degradation of subunit components to a certain extent and reduces the loss of gel strength.
1.3 The enzymatic method
The enzymatic method uses protease to catalyze the partial degradation of collagen so that the raw materials meet the requirements of gel extraction. The core is to break the chemical bonds of collagen cross-linking, and at the same time try not to damage or less damage the components of the collagen triple helix area. Therefore, the choice of the enzyme is the key to obtain high gel strength gelatin.
Studies have shown that enzymes that can specifically hydrolyze the non-helical regions of collagen ends are suitable for preparing high gel strength gelatin. For example, pepsin mainly acts on the terminal peptide of the collagen molecule, which can degrade the triple helix structure into a single α-chain, and has little effect on the peptide bond within the collagen molecule. The gelatin strength of tilapia fish skin gelatin prepared by the compound enzymatic method of acid protease and delipase can be as high as 899.3g. Among them, lipase can remove lipids in fish skin, which is beneficial to the separation of collagen and non-collagen substances. Acid protease selectively cuts the acidic amino acid peptide bonds in the non-helical region of collagen to make it into soluble collagen, and better maintains the integrity of the subunit components, which is conducive to obtaining gelatin with high gel strength. However, the enzymatic method has problems such as high cost, difficulty in controlling the point of enzymatic hydrolysis, and the endpoint.
At present, the only gelatinization method that can be applied in large-scale production is the traditional acid-base method. However, traditional methods generally have the disadvantages of the long production cycle, low efficiency, high resource consumption, environmental pollution, etc., and they are prone to very rupture of peptide bonds of collagen molecules, destroying the integrity of collagen subunits, and then affecting the quality of finished gelatin. Therefore, exploring new methods for the induction of collagen gelatinization and establishing new technologies for the preparation of green, clean, and efficient high-grade gelatin is of great practical significance to the healthy development of the gelatin industry.
2 Ultra high pressure induces collagen gelatinization
2.1 The theoretical basis of ultra-high pressure induced collagen gelatinization
In the process of collagen gelatinization, breaking the non-covalent bonds and covalent cross-links in collagen is the key to promoting collagen gelatinization. In addition, high-quality gelatin should be a subunit component of collagen. This requires that the peptide bond of collagen is destroyed as little as possible in the process of inducing collagen gelatinization to maintain the integrity of the subunit components.
Ultra-high pressure technology has been widely used in protein modification in recent years. Ultra-high pressure can destroy the tertiary and quaternary structure of the protein, break non-covalent bonds such as hydrophobic and ionic bonds, stretch the protein-peptide chain, and loosen the three-dimensional structure. Studies have found that excessive high pressure can destroy the non-covalent bond balance of proteins. Ultra-high pressure can enhance the hydration of the protein and promote its solubility.
It can be seen that the effect of ultra-high pressure mainly destroys the non-covalent bond structure of proteins, which is consistent with the purpose of acid-base acting on collagen. Compared with acid-base treatment, ultra-high pressure does not destroy the covalent bond of collagen and is more conducive to maintaining the integrity of subunits in collagen, which is more conducive to the production and preparation of high-quality gelatin. In addition, ultra-high pressure can increase the hydration of the protein, which will be more conducive to the effect of heat in the process of thermal gel extraction.
2.2 Related research on ultra-high pressure technology-induced collagen gelatinization
At present, there have been reports on the preparation of gelatin by ultra-high pressure-induced collagen gelatinization. A research scholar once used ultra-high pressure to prepare fish skin gelatin. After pre-treating the collagen with 50 mmol/L acetic acids for 3 hours, the ultra-high pressure treatment was carried out, and finally, the gelatin was extracted with hot water. The results show that 250MPa ultra-high pressure pretreatment can increase the product yield, and the gelatin yield will decrease under 400MPa pressure. But 400MPa treatment is more conducive to obtaining gelatin with higher gel strength and melting/freeze-thaw temperature. There is no further report on the relevant mechanism.
Another researcher has conducted research on the ultra-high pressure preparation process of fish gelatin. The results showed that the gelatin yield was the highest under the conditions of 300MPa pressure and 10min ultrahigh-pressure time, reaching 75.03%, which was higher than 66.13% of the traditional process. The gel strength of the product can reach 274g, which is better than the traditional 234g.
On this basis, some researchers have conducted further research on ultra-high pressure preparation of pigskin gelatin. The results show that because the pigskin structure contains more covalent cross-links, compared with the traditional acid treatment method, pure ultra-high pressure treatment cannot effectively improve the yield of pigskin gelatin. The combination of ultra-high pressure and low-concentration HCI can effectively induce the gelatinization of porcine skin collagen. Under the conditions of pressure 250MPa, ultra-high pressure time of 10 minutes, and HCI mass fraction of 0.75%, the extraction rate of pigskin gelatin is as high as 88.62%, and the gel strength can reach 384.43g.
Compared with the traditional method, the acid concentration used is reduced, and the pretreatment time is greatly shortened. From an industrial point of view, the use of ultra-high pressure methods to replace acid and alkali can effectively reduce environmental pollution caused by the gelatin production process, save production time, and improve production efficiency. The results of SDS-PAGE electrophoresis analysis showed that the acid/ultra-high pressure group gelatin has a higher content of α, β, γ, and other subunit components, which proves that it has good gel properties from a microscopic point of view. This shows that the use of ultra-high pressure in conjunction with a specific liquid environment can effectively induce collagen gelatinization, and has a beneficial effect on the gelation process of the gel. It can be seen that, whether it is aquatic raw materials or mammalian raw materials, ultra-high pressure technology can be applied to the preparation of gelatin, and the product has outstanding gel properties.