What are the characteristics of the action of antifreeze proteins of plant origin and their application in food?

Plant-derived antifreeze proteins
Plant-derived antifreeze proteins (AFPs) are functional proteins produced by plants to protect their cells from freezing when the external environment changes.AFPs are distributed differently in different plants, and all of them have certain antifreeze activity, which is a general term for a group of proteins that have the ability to improve the antifreeze activity of plant cells.
AFPs have three properties, namely thermal hysteresis activity (THA), modification of ice crystal growth morphology and inhibition of ice crystal recrystallization. Plant-derived AFPs have lower thermal hysteresis activity than fish- and insect-derived AFPs, but stronger ability to inhibit ice crystal recrystallization. Therefore, it is believed that the main pathway of plant-derived AFPs to regulate freezing resistance is to inhibit the growth of extracellular ice crystals and inhibit ice recrystallization.
By inhibiting the recrystallization of ice crystals in solution, plant-derived AFPs can reduce the mechanical damage caused by the fluctuation of ambient temperature below zero and inhibit the quality deterioration of food products due to repeated freezing and thawing during the process of refrigeration, which makes them more suitable for addition to refrigerated foods than other sources of AFPs.
Characterization of antifreeze proteins
2.1 Thermal Hysteresis Activity (THA) The freezing point of a general solution (e.g., NaCl, sucrose solution, etc.) is the temperature at which the vapor pressures of the solid and liquid phases are in equilibrium, and thus the freezing point should be equal to the melting point. Since AFPs only affect the icing process and hardly affect the melting process, making the freezing point lower than the melting point, the difference between its freezing point and melting point is called the thermal hysteresis difference, and this activity of AFPs is called THA.
2.2 Modification of ice crystal growth morphology effect at low temperatures, ice crystals due to the influence of AFPs, making the growth of ice crystal morphology change from the normal ice crystal of flat round type growth to hexagonal prismatic cone. With the concentration of AFPs and the prolongation of time, the morphology of ice crystals tends to be needle-like.
2.3 Inhibit ice crystal recrystallization in the solution at freezing temperature, the small ice crystals in the solution gradually disappeared and aggregated into large ice crystals, causing damage to the organizational structure of the product. The solution with AFPs can inhibit the recrystallization of ice crystals and make the small ice crystals evenly distributed.
Application of plant-derived AFPs in the food field
Freezing is one of the most important stages in the cold chain transportation of food, and refrigeration is a commonly used storage method in the food field. Temperature fluctuations are very likely to occur in the cold chain transportation of food, resulting in the freezing-thawing cycle of food. Therefore, it is inevitable that the ice crystal recrystallization phenomenon occurs, which in turn causes damage to the cellular tissue, leading to changes in the physicochemical properties of food, tissue morphology.
At the same time, the recrystallization phenomenon of water will also lead to changes in the internal components of cells, protein denaturation, starch regeneration and other phenomena. Plant-derived AFPs as frozen food additives can effectively improve the quality of frozen food.
3.1 Application in Ice Cream The formation and recrystallization of ice crystals in frozen sweet dairy products play a crucial role in the quality of the products. The formation process of ice crystals determines the growth of ice crystals during the hardening and storage of frozen dairy products, and influences the sensory qualities such as the creamy state of the ice cream, the roughness and hydration, the hardness, and the icy mouthfeel, etc.; and recrystallization determines the stability of the texture and the structure of the ice cream after the whole freezing operation.
The addition of AFPs can mechanically control the size of ice crystal nuclei, inhibit the growth of ice crystals in sucrose solution, and improve the stability under static storage conditions.
Lillford et al. extracted AFPs from winter wheatgrass and added them to ice cream, and observed through experimental studies that the average size of ice crystals could be induced to decrease when the concentration of AFPs reached 0.05%~0.1%.
Regand et al. added winter wheatgrass AFPs to ice cream and observed that winter wheatgrass AFPs showed significant antifreeze properties when 0.003% of crude protein was added by bright-field microscopy.
Sensory evaluation of ice cream after one month of low-temperature storage (-18 ℃) showed that the ice cream of the blank group at -18 ℃ and fluctuating conditions (-20 ℃~-10 ℃) was very rough and the ice crystals of the ice cream were in the form of flakes, whereas the ice cream with the addition of wintergrass AFPs had a smooth texture and the ice crystals were fine and homogeneous, which confirmed that the ice cream with the addition of wintergrass AFPs was capable of modifying the growth morphology of the ice crystals and inhibited recrystallization.
In addition, the activity of wintergrass AFPs was verified by heat treatment, and the activity of wintergrass AFPs was not affected by pasteurization, and the activity effect of wintergrass AFPs reached equilibrium at the addition level of 0.13%.
Zhang et al. extracted AFPs from cold domesticated oats and modified ice cream with 0.1% AFPs, which increased the glass transition temperature from -29.14 ℃ to -27.74 ℃, improved the melt resistance of ice cream and effectively inhibited its recrystallization. The addition of plant-derived AFPs mitigated the damage to ice cream caused by freezing and temperature fluctuations, resulting in a frozen food product with high quality and taste.
3.2 Application in frozen dough Starch is an indispensable processing raw material in many kinds of food processing, and the application of plant-derived AFPs in frozen dough is relatively large.
Plant-derived AFPs can regulate the amount of crystalline water precipitated from the dough, so that the water and gluten in the dough can be maintained in the state before freezing, and thus the gel stability of the dough, the pore size and uniformity of the fermented dough, and the texture and aroma of the dough after maturing will not be deteriorated.
Jia Chunli et al. added Ligustrum officinale AFPs to wheat starch to study the stability of starch gel after freezing and thawing, and found that with the increase of concentration of Ligustrum officinale AFPs, the precipitation rate of freezing and thawing starch and the freezable water content were significantly reduced, and the modification of the ultrastructure of the gel made the increase of starch pore size inhibited, and the homogeneity of starch was improved, and the increase of gel hardness and the decrease of gel elasticity were retarded, which improved the texture of the gel after freezing and thawing.
Kontogiorgos et al. isolated a kind of heat-stable AFPs from winter wheatgrass and added them to dough, and found that the gluten structure of blank dough was altered when stored under temperature fluctuation conditions (-20 ℃ to -10 ℃) for 30 days, whereas ice crystal recrystallization was reduced in the dough with the addition of 0.1% winter wheatgrass AFPs, and the gluten mesh structure of the dough was smaller and uniform in shape.
In addition, Liu Mei et al. added carrot AFPs to the dough and found that the addition of carrot AFPs reduced the increase in the freeze-thaw water content of the dough during the freeze-thaw cycle, attenuated the damage to the storage properties and ultrastructure of the frozen dough caused by the freeze-thaw cycle, and thus improved the characteristic volume and textural properties of the dough after maturation.
Xia Lu et al. used AFPs extracted from winter wheat bran as an additive to prepare quick-frozen soup dumplings at 1%, 2%, 2.5% and 3% additions relative to glutinous rice flour, and the experimental results showed that 2.5% AFPs additions significantly improved the quality of the dumplings, and the cooked dumplings had a smooth appearance, good elasticity, and clear and transparent broth.
3.3 Application in fresh fruits Frozen fruits have high water content and are prone to juice loss and soft deformation during freezing and refrigerating. Alginate and winter wheat AFPs mixed into fresh strawberries, then frozen by liquid nitrogen, after thawing to determine its cell activity and juice loss rate, the results show that AFPs significantly improve the freezing resistance of strawberries, and can retain its original form and the texture of the fruit itself, to maintain the sensory and flavor of the fruit.
Rui et al. used the vacuum impregnation method to react the bean curd leaves with AFPs in a closed storage tank, and after a short time of vacuum pressure and atmospheric pressure recovery process, the AFPs were in direct contact with the pores of bean curd leaves to avoid the damage to the leaf tissues and to reduce the loss of leaf juice.
The microstructure analysis showed that the ice crystals in bean curd leaves were small. Fresh fruits and vegetables antifreeze protection focuses on whether the cell morphology is intact after freezing and whether the cell viability of frostbitten tissues exists, and the applicability of different AFPs in the field of fruit and vegetable antifreeze to fruit and vegetable cellular foods also needs further research.
Conclusion and Outlook
The production of AFPs in plants is a complex process, and the existence of plant-derived AFPs is closely related to plant growth resistance. Changes in the external environment will directly affect the concentration of plant-derived AFPs, and factors such as chemical induction will also stimulate the production of AFPs in plants, among which the food field mostly adopts the cloning or transgenic route to obtain plant-derived AFPs.
Plant-derived AFPs can inhibit recrystallization during freezing and cryopreservation, and reduce the nutritional and quality degradation caused by the flow of juice during thawing. In addition, plant-derived AFPs are non-toxic, and their addition to a variety of food products will not produce negative effects, and their functional properties are not associated with any toxic proteins, so the study of plant-derived AFPs in the future application of the food field is very promising.
Currently, research on the mechanism of AFPs has revealed a new direction of antifreeze technology in food, and has also provided a theoretical basis for production practice. However, plant-derived AFPs are still limited in their large-scale application, mainly due to the small amount of AFPs extracted from plants, which prevents large-scale production, and the high cost and poor reproducibility of synthetic AFPs.
Therefore, it is important to use the properties of plant-derived AFPs, such as relative molecular mass, isoelectric point, thermohysteretic activity and its antifreeze mechanism, to develop efficient separation and purification methods for large-scale application, and to find the optimal conditions for the application of plant-derived AFPs in various types of food, which can provide protection for cold-chain food production, transportation, and storage. Meanwhile, utilizing gene technology to transfer antifreeze genes from plants into recipient cells to obtain a large number of molecules with antifreeze activity will also be a new direction for us in the application of plant-derived AFPs in food.