Low-temperature cold chain technology is one of the most common and effective means of long-term food storage. The growth of ice crystals and recrystallization caused by freezing, storage, transportation and freezing/thawing of food products in the low-temperature cold chain are the key constraints on product quality. Repeated fluctuations in temperature cause products to suffer from ice crystal growth, freezing and thawing and recrystallization, damaging cellular and tissue structures, thus causing products to lose their original quality, which leads to quality damage and huge economic losses that are of increasing concern to people.

Scientists in related fields worldwide are facing a serious challenge: how to control ice crystal growth and recrystallization, and how to realize the inhibition of ice crystal growth in the process of low-temperature cold chain, which is the key to ensure the quality of many food products.

Introduction of Antifreeze Protein

After a long period of selection in the natural environment, organisms in high temperature and high altitude areas produce a kind of active protein, antifreeze protein (AFP), to resist the cold environment.

The most important feature of antifreeze protein is that it can be adsorbed on the surface of ice crystals, thus restricting the growth of ice crystals, inhibiting the recrystallization of ice crystals and changing the morphology of ice crystals.

Along with the discovery of various antifreeze proteins and the deepening of research, the two key problems that restrict the research and application of natural antifreeze proteins in the food field have become more and more prominent:
(1) The quantity of antifreeze proteins obtained by natural isolation and purification is tiny, and the very limited quantity restricts the prospect of their large-scale application in the food industry;
(2) While scientists are working on transgenic technology to expand the production of antifreeze proteins from living organisms, the safety of GM antifreeze proteins in food applications has become a common concern for consumers, the European Union, and the U.S. Food and Drug Administration (FDA).

Sources of antifreeze peptides

Antifreeze proteins are mainly derived from fish, insects, bacteria, plants and other organisms grown under extreme conditions such as high temperature and high altitude.

Antifreeze proteins can be categorized into four groups according to their sources: fish-derived antifreeze proteins, insect-derived antifreeze proteins, bacterial-derived antifreeze proteins and plant-derived antifreeze proteins; and according to their activities, they can be categorized into: AFP I to IV, hyperactive-AFP and antifreeze glycoproteins.

Natural antifreeze proteins have very low content in organisms, high purification cost, and large loss of activity during purification, which limits the research and large-scale application of antifreeze proteins. In contrast, antifreeze peptides are mainly obtained from food-borne protein sources by hydrolysis of specific enzymatic sites, which is characterized by controllable and efficient preparation.

Currently reported food-borne antifreeze peptides are mostly prepared from edible gelatin or processing by-products such as animal skin and fish scales.

Properties of antifreeze peptides

3.1 Thermal Hysteresis Activity
Antifreeze proteins can specifically lower the freezing point of a solution without affecting its melting point, so that the difference between the freezing point and melting point is called thermal hysteresis activity.

Studies have shown that the antifreeze active fragments of antifreeze proteins exist only in localized specific polypeptide chain structure domains, and their antifreeze activity is not the overall protein in action.

Hong Jing and Wu Jinhong also found that collagen antifreeze peptides with specific amino acid lengths and structures were able to bind to the ice layer through hydrogen bonding, and then inhibit the formation of ice crystals through hydrophobic interactions, indicating that the Kelvin effect of antifreeze proteins is also applicable to antifreeze peptides, which are also known as thermal hysteresis activity.

3.2 Recrystallization inhibition activity
When the temperature is lower than the melting point, ice crystals have a tendency to aggregate, and the recrystallization effect is the aggregation between ice crystals, and the aggregation of small ice crystals to form large ice crystals.

The recrystallization inhibition effect of antifreeze peptide can regulate ice crystals and prevent ice crystal aggregation, so that the size and shape of ice crystals can be regulated and the ice crystals formed are fine and uniform.

Under the effect of hydrogen bonding, hydrophobic interaction and van der Waals force, antifreeze peptide can regulate ice crystals and reduce the mechanical damage caused by ice crystals to the organism.

3.3 Cell membrane protection
When cells are in a frozen or supercooled state, the ice crystals generated in the surrounding and internal environment of the cells will cause mechanical damage to the cells, and cold stress will induce apoptosis, which will accelerate the death of the cells.Hirano, Tatsuro, and Davies reported that antifreeze proteins in fish can protect cell membranes from low-temperature damage.

Application of antifreeze peptides in the food industry

With the growth of global trade and expansion of production and marketing spacing, the demand for processed foods in the cold chain has increased. The share of frozen food in the food industry is also growing.

As a new class of food additives, antifreeze peptides can effectively reduce the formation of ice crystals and recrystallization in food during the cold chain process, thus improving the quality of low-temperature cold-chained food.

4.1 Ice Cream
The growth of ice crystals during cold storage is one of the main challenges faced by manufacturers of frozen products such as ice cream, because fluctuations in temperature during storage and handling can promote the growth of ice crystals, which can affect the taste of ice cream and degrade the quality of the product.

It is well known that there is a direct and close relationship between the size of ice crystals and the degree of roughness and/or the formation of ice crystal structure. Therefore, efforts must be made to reduce the size of ice crystals and the occurrence of recrystallization in ice cream production formulations, processing, storage and distribution conditions, because both ice crystal size and recrystallization formation have a large impact on ice cream texture.

Wang Shaoyun and Damodaran et al. used collagen hydrolysis to sieve ice structure peptide, it was found that this type of ice structure peptide can significantly reduce the size of ice crystals in the ice cream, and through the hot and cold cycle system to simulate the temperature fluctuations in the cold chain process, it was found that this type of ice structure peptide can significantly inhibit the ice crystal recrystallization in ice cream.

4.2 Probiotics
Probiotics are beneficial bacteria commonly used in food processing and are also key carriers in molecular biology, structural biology, microbiology and infectious disease research. The continuous cultivation of strains for a long period of time is not only time-consuming and labor-intensive, but also impractical, therefore, cryopreservation and freeze-drying techniques are usually used in industrial applications and academic research.

Anti-freezing peptides can significantly improve the probiotic freezing survival rate, freezing stability and maintain the metabolic vitality of bacterial cells. In addition, the antifreeze peptide can protect the cell membrane by hydrogen bonding with the cell membrane to reduce the leakage of intracellular substances; on the other hand, the antifreeze peptide can enter into the cell to reduce the damage of ice crystals formed in the cell during the freezing process.

4.3 Frozen dough
Modern freezing technology is an effective means to solve the problems of traditional staple foods such as easy aging and short shelf life. As a new type of food antifreeze, antifreeze peptides or ice-structured peptides have been reported in recent years to be used in the cryopreservation of frozen dough products.

Among them, Wang Shaoyun’s team from Fuzhou University, Zhang Hui’s team from Jiangnan University and Huang Weining’s team successfully applied antifreeze peptide to frozen dough and frozen potato dough. After the addition of antifreeze peptide, the fermentation time of frozen dough was significantly shorter and the specific volume after freezing was significantly higher than that of the control group. In addition, it was found that the specific volume of steamed bread made from frozen dough with antifreeze peptide was significantly higher than that of the control group.

4.4 Frozen meat
With global meat exports currently totaling more than US$13 billion, freezing technology plays a vital role in ensuring the safety of meat products supplied to the world. However, the effect of freezing and thawing on meat quality remains a major issue.

Repeated freezing and thawing primarily affects the moisture content of meat. Since moisture is contained within and at the interstices of muscle fibers, when moisture freezes, the concentration of remaining solutes (proteins, lipids, carbohydrates, minerals, and vitamins) increases, and ice crystals grow and recrystallize during the freezing process and the cold chain, thereby disrupting the homeostasis of the complex meat system and damaging the original organization of the meat.

To summarize

Adding antifreeze is an effective way to reduce the quality degradation of frozen food, and new antifreeze such as antifreeze peptide to replace the traditional high sugar and high salt commercial antifreeze is an inevitable trend with the growth of consumers’ demand for quality of life and healthy food.

Food-derived antifreeze peptides with specific peptide chain length and structural domain composition are an effective way to solve the problem of limited research and application of natural antifreeze proteins.